Transcriptomics reveals a core transcriptional network of K-type cytoplasmic male sterility microspore abortion in wheat (Triticum aestivum L.).

BACKGROUND: Cytoplasmic male sterility (CMS) plays a crucial role in hybrid production. K-type CMS, a cytoplasmic male sterile line of wheat with the cytoplasms of Aegilops kotschyi, is widely used due to its excellent characteristics of agronomic performance, easy maintenance and easy restoration. However, the mechanism of its pollen abortion is not yet clear. RESULTS: In this study, wheat K-type CMS MS(KOTS)-90-110 (MS line) and it's fertile near-isogenic line MR (KOTS)-90-110 (MR line) were investigated. Cytological analysis indicated that the anthers of MS line microspore nucleus failed to divide normally into two sperm nucleus and lacked starch in mature pollen grains, and the key abortive period was the uninucleate stage to dinuclear stage. Then, we compared the transcriptome of MS line and MR line anthers at these two stages. 11,360 and 5182 differentially expressed genes (DEGs) were identified between the MS and MR lines in the early uninucleate and binucleate stages, respectively. Based on GO enrichment and KEGG pathways analysis, it was evident that significant transcriptomic differences were "plant hormone signal transduction", "MAPK signaling pathway" and "spliceosome". We identified 17 and 10 DEGs associated with the IAA and ABA signal transduction pathways, respectively. DEGs related to IAA signal transduction pathway were downregulated in the early uninucleate stage of MS line. The expression level of DEGs related to ABA pathway was significantly upregulated in MS line at the binucleate stage compared to MR line. The determination of plant hormone content and qRT-PCR further confirmed that hormone imbalance in MS lines. Meanwhile, 1 and 2 DEGs involved in ABA and Ethylene metabolism were also identified in the MAPK cascade pathway, respectively; the significant up regulation of spliceosome related genes in MS line may be another important factor leading to pollen abortion. CONCLUSIONS: We proposed a transcriptome-mediated pollen abortion network for K-type CMS in wheat. The main idea is hormone imbalance may be the primary factor, MAPK cascade pathway and alternative splicing (AS) may also play important regulatory roles in this process. These findings provided intriguing insights for the molecular mechanism of microspore abortion in K-type CMS, and also give useful clues to identify the crucial genes of CMS in wheat.

Low-Temperature-Mediated Promoter Methylation Relates to the Expression of TaPOR2D, Affecting the Level of Chlorophyll Accumulation in Albino Wheat (Triticum aestivum L.).

Chlorophyll is an indispensable photoreceptor in plant photosynthesis. Its anabolic imbalance is detrimental to individual growth and development. As an essential epigenetic modification, DNA methylation can induce phenotypic variations, such as leaf color transformation, by regulating gene expression. Albino line XN1376B is a natural mutation of winter wheat cultivar XN1376; however, the regulatory mechanism of its albinism is still unclear. In this study, we found that low temperatures induced albinism in XN1376B. The number of chloroplasts decreased as the phenomenon of bleaching intensified and the fence tissue and sponge tissue slowly dissolved. We identified six distinct TaPOR (protochlorophyllide oxidoreductase) genes in the wheat genome, and TaPOR2D was deemed to be related to the phenomenon of albinism based on the expression in different color leaves (green leaves, white leaves and returned green leaves) and the analysis of promoters' cis-acting elements. TaPOR2D was localized to chloroplasts. TaPOR2D overexpression (TaPOR2D-OE) enhanced the chlorophyll significantly in Arabidopsis, especially at two weeks; the amount of chlorophyll was 6.46 mg/L higher than in WT. The methylation rate of the TaPOR2D promoter in low-temperature albino leaves is as high as 93%, whereas there was no methylation in green leaves. Correspondingly, three DNA methyltransferase genes (TaMET1, TaDRM and TaCMT) were up-regulated in white leaves. Our study clarified that the expression of TaPOR2D is associated with its promoter methylation at a low temperature; it affects the level of chlorophyll accumulation, which probably causes the abnormal development of plant chloroplasts in albino wheat XN1376B. The results provide a theoretical basis for in-depth analysis of the regulation of development of plant chloroplasts and color variation in wheat XN1376B leaves.

