An aldose reductase homolog from the resurrection plant Xerophyta viscosa Baker

An aldose reductase homologue (ALDRXV4) was cloned fi om the resurrection p lant Xerophyta viscosa Baker using complementation by functional sufficienc y in Escherichia cull. A cDNA library constructed from X. viscosa leaves de hydrated to 85%, 37% and 5% relative water contents (RWC) was converted int o an infective phagemid library. Escherichia coli (sr1::Tn10) cells transfo rmed with ds-pBluescript phage mids were selected on minimal medium plates supplemented with 1 mM isopropyl beta -D-thiogalactopyranoside and 1.25 M s orbitol. Nine cDNA clones that conferred tolerance to the osmotically stres sed E. coli cells were selected. The phagemid fi om one clone contained the ALDRXV4 insert. The E. coli cells expressing ALDRXV4 were capable of toler ating the osmotic stress, whereas control cultures were not. The ALDRXV4 in sert contained an open reading frame that can code for 319 amino acids, and the predicted protein had a calculated M-r of 35,667. Amino acid sequence comparisons revealed significant similarity to several aldose reductases, w ith the highest similarity to aldose reductase proteins from Hordeum vulgar e, Bromus inermis and Avena fatua, in the order of 66%, 65% and 65% respect ively. Northern blot analysis revealed that ALDRXV4 was expressed only unde r dehydration conditions in X. viscosa leaves. Western blot analysis detect ed a protein of 36 1;Da. under dehydration conditions only. Aldose reductas e activity levels in X. viscosa leaves increased as the leaf RWC decreased, whereas there was no significant change in aldose reductase activity in Sp orobolus stafianus as the leaf RWC decreased.