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...

To gain insights into the molecular details of photosystem I (PSI) biogenesis, we characterized the PsbP-domain protein1 (ppd1) mutant of Arabidopsis thaliana that specifically lacks PSI activity. Deletion of PPD1 results in an inability of the mutant to grow photoautotrophically and a specific loss of the stable PSI complex. Unaltered transcription and translation of plastidencoded PSI genes indicate that PPD1 acts at the posttranslational level. In vivo protein labeling experiments reveal that the rate of synthesis of PSI reaction center proteins PsaA/B in ppd1 is comparable to that of wild-type plants, whereas the rate of turnover of PsaA/B proteins is higher in ppd1 than in wild-type plants. With increasing leaf age, PPD1 content decreases considerably, while PSI content remains constant. PPD1 is a nuclear-encoded thylakoid lumenal protein and is associated with PSI but is not an integral subunit of PSI. Biochemical and molecular analyses reveal that PPD1 interacts directly and specifically with PsaB and PsaA. Yeast two-hybrid experiments show that PPD1 interacts with some lumenal loops of PsaB and PsaA. Our results suggest that PPD1 is a PSI assembly factor that assists the proper folding and integration of PsaB and PsaA into the thylakoid membrane.

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to accumulate glycinebetaine was established. The wild type and transgenic plants were exposed to heat treatment (25-50 degrees C) for 4 h in the dark and under growth light intensity (300 mumol m(-2) s(-1)). The analyses of oxygen-evolving activity and chlorophyll fluorescence demonstrated that photosystem II (PSII) in transgenic plants showed higher thermotolerance than in wild type plants in particular when heat stress was performed in the light, suggesting that the accumulation of glycinebetaine leads to increased tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement on thermostability of the oxygen-evolving complex and the reaction center of PSII. The enhanced tolerance was caused by acceleration of the repair of PSII from heat-enhanced photoinhibition. Under heat stress, there was a significant accumulation of H(2)O(2), O (2) (-) and catalytic Fe in wild type plants but this accumulation was much less in transgenic plants. Heat stress significantly decreased the activities of catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase in wild type plants whereas the activities of these enzymes either decreased much less or maintained or even increased in transgenic plants. In addition, heat stress increased the activity of superoxide dismutase in wild type plants but this increase was much greater in transgenic plants. Furthermore, transgenic plants also showed higher content of ascorbate and reduced glutathione than that of wild type plants under heat stress. The results suggest that the increased thermotolerance induced by accumulation of glycinebetaine in vivo was associated with the enhancement of the repair of PSII from heat-enhanced photo inhibition, which might be due to less accumulation of reactive oxygen species in transgenic plants.

Carotenoids, a class of natural pigments found in all photosynthetic organisms, are involved in a variety of physiological processes, including coloration, photoprotection, biosynthesis of abscisic acid (ABA) and chloroplast biogenesis. Although carotenoid biosynthesis has been well studied biochemically, the genetic basis of the pathway is not well understood. Here, we report the characterization of two allelic Arabidopsis mutants, spontaneous cell death1-1 (spc1-1) and spc1-2. The weak allele spc1-1 mutant showed characteristics of bleached leaves, accumulation of superoxide and mosaic cell death. The strong mutant allele spc1-2 caused a complete arrest of plant growth and development shortly after germination, leading to a seedling-lethal phenotype. Genetic and molecular analyses indicated that SPC1 encodes a putative z-carotene desaturase (ZDS) in the carotenoid biosynthesis pathway. Analysis of carotenoids revealed that several major carotenoid compounds downstream of SPC1/ZDS were substantially reduced in spc1-1, suggesting that SPC1 is a functional ZDS. Consistent with the downregulated expression of CAO and PORB, the chlorophyll content was decreased in spc1-1 plants. In addition, expression of Lhcb1.1, Lhcb1.4 and RbcS was absent in spc1-2, suggesting the possible involvement of carotenoids in the plastid-to-nucleus retrograde signaling. The spc1-1 mutant also displays an ABA-deficient phenotype that can be partially rescued by the externally supplied phytohormone. These results suggest that SPC1/ZDS is essential for biosynthesis of carotenoids and plays a crucial role in plant growth and development.

To elucidate the molecular mechanism of photosystem II (PSII) assembly, we characterized the low psii accumulation2 (lpa2) mutant of Arabidopsis thaliana, which is defective in the accumulation of PSII supercomplexes. The levels and processing patterns of the RNAs encoding the PSII subunits are unaltered in the mutant. In vivo protein-labeling experiments showed that the synthesis of CP43 (for chlorophyll a binding protein) was greatly reduced, but CP47, D1, and D2 were synthesized at normal rates in the lpa2-1 mutant. The newly synthesized CP43 was rapidly degraded in lpa2-1, and the turnover rates of D1 and D2 were higher in lpa2-1 than in wild-type plants. The newly synthesized PSII proteins were assembled into PSII complexes, but the assembly of PSII was less efficient in the mutant than in wild-type plants. LPA2 encodes an intrinsic thylakoid membrane protein, which is not an integral subunit of PSII. Yeast two-hybrid assays indicated that LPA2 interacts with the PSII core protein CP43 but not with the PSII reaction center proteins D1 and D2. Moreover, direct interactions of LPA2 with Albino3 (Alb3), which is involved in thylakoid membrane biogenesis and cell division, were also detected. Thus, the results suggest that LPA2, which appears to form a complex with Alb3, is involved in assisting CP43 assembly within PSII.

Psb27 has been identified as a lumenal protein associated with photosystem II (PSII). To gain insight into the function of Psb27, we isolated a mutant Arabidopsis plant with a loss of psb27 function. The quantity of PSII complexes and electron transfer within PSII remained largely unaffected in the psb27 mutant. Our results also showed that under high-light-illumination, PSII activity and the content of the PSII reaction center protein D1 decreased more significantly in the psb27 mutant than in wild-type (WT) plant. Treatment of leaves with a chloroplast protein synthesis inhibitor resulted in similar light-induced PSII inactivation levels and D1 protein degradation rates in the WT and psb27 mutant plants. Recovery of PSII activity after photoinhibition was delayed in the psb27 mutant, suggesting that Psb27 is required for efficient recovery of the photodamaged PSII complex. Overall, these results demonstrated that Psb27 in Arabidopsis is not essential for oxygenic photosynthesis and PSII formation. Instead, our results provide evidence for the involvement of this lumenal protein in the recovery process of PSII.