A superior allele of the wheat gene TaGL3.3-5B, selected in the breeding process, contributes to seed size and weight.

KEY MESSAGE: A superior allele of wheat gene TaGL3.3-5B was identified and could be used in marker-assisted breeding in wheat. Identifying the main genes which mainly regulate the yield-associated traits can significantly increase the wheat production. In this study, gene TaGL3.3 was cloned from common wheat according to the sequence of OsPPKL3. A SNP in the 8th exon of TaGL3.3-5B, T/C in coding sequence (CDS), which resulted in an amino acid change (Val/Ala), was identified between the low 1000-kernel weight (TKW) wheat Chinese Spring and the high TKW wheat Xinong 817 (817). Subsequently, association analysis in the mini-core collection (MCC) and the recombinant inbred lines (RIL) revealed that the allele TaGL3.3-5B-C (from 817) was significantly correlated with higher TKW. The high frequency of TaGL3.3-5B-C in the Chinese modern wheat cultivars indicated that it was selected positively in wheat breeding programs. The overexpression of TaGL3.3-5B-C in Arabidopsis resulted in shorter pods and longer grains than those of wild-type counterparts. Additionally, TaGL3.3 expressed a tissue-specific pattern in wheat as revealed by qRT-PCR. We also found that 817 showed higher expression of TaGL3.3 than that in Chinese Spring (CS) during the seed development. These results demonstrate that TaGL3.3 plays an important role in the formation of seed size and weight. Allele TaGL3.3-5B-C is associated with larger and heavier grains that are beneficial to wheat yield improvement.

The Transcription Factors TaTDRL and TaMYB103 Synergistically Activate the Expression of TAA1a in Wheat, Which Positively Regulates the Development of Microspore in Arabidopsis.

Pollen fertility plays an important role in the application of heterosis in wheat (Triticum aestivum L.). However, the key genes and mechanisms underlying pollen abortion in K-type male sterility remain unclear. TAA1a is an essential gene for pollen development in wheat. Here, we explored the mechanism involved in its transcriptional regulation during pollen development, focusing on a 1315-bp promoter region. Several cis-acting elements were identified in the TAA1a promoter, including binding motifs for Arabidopsis thaliana AtAMS and AtMYB103 (CANNTG and CCAACC, respectively). Evolutionary analysis indicated that TaTDRL and TaMYB103 were the T. aestivum homologs of AtAMS and AtMYB103, respectively, and encoded nucleus-localized transcription factors containing 557 and 352 amino acids, respectively. TaTDRL and TaMYB103 were specifically expressed in wheat anthers, and their expression levels were highest in the early uninucleate stage; this expression pattern was consistent with that of TAA1a. Meanwhile, we found that TaTDRL and TaMYB03 directly interacted, as evidenced by yeast two-hybrid and bimolecular fluorescence complementation assays, while yeast one-hybrid and dual-luciferase assays revealed that both TaTDRL and TaMYB103 could bind the TAA1a promoter and synergistically increase its transcriptional activity. Furthermore, TaTDRL-EAR and TaMYB103-EAR transgenic Arabidopsis plants displayed abnormal microspore morphology, reduced pollen viability, and lowered seed setting rates. Additionally, the expression of AtMS2, a TAA1a homolog, was significantly lower in the two repressor lines than in the corresponding overexpression lines or WT plants. In summary, we identified a potential transcriptional regulatory mechanism associated with wheat pollen development.

Comparative Proteomic Analysis of Developmental Changes in P-Type Cytoplasmic Male Sterile and Maintainer Anthers in Wheat.

