Wiley: Plant Biotechnology Journal: Table of Contents

Issue Information

Fri, 22 May 2026 00:54:26 -0700

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30 years ago, China developed homegrown Bt cotton. It eradicated bollworms, now commanding >99% of the market on 510 million mu, cutting pesticide use by 650,000 tons and adding ¥65 billion—pioneering independent biotech innovation. The cover image highlights three decades of contributions to cotton harvests and the textile industry.

Challenges in Bringing Pangenome Research Into Breeding: A Case Study in Rice

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Crop breeding has entered the pangenomics era, unlocking a far more comprehensive view of genetic diversity than a single reference genome can capture. In rice (Oryza sativa), a staple crop critical to global food security, the construction of pangenome resources has uncovered extensive structural variations (SVs), presence/absence variations (PAVs) and novel genes that underpin key agronomic traits. As the rice pangenome matures from a research resource into a practical breeding tool, it promises to accelerate the development of higher-yielding, stress-resilient and disease-resistant varieties. This transition represents a pivotal advance toward sustainable agriculture and enhanced global food security, while also establishing a model for applying pangenomics to other crops. Here, we review how rice pangenome research, encompassing both cultivated and wild species, has advanced trait discovery from yield improvement and disease resistance to stress tolerance and enabled new molecular breeding strategies. Despite these advances, several challenges remain before pangenomic data can be routinely integrated into breeding pipelines. The complexity of graph-based data structures, difficulties in detecting multiallelic variants from population-wide resequencing data and the lack of breeder-friendly genotyping tools are significant barriers. Additionally, while artificial intelligence (AI) and machine learning (ML) approaches show great promise for interpreting complex pangenomic data and accelerating trait discovery by genomic selection, their practical adoption is hindered by the absence of breeder-oriented interfaces, integration challenges with multi-omics data and high computational demands. Overcoming these issues will require interdisciplinary collaboration, robust infrastructure and innovations focused on practical breeding needs across diverse crop species.

The BAHD Acyltransferase Gene Family: Evolutionary Dynamics, Biochemical Mechanisms, and Roles in Plant Stress Adaptation

Fri, 22 May 2026 00:54:26 -0700

BAHD acyltransferases drive metabolic diversification in plants by coupling conserved catalytic scaffolds with regulatory flexibility, enabling stress adaptation and ecological specialisation.

ABSTRACT

BAHD acyltransferases constitute one of the most versatile enzyme superfamilies in plants, catalysing the acylation of alcohols, amines, polyamines, and phenolic compounds to generate an extraordinary diversity of specialised metabolites. Initially identified through a limited number of anthocyanin- and alkaloid-modifying enzymes, BAHDs are now recognised as key regulators of phenylpropanoid flux, cutin and suberin polymerisation, volatile ester biosynthesis, and the stabilisation of acylated flavonoids. Comparative genomic analyses classify BAHD proteins into eight clades that share conserved catalytic motifs yet display pronounced functional divergence, reflecting a balance between deep evolutionary conservation and lineage-specific innovation. Recent structural and biochemical studies demonstrate how subtle active-site modifications govern substrate promiscuity and specialisation, enabling rapid metabolic reprogramming during environmental stress. Omics-based investigations further reveal widespread induction of BAHD genes under drought, salinity, heat stress, pathogen attack, and herbivory, linking BAHD activity to cell wall reinforcement, phenolamide biosynthesis, anthocyanin acylation, and ecological signalling. Beyond their physiological roles, BAHD acyltransferases have emerged as attractive targets for metabolic engineering, synthetic biology, and crop improvement, where manipulation of specific family members enhances stress tolerance, biomass quality, and nutritional or industrial value. Here, we integrate evolutionary, structural, and regulatory insights into BAHD function, highlight emerging translational opportunities, and discuss challenges associated with functional redundancy, substrate promiscuity, and biosafety considerations. Collectively, this synthesis positions BAHD acyltransferases as central mediators of plant adaptation and as promising tools for sustainable agriculture and biotechnological innovation.

Plant Peptides on the Rise: From Historical Insight to Future Applications

Shunxi Wang, Jinghua Zhang, Minghui Chen, Bokai Zhang, Huina Zhao, Liuji Wu — Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Plant peptides constitute a rapidly expanding class of signalling molecules essential to plant physiology, mediating key processes such as development, stress adaptation, and immune responses. This review traces the history of plant peptide research, from the seminal discovery of systemin to the recent identification of non-canonical peptides (NCPs) translated from small open reading frames (sORFs) in non-coding RNAs. We delineate the distinct biosynthetic pathways of canonical peptides (CPs), which undergo proteolytic processing and post-translational modifications, and NCPs, which are directly translated, often without further processing. The diverse biological functions of these peptides span development, reproduction, abiotic stress tolerance, biotic defence, and antimicrobial activity. Furthermore, we discuss emerging agricultural applications, including genetic engineering of peptides, exogenous peptide application, and trait optimization informed by natural peptide variation. Beyond agriculture, many plant peptides exhibit therapeutic potential due to their antimicrobial and anticancer properties. Despite significant advances, challenges remain in functional validation, field application, and scalable production. Future progress will depend on the integration of multi-omics approaches, artificial intelligence (AI)-driven prediction, and precision genome editing to fully harness the transformative potential of plant peptides for crop improvement and novel biopharmaceuticals.

Three Decades of China's Bt Cotton: Achievements and Insights

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Cotton is a vital natural fibre crop with significant economic value worldwide. In response to the threat of cotton bollworm (Helicoverpa armigera), the China government initiated a research project in 1992 to develop transgenic Bacillus thuringiensis (Bt) cotton. Through domestic research and development efforts, China achieved a significant milestone in 1994: the successful development of Bt cotton. The project has continued since then, consistently advancing agricultural plant breeding efforts and providing economically critical new cotton germplasm to combat insect infestation and other stressors. We here present an overview of the 30-year history of this project, highlighting eight important lessons, six significant achievements, and three valuable insights gleaned from the pioneering agricultural endeavour. Chinese Bt cotton revolutionised the country's cotton industry and contributed to the economic growth and sustainability of Chinese agriculture. Through careful analysis and reflection, this article provides guidance for future development and implementation of agricultural biotechnologies.

Mycovirus Vector‐Mediated RNAi for Effective Gene Knockdown in Pine Wood Nematodes

Fri, 22 May 2026 00:54:26 -0700

Plant Biotechnology Journal, Volume 24, Issue 6, Page 3531-3533, June 2026.

BarleyGVDB: A Comprehensive Barley Variome Database for Population Evolution and Molecular Breeding Research in Barley

Fri, 22 May 2026 00:54:26 -0700

Plant Biotechnology Journal, Volume 24, Issue 6, Page 3570-3572, June 2026.

Telomere to Telomere Genome Assembly and Efficient Transformation and Genome Editing in Populus euphratica

Fri, 22 May 2026 00:54:26 -0700

Plant Biotechnology Journal, Volume 24, Issue 6, Page 4282-4284, June 2026.

Transgenic Lepidopteran‐Pests‐Resistant and Herbicide‐Tolerant Cotton Through Transfer of Cry1Ab‐vip3Aa and Cp4‐epsps+bar Genes

Qi Mou, Jun Zhang, Zhanfeng Si, Shangkun Jin, Wanying Zhang, Tianzhen Zhang — Fri, 22 May 2026 00:54:26 -0700

Plant Biotechnology Journal, Volume 24, Issue 6, Page 4102-4104, June 2026.

In Vivo Maternal Haploid Induction by Disrupting KOKOPELLI in Medicago truncatula

Fri, 22 May 2026 00:54:26 -0700

Plant Biotechnology Journal, Volume 24, Issue 6, Page 4041-4043, June 2026.

A New Breeding Technique for F1 Hybrid Production From Self‐Incompatible Species

Fri, 22 May 2026 00:54:26 -0700

Plant Biotechnology Journal, Volume 24, Issue 6, Page 4061-4063, June 2026.

A Near Gap‐Free Haplotype‐Resolved Genome Assembly of Zoysia japonica Uncovers Intra‐Subgenomic Gene Expression and Regulatory Variation

Sae Hyun Lee, Preethi Purushotham, Ambika Chandra, Murukarthick Jayakodi — Fri, 22 May 2026 00:54:26 -0700

Plant Biotechnology Journal, Volume 24, Issue 6, Page 4136-4138, June 2026.

Efficient Transgene‐Free Multiplexed Germline Editing via Viral Delivery of an Engineered TnpB

Fri, 22 May 2026 00:54:26 -0700

Plant Biotechnology Journal, Volume 24, Issue 6, Page 4358-4360, June 2026.

Tuning Rice Gene Expression via In Situ Promoter Truncations Using a Prime Editing Library

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Promoter engineering holds immense potential for fine-tuning gene expression and optimising agronomic traits, yet conventional genome-editing tools face limitations in precision, scalability, and risk mitigation. Here, we develop Prime Editing-mediated Promoter Engineering (PEPE), a DSB-free platform integrating bidirectional Protospacer adjacent motif (PAM) recognition (NGG/CCN) with combinatorial duo-pegRNA strategies to achieve tiled, overlapping deletions across entire plant promoters. Applying PEPE to the 1.8-kb rice D53 promoter, we generated a mutant library with stepwise deletions. Edited alleles showed stable inheritance, and dual-method validation confirmed the precision at junctions. Quantitative profiling revealed functional modularity: core-region deletions suppressed D53 expression by 70%–85%, while a distal deletion (D53-G9C10) paradoxically upregulated transcription 2.2-fold, uncovering a cryptic repressor element. Phenotypic variation corresponded with transcriptional changes, establishing a direct link between cis-regulatory diversity and agronomic traits. By circumventing DSBs and enabling kilobase-scale CRE manipulation, PEPE establishes a robust framework for decoding promoter logic and accelerating trait pyramiding in crops. This study advances plant genome editing by merging precision with scalability, offering transformative potential for breeding climate-resilient, high-yield cultivars.

First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue

Susanne K. Vollmer, Markus G. Stetter, Götz Hensel — Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Grain amaranth is a nutritionally rich, stress-tolerant C₄ dicot with considerable potential for climate-resilient agriculture; however, efficient and reproducible protocols for stable transformation, regeneration, and CRISPR/Cas9-mediated editing have not yet been established. CRISPR/Cas-based genome editing is a cornerstone technology for accelerating the development of climate-resilient, high-yielding crops. Its effective application depends on robust, stable transformation procedures and CRISPR/Cas systems optimised for the target species. The absence of such tools remains a critical constraint for the genetic improvement of many promising yet underexplored crops. In this study, we edited key genes of the betalain biosynthesis pathway in grain amaranth (Amaranthus hypochondriacus L.) using the CasCADE modular cloning system, thereby demonstrating the feasibility of targeted mutagenesis in an orphan crop. We observed successful edits in up to 49% of transformed calli, resulting in deletions or insertions in the target genes. Our CRISPR/Cas9-mediated editing paves the way for targeted molecular research and breeding of grain amaranth.

The Rice E3 Ubiquitin Ligase Gene OsPUB77 Regulates Head Milled Rice Rate by Affecting Grain Starch Accumulation

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Head milled rice rate (HMRR) is a critical trait determining rice yield and economic value, yet its genetic basis remains poorly understood. Here, through genome-wide association study across multiple environments, we identified a major QTL, qHMRR4-2, and pinpointed OsPUB77, a U-box E3 ubiquitin ligase, as the causal gene. Haplotype analysis and transgenic validation revealed that both knockout and overexpression of OsPUB77 significantly reduce HMRR by disrupting starch accumulation and increasing grain chalkiness. We further demonstrated that OsMADS29 directly binds to the OsPUB77 promoter and represses its transcription. Transcriptomic analysis indicated that OsPUB77 maintains metabolic homeostasis essential for starch biosynthesis during grain filling. Our findings establish the OsMADS29-OsPUB77 module as a critical regulator of HMRR and provide a promising target for improving rice milling quality through precision breeding.

LUCID: An Integrative Approach for Target Discovery and dsRNA Design in Plant Fungal Pathogens

Fri, 22 May 2026 00:54:26 -0700

LUCID: A computational pipeline for RNAi-based biofungicide design.

ABSTRACT

Phytopathogenic fungi pose an escalating threat to global food security and ecosystem stability, as resistance and environmental concerns diminish the effectiveness of conventional fungicides. Double-stranded RNA (dsRNA)-based fungicides offer a species-specific, eco-friendly alternative. We introduce LUCID (Locating Uncovered, conserved, and Indispensable for pathogenicity Determinants), a computational pipeline that accelerates the development of RNAi-based biofungicides by integrating target identification with dsRNA design and off-target prediction. LUCID employs a dual-branch strategy to identify both Conserved Essential Proteins (CEPs) and Conserved Non-Annotated Proteins (CNAPs), leveraging transcriptomic data and comparative genomics across diverse fungal species. Validation in Botrytis cinerea demonstrated high efficacy, with 67% of proposed targets successfully silenced and an average silencing efficiency of 96%. Additionally, coupling LUCID with advanced protein language models (PLMs) revealed a novel pathogenicity determinant in B. cinerea: a putative mediator complex protein. LUCID offers a scalable, species-agnostic framework for designing sustainable fungicides, enabling rapid, targeted control of fungal diseases with minimal ecological impact.

Enhancing CRISPR/Cas‐Mediated Gene Knockout With Short Non‐Homologous Oligonucleotides

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Chlamydomonas reinhardtii is a model green microalga that has great industrial potential as a sustainable bio-factory for recombinant protein and high-value chemical production. Efficient genome editing tools are required to redesign this organism for synthetic biology applications. CRISPR-Cas editing technologies have already been adapted for gene knockout, transgene knock-in, and precise gene editing in C. reinhardtii. However, the efficacy of CRISPR/Cas-mediated gene knockout (KO) is low, which hampers pathway engineering and functional genomic studies. Here we report that co-delivery of CRISPR-Cas gene editing reagents with short double-stranded non-homologous oligodeoxynucleotides (dsNHO) increases gene knockout efficacy up to 100-fold in C. reinhardtii. This phenomenon, referred to as non-homologous oligonucleotide enhancement (NOE), is heavily affected by the length, structure, and chemical modifications of dsNHO, and is largely mediated by the DNA double-stranded break sensor KU70/80 (KU) heterodimer in a Cas nuclease-, locus-, and strain-independent manner. Our data suggest that dsNHOs disrupt the cell's double-stranded break (DSB) sensing pathways, consequently shifting the balance of DNA repair from canonical non-homologous end joining (c-NHEJ) towards the more error-prone, microhomology-mediated end joining (MMEJ), which could be harnessed as a strategy for improving gene KO efficiency in Chlamydomonas and beyond.

Synthetic Raphanobrassica Genome Reveals Functional and Evolutionary Insights Into Clubroot Resistance Genes on Chromosome R5

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Clubroot, a severe soil-borne disease caused by Plasmodiophora brassicae, poses a severe threat to global production of Brassicaceae oilseed crops and vegetables. To date, there has been a serious lack of clubroot-resistant germplasms in Brassica napus (AACC), necessitating the urgent development of novel disease-resistant germplasm. We present a high-quality genome assembly of an artificially synthesised allotetraploid Raphanobrassica (RRCC), which exhibits broad-spectrum immunity to diverse P. brassicae pathotypes. Using a sesquidiploid RACC as a genetic bridge, we developed B. napus-R. sativus (AACC-R) monosomic addition lines and mapped a major clubroot resistance (CR) locus on chromosome R5. Comparative genomic analysis identified 30 candidate CR genes in this region. Notably, overexpression of CRR5.5.11, which encodes a receptor-like protein, via hairy root transformation conferred significant resistance in susceptible B. napus. Evolutionary and functional analyses revealed conserved homologues of CRR5.5.11 in diploid Isatis tinctoria and triploid turnip ECD04. Furthermore, the ECD04 allele CRA3.2.2 also conferred clubroot resistance, indicating that CRR5.5.11 and CRA3.2.2 have originated from the same diploid ancestor. Our study elucidates the genetic and evolutionary basis of clubroot resistance in Raphanobrassica, providing novel germplasm, gene resources and theoretical insights for clubroot-resistance breeding in rapeseed and other Brassicaceae crops.

Rooting Conifer Genetic Research: An Accessible and Efficient Transformation System

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Conifers serve as the cornerstone of global forest ecosystems, yet their genetic transformation faces notorious challenges. To overcome the intrinsic resistance of conifer adventitious roots to Agrobacterium/Rhizobium-mediated transformation, we systematically and iteratively engineered the binary vector by introducing chimeric Ri plasmid derived T-DNA borders, a phosphomimetic VirG mutant (VirGN54D) and a hyperactive replication origin mutant (pVS1 R106H). This optimised system enabled efficient, cross-species genetic transformation in diverse plants, including the recalcitrant gymnosperm Ginkgo biloba, thus establishing a broadly applicable genetic toolkit for plant research. Leveraging this system, we established a non-aseptic efficient root transformation system for Pinus tabuliformis. Given that conifers are evolutionarily ancient gymnosperms, this readily accessible system provides an unprecedented window to probe fundamental questions in functional genomics and evolutionary mechanisms, such as the dynamics of long-distance macromolecular trafficking. Transgenic deployment of Arabidopsis florigen FT in Chinese pine roots uncovered conserved protein level long-distance mobility, despite lacking functional FT orthologs in conifer genomes. Conversely, the conifer age biomarker DAL1 exhibited no detectable mobility even when engineered fusion with tRNA-like sequence (TLS) tag, which has been shown to facilitate long-distance mRNA transport in angiosperms, exposing lineage-specific constraints on mobile mRNA. Our work provides a versatile genetic tool for challenging plant species and offers new insights into long-distance signalling evolution. The streamlined protocols and universal vector system address critical bottlenecks in conifer biotechnology and open avenues for functional genomics in other non-model plants.

Poplar miR1447 Is a Negative Regulator of Disease Resistance Through the SA‐Dependent Pathway

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Although microRNA1447 (miR1447) regulates poplar responses to abiotic stress and pest infestation, how miR1447 regulates poplar immunity against pathogens and its underlying molecular mechanisms remain to be elucidated. Here, we revealed that miR1447 functioned as a negative regulator of poplar disease resistance against fungal and bacterial pathogens using overexpression (OE) and short tandem target mimic (STTM) poplar lines of miR1447. Moreover, we demonstrated that PopTCTP contributed to poplar immunity as a target of miR1447 through integrative analysis of overexpression and RNAi lines, degradomes, transient co-expression assay and GFP fluorescence report system, and found that PopTCTP interacted with dnaJ A6. Further molecular and genetic analyses revealed that the promoters of miR1447 and PopTCTP were responsive to exogenous salicylic acid (SA) treatment. We showed that the negative regulatory role of miR1447 in SA signalling and poplar resistance was weakened with exogenous SA treatment. Notably, the miR1447-PopTCTP module contributed to PTI in poplar triggered by flg22 and associated with crosstalk between PTI and ETI via regulating MAPK signalling and scavenging ROS. Taken together, these findings unveil a novel pathway by which the miR1447-PopTCTP-SA signalling mediates disease resistance to diverse pathogens in poplar, offering promising genetic targets for tree breeding of disease resistance.

