Expression of heat shock protein and trehalose-6-phosphate synthase homologues induced during water deficit in cotton

Abstract

Tolerance to drought in plants is not a simple trait, but a complex of mechanisms working in combination to avoid or to resist water deficit. Genotypes that differ in tolerance to water deficit may show qualitative and quantitative differences in gene expression when submitted to drought periods. Four cotton (Gossypium hirsutum L.) genotypes (Siokra L-23, Stoneville 506, CS 50 and T-1521) with contrasting responses to water deficit stress were studied using the Differential Display (DD) technique to identify and isolate genes which may differ among them. Fifty-two cDNA fragments differentially expressed during water deficit were isolated, cloned and sequenced. Search of gene bank databases showed that two cDNA clones, A12B15-6 and A12B13-1, have high homology with a heat shock protein that binds to calmodulin found in Nicotiana tabacum (2.9e-32 P(N)) and with an Arabidopsis thaliana trehalose-6-phosphate synthase enzyme (9.0e-37 P(N)), respectively. One of the presumed functions of heat shock proteins is related to prevention of protein denaturation during cellular dehydration. Trehalose-6-phosphate synthase is involved in the production of trehalose, a disaccharide known to osmotically protect cell membranes during dehydration. The HSP homologue was found to be differentially expressed during the drought period in two drought tolerant genotypes but not in drought-sensitive genotypes. The trehalose-6-phosphate synthase homologue was also up-regulated during water deficit stress, however, all four genotypes were induced to express this homologue. Ribonuclease protection assays confirmed these results. This is an important finding since there are only few reports of trehalose presence in higher plants and none in cotton.

Isolation and characterization of drought-related trehalose 6-phosphate-synthase gene from cultivated cotton (Gossypium hirsutum L.)

“…Based on a 323-bp cDNA fragment with significant homology to the A. thaliana TPS gene, already reported by Nepomuceno et al (2002), two specific primers (SK1 and SK2) were designed in order to amplify the respective gene fragment in G. hirsutum. Furthermore, two degenerate primers (T6PS6 and T6PS7) were designed, based on the conserved TPS regions after the alignment of four TPS amino acid sequences from A. thaliana (NP_177979.1, NP_173143.1, NP_177186.2 and NP_176221.1).…”

“…Polymerase chain reaction primers (Microchemistry Laboratory of the Institute of Molecular Biology and Biotechnology Heraklion, Greece) were designed using the data provided by Nepomuceno et al (2002), and the multiple sequence alignment of homologous amino acid sequences to TPS from A. thaliana (NP_177979.1, NP_173143.1, NP_177186.2 and NP_176221.1). Adaptor primers used in chromosome walking were: AP1 5¢-GTAATACGACTCACTATAGGGC-3¢ (primary) and AP2 5¢-ACTATAGGGCACGCGTGGT-3¢ (nested).…”

“…Since drought is a major concern in cotton productivity and therefore in cotton breeding, isolation of genes that are uniquely expressed in drought-tolerant cotton genotypes will be helpful in understanding the mechanisms of drought tolerance. Nepomuceno et al (2002) isolated a DNA fragment (323 bp, AF056946) with high homology to TPS gene from A. thaliana, which was found as a transcript under water stress conditions in four cotton genotypes. The objective of this study is to isolate and characterize the TPS gene from cultivated cotton, the most important crop for fibre production.…”

“…Cloning this gene will enable us to study the effects of TPS in drought tolerance. Since there is strong evidence that T6P plays an indispensable role in plants carbon metabolism and development (Eastmond et al 2002;Schluepmann et al 2003), and overexpression of TPS gene in transgenic plants confers drought tolerance, we first examined the presence of transcripts in different plant tissues under well-water and water-stressed conditions, as Nepomuceno et al (2002) reported detection of TPS transcripts in leaves.…”

Differential gene expression and mitotic cell analysis of the drought tolerant soybean (Glycine max L. Merrill Fabales, Fabaceae) cultivar MG/BR46 (Conquista) under two water deficit induction systems

“…The identification and understanding of tolerance mechanisms is crucial not only in soybean but also in other crops (Somerville and Koornneef, 2002;Nepomuceno et al, 2002). Since roots play a key role in stress detection and tolerance response, a reliable system for molecular analysis in roots should be used to ensure consistency among studies.…”