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Summary: Plant breeding is constrained by trade‐offs among different agronomic traits by the pleiotropic nature of many genes. Genes that contribute to two or more favourable traits with no penalty on yield are rarely reported, especially in wheat. Here, we describe the editing of a wheat auxin response factor TaARF12 by using CRISPR/Cas9 that rendered shorter plant height with larger spikes. Changes in plant architecture enhanced grain number per spike up to 14.7% with significantly higher thousand‐grain weight and up to 11.1% of yield increase under field trials. Weighted Gene Co‐Expression Network Analysis (WGCNA) of spatial–temporal transcriptome profiles revealed two hub genes: RhtL1, a DELLA domain‐free Rht‐1 paralog, which was up‐regulated in peduncle, and TaNGR5, an organ size regulator that was up‐regulated in rachis, in taarf12 plants. The up‐regulation of RhtL1 in peduncle suggested the repression of GA signalling, whereas up‐regulation of TaNGR5 in spike may promote GA response, a working model supported by differential expression patterns of GA biogenesis genes in the two tissues. Thus, TaARF12 complemented plant height reduction with larger spikes that gave higher grain yield. Manipulation of TaARF12 may represent a new strategy in trait pyramiding for yield improvement in wheat. [ABSTRACT FROM AUTHOR]

Summary: Phased, small interfering RNAs (phasiRNAs) are important for plant anther development, especially for male sterility. PhasiRNA biogenesis is dependent on genes like RNA polymerase 6 (RDR6), DICER‐LIKE 4 (DCL4), or DCL5 to produce 21‐ or 24 nucleotide (nt) double‐strand small RNAs. Here, we generated mutants of DCL4, DCL5 and RDR6 using CRISPR/Cas9 system and studied their effects on plant reproductive development and phasiRNA production in wheat. We found that RDR6 mutation caused sever consequence throughout plant development starting from seed germination and the dcl4 mutants grew weaker with thorough male sterility, while dcl5 plants developed normally but exhibited male sterility. Correspondingly, DCL4 and DCL5, respectively, specified 21‐ and 24‐nt phasiRNA biogenesis, while RDR6 contributed to both. Also, the three key genes evolved differently in wheat, with TaDCL5‐A/B becoming non‐functioning and TaRDR6‐A being lost after polyploidization. Furthermore, we found that PHAS genes (phasiRNA precursors) identified via phasiRNAs diverged rapidly among sub‐genomes of polyploid wheat. Despite no similarity being found among phasiRNAs of grasses, their targets were enriched for similar biological functions. In light of the important roles of phasiRNA pathways in gametophyte development, genetic dissection of the function of key genes may help generate male sterile lines suitable for hybrid wheat breeding. [ABSTRACT FROM AUTHOR]

Summary: Reactive oxygen species (ROS) play important roles during anther and pollen development. DNA damage may cause chromosome fragmentation that is considered to underlie chromosome elimination for haploid induction by matrilineal pollen, a key step in MATRILINEAL‐based double haploid breeding technology. But when and how DNA damage occurs is unknown.We performed comparative studies of wheat pollens from the wild‐type and the CRISPR/Cas9 edited matrilineal mutant (mMTL).Chemical assays detected a second wave of ROS in mMTL pollen at the three‐nuclei‐stage and subsequently, along with reduced antioxidant enzyme activities. RNA‐seq analysis revealed disturbed expression of genes for fatty acid biosynthesis and ROS homoeostasis. Gas chromatography‐mass spectrometry measurement identified abnormal fatty acid metabolism that may contribute to defective mMTL pollen walls as observed using electron microscopy, consistent with the function of MTL as a phospholipase. Moreover, DNA damage was identified using TdT‐mediated dUTP nick‐end labelling and quantified using comet assays. Velocity patterns showed that ROS increments preceded that of DNA damage over the course of pollen maturation.Our work hypothesises that mMTL‐triggered later‐stage‐specific ROS causes DNA damage that may contribute to chromosome fragmentation and hence chromosome elimination during haploid induction. These findings may provide more ways to accelerate double haploid‐based plant breeding. [ABSTRACT FROM AUTHOR]

Summary: The AGAMOUS‐LIKE6 (AGL6)‐like genes are ancient MADS‐box genes and are functionally studied in a few model plants. The knowledge of these genes in wheat remains limited. Here, by studying a 'double homoeolog mutant' of the AGL6 gene in tetraploid wheat, we showed that AGL6 was required for the development of all four whorls of floral organs with dosage‐dependent effect on floret fertility. Yeast two‐hybrid analyses detected interactions of AGL6 with all classes of MADS‐box proteins in the ABCDE model for floral organ development. AGL6 was found to interact with several additional proteins, including the G protein β and γ (DEP1) subunits. Analysis of the DEP1‐B mutant showed a significant reduction in spikelet number per spike in tetraploid wheat, while overexpression of AGL6 in common wheat increased the spikelet number per spike and hence the grain number per spike. RNA‐seq analysis identified the regulation of several meristem activity genes by AGL6, such as FUL2 and TaMADS55. Our work therefore extensively updated the wheat ABCDE model and proposed an alternative approach to improve wheat grain yield by manipulating the AGL6 gene. [ABSTRACT FROM AUTHOR]