Host‐Induced Silencing of Rhizoctonia Solani 5‐Enolpyruvylshikimate‐3‐Phosphate Synthase Impairs Its Virulence in Rice

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Sheath blight disease of rice caused by the fungal pathogen R. solani AG1-IA remains a big threat to rice production worldwide. A limited genetic variation in rice for tolerance to this pathogen and little success in understanding how it defeats host defence are major reasons behind it. In this study, we attempted to decode the virulence spectrum of R. solani AG1-IA in rice using time-course transcriptome analysis and functional genomics tools. Several stage-specific and commonly expressed genes were identified. Notably, the shikimate pathway emerged as an important pathway and was implicated in the virulence of R. solani AG1-IA. Inhibition of the shikimate pathway by glyphosate, a known inhibitor of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, reduced the vegetative growth of R. solani AG1-IA and several other phytopathogens. Our results were complemented by in vitro inhibition studies of RsEPSP synthase using a recombinantly expressed protein. Comparative sequence analysis of RsEPSP synthase with plants and known major phytopathogens revealed a distinct region in RsEPSP synthase. Using Nicothaiana benthamiana as a model system and stable rice transgenic lines, we demonstrated that targeting this distinct region through host-induced gene silencing (HIGS) compromises the growth and virulence of R. solani AG1-IA. The study lays a foundation for a deeper understanding of the identified virulence genes and establishes the shikimate pathway as a central target to control phytopathogens.

Ubiquitination‐Mediated Degradation of SlVPS29 by the SlHSFA7‐SlRNF185 Module Enhances Tomato Pollen Thermotolerance

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

High temperature has posed significant challenges to global agriculture, markedly leading to reduced fertility and yield losses in tomato (Solanum lycopersicum). Therefore, thermotolerance-conferring genes and loci are needed to further improve cultivated tomatoes. Here we identified an E3 ubiquitin ligase SlRNF185, containing a C3HC4-type RING-HC domain, that confers tomato pollen thermotolerance. As a predominant ubiquitination member, SlRNF185 could degrade the SlVPS29 protein induced by heat stress to enhance thermotolerance. Mechanistically, we found that a heat shock transcription factor SlHSFA7 dramatically activates the expression of SlRNF185 under heat stress and acts as a positive regulator of tomato pollen thermotolerance. Collectively, our findings reveal that a SlHSFA7-SlRNF185 genetic module regulates ubiquitination-mediated degradation of SlVPS29 under heat stress, providing the strategy for breeding thermotolerance tomato varieties.

An Elite Haplotype of Nitrogen‐Use‐Efficiency Gene LHT5 Enhances Salt Tolerance in Rice

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Amino acids serve as fundamental building blocks and signalling molecules in plants, orchestrating stress adaptation mechanisms against diverse biotic and abiotic environmental challenges. However, the mechanism by which plants alter their nutrient metabolism processes to coordinate nitrogen use efficiency (NUE) and salt tolerance remains elusive. Here, we identified a Lysine-Histidine-type transporter 5 (LHT5) gene through genome-wide association studies (GWAS) that enhances NUE via amino acid accumulation regulation. Further research showed that OsLHT5 also confers salt tolerance in rice by promoting proline biosynthesis through transcriptional upregulation of OsP5CS1 and OsP5CS2 genes, thereby increasing cellular proline levels for osmotic adjustment. Notably, we identified a functionally critical 30-bp deletion in the OsLHT5 coding region, designated as the elite haplotype LHT5 ^HapA, which substantially enhances amino acid transport capacity and consequently improves both NUE and salt tolerance. Functional validation demonstrated that overexpression of LHT5 ^HapA significantly increases amino acid content, nitrogen accumulation, grain yield and salt stress tolerance compared to the wildtype allele. This study establishes a novel molecular framework linking amino acid transport to the coordination of nutrient utilisation and stress tolerance, offering valuable genetic resources and breeding strategies for developing climate-resilient rice cultivars with enhanced productivity under both optimal and saline conditions.

Hydrogen Gas Enhances Salinity Tolerance in Tomato Seedlings by Regulating the S‐Nitrosylation of MEK1

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Hydrogen gas (H₂) effectively alleviates abiotic stress in horticultural plants. Protein S-nitrosylation, a key post-translational modification, serves as a critical mechanism for nitric oxide (NO) to exert its biological function under adverse conditions. However, the relationship among H₂, NO and S-nitrosylation in response to salt stress remains poorly understood. In this study, we demonstrate that NO participated in H₂-enhanced salt tolerance in tomato seedlings (Solanum lycopersicum cv. Micro-Tom). H₂ triggered an increase in NO levels and S-nitrosothiol (SNO) content under salt stress, enhancing the enrichment of S-nitrosylated proteins. S-nitrosoproteomic analysis revealed that MEK1, a conserved MAPK component, was notably induced and S-nitrosylated by NO and H₂ under salt stress. Furthermore, SlMEK1-overexpressing tomato lines exhibited enhanced salt stress tolerance, while knockout lines showed reduced tolerance, indicating the positive regulatory role of SlMEK1 in salt tolerance. Additionally, SlMEK1 may contribute to H₂-enhanced salt tolerance. Meanwhile, MEK1 was shown to undergo S-nitrosylation at Cys 172, and this modification was involved in H₂-enhanced salinity tolerance. Moreover, site-specific mutation analysis at Cys 172 confirmed that MEK1 S-nitrosylation positively contributed to both intrinsic and H₂-facilitated salt tolerance. Finally, S-nitrosylation of MEK1 increased its interaction with GSNOR, and H₂ further promoted this interaction under salt stress. Collectively, our findings indicate that H₂ may enhance the salinity tolerance in tomato seedlings by orchestrating the interplay between S-nitrosylated MEK1 and GSNOR.

PlantCTCIP: Chromatin Interaction Prediction Using Convolutional Neural Network and Transformer in Plants

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Chromatin interactions establish spatial proximity between distant regulatory elements and their target genes, significantly influencing gene expression, and phenotypic traits. In this study, we present a plant chromatin interaction prediction model called PlantCTCIP based on Convolutional Neural Networks and Transformer. PlantCTCIP demonstrated superior performance compared to the conventional models. Specifically, PlantCTCIP improved the average AUC of chromatin interaction predictions by 14.56% across the four species in PPI (proximal promoter interaction) mode. Similarly, PlantCTCIP improved the average AUC of chromatin interaction predictions by 9.6% in the PDI (distal promoter interaction) mode. We constructed genome-wide chromatin interaction maps for four plants (maize, rice, cotton and wheat) through PlantCTCIP, further used the Hi-C experiment to validate correctness of the predicted PPIs and PDIs. Some key motifs that influence chromatin interactions are identified, and they are significantly enriched in expression quantitative trait loci (eQTLs) and open chromatin regions. We also analysed the enrichment and species specificity of the transcription factors (TF) and synergistic network of TFs that affect PPIs and PDIs of four crops. Using cloned genes (ZmRAVL1, ZmRPG, ZmRap2.7 and GaFZ) of maize and cotton as examples, PlantCTCIP can assist in identifying target genes regulated by distal elements and mining functional sites combined with chromatin conformation capture (3C) experiments. This research helps to analyse the regulatory mechanism of gene expression and provides novel perspectives for intelligent design breeding of diverse crops. PlantCTCIP is available at http://www.plantctcip.com.

The Structure of Carboxyl Methyltransferase Provides Insights Into the Substrate Specificity and Divergent Evolution of Iridoid

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Iridoids constitute a prominent class of plant-specialised metabolites, with carbocyclic iridoids (e.g., geniposide) and secoiridoids (e.g., loganin) diverging early in their biosynthetic pathways. This divergence is marked by substrate-specific carboxyl methyltransferases—GjGAMT and CrLAMT—that catalyse the decisive methylation step in Gardenia jasminoides and Catharanthus roseus, respectively. However, the molecular determinants responsible for their strict substrate specificity and evolutionary relationship remain unclear. In this study, the substrate recognition mechanism of GjGAMT was elucidated through a combination of X-ray crystallography, phylogenetic analysis, site-directed mutagenesis and biochemical assays. GjGAMT forms an asymmetric homodimer, wherein one active site is occupied by both the cofactor product SAH and substrate geniposidic acid, while the other site contains only SAH, suggesting a half-of-the-sites catalysis mechanism. Structural comparison between GjGAMT and CrLAMT suggested that residues Phe311 and Phe315 in GjGAMT (corresponding to Val317 and Met321 in CrLAMT) are critical for substrate discrimination. Consistently, the double mutation F311V/F315M in GjGAMT conferred activity toward loganic acid, whereas reciprocal mutations in CrLAMT enabled recognition of geniposidic acid. Furthermore, functional charaterisation of cytochrome P450 enzymes (CYP72As) showed that CrSLS1 (CrCYP72A1) catalyses ring cleavage of loganin but not geniposide, whereas homologous GjCYP72As lack cleavage activity. Altogether our study elucidates the structural mechanism driving substrate specificity in iridoid methyltransferases, thereby deepening our understanding of how catalytic plasticity shapes metabolic diversity in plants and offering a framework for rational enzyme engineering.

OsbHLH064, an IVb bHLH Transcription Factor, Regulates Iron Homeostasis and Enhances Grain Fe Accumulation in Rice

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Iron (Fe) is an essential micronutrient for plant growth and development. The maintenance of Fe homeostasis relies on sophisticated regulatory networks where bHLH transcription factors play a key role. However, how these factors coordinate to regulate this vital process is not fully understood. Here, we characterise OsbHLH064, a previously unstudied IVb bHLH transcription factor, and reveal its critical role in iron homeostasis in rice. Loss of OsbHLH064 results in constitutive activation of Fe homeostasis-related genes under Fe-sufficient conditions, whereas its overexpression strongly suppresses their expression. Remarkably, OsbHLH064 overexpression leads to excessive Fe accumulation in roots, shoots and brown rice. Under Fe deficiency, it also triggers ROS overproduction, highlighting its essential role in balancing Fe homeostasis and oxidative stress. Mechanistically, OsbHLH064 forms heterodimers with IVc bHLH transcription factors, and these interactions facilitate its nuclear localisation. OsbHLH064 represses OsIRO2 and OsIRO3 by competing with or sequestering IVc activators at shared promoters, thereby limiting downstream transcriptional activation. Furthermore, OsbHLH064 directly binds not only canonical IVb/IVc targets but also a broader set of genes involved in Fe uptake, translocation and signalling. Collectively, these findings establish OsbHLH064 as a central upstream regulator integrating multiple pathways to maintain Fe homeostasis and suggest its potential as a target for biofortification strategies aimed at enhancing iron content in rice grains.

VqLecRKV.4 and VqBAK1 Modulate Grapevine Resistance to Powdery Mildew by Regulating Dynamic Balance of ROS

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Grapevine powdery mildew, caused by the fungal pathogen Erysiphe necator, severely impacts plant growth and berry quality. However, the grapevine receptors and molecular mechanisms underlying grapevine resistance to E. necator remain poorly understood. In this study, we identify a G-type Lectin receptor-like kinase (LecRK), VqLecRKV.4, identified from the wild Chinese grapevine Vitis quinquangularis, whose expression is significantly upregulated in response to E. necator infection. Overexpression of VqLecRKV.4 in the susceptible cultivar V. vinifera ‘Thompson Seedless’ confers enhanced resistance against the pathogen, as evidenced by significantly reduced fungal colonisation and sporulation. Through TurboID-mediated proximity labelling, we demonstrate that VqLecRKV.4 interacts with VqCu/ZnSOD1. Further analysis reveals that VqLecRKV.4 promotes ROS accumulation and cell death by upregulating VqCu/ZnSOD1 expression, thereby inhibiting E. necator colonisation. A critical challenge in plant immunity is balancing immune responses to avoid overactivation. Here, we discover that VqBAK1 interacts with VqLecRKV.4 and attenuates its overreaction. Collectively, our findings reveal that the VqLecRKV.4-VqBAK1 module fine-tunes grapevine resistance to powdery mildew by maintaining ROS homeostasis, providing novel insights into the molecular mechanisms of grapevine immunity and its regulation to prevent detrimental overreaction.

Chromosome‐Level Genome Assembly of the Allotetraploid Gynostemma pentaphyllum Provides Novel Insights Into the Biosynthesis of Ginsenoside and Gypenoside LVI

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Gynostemma pentaphyllum, a herb used in tea and traditional Chinese medicine, shows geographic variation in its production of valuable dammarane-type ginsenosides and gypenoside LVI between populations from Suining (SN) and Nanning (NN). To elucidate the mechanisms underlying this differential metabolite accumulation, a chromosome-level genome for G. pentaphyllum (SN population) was assembled. The analysis revealed that SN is a tetraploid (~1.2 Gb), resulting from a recent whole-genome duplication event in a diploid ancestor. Phylogenetic analysis indicates SN and diploid NN share a recent common ancestor, diverging approximately 4.95 million years ago. Chromosome evolution analysis confirmed SN is an allotetraploid with clear subgenomic differentiation. This genome, combined with multi-omics data, enabled the screening of candidate P450 genes involved in ginsenoside/gypenoside LVI biosynthesis. In vivo and in vitro experiments confirmed that GpCYP88AB3 functions as a bifunctional enzyme by first hydroxylating dammarenediol-II at C-12 to yield protopanaxadiol (PPD), and then hydroxylating PPD at C-2 to form 2α-OH-PPD. Phylogenetically, GpCYP88AB3 and similar enzymes from Araliaceae belong to distinct CYP subfamilies, demonstrating convergent evolution of this function between the two plant families and highlighting the functional plasticity of P450s. Evolutionary analysis suggests that GpCYP88AB3 emerged from a CYP88 gene family expansion in the tetraploid G. pentaphyllum. This expansion occurred after, but was not directly caused by, the whole-genome duplication event. This study elucidates the biosynthetic pathway for the key metabolites in G. pentaphyllum, providing a foundation for future metabolic engineering and synthetic biology applications.

Gallic Acid‐Responsive microRNAs Reprogram Lignification During Drought Acclimation Process in Spearmint

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Mentha spicata L. (spearmint) is a high-value aromatic and medicinal species, whose productivity is strongly affected by water deficit. Nevertheless, the molecular mechanisms underlying drought acclimation in this mint remain largely unexplored. Thus, here, we investigated the microRNA-mediated regulatory processes triggered in M. spicata under drought stress (DS) and following treatment with gallic acid (GA), a natural phenolic compound that our research group has already documented to be a potential biostimulant for spearmint. A small-RNA sequencing approach revealed that both DS and GA induced substantial changes of the expressed miRNome, modulating 35 microRNAs (e.g., miR397a, miR159a, miR172b) whose predicted targets (e.g., Laccase-2, MYB transcription factors) are known to be involved also in lignin production. In detail, DS induced upregulation of lignin biosynthetic genes, enhancement of Laccase activity, and shifting in lignin monomer composition, promoting the putative reinforcement of the cell wall as expected during water deficiency. Conversely, GA treatment attenuated DS-induced stress, regulating microRNA-mRNA modules which balanced phytochemical and hormonal response while maintaining controlled lignification and optimising xylem function. These results highlight the pivotal role of microRNAs in orchestrating drought acclimation in M. spicata and identify GA as a compensatory agent under water-limiting conditions, capable of fine-tuning growth, cell wall remodelling, and redox homeostasis. Collectively, our findings provide molecular insights into biostimulant-mediated stress resilience and identify GA treatment as a promising biotechnological strategy to improve drought tolerance in Lamiaceae crops.

The Advantaged Salt Inducible Suaeda salsa SsNRT2.5 and Its Promoter Significantly Enhance Nitrate Transport Efficiency and Salt Tolerance in Transgenic Arabidopsis and Rice

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Efficient nitrogen (N) uptake is critical for crop yield, but soil salinization inhibits plant nitrogen acquisition. In this study, the nitrate (NO₃ ⁻) transporter gene SsNRT2.5 and its promoter from the halophyte Suaeda salsa was investigated to elucidate the functional role in NO₃ ⁻ transport under salinity and low NO₃ ⁻–N conditions. SsNRT2.5 and its promoter were cloned and transformed into Arabidopsis thaliana and rice (Oryza sativa L.) for functional identification, which included analyses of expression patterns, promoter cis-element characterisation and phenotypic assessments under salt and low NO₃ ⁻–N conditions. SsNRT2.5 expression was significantly upregulated under salt stress and low NO₃ ⁻–N conditions in S. salsa, which improved NO₃ ⁻ transport in trangenic Arabidopsis thaliana and rice. Its promoter contained salt-responsive (e.g., GT-1, DRE) and N-related (e.g., GATABOX) elements, which drove stronger salt-induced NRT2.5 expression than AtNRT2.5 promoter. Transgenic Arabidopsis and rice with SsNRT2.5 and its promoter showed enhanced NO₃ ⁻ accumulation, reduced Na⁺ toxicity, and higher salt tolerance, as well as improved seed NO₃ ⁻ storage and viability compared to WT. SsNRT2.5 plays a key role in the adaptation of S. salsa to high saline and nitrogen-limited environments, offering valuable genetic resources and theoretical insights for breeding salt-tolerant crops and developing sustainable saline agriculture.

The miR171a‐TaSCL6‐1 Module Acts Downstream of miR164‐Targeted TaNAC21/22 to Regulate Leaf Rust Resistance in Wheat

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Wheat leaf rust, caused by Puccinia triticina, poses a significant threat to global wheat production. MicroRNAs (miRNAs) are critical regulators of plant growth, development and stress responses; however, their role in wheat resistance to leaf rust remains poorly understood. In this study, we identified the miR171a-TaSCL6-1 module as a key regulator of leaf rust resistance in wheat. Expression of miR171a was upregulated in wheat seedlings following pathogen inoculation. Transgenic assays, including overexpression and short tandem target mimic approaches, demonstrated that miR171a negatively regulates resistance to leaf rust. Degradome sequencing confirmed TaSCL6-1 as the primary target of miR171a, and both overexpression and knockout of TaSCL6-1 established its role as a positive regulator of resistance. Transcriptome analysis revealed that TaSCL6-1 upregulates defence-related genes encoding peroxidases and transcription factors. TaSCL6-1 can activate the transcription of downstream genes by binding to the GAA motif in the promoters, and silencing of downstream genes (TaPOD2, TaPOD35 and TaERF114) increased wheat susceptibility to leaf rust. Furthermore, miR164-targeted TaNAC21/22 acts as an upstream regulator of MIR171a by binding to its promoter and activating its expression. Overexpression of TaNAC21/22 increased susceptibility to leaf rust, whereas transient silencing of TaNAC21/22 or overexpression of miR164 enhanced seedling resistance to leaf rust. Our findings elucidate the role of the miR171a-TaSCL6-1 module in regulating wheat defence against leaf rust, providing potential genetic targets for breeding resistant wheat varieties.

