To gain insight into the processes involved in photosystem II (PSII) biogenesis and maintenance, we characterized the low psii accumulation1 (lpa1) mutant of Arabidopsis thaliana, which generally accumulates lower than wild-type levels of the PSII complex. In vivo protein labeling experiments showed that synthesis of the D1 and D2 proteins was greatly reduced in the lpa1 mutant, while other plastid-encoded proteins were translated at rates similar to the wild type. In addition, turnover rates of the PSII core proteins CP47, CP43, D1, and D2 were higher in lpa1 than in wild-type plants. The newly synthesized PSII proteins were assembled into functional protein complexes, but the assembly was less efficient in the mutant. LPA1 encodes a chloroplast protein that contains two tetratricopeptide repeat domains and is an intrinsic membrane protein but not an integral subunit of PSII. Yeast two-hybrid studies revealed that LPA1 interacts with D1 but not with D2, cytochrome b6, or Alb3. Thus, LPA1 appears to be an integral membrane chaperone that is required for efficient PSII assembly, probably through direct interaction with the PSII reaction center protein D1.

SummaryPre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice (Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants (phs). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), f-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene b-cyclase (b-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso/phs3-1mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice.

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...