Pleiotropic phenotypic effects of the TaCYP78A family on multiple yield-related traits in wheat.

Increasing crop yield depends on selecting and utilizing pleiotropic genes/alleles to improve multiple yield-related traits (YRTs) during crop breeding. However, synergistic improvement of YRTs is challenging due to the trade-offs between YRTs in breeding practices. Here, the favourable haplotypes of the TaCYP78A family are identified by analysing allelic variations in 1571 wheat accessions worldwide, demonstrating the selection and utilization of pleiotropic genes to improve yield and related traits during wheat breeding. The TaCYP78A family members, including TaCYP78A3, TaCYP78A5, TaCYP78A16, and TaCYP78A17, are organ size regulators expressed in multiple organs, and their allelic variations associated with various YRTs. However, due to the trade-offs between YRTs, knockdown or overexpression of TaCYP78A family members does not directly increase yield. Favourable haplotypes of the TaCYP78A family, namely A3/5/16/17Ap-Hap II, optimize the expression levels of TaCYP78A3/5/16/17-A across different wheat organs to overcome trade-offs and improve multiple YRTs. Different favourable haplotypes have both complementary and specific functions in improving YRTs, and their aggregation in cultivars under strong artificial selection greatly increase yield, even under various planting environments and densities. These findings provide new support and valuable genetic resources for molecular breeding of wheat and other crops in the era of Breeding 4.0.

TaBZR1 enhances wheat salt tolerance via promoting ABA biosynthesis and ROS scavenging.

Brassinosteroids (BRs) are vital plant steroid hormones involved in numerous aspects of plant life including growth, development, and responses to various stresses. However, the underlying mechanisms of how BR regulates abiotic stress responses in wheat (Triticum aestivum L.) remain to be elucidated. Here, we find that BR signal core transcription factor BRASSINAZOLE-RESISTANT1 (TaBZR1) is significantly up-regulated by salt treatment. Overexpression of Tabzr1-1D (a gain-of-function TaBZR1 mutant protein) improves wheat salt tolerance. Furthermore, we show that TaBZR1 binds directly to the G-box motif in the promoter of ABA biosynthesis gene TaNCED3 to activate its expression and promotes ABA accumulation. Moreover, TaBZR1 associates with the promoters of ROS-scavenging genes TaGPX2 and TaGPX3 to activate their expression. Taken together, our results elucidate that TaBZR1 improves salt-stress tolerance by activating some genes involved in the biosynthesis of ABA and ROS scavenging in wheat, which gives us a new strategy to improve the salt tolerance of wheat.

Two alternative splicing variants of a wheat gene TaNAK1, TaNAK1.1 and TaNAK1.2, differentially regulate flowering time and plant architecture leading to differences in seed yield of transgenic Arabidopsis.

In wheat production, appropriate flowering time and ideal plant architecture are the prerequisites for high grain yield. Alternative splicing (AS) is a vital process that regulates gene expression at the post-transcriptional level, and AS events in wheat have been found to be closely related to grain-related traits and abiotic stress tolerance. However, AS events and their biological roles in regulating flowering time and plant architecture in wheat remain unclear. In this study, we report that TaNAK1 undergoes AS, producing three splicing variants. Molecular characterization of TaNAK1 and its splicing variants demonstrated that all three protein isoforms have a conserved NB-ARC domain and a protein kinase domain, but the positions of these two domains and the length of the protein kinase domains are different among them, implying that they may have different three-dimensional structures and therefore have different functions. Further investigations showed that the two splicing variants of TaNAK1, TaNAK1.1 and TaNAK1.2, exhibited different expression patterns during wheat growth and development, while the other one, TaNAK1.3, was not detected. Subcellular localization demonstrated that TaNAK1.1 was mainly localized in the cytoplasm, while TaNAK1.2 was localized in the nucleus and cytoplasm. Both TaNAK1.1 and TaNAK1.2 exhibit protein kinase activity in vitro. Ectopic expression of TaNAK1.1 and TaNAK1.2 in Arabidopsis demonstrated that these two splicing variants play opposite roles in regulating flowering time and plant architecture, resulting in different seed yields. TaNAK1.2 positive regulates the transition from vegetative to reproductive growth, plant height, branching number, seed size, and seed yield of Arabidopsis, while TaNAK1.1 negatively regulates these traits. Our findings provide new gene resource for regulating flowering time and plant architecture in crop breeding for high grain yield.