Dynamics of Gene and Allelic Expression During Modern Hybrid Maize Breeding

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Maize breeding has greatly improved yield through single-cross hybrids, but the underlying gene regulatory changes remain unclear. This study analysed transcriptomes of landmark maize hybrids and their parents across developmental stages and planting densities. Compared with their parents, hybrids showed a trade-off in the expression of photosynthesis-related genes and stress-responsive genes. This expression rebalancing suggested a strategy that prioritises photosynthetic efficiency and growth vigour over stress defence mechanisms. Allele-specific expression (ASE) analysis identified 19.9% of heterozygous loci exhibiting significant allelic imbalance, with notable enrichment in photosynthesis and stress response pathways. Importantly, the suppressed expression of deleterious alleles in hybrids not only correlated with phenotypic performance but also exhibited progressive enhancement through decades of breeding, indicating this regulatory mechanism has been selected during improvement. Consistent with this finding, breeding selection preferentially acted on cis-regulatory regions, with stronger correlation between cis-regulatory complementation of deleterious variants and hybrid release year compared to coding regions. Transcriptomic plasticity across environments was evaluated using the concept of entropy. Results showed that hybrids had lower transcriptomic entropy than their parental lines, and this reduction in entropy was significantly associated with heterosis. These findings highlight the critical role of allelic expression optimization in maize hybrid breeding and provide insights into the transcriptomic dynamics that underlie heterosis.

Systemic Delivery of Functional Proteins Into Plants Using an Engineered Membrane Translocation Domain

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Protein-based biopesticides and biostimulants are critical for the future of sustainable agriculture, yet their utility is severely limited by inefficient delivery into plant cells. Traditional cell-penetrating peptides enable protein uptake but lack the efficiency, systemic activity and scalability required for crop production. Here, we present a potential general solution using a novel engineered membrane translocation domain, MTD4, to enable robust, systemic protein delivery into crops and it is suitable for large-scale application. We first demonstrate that MTD4 enables rapid foliar delivery of a model protein (SEP) and, importantly, facilitates its systemic translocation from lower to upper leaves and from roots to shoots, a key requirement for whole-plant protection. To prove the platform's utility, we fused MTD4 to the harpin protein HrpZ, a known defence elicitor. MTD4-HrpZ delivered into tobacco and tomato plants triggered a potent hypersensitive response and systemic acquired resistance, resulting in significant reductions in disease severity from bacterial and fungal pathogens. Remarkably, the MTD4-HrpZ fusion protein was over five times more effective than HrpZ alone, highlighting MTD4's capacity to dramatically enhance protein efficacy. This work introduces MTD4 as a transformative tool for overcoming protein delivery barriers in plants, paving the way for a new generation of high-potency biotherapeutics that can advance sustainable crop protection and reduce dependence on chemical applications.

AhBWR15, A Novel RLK Gene, Confers Resistance to Ralstonia solanacearum in Peanut

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Bacterial wilt (BW), a severe soil-borne disease caused by Ralstonia solanacearum, significantly impedes global peanut production. Despite its impact, the mechanisms underlying BW resistance in peanut remain unclear. Herein, we selected the highly resistant variety Nongdahua108 (H108) and the susceptible variety Nongdahua107 (H107) to develop a genetically segregating population. Bulked segregant analysis sequencing (BSA-seq) of the F₂ population (n = 432) revealed a novel major quantitative trait locus (QTL) qBWR15, located within a 5.9-Mb region on Chromosome 15 (B05). Subsequently, 25 new molecular markers were developed within this region, allowing for the fine mapping of qBWR15 to a 668-kb region containing four genes with non-synonymous mutations. Notably, one of these genes, a leucine-rich repeat receptor-like kinase (LRR-RLK) designated as AhBWR15, exhibits a non-synonymous G > A mutation in the first exon. This mutation results in alterations to the encoded amino acid and the three-dimensional structure of the protein. The 238 natural germplasms analysis also identified this novel mutation occurring exclusively in the peanut variety H108. AhBWR15 is localised on the plasma membrane and is responsive to R. solanacearum infection and phytohormone inductions. Overexpression of the LRR-RLK gene AhBWR15 can significantly enhance resistance to BW by activating abscisic acid (ABA)-signalling pathways. Our research provides valuable insights into a novel LRR-RLK-mediated disease resistance pathway, which has significant implications for enhancing peanut resistance to BW.

Multi‐Omics Insights Into Anthraquinone Biosynthesis in Rheum tanguticum

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Rheum tanguticum is renowned for its medicinal properties, including purgative, anti-inflammatory and hepatoprotective effects, primarily attributed to anthraquinones (AQs). However, the molecular mechanisms of AQs biosynthesis have largely been hindered by insufficient genomic resources and functional genomics investigations. Here, we employed multi-omics approaches to address this knowledge gap. The high-quality T2T-level genome was constructed with a size of 2.68 Gb and a contig N50 of 233.65 Mb. Functional annotation revealed that the specific and expanded gene families of R. tanguticum are involved in efficient energy metabolism and secondary metabolite biosynthesis, providing a molecular basis for its stress adaptation and medicinal value. Integrated widely targeted metabolomics, spatial metabolomics and targeted quantification of AQs, we successfully elucidate the AQs spatiotemporal accumulation patterns. Seeds and leaves are key sites for the synthesis and transport of free AQs, while the root core serves as the primary location for the synthesis and accumulation of conjugated AQs. Through integrated genomic, transcriptomic, metabolomic and functional validation analyses, we preliminarily characterised the positive regulatory role of RtPKSIII-8 in the synthesis of aloe-emodin and emodin, as well as the potential function of RtUGT85AD11 in the biosynthesis of AQs and flavonoids. These findings provide essential genomic and functional data for deciphering AQs biosynthetic pathways and lay a theoretical foundation for the medicinal development and genetic improvement of R. tanguticum.

Engineering a Gibberellin‐Switchable Dual‐Use Line Based on Ent‐Kaurene Oxidase Gene ZmKO1 Enables Two‐Line Hybrid Seed Production in Maize

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Male-sterile systems are pivotal for maize hybrid seed production, yet existing systems are constrained by significant limitations. Here, we identified a novel single-gene-controlled recessive genic male sterility (GMS) mutant, defective in filament elongation 1 (def1), which exhibits non-elongated filaments, unopened glumes, and non-exserted anthers, yet undergoes normal microsporogenesis and produces viable pollen. Map-based cloning revealed that def1 is caused by a loss-of-function mutation in the ZmKO1 gene. Previous studies have identified ZmKO1 and ZmKO2 as the two putative KO-encoding genes in maize. Although heterologous expression evidence suggested that only ZmKO1 possesses KO activity, we demonstrate that both ZmKO1 and ZmKO2 are functional KOs for GA biosynthesis. The def1 mutant or ZmKO1 knockout lines exhibited reduced plant height during early seedling development but recovered rapidly to wild-type levels. In contrast, ZmKO2 knockout lines displayed severe dwarfism and pollen-less male sterility. These contrasting phenotypes demonstrate a clear functional divergence between the two genes. Utilising marker-assisted backcrossing and CRISPR/Cas9 editing, we generated ZmKO1-based GMS lines across diverse genetic backgrounds. These lines maintained stable male sterility and could be efficiently propagated via exogenous GA₃ application under different genetic backgrounds and environments, demonstrating robust “dual-use” properties. Data indicated that this dual-use trait has no significant effect on other agronomic traits of the male sterile line and its derived hybrid combinations. Based on this, we established a novel two-line hybrid seed production system in maize, termed the Gibberellin-switchable Dual-use Line (GDL), and successfully applied it in the hybrid seed production of Chuandan 99.

Psyllid Cysteine Cathepsins Directly Cleave the Outer Membrane Protein BamD of Citrus Huanglongbing Pathogen

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Candidatus Liberibacter asiaticus (CLas), the causative agent of citrus Huanglongbing (HLB), is transmitted by Asian citrus psyllid, Diaphorina citri; however, the role of the insect's immune effectors in defending against CLas remains poorly understood. This study reveals that D. citri cathepsin L (DcCTSL1), a member of the cysteine protease family, serves as a key immune effector directly cleaving CLas outer membrane protein BamD, thereby defending against CLas infection in insect vectors. Upon CLas infection, DcCTSL1 expression is upregulated. This protease targets the CLas membrane by specifically interacting with BamD, which in turn enables DcCTSL1 to recognise the conserved Ile145-Arg146 motif of BamD. Following this recognition, DcCTSL1 cleaves the peptide bond between Arg146 and Asp147 of BamD, an action that is mechanistically dependent on the conserved catalytic triad (Cys185, His325, and Asn350) of DcCTSL1. Heterologous application of DcCTSL1 in citrus plants confers resistance to CLas. Furthermore, a homologous citrus cysteine protease also cleaves BamD via the same mechanism. Our study reveals an evolutionarily conserved defence mechanism mediated by cysteine proteases against CLas in both insect vectors and plant hosts, thus offering a novel foundation for developing protease-based strategies against HLB.

A Near Telomere‐to‐Telomere Genome of Belamcanda chinensis Provides Insights Into Genome Evolution and the Biosynthesis of Characteristic Isoflavones

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Belamcanda chinensis is a non-leguminous medicinal plant rich in bioactive isoflavones; however, the lack of a high-quality reference genome has limited elucidation of its isoflavone biosynthetic and modification network. Here, we present the first near telomere-to-telomere genome assembly of B. chinensis (4.18 Gb), generated using Illumina survey reads, PacBio HiFi and Oxford Nanopore long reads, and Hi-C scaffolding, achieving high completeness and accuracy (assembly BUSCO: 98.70%; LAI: 17.2). Ks/synteny-depth analyses and fossil-calibrated dating, with calibration at four fossil nodes, indicate two lineage-specific WGD events (~54.6 and ~27.3 MYA). These events drove significant expansions of key gene families involved in stress response and secondary metabolism. Leveraging this genome, we identified two key O-methyltransferases (BcOMT03 and BcOMT33), which are responsible for catalysing the biosynthesis of quality-marker compound irisflorentin. Meanwhile, BcUGT009, BcUGT119, BcUGT124, and BcUGT032 were characterised as glycosyltransferases with 7-O catalytic activity. Structural modelling and site-directed mutagenesis further elucidated the catalytic mechanism of BcUGT009, and its K404A mutant exhibited a significant increase in relative activity. Cross-species comparative analyses further revealed that convergent expansion of these key enzyme families underlies isoflavone biosynthetic capacity in both leguminous and non-leguminous plants. This study not only reveals the ancient polyploidization events of B. chinensis, the amplification of lineage-specific gene families and the biosynthetic pathway of characteristic isoflavones, but also provides a reference genome and functionally validated tailoring enzymes that will facilitate future heterologous pathway reconstruction and metabolic engineering of these isoflavones.

The Rice Cis‐Natural Antisense Transcript NAT1850 of Pri‐miR1850 Negatively Regulates Cold Tolerance by Repressing NPR3

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Natural antisense transcripts (NATs) correspond to nearly 60% of annotated rice loci, however their functions are largely unknown. In this study, we characterise a rice cis-NAT (NAT1850) that completely overlaps with a rice-specific primary miRNA, pri-miR1850. Pri-miR1850, but not its mature miR1850 products, promotes the accumulation of NAT1850, while NAT1850 overexpression in turn reduces the accumulation of pri-miR1850 transcripts. A 21-nt siRNA (siR1850) derived from the pri-miR1850 transcripts is generated by cleavage of pri-miR1850-NAT1850 dsRNA and overlaps in sequence with miR1850.1 and miR1850.2. Both NAT1850 and siR1850 negatively regulate cold tolerance at both the young-seedling and booting stages. Interestingly, siR1850 targets and represses NPR3, which is also a target of miR1850.1. NPR3 interacts with the WRKY76 transcription factor and acts as a co-transcriptional activator of WRKY76 to trigger DREB1B under cold stress. Genetic evidence shows the NAT1850-siR1850 module functions in cold stress response via an NPR3-dependent manner. Furthermore, NAT1850 and siR1850 control nitrogen assimilation and rice yield in a miR1850.1-NPR3-independent pathway. Our findings reveal a regulatory mode for a pri-miRNA and its cis-NAT, and uncover their roles in balancing the cold-stress response and rice yields.

Natural Variations in the Activity of Hydroxycinnamoyl Transferases Promote Accumulation of Metabolites Conferring Rice Resistance

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Plant-derived secondary metabolites act as regulators of in planta defence. Plant phenylpropanoid metabolic compounds, including final metabolites such as lignin and flavonoids, and a few intermediate metabolites play critical roles in defence. Here, we functionally characterised two rice hydroxycinnamoyl transferases OsSHT1 and OsSHT2, which are responsible for catalysing branches of the phenylpropanoid metabolic pathway and regulating resistance to bacterial pathogens. Both OsSHT1 and OsSHT2 can use p-Coumaroyl-CoA and feruloyl-CoA as acyl donors and prefer spermidine as an acyl acceptor, as verified by in vitro biochemical assays and in vivo metabolic analysis. Knocking out OsSHT1 and OsSHT2 blocked the branching of the phenylpropanoid metabolic pathway, resulting in an accumulation of intermediate metabolites p-Coumaric acid and ferulic acid and final metabolites lignin and flavonoids. These intermediate metabolites exhibit antimicrobial activity towards phytopathogenic bacteria and final metabolites enhance rice immunity. Haplotype analysis and population genetics revealed rice accessions with lower expression levels of OsSHT1 and OsSHT2 accumulate higher concentrations of phenolic compounds and exhibit enhanced resistance. Furthermore, OsMYB30 binds directly to the promoters of OsSHT1 and OsSHT2, thereby suppressing their expression and negatively regulating rice resistance to bacterial pathogens. The OsMYB30^D239 variant is considered to be an elite haplotype due to its greater stability and stronger inhibitory effect on the expression of OsSHT1 and OsSHT2. Taken together, these findings demonstrate that there is a link between phenylpropanoid metabolism and immune responses via the OsMYB30-OsSHTs module, advancing our understanding of phenylpropanoid metabolites-mediated resistance and providing elite haplotypes for rice breeding.

Methionine Synthase Positively Regulates Plant Defence to Both RNA and DNA Viruses and Is Useful for Developing Broad‐Spectrum Antiviral Resistance in Crops

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Plant viruses frequently cause severe economic losses in worldwide crop production. Developing broad-spectrum resistance is the most efficient approach for controlling plant viral diseases. In this work, we found that the 17K protein of barley yellow dwarf viruses (BYDVs), which has multiple functions in viral pathogenesis including acting as a viral suppressor of gene silencing (VSR), interacted with plant methionine synthase (MS), the last enzyme in the methionine cycle. Silencing HvMS gene expression enhanced BYDV symptoms and viral gene expression in barley. In contrast, overexpressing HvMS1 in wheat, another important host of BYDVs, attenuated disease symptoms and decreased viral genome proliferation. Interestingly, the γb VSR of barley stripe mosaic virus (BSMV) also interacted with HvMS protein, and HvMS1 overexpression lines likewise exhibited improved BSMV resistance. Further investigations uncovered that the VSRs of potato virus X (PVX) and tobacco rattle virus (TRV) could interact with the MS protein of Nicotiana benthamiana; lowering NbMS gene expression by genome editing reduced tobacco resistance to PVX and TRV, whereas the reverse was observed in HvMS1 overexpression tobacco lines. Finally, we showed that HvMS1 could counteract the VSR function of 10 distinct RNA and DNA viruses by obstructing their ability to revive GFP expression in 16c tobacco, suggesting that plant MS protein may act broadly in disrupting the anti-gene silencing activities of VSRs. Altogether, our data suggest that plant MS protein positively regulates host defence to diverse viruses through inhibiting their VSRs, thus providing a promising target for engineering broad-spectrum antiviral resistance in crops.

DNAwhisper: An Integrated Deep Learning Pyramidal Framework for Multi‐Trait Genomic Prediction and Adaptive Marker Prioritisation

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Genomic selection (GS) is critical for accelerating genetic gain in modern plant breeding. Deep learning approaches offer powerful non-linear representation capabilities for modelling non-additive effects. However, their application in GS remains restricted, as high-dimensional, low-sample and noisy data hinder the identification of informative markers. The present study proposes DNAwhisper, a deep learning framework designed for multi-trait prediction and adaptive marker prioritisation. The framework integrates a cascaded architecture, GFIformer, employing shared network parameters across partitioned marker blocks to adaptively compress genetic features within a hierarchical pyramid. Pre-training on population genetic structure regularises feature learning to establish a generalisable latent representation. During trait modelling, importance scores for aggregated genomic regions at multi-resolutions are extracted from the distinct pyramid levels under trait-guided deep supervision, enhancing interpretability and supporting marker prioritisation. DNAwhisper was evaluated on maize, wheat, tomato and grape datasets for marker prioritisation and phenotypic prediction, achieving prediction accuracy approximately 3.0% to 10.0% higher than the baseline model. Furthermore, DNAwhisper identifies major QTLs (e.g., VGT1, ZCN8) and epistatic signals within the gibberellin metabolic pathway across maize flowering traits. This framework provides a new strategy for dissecting the genetic architecture of complex traits.

AaSIZ1‐Mediated SUMOylation of AaMYB31 Positively Regulates Freezing Tolerance in Actinidia arguta

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Several studies revealed that wax accumulation enhances plant tolerance to cold stress. However, the molecular regulation mechanism of wax synthesis under cold stress is still unclear in Actinidia arguta, a hardy kiwifruit species. Here, we found that cold stress significantly induces wax accumulation in Actinidia arguta leaves, which is driven by the robust upregulation of wax biosynthetic genes, particularly AaKCS2 and AaKCS6.1. Overexpression of AaKCS2/6.1 in Actinidia chinensis not only elevated wax biosynthesis but also enhanced freezing tolerance. Furthermore, we identified that the R2R3-MYB transcription factor AaMYB31.1 and AaMYB31.2 are transcriptionally activated by cold stress and directly bind to the promoters of AaKCS2/6.1 to activate their expression. Overexpression of AaMYB31s in Actinidia chinensis significantly promoted wax biosynthesis under cold stress, thus enhancing the freezing tolerance of kiwifruit. Besides, we identified that AaSIZ1, a small ubiquitin-like modifier (SUMO) E3 ligase from Actinidia arguta, interacts with AaMYB31s to mediate the SUMOylation of AaMYB31s to enhance their protein stability. The functional validation in Actinidia chinensis further revealed that overexpression of AaSIZ1 robustly upregulated the cuticular wax biosynthesis and enhanced freezing tolerance. Importantly, our evidence suggests that the interaction between AaMYB31s and AaSIZ1 and the AaSIZ1-mediated SUMOylation of AaMYB31s were significantly enhanced under cold stress. Therefore, we conclude that the AaSIZ1-AaMYB31s-AaKCSs module positively regulates wax synthesis in response to freezing stress.

