Studies on Drought-Responsive Genes in Citrullus colocynthis

Abstract

Citrullus colocynthis (L.) Schrad, closely related to watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai.), is a member of the Cucurbitaceae family. This plant is a drought tolerant species with a deep root system, widely distributed in the Sahara-Arabian deserts in Africa and the Mediterranean region. cDNA Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to isolate drought responsive genes in roots of seedlings following a 20% polyethylene glycol (PEG8000) treatment to induce drought stress. Eighteen genes which show similarity to known function genes were confirmed by relative quantitative (RQ) real-time RT-PCR to be differentially regulated. The expression of these genes was quantified in root and shoot tissues at five time points (4, 8, 12, 24, and 48 h, respectively) following PEG treatment. In general, the highest induction levels in roots occurred earlier than in shoots, since the highest expression levels were detected in roots following 4 h and 12 h, in shoots following 12 h and 48 h of drought. Some genes showed tissue specific expression patterns and were induced in shoots, but suppressed in roots. Seedlings were treated with salicylic acid (SA), jasmonic acid (JA) or abscisic acid (ABA) and analyzed using RQ real-time RT-PCR. A complex interplay between hormone signaling pathways regulate plant gene expression during adaptive responses to abiotic stress. The full length Ccrboh gene, encoding respiratory burst oxidase protein, was cloned using RACE (Rapid Amplication cDNA Ends). Sequence comparisons showed that the Ccrboh protein contains a Ca2+-binding motif of the EF-hand loop type at the N-terminal, and cytosolic FAD-and NADPH-binding domains at the C-terminal, characteristic of the RBO protein family. Southern blot analysis indicated that this gene exists as one or two copies in C. colocynthis. The subcellular location of Ccrboh was investigated by transient expression of Ccrboh::GFP fusion protein in protoplasts, and the result confirmed that Ccrboh is located at the plasma membrane. RQ Real-time RT-PCR analysis showed that expression of Ccrboh was rapidly and strongly induced by abiotic stress imposed by PEG, ABA, SA, and JA treatment in C. colocynthis, but it did not change in domesticated watermelon (C. lanatus var. lanatus CLL) under these treatments. Grafting of C. colocynthis (CC) and CLL was conducted. Ccrboh gene expression in CLL scion with CC as a rootstock was induced, but induction levels were less as compared to non-grafted CC plants. Ccrboh gene levels did not change in CC scion grafted on CLL rootstock. Gene expression changes also occurred following vegetative growth and root growth. Our data suggest that Ccrboh plays a broader role during stress and in plant development, and may hold great promise for improving stress tolerance of other cucurbit species.