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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. [ABSTRACT FROM AUTHOR]

Microtubules play a fundamental role in plant development, morphogenesis, and cytokinesis; they are assembled from heterodimers containing an α-tubulin (TUA) and a β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Multi-ovary wheat is a unique variety of wheat that has one to three pistils and three stamens, and can stably set one to three grains in each floret. By observing the developmental process of additional pistils, we found that the additional pistil was derived from a protrusion generated at the base of the main pistil, between the frontal stamen and lateral stamen. The additional pistil's development was greatly delayed compared with the main pistil at an early stage. However, after the awn exposed stage, it developed very rapidly to a mature pistil within the maturity time of the main pistil. Generally, the grains originating from additional pistils were smaller than the grains from the main pistil. By studying the penetrance and germination conditions of multi-ovary wheat, we found that no matter which ovary the grains originated from, they had the same penetrance. However, the germination ability of grains generated from the main pistil was significantly higher than that of grains from additional pistils. Our results showed that multi-ovary wheat was an excellent variety, not only for studying the mechanisms of the multi-ovary trait and floral development in wheat, but also for improving the propagation coefficient and promoting the progress of wheat breeding. This paper lays a theoretical foundation for the practical application of multi-ovary trait in hybrid wheat; our results could be implemented in fostering future breeding activities focussed on the development of high yield wheat cultivars. Applying the multi-ovary trait to hybrid wheat can effectively improve the propagation coefficient and potentially promote the development of hybrid wheat. This study investigated the grain developmental process and explored the penetrance and seed vigour of different grains in multi-ovary wheat. This paper lays a theoretical foundation for the practical application of the multi-ovary trait in hybrid wheat, and our results could be implemented in fostering future breeding activities focussed on the development of high yield wheat cultivars. [ABSTRACT FROM AUTHOR]

Male sterility in plants has been strongly linked to mitochondrial dysfunction. Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. Therefore, it is important to better understand the relationship between mitochondria and CHA-induced male sterility in wheat. This study reports on the impairment of mitochondrial function duo to CHA-SQ-1, which occurs by decreasing cytochrome oxidase and adenosine triphosphate synthase protein levels and theirs activities, respiratory rate, and in turn results in the inhibition of the mitochondrial electron transport chain (ETC), excessive production of reactive oxygen species (ROS) and disruption of the alternative oxidase pathway. Subsequently, excessive ROS combined with MnSOD defects results in damage to the mitochondrial membrane, followed by ROS release into the cytoplasm. The microspores underwent severe oxidative stress during pollen development. Furthermore, chronic oxidative stress, together with the overexpression of type II metacaspase, triggered premature tapetal apoptosis, which resulted in pollen abortion. Accordingly, we propose a metabolic pathway for mitochondrial-mediated male sterility in wheat, which provides information on the molecular events underlying CHA-SQ-1-induced abortion of anthers and may serve as an additional guide to the practical application of hybrid breeding. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We investigated the relationship between a pollen-specific protein homolog and pollen abortion in an SQ-1-induced male sterile line of wheat ( Triticum aestivum L.). We previously determined that EST S414 (Ta20678) was obviously down-regulated in a line of Xinong 1376 wheat in which male sterility was induced by SQ-1 (a chemical hybridizing agent). In the present study, we obtained the full sequence of F8- 1 from wheat. The open reading frame sequence of the F8- 1 gene was 495 bp, encoding 165 amino acid residues with a predicted molecular weight of 18.2759 kDa and an isoelectric point of 4.86. This sequence was identified as a Secale cereale × Triticum durum F8- 4 homolog with 100 % identity. The amino acids represent a conserved domain in pollen Ole e I that contains the consensus sequence Q-G-R-V-Y-C-D-T-C-R. The protein has a close evolutionary relationship with orthologs in S. cereale × T. durum and Triticum urartu, consisting of 9.09 % alpha helix, 24.85 % extended strand, and 66.06 % random coil. F8- 1 was only expressed in anthers during the binucleate and trinucleate stages and expression gradually decreased from the binucleate stage to the trinucleate stage. The expression of F8- 1 gene of physiological male sterility 1376 (PHYMS-1376) was lower than that of 1376 at both the binucleate and trinucleate stages. Subcellular localization showed that the F8-1 protein was expressed at the nucleus. RNAi tests showed that down-regulation of the F8- 1 was associated with an increase in the percentage of pollen abortion. Therefore, F8- 1 is a positive regulator of physiological male sterility. [ABSTRACT FROM AUTHOR]

