Appropriate reduction of importin-α gene expression enhances yellow dwarf disease resistance in common wheat.

Barley yellow dwarf viruses (BYDVs) cause widespread damage to global cereal crops. Here we report a novel strategy for elevating resistance to BYDV infection. The 17K protein, a potent virulence factor conserved in BYDVs, interacted with barley IMP-α1 and -α2 proteins that are nuclear transport receptors. Consistently, a nuclear localization signal was predicted in 17K, which was found essential for 17K to be transported into the nucleus and to interact with IMP-α1 and -α2. Reducing HvIMP-α1 and -α2 expression by gene silencing attenuated BYDV-elicited dwarfism, accompanied by a lowered nuclear accumulation of 17K. Among the eight common wheat CRISPR mutants with two to four TaIMP-α1 and -α2 genes mutated, the triple mutant α1aaBBDD /α2AAbbdd and the tetra-mutant α1aabbdd /α2AAbbDD displayed strong BYDV resistance without negative effects on plant growth under field conditions. The BYDV resistance exhibited by α1aaBBDD /α2AAbbdd and α1aabbdd /α2AAbbDD was correlated with decreased nuclear accumulation of 17K and lowered viral proliferation in infected plants. Our work uncovers the function of host IMP-α proteins in BYDV pathogenesis and generates the germplasm valuable for breeding BYDV-resistant wheat. Appropriate reduction of IMP-α gene expression may be broadly useful for enhancing antiviral resistance in agricultural crops and other economically important organisms.

Genome-wide analysis and functional characterization of CHYR gene family associated with abiotic stress tolerance in bread wheat (Triticum aestivum L.).

BACKGROUND: CHY zinc-finger and RING finger (CHYR) proteins have been functionally characterized in plant growth, development and various stress responses. However, the genome-wide analysis was not performed in wheat. RESULTS: In this study, a total of 18 TaCHYR genes were identified in wheat and classified into three groups. All TaCHYR genes contained CHY-zinc finger, C3H2C3-type RING finger and zinc ribbon domains, and group III members included 1-3 hemerythrin domains in the N-terminus regions. TaCHYR genes in each group shared similar conserved domains distribution. Chromosomal location, synteny and cis-elements analysis of TaCHYRs were also analyzed. Real-time PCR results indicated that most of selected 9 TaCHYR genes exhibited higher expression levels in leaves during wheat seedling stage. All these TaCHYR genes were up-regulated after PEG treatment, and these TaCHYRs exhibited differential expression patterns in response to salt, cold and heat stress in seedling leaves. The growth of yeast cells expressing TaCHYR2.1, TaCHYR9.2 and TaCHYR11.1 were inhibited under salt and dehydration stress. Moreover, gene ontology (GO) annotation, protein interaction and miRNA regulatory network of TaCHYR genes were analyzed. CONCLUSIONS: These results increase our understanding of CHYR genes and provide robust candidate genes for further functional investigations aimed at crop improvement.

Exogenous melatonin mitigates cadmium toxicity through ascorbic acid and glutathione pathway in wheat.

Cadmium (Cd) is a dispensable element that can be absorbed by crops, posing a threat to human health through the food chains. Melatonin (MT), as a plant growth regulator, has been used to alleviate Cd toxicity in many plant species; however, the underlying molecular mechanisms responsible for Cd toxicity in wheat are still poorly understood. In this study, the suitable exogenous MT concentration (50 µM) was screened to mitigate Cd toxicity of wheat plants by increasing the plant height, root length, fresh or dry weight and chlorophyll content, or decreasing the malondialdehyde (MDA) content. In addition, MT application significantly increased ascorbic acid (ASA) and glutathione (GSH) content by reducing ROS production, especially in roots, further decreasing Cd content in fraction of organelles. Moreover, the expression levels of ASA-GSH synthesis genes, APX, GR, and GST were significantly increased by 171.5%, 465.2%, and 256.8% in roots, respectively, whereas GSH, DHAR, or MDHAR were significantly decreased by 48.5%, 54.3%, or 60.0% in roots under MT + Cd stress. However, the expression levels of Cd-induced metal transporter genes TaNramp1, TaNramp5, TaHMA2, TaHMA3, and TaLCT1 were significantly decreased by 53.7%, 50.1%, 86.5%, 87.2%, and 94.5% in roots under MT + Cd stress compared with alone Cd treatment, respectively. In conclusion, our results suggesting that MT alleviate Cd toxicity in wheat by enhancing ASA-GSH metabolism, suppressing Cd transporter gene expression, and regulating Cd uptake and translocation in wheat plants.