The miR319/TaGAMYB3 module regulates plant architecture and improves grain yield in common wheat (Triticum aestivum).

Plant architecture is a key determinant of crop productivity and adaptation. The highly conserved microRNA319 (miR319) family functions in various biological processes, but little is known about how miR319 regulates plant architecture in wheat (Triticum aestivum). Here, we determined that the miR319/TaGAMYB3 module controls plant architecture and grain yield in common wheat. Repressing tae-miR319 using short tandem target mimics resulted in favorable plant architecture traits, including increased plant height, reduced tiller number, enlarged spikes and flag leaves, and thicker culms, as well as enhanced grain yield in field plot tests. Overexpressing tae-miR319 had the opposite effects on plant architecture and grain yield. Although both TaPCF8 and TaGAMYB3 were identified as miR319 target genes, genetic complementation assays demonstrated that only miR319-resistant TaGAMYB3 (rTaGAMYB3) abolished tae-miR319-mediated growth inhibition of flag leaves and spikes. TaGAMYB3 functions as a transcriptional activator of downstream genes, including TaPSKR1, TaXTH23, TaMADS5 and TaMADS51, by binding to their promoters. Furthermore, TaGAMYB3 physically interacts with TaBA1, an important regulator of spike development, to additively activate the transcription of downstream genes such as TaMADS5. Our findings provide insight into how the miR319/TaGAMYB3 module regulates plant architecture and improves grain yield in common wheat.

TaKLU Plays as a Time Regulator of Leaf Growth via Auxin Signaling.

The growth of leaves is subject to strict time regulation. Several genes influencing leaf growth have been identified, but little is known about how genes regulate the orderly initiation and growth of leaves. Here, we demonstrate that TaKLU/TaCYP78A5 contributes to a time regulation mechanism in leaves from initiation to expansion. TaKLU encodes the cytochrome P450 CYP78A5, and its homolog AtKLU has been described whose deletion is detrimental to organ growth. Our results show that TaKLU overexpression increases leaf size and biomass by altering the time of leaf initiation and expansion. TaKLU-overexpressing plants have larger leaves with more cells. Further dynamic observations indicate that enlarged wheat leaves have experienced a longer expansion time. Different from AtKLU inactivation increases leaf number and initiation rates, TaKLU overexpression only smooths the fluctuations of leaf initiation rates by adjusting the initiation time of local leaves, without affecting the overall leaf number and initiation rates. In addition, complementary analyses suggest TaKLU is functionally conserved with AtKLU in controlling the leaf initiation and size and may involve auxin accumulation. Our results provide a new insight into the time regulation mechanisms of leaf growth in wheat.

BnERF114.A1, a Rapeseed Gene Encoding APETALA2/ETHYLENE RESPONSE FACTOR, Regulates Plant Architecture through Auxin Accumulation in the Apex in Arabidopsis.

Plant architecture is crucial for rapeseed breeding. Here, we demonstrate the involvement of BnERF114.A1, a transcription factor for ETHYLENE RESPONSE FACTOR (ERF), in the regulation of plant architecture in Brassica napus. BnERF114.A1 is a member of the ERF family group X-a, encoding a putative 252-amino acid (aa) protein, which harbours the AP2/ERF domain and the conserved CMX-1 motif. BnERF114.A1 is localised to the nucleus and presents transcriptional activity, with the functional region located at 142-252 aa of the C-terminus. GUS staining revealed high BnERF114.A1 expression in leaf primordia, shoot apical meristem, leaf marginal meristem, and reproductive organs. Ectopic BnERF114.A1 expression in Arabidopsis reduced plant height, increased branch and silique number per plant, and improved seed yield per plant. Furthermore, in Arabidopsis, BnERF114.A1 overexpression inhibited indole-3-acetic acid (IAA) efflux, thus promoting auxin accumulation in the apex and arresting apical dominance. Therefore, BnERF114.A1 probably plays an important role in auxin-dependent plant architecture regulation.

Modified expression of TaCYP78A5 enhances grain weight with yield potential by accumulating auxin in wheat (Triticum aestivum L.).

