Genome wide expression profiling of two accession of G. herbaceum L. in response to drought.

BACKGROUND: Genome-wide gene expression profiling and detailed physiological investigation were used for understanding the molecular mechanism and physiological response of Gossypium herbaceum, which governs the adaptability of plants in drought conditions. Recently, microarray-based gene expression analysis is commonly used to decipher genes and genetic networks controlling the traits of interest. However, the results of such an analysis are often plagued due to a limited number of genes (probe sets) on microarrays. On the other hand, pyrosequencing of a transcriptome has the potential to detect rare as well as a large number of transcripts in the samples quantitatively. We used Affymetrix microarray as well as Roche's GS-FLX transcriptome sequencing for a comparative analysis of cotton transcriptome in leaf tissues under drought conditions. RESULTS: Fourteen accessions of Gossypium herbaceum were subjected to mannitol stress for preliminary screening; two accessions, namely Vagad and RAHS-14, were selected as being the most tolerant and most sensitive to osmotic stress, respectively. Affymetrix cotton arrays containing 24,045 probe sets and Roche's GS-FLX transcriptome sequencing of leaf tissue were used to analyze the gene expression profiling of Vagad and RAHS-14 under drought conditions. The analysis of physiological measurements and gene expression profiling showed that Vagad has the inherent ability to sense drought at a much earlier stage and to respond to it in a much more efficient manner than does RAHS-14. Gene Ontology (GO) studies showed that the phenyl propanoid pathway, pigment biosynthesis, polyketide biosynthesis, and other secondary metabolite pathways were enriched in Vagad under control and drought conditions as compared with RAHS-14. Similarly, GO analysis of transcriptome sequencing showed that the GO terms responses to various abiotic stresses were significantly higher in Vagad. Among the classes of transcription factors (TFs) uniquely expressed in both accessions, RAHS-14 showed the expression of ERF and WRKY families. The unique expression of ERFs in response to drought conditions reveals that RAHS-14 responds to drought by inducing senescence. This was further supported by transcriptome analysis which revealed that RAHS-14 responds to drought by inducing many transcripts related to senescence and cell death. CONCLUSION: The comparative genome-wide gene expression profiling study of two accessions of G.herbaceum under drought stress deciphers the differential patterns of gene expression, including TFs and physiologically relevant processes. Our results indicate that drought tolerance observed in Vagad is not because of a single molecular reason but is rather due to several unique mechanisms which Vagad has developed as an adaptation strategy.

The histone H1 variant accumulates in response to water stress in the drought tolerant genotype of Gossypium herbaceum L.

We have optimized and improved the protocol for extraction of histone proteins from Gossypium herbaceum. Histone proteins were isolated by acid extraction method and fractionation of histone proteins were performed using RP-HPLC (reverse-phase high performance liquid chromatography). Analysis of histones from drought tolerant (Vagad) and drought sensitive genotype (RAHS-14) indicated that the tolerant genotype Vagad encodes a 29 kDa protein. Protein sequencing on MALDI TOF/TOF revealed that the 29 kDA protein shared sequence similarity with another drought-inducible linker histone-H1.S reported in tomato. This H1.S like linker histone was not found in RAHS-14 in our study. We further examined the expression of H1 variant at the transcript and protein levels and found that it was induced specifically in the tolerant genotype Vagad.

Analysis of histones and histone variants in plants.

Histone proteins are the major protein components of chromatin - the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. For many years, histones were considered passive structural components of eukaryotic chromatin. In recent years, it has been demonstrated that dynamic association of histones and their variants to the genome plays a very important role in gene regulation. Histones are extensively modified during posttranslation viz. acetylation, methylation, phosphorylation, ubiquitylation, etc., and the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Different biochemical techniques have been developed to purify and separate histone proteins; here, we describe techniques for analysis of histones from plant tissues.

Analysis of chromatin structure in plant cells.

A vast body of evidence in the literature indicates that nucleosomes can act as barriers to transcriptional initiation. The nucleosome at the promoter inhibits association of transcription factors disallowing active transcription of the gene. We have found a nucleosome on tobacco pathogenesis-related gene-1a (PR-1a) core promoter and mapped its boundaries and extension to find its span. The nucleosome covers the TATA box and Inr region of the core promoter and gets disassembled upon induction. Prior to its removal, modifications (i.e., acetylation and methylation of histones) occur at the nucleosome, proving a role of epigenetic modifications in transcriptional regulation. We summarize here various methodologies to analyze promoter chromatin structure in plants using the PR-1a core promoter as an example.

Efficient production of gossypol from hairy root cultures of cotton (Gossypium hirsutum L.).

A protocol for induction and establishment of Agrobacterium rhizogenes mediated hairy root culture of Gossypium hirsutum was developed through infection with the A4 strain and co-cultivation on hormone-free semi-solid MS medium with B5 vitamins. It resulted in the emergence of hairy roots from the leaf explants, 21 days after infection. The transformation of hairy roots was established by PCR amplification of rol B and rol C genes of the Ri plasmid. All root lines expressed gossypol, although distinct inter-clonal quantitative variations were noticed. Five independent hairy root lines were studied for their growth kinetics as well as gossypol production. The yield potentials of one of them superseded others, as well as the non-transformed, in-vitro grown control roots. The content of gossypol in hairy roots reached a level of 2.43 mg/g DW. It was 4.5 times higher than in vitro and 1.47 times higher than in vivo grown roots of G. hirsutum. Selection of high gossypol producing hairy root lines of G. hirsutum can provide an alternative source for large-scale production of gossypol.