Role of GhHDA5 in H3K9 deacetylation and fiber initiation in Gossypium hirsutum.

Cotton fibers are single-celled trichomes that initiate from the epidermal cells of the ovules at or before anthesis. Here, we identified that the histone deacetylase (HDAC) activity is essential for proper cotton fiber initiation. We further identified 15 HDACs homoeologs in each of the A- and D-subgenomes of Gossypium hirsutum. Few of these HDAC homoeologs expressed preferentially during the early stages of fiber development [-1, 0 and 6 days post-anthesis (DPA)]. Among them, GhHDA5 expressed significantly at the time of fiber initiation (-1 and 0 DPA). The in vitro assay for HDAC activity indicated that GhHDA5 primarily deacetylates H3K9 acetylation marks. Moreover, the reduced expression of GhHDA5 also suppresses fiber initiation and lint yield in the RNA interference (RNAi) lines. The 0 DPA ovules of GhHDA5RNAi lines also showed alterations in reactive oxygen species homeostasis and elevated autophagic cell death in the developing fibers. The differentially expressed genes (DEGs) identified through RNA-seq of RNAi line (DEP12) and their pathway analysis showed that GhHDA5 modulates expression of many stress and development-related genes involved in fiber development. The reduced expression of GhHDA5 in the RNAi lines also resulted in H3K9 hyper-acetylation on the promoter region of few DEGs assessed by chromatin immunoprecipitation assay. The positively co-expressed genes with GhHDA5 showed cumulative higher expression during fiber initiation, and gene ontology annotation suggests their involvement in fiber development. Furthermore, the predicted protein interaction network in the positively co-expressed genes indicates HDA5 modulates fiber initiation-specific gene expression through a complex involving reported repressors.

GhMYB1 regulates SCW stage-specific expression of the GhGDSL promoter in the fibres of Gossypium hirsutum L.

Secondary cell wall (SCW) biosynthesis is an important stage of the cotton fibre development, and its transcriptional regulation is poorly understood. We selected the Gossypium hirsutum GDSL (GhGDSL) lipase/hydrolase gene (CotAD_74480), which is expressed during SCW biosynthesis (19 through to 25 days postanthesis; DPA), for study. T1 -transgenic cotton lines expressing the ß-glucuronidase (gus) reporter under the control of a 1026-bp promoter fragment of GhGDSL (PGhGDSL ) showed 19 DPA stage-specific increase in GUS expression. 5' deletion indicated that the 194-bp fragment between -788 and -594 relative to the transcription start site was essential for this stage-specific expression. Site-directed mutagenesis of eight transcription factor binding sites within PGhGDSL demonstrated that the MYB1AT motif (AAACCA) at -603/-598 was critical for the 19 DPA-specific reporter gene expressions. Yeast one-hybrid (Y1H) analysis identified nine proteins, including GhMYB1 (CotAD_64719) that bound to the PGhGDSL promoter. Further, Y1H experiments using the 5' promoter deletions and individually mutated promoter motifs indicated that GhMYB1 interacted with PGhGDSL at MYB1AT sequence. GhMYB1 was expressed specifically in fibre from 19 DPA, overlapping with the sharp rise in GhGDSL expression, indicating that it could regulate GhGDSL during fibre development. Analysis of genes co-expressed with GhMYB1 showed that it potentially regulates a number of other 19-25 DPA-specific genes in networks including those functioning in the cell wall and precursor synthesis, but not the major polysaccharide and protein components of the fibre SCW. GhGDSL and its promoter are therefore potential tools for the improvement of cotton fibre quality traits.

Inhibition of Heat Shock proteins HSP90 and HSP70 induce oxidative stress, suppressing cotton fiber development.

Cotton fiber is a specialized unicellular structure useful for the study of cellular differentiation and development. Heat shock proteins (HSPs) have been shown to be involved in various developmental processes. Microarray data analysis of five Gossypium hirsutum genotypes revealed high transcript levels of GhHSP90 and GhHSP70 genes at different stages of fiber development, indicating their importance in the process. Further, we identified 26 and 55 members of HSP90 and HSP70 gene families in G. hirsutum. The treatment of specific inhibitors novobiocin (Nov; HSP90) and pifithrin/2-phenylethynesulfonamide (Pif; HSP70) in in-vitro cultured ovules resulted in a fewer number of fiber initials and retardation in fiber elongation. The molecular chaperone assay using bacterially expressed recombinant GhHSP90-7 and GhHSP70-8 proteins further confirmed the specificity of inhibitors. HSP inhibition disturbs the H2O2 balance that leads to the generation of oxidative stress, which consequently results in autophagy in the epidermal layer of the cotton ovule. Transmission electron microscopy (TEM) of inhibitor-treated ovule also corroborates autophagosome formation along with disrupted mitochondrial cristae. The perturbations in transcript profile of HSP inhibited ovules show differential regulation of different stress and fiber development-related genes and pathways. Altogether, our results indicate that HSP90 and HSP70 families play a crucial role in cotton fiber differentiation and development by maintaining cellular homeostasis.