Increasing grain yield has always been the primary goal of crop breeding. KLUH/CYP78A5 has been shown to affect seed size in several plant species, but the relevant molecular mechanism is still unclear and there are no reports of this gene contributing to yield. Here, we demonstrate that modified expression of TaCYP78A5 can enhance wheat grain weight and grain yield per plant by accumulating auxin. TaCYP78A5 is highly expressed in maternal tissues, including ovary and seed coat during wheat development. The constitutive overexpression of TaCYP78A5 leads to significantly increased seed size and weight but not grain yield per plant due to the strengthening of apical dominance. However, localized overexpression of TaCYP78A5 in maternal integument enhances grain weight and grain yield per plant by 4.3%-18.8% and 9.6%-14.7%, respectively, in field trials. Transcriptome and hormone metabolome analyses reveal that TaCYP78A5 participates in auxin synthesis pathway and promotes auxin accumulation and cell wall remodelling in ovary. Phenotype investigation and cytological observation show that localized overexpression of TaCYP78A5 in ovary results in delayed flowering and prolonged proliferation of maternal integument cells, which promote grain enlargement. Moreover, naturally occurring variations in the promoter of TaCYP78A5-2A contribute to thousand-grain weight (TGW) and grain yield per plant of wheat;TaCYP78A5-2A haplotype Ap-HapII with higher activity is favourable for improving grain weight and grain yield per plant and has been positively selected in wheat breeding. Then, a functional marker of TaCYP78A5 haplotype Ap-HapII is developed for marker-assisted selection in wheat grain and yield improvement.

The transcription factor TaLAX1 interacts with Q to antagonistically regulate grain threshability and spike morphogenesis in bread wheat.

The domestication gene Q is largely responsible for the widespread cultivation of wheat because it confers multiple domestication traits. However, the underlying molecular mechanisms of how Q regulates these domestication traits remain unclear. In this study, we identify a Q-interacting protein TaLAX1, a basic helix-loop-helix transcription factor, through yeast two-hybrid assays. Using biochemical and genetic approaches, we explore the roles of TaLAX1 in regulating wheat domestication traits. Overexpression of TaLAX1 produces phenotypes, reminiscent of the q allele; loss-of-function Talax1 mutations confer compact spikes, largely similar to the Q-overexpression wheat lines. The two transcription factors TaLAX1 and Q disturb each other's activity to antagonistically regulate the expression of the lignin biosynthesis-related gene TaKNAT7-4D. More interestingly, a natural variation (InDel, +/- TATA), which occurs in the promoter of TaLAX1, is associated with the promoter activity difference between the D subgenome of bread wheat and its ancestor Aegilops tauschii accession T093. This study reveals that the transcription factor TaLAX1 physically interacts with Q to antagonistically regulate wheat domestication traits and a natural variation (InDel, +/- TATA) is associated with the diversification of TaLAX1 promoter activity.

The HD-ZIP II Transcription Factors Regulate Plant Architecture through the Auxin Pathway.

The homeodomain-leucine zipper (HD-ZIP) family transcription factors play important roles in plant growth and development. However, the underlying mechanisms remain largely unclear. Here we found that ATHB2, encoding a HD-ZIP transcription factor, is an early auxin responsive gene. Phenotypic analyses show that overexpression of ATHB2 impairs plant architecture, including reduced plant height and small leaves, and also reduces auxin response in leaves when grown in soil. Simultaneously, the seedlings with chemical induction of ATHB2 exhibit abnormal root gravitropism, a typical auxin-related phenotype. We further show that the auxin response pattern is altered in roots of the inducible ATHB2 seedlings. Consistently, the transcript levels of some auxin biosynthetic and transport genes are significantly decreased in these transgenic seedlings. Further, protein and promoter sequence analyses in common wheat showed that the HD-ZIP II subfamily transcription factors have highly conserved motifs and most of these encoding gene promoters contain the canonical auxin-responsive elements. Expression analyses confirm that some of these HD-ZIP II genes are indeed regulated by auxin in wheat. Together, our results suggest that the HD-ZIP II subfamily transcription factors regulate plant development possibly through the auxin pathway in plants.

Global transcriptome analysis uncovers the gene co-expression regulation network and key genes involved in grain development of wheat (Triticum aestivum L.).