MdRLKT1–MdRAX2–MdMKS1 Module Positively Regulating Resistance to Cytospora mali in Apple

Yanan Tang, Guangyao Li, Yang Li, Yuzhu Wang, Hao Feng, Lili Huang — Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Valsa canker (caused by Cytospora mali = Valsa mali. C. mali) is one of the most destructive diseases affecting apple cultivation. The scarcity of natural germplasm resources with high resistance and immunity underscores the importance of exploring plant immune regulation factors of disease-resistant breeding. Protein post-translational modifications, particularly phosphorylation, are critical regulatory mechanisms in plant immunity. This study investigates how the apple receptor-like kinase MdRLKT1 modulates resistance to Valsa canker through the phosphorylation of the transcription factor MdRAX2. We found that MdRLKT1-interference (RNAi) transgenic lines exhibit increased susceptibility to C. mali infection compared to wild-type controls, indicating that MdRLKT1 positively regulates apple immune responses. Notably, MdRLKT1 interacts with the MYB transcription factor MdRAX2, facilitating its translocation into the nucleus. In vitro phosphorylation assays identified serine 147 (Ser147) as the phosphorylation site of MdRAX2 by MdRLKT1. Mutant MdRAX2^S147A, with this phosphorylation site inactivated, demonstrated reduced resistance to C. mali. Further analysis revealed that MdRAX2 binds to the promoter region of MdMKS1, transcriptionally repressingits expression, whereas MdRAX2^S147A failed to regulate MdMKS1 transcriptionally. Overexpression of MdMKS1 in apple resulted in reduced resistance to C. mali, suggesting that MdMKS1 negatively regulates apple immunity. These findings establish that the MdRLKT1-MdRAX2-MdMKS1 module plays a positive regulatory role in enhancing apple resistance to C. mali. In conclusion, MdRLKT1 activates the transcriptional repressor function of MdRAX2 through phosphorylation, thereby alleviating the negative regulatory effect of MdMKS1 on disease resistance and ultimately boosting the defensive capabilities of apple against pathogens.

OsRALF26 Serves as an Endogenous Signal Recognised by XA21 to Promote Robust and Distal Resistance in Rice

Oh‐Kyu Kwon, A‐Ram Jeong, Chang‐Jin Park — Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Plant immune receptors detect both microbe-derived and endogenous signals to activate defences. XA21, a rice immune receptor, confers strong race-specific resistance to a subset of Xanthomonas oryzae pv. oryzae (Xoo) strains by recognising the microbial sulphated peptide RaxX. However, the molecular basis for the notably robust XA21-mediated immune response has remained unclear. Here, we report that the small secreted peptide OsRALF26, previously identified as an Oryza-specific ligand for FERONIA-like receptor 1 (OsFLR1), is also directly perceived by XA21. Recognition of OsRALF26 by XA21 triggers a pronounced reactive oxygen species (ROS) burst, pathogenesis-related (PR) gene induction, and enhanced resistance to Xoo. Notably, silencing OsRALF26 leads to a spatially biased reduction in XA21-mediated resistance, particularly in distal tissues. These findings identify OsRALF26 as a host-derived ligand of XA21 that is required for full activation of XA21-mediated immunity in distal tissues, consistent with a role for OsRALF26 in spatial propagation of XA21-dependent defence. By integrating microbe-derived and endogenous signals, XA21 exemplifies a versatile immune strategy in rice. This dual recognition may have arisen through the introgression of XA21, which unintentionally conferred OsRALF26 responsiveness—thereby reinforcing immune robustness in rice varieties.

Mechanical Strength: An Unrecognised Target in the Genetic Improvement of Crops

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Leaf angle (LA) is a crucial agronomic trait influencing planting density and crop yield. Previous research highlighted the importance of cellular variations in the ligular region for determining LA, but the underlying regulatory mechanisms remain unclear. Here, we demonstrate LA is not a static trait, but rather represents a dynamic equilibrium between mechanical forces maintaining leaf erectness and those promoting blade drooping. To quantify the drooping tendency, we introduce gravitational moments, which show positive correlations with LA, blade length (BL) and blade weight (BWt). Notably, the mechanical forces are tightly regulated by the sheath layers surrounding the stalk and by the thickness and lignin deposition on the ligular region of sclerenchyma (SC) cells. Furthermore, we applied single-nucleus transcriptome analyses (snRNA-seq) to construct a comprehensive transcriptional atlas spanning the ligular regions of compact-type (Z58), intermediate-type (B73) and expanded-type (W22) inbred lines. Through the comparative analysis of snRNA-seq and RNA-seq of three inbred lines, we identified the adaxial hypodermis (HP) cells as pivotal sites where lignin biogenesis and metabolism genes were specifically expressed in compact-type Z58, consistent with the lignin deposition pattern. Notably, we discovered that the NAM, ATAF and CUC (NAC) transcription factor-encoding genes NAC secondary wall thickening promoting factor 2 (NST2) and NST3, which mediate lignin biogenesis in the ligular region, especially on the adaxial side, play key roles in reinforcing mechanical support and reducing LA. Collectively, this study advances our understanding of ligular development and LA regulatory mechanisms and provides strategic insights for breeding crops with improved agricultural productivity.

ZmWRKY29 Transcriptionally Represses ZmRBOHC to Attenuate ROS Production and Facilitates Gibberella Stalk Rot Susceptibility in Maize

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Gibberella stalk rot (GSR), caused by the fungal pathogen Fusarium graminearum, severely threatens maize production. However, the molecular mechanisms underlying maize resistance to GSR remain poorly understood. Here, we have identified ZmWRKY29 as a transcriptional repressor induced by F. graminearum infection, which negatively regulates maize resistance to GSR. Overexpression of ZmWRKY29 significantly compromised GSR resistance with a marked reduction in reactive oxygen species (ROS) production and callose deposition, while its mutation enhanced resistance and elevated ROS accumulation. RNA-seq revealed that the expression levels of two respiratory burst oxidase homologues, ZmRBOHA and ZmRBOHC, were decreased in ZmWRKY29-OE lines following F. graminearum challenge. ZmWRKY29 directly binds to the W-boxes in the promoters of ZmRBOHA and ZmRBOHC to repress their transcription. Moreover, mutation of ZmRBOHC similarly led to increased susceptibility and diminished ROS accumulation, confirming that ZmWRKY29-targeted ZmRBOHC is required for ROS production and GSR resistance. Thus, ZmWRKY29 acts as a susceptibility factor, repressing GSR resistance possibly by inhibiting ZmRBOHC-mediated ROS production. This highlights a pathogen strategy of exploiting host transcriptional machinery to suppress ROS defences and facilitate infection.

LsMAPK6 Phosphorylates the LsCO Protein to Enhance Its Stability and Transcriptional Activity, Promoting Floral Transition Upon High Temperatures in Lettuce

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

High temperatures significantly accelerate the timing of floral transition, namely, bolting and flowering, in lettuce, which results in severe loss of marketable yield. Thus, understanding the genetic regulation of floral transition is of great interest to plant biologists and lettuce breeders. Here, we show that mitogen-activated protein kinase (LsMAPK6), whose expression and phosphorylation are stimulated by elevated temperatures, plays a positive role in the floral transition. The lsmapk6 mutants exhibit delayed bolting and flowering, whereas LsMAPK6 overexpression accelerates lettuce bolting. LsMAPK6 physically interacts with LsCO. Knockout of LsCO, to a large extent, phenocopies lsmapk6 mutants, strongly indicating that they function in the same genetic pathway. Mechanistically, LsMAPK6 phosphorylates LsCO at residue serine-258, resulting in the enhanced transcriptional activity and protein stability of LsCO and consequently the activation of LsFT-mediated flowering signalling pathways, which was confirmed by phospho-inactive and mimic analyses. Collectively, these findings reveal that the LsMAPK6-LsCO signalling module fine-tunes the timing of floral transition upon high temperatures, demonstrating that LsMAPK6 is a potential target for breeding lettuce cultivars adapted to global warming.

Metabolic Enzyme MeHNL11 Regulates MeCAS1b Transcription for Cyanide Reutilization in Response to Nitrate Deficiency in Cassava

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Cassava (Manihot esculenta Crantz) exhibits exceptional tolerance to infertile soils and contains abundant cyanogenic glucosides (CGs). Previous research has indicated that CGs can serve as a significant reservoir of organic nitrogen in plants. However, the extent to which its high-CG content contributes to efficient nitrogen utilisation and adaptation to low nitrogen (N) in cassava remains to be further elucidated. This study represents the first identification of MeHNL11 as a bifunctional protein. In response to N deficiency, the hydroxynitrile lyase activity of MeHNL11 promotes the generation and accumulation of cyanide and the Cys245 residue of MeHNL11 is critical for its nuclear oligomerization, in which the protein functions as a transcription factor. Following the cyanide transmission into the nucleus, the oligomeric form of MeHNL11 dissociates into monomers, leading to a dramatic upregulation of MeCAS1b transcription. This regulatory mechanism helps sustain intracellular cyanide homeostasis within cassava and facilitates the synthesis of primary N metabolites, thereby alleviating N deficiency. The exogenous application of the cyanide antidote hydroxocobalamin (COB) inhibited cyanide assimilation by MeCAS1b, leading to exacerbated N deficiency symptoms, such as leaf yellowing and a significant reduction in the contents of NH₄ ⁺ and free amino acids (AA) in cassava seedlings under low-N conditions (LN). Our research demonstrates that the MeHNL11-MeCAS1b module plays a pivotal role in CG recycling, offering new insights into the underlying mechanisms governing cassava's exceptional tolerance to low N stress.

SlMYB17 Antagonises the SlCBF Pathway to Negatively Regulate Tomato Chilling Tolerance

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Low temperature is a significant environmental constraint, impeding the extensive cultivation of tropical plants. Here, we identify the transcription factor SlMYB17 as an important negative regulator of tomato chilling tolerance. Overexpression of SlMYB17 significantly reduced chilling tolerance, whereas slmyb17 mutants exhibited enhanced tolerance. ChIP-seq analysis revealed that SlMYB17 targets genes involved in diverse biological processes and stress responses, suggesting its role in coordinating plant development and stress adaptation. SlMYB17 directly binds to the promoters of cold-regulated (COR) genes, such as SlCOR27b and WCOR413, thereby inhibiting their expression. Crucially, protein–protein interaction studies and Dual-LUC assays demonstrated that SlMYB17 interacts with SlCBF1, SlCBF2, and SlCBF3 in the nucleus to inhibit SlCBF-mediated activation of COR genes. Using virus-induced gene silencing (VIGS) targeting SlCBF1-3 in wild-type and slmyb17 mutants, we demonstrate genetically that SlMYB17's function in chilling tolerance is dependent on SlCBFs. Collectively, SlMYB17 antagonises the SlCBF pathway at both transcriptional and protein activity levels, ultimately suppressing tomato chilling tolerance. Our work establishes slmyb17 mutants as valuable genetic resources for developing chilling-tolerant tomato varieties, and the ChIP-seq data provide important insights for studying MYB transcription factors in stress responses and development.

A Hierarchical VvbHLH30‐VvERF70‐VvACS2 Module Orchestrates Ethylene Biosynthesis and Cold Adaptation in Grapevine

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Ethylene is a key gaseous phytohormone that plays crucial roles in regulating plant growth, development and stress responses. However, ethylene-associated biosynthetic and transcriptional regulatory mechanisms governing cold-adaptation responses in plants remain poorly understood. In this work, genome-wide analysis from grapevines (Vitis vinifera) identified nine ACS family members, of which VvACS2, VvACS4 and VvACS6 exhibited the most dynamic transcriptional responses to cold stress and were chosen for functional validation. CRISPR-Cas9-mediated knockout and overexpression experiments revealed that VvACS2 is the major contributor to ethylene biosynthesis during cold stress in grapevine roots. Screening time-course cold treatment data from Vitis vinifera and Vitis amurensis roots identified VvERF70 and VvbHLH30 as the only two TFs, among six candidates, that directly regulate VvACS2 expression. Overexpression and CRISPR-Cas9-mediated knockout of VvERF70 or VvbHLH30 in roots further confirmed their contribution to enhanced ethylene production and cold tolerance under low-temperature treatment. Furthermore, the induction of VvACS2 was greatly enhanced when VvERF70 dimerized with VvbHLH30. Notably, VvbHLH30 further positively regulates ethylene biosynthesis under cold stress by interacting with VvERF70 and binding to its promoter. Taken together, we define a hierarchical transcriptional regulatory network where the VvbHLH30-VvERF70-VvACS2 module is pivotal for ethylene biosynthesis and underpins grapevine cold tolerance. This work provides new mechanistic insights into cold adaptation mechanisms and offers novel strategies to mitigate frost damage in agricultural crops.

The ZmCOP1s–ZmCOL3 Module Enhances Late Flowering, Grain Yield and Grain Quality in Maize

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Flowering time is a key determinant of yield and regional adaptation in crops and is largely controlled by light signalling. In this study, we identified two maize orthologs of Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), designated ZmCOP1a and ZmCOP1b, which activate light signalling and reduce plant height. Loss-of-function mutants of ZmCOP1a and ZmCOP1b flowered early, whereas overexpression of either gene significantly delayed flowering under both long- and short-day conditions. Mechanistically, both ZmCOP1a and ZmCOP1b interacted with and stabilised the C2C2-CO-like transcription factor ZmCOL3. Together, they upregulated key repressors of flowering (ZmPRR37, ZmPRR95, ZmCCA1 and ZmCCT9), leading to suppression of florigen genes (ZCN7, ZCN8 and ZCN12) and floral meristem identity genes (ZMM3, ZMM4 and ZMM15). Field trials demonstrated that overexpression of ZmCOP1a or ZmCOP1b reduced starch content but increased protein levels, kernel numbers and kernel length, width and weight. In contrast, overexpression of ZmCOL3 decreased both starch and protein content. Collectively, our results demonstrate that the ZmCOP1s–ZmCOL3 module is a key regulator of flowering time and source–sink reallocation, providing valuable genetic targets for breeding high-yield, high-quality maize with optimised flowering time and enhanced carbon and nitrogen allocation.

CpERF‐WRI1 Manipulates Ethylene Sensing by Regulating the Expression of CpERS1 and Fruit Ripening in Papaya

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Ethylene plays a crucial role in fruit ripening and is perceived by specialised receptor proteins embedded in the endoplasmic reticulum (ER) membrane. As an ethylene antagonist, 1-methylcyclopropene (1-MCP) binds to these receptors and delays papaya ripening, but improper use can cause ripening disorders. The expression of CpERS1 and CpERF-WRI1 is markedly upregulated during ripening yet is suppressed by inappropriate 1-MCP treatments. Similar ripening disorders and expression patterns of CpERS1 and CpERF-WRI1 were observed in immature, chilled and heat-stressed papaya fruits, indicating that these genes may play key roles in ripening disorder. CpERF-WRI1 binds to and activates the CpERS1 promoter both in vitro and in vivo. Transient overexpression of CpERF-WRI1 in papaya and ectopic expression in tomato accelerated fruit ripening, increased CpERS1 expression and upregulated ripening-related pathways, whereas virus-induced gene silencing (VIGS) of CpERF-WRI1 in papaya delayed ripening and downregulated those pathways. Similarly, overexpressing CpERS1 in papaya and tomato accelerated ripening and boosted the expression of ripening-associated genes, while VIGS-mediated silencing of CpERS1 delayed ripening and suppressed these genes. Together, these results indicate that CpERF-WRI1 regulates CpERS1 expression and modulates ethylene sensing within the system II ethylene signalling pathway to control papaya ripening.

A Truncated WRKY Protein Enhances Drought Resistance in Wild Tomatoes Through the SlWRKY16‐CIP2b‐SlSYP121 Module

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Drought stress is a major abiotic factor that severely affects plant growth and food production. Identifying drought-resistant genes and their regulatory mechanisms is essential for mitigating the negative impacts of drought on plants. In this study, we identified a natural single nucleotide polymorphism (SNP) mutation in SlWRKY16 that is closely linked to drought tolerance in tomato. This SNP leads to the expression of a truncated SlWRKY16 protein. The CRISPR/Cas9 knockout of SlWRKY16, which produces this truncated SlWRKY16 protein, exhibits enhanced drought tolerance, whereas the overexpression lines demonstrate the opposite effect. Yeast two-hybrid screening demonstrated that SlWRKY16 physically interacted with CONSTANS Interacting Protein 2b (CIP2b). The CIP2b knockout mutants displayed increased sensitivity to drought stress. Importantly, this drought-sensitive phenotype was rescued in double mutants (cip2b/slwrky16). RNA-seq analysis revealed that a syntaxin gene (SlSYP121) co-expressed with both SlWRKY16 and CIP2b. Electrophoretic mobility shift assays confirmed that SlWRKY16 directly binds to the promoter of SlSYP121 and represses its expression, while the truncated SlWRKY16 protein failed to bind. Moreover, SlSYP121 acts as a positive regulator of drought tolerance. Our findings further demonstrate that the interaction between CIP2b and SlWRKY16 reduces the binding affinity of SlWRKY16 to the SlSYP121 promoter. This study identified a key SNP associated with differences in drought tolerance between wild and cultivated tomato, elucidated the regulatory function of the SlWRKY16-CIP2b-SlSYP121 module in the tomato drought response, and enhanced our understanding of the molecular mechanisms underlying plant drought resistance.

Distinct Defence Mechanisms of Allelopathic Rice Against Quinclorac‐Susceptible and ‐Resistant Barnyardgrass: Involvement of Specific Metabolites and Rhizosheath Microbiota

Shuyan Li, Qiling Yan, Jianhua Tong, Yongfeng Li, Lianyang Bai, Qiong Peng — Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Allelopathic rice is increasingly recognised as a promising strategy for sustainable weed management. Resistance to the herbicide quinclorac is widespread in barnyardgrass, but it remains unclear whether allelopathic rice exerts the same defence against herbicide-susceptible and -resistant barnyardgrass. We conducted integrated transcriptomic, metabolomic, and metagenomic analyses to investigate the responses of allelopathic rice to quinclorac-susceptible (S) and -resistant barnyardgrass (R) lines. Distinct chemical strategies were identified in allelopathic rice: Terpenoids (e.g., carnosic acid, phytocassane B, and ipomeatetrahydrofuran) mainly suppressed S, while amino acids (e.g., pipecolic acid, L-glutamate, and L-histidine) were key against R. Correspondingly, terpenoid biosynthesis and nitrogen metabolism were the most enriched pathways under S and R stress, respectively. Additionally, terpenoid accumulation correlated positively with salicylic acid (SA) and jasmonic acid (JA) concentrations in roots under S. Both terpenoids and amino acids formed the stable ecological networks with rhizosheath microbiota. Functional metagenomic analysis further showed that ABC transporter and quorum sensing pathways were upregulated under S, whereas nitrogen fixation predominated under R. Notably, amino acids formed a nitrogen-related ecological network with nitrogen-metabolising microbiota, contributing to improved plant-available soil nitrogen and total nitrogen content in rice plants. Bioassays showed that exogenous pipecolic acid (≥ 40 μM) and L-histidine (80 μM) inhibited barnyardgrass seedling growth without affecting allelopathic and non-allelopathic rice. These findings demonstrate that allelopathic rice employs divergent chemical-microbial defence strategies against S and R barnyardgrass, highlight the dual role of amino acids, and provide a basis for precision weed management, particularly for herbicide-resistant weeds in paddy fields.

