An aldose reductase homolog from the resurrection plant Xerophyta viscosa Baker

Abstract.

An aldose reductase homologue (ALDRXV4) was cloned from the resurrection plant Xerophyta viscosa Baker using complementation by functional sufficiency in Escherichia coli. A cDNA library constructed from X. viscosa leaves dehydrated to 85%, 37% and 5% relative water contents (RWC) was converted into an infective phagemid library. Escherichia coli (srl::Tn10) cells transformed with ds-pBluescript phagemids were selected on minimal medium plates supplemented with 1 mM isopropyl β-d-thiogalactopyranoside and 1.25 M sorbitol. Nine cDNA clones that conferred tolerance to the osmotically stressed E. coli cells were selected. The phagemid from one clone contained the ALDRXV4 insert. The E. coli cells expressing ALDRXV4 were capable of tolerating the osmotic stress, whereas control cultures were not. The ALDRXV4 insert 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, with the highest similarity to aldose reductase proteins from Hordeum vulgare, Bromus inermis and Avena fatua, in the order of 66%, 65% and 65% respectively. Northern blot analysis revealed that ALDRXV4 was expressed only under dehydration conditions in X. viscosa leaves. Western blot analysis detected a protein of 36 kDa under dehydration conditions only. Aldose reductase activity levels in X. viscosa leaves increased as the leaf RWC decreased, whereas there was no significant change in aldose reductase activity in Sporobolus stafianus as the leaf RWC decreased.