Cytoplasmic male sterility (CMS) plays an important role in the application of heterosis in wheat (Triticum aestivum L.). However, the molecular mechanism underlying CMS remains unknown. This study provides a comprehensive morphological and proteomic analysis of the anthers of a P-type CMS wheat line (P) and its maintainer line, Yanshi 9 hao (Y). Cytological observations indicated that the P-type CMS line shows binucleate microspore abortion. In this line, the tapetum degraded early, leading to anther cuticle defects, which could not provide the nutrition needed for microspore development in a timely manner, thus preventing the development of the microspore to the normal binucleate stage. Proteomic analysis revealed novel proteins involved in P-type CMS. Up to 2576 differentially expressed proteins (DEPs) were quantified in all anthers, and these proteins were significantly enriched in oxidative phosphorylation, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), starch and sucrose metabolism, phenylpropanoid biosynthesis, and pyruvate metabolism pathways. These proteins may comprise a network that regulates male sterility in wheat. Based on the function analysis of DEPs involved in the complex network, we concluded that the P-type CMS line may be due to cellular dysfunction caused by disturbed carbohydrate metabolism, inadequate energy supply, and disturbed protein synthesis. These results provide insights into the molecular mechanism underlying male sterility and serve as a valuable resource for researchers in plant biology, in general, and plant sexual reproduction, in particular.

Transcriptome response comparison between vector and non-vector aphids after feeding on virus-infected wheat plants.

BACKGROUND: Plant viruses maintain intricate interactions with their vector and non-vector insects and can impact the fitness of insects. However, the details of their molecular and cellular mechanisms have not been studied well. We compared the transcriptome-level responses in vector and non-vector aphids (Schizaphis graminum and Rhopalosiphum padi, respectively) after feeding on wheat plants with viral infections (Barley Yellow Dwarf Virus (BYDV) and Wheat dwarf virus (WDV), respectively). We conducted differentially expressed gene (DEG) annotation analyses and observed DEGs related to immune pathway, growth, development, and reproduction. And we conducted cloning and bioinformatic analyses of the key DEG involved in immune. RESULTS: For all differentially expressed gene analyses, the numbers of DEGs related to immune, growth, development, reproduction and cuticle were higher in vector aphids than in non-vector aphids. STAT5B (signal transducer and activator of transcription 5B), which is involved in the JAK-STAT pathway, was upregulated in R. padi exposed to WDV. The cloning and bioinformatic results indicated that the RpSTAT5B sequence contains a 2082 bp ORF encoding 693 amino acids. The protein molecular weight is 79.1 kD and pI is 8.13. Analysis indicated that RpSTAT5B is a non-transmembrane protein and a non-secreted protein. Homology and evolutionary analysis indicated that RpSTAT5B was closely related to R. maidis. CONCLUSIONS: Unigene expression analysis showed that the total number of differentially expressed genes (DEGs) in the vector aphids was higher than that in the non-vector aphids. Functional enrichment analysis showed that the DEGs related to immunity, growth and reproduction in vector aphids were higher than those in non-vector aphids, and the differentially expressed genes related to immune were up-regulated. This study provides a basis for the evaluation of the response mechanisms of vector/non-vector insects to plant viruses.

Cyclin-Dependent Kinase Inhibitor Gene TaICK1 acts as a Potential Contributor to Wheat Male Sterility induced by a Chemical Hybridizing Agent.

Heterosis has been widely accepted as an effective strategy to increase yields in plant breeding. Notably, the chemical hybridization agent SQ-1 induces male sterility in wheat, representing a critical potential tool in hybrid seed production. However, the mechanisms underlying the male sterility induced by SQ-1 still remain poorly understood. In this study, a cyclin-dependent kinase inhibitor gene, TaICK1, which encodes a 229 amino acid protein, was identified as a potential contributor to male sterility in common wheat. The expression of TaICK1 was upregulated during the development of anthers in Xinong1376 wheat treated with SQ-1. Meanwhile, the seed setting rate was found to be significantly decreased in TaICK1 transgenic rice. Furthermore, we identified two cyclin proteins, TaCYCD2;1 and TaCYCD6;1, as interactors through yeast two-hybrid screening using TaICK1 as the bait, which were validated using bimolecular fluorescence complementation. Subcellular localization revealed that the proteins encoded by TaICK1, TaCYCD2;1, and TaCYCD6;1 were localized in the cell nucleus. The expression levels of TaCYCD2;1 and TaCYCD6;1 were lower in Xinong1376 treated with SQ-1. A further analysis demonstrated that the expression levels of OsCYCD2;1 and OsCYCD6;1 were lower in transgenic TaICK1 rice lines as well. Taken together, these results suggest that the upregulation of TaICK1, induced by SQ-1, may subsequently suppress the expression of TaCYCD2;1 and TaCYCD6;1 in anthers, resulting in male sterility. This study provides new insights into the understanding of SQ-1-induced wheat male sterility, as well as the developmental mechanisms of anthers.

