Hailiang Zhao

Grain number and size are important agronomic traits determining grain yield, and yield improvement depends on exploring functional variations of key regulatory genes. Mitogen-activated protein kinase 6 (MAPK6) plays a key role in crop development; however, its function and variation in wheat remain largely unclear. In this study, we aimed to characterize the function and haplotype variations of TaMAPK6-7A in wheat and develop functional molecular markers for marker assisted breeding. We identified three TaMAPK6 homoeologs on 7A, 7B, and 7D in wheat through bioinformatics analysis and revealed their evolutionary trajectory by phylogenetic analysis, with clear monocot-dicot lineage divergence and TaMAPK6 homoeolog clustering matching with hexaploid wheat’s allopolyploid origin. Spatiotemporal expression analysis showed that the TaMAPK6 homoeologs constitutively expressed in wheat tissues and were highly abundant in endosperm, spike, grain, and anther, with TaMAPK6-7A showing slightly higher transcript levels. In an ethyl methanesulfonate (EMS)-induced Jing411 mutant library, we identified a loss-of-function mutant of TaMAPK6-7A (J7633452), which exhibited severely reduced grain number per spike, impaired anther fertility, and increased grain size. Natural variation analysis of a large set of wheat accessions identified two major haplotypes of TaMAPK6-7A, with Type I was identical to the reference genome cultivar ‘Chinese Spring’, and Type II was consistent with the elite wheat cultivar ‘AK58’. We developed a PCR marker to accurately distinguish the two haplotypes and genotyped 192 wheat cultivars and elite breeding lines. Phenotypic evaluation indicated that Type II was an elite haplotype significantly associated with higher grain number per spike. This study characterizes TaMAPK6-7A as a key regulator of grain number per spike, providing a gene and molecular marker for marker-assisted breeding to improve grain yield.

The photosynthetic system of maize (Zea mays) leaves is sensitive to low temperatures and suffers from irreversible damage induced by cold exposure, making cold stress a major factor limiting maize yield. Identifying genes that improve the recovery of photosynthesis from low temperatures in maize will help enhance the cold tolerance of this crop and ensure stable yields. Here, we demonstrate the role of starch phosphorylase 2 (ZmPHOH) in promoting photosynthetic recovery from cold damage. Chlorotic leaf3 (chl3), a null mutant of ZmPHOH, which undergoes chlorophyll degradation and chlorosis earlier than under normal growth conditions after brief exposure to 8 °C and restoration to normal. We determined that chl3 plants could not repair the damage to their photosynthetic system caused by short-term cold exposure after the temperature returned to normal. Metabolome and transcriptome profiling indicated that the soluble sugar content in chl3 leaves was significantly increased after cold treatment and could not be catabolized promptly, leading to repression of photosynthetic gene expression. Our results reveal that ZmPHOH enhances post-cold photosynthetic recovery by promoting the decomposition and metabolism of soluble sugars, thereby regulating the low-temperature resilience in maize, which provides new insights into the chilling tolerance mechanism of maize.

Carbohydrate partitioning is essential for plant growth and development, and its hindrance will result in excess accumulation of carbohydrates in source tissues. Most of the related mutants in maize (Zea mays L.) display impaired whole‐plant sucrose transport, but other mechanisms affecting carbohydrate partitioning have seldom been reported. Here, we characterized chlorotic leaf3 (chl3), a recessive mutation causing leaf chlorosis with starch accumulation excessively in bundle sheath chloroplasts, suggesting that chl3 is defective in carbohydrate partitioning. Positional cloning revealed that the chl3 phenotype results from a frameshift mutation in ZmPHOH, which encodes starch phosphorylase 2. Two mutants in ZmPHOH exhibited the same phenotype as chl3, and both alleles failed to complement the chl3 mutant phenotype in an allelism test. Inactivation of ZmPHOH in chl3 leaves reduced the efficiency of transitory starch conversion, resulting in increased leaf starch contents and altered carbohydrate metabolism patterns. RNA‐seq revealed the transcriptional downregulation of genes related to photosynthesis and carbohydrate metabolism in chl3 leaves compared to the wild type. Our results demonstrate that transitory starch remobilization is very important for cellular carbohydrate partitioning in maize, in which ZmPHOH plays an indispensable role.

