Characterisation of Brassinosteroid Effects and Brassinosteroid-Responsive Genes in Cotton for Growth and Stress Tolerance Enhancement Against Abiotic and Biotic Stresses

Abstract

<p>Brassinosteroids (BRs) are plant steroid hormones that not only play vital roles in plant growth and development, but also in mediating stress responses. A group of calmodulin-binding proteins, known as CBP60s are also involved in mediating the response of plants to stress. The aims of the present study were: (1) to investigate the effect of exogenous 24-epibrassinolide (EBR) on the phenotype of cotton (<i>Gossypium hirsutum</i>) seedlings under mild to moderate biotic and abiotic stresses, (2) to find and characterise cotton CBP60-encoding genes, orthologous to Arabidopsis CBP60s with known involvement in stress responses, and to investigate whether EBR may act by modulating the expression of <i>GhCBP60</i> genes in cotton leaf tissue under salt stress. Experiments were designed to demonstrate the effects of EBR application from 0.1 to 2 µM on the phenotypic responses of cotton seedlings to mild/moderate salt, drought and pathogen (<i>Verticillium dahliae</i>) stresses. Results show that the exogenous application of EBR at low concentrations of 0.1 and 0.2 µM had no positive effect on seedling growth under all stresses. In addition, EBR at a higher concentration (0.5 µM) or with the surfactant Tween 20 caused toxic effects. Bioinformatics approaches revealed the presence of <i>GhCBP60</i> orthologues of AtCBP60. Phylogenetic analysis indicated that <i>CBP60a</i>, <i>CBP60g</i>, and<i>SARD1</i> from Arabidopsis each have four co-orthologues in cotton. <i>AtCBP60f</i> has two coorthologues, whereas <i>CBP60b/c/d</i> have nine co-orthologues. Multiple amino acid sequence alignments indicate that the DNA-binding and CaM-binding domains of AtCBP60 are highly conserved in <i>GhCBP60</i>, suggesting similar protein structures to AtCBP60. Prediction of subcellular localisation suggested that all <i>GhCBP60</i> proteins contain a nuclear localisation signal. This, together with the highly conserved putative DNA binding region, suggests that all <i>GhCBP60</i> are transcription factors. The results of qRT-PCR demonstrated that EBR treatment of cotton up-regulated the expression of <i>GhCBP60</i>a/f/g. On the other hand, salt down-regulated the expression of <i>GhCBP60</i>a but up-regulated the expression of <i>GhCBP60</i>f/g. Interestingly, treatment with EBR in the absence of salt restored the expression of <i>GhCBP60</i>a to levels similar to the control tissue. Analysis of promoters of <i>GhCBP60</i> genes for putative BR-related transcription factor binding motifs indicated the presence of CANNTG and GGTCC elements. However, these were not significantly enriched in stress-regulated genes. Furthermore, higher stringency BR-signalling-related elements: BRRE (CGTGTG/CGTGCG), G-box (CACGTG) and transcription factors TGA 1/TGA4 (TGACG) sense strands were absent in stress responsive genes <i>GhCBP60</i>a/f/g and <i>GhSARD1</i> as compared to other groups. In the light of these results, I concluded that brassinosteroids (BRs) positively regulates the expression of novel <i>GhCBP60</i> genes suggesting a possible connection between BR signalling and <i>GhCBP60</i> transcription factors in mediating abiotic stress responses in cotton. However, the results from the cis-element search suggest that this connection is likely to be indirect rather than via a direct interaction with the BR signal transduction pathway.</p>