Isolation and Identification of a TaTDR-Like Wheat Gene Encoding a bHLH Domain Protein, Which Negatively Regulates Chlorophyll Biosynthesis in Arabidopsis.

Chlorophyll biosynthesis plays a vital role in chloroplast development and photosynthesis in plants. In this study, we identified an orthologue of the rice gene TDR (Oryza sativa L., Tapetum Degeneration Retardation) in wheat (Triticum aestivum L.) called TaTDR-Like (TaTDRL) by sequence comparison. TaTDRL encodes a putative 557 amino acid protein with a basic helix-loop-helix (bHLH) conserved domain at the C-terminal (295-344 aa). The TaTDRL protein localised to the nucleus and displayed transcriptional activation activity in a yeast hybrid system. TaTDRL was expressed in the leaf tissue and expression was induced by dark treatment. Here, we revealed the potential function of TaTDRL gene in wheat by utilizing transgenic Arabidopsis plants TaTDRL overexpressing (TaTDRL-OE) and TaTDRL-EAR (EAR-motif, a repression domain of only 12 amino acids). Compared with wild-type plants (WT), both TaTDRL-OE and TaTDRL-EAR were characterized by a deficiency of chlorophyll. Moreover, the expression level of the chlorophyll-related gene AtPORC (NADPH:protochlorophyllide oxidoreductase C) in TaTDRL-OE and TaTDRL-EAR was lower than that of WT. We found that TaTDRL physically interacts with wheat Phytochrome Interacting Factor 1 (PIF1) and Arabadopsis PIF1, suggesting that TaTDRL regulates light signaling during dark or light treatment. In summary, TaTDRL may respond to dark or light treatment and negatively regulate chlorophyll biosynthesis by interacting with AtPIF1 in transgenic Arabidopsis.

Identification of the WUSCHEL-Related Homeobox (WOX) Gene Family, and Interaction and Functional Analysis of TaWOX9 and TaWUS in Wheat.

The WUSCHEL-related homeobox (WOX) is a family of plant-specific transcription factors, with important functions, such as regulating the dynamic balance of division and differentiation of plant stem cells and plant organ development. We identified 14 distinct TaWOX genes in the wheat (Triticum aestivum L.) genome, based on a genome-wide scan approach. All of the genes under evaluation had positional homoeologs on subgenomes A, B and D except TaWUS and TaWOX14. Both TaWOX14a and TaWOX14d had a paralogous copy on the same genome due to tandem duplication events. A phylogenetic analysis revealed that TaWOX genes could be divided into three groups. We performed functional characterization of TaWOX genes based on the evolutionary relationships among the WOX gene families of wheat, rice (Oryza sativa L.), and Arabidopsis. An overexpression analysis of TaWUS in Arabidopsis revealed that it affected the development of outer floral whorl organs. The overexpression analysis of TaWOX9 in Arabidopsis revealed that it promoted the root development. In addition, we identified some interaction between the TaWUS and TaWOX9 proteins by screening wheat cDNA expression libraries, which informed directions for further research to determine the functions of TaWUS and TaWOX9. This study represents the first comprehensive data on members of the WOX gene family in wheat.

Comparative proteomic analysis of multi-ovary wheat under heterogeneous cytoplasm suppression.

