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...

Numerous attempts have been made to identify and engineer sequence-specific RNA endonucleases, as these would allow for efficient RNA manipulation. However, no natural RNA endonuclease that recognizes RNA in a sequence-specific manner has been described to date. Here, we report that SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), an Arabidopsis pentatricopeptide repeat (PPR) protein with a small MutS-related (SMR) domain, has RNA endonuclease activity. We show that the SMR moiety of SOT1 performs the endonucleolytic maturation of 23S and 4.5S rRNA through the PPR domain, specifically recognizing a 13-nucleotide RNA sequence in the 5′ end of the chloroplast 23S-4.5S rRNA precursor. In addition, we successfully engineered the SOT1 protein with altered PPR motifs to recognize and cleave a predicted RNA substrate. Our findings point to SOT1 as an exciting tool for RNA manipulation.photosynthesis | PPR-SMR protein | RNA endonuclease | rRNA biogenesis S equence-specific RNA endonucleases are crucial to establishing RNA manipulation technology (1). Compared with DNA editing, RNA manipulation could be more useful and reversible because it does not result in permanent changes to the genome. In addition, sequence-specific RNA endonucleases could potentially be used as an RNA silencing tool to complement RNAi, because RNAi is sometimes ineffective in certain organisms and RNAi machinery is not present in cellular compartments such as chloroplasts and mitochondria. Despite extensive investigations, a natural RNA endonuclease that recognizes RNA in an intrinsic sequence-specific manner has not yet been identified.Pentatricopeptide repeat (PPR) proteins exist in eukaryotes, have greatly expanded in terrestrial plants, and take part in most RNA metabolic processes in organelles (2-5). The PPR domain can specifically recognize RNAs in an intrinsic sequence-specific manner (5-8). The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9-11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).The small MutS-related (SMR) domain was originally identified at the C terminus of MutS2 in the cyanobacterium Synechocystis (18). SMR proteins are widely distributed in almost all organisms (19). Recent studies demonstrated the SMR domain exhibits DNA nicking nuclease activity in vitro (20-25). Furthermore, a C-terminal SMR domain in Leishmania donovani S-phase mRNA cycling sequence binding protein (CSBP) has RNA cleavage activity in vitro (26). These findings suggest that the SMR domain has nuclease activity.Interestingly, a small protein family containing both PPR and SMR domains was recently described (27). PPR-SMR proteins are found mainly in land plants. Arabidopsis thaliana contains eight PPR-SMR proteins localized to the organelles, including mitochondria and chloroplasts (27). Currently, four PPR-SMR proteins have been characterized. They play ...

RNA polymerase transcriptional pausing represents a major checkpoint in transcription in bacteria and metazoans, but it is unknown whether this phenomenon occurs in plant organelles. Here, we report that transcriptional pausing occurs in chloroplasts. We found that mTERF5 specifically and positively regulates the transcription of chloroplast psbEFLJ in Arabidopsis thaliana that encodes four key subunits of photosystem II. We found that mTERF5 causes the plastid-encoded RNA polymerase (PEP) complex to pause at psbEFLJ by binding to the +30 to +51 region of double-stranded DNA. Moreover, we revealed that mTERF5 interacts with pTAC6, an essential subunit of the PEP complex, although pTAC6 is not involved in the transcriptional pausing at psbEFLJ. We showed that mTERF5 recruits additional pTAC6 to the transcriptionally paused region of psbEFLJ, and the recruited pTAC6 proteins could be assembled into the PEP complex to regulate psbEFLJ transcription. Taken together, our findings shed light on the role of transcriptional pausing in chloroplast transcription in plants.