FvMAPK6‐Mediated FvMYB44s/FvSWEET1 Dual‐Layer Regulation Modulates Sugar Accumulation in Strawberry Fruit, With FvSPS3 Enabling Quality–Yield Balance

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Sugar content is a key determinant of fruit quality, and sugars also act as signalling molecules that regulate ripening processes, including anthocyanin accumulation. However, the molecular mechanisms underlying sugar accumulation and sugar signal-mediated ripening remain incompletely understood. In this study, we identify FvMAPK6 as an important phosphorylation hub that coordinates both sugar and anthocyanin accumulation in strawberry fruit. FvMAPK6 forms a phosphorylation cascade with FvMAPKK4, which directly phosphorylates the transcription factors FvMYB44.1 and FvMYB44.2. This phosphorylation reduces the stability and transcriptional activity of these proteins, attenuates their repression of downstream target genes such as FvCHI, FvSPS3 and FvSWEET1, thereby coordinating anthocyanin and sugar accumulation. Furthermore, FvMAPK6 increases the protein abundance of the hexose transporter FvSWEET1 in strawberry fruits and alters its transport activity through phosphorylation. We demonstrate that sucrose treatment activates FvMAPK6, reinforcing its regulation of FvMYB44s and FvSWEET1 and thus amplifying sugar and anthocyanin accumulation. These findings establish FvMAPK6 as a key regulator that integrates both sugar accumulation and signalling at both transcriptional and post-transcriptional levels. Although FvMAPK6 promotes sugar accumulation, it significantly reduces fruit yield and vegetative growth. To overcome this limitation, we screen for downstream targets of FvMAPK6 and identify FvSPS3 as a promising breeding target: modulating FvSPS3 improves fruit quality without compromising vegetative growth or yield. Collectively, our findings reveal novel regulatory pathways modulating sugar accumulation and signalling in strawberry while providing a valuable molecular target for the simultaneous improvement of fruit quality and agricultural productivity.

A Conserved Magnaporthe oryzae Effector Counteracts the Rice Ubiquitin‐Proteasome System by Disrupting the E2 Function to Suppress Immunity

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Pathogens commonly secrete effectors into host cells to facilitate invasion. In the host ubiquitin-proteasome system (UPS), E3 ubiquitin ligases often target pathogen effectors for degradation, thereby enhancing immune responses. In turn, pathogen effectors frequently disrupt E3 ligase function to promote virulence. However, it remains largely unclear whether pathogen effectors also interfere with other enzymes of the UPS, such as E2 ubiquitin-conjugating enzymes. In this study, we identified a conserved effector, MoCE1, that is essential for the pathogenicity of Magnaporthe oryzae. MoCE1 is secreted into rice cells, where it interacts with the rice E3 ligase OsRING10 and the E2 enzyme OsUBC11. Upon M. oryzae infection, OsRING10 and OsUBC11 act synergistically to degrade MoCE1 through K48-linked polyubiquitination. Overexpression of either OsRING10 or OsUBC11 enhances resistance to M. oryzae. To counteract this defence, MoCE1 inhibits the enzymatic activity of OsUBC11. Collectively, these findings reveal a nuanced mechanism in which a pathogen effector, regulated by a host E2–E3 pair, disrupts E2 function to escape UPS-mediated immunity in plants.

Cross‐Species Reprogramming of Developmental Plasticity and Metabolic Rewiring via Banana‐Derived WUS2 Developmental Regulator

Roni Chaudhary, Surender Singh, Usman Ali, Siddharth Tiwari — Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Plant regeneration is governed by intrinsic gene regulation and phytohormonal cues. WUSCHEL (WUS) gene promotes regeneration, but its broader functional role remains unexplored. Here, we demonstrate that the constitutive and inducible expression of banana-derived WUS2 (GN-WUS2) enhances regeneration in Nicotiana tabacum (tobacco) and Musa acuminata (banana) cv. Grand Naine, even in hormone-free MS medium. Constitutive (CaMV35S::GN-WUS2) expression promoted shoot formation and modulated hormonal and morphogenic gene expression, as evidenced by molecular, biochemical and histological analyses. However, it caused some pleiotropic effects. To overcome this, glucocorticoid receptor-based inducible GN-WUS2 expression enabled healthy shoot development. The upregulated expression of HMGR1, IPPI2 and SMT1-2 in transgenic tobacco lines boosted isoprenoid and phytosterol biosynthesis and correlated with increased cell division, biomass, pod size and seed yield. Proteomic analysis of seeds from transgenic tobacco lines revealed an enrichment of lipid-associated proteins and the accumulation of the novel lipid adipic acid, supported by expression profiling of NtKA and NtSA genes. Collectively, these results establish GN-WUS2 as a master regulator that integrates developmental reprogramming with novel phytosterol biosynthesis and yield enhancement, presenting its versatile role in next-generation regeneration and crop improvement.

Analysis of BpbHLH Gene Family Responsive to MeJA Signalling in Betula platyphylla Suk. and Functional Mechanisms of BpbHLH42/44 in Genetic Improvement and Triterpenoid Biosynthesis

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

The basic helix–loop–helix (bHLH) transcription factor family regulates plant secondary metabolism, development and stress responses. Although triterpenoids such as betulinic acid (BA), betulin (BT) and oleanolic acid (OA) from Betula platyphylla Suk. are of pharmacological importance, the methyl jasmonate (MeJA)-induced regulation of IVa bHLHs remains unclear. A total of 131 BpbHLH genes were identified, and functional characterisation was performed for two IVa members, BpbHLH42 and BpbHLH44, in yeast, tobacco, birch cells and transgenic plants. Both enhanced triterpenoid biosynthesis but exhibited distinct specificities: BpbHLH42 primarily promoted OA accumulation by activating BpHMGR, BpSE1/3, BpW and BpY6, while BpbHLH44 favoured BT production through BpW, BpY6 and BpY11. Both indirectly regulated BpY9 via BpMYB21, forming a bHLH–MYB complex, and the MeJA repressor BpJAZ2 disrupted the BpbHLH42–MYB21 interaction. Transgenic birch lines further revealed divergent phenotypes: BpbHLH42 overexpression enlarged and smoothed leaves and increased OA levels, whereas BpbHLH44 enhanced alkali stress tolerance by elevating secondary metabolite accumulation. These results uncover the differential regulatory roles of BpbHLH42 and BpbHLH44 in triterpenoid biosynthesis and demonstrate their potential for metabolic engineering and genetic improvement in woody plants.

A Natural LTR Retrotransposon Insertion in the Promoter of GhNAC140‐Dt Boosts Cotton Lint Yield

Fri, 22 May 2026 00:54:26 -0700

ABSTRACT

Transposable elements (TEs) are fundamental drivers of crop evolution and domestication. Whereas the underlying mechanisms of TE-mediated gene activation remain poorly understood. Lint percentage is an important yield component in cotton. Here, we report a retrotransposon insertion in the promoter of GhNAC140-Dt, a secondary wall NAC encoding gene, to promote the lint production by elevating its expression. We confirm that a 60 bp core cis-regulatory module within the TE's LTR (long terminal repeat) specifically recruits the transcription factor GhMYB46 and increases downstream genes' expression. GhNAC140-Dt overexpression activates the expressions of secondary cell wall development related genes, including GhCESA4-Dt, GhCESA4-At, and GhCOBL9-At, promotes cellulose deposition, and enhances lint percentage. This retrotransposon insertion massively emerges on the domestication transition from Gossypium hirsutum races to cultivated cotton accessions, with > 80% fixation in modern cultivars. This work deepens our understanding of TE-mediated gene activation; it also provides direct molecular evidence for “transposon-driven yield evolution” in crop domestication.

Post‐Translational Control of TaFT1 by WAPO1 Ubiquitination Shapes Spike Architecture and Yield in Wheat

Thu, 21 May 2026 06:14:55 -0700

ABSTRACT

The florigen protein TaFT1 coordinately regulates heading time and spikelet number per spike (SNS), serving as a key yield determinant in wheat. However, how its stability is post-translationally controlled in the shoot apical meristem remains unclear. Here, we identify the F-box protein WHEAT ORTHOLOG OF APO1 (WAPO1), allelic to a major SNS quantitative trait locus (QSns.cau-7A), as a direct ubiquitin E3 ligase targeting TaFT1 for degradation. A crucial missense mutation (C47F) in the F-box domain of WAPO1 has a significant impact on the SNS. The elite allele WAPO-A1b (from large-spike germplasm AS420, encoding 47F) exhibits stronger binding affinity and ubiquitination activity toward TaFT1 compared with the allele WAPO-A1f (from cultivar Lunxuan987, encoding 47C). Enhanced degradation of TaFT1 by WAPO-A1b in the shoot apical meristem impairs the TaFT1–TaFDL transcriptional complex, thereby downregulating the floral identity gene VRN1/WAP1 and increasing SNS without delaying heading. Notably, the favourable WAPO-A1b allele has been positively selected in modern breeding, and its ectopic activation significantly boosts grain yield in field trials. Our work elucidates a post-translational pathway that fine-tunes spike architecture and highlights WAPO-A1b as a valuable genetic target for high-yield wheat breeding.

A Novel AP2/ERF Transcription Factor Controls Trichome Initiation and Fruit Development in Cucumber

Wed, 20 May 2026 03:22:09 -0700

ABSTRACT

Trichomes, the hair-like outgrowths of the plant epidermis, are critical for defence, reproduction and development. However, the underlying molecular mechanisms governing their initiation remain elusive. Here, we identified CsGL2, an AP2/ERF transcription factor, as a key integrator controlling trichome initiation and fruit development in cucumber. Genetic and molecular studies indicate that CsGL2 activates CsGL1/Mict, subsequently triggering the morphogenesis of multicellular trichomes. Beyond epidermal fate, CsGL2 upregulates the tubercle regulator Tu together with the cytokinin rate-limiting enzyme gene IPT1, thereby initiating fruit tubercles through cytokinin-linked pathways. Notably, we found that ovule-specific CsGL2 activates the parthenocarpy gene CsNPF1, and CsGL2 overexpression markedly increases the frequency of seedless fruit set. Thus, our findings establish a switch-based mechanism for progressive cell-fate specification in multicellular epidermal organs. This reveals how a single transcription factor integrates transcriptional and hormonal pathways to coordinate distinct developmental programmes.

The Dual‐Function of CtrNAC019‐CtrNPF2.1 Module in Salt Tolerance and Nitrogen Use Efficiency Via Enhancing Vacuolar Chloride Sequestration and Nitrate Efflux in Citrus trifoliata

Mon, 18 May 2026 05:47:04 -0700

ABSTRACT

Salt stress and nitrogen utilisation efficiency (NUE) represent critical constraints affecting worldwide crop productivity. While nitrate transporter proteins (NPFs) have been implicated in saline chloride (Cl⁻) ion transport, the mechanistic linkage between chloride stress and NUE remains poorly understood. Through comparative transcriptomic profiling of Citrus trifoliata, we identified CtrNPF2.1 as a dual-responsive gene significantly upregulated in root tissues under both chloride salt and high nitrate conditions. The CtrNPF2.1 protein is localized to the vacuole membrane and highly expressed in root cortical cells. Transport functional characterisation combined with transgenic phenotypic analyses established that CtrNPF2.1 mediates dual transport mechanisms: facilitating vacuolar Cl⁻ compartmentalisation while enabling nitrate efflux. This coordinated process enhances cellular ion detoxification and promotes nitrate redistribution for optimal root development under saline conditions. Molecular investigations further uncovered that CtrNAC019 transcriptionally regulates CtrNPF2.1 expression through direct binding to its promoter, forming a chloride/nitrate-responsive regulatory module. Notably, physiological validation reveals coordinated responses in which nitrate supplementation mitigates chloride toxicity while chloride availability enhances nitrogen utilisation in trifoliate orange, which may be explained by the CtrNAC019-CtrNPF2.1 expression regulation module. These results provide new insights into vacuolar chloride sequestration and nitrate efflux by CtrNAC019-CtrNPF2.1 under saline and high-nitrate conditions, offering a promising strategy for breeding salt-resilient and resource-efficient crops.

Gene Editing of Nicotiana benthamiana Architecture for Space‐Efficient Production of Recombinant Proteins in Closed Environments

Thu, 14 May 2026 22:45:24 -0700

ABSTRACT

Indoor vertical farming (VF) offers practical advantages for the cultivation of plant protein bio-factories including plant uniformity, product consistency, water/nutrient recycling and production cycles on a year-round basis. Much progress has been achieved toward the development of innovative systems for artificial lighting, automated irrigation, plant handling, environmental control and space use optimization in VF systems. Here, we used a CRISPR/Cas9 gene editing approach to generate mutant lines of protein expression host Nicotiana benthamiana presenting a compact, space-efficient phenotype suited to VF systems. Our strategy consisted of altering apical dominance by suppressing the synthesis of strigolactone, a negative regulator of axillary bud outgrowth. Strigolactone-depleted lines were generated by hindering the expression of either Carotenoid cleavage dioxygenase 7 (CCD7) or Carotenoid cleavage dioxygenase 8 (CCD8), two key enzymes of the strigolactone synthetic pathway. Knocking out of either enzyme had no impact on the plant's growth rate but drastically influenced its auxin/cytokinin ratio, leaf proteome and overall architecture. The ΔCCD mutants exhibited an axillary growth-oriented development pattern, altered glycolytic and malate-processing metabolic fluxes to drive pyruvate and cytokinin production, and a spatial footprint reduced by 45%–50% compared to the LAB strain commonly used for protein expression. Most importantly, recombinant protein yields per plant were maintained in the mutant lines, as illustrated with GFP and rituximab, a chimeric antibody of therapeutic value. Our data confirm the usefulness of ΔCCD7 and ΔCCD8 knockouts leading to strigolactone depletion for the generation of compact, space-efficient N. benthamiana lines better suited to VF systems.

GmDOF3.1‐GmCPX Module Regulates Nodulation and Nitrogen Fixation Abilities in Soybean

Thu, 14 May 2026 06:01:19 -0700

ABSTRACT

Soybean nodule nitrogen fixation is very important, which can provide a large amount of nitrogen supply for its own growth and that of other crops, but the mechanism is largely unclear. In the present study, a coproporphyrinogen oxidase gene, GmCPX, was identified to facilitate soybean nodulation and nitrogen-fixation under the regulation of transcription factor GmDOF3.1. GmCPX was predominantly expressed in nodule which was highly induced after the inoculation of rhizobium. Overexpression of GmCPX significantly increased nodule numbers, fresh weights, nitrogenase activities, total nitrogen and ammonium nitrogen contents in transgenic soybeans, and also significantly increased the infection cell numbers and areas, heme contents and haemoglobin contents, while significantly decreased the ROS contents. Oppositely, the decreased corresponding characteristics and increased ROS contents were observed in RNAi transgenic soybeans and EMS mutant. The allelic variation analysis of GmCPX in 547 resequencing accessions found that three upstream SNPs were associated with the related traits of nodulation and nitrogen fixation, which further induced a copy number variation of recognition element for transcription factor GmDOF3.1. Further analyses found that GmDOF3.1 negatively regulated the expression of GmCPX, and the opposite phenomena to GmCPX transgenic lines were found in GmDOF3.1 overexpression and RNAi transgenic soybeans. Moreover, the genetic variation analysis of GmDOF3.1 in resequencing soybeans demonstrated its negative regulation for nodulation and nitrogen fixation. Thus, the regulation of GmDOF3.1-GmCPX module to soybean nodulation and nitrogen fixation was fully verified via forward- and reverse-genetics strategies and could be applied in the genetic improvements of corresponding characteristics in soybean.

Revisiting the Molecular Roadmap for Sugar Crops: Genome Reading, Trait Writing and Variety Redesigning

Wed, 13 May 2026 06:10:30 -0700

ABSTRACT

Sugar crops, including but not limited to sugarcane, sugar beet, sweet sorghum and stevia, are major sources of sugar production in the world. However, conventional breeding approaches, limited by long breeding cycles, low efficiency and restricted capacity to improve complex traits in sugar crops, are increasingly insufficient to address the challenges posed by climate change and the demands of sustainable agriculture. This review systematically summarizes recent advances in biotechnology and molecular breeding that have transformed sugar crop improvement. Recently, high-throughput sequencing technologies have generated extensive multi-omics resources. Concurrently, numerous functional genes and genetic elements with substantial breeding potential have been identified and cloned, offering precise targets for the key agronomic traits in sugar crops. Marker-assisted selection has been successfully implemented to enhance disease resistance, while genomic selection has demonstrated well for the evaluation and selection of complex quantitative traits. Importantly, genetic transformation systems have enabled precise manipulation of target genes and facilitated the creation of novel germplasm. In the future, the integration of multi-omics data, artificial intelligence, high-throughput phenotyping and precision genome editing into an intelligent breeding framework will be essential for achieving breeding by design and developing climate-adaptive and smart cultivars. Ultimately, these technological innovations will expand the role of sugar crops beyond traditional sugar production, positioning them as a central platform for sustainable biomanufacturing and providing critical support for global sugar security, energy transition and the development of the bioeconomy.

KAS‐Seq Captures Global Transcription Dynamics and Active Single‐Stranded Enhancers in Rice

Wed, 13 May 2026 00:00:00 -0700

Plant Biotechnology Journal, EarlyView.

Negative Regulators of Rice Agronomic Traits: Functional Insights and Applications in Genome Editing‐Based Breeding

Wenhao Wu, Feng Jin, Huaxiang Xu, Ranran Liao, Zhongming Fang — Tue, 12 May 2026 06:36:12 -0700

ABSTRACT

Rice is the staple crop for more than half of the global population, and improving grain yield, grain quality, and stress resistance remain central goals of modern rice breeding. Among current precision breeding strategies, genome editing has created new opportunities for crop improvement, but its success depends heavily on the selection of effective target genes. In this context, negative regulators of agronomic traits are particularly valuable because their disruption or attenuation can relieve constraints on desirable phenotypes and generate beneficial variation. In this review, we summarize recent progress in the identification and functional characterization of negative regulatory genes associated with rice grain yield, grain quality and stress resistance. We further integrate the current knowledge of their molecular functions, regulatory mechanisms, and genetic networks and discuss their potential applications in genome editing-assisted breeding. This review provides a target-oriented framework for understanding negative regulation in rice and facilitating the development of improved varieties with increased productivity, quality and stress resistance.

