Identification and functional evaluation of a drought-induced "late embryogenesis abundant" gene from cowpea plants

Abstract

Crop breeding for drought tolerance is very important in South Africa. Biotechnology can be applied to improve a crop’s drought tolerance through candidate gene identification, followed by transformation or molecular breeding strategies. Cowpea was utilised as a gene source due to its inherent drought tolerance. A cDNA library, containing 4160 clones and enriched for drought responsive cowpea genes, was generated following suppression subtractive hybridisation (SSH) on two lines differing in their drought tolerance. Microarray hybridisation was used to screen the library for differentially expressed genes. Microarray data was analysed with a custom software package called SSHscreen. The SSH procedure was successful, since it enriched the library for transcripts differentially expressed by drought stress and rare transcripts. Selected clones were sequenced and several cowpea genes responding to drought stress were identified. Among these was a gene from the well-studied late embryogenesis abundant (LEA) family. Other stress-induced genes identified included a glutathione S-transferase, a thaumatin, a universal stress protein and a wound induced protein. A lipid transfer protein and several components of photosynthesis were down-regulated by the drought stress. SSHdb, a web-accessible database, was used to manage the clone sequences, group redundant clones, annotate the sequence information, and combine the SSHscreen data with sequence annotations obtained from BLAST and Blast2GO. A high frequency of redundancy existed in the library, with only 39 unique sequences among the 118 sequenced clones. SSHscreen plots proved to be a useful tool for choosing anonymous clones for sequencing, since redundant clones cluster together on the enrichment ratio plots. The gene expression patterns of selected cowpea genes were verified with reverse-transcriptase quantitative PCR (RT-qPCR). The gene encoding an atypical group 5 LEA protein (VuLEA5) was selected for further characterisation. LEA proteins play an important protective role during drought and other abiotic stresses of plants, but group 5 proteins are not as well studied as the other LEA groups. VuLEA5 was shown to be highly up-regulated by drought, abscisic acid (ABA), salt and cold in cowpea leaves. VuLEA5 was over-expressed in the model plant Arabidopsis thaliana to study its role in tolerance to drought. The integration of the transgene into the genome was analysed by PCR and Southern blot. mRNA expression was detected by RT-PCR and RT-qPCR, and recombinant protein expression by Western blot. Transgenic A. thaliana lines were investigated for improved drought tolerance compared to the wild type. Seedling stress tests were performed in vitro in the presence of osmotic agents (high molecular weight polyethylene glycol) that simulate drought conditions in the soil. Quantitative growth parameters (hypocotyl and primary root elongation) of homozygous transgenic T4 seedlings were measured. No statistically significant improvement in growth and recovery after stress could, however, be attributed to the cowpea LEA5. A number of progressive drought trials of plants growing in a growth room did not indicate any quantitative improvement in their ability to withstand drought. This LEA5 protein alone could, therefore, not improve the drought tolerance of transgenic A. thaliana lines. Its possible role in cold stress protection is suggested as a follow-up study.