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

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

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.

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.

Root Proteomics Reveals the Effects of Wood Vinegar on Wheat Growth and Subsequent Tolerance to Drought Stress.

Wood vinegar (WV) or pyroligneous acid (PA) is a reddish-brown liquid created during the dry distillation of biomass, a process called pyrolysis. WV contains important biologically active components, which can enhance plant growth and tolerance to drought stress. However, its mechanism of action remains unknown. Our results after presoaking wheat seeds with various concentrations of WV indicate that a 1:900 WV concentration can significantly enhance growth. To investigate the response of wheat roots to drought stress, we compared quantitative proteomic profiles in the roots of wheat plants grown from seeds either presoaked (treatment) or non-presoaked (control) with WV. Our results indicated that the abscisic acid (ABA) content of wheat roots in the WV treatment was significantly increased. Reactive oxygen species (ROS) and malonaldehyde (MDA) levels roots were significantly lower than in the control treatment under drought stress, while the activity of major antioxidant enzymes was significantly increased. Two-dimensional electrophoresis (2D-PAGE) identified 138 differentially accumulated protein (DAP) spots representing 103 unique protein species responding to drought stress in wheat roots of the control and WV-treated groups. These DAPs are mostly involved in the stress response, carbohydrate metabolism, protein metabolism, and secondary metabolism. Proteome profiles showed the DAPs involved in carbohydrate metabolism, stress response, and secondary metabolism had increased accumulation in roots of the WV-treated groups. These findings suggest that the roots from wheat seeds presoaked with WV can initiate an early defense mechanism to mitigate drought stress. These results provide an explanation of how WV enhances the tolerance of wheat plants to drought stress.

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.

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.

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.

Genome-wide identification, characterization, and expression patterns analysis of the SBP-box gene family in wheat (Triticum aestivum L.).

SQUAMOSA promoter-binding protein (SBP)-box genes encode a family of plant-specific transcription factors that play roles in plant growth and development. The characteristics of SBP-box genes in rice (Oryza sativa) and Arabidopsis have been reported, but their potential roles in wheat (Triticum aestivum) are not fully understood. In this study, 48 SBP-box genes (TaSBPs) were identified; they were located in all wheat chromosomes except for 4B and 4D. Six TaSBPs were identified as tandem duplication genes that formed three tandem duplication pairs, while 22 were segmentally duplicated genes that formed 16 segmental duplication pairs. Subcellular localization prediction showed TaSBPs were located in nucleus. Among the 48 TaSBPs, 24 were predicted to be putative targets of TamiR156. Phylogenetic analysis showed that TaSBPs, AtSBPs, and OsSBPs that shared similar functions were clustered into the same subgroups. The phylogenetic relationships between the TaSBPs were supported by the identification of highly conserved motifs and gene structures. Four types of cis-elements--transcription-related, development-related, hormone-related, and abiotic stress-related elements--were found in the TaSBP promoters. Expression profiles indicated most TaSBPs participate in flower development and abiotic stress responses. This study establishes a foundation for further investigation of TaSBP genes and provides novel insights into their biological functions.

Morphological and proteomic analysis of young spikes reveals new insights into the formation of multiple-pistil wheat.

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.

Mitochondrial Dysfunction Causes Oxidative Stress and Tapetal Apoptosis in Chemical Hybridization Reagent-Induced Male Sterility in Wheat.

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.

Comparative proteomic analysis of a membrane-enriched fraction from flag leaves reveals responses to chemical hybridization agent SQ-1 in wheat.

The induction of wheat male fertile lines by using the chemical hybridizing agent SQ-1 (CHA-SQ-1) is an effective approach in the utilization of heterosis; however, the molecular basis of male fertility remains unknown. Wheat flag leaves are the initial receptors of CHA-SQ-1 and their membrane structure plays a vital role in response to CHA-SQ-1 stress. To investigate the response of wheat flag leaves to CHA-SQ-1 stress, we compared their quantitative proteomic profiles in the absence and presence of CHA-SQ-1. Our results indicated that wheat flag leaves suffered oxidative stress during CHA-SQ-1 treatments. Leaf O2 (-), H2O2, and malonaldehyde levels were significantly increased within 10 h after CHA-SQ-1 treatment, while the activities of major antioxidant enzymes such as superoxide dismutase, catalase, and guaiacol peroxidase were significantly reduced. Proteome profiles of membrane-enriched fraction showed a change in the abundance of a battery of membrane proteins involved in multiple biological processes. These variable proteins mainly impaired photosynthesis, ATP synthesis protein mechanisms and were involved in the response to stress. These results provide an explanation of the relationships between membrane proteomes and anther abortion and the practical application of CHA for hybrid breeding.

Abnormal development of tapetum and microspores induced by chemical hybridization agent SQ-1 in wheat.

Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.

[Analysis of three wheat cytoplasmic male sterile lines mitochondrial DNA by AFLP].

Cytoplasmic male sterility is an important way to utilize wheat heterosis. The purpose of thisstudy was to identify cytoplasmic type of three wheat male sterile lines. Amplified fragment length polymorphism (AFLP) marker technique was used to analyze the wheat mitochondrial DNA. We isolated mitochondria by differential centrifugation and density gradient ultracentrifugation. The results show that the extracted mitochondrial DNA was pure. It was suitable for PCR and genetic analysis. We got 4 pairs of specific primers from 64 primers combinations. Primer E1/M7 amplified 3 specific fragments in ms(Kots)-90-110. Primer E4/M2 generated 2 specific fragments in ms(Ven)-90-110. Primer E7/M6 amplified 2 specific fragments in ms(S)-90-110. Primer E6/M4 produced 2 specific fragments in ms(Kots)-90-110. Four specific primers could be used to identify three cytoplasmic types of Aegilops kotschyi, Ae. ventricosa and Triticum spelta. It provided the molecular basis to further study the mechanism of wheat cytoplasmic male sterility.