Genome-Wide Identification and Analysis of FKBP Gene Family in Wheat (Triticum asetivum).

FK506-binding protein (FKBP) genes have been found to play vital roles in plant development and abiotic stress responses. However, limited information is available about this gene family in wheat (Triticum aestivum L.). In this study, a total of 64 FKBP genes were identified in wheat via a genome-wide analysis involving a homologous search of the latest wheat genome data, which was unevenly distributed in 21 chromosomes, encoded 152 to 649 amino acids with molecular weights ranging from 16 kDa to 72 kDa, and was localized in the chloroplast, cytoplasm, nucleus, mitochondria, peroxisome and endoplasmic reticulum. Based on sequence alignment and phylogenetic analysis, 64 TaFKBPs were divided into four different groups or subfamilies, providing evidence of an evolutionary relationship with Aegilops tauschii, Brachypodium distachyon, Triticum dicoccoides, Arabidopsis thaliana and Oryza sativa. Hormone-related, abiotic stress-related and development-related cis-elements were preferentially presented in promoters of TaFKBPs. The expression levels of TaFKBP genes were investigated using transcriptome data from the WheatExp database, which exhibited tissue-specific expression patterns. Moreover, TaFKBPs responded to drought and heat stress, and nine of them were randomly selected for validation by qRT-PCR. Yeast cells expressing TaFKBP19-2B-2 or TaFKBP18-6B showed increased influence on drought stress, indicating their negative roles in drought tolerance. Collectively, our results provide valuable information about the FKBP gene family in wheat and contribute to further characterization of FKBPs during plant development and abiotic stress responses, especially in drought stress.

TaPHT1;9-4B and its transcriptional regulator TaMYB4-7D contribute to phosphate uptake and plant growth in bread wheat.

Efficient phosphate (Pi) uptake and utilisation are essential for promoting crop yield. However, the underlying molecular mechanism is still poorly understood in complex crop species such as hexaploid wheat. Here we report that TaPHT1;9-4B and its transcriptional regulator TaMYB4-7D function in Pi acquisition, translocation and plant growth in bread wheat. TaPHT1;9-4B, a high-affinity Pi transporter highly upregulated in roots by Pi deficiency, was identified using quantitative proteomics. Disruption of TaPHT1;9-4B function by BSMV-VIGS or CRISPR editing impaired wheat tolerance to Pi deprivation, whereas transgenic expression of TaPHT1;9-4B in rice improved Pi uptake and plant growth. Using yeast-one-hybrid assay, we isolated TaMYB4-7D, a R2R3 MYB transcription factor that could activate TaPHT1;9-4B expression by binding to its promoter. Silencing TaMYB4-7D decreased TaPHT1;9-4B expression, Pi uptake and plant growth. Four promoter haplotypes were identified for TaPHT1;9-4B, with Hap3 showing significant positive associations with TaPHT1;9-4B transcript level, growth performance and phosphorus (P) content in wheat plants. A functional marker was therefore developed for tagging Hap3. Collectively, our data shed new light on the molecular mechanism controlling Pi acquisition and utilisation in bread wheat. TaPHT1;9-4B and TaMYB4-7D may aid further research towards the development of P efficient crop cultivars.

Melatonin promotes potassium deficiency tolerance by regulating HAK1 transporter and its upstream transcription factor NAC71 in wheat.

Melatonin (MT) is involved in various physiological processes and stress responses in animals and plants. However, little is known about the molecular mechanisms by which MT regulates potassium deficiency (DK) tolerance in crops. In this study, an appropriate concentration (50 µmol/L) was found to enhance the tolerance of wheat plants against DK. RNA-seq analysis showed that a total of 6253 and 5873 differentially expressed genes (DEGs) were separately identified in root and leaf tissues of the DK + MT-treated wheat plants. They functionally involved biological processes of secondary metabolite, signal transduction, and transport or catabolism. Of these, an upregulated high-affinity K transporter 1 (TaHAK1) gene was next characterized. TaHAK1 overexpression markedly enhanced the K absorption, while its transient silencing exhibited the opposite effect, suggesting its important role in MT-mediated DK tolerance. Moreover, yeast one-hybrid (Y1H) was used to screen the upstream regulators of TaHAK1 gene and the transcription factor TaNAC71 was identified. The binding between TaNAC71 and TaHAK1 promoter was evidenced by using Y1H, LUC, and EMSA assays. Transient overexpression of TaNAC71 in wheat protoplasts activated the TaHAK1 expression, whereas its transient silencing inhibited the TaHAK1 expression and aggravated the sensitivity to DK. Exogenous MT application greatly upregulated the expression of TaHAK1 in both transient overexpression and silencing systems. Our findings revealed some molecular mechanisms underlying MT-mediated DK tolerance and helped broaden its practical application in agriculture.