Designed Alleles of ZmRap2.7 Decouple the Trade‐Off Between Early Flowering and Yield Penalty

Mon, 11 May 2026 06:20:22 -0700

ABSTRACT

Flowering time genes often exhibit pleiotropic effects. In particular, early flowering is frequently associated with reduced grain yield due to a shorter growth period. This trade-off poses a major challenge for developing early-maturing and high-yielding varieties, a key breeding objective in modern maize production. Here, we demonstrate that ZmRap2.7, a well-known flowering repressor in maize, positively regulates ear and kernel development. Knocking out ZmRap2.7 promoted flowering but reduced ear size and kernel weight. Integrated genetic and molecular analyses revealed a multi-pathway regulatory network: ZmRap2.7 delays flowering by directly repressing the florigen ZCN8 in leaves and regulating several flowering time genes in the shoot apical meristem (SAM); it increases ear size by inhibiting ZmMADS4 to sustain inflorescence meristem activity; and it enhances kernel weight by activating ZmGRAS11 to promote cell expansion during kernel development. A comprehensive transcriptomic comparison across four tissues showed that SAM and ear exhibited a greater overlap in differentially expressed genes, providing a potential molecular basis for the flowering-yield trade-off. To mitigate this trade-off, we edited the promoter and upstream enhancer of ZmRap2.7 and obtained three edited lines with specific downregulation of ZmRap2.7 in the SAM. This tissue-specific alteration likely underpins the decoupling of flowering time from yield-related traits, enabling early flowering without compromising yield. Our findings highlight the utility of cis-regulatory editing as a promising strategy for decoupling pleiotropic trade-offs in crop improvement, particularly for genes with tissue-differential regulatory architecture.

Engineering Herbicide Cross‐Resistance in Rapeseed by Generating Stacked BnaALS Mutations via Sequential CBE and ABE8e‐SpRY Editing

Fri, 08 May 2026 04:56:52 -0700

Plant Biotechnology Journal, EarlyView.

OsMYB306‐OsRAV11 Regulates Resistance of Rice to Striped Stem Borer by Modulating Serotonin Biosynthesis

Thu, 07 May 2026 00:00:00 -0700

ABSTRACT

Striped stem borer (SSB; Chilo suppressalis Walker) is one of the most destructive pests in rice production. Previous studies have demonstrated that SSB infestation induces transcription of OsT5H (tryptamine-5-hydroxylase) and biosynthesis of serotonin, a newly recognised phytohormone, and that disruption of serotonin biosynthesis significantly increases SSB resistance. However, the regulatory module modulating serotonin biosynthesis remains to be identified and characterised. Here, we reveal an OsMYB306-OsRAV11 module that regulates OsT5H transcription and serotonin biosynthesis in response to SSB infestation in rice. OsMYB306 and OsRAV11 can bind to the OsT5H promoter and repress its transcription. In the module, OsRAV11 interacts with OsMYB306 and enhances its inhibitory effect on OsT5H transcription. CRISPR/Cas9-generated knockout mutants (myb306, rav11 and myb306 rav11) exhibited elevated OsT5H expression, increased serotonin accumulation and reduced SSB resistance. Conversely, OsRAV11 overexpression reduced OsT5H transcription. Our findings establish a transcriptional regulatory framework for the biosynthesis of serotonin in response to SSB infestation. These findings inform the development of new strategies for producing SSB-resistant rice by genome editing, potentially reducing reliance on chemical pesticides for SSB control.

Development and Application of Prime Editors for the Induction of Site‐Specific, Heritable Edits in Soybean [Glycine max (L.) Merr.]

Nirmal Khadka, Hien Thuy Bui, Ajay Gupta, Steve A. Whitham, Bing Yang — Tue, 05 May 2026 21:44:07 -0700

Plant Biotechnology Journal, EarlyView.

A Modified Cas9 Scaffold Allows Extension of the Virus‐Induced Gene Editing Technology to the Large Potyvirus Genus

Fernando Merwaiss, Verónica Aragonés, Arcadio García, José‐Antonio Daròs — Mon, 04 May 2026 06:26:16 -0700

ABSTRACT

Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus) comprise the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompasses most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I (CHLI). Second, we developed a tobacco etch virus (Potyvirus nicotianainsculpentis)-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed us to obtain plants exhibiting a white phenotype with their four alleles edited through in vitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus, these findings may be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.

Ectopic Expression of ScALDH21 From a Desert Moss Enhances Cotton Resistance to Verticillium Wilt via the Modulation of Jasmonates and Phenylpropanoid Pathways

Tue, 28 Apr 2026 00:13:12 -0700

ABSTRACT

Biotic stresses, particularly Verticillium wilt (VW), lead to a global decline in cotton yields. Here, we demonstrate that ectopic expression of ScALDH21, a gene from the desiccation-tolerant moss Syntrichia caninervis Mitt. and absent in angiosperms, enhances cotton's resistance to VW. Multi-year, multiple location field evaluations showed that transgenic cotton lines consistently exhibited two major advantages: markedly improved resistance to VW, and significantly reduced yield loss, with an approximate 23.8% yield increase relative to non-transgenic counterparts under pathogen infection conditions. This disease resistance is associated with enhanced capacity of the transgenic lines to scavenge reactive oxygen species (ROS), induced by pathogen infection. This finding aligns with the ScALDH21-conferred detoxification function. Transcriptome analyses revealed a significant alteration in expression pattern of those genes that regulate phenylpropanoid and jasmonic acid (JA) pathways. Correspondingly, the accumulation of lignin and defence-related metabolites (e.g., rutin, cyanidin and jasmonates) significantly increased, suggesting that ScALDH21-mediated activation of the phenylpropanoid and JA pathways contributes to enhanced resistance. Analyses of ScALDH21 binding activity using CUT&Tag and EMSA assays showed that it can bind to specific gene promoters within the cotton genome, highlighting that ScALDH21 not only catalyses the detoxification of aldehydes but also gains transcriptional regulatory roles. In summary, we demonstrate that expression of the heterologous ScALDH21 in cotton leads to enhancement of resistance to VW and elucidated the mechanism. Our findings further demonstrate a promising strategy to improve biotic resistance in crops by utilizing unique functional genes from evolutionarily distant species in extreme environments.

PbrIDD2‐PbrAPX14 Module Functions in the Ethylene‐Mediated Ripening and Senescence Process of Pear Fruit

Mon, 27 Apr 2026 06:21:17 -0700

ABSTRACT

Endogenous H₂O₂ participated in the ethylene-dependent ripening and senescence process of horticultural fruit as a secondary messenger; however, the molecular mechanism beneath such a phenomenon has not been fully clarified until recently. By a conjoint analysis of metabolite, enzyme activities, gene expression profiles in AsA-GSH cycle of ‘Kolar’ pear, PbrAPX14 might act as a negative factor in the ethylene-mediated H₂O₂ accumulation. PbrAPX14, located in cytosol, would reduce H₂O₂ in vitro and in vivo, inhibit ethylene production, and thus fruit ripening and senescence. After analysing the expression profiles of the differentially expressed transcription factors (TFs) followed by experimental validation, the nuclear PbrIDD2 could directly bind to the cis-acting element (core motif: TTTGTCG) in PbrAPX14 promoter, activate its expression and thus enhance the H₂O₂-scavenging capacity of fruit/calli, which was associated with the mitigated ethylene evolution and fruit ripening and senescence. Further study explored that the H₂O₂-mediated post-translational S-sulfenylation of Cys⁴⁸ residue in PbrAPX14, which mitigated its function, existed in vitro and in vivo, and was upregulated by ethylene, facilitating endogenous H₂O₂ accumulation. Overall, our results implied that both transcriptional and post-translational regulation of PbrAPX14, which were (in)directly under the control of ethylene, functioned in pear ripening and senescence process via regulating endogenous H₂O₂ level.

Spatiotemporal Metabolome and Single‐Nucleus Transcriptome Integration Illuminates an Auxin Gradient Orchestrated by NtTAC1 Underlying Leaf Angle Regulation in Tobacco

Mon, 27 Apr 2026 02:08:55 -0700

Proposed model of TAC1-mediated leaf angle regulation in tobacco.

ABSTRACT

Plant architecture is key to crop yield, with leaf angle being critical for high-density cultivation. Although TAC1 represents a promising regulator of leaf angle for breeding, its molecular mechanism remains poorly understood, particularly at single-nucleus resolution. Here, we performed single-nucleus RNA sequencing on NtTAC1 knockdown lines exhibiting reduced leaf angle. This analysis generated a transcriptional atlas comprising 20 distinct clusters corresponding to 14 cell types and identified the endodermis as a central regulatory hub. Weighted gene co-expression network analysis and trajectory inference revealed that the auxin transporter NtPIN3 acts as a key downstream effector of NtTAC1. The two genes are co-expressed in endodermal cells and promote their differentiation from meristematic cells. Spatial metabolomics further demonstrated that NtTAC1 suppression elevates auxin levels and alters its spatial distribution, resulting in asymmetric auxin accumulation preferentially in the abaxial region and consequent reduction in leaf angle. Silencing NtPIN3 recapitulated the NtTAC1 disruption phenotype, confirming that the NtTAC1-NtPIN3 axis regulates both auxin asymmetry and cell wall remodelling. Consistently, both knockdown lines exhibited enhanced lignin deposition, linking disrupted auxin flow to secondary wall thickening. Moreover, CRISPR/Cas9-mediated editing of SlTAC1 in tomato suppressed SlPIN3 expression, indicating evolutionary conservation of this module. Collectively, our findings uncover a cell-type-resolved mechanism underlying leaf angle regulation and provide a mechanistic framework for precision engineering of crop architecture adapted to high-density cultivation.

DcH3.3 and DcNAC1 Regulate the Expression of UGT73A93 Involved in the Changes in Flower Colour and Fungal Resistance in Carnation

Sat, 25 Apr 2026 05:50:52 -0700

ABSTRACT

Carnation (Dianthus caryophyllus) contains abundant flavonoid glycosides (FGs), which are important natural functional and colour components. However, there are few reports on the modification of UDP-glycosyltransferases (UGTs) in relation to flavonoids in carnation. In this study, we cloned and characterised a flavonoid 3′-O-glucosyltransferase (UGT73A93) in carnation in vitro. Overexpression of UGT73A93 in carnation and tobacco increased flavonoid glycoside accumulation, particularly kaempferol glycosides, while decreasing anthocyanin content and lightening flower colour. UGT73A93 also enhanced fungal resistance, antioxidant capacity, anti-amylase and anti-pancreatic lipase activities. Yeast one-hybrid and dual-luciferase assays revealed that the UGT73A93 promoter interacted with DcH3.3 and DcNAC1, key regulatory proteins involved in flavonoid biosynthesis. We predicted the interaction between DcLON2 and DcNAC1 using AlphaFold 3 and confirmed this hypothesis through yeast two-hybrid assay and bimolecular fluorescence complementation assays. These findings suggest an epigenetic-transcriptional cascade (DcH3.3–DcNAC1–UGT73A93) wherein DcH3.3 opens chromatin for DcNAC1-mediated UGT73A93 activation, while DcLON2 potentially degrades DcNAC1 to form a feedback loop. These results provide new insights into flavonoid 3′-O-glucosyltransferase and may contribute to future strategies aimed at improving the benefits of flavonoid biosynthesis for both plants and humans. It also demonstrates that AI can be applied in the field of plant biosynthesis, accelerating the process of plant breeding.

Engineering of Amygdalin Biosynthesis in Rice Endosperm for Pharmaceutical Production and Sitophilus oryzae Resistance

Tue, 21 Apr 2026 06:47:10 -0700

Plant Biotechnology Journal, EarlyView.

Mapping and Functional Characterization of Homologous Genes AhSUCA06 and AhSUCA16 Underlying Sucrose, Oil and Protein Contents in Peanut (Arachis hypogaea L.)

Sat, 18 Apr 2026 00:25:31 -0700

ABSTRACT

Cultivated peanut (Arachis hypogaea L.) is an important oilseed and cash crop, and seed sucrose content (SSC), seed oil content (SOC) and seed protein content (SPC) are key determinants of seed flavour, texture, and overall quality. Identifying quantitative trait loci (QTLs) and candidate genes associated with SSC, SOC and SPC is therefore of considerable importance for peanut genetics and breeding. In this study, two recombinant inbred line (RIL) populations derived from reciprocal crosses between the lines Jihuatian1 (JHT1) and PI478819 (PI) were used to detect major QTLs for SSC, SOC and SPC through bulked segregant analysis combined with whole-genome sequencing (BSA-seq). Multiple lines of evidence supported the homologous gene pair AhSUCA06 and AhSUCA16 as candidate genes underlying the epistatic QTLs qA06.1 and qA16.1, which exhibited major and stable effects across multiple phenotypic evaluations. Furthermore, the function of AhSUCA06 was validated through CRISPR/Cas9-mediated genetic transformation. Subcellular localization assays using GFP fusion proteins, together with dual-luciferase reporter assays, demonstrated that AhSUCA06 and AhSUCA16—both containing a DUF7950 domain of previously unknown function—localize to the nucleus and act as transcriptional repressors. In addition, DAP-seq analysis suggested that these genes may regulate pathways related to glycolysis and gluconeogenesis. Overall, this study provides new insights into the molecular mechanisms underlying the regulation of SSC, SOC and SPC in peanut and offers valuable information to support the genetic improvement of seed quality traits in peanut breeding programs.

Transcriptional Regulation of the Novel Theacrine Synthase Gene CsTcS2 by the CsTINY–CsWRKY33 Module Underpins Theacrine Biosynthesis in Camellia sinensis

Wed, 15 Apr 2026 06:20:38 -0700

ABSTRACT

Theacrine (1,3,7,9-tetramethyluric acid) is a purine alkaloid detected in multiple wild and specialised tea germplasms (Camellia sinensis) from South China, including Kucha. However, the molecular mechanisms governing its biosynthesis remain poorly understood. Here, we identify CsTcS2 as a novel theacrine synthase in tea plant. Functional assays involving heterologous expression in Nicotiana benthamiana, antisense oligonucleotide-mediated gene silencing, transient overexpression in tea plants and co-expression with caffeine dehydrogenase (CsCDH) confirm its catalytic role in converting caffeine to theacrine. Transcription factors CsTINY and CsWRKY33 directly bind the CsTcS2 promoter and activate its transcription, as demonstrated by yeast one-hybrid, dual-luciferase reporter and electrophoretic mobility shift assays. Further molecular docking, yeast two-hybrid, bimolecular fluorescence complementation, luciferase complementation, co-immunoprecipitation and antisense inhibition experiments reveal a synergistic interaction between CsTINY and CsWRKY33 that regulates CsTcS2 expression and thus controls theacrine biosynthesis. Together, our findings unravel the transcriptional regulatory network underlying theacrine biosynthesis and provide a molecular foundation for breeding tea cultivars with elevated theacrine levels for health-promoting applications.

Unfolding Plant Defence: Endoplasmic Reticulum Stress Signalling at the Plant‐Pathogen Interface

Sat, 11 Apr 2026 03:59:18 -0700

ABSTRACT

The endoplasmic reticulum (ER) stress response, a conserved proteostasis network, has emerged as a central hub that reprograms plant immunity during pathogen attack. This review synthesises how plants harness ER-stress signalling to mount multilayered defences and how pathogens have evolved counterstrategies to subvert these pathways. We delineate the molecular integration of the unfolded protein response (UPR) with canonical immune layers including pattern-triggered immunity (PTI), effector-triggered immunity (ETI) and systemic defences, highlighting salicylic acid (SA) and jasmonic acid (JA) as rheostats that fine-tune ER stress-immune crosstalk. Functionally, the UPR bolsters immunity by coordinating protein folding and secretion, reprogramming transcription and translation, activating ER-dependent programmed cell death (ER-PCD), and orchestrating ER-associated degradation (ERAD) and selective autophagy. Pathogens such as bacteria, oomycetes and viruses in turn deploy virulence factors that target UPR sensors and transcription factors, thereby attenuating ER-driven immunity. We propose a conceptual framework in which the outcome of UPR activation—resistance versus susceptibility—is determined by pathogen lifestyle, ER stress dynamics, subcellular compartmentalisation and pathogen effector intervention. We also consider biotechnological contexts in which strong transgene expression can itself provoke the UPR, and outline diagnostic experimental strategies to distinguish UPR-mediated effects from intended transgene functions. By integrating molecular mechanisms with pathogen counterstrategies, this review underscores the dynamic interplay between ER stress and immune signalling in plants and highlights opportunities to enhance crop resilience under global climate challenges.

Rice EMF3 Alleles Adjust Flower Opening Time to Enhance the Seed Setting Rate Under High Temperature Stress

Thu, 09 Apr 2026 05:29:47 -0700

ABSTRACT

To safeguard global food security against rapid population growth and a warming world, the effective genetic improvement of cereals is imperative. Flower opening time (FOT) critically affects the seed setting rate. In this study, we identified a gene, EARLY-MORNING FLOWERING 3 (EMF3), in which single-nucleotide substitutions strongly modulate FOT in rice in a semi-dominant manner, resulting in wide variation in FOT from earlier to later FOT than the wild-type. EMF3 knock-out mutants showed significantly reduced FOT synchrony and disrupted anther dehiscence, leading to fertilisation failure. EMF3 encodes a plasma membrane-localised polypeptide of 723 amino acids with an armadillo repeat fold and four transmembrane segments. Furthermore, EMF3 is specifically expressed in the anthers starting from nighttime on the day of flowering, with substantial impacts on the transcriptomes of both anther and lodicule, which suggested an exclusive role of EMF3 in flowering events. Modifying EMF3 alleles of O. sativa enabled the adjustment of FOT among Oryza species and subspecies, potentially facilitating cross-fertilisation by overcoming one of the major challenges of inter-specific hybridisation to exploit heterosis. Introducing the EMF3 alleles with the earlier FOT into popular rice cultivars resulted in flowering at an earlier time of day when the temperature was cooler, efficiently increasing seed setting rate under heat stress. This discovery unveils the novel mechanism of anther control of flower opening time through the EMF3 gene, while also enabling the use of EMF3 alleles in breeding strategies for efficient fertilisation for increasing hybrid rice seed production and mitigating future heat-stress damage at flowering.

Spatial Regulation of Silicon Accumulation in Peduncle Confers Sheathed Spike in Barley

Wed, 08 Apr 2026 04:36:23 -0700

ABSTRACT

Peduncle, the uppermost internode in cereals, connects the stem to the inflorescence and is critical for the transport of water, nutrients and photosynthetic assimilates. While peduncle length associates with plant height and its elongation is primarily regulated by phytohormones, we report a previously unrecognized mechanism involving the spatial distribution of silicon (Si). We identified a barley mutant, sheathed spike 2 (ss2), characterized by a specifically shortened peduncle that traps the spike within the flag leaf sheath. Positional cloning and analysis of allelic mutants revealed that the wild-type SS2 gene encodes a putative silicon efflux transporter. SS2 is expressed throughout the lifecycle, with higher transcriptional levels in the rachis and stem internodes, and its encoded protein localizes to the plasma membrane. We demonstrate that SS2 is required for polarized Si partitioning. Unlike wild-type plants, which ultimately deposit Si in spikes, the ss2 mutant exhibits an 8-fold increase in Si accumulation in the peduncle and a significant increase in the flag leaf. Hydroponic experiments without Si supply restored normal peduncle elongation in the ss2 mutant, demonstrating that local Si hyper-accumulation directly inhibits elongation. The conserved role of SS2 was supported by diversity analysis across barley and common wheat, as well as by the similar sheathed spike phenotype in tetraploid wheat lines carrying non-functional SS2 homologues. Collectively, our findings uncover an evolutionarily conserved, silicon-dependent mechanism that regulates peduncle elongation and spike emergence in Triticeae crops like barley and wheat.

