Compared with small heat shock proteins (sHSPs) in other organisms, those in plants are the most abundant and diverse. However, the molecular mechanisms by which sHSPs are involved in cell protection remain unknown. Here, we characterized the role of HSP21, a plastid nucleoid-localized sHSP, in chloroplast development under heat stress. We show that an Arabidopsis thaliana knockout mutant of HSP21 had an ivory phenotype under heat stress. Quantitative real-time RT-PCR, run-on transcription, RNA gel blot, and polysome association analyses demonstrated that HSP21 is involved in plastidencoded RNA polymerase (PEP)-dependent transcription. We found that the plastid nucleoid protein pTAC5 was an HSP21 target. pTAC5 has a C 4 -type zinc finger similar to that of Escherichia coli DnaJ and zinc-dependent disulfide isomerase activity. Reduction of pTAC5 expression by RNA interference led to similar phenotypic effects as observed in hsp21. HSP21 and pTAC5 formed a complex that was associated mainly with the PEP complex. HSP21 and pTAC5 were associated with the PEP complex not only during transcription initiation, but also during elongation and termination. Our results suggest that HSP21 and pTAC5 are required for chloroplast development under heat stress by maintaining PEP function.
INTRODUCTIONThe small heat shock proteins (sHSPs) and the related a-crystallins are virtually ubiquitous proteins that are strongly induced not only by heat stress but also by a variety of other stresses in prokaryotic and eukaryotic cells (Sun et al., 2002;Basha et al., 2012). The sHSPs are characterized by a core a-crystallin domain of ;100 amino acids, which is flanked by an N-terminal arm of variable length and divergent sequence and a short C-terminal extension (Haslbeck et al., 2005). Although sHSP monomers are relatively small, ranging in size from ;15 to 42 kD, the majority of these proteins exist as oligomers of between 12 and >48 subunits in their native state (Lambert et al., 2011;Basha et al., 2012). Moreover, sHSPs vary at the secondary, tertiary, and quaternary levels of protein organization, with dynamic exchange of subunits between sHSP oligomers (Stengel et al., 2010;Baldwin et al., 2011). In addition, sHSPs show extensive sequence variation and evolutionary divergence unlike other families of HSPs, such as the HSP90 and HSP70 chaperone families (Basha et al., 2012). Although the molecular mechanisms by which sHSPs and a-crystallins are involved in cell protection in many organisms remain largely unknown, many studies have demonstrated that both mammalian and plant sHSPs act as ATP-independent molecular chaperones by binding proteins that are unfolding or denaturing and thereby preventing their aggregation and facilitating subsequent substrate refolding by ATP-dependent chaperone systems (Lee et al., 1997;Haslbeck et al., 2005;Sun and MacRae, 2005;McHaourab et al., 2009). Due to their molecular chaperone characteristics, sHSPs are considered important components of the protein quality control network (Basha et al., 2012).Plant...