Wheat grain development is a robust biological process that largely determines grain quality and yield. In this study, we investigated the grain transcriptome of winter wheat cv. Xiaoyan-6 at four developmental stages (5, 10, 15, and 20 days post-anthesis), using high-throughput RNA sequencing (RNA-Seq). We identified 427 grain-specific transcription factors (TFs) and 1653 differentially expressed TFs during grain development as well as a grain co-expression regulation network (GrainNet) of the TFs and their predicted co-expressed genes. Our study identified ten putative key TFs and the predicted regulatory genes of these TFs in wheat grain development of Xiaoyan-6. The analysis was given a firm basis through the study of additional wheat tissues, including root, stem, leaf, flag leaf, grain, spikes (from wheat plants at booting or heading stages) to provide a dataset of 92,478 high-confidence protein-coding genes that were mostly evenly distributed among subgenomes, but unevenly distributed across each of the chromosomes or each of the seven homeologous groups. Within this larger framework of the transcriptomes, we identified 4659 grain-specific genes (SEGs) and 26,500 differentially expressed genes (DEGs) throughout grain development stages tested. The SEGs identified mainly associate with regulation and signaling-related biological processes, while the DEGs mainly involve in cellular component organization or biogenesis and nutrient reservoir activity during grain development of Xiaoyan-6. This study establishes new targets for modifying genes related to grain development and yield, to fine-tune expression in different varieties and environments.

Identification and Functional Characterization of Squamosa Promoter Binding Protein-Like Gene TaSPL16 in Wheat (Triticum aestivum L.).

Wheat (Triticum aestivum L.) is one of the most important crops in the world. Squamosa promoter binding protein-like (SPL) proteins are plant-specific transcript factors and play critical roles in plant growth and development. The functions of many SPL gene family members were well characterized in Arabidopsis and rice, in contrast, research on wheat SPL genes is lagging behind. In this study, we cloned and characterized TaSPL16, an orthologous gene of rice OsSPL16, in wheat. Three TaSPL16 homoeologs are located on the short arms of chromosome 7A, 7B, and 7D, and share more than 96% sequence identity with each other. All the TaSPL16 homoeologs have three exons and two introns, with a miR156 binding site in their last exons. They encode putative proteins of 407, 409, and 414 amino acid residues, respectively. Subcellular localization showed TaSPL16 distribution in the cell nucleus, and transcription activity of TaSPL16 was validated in yeast. Analysis of the spatiotemporal expression profile showed that TaSPL16 is highly expressed in young developing panicles, lowly expressed in developing seeds and almost undetectable in vegetative tissues. Ectopic expression of TaSPL16 in Arabidopsis causes a delay in the emergence of vegetative leaves (3-4 days late), promotes early flowering (5-7 days early), increases organ size, and affects yield-related traits. These results demonstrated the regulatory roles of TaSPL16 in plant growth and development as well as seed yield. Our findings enrich the existing knowledge on SPL genes in wheat and provide valuable information for further investigating the effects of TaSPL16 on plant architecture and yield-related traits of wheat.

Male Sterility of an AHAS-Mutant Induced by Tribenuron-Methyl Solution Correlated With the Decrease of AHAS Activity in Brassica napus L.

Tribenuron-methyl (TBM), an acetohydroxyacid synthase (AHAS)-inhibiting herbicide, can be used as an efficient chemical hybridization agent to induce male sterility for practical utilization of heterosis in rapeseed (Brassica napus L.). Utilization of rapeseed mutants harboring herbicide-resistant AHAS alleles as the male parent can simplify the hybrid seed production protocol. Here we characterized a novel TBM-resistant mutant K5 derived from an elite rapeseed variety, Zhongshuang No. 9 (ZS9), by ethyl methyl sulfonate mutagenesis. Comparative analysis of three BnAHAS genes (BnAHAS1, BnAHAS2, and BnAHAS3) between the mutant K5 and ZS9 identified a C-to-T transition at 544 from the translation start site in BnAHAS1 in K5 (This resistant allele is referred to as BnAHAS1544T ), which resulted in a substitution of proline with serine at 182 in BnAHAS1. Both ZS9 and K5 plants could be induced complete male sterility under TBM treatment (with 0.10 and 20 mg⋅L-1 of TBM, respectively). The relationship between TBM-induced male sterility (Y) and the relative AHAS activity of inflorescences (X) could be described as a modified logistic function, Y = 100-A/(1+Be(-KX)) for the both genotypes, although the obtained constants A, B, and K were different in the functions of ZS9 and K5. Transgenic Arabidopsis plants expressing BnAHAS1544T exhibited a higher TBM resistance of male reproductive organ than wild type, which confirmed that the Pro-182-Ser substitution in BnAHAS1 was responsible for higher TBM-resistance of male reproductive organs. Taken together, our findings provide a novel valuable rapeseed mutant for hybrid breeding by chemical hybridization agents and support the hypothesis that AHAS should be the target of the AHAS-inhibiting herbicide TBM when it is used as chemical hybridization agent in rapeseed.