Summary: Auxin signaling is essential for the development of grain size and grain weight, two important components for crop yield. However, no auxin/indole acetic acid repressor (Aux/IAA) has been functionally characterized to be involved in the development of wheat (Triticum aestivum L.) grains to date. Here, we identified a wheat Aux/IAA gene, TaIAA21, and studied its regulatory pathway. We found that TaIAA21 mutation significantly increased grain length, grain width, and grain weight. Cross‐sections of mutant grains revealed elongated outer pericarp cells compared to those of the wild type, where the expression of TaIAA21 was detected by in situ hybridization. Screening of auxin response factor (ARF) genes highly expressed in early developing grains revealed that TaARF25 interacts with TaIAA21. In contrast, mutation of the tetraploid wheat (Triticum turgidum) ARF25 gene significantly reduced grain size and weight. RNA sequencing analysis revealed upregulation of several ethylene response factor genes (ERFs) in taiaa21 mutants which carried auxin response cis‐elements in their promoter. One of them, ERF3, was upregulated in the taiaa21 mutant and downregulated in the ttarf25 mutant. Transactivation assays showed that ARF25 promotes ERF3 transcription, while mutation of TtERF3 resulted in reduced grain size and weight. Analysis of natural variations identified three TaIAA21‐A haplotypes with increased allele frequencies in cultivars relative to landraces, a signature of breeding selection. Our work demonstrates that TaIAA21 works as a negative regulator of grain size and weight development via the ARF25–ERFs module and is useful for yield improvement in wheat. [ABSTRACT FROM AUTHOR]

Summary: The wheat AP2‐like transcription factor gene Q has played a major role in domestication by conferring the free‐threshing character and pleiotropically affecting numerous other traits. However, little information is known regarding the molecular mechanisms associated with the regulation of these traits by Q, especially for the structural determination of threshability. Here, transcriptome analysis of immature spike tissues in three lines nearly isogenic for Q revealed over 3000 differentially expressed genes (DEGs) involved in a number of pathways. Using phenotypic, microscopic, transcriptomic, and tissue‐specific gene expression analyses, we demonstrated that Q governs threshability through extensive modification of wheat glumes including their structure, cell wall thickness, and chemical composition. Critical DEGs and pathways involved in secondary cell wall synthesis and regulation of the chemical composition of glumes were identified. We also showed that the mutation giving rise to the Q allele synchronized the expression of genes for micro‐sporogenesis that affected pollen fertility, and may determine the final grain number for wheat spikes. Transcriptome dissection of genes and genetic pathways regulated by Q should further our understanding of wheat domestication and improvement. Significance Statement: The Q gene has played a major role in the domestication of wheat, and contributed to the rise of modern human civilization. The knowledge gained in this study regarding the genes, pathways, and physiological mechanisms underpinning seed threshability and pollen fertility, two important traits pleiotropically regulated by Q, provides a better understanding of wheat domestication and improvement. [ABSTRACT FROM AUTHOR]

Summary: DNA methylation is dynamically involved in plant immunity, but little information is known about its roles in plant interactions with biotrophic fungi, especially in temperate grasses such as wheat (Triticum aestivum).Using wheat diploid progenitor Aegilops tauschii accession AL8/78, the genome of which has been sequenced, we assessed the extent of DNA methylation in response to infection with Blumeria graminis f. sp. tritici (Bgt), which causes powdery mildew.Upon Bgt infection, ARGONAUTE4a (AGO4a) was significantly downregulated in A. tauschii, which was accompanied by a substantial reduction in AGO4a‐sorted 24‐nt siRNA levels, especially for genes near transposable elements (TAGs). Bisulfite sequencing revealed abundant differentially methylated regions (DMRs) with CHH hypomethylation. TAGs bearing CHH‐hypomethylated DMRs were enriched for 'response to stress' functions, including receptor kinase, peroxidase, and pathogenesis‐related genes. Virus‐induced gene silencing (VIGS) of a DOMAINS REARRANGED METHYLASE 2 (DRM2) homolog enhanced plant resistance to Bgt. The effect of CHH hypomethylation was exemplified by the upregulation of a pathogenesis‐related β‐1,3‐glucanse gene implicated in Bgt defense.These findings support the idea that dynamic DNA methylation represents a regulatory layer in the complex mechanism of plant immunity, which could be exploited to improve disease resistance in common wheat. [ABSTRACT FROM AUTHOR]