Measurements of Proline and Malondialdehyde Content and Antioxidant Enzyme Activities in Leaves of Drought Stressed Cotton

Cotton is an important cash crop worldwide, and is a significant source of fiber, feed, foodstuff, oil and biofuel products. Considerable effort has been expended to increase sustainable yield and quality through molecular breeding and genetic engineering of new cotton cultivars. Given the recent availability of the whole-genome sequence of cotton, it is necessary to develop molecular tools and resources for large-scale analysis of gene functions at the genome-wide level. We have successfully developed an Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in several cotton cultivars with various genetic backgrounds. The genes of interest were potently and readily silenced within 2 weeks after inoculation at the seedling stage. Importantly, we showed that silencing GhNDR1 and GhMKK2 compromised cotton resistance to the infection by Verticillium dahliae, a fungal pathogen causing Verticillium wilt. Furthermore, we developed a cotton protoplast system for transient gene expression to study gene functions by a gain-of-function approach. The viable protoplasts were isolated from green cotyledons, etiolated cotyledons and true leaves, and responded to a wide range of pathogen elicitors and phytohormones. Remarkably, cotton plants possess conserved, but also distinct, MAP kinase activation with Arabidopsis upon bacterial elicitor flagellin perception. Thus, using gene silencing assays, we have shown that GhNDR1 and GhMKK2 are required for Verticillium resistance in cotton, and have developed high throughput loss-of-function and gain-of-function assays for functional genomic studies in cotton. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Drought stress negatively affects plant growth and limits plant productivity. Genes functioning in plant responses to drought stress are essential for the development of drought-tolerant crops. Here, we report that an R2R3-type MYB transcription factor gene in Gossypium barbadense, GbMYB5, confers drought tolerance in cotton and transgenic tobacco. Virus-induced gene silencing of GbMYB5 compromised the tolerance of cotton plantlets to drought stress and reduced the post-rewatering water recovery survival rate to 50% as compared with the 90% survival rate in the wild type (WT). Silencing GbMYB5 decreased proline content and antioxidant enzyme activities and increased malondialdehyde (MDA) content in cotton under drought stress. The expression levels of drought-inducible genes NCED3, RD22 and RD26 were not affected by the silencing of GbMYB5. However, GbMYB5-overexpressing tobacco lines displayed hypersensitivity to ABA and improved survival rates as well as reduced water loss rates under drought stress. Furthermore, stomatal size and the rate of opening of stomata were markedly decreased in transgenic tobacco. The overexpression of GbMYB5 enhanced the accumulation of proline and antioxidant enzymes while it reduced production of MDA in transgenic tobacco as compared with the WT under drought stress. The transcript levels of the antioxidant genes SOD, CAT and GST, polyamine biosynthesis genes ADC1 and SAMDC, the late embryogenesis abundant protein-encoding gene ERD10D and drought-responsive genes NCED3, BG and RD26 were generally higher in GbMYB5-overexpressing tobacco than in the WT under drought stress. Collectively, our data suggested that GbMYB5 was positively involved in the plant adaptive response to drought stress.

Cotton is an important economic crop worldwide. Due to its long growth cycle, large genome size and recalcitrance to stable transformation, traditional methods for the analysis of gene function in this crop are difficult and labour intensive. Here, we report a cotton leaf crumple virus (CLCrV)-based vector and its application in gene function analysis through virus-induced gene silencing (VIGS) and overexpression of microRNAs (miRNAs), small tandem target mimic (STTM) and artificial miRNA (amiRNA) in cotton via an Agrobacterium-mediated infiltration approach. Using this system, we were able to efficiently silence two endogenous genes, magnesium chelatase subunit I (CHLI) and elongation factor-1α (EF-1α), in Gossypium species and the Bacillus thuringiensis cry1A gene in transgenic cotton. Furthermore, our results show that this vector can be used to ectopically express endogenous miR156 in G. hirsutum, causing a reduction in miR156-targeted RNA transcripts resulting in the development of abnormal leaf phenotypes. Ectopic expression of miR165/166 STTM with this vector led to downward curling and crumpled leaves, and a significant increase in the miR165/166 target mRNAs. This versatile system is easy to use and can provide more uniform and persistent gene silencing in cotton, thereby providing a powerful approach for gene discovery in cotton. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.