Virus-Based MicroRNA Silencing and Overexpressing in Common Wheat (Triticum aestivum L.).

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that arise from large RNA precursors with a stem-loop structure and play important roles in plant development and responses to environmental stress. Although a hundred and nineteen wheat miRNAs have been identified and registered in the miRBase (Release 21.0, June, 2014; http://www.mirbase.org), the functional characterization of these miRNAs in wheat growth and development is lagging due to lack of effective techniques to investigate endogenous miRNA functions in wheat. Here we report barley stripe mosaic virus(BSMV)-based miRNA overexpression and silence systems that can be applied to study miRNA functions in wheat. By utilizing the BSMV system, we successfully knocked down endogenous miR156 and miR166 levels and over-expressed endogenous miR156 and artificial miRNA against phytoene desaturase gene PDS (amiR-PDS) in wheat. amiR-PDS expression caused a great reduction in endogenous mRNA abundance of PDS gene in wheat plant, leading to leaf obviously photobleaching. miR156 silencing led to a great increase in the mRNA level of its target gene SPL2, resulting in a leaf-curl phenotype in wheat seedlings. In contrast, overexpression of miR156 led to a significant reduction in the mRNA level of SPL2 in wheat seedlings, resulting in serious delay of the vegetative phase transitions as well as booting and flowering in wheat. These confirmed that miR156 regulates wheat development and booting time through SPL genes. In summary, the BSMV-based miRNA overexpression and silence systems have extraordinary potential not only for functional study of protein-encoding genes but also for miRNA genes in wheat.

Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage.

bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage.

Global Dynamic Transcriptome Programming of Rapeseed (Brassica napus L.) Anther at Different Development Stages.

Rapeseed (Brassica napus L.) is an important oil crop worldwide and exhibits significant heterosis. Effective pollination control systems, which are closely linked to anther development, are a prerequisite for utilizing heterosis. The anther, which is the male organ in flowering plants, undergoes many metabolic processes during development. Although the gene expression patterns underlying pollen development are well studied in model plant Arabidopsis, the regulatory networks of genome-wide gene expression during rapeseed anther development is poorly understood, especially regarding metabolic regulations. In this study, we systematically analyzed metabolic processes occurring during anther development in rapeseed using ultrastructural observation and global transcriptome analysis. Anther ultrastructure exhibited that numerous cellular organelles abundant with metabolic materials, such as elaioplast, tapetosomes, plastids (containing starch deposits) etc. appeared, accompanied with anther structural alterations during anther development, suggesting many metabolic processes occurring. Global transcriptome analysis revealed dynamic changes in gene expression during anther development that corresponded to dynamic functional alterations between early and late anther developmental stages. The early stage anthers preferentially expressed genes involved in lipid metabolism that are related to pollen extine formation as well as elaioplast and tapetosome biosynthesis, whereas the late stage anthers expressed genes associated with carbohydrate metabolism to form pollen intine and to accumulate starch in mature pollen grains. Finally, a predictive gene regulatory module responsible for early pollen extine formation was generated. Taken together, this analysis provides a comprehensive understanding of dynamic gene expression programming of metabolic processes in the rapeseed anther, especially with respect to lipid and carbohydrate metabolism during pollen development.

TaCYP78A5 regulates seed size in wheat (Triticum aestivum).

Seed size is an important agronomic trait and a major component of seed yield in wheat. However, little is known about the genes and mechanisms that determine the final seed size in wheat. Here, we isolated TaCYP78A5, the orthologous gene of Arabidopsis CYP78A5/KLUH in wheat, from wheat cv. Shaan 512 and demonstrated that the expression of TaCYP78A5 affects seed size. TaCYP78A5 encodes the cytochrome P450 (CYP) 78A5 protein in wheat and rescued the phenotype of the Arabidopsis deletion mutant cyp78a5. By affecting the extent of integument cell proliferation in the developing ovule and seed, TaCYP78A5 influenced the growth of the seed coat, which appears to limit seed growth. TaCYP78A5 silencing caused a 10% reduction in cell numbers in the seed coat, resulting in a 10% reduction in seed size in wheat cv. Shaan 512. By contrast, the overexpression of TaCYP78A5 increased the number of cells in the seed coat, resulting in seed enlargement of ~11-35% in Arabidopsis. TaCYP78A5 activity was positively correlated with the final seed size. However, TaCYP78A5 overexpression significantly reduced seed set in Arabidopsis, possibly due to an ovule development defect. TaCYP78A5 also influenced embryo development by promoting embryo integument cell proliferation during seed development. Accordingly, a working model of the influence of TaCYP7A5 on seed size was proposed. This study provides direct evidence that TaCYP78A5 affects seed size and is a potential target for crop improvement.

