Identification and Functional Analysis of microRNAs Involved in the Anther Development in Cotton Genic Male Sterile Line Yu98-8A.

Hybrid vigor contributes in a large way to the yield and quality of cotton (Gossypium hirsutum) fiber. Although microRNAs play essential regulatory roles in flower induction and development, it is still unclear if microRNAs are involved in male sterility, as the regulatory molecular mechanisms of male sterility in cotton need to be better defined. In this study, two independent small RNA libraries were constructed and sequenced from the young buds collected from the sporogenous cell formation to the meiosis stage of the male sterile line Yu98-8A and the near-isogenic line. Sequencing revealed 1588 and 1536 known microRNAs and 347 and 351 novel miRNAs from male sterile and male fertile libraries, respectively. MicroRNA expression profiles revealed that 49 conserved and 51 novel miRNAs were differentially expressed. Bioinformatic and degradome analysis indicated the regulatory complexity of microRNAs during flower induction and development. Further RT-qPCR and physiological analysis indicated that, among the different Kyoto Encyclopedia Gene and Genomes pathways, indole-3-acetic acid and gibberellic acid signaling transduction pathways may play pivotal regulatory functions in male sterility.

Identification of early salt stress responsive proteins in seedling roots of upland cotton (Gossypium hirsutum L.) employing iTRAQ-based proteomic technique.

Soil salinity is a major abiotic stress that limits plant growth and agricultural productivity. Upland cotton (Gossypium hirsutum L.) is highly tolerant to salinity; however, large-scale proteomic data of cotton in response to salt stress are still scant. Here, an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic technique was employed to identify the early differentially expressed proteins (DEPs) from salt-treated cotton roots. One hundred and twenty-eight DEPs were identified, 76 of which displayed increased abundance and 52 decreased under salt stress conditions. The majority of the proteins have functions related to carbohydrate and energy metabolism, transcription, protein metabolism, cell wall and cytoskeleton metabolism, membrane and transport, signal transduction, in addition to stress and defense. It is worth emphasizing that some novel salt-responsive proteins were identified, which are involved in cell cytoskeleton metabolism (actin-related protein2, ARP2, and fasciclin-like arabinogalactan proteins, FLAs), membrane transport (tonoplast intrinsic proteins, TIPs, and plasma membrane intrinsic proteins, PIPs), signal transduction (leucine-rich repeat receptor-like kinase encoding genes, LRR-RLKs) and stress responses (thaumatin-like protein, TLP, universal stress protein, USP, dirigent-like protein, DIR, desiccation-related protein PCC13-62). High positive correlation between the abundance of some altered proteins (superoxide dismutase, SOD, peroxidase, POD, glutathione S-transferase, GST, monodehydroascorbate reductase, MDAR, and malate dehydrogenase, MDH) and their enzyme activity was evaluated. The results demonstrate that the iTRAQ-based proteomic technique is reliable for identifying and quantifying a large number of cotton root proteins. qRT-PCR was used to study the gene expression levels of the five above-mentioned proteins; four patterns are consistent with those of induced protein. These results showed that the proteome of cotton roots under NaCl stress is complex. The comparative protein profiles of roots under salinity vs control improves the understanding of the molecular mechanisms involved in the tolerance of plants to salt stress. This work provides a good basis for further functional elucidation of these DEPs using genetic and/or other approaches, and, consequently, candidate genes for genetic engineering to improve crop salt tolerance.

Comparative Proteomic Analysis of Gossypium thurberi in Response to Verticillium dahliae Inoculation.

Verticillium wilt is threatening cotton productivity globally. This disease is caused by soil-borne Verticillium dahliae which directly infects cotton roots, and exclusively colonizes and occludes xylem vessels, finally resulting in necrosis, defoliation, and most severely, plant death. For the first time, iTRAQ (isobaric tags for relative and absolute quantification) was applied to screen the differentially expressed proteins of Gossypium thurberi inoculated with V. dahliae. A total of 6533 proteins were identified from the roots of G. thurberi after inoculation with V. dahliae, and 396 showed up- and 279 down-regulated in comparison to a mock-inoculated roots. Of these identified proteins, the main functional groups were those involved in cell wall organization and reinforcement, disease-resistant chemicals of secondary metabolism, phytohormone signaling, pathogenesis-related proteins, and disease-resistant proteins. Physiological and biochemical analysis showed that peroxidase activity, which promotes the biosynthesis and accumulation of lignin, was induced early in the hypocotyl after inoculation with V. dahliae. Similarly, salicylic acid also accumulated significantly in hypocotyl of the seedlings after inoculation. These findings provide an important knowledge of the molecular events and regulatory networks occurring during G. thurberi-V. dahliae interaction, which may provide a foundation for breeding disease-resistance in cotton.

Transcriptomic Profiling Reveals Complex Molecular Regulation in Cotton Genic Male Sterile Mutant Yu98-8A.

Although cotton genic male sterility (GMS) plays an important role in the utilization of hybrid vigor, its precise molecular mechanism remains unclear. To characterize the molecular events of pollen abortion, transcriptome analysis, combined with histological observations, was conducted in the cotton GMS line, Yu98-8A. A total of 2,412 genes were identified as significant differentially expressed genes (DEGs) before and during the critical pollen abortion stages. Bioinformatics and biochemical analysis showed that the DEGs mainly associated with sugars and starch metabolism, oxidative phosphorylation, and plant endogenous hormones play a critical and complicated role in pollen abortion. These findings extend a better understanding of the molecular events involved in the regulation of pollen abortion in genic male sterile cotton, which may provide a foundation for further research studies on cotton heterosis breeding.

Proteomic identification of differentially expressed proteins in Gossypium thurberi inoculated with cotton Verticillium dahliae.

Thurber's cotton (Gossypium thurberi) is the wild relative of cultivated cotton. It is highly resistant to cotton Verticillium wilt, a disease that significantly affects cotton yield and quality. To reveal the mechanism of disease resistance in G. thurberi and to clone resistance-related genes, we used two-dimensional electrophoresis (2-DE) and tandem time-of-flight mass spectrometry (MALDI-TOF-MS) to identify differentially expressed proteins in Thurber's cotton after inoculation with Verticillium dahliae. A total of 57 different protein spots were upregulated, including 52 known proteins representing 11% of the total protein spots. These proteins are involved in resistance to stress and disease, transcriptional regulation, signal transduction, protein processing and degradation, photosynthesis, production capacity, basic metabolism, and other processes. In addition, five disease resistance proteins showed intense upregulation, indicating that resistance genes (R genes) may play a critical role in resistance to Verticillium wilt in Thurber's cotton. Our results suggest that disease and stress resistance are the combined effects of multiple co-expressed genes. This provides a basis for further, detailed investigation into the mechanisms underlying Verticillium wilt resistance of G. thurberi and for cloning essential genes into cotton cultivars to produce Verticillium wilt resistant plants.