First Tetraploa Genome and Multi‐Omics Analysis Reveal Key Plant‐Microbe‐Soil Interactions for Salt Tolerance and Yield Improvement of Wheat

Fri, 03 Apr 2026 21:59:23 -0700

ABSTRACT

Salinity is a major threat to global agricultural productivity of staple crops such as wheat. Although microbial-based solutions hold promise for alleviating salinity stress, practical implementation is hindered by insufficient mechanistic characterization of bioinoculants and their interactions with plants. Here, we assembled the first complete reference genome of a halotolerant strain within the genus Tetraploa—the endophytic fungus Tetraploa sp. E00680. This novel genomic resource serves as a foundation for exploring previously uncharacterised salt tolerance mechanisms in this potential fungal inoculant. Our research demonstrates that E00680 enhances wheat yield under both controlled and field saline conditions. We found that E00680 systematically modulates the plant-microbe-soil interactions by optimizing rhizosphere microbial communities, increasing nutrient bioavailability, and triggering coordinated transcriptional and metabolic reprogramming in wheat. Notably, E00680 expands tryptophan metabolism to synergistically boost auxin biosynthesis in wheat by supplying precursors and activating relevant metabolic pathways. This cross-kingdom metabolic coupling facilitates better growth and salt tolerance in wheat plants. Our findings offer multi-omics and rhizosphere-level insights that can guide the development of microbial inoculants to enhance climate-resilient and sustainable crop production.

Regulatory Mechanism of CsMYB1‐CsMYB82/CsbHLH48‑CsCAD4 Model for Resistance Against Colletotrichum gloeosporioides in Camellia sinensis

Fri, 03 Apr 2026 21:29:58 -0700

ABSTRACT

Anthracnose caused by Colletotrichum gloeosporioides is a major threat to tea cultivation; however, the molecular mechanism underlying different resistance among tea cultivars remains unclear. We identified distinct expression patterns of CsMYB82 between anthracnose-resistant and susceptible varieties after infection with anthracnose from previous RNA-seq data. We further investigated the role of CsMYB82 within a lignin-associated regulatory network during anthracnose responses. We found that CsMYB1 negatively regulates CsMYB82 expression by Y1H screen. Additionally, we identified the interaction between CsMYB82 and CsbHLH48 both in vitro and in vivo. DNA-affinity purification sequencing (DAP-seq) revealed that CsMYB82 directly binds to the promoter of CsCAD4, and this binding activity is enhanced in the presence of CsbHLH48. Functional analyses indicated that overexpression of CsMYB82 or CsCAD4 in tobacco and tea leaves was associated with increased susceptibility to anthracnose, whereas transient silencing of CsMYB82 or CsCAD4 via virus-induced gene silencing (VIGS) in tea leaves resulted in reduced disease symptoms accompanied by elevated lignin accumulation. The functional analysis of CsMYB1 showed the opposite phenotype. Collectively, these results suggest that CsMYB82 participates in a transcriptional regulatory module involving CsMYB1, CsbHLH48, and CsCAD4, which modulates lignin biosynthesis and influences anthracnose responses in tea plants. This study provides mechanistic insights into the transcriptional regulation of lignin-associated defence responses and contributes to a better understanding of anthracnose resistance in tea.

Establishment of an Agrobacterium‐mediated CRISPR/Cas9 Genome Editing System for Kenaf (Hibiscus cannabinus)

Fri, 03 Apr 2026 21:08:53 -0700

Plant Biotechnology Journal, EarlyView.

Precise Creation of Elite Multilocular Germplasm Using a CBE NG System in Brassica napus

Fri, 03 Apr 2026 04:41:30 -0700

Plant Biotechnology Journal, EarlyView.

Redundant Glycerol‐3‐Phosphate Acyltransferases Regulate Thermo‐Sensitive Genic Male Sterility in Rice

Wed, 01 Apr 2026 06:31:17 -0700

ABSTRACT

Thermo-sensitive genic male sterility (TGMS) lines play a pivotal role in two-line hybrid rice breeding. However, the availability of elite TGMS germplasm remains limited, and the molecular mechanisms underlying fertility transition of TGMS lines are still poorly understood. Here, we identified a novel TGMS line, Osgpat6.1, which exhibits temperature-dependent fertility transition, with male sterility under high-temperature (HT) and restored fertility under low-temperature (LT). Cytological observations revealed that the TGMS line exhibits disrupted programmed cell death (PCD) of the tapetum under HT conditions. This disruption leads to delayed tapetum degradation and defective pollen exine formation. Map-based cloning identified a premature termination mutation in glycerol-3-phosphate acyltransferase 6.1 (OsGPAT6.1), which is highly expressed in anthers and essential for lipid biosynthesis. Lipidomic profiling revealed that disruption of OsGPAT6.1 perturbed lipid metabolism during pollen development. Genetic studies further revealed that male sterility in Osgpat6.1 mutant was restored through functional redundancy with its homologues OsGPAT6.2 and OsGPAT6.3 under LT. Overexpression of OsGPAT6.2 or OsGPAT6.3 partially restored the fertility of Osgpat6.1 mutant under HT. However, simultaneous disruption of all three homologues abolished fertility restoration even under LT. Notably, introgression of the Osgpat6.1 allele into diverse rice cultivars produced stable TGMS lines without compromising hybrid vigor. Collectively, these findings demonstrate that dysfunction of GPAT confers TGMS in rice by disrupting glycerolipid metabolism and provides TGMS germplasm to facilitate two-line hybrid rice breeding.

Field Trials and Baking Studies of Ultra‐Low Asparagine, Genome Edited (CRISPR/Cas9) and Mutant (TILLING) Wheat

Wed, 01 Apr 2026 06:26:02 -0700

ABSTRACT

Field trials were conducted of wheat (Triticum aestivum) cv. Cadenza in which asparagine synthetase gene, TaASN2, had been knocked out, either on its own or together with a partial knockout of the related gene, TaASN1, using CRISPR/Cas9. Chemical mutagenesis (TILLING) TaASN2 nulls in the Claire background were also included. The main aim was to assess the free asparagine content of the grain and the conversion of free asparagine to acrylamide, a toxic contaminant, in bread, toast and biscuits. Over 2 years of trials combined, the TaASN2 and TaASN1/2 CRISPR knockouts resulted in a reduction of free asparagine in the grain of 59% and 93%, respectively, compared with Cadenza. The reduction in the TaASN2 total knockout TILLING line compared with Claire was 50%. Yield was not affected in the edited lines but was reduced in the TILLING lines. Acrylamide in bread made from a TaASN1/2 CRISPR line was below detection levels, while in a TaASN2 CRISPR line it was 14% of the Cadenza control. Even after 4 min of toasting, acrylamide levels remained at 8% and 23%, respectively, of the control. The concentration in bread made from the TILLING TaASN2 knockout was 21% that for the Claire control, rising to 46% after 4 min of toasting. Acrylamide in biscuits made from a TaASN1/2 CRISPR line was reduced by 93% compared with the control. The relationship between acrylamide and colour was altered in the edited and mutant lines compared with the controls, with less acrylamide forming for the same degree of colour.

Metabolic Reprogramming of a Phenolic Acid by a Plant P450 Monooxygenase Reverses Bacterial Immunosuppression

Tue, 31 Mar 2026 01:34:38 -0700

Ralstonia solanacearum suppresses P450 activity to block VA-to-VanA conversion, leading to VA accumulation and compromised immunity.

ABSTRACT

Soil acidification often exacerbates plant diseases caused by soil-borne pathogens like Ralstonia solanacearum, but the underlying molecular mechanisms remain elusive. This study unveils a sophisticated metabolic game in the tobacco-R. solanacearum pathosystem, where the pathogen manipulates host metabolism to suppress immunity, and the plant counteracts by enzymatically reprogramming a key metabolic signal. Using multi-omics approaches, we discovered that R. solanacearum infection induces a significant accumulation of veratric acid (VA) in tobacco. We demonstrated that VA acts as a potent immunosuppressant rather than a nutrient for the pathogen. It broadly inhibits plant pattern-triggered immunity, including flg22-induced ROS burst, and transcriptionally represses a suite of nucleotide-binding leucine-rich repeat (NLR) receptors, crucially including NtG28897 (NtTAO1). Silencing of NtTAO1 confirmed its pivotal role in resistance against Tobacco Mosaic Virus (TMV). Facing this metabolic sabotage, tobacco engages a counter-defence mechanism. We identified a specific cytochrome P450 monooxygenase (CYP86A22) that catalyses the conversion of the disease-promoting VA into vanillic acid (VanA). Transient overexpression of this P450 in Nicotiana benthamiana enhanced the in vivo conversion of VA to VanA. Crucially, this enzymatic conversion conferred strong resistance against TMV, whereas the P450 or VA alone did not. Our findings reveal a novel plant immune strategy ‘metabolic signal reprogramming’ where a P450 enzyme detoxifies a susceptibility metabolite into a defensive compound. This work provides a new paradigm for plant–pathogen interactions and identifies promising targets for metabolic engineering or green chemical strategies to achieve sustainable disease control.

The Transcription Factor OsWRKY64 Interacts With OsART1 to Positively Regulate Al Resistance in Rice

Tue, 31 Mar 2026 01:17:22 -0700

A proposed working model illustrating the cooperative regulation of Al tolerance by OsWRKY64 and OsART1 in rice. Under Al stress conditions in acidic soil, the transcription factor OsART1 directly binds to the promoter of OsWRKY64 to activate its transcription. The synthesized OsWRKY64 protein then physically interacts with OsART1 in the nucleus. This interaction positively regulates Al tolerance through two synergistic mechanisms: (1) OsWRKY64 stabilizes OsART1 protein by inhibiting its degradation via the 26S proteasome pathway; and (2) the OsWRKY64-OsART1 complex enhances the transcriptional activation of downstream target genes, including OsALS1, OsSTAR1, and OsFRDL4. The robust expression of these genes promotes cell wall modification and Al exclusion mechanisms, ultimately resulting in reduced Al accumulation in root tips and enhanced Al tolerance.

ABSTRACT

Rice (Oryza sativa) exhibits notable aluminium (Al) tolerance, as the C₂H₂-transcription factor OsART1 plays a crucial role in regulating Al tolerance in rice by modulating the expression of specific genes. However, the posttranscriptional regulation of OsART1 remains poorly understood. In this study, we identified and characterised OsWRKY64, which interacts with OsART1. OsWRKY64 was localised in the nucleus. Tissue expression analysis indicated that OsWRKY64 was primarily expressed in the roots, and its transcription and protein accumulation were significantly induced by Al. The oswrky64 mutants and the OsWRKY64 overexpression lines exhibited decreased or increased expression of genes regulated by OsART1, which consequently led to phenotypes sensitive or resistant to Al, respectively. Further analysis indicated that OsWRKY64 interacted with OsART1 to promote its stability, thereby accelerating the transcriptional activity of OsART1 on its downstream genes. Additionally, OsART1 upregulated OsWRKY64 expression by directly binding to the OsWRKY64 promoter, thus forming a negative feedback loop between OsART1 and OsWRKY64. Overall, our results demonstrated that OsWRKY64 influences Al resistance by interacting with and stabilising OsART1 to accelerate its transcriptional activity.

Functional Characterisation of HAIRPLUS (NtHAP) Genes Involved in Trichome Development and Specialised Metabolism of Tobacco

Mon, 30 Mar 2026 03:22:05 -0700

ABSTRACT

Plant glandular trichomes are specialised epidermal structures capable of synthesising, storing and secreting numerous varieties of secondary metabolites in different classes and are central to plant defence and the biosynthesis of high-value metabolites. In this study, we characterised the HAIRPLUS (HAP) gene family and uncovered its role as a conserved regulator of trichome development and metabolism in tobacco. Four homologues, NtHAP1a, NtHAP1b, NtHAP2a and NtHAP2b, were identified and functional studies using RNAi and CRISPR-Cas9 revealed that NtHAPs act as negative regulators of glandular trichome development. Suppression of NtHAPs resulted in increased trichome density and enlarged glandular heads, as well as enhanced accumulation of diterpenoids (e.g., neophytadiene) and increased nicotine levels. Additionally, NtHAP1 appeared to have a stronger effect on trichome density. This study establishes the NtHAP genes as key negative regulators of glandular trichome development in tobacco, expanding their functional scope from trichome morphogenesis to metabolic regulation and highlighting their evolutionary conservation across Solanaceae. These findings pave the way for both fundamental research into trichome biology and practical applications in metabolic engineering and crop improvement, such as pest resistance.

Beyond Starch: Towards a Scalable Potato Platform for Molecular Farming

Izabela Anna Chincinska, Dorota Sołtys‐Kalina, Audrey Y.‐H. Teh — Sun, 29 Mar 2026 00:00:00 -0700

Re-engineering potato as a biosafe and host-optimised platform for plant molecular farming by integrating intrinsic biological traits with targeted engineering strategies.

ABSTRACT

Thirty-five years after the first recombinant protein was produced in potato and 30 years after clinical trials of edible vaccines from its tubers, the crop is being reconsidered as a molecular farming chassis. Potatoes can accumulate recombinant proteins in tubers, enabling long-term storage and simplified logistics. Clonal propagation, access to minitubers and microtubers, and an established production infrastructure further distinguish the platform. Limited pollen dispersal and reliance on vegetative propagation also provide biosafety advantages. Despite these features, potato lost ground to other hosts due to low expression levels, high downstream processing (DSP) costs in water- and starch-rich tissues and limited scalability relative to seed-propagated crops. We review the technical and economic factors behind this decline and assess new opportunities to overcome them. Recent advances include refined expression cassettes, ER and secretory pathway engineering (including targeted glycoengineering), multigene stacking, genome editing and enzyme-assisted DSP. Seed-propagated diploid potatoes and alternative Solanum germplasm offer additional chassis options. Together with the emergence of start-ups revisiting potato as a production platform, these advances point to practical routes for re-evaluating its role in plant molecular farming. We argue that a rationally engineered, diploid-based ‘bioreactor potato’ could complement existing hosts and re-establish relevance in specific niches of next-generation biomanufacturing.

Deciphering Cold Stress Resilience: Multiomics Insights in Contrasting Wheat Genotypes From the Western Himalayas

Fri, 27 Mar 2026 21:28:05 -0700

ABSTRACT

Cold stress threatens wheat productivity, particularly in regions with extreme climatic conditions. To elucidate the molecular mechanisms underlying wheat's response to cold stress, we performed a multiomics analysis integrating lipidomics, transcriptomics, proteomics and metabolomics. Our study focused on two wheat genotypes with contrasting cold tolerance levels, SKAU_52 (tolerant) and SKAU_4301 (susceptible) to capture genotype-specific responses under cold stress. Lipidomic analysis revealed significant changes in lipid composition, with unsaturated lipids such as digalactosyldiacyl glycerols (DGDGs) and monogalactosyldiacylglycerols (MGDGs) upregulated in response to cold stress. These lipids are associated with maintaining membrane fluidity, whereas saturated lipids were downregulated in the cold-tolerant genotype. Transcriptomics analysis provides a strong evidence that cold tolerance in wheat is governed by coordinated activation of the ICE-CBF-COR regulatory cascade, with the cold-tolerant genotype ‘SKAU_52’ showing stronger and more sustained induction across pathway tiers than the cold susceptible wheat genotype ‘SKAU_4301’. Similarly, proteomic data highlighted differential abundance of proteins involved in antioxidative defence, osmotic adjustment and signal transduction, including late embryogenesis abundant (LEA) proteins. Metabolome assessment revealed substantial alterations in carbohydrate and amino acid metabolism, with sucrose and amino acids such as hydroxyproline identified as key contributors to cold tolerance. Additionally, defence hormones such as salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) exhibited genotype-specific regulation with higher accumulation in cold-tolerant genotype. Overall, this integrated multi-omics approach provides novel insights into the complex molecular mechanisms underlying cold stress adaptation in wheat, supporting the development of resilient wheat varieties capable of thriving in challenging cold environments.

Genomic and Transcriptomic Analyses Provide Insights Into Erysiphe necator Pathogenicity and Grapevine Response

Fri, 27 Mar 2026 20:34:34 -0700

ABSTRACT

Grapevine powdery mildew, caused by the fungus Erysiphe necator, is one of the most prevalent obligate biotrophic pathogens in vineyards, posing a significant threat to grape production. Despite its impact, research on E. necator pathogenicity and grapevine responses remains limited. In this study, we assembled a high-quality 69.93 Mb genome for E. necator isolate NAFU1, identifying 248 candidate-secreted effector proteins (CSEPs). RNA-Seq analysis of E. necator NAFU1 and the grapevine host during various infection stages revealed that expression of many genes, especially those encoding CSEPs, was induced in planta to facilitate host colonisation. Detailed analysis identified CSEP118 as a key highly induced effector gene, plays an important role in suppression of host defence. CSEP118 appears to interfere with the grapevine defence response to infection by targeting VviTrxz, a grapevine thioredoxin. Comparative transcriptome analysis of susceptible (Vitis vinifera cv. Cabernet Sauvignon) and resistant (Vitis piasezkii accession Baishui-40) grapevines upon infection by E. necator identified VviTCP14, encoding a transcription factor, to be a likely negative regulator of grapevine resistance against E. necator. Supporting this, VviTCP14-silenced plants exhibited increased expression of resistance-related genes such as those encoding stilbene synthases (STSs) and elevated stilbene contents when compared with the wild type grapevine. In summary, this study utilised a multi-omics approach to understand mechanisms underlying E. necator effector-triggered suppression of grapevine immunity and transcriptional regulation of host defence response during grapevine-powdery mildew interaction. The regulatory mechanisms uncovered in this study should provide valuable insights for improving grapevine resistance to powdery mildew.

Multisite Field Evaluation of Oil Accumulation and Agronomic Performance in Grain and Sweet Sorghums Engineered for Lipid Hyperaccumulation

Wed, 25 Mar 2026 09:25:38 -0700

ABSTRACT

Oil sorghum (OS) has been developed by engineering grain (TX430) and sweet (Ramada) genetic backgrounds to accumulate triacylglycerols (TAG) in vegetative tissues as an energy-dense feedstock for sustainable aviation fuel (SAF) and other biofuels. This study evaluated two TX430 OS lines (TxHO-2, TxHO-3) and two Ramada OS lines (RmHO-1, RmHO-2) alongside wild-type (WT) lines in NE and IL over 2 years (2023–2024) to quantify genotype × environment effects on agronomic performance and TAG accumulation. Across four environments, TX430 OS lines showed average TAG concentrations of 15.0 g kg⁻¹ in leaves and 12.8 g kg⁻¹ in stems, approximately 19-fold higher than WT. Ramada OS lines accumulated 26.1 g kg⁻¹ in leaves and 12.3 g kg⁻¹ in stems, approximately 25-fold and 13-fold increases over WT, respectively. OS lines in TX430 exhibited an 18% reduction in biomass (8.4 vs. 9.9 Mg ha⁻¹ for WT), while Ramada OS lines had similar WT biomass (18.3 vs. 19.9 Mg ha⁻¹ for WT). Among TX430 OS lines, TxHO-2 achieved the highest TAG yield (190 kg ha⁻¹), while RmHO-1 led the Ramada lines (335 kg ha⁻¹) due to higher biomass and similar TAG concentration. Enhanced TAG accumulation increased N, P, and K removal in TX430 lines but not in Ramada lines. Structural carbohydrate and ash concentration were unaffected. Overall, results confirm vegetative lipid accumulation as a viable strategy for high-biomass sorghum, supporting its potential as a dual-purpose feedstock for SAF. Future work should focus on minimizing biomass yield penalties and improving nutrient use efficiency in oil sorghum systems.