BACKGROUND: DUOII is a multi-ovary wheat (Triticum aestivum L.) line with two or three pistils and three stamens in each floret. The multi-ovary trait of DUOII is controlled by a dominant gene, whose expression can be suppressed by the heterogeneous cytoplasm of TeZhiI (TZI), a line with the nucleus of common wheat and the cytoplasm of Aegilops. Crosses between female DUOII plants and male TZI plants resulted in multi-ovary F1s; whereas, the reciprocal crosses resulted in mono-ovary F1s. Although the multi-ovary trait is inherited as single trait controlled by a dominant allele in lines with a Triticum cytoplasm, the mechanism by which the special heterogeneous cytoplasm suppresses the expression of multi-ovary is not well understood. RESULTS: Observing the developmental process, we found that the critical stage of additional pistil primordium development was when the young spikes were 2-6 mm long. Then, we compared the quantitative proteomic profiles of 2-6 mm long young spikes obtained from the reciprocal crosses between DUOII and TZI. A total of 90 differentially expressed proteins were identified and analyzed based on their biological functions. These proteins had obvious functional pathways mainly implicated in chloroplast metabolism, nuclear and cell division, plant respiration, protein metabolism, and flower development. Importantly, we identified two key proteins, Flowering Locus K Homology Domain and PEPPER, which are known to play an essential role in the specification of pistil organ identity. By drawing relationships between the 90 differentially expressed proteins, we found that these proteins revealed a complex network which is associated with multi-ovary gene expression under heterogeneous cytoplasmic suppression. CONCLUSIONS: Our proteomic analysis has identified certain differentially expressed proteins in 2-6 mm long young spikes, which was the critical stage of additional primordium development. This paper provided a universal proteomic profiling involved in the cytoplasmic suppression of wheat floral meristems; and our findings have laid a solid foundation for further mechanistic studies on the underlying mechanisms that control the heterogeneous cytoplasm-induced suppression of the nuclear multi-ovary gene in wheat.

High-voltage electrostatic field-induced oxidative stress: Characterization of the physiological effects in Sitobion avenae (Hemiptera: Aphididae) across multiple generations.

In recent decades, man-made electric fields have greatly increased the intensity of electrostatic fields that are pervasively present in the environment. To better understand the physiological alterations exhibited by herbivorous insects in response to changing electric environments, we determined the activities of anti-oxidative enzymes and the metabolic rate of Sitobion avenae Fabricius (Hemiptera: Aphididae) over multiple generations in response to direct and host-seed exposure to a high-voltage electrostatic field (HVEF) of varying strength for different durations. Under controlled greenhouse conditions, 20-min direct exposure of S. avenae and wheat seeds to a 2- or 4-kV/cm HVEF resulted in significantly increased superoxide dismutase (SOD) activity in the sixth, 11th, 16th, and 21st generations relative to the control activities, whereas significantly decreased SOD activity was detected in the second generation. In addition, the activities of catalase (CAT) and peroxidase (POD) in S. avenae showed significant decreases over multiple generations. We also examined the suppressive effects of the duration of 4-kV/cm treatment on aphid physiology. The results showed that exposure to the 4-kV/cm HVEF for 20 min exerted adverse effects on CAT and POD activities and significantly decreased the metabolic rates of S. avenae, as demonstrated through evaluations of CO2 production rate, and these parameters were not significantly affected by higher HVEF durations. Overall, these findings increase our understanding of plant-pest interactions under novel HVEF environments and provide information that can improve integrated management strategies for S. avenae. Bioelectromagnetics. 40:52-61, 2019. © 2018 Wiley Periodicals, Inc.

Identification and Functional Analysis of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) Gene Family in Wheat.

CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides are post-translationally cleaved and modified peptides from their corresponding pre-propeptides. Although they are only 12 to 13 amino acids in length, they are important ligands involved in regulating cell proliferation and differentiation in plant shoots, roots, vasculature, and other tissues. They function by interacting with their corresponding receptors. CLE peptides have been studied in many plants, but not in wheat. We identified 104 TaCLE genes in the wheat genome based on a genome-wide scan approach. Most of these genes have homologous copies distributed on sub-genomes A, B, and D. A few genes are derived from tandem duplication and segmental duplication events. Phylogenetic analysis revealed that TaCLE genes can be divided into five different groups. We obtained functional characterization of the peptides based on the evolutionary relationships among the CLE peptide families of wheat, rice, and Arabidopsis, and expression pattern analysis. Using chemically synthesized peptides (TaCLE3p and TaCLE34p), we found that TaCLE3 and TaCLE34 play important roles in regulating wheat and Arabidopsis root development, and wheat stem development. Overexpression analysis of TaCLE3 in Arabidopsis revealed that TaCLE3 not only affects the development of roots and stems, but also affects the development of leaves and fruits. These data represent the first comprehensive information on TaCLE family members.