Successive biochar amendment affected crop yield by regulating soil nitrogen functional microbes in wheat-maize rotation farmland.

Biochar has attracted increased attention because of its potential benefits for carbon sequestration, soil fertility, and contaminant immobilization. However, mechanism of long-term successive biochar amendment affected crop yield by regulating soil properties and nitrogen (N) functional microbes is still unclear by now. A field fixed experiment was carried out from 2011 to 2018 that aimed to study the effects of successive biochar on soil properties, soil nitrogen functional microbial genes, and grain yield in wheat and maize rotation farmland in Northern China. Four straw biochar treatments were tested in this study: 0 (BC0, CK), 2.25 (BC2.25), 6.75 (BC6.75), and 11.25 (BC11.25) Mg ha-1. The results showed that, after seven wheat-maize rotations, the total organic carbon (TOC), total N (TN), NO3-, available potassium (AK), and the C/N ratio in 0-20 cm topsoil were increased significantly following biochar application; however, there were no obvious differences in available phosphorus (AP) and NH4+ among biochar treatments. Biochar also resulted in a significant increase in crop yield and NO3- accumulation in 0-200 cm soil layer, with the highest yield in BC6.75. Furthermore, a marked increase was found in the amoA gene abundance in topsoil; however, it decreased significantly with excessive biochar application (BC11.25). At wheat maturity, the nirS gene abundance consistently decreased following biochar application, whereas the nosZ gene abundance initially increased and then decreased (peaking in BC6.75); however, no obvious changes in the nirK gene were observed. At maize maturity, biochar significantly increased the nirS and nosZ gene abundance in topsoil, especially in BC6.75. In addition, redundancy analysis indicated that the soil moisture content, AP, AK, TN, TOC, NO3-, NH4+, pH, and C/N ratio had markedly effects on the abundance of the amoA, nirK, nirS, and nosZ genes. In general, biochar-induced alterations of soil properties resulted in changes of gene abundance of soil nitrifying and denitrifying bacteria, and eventually affecting crop yields.

TaWRKY74 participates copper tolerance through regulation of TaGST1 expression and GSH content in wheat.

Glutathione S-transferase (GST) is the key enzyme in glutathione (GSH) synthesis, and plays a crucial role in copper (Cu) detoxification. Nonetheless, its regulatory mechanisms remain largely unclear. In this study, we identified a Cu-induced glutathione S-transferase 1 (TaGST1) gene in wheat. Yeast one-hybrid (Y1H) screened out TaWRKY74, which was one member from the WRKY transcription factor family. The bindings between TaGST1 promoter and TaWRKY74 were further verified by using another Y1H and luciferase assays. Expression of TaWRKY74 was induced more than 30-folds by Cu stress. Functions of TaWRKY74 were tested by using transiently silence methods. In transiently TaWRKY74-silenced wheat plants, TaWRKY74 and TaGST1 expression, GST activity, and GSH content was significantly inhibited by 25.68%, 19.88%, 27.66%, and 12.68% in shoots, and 53.81%, 52.11%, 23.47%, and 17.11% in roots, respectively. However, contents of hydrogen peroxide, malondialdehyde, or Cu were significantly increased by 2.58%, 12.45%, or 37.74% in shoots, and 25.24%, 53.84%, and 103.99% in roots, respectively. Notably, exogenous application of GSH reversed the adverse effects of transiently TaWRKY74-silenced wheat plants during Cu stress. Taken together, our results suggesting that TaWRKY74 regulated TaGST1 expression and affected GSH accumulation under Cu stress, and could be useful to ameliorate Cu toxicity for crop food safety.

Exogenous Glutathione Alleviates Cadmium Toxicity in Wheat by Influencing the Absorption and Translocation of Cadmium.

Cadmium (Cd), a toxic heavy metal, is harmful to plants and human health. Glutathione (GSH) could alleviate Cd toxicity of plant species, whereas its mechanism responsible for wheat remains poorly understood. Here, we found that exogenous GSH application significantly increased the fresh and dry weight, root elongation, chlorophyll contents, while decreased the contents of malondialdehyde (MDA) and GSH, and translocation factor of Cd compared with Cd treatment. Moreover, GSH application significantly increased activities of antioxidant enzymes and expression of related genes, which involved in GSH synthesis, especially in roots. In addition, we found that GSH application suppressed Cd-induced expression of metal transporter genes TaNramp1, TaNramp5, TaHMA2, TaHMA3, TaLCT1 and TaIRT2 in roots. Taken together, our results suggested that GSH could alleviate Cd toxicity in wheat by increasing GSH synthesis gene expression or suppressing Cd transporter genes expression, and further affecting Cd uptake and translocation in wheat plants.

Functional characterization and regulatory mechanism of wheat CPK34 kinase in response to drought stress.

BACKGROUND: Drought is one of the most adverse environmental factors limiting crop productions and it is important to identify key genetic determinants for food safety. Calcium-dependent protein kinases (CPKs) are known to be involved in plant growth, development, and environmental stresses. However, biological functions and regulatory mechanisms of many plant CPKs have not been explored. In our previous study, abundance of the wheat CPK34 (TaCPK34) protein was remarkably upregulated in wheat plants suffering from drought stress, inferring that it could be involved in this stress. Therefore, here we further detected its function and mechanism in response to drought stress. RESULTS: Transcripts of the TaCPK34 gene were significantly induced after PEG-stimulated water deficiency (20% PEG6000) or 100 µM abscisic acid (ABA) treatments. The TaCPK34 gene was transiently silenced in wheat genome by using barley stripe mosaic virus-induced silencing (BSMV-VIGS) method. After 14 days of drought stress, the transiently TaCPK34-silenced wheat seedlings showed more sensitivity compared with control, and the plant biomasses and relative water contents significantly decreased, whereas soluble sugar and MDA contents increased. The iTRAQ-based quantitative proteomics was employed to measure the protein expression profiles in leaves of the transiently TaCPK34-silenced wheat plants after drought stress. There were 6103 proteins identified, of these, 51 proteins exhibited significantly altered abundance, they were involved in diverse function. And sequence analysis on the promoters of genes, which encoded the above identified proteins, indicated that some promoters harbored some ABA-responsive elements. We determined the interactions between TaCPK34 and three identified proteins by using bimolecular fluorescent complementation (BiFC) method and our data indicated that TaCPK34directly interacted with the glutathione S-transferase 1 and prx113, respectively. CONCLUSIONS: Our study suggested that the TaCPK34 gene played positive roles in wheat response to drought stress through directly or indirectly regulating the expression of ABA-dependent manner genes, which were encoding identified proteins from iTRAQ-based quantitative proteomics. And it could be used as one potential gene to develop crop cultivars with improved drought tolerance.

Large-scale Proteomics Combined with Transgenic Experiments Demonstrates An Important Role of Jasmonic Acid in Potassium Deficiency Response in Wheat and Rice.

Potassium (K+) is the most abundant inorganic cation in plants, and molecular dissection of K+ deficiency has received considerable interest in order to minimize K+ fertilizer input and develop high quality K+-efficient crops. However, the molecular mechanism of plant responses to K+ deficiency is still poorly understood. In this study, 2-week-old bread wheat seedlings grown hydroponically in Hoagland solution were transferred to K+-free conditions for 8 d, and their root and leaf proteome profiles were assessed using the iTRAQ proteome method. Over 4000 unique proteins were identified, and 818 K+-responsive protein species showed significant differences in abundance. The differentially expressed protein species were associated with diverse functions and exhibited organ-specific differences. Most of the differentially expressed protein species related to hormone synthesis were involved in jasmonic acid (JA) synthesis and the upregulated abundance of JA synthesis-related enzymes could result in the increased JA concentrations. Abundance of allene oxide synthase (AOS), one key JA synthesis-related enzyme, was significantly increased in K+-deficient wheat seedlings, and its overexpression markedly increased concentrations of K+ and JA, altered the transcription levels of some genes encoding K+-responsive protein species, as well as enhanced the tolerance of rice plants to low K+ or K+ deficiency. Moreover, rice AOS mutant (osaos) exhibited more sensitivity to low K+ or K+ deficiency. Our findings could highlight the importance of JA in K+ deficiency, and imply a network of molecular processes underlying plant responses to K+ deficiency.

PDIL1-2 can indirectly and negatively regulate expression of the AGPL1 gene in bread wheat.

BACKGROUND: ADP-glucose pyrophosphorylase (AGPase), the key enzyme in plant starch biosynthesis, is a heterotetramer composed of two identical large subunits and two identical small subunits. AGPase has plastidial and cytosolic isoforms in higher plants, whereas it is mainly detected in the cytosol of grain endosperms in cereal crops. Our previous results have shown that the expression of the TaAGPL1 gene, encoding the cytosolic large subunit of wheat AGPase, temporally coincides with the rate of starch accumulation and that its overexpression dramatically increases wheat AGPase activity and the rate of starch accumulation, suggesting an important role. METHODS: In this study, we performed yeast one-hybrid screening using the promoter of the TaAGPL1 gene as bait and a wheat grain cDNA library as prey to screen out the upstream regulators of TaAGPL1 gene. And the barley stripe mosaic virus-induced gene-silencing (BSMV-VIGS) method was used to verify the functional characterization of the identified regulators in starch biosynthesis. RESULTS: Disulfide isomerase 1-2 protein (TaPDIL1-2) was screened out, and its binding to the TaAGPL1-1D promoter was further verified using another yeast one-hybrid screen. Transiently silenced wheat plants of the TaPDIL1-2 gene were obtained by using BSMV-VIGS method under field conditions. In grains of BSMV-VIGS-TaPDIL1-2-silenced wheat plants, the TaAGPL1 gene transcription levels, grain starch contents, and 1000-kernel weight also significantly increased. CONCLUSIONS: As important chaperones involved in oxidative protein folding, PDIL proteins have been reported to form hetero-dimers with some transcription factors, and thus, our results suggested that TaPDIL1-2 protein could indirectly and negatively regulate the expression of the TaAGPL1 gene and function in starch biosynthesis.

Transcriptomic and Metabolomics Analysis of Different Endosperm Region under Nitrogen Treatments.

Storage protein distribution in wheat-grain endosperm is heterogeneous, but the underlying molecular mechanism remains unclear. Two parts of the endosperm region, the innermost endosperm (IE) region and the remaining endosperm (RE) region, grown under low nitrogen (LN) and high nitrogen (HN) treatments were used to perform metabolomic and transcriptomic analysis. We identified 533 and 503 differentially expressed genes (DEGs) with at least a two-fold expression change (p < 0.05) between IE and RE, among which 81 and 78 transcripts under LN and HN, respectively, related to carbon and nitrogen metabolism, and encoded transcription factors or proteins involved in post-translational modification (PTM). The significantly differentially abundant metabolites between IE and RE were mainly amino acids, N-compounds, carbohydrates, and nucleic acids. More upregulated transcripts and metabolites were identified in RE than IE under HN conditions, indicating that HN activates metabolism in the endosperm periphery. In addition to carbon and nitrogen metabolism, transcription factors and protein PTMs, such as phosphorylation and acetylation, might determine the protein heterogeneous distribution between IE and RE and its response to nitrogen fertilizer supply.

Proteomics analysis reveals that nitric oxide regulates photosynthesis of maize seedlings under water deficiency.

Nitric oxide (NO) is an important bioactive molecule that functions in regulating diverse abiotic stresses in plants, whereas its molecular mechanism remains obscure. In this study, treatment with 0.1 mM NO donor (sodium nitroprusside, SNP) significantly alleviated the inhibited growth induced by 15% polyethyleneglycol (PEG)-stimulated water deficiency (WD) for 3 days in maize seedlings, manifested by less decreased plant total fresh weight and dry weight. Comprehensive proteome analysis was further used to measure the expression profiles of leaf proteins of SNP-pretreated maize seedlings under WD conditions to explore the molecular mechanisms of NO-induced WD tolerance. Using 2-DE method, 135 protein spots showed significantly enhanced or reduced abundance, of which 102 spots were successfully identified MALDI-TOF/TOF MS. The identified protein species were associated with diverse functions, and most (52/83, 62.7%) of known protein species were related to photosynthetic processes. Compared to alone PEG treatment, the abundance of 25 identified protein species in SNP + PEG treatment were enhanced among the identified photosynthesis-related protein species. In addition, exogenous SNP application dramatically regulated chlorophyll α fluorescence kinetics e.g. the increase of maximum quantum yield of PSII (Fv/Fm), photosynthetic performance index (PI), and IP phase, whereas it remarkably reduced the polyphasic OJIP fluorescence transient, the accumulation of reactive oxygen species (H2O2 and O2•-) and malondialdehyde (MDA). These findings suggest that the NO-induced WD tolerance could be associated with improved photosynthetic capability in higher plants.

Nitric Oxide Enhancing Resistance to PEG-Induced Water Deficiency is Associated with the Primary Photosynthesis Reaction in Triticum aestivum L.

Photosynthesis is affected by water-deficiency (WD) stress, and nitric oxide (NO) is a free radical that participates in the photosynthesis process. Previous studies have suggested that NO regulates excitation-energy distribution of photosynthesis under WD stress. Here, quantitative phosphoproteomic profiling was conducted using iTRAQ. Differentially phosphorylated protein species (DEPs) were identified in leaves of NO- or polyethylene glycol (PEG)-treated wheat seedlings (D), and in control seedlings. From 1396 unique phosphoproteins, 2257 unique phosphorylated peptides and 2416 phosphorylation sites were identified. Of these, 96 DEPs displayed significant changes (≥1.50-fold, p < 0.01). These DEPs are involved in photosynthesis, signal transduction, etc. Furthermore, phosphorylation of several DEPs was upregulated by both D and NO treatments, but downregulated only in NO treatment. These differences affected the chlorophyll A⁻B binding protein, chloroplast post-illumination chlorophyll-fluorescence-increase protein, and SNT7, implying that NO indirectly regulated the absorption and transport of light energy in photosynthesis in response to WD stress. The significant difference of chlorophyll (Chl) content, Chl a fluorescence-transient, photosynthesis index, and trapping and transport of light energy further indicated that exogenous NO under D stress enhanced the primary photosynthesis reaction compared to D treatment. A putative pathway is proposed to elucidate NO regulation of the primary reaction of photosynthesis under WD.

Function of the ERFL1a Transcription Factor in Wheat Responses to Water Deficiency.

The APETALA2/ethylene response factor (AP2/ERF) superfamily is involved in the responses of plants to biotic and abiotic stresses; however, the functions and mechanisms of some members of this family in plants are unclear. In our previous study, expression of TaERFL1a, a member of the AP2/ERF family, was remarkably induced in wheat seedlings suffering freezing stress. In this study, we show that its expression was rapidly upregulated in response to salt, cold, and water deficiency, suggesting roles in the responses to abiotic stresses. Further, transient barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) resulted in significantly reduced tolerance to 20% PEG6000-stimulated water deficiency. Subcellular localization and transcriptional activation assays separately showed that TaERFL1a was targeted to the nucleus and possessed transcriptional activation activity. Yeast two-hybrid library screening identified six interacting proteins, and of these, the interactions between TaERFL1a and TaSGT1, and TaERFL1a and TaDAD2 proteins were further confirmed by yeast co-transformation and bimolecular fluorescent complementation (BiFC). Collectively, our results suggest that TaERFL1a is a stress-responsive transcription factor, which could be functionally related to proteins involved in the abiotic stress responses of plants.

Functional Analysis of a Wheat AGPase Plastidial Small Subunit with a Truncated Transit Peptide.

ADP-glucose pyrophosphorylase (AGPase), the key enzyme in starch synthesis, consists of two small subunits and two large subunits with cytosolic and plastidial isoforms. In our previous study, a cDNA sequence encoding the plastidial small subunit (TaAGPS1b) of AGPase in grains of bread wheat (Triticum aestivum L.) was isolated and the protein subunit encoded by this gene was characterized as a truncated transit peptide (about 50% shorter than those of other plant AGPS1bs). In the present study, TaAGPS1b was fused with green fluorescent protein (GFP) in rice protoplast cells, and confocal fluorescence microscopy observations revealed that like other AGPS1b containing the normal transit peptide, TaAGPS1b-GFP was localized in chloroplasts. TaAGPS1b was further overexpressed in a Chinese bread wheat cultivar, and the transgenic wheat lines exhibited a significant increase in endosperm AGPase activities, starch contents, and grain weights. These suggested that TaAGPS1b subunit was targeted into plastids by its truncated transit peptide and it could play an important role in starch synthesis in bread wheat grains.

Virus-Induced Gene Silencing Identifies an Important Role of the TaRSR1 Transcription Factor in Starch Synthesis in Bread Wheat.

The function of a wheat starch regulator 1 (TaRSR1) in regulating the synthesis of grain storage starch was determined using the barley stripe mosaic virus-virus induced gene-silencing (BSMV-VIGS) method in field experiments. Chlorotic stripes appeared on the wheat spikes infected with barley stripe mosaic virus-virus induced gene-silencing- wheat starch regulator 1 (BSMV-VIGS-TaRSR1) at 15 days after anthesis, at which time the transcription levels of the TaRSR1 gene significantly decreased. Quantitative real-time PCR was also used to measure the transcription levels of 26 starch synthesis-related enzyme genes in the grains of BSMV-VIGS-TaRSR1-silenced wheat plants at 20, 27, and 31 days after anthesis. The results showed that the transcription levels of some starch synthesis-related enzyme genes were markedly induced at different sampling time points: TaSSI, TaSSIV, TaBEIII, TaISA1, TaISA3, TaPHOL, and TaDPE1 genes were induced at each of the three sampling time points and TaAGPS1-b, TaAGPL1, TaAGPL2, TaSSIIb, TaSSIIc, TaSSIIIb, TaBEI, TaBEIIa, TaBEIIb, TaISA2, TaPHOH, and TaDPE2 genes were induced at one sampling time point. Moreover, both the grain starch contents, one thousand kernel weights, grain length and width of BSMV-VIGS-TaRSR1-infected wheat plants significantly increased. These results suggest that TaRSR1 acts as a negative regulator and plays an important role in starch synthesis in wheat grains by temporally regulating the expression of specific starch synthesis-related enzyme genes.

Hg-responsive proteins identified in wheat seedlings using iTRAQ analysis and the role of ABA in Hg stress.

Wheat seedlings exposed to 100 µM HgCl2 for 3 days exhibited high-level mercury (Hg) accumulation, which led to inhibited growth, increased lipid peroxidation, and disrupted cellular ultrastructures. And root growth and ultrastructural changes of wheat seedlings were inhibited more severely than those of leaves. To identify the wheat protein response to Hg stress, the iTRAQ method was used to determine the proteome profiles of the roots and leaves of wheat seedlings exposed to high-Hg conditions. 249 proteins were identified with significantly altered abundance. 117 were found in roots and 132 in leaves. These proteins were classified into signal transduction, stress defense, carbohydrate metabolism, protein metabolism, energy production, and transport functional groups. The majority of proteins identified in Hg-stressed roots and leaves displayed differently altered abundance, revealing organ-specific differences in adaption to Hg stress. Pathway Studio software was used to identify the Hg-responsive protein interaction network that included 49 putative key proteins, and they were potentially regulated by abscisic acid (ABA). Exogenous ABA application conferred protection against Hg stress and increased activities of peroxidase enzyme, suggesting that it may be an important factor in the Hg signaling pathway. These findings can provide useful insights into the molecular mechanisms of Hg responses in higher plants.

Ultrastructural observation of mesophyll cells and temporal expression profiles of the genes involved in transitory starch metabolism in flag leaves of wheat after anthesis.

Transitory starch in cereal plant leaves is synthesized during the day and remobilized at night to provide a carbon source for growth and grain filling, but its mechanistic basis is still poorly understood. The objective of this study is to explore the regulatory mechanism for starch biosynthesis and degradation in plant source organs. Using transmission electron microscopy, we observed that during the day after anthesis, starch granules in mesophyll cells of wheat flag leaves accumulated in chloroplasts and the number of starch granules gradually decreased with wheat leaf growth. During the night, starch granules synthesized in chloroplasts during the day were completely or partially degraded. The transcript levels of 26 starch synthesis-related genes and 16 starch breakdown-related genes were further measured using quantitative real-time reverse transcription polymerase chain reaction. Expression profile analysis revealed that starch metabolism genes were clustered into two groups based on their temporal expression patterns. The genes in the first group were highly expressed and presumed to play crucial roles in starch metabolism. The genes in the other group were not highly expressed in flag leaves and may have minor functions in starch metabolism in leaf tissue. The functions of most of these genes in leaves were further discussed. The starch metabolism-related genes that are predominantly expressed in wheat flag leaves differ from those expressed in wheat grain, indicating that two different pathways for starch metabolism operate in these tissues. This provides specific information on the molecular mechanisms of transitory starch metabolism in higher plants.