Wheat (Triticum aestivum L.), one of the world’s most important food crops, is a strictly autogamous (self-pollinating) species with exclusively perfect flowers. Male sterility induced by chemical hybridizing agents has increasingly attracted attention as a tool for hybrid seed production in wheat; however, the molecular mechanisms of male sterility induced by the agent SQ-1 remain poorly understood due to limited whole transcriptome data. Therefore, a comparative analysis of wheat anther transcriptomes for male fertile wheat and SQ-1–induced male sterile wheat was carried out using next-generation sequencing technology. In all, 42,634,123 sequence reads were generated and were assembled into 82,356 high-quality unigenes with an average length of 724 bp. Of these, 1,088 unigenes were significantly differentially expressed in the fertile and sterile wheat anthers, including 643 up-regulated unigenes and 445 down-regulated unigenes. The differentially expressed unigenes with functional annotations were mapped onto 60 pathways using the Kyoto Encyclopedia of Genes and Genomes database. They were mainly involved in coding for the components of ribosomes, photosynthesis, respiration, purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, RNA transport and signal transduction, reactive oxygen species metabolism, mRNA surveillance pathways, protein processing in the endoplasmic reticulum, protein export, and ubiquitin-mediated proteolysis. This study is the first to provide a systematic overview comparing wheat anther transcriptomes of male fertile wheat with those of SQ-1–induced male sterile wheat and is a valuable source of data for future research in SQ-1–induced wheat male sterility. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract This study used cytology, cytochemistry, and non-targeted metabolomics to investigate the distribution characteristic of polysaccharides, lipids, and all the metabolites present during five wheat (Triticum aestivum L.) anther developmental stages to provide insights into wheat anther development. Anthers were collected from the tetrad through trinucleate stages, and 1.5% (w/v) acetocarmine and 4′,6-diamidino-2-phenylindole staining were used to confirm the developmental stage and visualize the nuclei, respectively. Polysaccharides and lipids were detected by staining with periodic acid-Schiff and Sudan Black B, respectively. The integrated optical density of the tapetum and microspores were calculated using IPP6.0 software. Furthermore, the metabolites were identified by gas chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer (GC-TOF-MS). The results indicated that the interior and exterior surface cells of anthers are orderly. Pollen was rich in numerous nutrient substances (e.g., lipids, insoluble carbohydrates, and others), and formed a normal sperm cell that contained three nuclei, i.e., one vegetative nuclei and two reproductive nuclei in the mature pollen. Semi-thin sectioning indicated that the tapetum cells degraded progressively from the tetrad to late uninucleate stage and disappeared from the bi-to trinucleate stages. Moreover, nutrient substances (lipids and insoluble carbohydrates) accumulated, were synthesized in the pollen, and gradually increased from the tetrad to trinucleate stages. Finally, the metabolomics results identified that 146 metabolites were present throughout the wheat anther developmental stages. Principal component analysis, hierarchical cluster analysis, and metabolite-metabolite correlation revealed distinct dynamic changes in metabolites. The metabolism of organic acids, amino acids, sugars, fatty acids, amines, polyols, and nucleotides were interrelated and involved in the tricarboxylic acid (TCA) cycle and glycolysis. Furthermore, their interactions were revealed using an integrated metabolic map, which indicated that the TCA cycle and glycolysis were very active during anther development to provide the required energy for anther and pollen development. Our study provides valuable insights into the mechanisms of substance metabolism in wheat anthers and can be used for possible application by metabolic engineers for the improvement of cell characteristics or creating new compounds and molecular breeders in improving pollen fertility or creating the ideal male sterile line, to improve wheat yield per unit area to address global food security. Highlights • This study investigated the physiological and metabolomic changes that occur during anther development in Xinong 1376 wheat (Triticum aestivum L.) using non-targeted metabolomics analysis. • A total of 120 metabolites were altered during anther development, leading to an increase in the supply of insoluble polysaccharides, lipids, proteins, and other substances and a change in the normal intracellular structure, ultimately resulting in pollen fertility in Xinong 1376. • These findings in this article will provide a basis for application by metabolic engineers and molecular breeders to improve wheat yield per unit area in order to increase global food security. [ABSTRACT FROM AUTHOR]

• The multiple-pistil wheat possesses 2–3 pistils in a floret without other floral organs defect. • The multiple-pistil wheat has obvious grain number advantage. • The secondary pistils are derived from extra stem cells that do not normally terminate activity in multiple-pistil wheat. • The DAPs were mainly involved in the translation and the metabolisms of carbohydrate, nucleotide and amino acid. • TaHUA2, TaRF2a, TaCHR12 and TaHEN2 may play vital roles in the regulation of wheat flower organ number. A new multiple-pistil wheat mutant germplasm with more than one pistil in a floret was obtained from natural mutagenesis. This mutant can develop 2–3 grains in a glume after pollination and has a significant grain number advantage compared with normal wheat. However, the basis of the formation of multiple-pistil wheat has thus far not been well established. In this study, we first performed a continuous phenotypic observation of the floral meristem (FM) in multiple-pistil wheat. The results indicated that the secondary pistils are derived from extra stem cells that fail to terminate normally between the carpel primordium and the lodicule primordium. To further probe the potential molecular basis for the formation of secondary pistils, comparative proteomic analyses were conducted. A total of 334 differentially abundant proteins (DAPs) were identified using isobaric tags for relative and absolute quantification (iTRAQ), among which 131 proteins were highly abundant and 203 proteins were less abundant in the young spikes of multiple-pistil wheat. The DAPs, located primarily in the cell, were involved in the translation and the metabolisms of carbohydrate, nucleotide, and amino acid. Differential expression analysis showed that TaHUA2, TaRF2a, TaCHR12 and TaHEN2 may play vital roles in the regulation of wheat flower organ number. In general, the DAPs support the phenotypic analysis results at the molecular level. In combination, these results reveal new insights into the formation of multiple-pistil wheat and provide possible targets for further research on the regulation of floral organ number in wheat. [ABSTRACT FROM AUTHOR]