Cloning, expression, and molecular characterization of a small pea gene family regulated by low levels of ultraviolet B radiation and other stresses

Abstract

A pea (Pisum sativum) DNA fragment (termed MB3) was isolated by differential display of cDNAs obtained from total leaf RNA of ultraviolet B (UV-B) radiation-treated plants. Longer cDNAs were cloned by rapid amplification of cDNA ends in the 3' to 5' direction. Three different, but very similar, cDNAs were cloned, sadA, sadB, and sadC, the major difference between them being a 36-bp deletion in the coding region of sadB. Southern blotting confirmed the occurrence of at least three genes in the pea genome. Database comparisons of the SAD protein sequences revealed high identity (46%) and similarity (77%) with a putative tomato (Lycopersicon esculentum) short-chain alcohol dehydrogenase. Very low levels of UV-B radiation (the biologically effective radiation normalized to 300 nm= 0.08 W m-2) was shown to up-regulate expression, a dose considerably lower than that needed to induce expression of the well-known UV-B defensive chalcone synthase and phenylalanine ammonia lyase genes. RNase protection assay revealed that primarily sadA and sadC mRNA accumulation was enhanced by UV-B. In addition to UV-B irradiation, ozone fumigation, wounding, aluminum stress, and salt stress induced increased transcript levels of the sad genes in pea.