Structural Elucidation and Engineering of the (S)‐scoulerine 2‐O‐Methyltransferase Enabling Regioselective Epiberberine Biosynthesis in Coptis chinensis

Wed, 25 Mar 2026 04:57:23 -0700

ABSTRACT

Protoberberine alkaloids are a characteristic group of natural products in Coptis plants known for their notable pharmacological activities. However, the structural similarity and the substrate promiscuity of their biosynthetic enzymes have left the precise synthetic pathways remain unclarified, posing challenges to regulate product formation. In this study, we identified CcOMT8, a key enzyme responsible for C2-methoxylation in the biosynthesis of epiberberine in C. chinensis, through methyl jasmonate elicitation analysis and comparative genomics-based microsynteny analysis. Functional characterisation demonstrated that CcOMT8 specifically catalyses 2-O-methylation of (S)-scoulerine, as verified by heterologous expression in both microbial and plant systems. Its lack of activity toward (S)-cheilanthifoline further confirmed the specific route for epiberberine biosynthesis. Structural investigations of CcOMT8 and its complexes revealed key aspects of substrate recognition and a catalytic mechanism mediated by the His253-Asp254-Glu312 triad. Comparative structural analysis with 9-O-methyltransferases indicated that hydrophilic residues and reduced steric hindrance in the substrate binding pocket govern the regioselectivity of CcOMT8. Using focused rational iterative site-specific mutagenesis (FRISM), we developed an optimised mutant, S109L/C250A/L300A, with 4.88-fold enhanced catalytic efficiency. This study elucidates the biosynthetic pathway of epiberberine in Coptis, clarifies the molecular basis of enzyme-directed metabolic flux, and provides efficient biocatalysts for the synthetic biosynthesis of protoberberine alkaloids.

CitPH4 Confers Resistance to Citrus Canker by Activating Papain‐Like Cysteine Protease

Wed, 25 Mar 2026 04:40:15 -0700

ABSTRACT

Citrus canker, a devastating disease caused by Xanthomonas citri subsp. citri (Xcc), poses a significant threat to global citrus production due to the high susceptibility of nearly all commercial citrus cultivars to it. Although transcription factor Citrus PH4 (CitPH4) is well known for regulating fruit acidity, its potential role in plant immunity remains elusive. Here, we demonstrate that CitPH4 positively regulates citrus defence against Xcc. Transcriptomic and biochemical analyses indicate that CitPH4 binds to the promoter of the responsive to dehydration 21a (CsRD21a), a gene encoding a papain-like cysteine protease (PLCP), thus modulating its expression. Functional characterisation further reveals that CsRD21a is essential for CitPH4-mediated resistance to Xcc. Mechanistically, CsRD21a interacts with pathogenesis-related protein 5 (CsPR5) to promote salicylic acid (SA) accumulation. In addition, reciprocal activation of PLCP activity and SA signalling enhances immune defences against Xcc. Collectively, our findings reveal a CitPH4-mediated citrus immunity mechanism in which the CitPH4–CsRD21a–CsPR5 module constitutes a defence loop by integrating reciprocal activation effects of PLCP activity and SA signalling. This study provides a novel insight into the regulatory network of citrus resistance against Xcc, offering potential targets for breeding citrus cultivars with enhanced disease resistance and superior fruit quality.

Sweet Potato Gene Clusters Control Anthocyanin Biosynthesis and Leaf Morphology

Tue, 24 Mar 2026 23:37:13 -0700

ABSTRACT

Sweet potato (Ipomoea batatas) exhibits diversity in pigmentation and leaf morphology, yet the genetic architecture and regulatory organisation underlying these traits remain poorly resolved, particularly with respect to organ-specific control. We hypothesised that phenotypic variation is governed by clustered genetic modules comprising regulatory and structural genes operating in an organ-specific manner. To test this, we conducted genome-wide association studies (GWAS) using 4.6 million SNPs across 260 diverse accessions, integrated with transcriptomic, haplotype and functional analyses. GWAS identified two tandem clusters of MYB transcription factors on chromosome 5 as the primary regulators of leaf anthocyanin accumulation. Expression profiling, heterologous expression and transcriptional activation assays demonstrated that IbMYB2 and IbMYB3 function as key transcriptional activators and form a mutually reinforcing regulatory module. In contrast, pigmentation in storage roots was associated with a spatially distinct genomic region enriched in anthocyanin biosynthetic genes, including IbAOMT, Ib3GGT and IbLDOX, indicating different regulations between aerial and underground organs. Comparative genomic analysis further revealed expansion and conservation of MYB clusters in sweet potato, suggesting evolutionary selection for enhanced transcriptional control. In addition, GWAS uncovered a major locus on chromosome 7 controlling leaf shape variation. Functional analyses demonstrated that conserved developmental regulators, including BEL1-like (g29974), WD40 (g26165) and LMI1-like (g29859) genes, play causal roles in leaf margin development. CRISPR/Cas9-mediated knockout of g26165 directly reduced leaf lobing, confirming its functional importance. These findings reveal clustered regulatory and structural gene modules underlying key agronomic traits and provide insights into the genetic and evolutionary mechanisms driving phenotypic diversification in sweet potato.

A Bioluminescent Reporter System for Real‐Time Monitoring of the Unfolded Protein Response in Plants

Tue, 24 Mar 2026 05:30:12 -0700

ABSTRACT

The unfolded protein response (UPR) is a critical mechanism for maintaining endoplasmic reticulum (ER) homeostasis under stress. Here, we developed a bioluminescent reporter system, AtbZIP60-LUC, in Arabidopsis to dynamically monitor ER stress by coupling IRE1-mediated splicing of bZIP60 mRNA to firefly luciferase (LUC) expression. Under ER stress, IRE1 removes a 23-bp sequence from bZIP60u, producing a spliced bZIP60s transcript in-frame with LUC, enabling luciferin-dependent luminescence. Transgenic AtbZIP60-LUC lines exhibited specificity for canonical ER stressors (heat, DTT, tunicamycin) but not osmotic stressors (NaCl, mannitol), confirmed by bioluminescence, qPCR, and immunoblotting. Time-course assays revealed rapid LUC induction by DTT (peak at 1 h) and delayed activation by tunicamycin (peak at 1–2 h), followed by signal decline, reflecting adaptive UPR dynamics. Heat stress optimization identified 38°C as optimal, inducing robust LUC expression after 2–3 h without compromising viability, while 42°C caused irreversible damage. Genetic validation in ire1a ire1b mutants abolished LUC induction, confirming IRE1 dependency, whereas constitutive UPR activation via maize 16-kDa γ-zein (16γz) overexpression triggered LUC expression without stress. Extending this system to tobacco and tomato, we engineered NbbZIP60-LUC and SlbZIP60-LUC, which similarly responded to heat (38°C), DTT, tunicamycin, and ER-localized protein aggregation (16γz, zeolin) in transient and stable assays. This work establishes bZIP60-LUC as versatile, non-invasive tools for real-time UPR monitoring in plants, offering insights into ER stress dynamics and enabling cross-species studies of stress adaptation mechanisms.

Tuning Xylan Polymerisation Enhanced Fibre Digestibility Without Biomass Loss in Sheepgrass (Leymus chinensis)

Tue, 24 Mar 2026 04:54:17 -0700

ABSTRACT

Leymus chinensis (sheepgrass), a dominant perennial grass of the Eurasian Steppe, is a crucial source of carbohydrates and energy for ruminants. However, the lignocellulose recalcitrance severely limits its digestibility. Here, we targeted xylan, a major hemicellulose interacting with cellulose and lignin in cell wall. To improve digestibility, we knocked out two IRREGULAR XYLEM 14-LIKE (LcIRX14L) genes, which encode key enzymes for xylan biosynthesis. The Lcirx14L knockout mutants exhibited reduced xylan molecular weight but produced similar amounts of biomass to wild-type plants. Simon's staining and cellulase binding assays indicated enhanced cellulose accessibility in the Lcirx14L mutant. In addition, the Lcirx14L plant showed increased saccharification efficiency, dry matter and NDF digestibility, and production of volatile fatty acids by in vitro fermentation. This study thus provides novel insight for engineering forage grasses with both high yield and quality by modifying xylan polymerization.

Two Aquaporins Mitigate Growth‐Defence Trade‐Offs by Facilitating CO2 and H2O2 Transport in Wheat

Tue, 24 Mar 2026 02:46:08 -0700

ABSTRACT

The growth-defence trade-offs pose a major challenge to breeding high-yield and disease-resistant crops. Aquaporins are membrane channels that facilitate the transport of water and other small compounds, therefore regulating the growth-defence trade-offs. However, the molecular mechanism that governs the function of aquaporins in the trade-offs remains unclear. Here, we report that Triticum aestivum TaPIP1;6 and TaPIP2;10, aquaporins of the plasma membrane intrinsic protein (PIP) family, function as dual substrate channels that concurrently enhance plant growth and resistance to powdery mildew and English grain aphid. In wheat plants growing under normal conditions, TaPIP1;6 and TaPIP2;10 facilitate CO₂ transport from the atmosphere into wheat cells and promote photosynthesis, which leads to growth enhancement and grain yield increase. In wheat plants under attack by powdery mildew pathogen or English grain aphid, TaPIP1;6 and TaPIP2;10 function as concurrent H₂O₂ transport channels, mediating the influx of apoplastic H₂O₂ into the cytoplasm. In turn, the transported H₂O₂ activates innate immunity, including the MAPK cascade, callose deposition and defence gene expression, and thereby enhances wheat resistance to powdery mildew and the English grain aphid. In essence, co-overexpression of TaPIP1;6 and TaPIP2;10 exhibits synergistic effects on CO₂ and H₂O₂ transports, further amplifying both yield and resistance traits. Taken together, our results suggest that TaPIP1;6 and TaPIP2;10 function as dual-substrate transporting channels to promote growth by regulating CO₂ transport and to enhance resistance against pathogens and insects via H₂O₂-mediated immune responses. This finding provides crucial genetic targets for breeding crop varieties combining high yield with resistance traits.

Disruption of Asparagine Synthetase Is Associated to Increased Biomass in Lotus japonicus

Tue, 24 Mar 2026 02:15:34 -0700

ABSTRACT

Asparagine (Asn) constitutes the major form of nitrogen translocated within Lotus japonicus plants. In this work we use knock-out (KO) LORE1 mutants-deficient in the asparagine synthetase gene (LjASN1), which is the most highly expressed ASN gene in plants grown under non-symbiotic (NS) conditions, but much less expressed under symbiotic (S) conditions. The analysis of two different Ljasn1 homozygous mutant lines grown under NS or S conditions indicated that a much higher biomass was produced in Ljasn1 mutants grown under NS conditions compared to the WT (wild-type), whereas little difference with the WT was observed in mutant plants under S conditions. Metabolomic analysis revealed that Ljasn1 mutant plants are quite distinct to WT plants when grown under NS conditions, but not under S conditions. Asn levels were considerably reduced in Ljasn1 mutant plants compared to the WT when plants were grown under NS but not under S conditions. A general decrease in amino acids and an increase in carbon compounds, such as sugars and oxo-acids, was detected in NS roots and shoots, respectively, which may explain the growth phenotypes observed. RNAseq analysis showed changes related to oxidative metabolism under NS conditions, and C/N metabolism under S conditions. The data indicate that the LjASN1 deficiency produces important changes in the C/N balance and metabolite allocation of L. japonicus plants resulting in higher biomass content and lower Asn levels, two interesting traits for biotechnological crops engineering.

Production of the High Value Pharmaceutical Target, Human Granulocyte‐Colony Stimulating Factor, in Nicotiana benthamiana Is Improved by Co‐Expression of the Transcriptional Regulator of the Unfolded Protein Response, bZIP60

Fri, 20 Mar 2026 04:51:52 -0700

Plant Biotechnology Journal, EarlyView.

Single‐Cell CRISPR: An Efficient Strategy for Decoding Plant Cis‐Regulatory Complexity

Thu, 19 Mar 2026 05:49:22 -0700

ABSTRACT

The generation of complex traits involves the coordinated interplay of multiple gene networks. Elucidating the function of transcriptional cis-regulatory elements (CREs) in regulating gene expression is crucial for understanding complex regulatory pathways and improving our ability to modify macro-phenotypes. While traditional bulk sequencing approaches rely on tissue or cell population aggregates, single-cell transcriptomics provides a more precise perspective by capturing cell-type-specific information. The integration of single-cell technology with genome-wide genetic screening, particularly through the single-cell CRISPR (scCRISPR) system, enables the identification of critical regulatory elements and provides novel insights into gene-expression control mechanisms. Here, we summarise recent advances in diverse strategies for functional genome analysis using the scCRISPR system, with an emphasis on its potential to revolutionise single-cell genetic screening of CREs. We also explore the challenges and opportunities for applying these approaches in plant research.

StALKBH10B‐Mediated RNA m6A Modification Inhibits Potato Salt Tolerance by Targeting Flavonoids and ABA Signalling Pathways

Thu, 19 Mar 2026 01:21:40 -0700

ABSTRACT

N⁶-methyladenosine (m⁶A) is the most prevalent methylation modification present in mRNAs, which has been confirmed to participate in many developmental and biological processes. However, the biological function and specific regulatory mechanism of m⁶A modification in relation to salt tolerance of potato remain obscure. Here, we generated a transcriptome-wide m⁶A map using salt-resistant and salt-sensitive potato varieties under salt stress conditions to uncover patterns of m⁶A methylation in the potato response to salt stress. MeRIP-seq revealed that m⁶A is significantly enriched in the CDS region in potato, by recognising the conserved motifs including RRACH and URRUAY. Numerous differential m⁶A-deposited transcripts have been identified, which were significantly enriched in ABA-signalling and flavonoids biosynthesis pathway in two potato varieties after salt stress. Notably, a positive correlation was observed between the m⁶A enrichment and mRNA abundance based on combined analysis of MeRIP-seq and mRNA-seq. StALKBH10B was identified as an m⁶A demethylase for decreasing m⁶A modification levels, inhibiting mRNA stability and translation efficiency of ABA signal-related genes (StABF3, StAAO3, and StZEP7) and flavonoids biosynthesis genes (StPAL3, StCHS, and StFLS), and overexpression of StALKBH10B suppressed salt resistance in potato. Collectively, we uncover a novel mechanism of post-transcriptional modification involved in affecting salt stress response in potato, via StALKBH10B-mediated m⁶A demethylation on targeted transcripts in the ABA signalling and flavonoids biosynthesis pathway, thereby providing candidate genes for the breeding of stress-tolerant potato cultivars.

Knocking Out Two Polyphenol Oxidase Genes Significantly Improves Recombinant Protein Purification in Nicotiana benthamiana

Sun, 08 Feb 2026 21:23:04 -0800

ABSTRACT

Efficient purification remains a key technical challenge affecting the recovery efficiency of recombinant proteins in plant-based systems. Complex metabolites, particularly polyphenols, often cause recombinant protein aggregation during purification. In this study, we identified two key polyphenol oxidase genes, PPOa and PPOb from Nicotiana benthamiana, as responsible for these effects. Using CRISPR-Cas9, we generated two ppoa;ppob double knockout lines that significantly improved the purification of virus surface proteins like SARS-CoV-2 Spike trimer and influenza HA trimer. These lines showed reduced polyphenol–protein interactions, minimised aggregation, and higher purification yields. Our work establishes a clean, high-efficiency N. benthamiana chassis for scalable recombinant protein production.

Harnessing Bulk‐Segregant Mapping to Identify Trait‐Associated Genes in the Allopolyploid Model Plant Nicotiana benthamiana

Thu, 29 Jan 2026 04:15:12 -0800

ABSTRACT

Forward genetics has been instrumental in identifying genes underlying desirable traits, yet its application to polyploid plants, many of which are key agricultural crops, remains challenging due to their genomic complexity. Therefore, we developed BenthMap, a bulk segregant analysis platform for high-throughput trait mapping and gene discovery, in the allotetraploid model plant Nicotiana benthamiana. BenthMap leverages high-quality genome assemblies of two genetically and phenotypically distinct strains, LAB and QLD. To validate the pipeline, we investigated their contrasting anthocyanin responses. Transient overexpression of AcMYB110, an activation regulator of anthocyanin biosynthesis, induces robust anthocyanin production in QLD leaves but gives a detrimental, often necrotic, response in LAB. Using BenthMap and a population derived from selfing the F1 hybrid of a LAB × QLD cross (F1S1 population), with genome coverage as low as 10×, we mapped the necrotic LAB response to a 3.5 Mb homozygous region on chromosome 10. This region contains a leucoanthocyanidin dioxygenase gene. Transiently expressing the QLD version of this gene, along with AcMYB110, restored robust anthocyanin accumulation in LAB, validating the causal gene. These findings demonstrate BenthMap's utility for rapid trait-gene identification in N. benthamiana and have potential for application to other allopolyploid plants.

Structural Variations Contribute to Subspeciation and Yield Heterosis in Rice

Zhiwu Dan, Yunping Chen, Wenchao Huang — Wed, 03 Dec 2025 04:50:26 -0800

ABSTRACT

Yield heterosis has been extensively exploited in hybrid breeding, with intersubspecific hybrids often exhibiting the most pronounced effects. However, developing elite hybrids remains a laborious and time-consuming process. The genetic basis of heterosis has been debated for over a century, hindered largely by the lack of high-quality genomes. Here, we assembled genomes for 12 representative indica, intermediate type and japonica rice accessions. Using sequence variants of the Phr1 gene, we functionally validated two deletions responsible for phenol reaction variation between the subspecies. Comparative genomic analyses revealed extensive sequence variation among these inbred lines and highlighted the pivotal role of structural variants (SVs) in rice subspeciation. Importantly, the number of SVs between parental inbred lines significantly correlated with heterosis across 17 agronomic traits, with distinct correlation patterns for intra- and intersubspecific F₁ hybrids. We identified SVs associated with S5-ORF5 and OsBZR1 and validated their function to heterosis for seed setting rate and yield heterosis, respectively, underscoring the importance of SVs in breeding intersubspecific hybrids. The genomic SVs altered gene expression and these transcriptional changes effectively explained the variance in heterosis. Furthermore, translocations outperformed other SVs and their heterozygous haplotypes exhibited heterosis over homozygous ones. Our findings establish SVs as pivotal drivers in subspeciation and highlight the overdominance model for harnessing rice heterosis.