RNA polymerase III (RNAPIII) contains 17 subunits forming 4 functional domains that control the different stages of RNAPIII transcription and are dedicated to the synthesis of small RNAs such as 5S rRNA and tRNAs. Here, we identified 23 genes encoding these subunits in Arabidopsis (Arabidopsis thaliana) and further analyzed 5 subunits (NRPC2, NRPC3, NRPC8, NRPABC1, and NRPABC2) encoded by 6 genes with different expression patterns and belonging to different sub-complexes. The knockdown of these genes repressed the expression of 5S rRNA and tRNAs, causing seed developmental arrest at different stages. Among these knockdown mutants, RNA-seq analysis revealed 821 common differentially expressed genes (DEGs), significantly enriched in response to stress, abscisic acid, cytokinins, and the jasmonic acid signaling pathway. Weighted gene co-expression network analysis (WGCNA) revealed several hub genes involved in embryo development, carbohydrate metabolic and lipid metabolic processes. We identified numerous unique DEGs between the mutants belonging to pathways, including cell proliferation, ribosome biogenesis, cell death, and tRNA metabolic processes. Thus, NRPC2, NRPC3, NRPC8, NRPABC1, and NRPABC2 control seed development in Arabidopsis by influencing RNAPIII activity and, thus, hormone signaling. Reduced expression of these subunit genes causes an insufficient accumulation of the total RNAPIII, leading to the phenotypes observed following the genetic knockdown of these subunits.

ZmNRPC2 affects RNAPIII activity and regulates the expression of genes involved in cell proliferation and endoreduplication to control maize kernel de

Starch, the main form of stored energy in plants, plays an important role in maize (Zea mays L.) kernel development. The Shrunken-2 (Sh2) gene encodes the large subunit of the rate-limiting starch biosynthetic enzyme ADP-glucose pyrophosphorylase (AGPase). The sh2 mutant exhibits impaired AGPase activity, resulting in the partial or complete loss of starch synthesis. Here, we investigated the transcriptional regulatory framework of sh2 through transcriptome and co-expression network analysis using an F₂ population derived from the maize reference line B73 and sweet corn inbred line HZ508. We identified 5175 differentially expressed genes (DEGs), including 2878 upregulated and 2297 downregulated genes in sh2 mutant lines. DEGs are associated with various biological processes including nutrient reservoir activity, transferase activity, catalytic activity, water deprivation and glycogen metabolism. At the genetic level, 2465 DEGs, including 357 transcription factors, were involved in transcription. In addition, the maize floury and opaque mutant genes fl1, ndk2, o7 and o2, which regulate the biosynthesis of 22KD zein, were co-expressed with the differential expressed transcription factor genes, thus suggesting that zein content might be a key regulator coordinating the expression of genes determining starch accumulation in maize endosperm.

Plant mitochondrial transcription termination factor (mTERF) genes comprise a large family with important roles in regulating organelle gene expression. In this study, a comprehensive database search yielded 31 potential mTERF genes in maize (Zea mays L.) and most of them were targeted to mitochondria or chloroplasts. Maize mTERF were divided into nine main groups based on phylogenetic analysis, and group IX represented the mitochondria and species-specific clade that diverged from other groups. Tandem and segmental duplication both contributed to the expansion of the mTERF gene family in the maize genome. Comprehensive expression analysis of these genes, using microarray data and RNA-seq data, revealed that these genes exhibit a variety of expression patterns. Environmental stimulus experiments revealed differential up or down-regulation expression of maize mTERF genes in seedlings exposed to light/dark, salts and plant hormones, respectively, suggesting various important roles of maize mTERF genes in light acclimation and stress-related responses. These results will be useful for elucidating the roles of mTERF genes in the growth, development and stress response of maize.