Characterization of 5′UTR of rice ClpB-C/Hsp100 gene: evidence of its involvement in post-transcriptional regulation

Rice (Oryza sativa) ClpB-C (OsClpB-C) protein is expressed upon heat stress in vegetative tissues and constitutively in seeds. We produced stably transformed Arabidopsis plants carrying β-glucuronidase (Gus) reporter gene downstream to 1-kb OsClpB-C promoter (1kbPro plants). In the 1kbPro plants, expression of Gus transcript and protein followed the expression pattern of OsClpB-C gene in rice plants, i.e., heat induced in vegetative tissues and constitutive in seeds. Next, we produced transgenic Arabidopsis plants containing Gus downstream to 862-bp fragment of OsClpB-C promoter [lacking 138 nucleotides from 3′ end of the 5′untranslated region (5′UTR); ∆UTR plants). In ∆UTR plants, Gus transcript was expressed in heat-inducible manner, but strikingly, Gus protein levels were negligible after heat treatment. However, Gus protein was expressed in ∆UTR seedlings at levels comparable to 1kbPro seedlings when recovery treatment of 22 °C/2 h was given post heat stress (38 °C/15 min). This suggests that 5′UTR of OsClpB-C gene is involved in its post-transcriptional regulation and is an obligate requirement for protein expression during persistent heat stress. Furthermore, the Gus transcript levels were higher in the polysomal RNA fraction in heat-stressed seedlings of 1kbPro plants as compared to ∆UTR plants, indicating that 5′UTR aids in assembly of ribosomes onto the Gus transcript during heat stress. Unlike the case of seedlings, Gus protein was formed constitutively in ∆UTR seeds at levels comparable to 1kbPro seeds. Hence, the function of 5′UTR of OsClpB-C is dispensable for expression in seeds.