Cytological and Proteomic Analysis of Wheat Pollen Abortion Induced by Chemical Hybridization Agent.

In plants, pollen grain transfers the haploid male genetic material from anther to stigma, both between flowers (cross-pollination) and within the same flower (self-pollination). In order to better understand chemical hybridizing agent (CHA) SQ-1-induced pollen abortion in wheat, comparative cytological and proteomic analyses were conducted. Results indicated that pollen grains underwent serious structural injury, including cell division abnormality, nutritional deficiencies, pollen wall defect and pollen grain malformations in the CHA-SQ-1-treated plants, resulting in pollen abortion and male sterility. A total of 61 proteins showed statistically significant differences in abundance, among which 18 proteins were highly abundant and 43 proteins were less abundant in CHA-SQ-1 treated plants. 60 proteins were successfully identified using MALDI-TOF/TOF mass spectrometry. These proteins were found to be involved in pollen maturation and showed a change in the abundance of a battery of proteins involved in multiple biological processes, including pollen development, carbohydrate and energy metabolism, stress response, protein metabolism. Interactions between these proteins were predicted using bioinformatics analysis. Gene ontology and pathway analyses revealed that the majority of the identified proteins were involved in carbohydrate and energy metabolism. Accordingly, a protein-protein interaction network involving in pollen abortion was proposed. These results provide information for the molecular events underlying CHA-SQ-1-induced pollen abortion and may serve as an additional guide for practical hybrid breeding.

Chemical hybridizing agent SQ-1-induced male sterility in Triticum aestivum L.: a comparative analysis of the anther proteome.

BACKGROUND: Heterosis is widely used to increase the yield of many crops. However, as wheat is a self-pollinating crop, hybrid breeding is not so successful in this organism. Even though male sterility induced by chemical hybridizing agents is an important aspect of crossbreeding, the mechanisms by which these agents induce male sterility in wheat is not well understood. RESULTS: We performed proteomic analyses using the wheat Triticum aestivum L.to identify those proteins involved in physiological male sterility (PHYMS) induced by the chemical hybridizing agent CHA SQ-1. A total of 103 differentially expressed proteins were found by 2D-PAGE and subsequently identified by MALDI-TOF/TOF MS/MS. In general, these proteins had obvious functional tendencies implicated in carbohydrate metabolism, oxidative stress and resistance, protein metabolism, photosynthesis, and cytoskeleton and cell structure. In combination with phenotypic, tissue section, and bioinformatics analyses, the identified differentially expressed proteins revealed a complex network behind the regulation of PHYMS and pollen development. Accordingly, we constructed a protein network of male sterility in wheat, drawing relationships between the 103 differentially expressed proteins and their annotated biological pathways. To further validate our proposed protein network, we determined relevant physiological values and performed real-time PCR assays. CONCLUSIONS: Our proteomics based approach has enabled us to identify certain tendencies in PHYMS anthers. Anomalies in carbohydrate metabolism and oxidative stress, together with premature tapetum degradation, may be the cause behind carbohydrate starvation and male sterility in CHA SQ-1 treated plants. Here, we provide important insight into the mechanisms underlying CHA SQ-1-induced male sterility. Our findings have practical implications for the application of hybrid breeding in wheat.

Comparative studies of mitochondrial proteomics reveal an intimate protein network of male sterility in wheat (Triticum aestivum L.).

Plant male sterility has often been associated with mitochondrial dysfunction; however, the mechanism in wheat (Triticum aestivum L.) has not been elucidated. This study set out to probe the mechanism of physiological male sterility (PHYMS) induced by the chemical hybridizing agent (CHA)-SQ-1, and cytoplasmic male sterility (CMS) of wheat at the proteomic level. A total of 71 differentially expressed mitochondrial proteins were found to be involved in pollen abortion and further identified by MALDI-TOF/TOF MS (matrix-assisted laser desorption/ionization-time of fight/time of flight mass spectrometry). These proteins were implicated in different cellular responses and metabolic processes, with obvious functional tendencies toward the tricarboxylic acid cycle, the mitochondrial electron transport chain, protein synthesis and degradation, oxidation stress, the cell division cycle, and epigenetics. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways involved in anther abortion and pollen defects. Accordingly, a mitochondria-mediated male sterility protein network in wheat is proposed; this network was further confirmed by physiological data, RT-PCR (real-time PCR), and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. The results provide intriguing insights into the metabolic pathway of anther abortion induced by CHA-SQ-1 and also give useful clues to identify the crucial proteins of PHYMS and CMS in wheat.