Expression of TaCYP78A3, a gene encoding cytochrome P450 CYP78A3 protein in wheat (Triticum aestivum L.), affects seed size.

Several studies have described quantitative trait loci (QTL) for seed size in wheat, but the relevant genes and molecular mechanisms remain largely unknown. Here we report the functional characterization of the wheat TaCYP78A3 gene and its effect on seed size. TaCYP78A3 encoded wheat cytochrome P450 CYP78A3, and was specifically expressed in wheat reproductive organs. TaCYP78A3 activity was positively correlated with the final seed size. Its silencing caused a reduction of cell number in the seed coat, resulting in an 11% decrease in wheat seed size, whereas TaCYP78A3 over-expression induced production of more cells in the seed coat, leading to an 11-48% increase in Arabidopsis seed size. In addition, the cell number in the final seed coat was determined by the TaCYP78A3 expression level, which affected the extent of integument cell proliferation in the developing ovule and seed. Unfortunately, TaCYP78A3 over-expression in Arabidopsis caused a reduced seed set due to an ovule developmental defect. Moreover, TaCYP78A3 over-expression affected embryo development by promoting embryo integument cell proliferation during seed development, which also ultimately affected the final seed size in Arabidopsis. In summary, our results indicated that TaCYP78A3 plays critical roles in influencing seed size by affecting the extent of integument cell proliferation. The present study provides direct evidence that TaCYP78A3 affects seed size in wheat, and contributes to an understanding of the cellular basis of the gene influencing seed development.

Comparative transcriptome analysis reveals carbohydrate and lipid metabolism blocks in Brassica napus L. male sterility induced by the chemical hybridization agent monosulfuron ester sodium.

BACKGROUND: Chemical hybridization agents (CHAs) are often used to induce male sterility for the production of hybrid seeds. We previously discovered that monosulfuron ester sodium (MES), an acetolactate synthase (ALS) inhibitor of the herbicide sulfonylurea family, can induce rapeseed (Brassica napus L.) male sterility at approximately 1% concentration required for its herbicidal activity. To find some clues to the mechanism of MES inducing male sterility, the ultrastructural cytology observations, comparative transcriptome analysis, and physiological analysis on carbohydrate content were carried out in leaves and anthers at different developmental stages between the MES-treated and mock-treated rapeseed plants. RESULTS: Cytological analysis revealed that the plastid ultrastructure was abnormal in pollen mother cells and tapetal cells in male sterility anthers induced by MES treatment, with less material accumulation in it. However, starch granules were observed in chloroplastids of the epidermis cells in male sterility anthers. Comparative transcriptome analysis identified 1501 differentially expressed transcripts (DETs) in leaves and anthers at different developmental stages, most of these DETs being localized in plastid and mitochondrion. Transcripts involved in metabolism, especially in carbohydrate and lipid metabolism, and cellular transport were differentially expressed. Pathway visualization showed that the tightly regulated gene network for metabolism was reprogrammed to respond to MES treatment. The results of cytological observation and transcriptome analysis in the MES-treated rapeseed plants were mirrored by carbohydrate content analysis. MES treatment led to decrease in soluble sugars content in leaves and early stage buds, but increase in soluble sugars content and decrease in starch content in middle stage buds. CONCLUSIONS: Our integrative results suggested that carbohydrate and lipid metabolism were influenced by CHA-MES treatment during rapeseed anther development, which might responsible for low concentration MES specifically inducing male sterility. A simple action model of CHA-MES inducing male sterility in B. napus was proposed. These results will help us to understand the mechanism of MES inducing male sterility at low concentration, and might provide some potential targets for developing new male sterility inducing CHAs and for genetic manipulation in rapeseed breeding.

SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-Type Protein Kinase, Is Important for Abscisic Acid Responses in Arabidopsis through Phosphorylation of ABSCISIC ACID-INSENSITIVE5.

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-related protein kinase3-type protein kinase, SOS2-like protein kinase5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, abscisic acid-insensitive5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-like calcium binding proteins) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana).