CRISPR/Cas9-Induced Dwarfism in Barley: Impacts on Yield-Related Traits and Root Architecture

A Wild Emmer Wheat B3 Transcription Factor Regulates Seed Growth and Nutrient Allocation.

Seed weight (SW) and nutrient allocation are key determinants of yield and grain quality in wheat, yet the regulatory basis of naturally occurring variation in these traits remains poorly resolved. Wild emmer wheat (Triticum dicoccoides), the progenitor of modern wheat, retains extensive eco-geographically structured genetic diversity that was largely eroded during domestication. Here, we identify a B3-domain transcription factor (B3TF) as a key regulator of seed growth and metabolic partitioning in wheat. Genome-wide association analysis of ~460 wild emmer accessions reveals a major locus on chromosome 2BL associated with SW, seed area and nitrogen (N) content, displaying pronounced climatic differentiation across environmental gradients. Introgression of the 2BL wild segment into the hexaploid wheat cultivars Chinese Spring and Bethlehem increases SW in cultivated backgrounds. Independent loss-of-function alleles generated by EMS mutagenesis in the tetraploid wheat cultivar Kronos produce larger seeds. Further, RNA-seq of EMS mutants revealed metabolic reprogramming with upregulated fatty acid, nitrogen and phenylpropanoid pathways and downregulated carbohydrate metabolism and sugar transport. Metabolomic, lipidomic and ICP-MS data showed increased essential amino acids, sugars, lipids, N content and minerals (Zn, Fe, Mo). Furthermore, CRISPR/Cas9-mediated editing in the hexaploid wheat cultivar Fielder produced similar increases in SW and N content as observed in the EMS mutants, establishing this gene as a negative regulator of seed growth across ploidy levels. In addition, natural haplotypes show reciprocal climatic distributions, linking regulatory variation to environmental adaptation. Our findings uncover a TF underlying natural seed trait variation in wild wheat, providing a framework for exploiting regulatory alleles to enhance yield and nutritional quality in modern wheat.

SiDT1 Defines Plant Architecture Reminiscent of Green Revolution in Foxtail Millet

ABSTRACT Foxtail millet (Setaria italica) is a drought‐tolerant C4 cereal that grows on marginal lands and serves as a nutrient‐rich food for millions in Asia and Africa. Despite its resilience and nutritional value, the genetic basis underlying plant height variation in foxtail millet remains incompletely understood, thereby constraining the effective implementation of semi‐dwarfing strategies analogous to those that drove the Green Revolution in major cereals. This study identified sidt1, a semi‐dwarf, high‐tillering mutant exhibiting a compact architecture and enhanced lodging resistance. It is demonstrated that SiDT1 encodes a GA3‐oxidase orthologous to rice D18/XIAOWEI. A single A‐to‐T mutation disrupted its catalytic function, reducing bioactive gibberellin biosynthesis. CRISPR‐Cas9 knockout lines recapitulated the sidt1 phenotype, confirming SiDT1’s functional role. Combined transcriptomic profiling and SiD53 immunoblot analysis indicated that strigolactone‐related signaling is perturbed in sidt1, in agreement with its enhanced tillering phenotype. Notably, under high‐density planting conditions, sidt1 maintained grain yield and quality while exhibiting superior lodging resistance. These findings identify SiDT1 as a key regulator of plant architecture and establish a semi‐dwarf ideotype reminiscent of the rice Green Revolution, providing a valuable genetic resource for high‐density and mechanized foxtail millet production.

A Gamma Ray–Induced Dwarf and Early Mutant of Brassica juncea With Favourable Traits for Developing High‐Yielding, Dwarf/Semi‐Dwarf Genotypes

Introduction of dwarfism to cereals has set a new paradigm in crop improvement programmes. To date, very little progress has been made in the development of high‐yielding dwarf/semi‐dwarf genotypes in oilseed Brassica . Previous attempts using induced mutagenesis employed in the induction of dwarfism in Brassica crops yielded limited success, and many mutants had markedly unfavourable modifications in the plant. In the present study, we characterised a gamma radiation–induced dwarf and early mutant, Trombay juncea dwarf‐1 ( tjd1 ), in Indian mustard, Brassica juncea cv. Varuna, that showed improved yield‐contributing traits like total siliquae number and number of branches. This mutant offers a prominent source of dwarfism and earliness for crop improvement in Indian mustard, B. juncea . Nature of dwarfism in tjd1 was assessed in terms of major components of plant architecture contributing to plant height. The mutant showed about 45.09% reduction in plant height compared to the parent cultivar due to the reduction in the height of the first branch, length of main fruiting axis and number and length of the internodes. At anatomical level, mutant tjd1 showed reduction in both cell size and number of cells/mm. The genetic analysis using F 1 , F 2 and BC 1 F 1 populations indicate that the dwarf plant height is controlled by a single recessive gene with incomplete dominance. The mutant responded towards auxin treatment at both seedling and late vegetative phase and also showed significant downregulation of the key genes involved in phytohormone pathways. Overall, the B. juncea mutant tjd1 will be very useful as a source for ideotype breeding to develop early and dwarf/semi‐dwarf high‐yielding genotypes.

Breaking the growth-defense trade-off in cereal crops: CRISPR/Cas and moonlighting proteins in biotic stress resistance

Molecular Mapping and CRISPR/Cas9 Validation Reveal That SlVQ10 Regulates Seed Size in Tomato

Seed size affects seed vigor, seedling establishment, and seed utilization in tomato. Yet the genetic basis of this trait remains poorly defined in tomato itself. Here, we describe a stable small–seed mutant, T31, isolated from an ethyl methanesulfonate (EMS)–mutagenized population of the elite inbred line DL5. Relative to the wild type, T31 showed a 24% reduction in seed width, whereas vegetative growth and major fruit traits were largely unchanged. Throughout this study, ‘seed size’ refers to seed width, which was used as the principal index of seed size because tomato seeds are oblate. Genetic analysis of six populations (P1, P2, F1, F2, BC1, and BC2) indicated that the phenotype is controlled by a single recessive locus, designated ssm. Bulked segregant analysis sequencing (BSA–seq) placed ssm within a 1.77 –Mb interval on chromosome 4. KASP–based fine mapping reduced this interval to 390 kb and identified six EMS–type SNPs. Only one of these SNPs was located in an exon of Solyc04g073950.2, where it caused a Pro337Ser substitution. This gene encodes a VQ motif–containing protein and was designated SlVQ10. To test gene function, we generated CRISPR/Cas9 knockout lines in the DL5 background. Two independent homozygous knockout lines reproduced the small–seed phenotype. Seed size was reduced by 31–33%, and thousand–seed weight decreased by 33–35%. Histological analysis further showed reduced seed–coat cell expansion in the mutants. Together, the genetic and genome–editing data support SlVQ10 as the gene underlying ssm and indicate that it promotes seed size in tomato.

TaGAD2 is a potential downstream effector of Rht5 in controlling wheat plant height

Plant height is a key determinant of wheat plant architecture that affect lodging resistance and grain yield. The GA-responsive (GAR) dwarfing gene Rht5 was previously shown to decrease plant height without compromise of wheat seedling vigor and considered as a promising candidate gene for breeding wheat varieties in water-limited conditions. However, the mechanisms underlying Rht5-mediated dwarfism are unclear. In this study, we investigated the genetic effects of Rht5 on wheat growth and development using recombinant inbred lines (RILs) and found that Rht5 reduces plant height through inhibition of cell proliferation while it promotes cell elongation. The dual functions of Rht5 on cell growth during wheat stem elongation were associated with the alteration of the homeostasis of endogenous growth-promoting phytohormones cytokinins and gibberellins. Transcriptome analysis of Rht5 RILs and their parental lines identified TaGAD2 (glutamate decarboxylase), encoding a functional glutamate decarboxylase localized at the plasma membrane that catalyzes γ-aminobutyric acid (GABA) biosynthesis, as a potential downstream regulator of Rht5-mediated dwarfism. Functional assays demonstrated that overexpression of TaGAD2 could reduce plant height while TaGAD2 knockdown increased plant height and improved lodging resistance, indicating a negative role of TaGAD2 in controlling wheat plant height. We also conducted haplotype analysis of TaGAD2 in a natural wheat population and identified TaGAD2H1 as a potential favorable allele for wheat dwarfing breeding without compromising grain number. Our study provides new insights into the molecular mechanism of the Rht5-mediated plant height regulatory pathway and valuable gene resource for the genetic improvement of wheat plant architecture. Rht5 regulates wheat plant height and yield-related traits partly through modulation of a downstream gene TaGAD2, which controls GABA biosynthesis and influences stem elongation, lodging resistance, and photosynthetic performance.