Candidate Genes Expressed in Tolerant Common Wheat With Resistant to English Grain Aphid.

The English grain aphid, Sitobion avenae (F.) (Hemiptera: Aphididae), is a common worldwide pest of wheat (Triticum aestivum L.). The use of improved resistant cultivars by the farmers is the most effective and environmentally friendly method to control this aphid in the field. The winter wheat genotypes 98-10-35 and Amigo are resistant to S. avenae. To identify genes responsible for resistance to S. avenae in these genotypes, differential-display reverse transcription-polymerase chain reaction was used to identify the corresponding differentially expressed sequences in current study. Two backcross progenies were obtained by crossing the two resistant genotypes with the susceptible genotype 1376. Six potential expected-differential bands were sequenced. Lengths of the expressed sequence tags ranged from 128 to 532 bp. Although these expressed sequences were likely associated with S. avenae resistance, there was one expressed sequence tag located on 7DL chromosome, and its potential function may associate with the ability to maintain photosynthesis in wheat. That serves as an active way for tolerant common wheat with resistant to S. avenae. Cloning the full length of these sequences would help us thoroughly understand the mechanism of wheat resistance to S. avenae and be valuable for breeding cultivars with S. avenae resistance.

Active oxygen generation induced by the glucose sensor TaHXK7-1A decreased the drought resistance of transgenic Arabidopsis and wheat (Triticum aestivum L.).

Improving wheat drought resistance is of great significance for grain production and food security. Hexokinases (HXKs) play a role in sugar signal transduction and are involved in abiotic stress responses in wheat. To clarify the relationship between HXKs and drought stress in wheat, we used the rice active oxygen induction gene OsHXK1 as a reference sequence and the homologously cloned wheat TaHXK7-1A gene. TaHXK7-1A was localized in the nucleus and cell membrane. Under drought stress, over-expression of TaHXK7-1A increased the contents of O2·ï¼ and malondialdehyde (MDA) and significantly up-regulated the respiratory burst oxidative homologue (RBOHs) genes in transgenic Arabidopsis. In addition, the over-expression of TaHXK7-1A inhibited the growth of Arabidopsis seedlings and increased ROS accumulation under 6 % exogenous glucose treatment. Gene silencing of TaHXK7-1 decreased the contents of O2·ï¼ and MDA in wheat leaves under drought stress, and the RBOHs was significantly down-regulated, which improved the drought resistance of wheat. The results of yeast one-hybrid, EMSA, and dual-luciferase assays showed that TabHLH148-5A bound to the E-box motif of the TaHXK7-1A promoter and inhibited the expression of TaHXK7-1A. In addition, yeast two-hybrid and luciferase complementation imaging assays showed that TaHXK7-1A interacted with TaGRF3-4A. These results indicate that the glucose sensor TaHXK7-1A was negatively regulated by TabHLH148-5A, interacted with TaGRF3-4A, and negatively regulated wheat drought resistance by regulating RBOHs expression and inducing ROS production, thus providing a theoretical basis for revealing the molecular mechanism of wheat drought resistance.

Carbohydrate metabolism and cytology of S-type cytoplasmic male sterility in wheat.

Introduction: Cytoplasmic male sterility (CMS) is an important tool for hybrid heterosis utilization. However, the underlying mechanisms still need to be discovered. An adequate supply of nutrients is necessary for anther development; pollen abortion would occur if the metabolism of carbohydrates were hampered. Methods: In order to better understand the relationship between carbohydrate metabolism disorder and pollen abortion in S-CMS wheat, the submicroscopic structure of wheat anthers was observed using light microscopy and transmission electron microscopy; chloroplast proteome changes were explored by comparative proteomic analysis; sugar measuring and enzyme assays were performed; and the expression patterns of carbohydrate metabolism-related genes were studied using quantitative real-time PCR (qRT-PCR) method. Results: These results indicated that the anther and microspore in S-CMS wheat underwent serious structural damage, including premature tapetum degeneration, nutritional shortage, pollen wall defects, and pollen grain malformations. Furthermore, the number of chloroplasts in the anthers of S-CMS lines decreased significantly, causing abnormal carbohydrate metabolism, and disintegration of osmiophilic granules and thylakoids. Meanwhile, some proteins participating in the Calvin cycle and carbohydrate metabolism were abnormally expressed in the chloroplasts of the S-CMS lines, which might lead to chloroplast dysfunction. Additionally, several key enzymes and genes related to carbohydrate metabolism were significantly inhibited in S-CMS. Discussion: Based on these results, we proposed a carbohydrate metabolism pathway for anther abortion in S-type cytoplasmic male sterility, which would encourage further exploration of the pollen abortion mechanisms for CMS wheat.

Genome-Wide Identification and Expression Analysis of TUA and TUB Genes in Wheat (Triticum aestivum L.) during Its Development.

Microtubules play a fundamental role in plant development, morphogenesis, and cytokinesis; they are assembled from heterodimers containing an α-tubulin (TUA) and a ß-tubulin (TUB) protein. However, little research has been conducted on the TUA and TUB gene families in hexaploid wheat (Triticum aestivum L.). In this study, we identified 15 TaTUA and 28 TaTUB genes in wheat. Phylogenetic analysis showed that 15 TaTUA genes were divided into two major subfamilies, and 28 TaTUB genes were divided into five major subfamilies. Mostly, there were similar motif compositions and exon-intron structures among the same subfamilies. Segmental duplication of genes (WGD/segmental) is the main process of TaTUA and TaTUB gene family expansion in wheat. It was found that TaTUA and TaTUB genes presented specific temporal and spatial characteristics based on the expression profiles of 17 tissues during wheat development using publicly available RNA-seq data. It was worth noting, via qRT-PCR, that two TaTUA and five TaTUB genes were highly expressed in fertile anthers compared to male sterility. These were quite different between physiological male sterile lines and S-type cytoplasmic male sterile lines at different stages of pollen development. This study offers fundamental information on the TUA and TUB gene families during wheat development and provides new insights for exploring the molecular mechanism of wheat male sterility.

Dissecting quantitative resistance against blast disease using heterogeneous inbred family lines in rice.

SHZ-2 is an indica rice cultivar that exhibits broad-spectrum resistance to rice blast; it is widely used as a resistance donor in breeding programs. To dissect the QTL responsible for broad-spectrum blast resistance, we crossed SHZ-2 to TXZ-13, a blast susceptible indica variety, to produce 244 BC(4)F(3) lines. These lines were evaluated for blast resistance in greenhouse and field conditions. Chromosomal introgressions from SHZ-2 into the TXZ-13 genome were identified using a single feature polymorphism microarray, SSR markers and gene-specific primers. Segregation analysis of the BC(4)F(3) population indicated that three regions on chromosomes 2, 6, and 9, designated as qBR2.1, qBR6.1, and qBR9.1, respectively, was associated with blast resistance and contributed 16.2, 14.9, and 22.3%, respectively, to the phenotypic variance of diseased leaf area (DLA). We further narrowed the three QTL regions using pairs of sister lines extracted from heterogeneous inbred families (HIF). Pairwise comparison of these lines enabled the determination of the relative contributions of individual QTL. The qBR9.1 conferred strong resistance, whereas qBR2.1 or qBR6.1 individually did not reduce disease under field conditions. However, when qBR2.1 and qBR6.1 were combined, they reduced disease by 19.5%, suggesting that small effect QTLs contribute to reduction of epidemics. The qBR6.1 and qBR9.1 regions contain nucleotide-binding sites and leucine rich repeats (NBS-LRR) sequences, whereas the qBR2.1 did not. In the qBR6.1 region, the patterns of expression of adjacent NBS-LRR genes were consistent in backcross generations and correlated with blast resistance, supporting the hypothesis that multiple resistance genes within a QTL region can contribute to non-race-specific quantitative resistance.