[Show abstract][Hide abstract] ABSTRACT: The chloroplast ribosome is a large and dynamic ribonucleoprotein machine that is composed of the 30S and 50S subunits. Although the components of the chloroplast ribosome have been identified in the last decade, the molecular mechanisms driving chloroplast ribosome biogenesis remain largely elusive. Here, we show that RNA helicase 22 (RH22), a putative DEAD RNA helicase, is involved in chloroplast ribosome assembly in Arabidopsis (Arabidopsis thaliana). A loss of RH22 was lethal, whereas a knockdown of RH22 expression resulted in virescent seedlings with clear defects in chloroplast ribosomal RNA (rRNA) accumulation. The precursors of 23S and 4.5S, but not 16S, rRNA accumulated in rh22 mutants. Further analysis showed that RH22 was associated with the precursors of 50S ribosomal subunits. These results suggest that RH22 may function in the assembly of 50S ribosomal subunits in chloroplasts. In addition, RH22 interacted with the 50S ribosomal protein RPL24 through yeast two-hybrid and pull-down assays, and it was also bound to a small 23S rRNA fragment encompassing RPL24-binding sites. This action of RH22 may be similar to, but distinct from, that of SrmB, a DEAD RNA helicase that is involved in the ribosomal assembly in Escherichia coli, which suggests that DEAD RNA helicases and rRNA structures may have coevolved with respect to ribosomal assembly and function.
[Show abstract][Hide abstract] ABSTRACT: Higher plants require chloroplasts for essential functions in photosynthesis and other important physiological processes, such as sugar, lipid and amino-acid biosynthesis. Most chloroplast proteins are nuclear-encoded proteins that are synthesized in the cytosol as precursors, and imported into chloroplasts by protein translocases in the outer and inner chloroplast envelope. The imported chloroplast proteins are then translocated into or across the thylakoid membrane by four distinct pathways. However, the mechanisms by which the imported nuclear-encoded proteins are delivered to these pathways remain largely unknown. Here we show that an Arabidopsis ankyrin protein, LTD (mutation of which causes the light-harvesting chlorophyll-binding protein translocation defect), is localized in the chloroplast and using yeast two-hybrid screens demonstrate that LTD interacts with both proteins from the signal recognition particle (SRP) pathway and the inner chloroplast envelope. Our study shows that LTD is essential for the import of light-harvesting chlorophyll-binding proteins and subsequent routing of these proteins to the chloroplast SRP-dependent pathway.
[Show abstract][Hide abstract] ABSTRACT: A photosensitive (phs1) mutant of Arabidopsis thaliana was isolated and characterized. The PHS1 gene was cloned using a map-based approach. The gene was found to encode a protein containing a deaminase-reductase domain that is involved in the riboflavin pathway. The phenotype and growth of the phs1 mutant were comparable to that of the wild-type when the plants were grown under low light conditions. When the light intensity was increased, the mutant was characterized by stunted growth and bleached leaves as well as a decrease in FNR activity. The NADPH levels declined, whereas the NADP(+) levels increased, leading to a decrease in the NADPH/NADP(+) ratio. The mutant suffered from severe photooxidative damage with an increase in antioxidant enzyme activity and a drastic reduction in the levels of chlorophyll and photosynthetic proteins. Supplementing the mutant with exogenous FAD rescued the photosensitive phenotype, even under increasing light intensity. The riboflavin pathway therefore plays an important role in protecting plants from photooxidative damage.
[Show abstract][Hide abstract] ABSTRACT: The pentatricopeptide-repeat (PPR) protein DELAYED GREENING 1 (DG1) has been shown to be involved in the regulation of early chloroplast development and chloroplast gene expression in Arabidopsis. To gain insight into the mode of DG1 action, we used a yeast two-hybrid screening approach and identified a partner, SIG6, which is a chloroplast sigma factor responsible for the transcription of plastid-encoded RNA polymerase (PEP)-dependent chloroplast genes in cotyledons. Further analysis showed that the C-terminal region of DG1 and the N-terminal region of SIG6 are responsible for such interactions. High-level expression of a truncated C-terminal DG1 in wild-type Arabidopsis caused a dominant-negative phenotype. The sig6 dg1 double mutant displayed a more severe chlorotic phenotype, and the PEP-dependent chloroplast gene transcripts were greatly reduced compared with transcript levels in the single mutants. Overexpression of SIG6 rescued the chlorophyll deficiency in dg1 cotyledons but not in young leaves. In addition, increased SIG6 promoted PEP-dependent chloroplast gene transcript accumulation in the dg1 mutant background. These results suggest that the interaction of DG1 and SIG6 is functionally significant in the regulation of PEP-dependent chloroplast gene transcription in Arabidopsis cotyledons.
The Plant Journal 10/2010; 64(1):14-25. DOI:10.1111/j.1365-313X.2010.04304.x · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To gain insight into the functions of the nuclear-encoded factors involved in chloroplast development, we characterized the high chlorophyll fluorescence and pale green mutant 108-1 (designated as hfp108-1) of Arabidopsis thaliana. Map-based cloning revealed that the mutant contains a tandem repeat of part of the sequence (including 116 nucleotides from 631 to 746 bp downstream of the ATG) of At3g63190, which encodes a chloroplast ribosome recycling factor homologue and was named AtcpRRF. The chloroplasts of hfp108-1 plants contain few internal thylakoid membranes and are severely defective in the accumulation of chloroplast-encoded proteins. In vivo labeling experiments showed a drastic decrease in the synthesis of the chloroplast-encoded proteins, which may be attributed primarily to reduced translation of the corresponding mRNA molecules. The level of the HFP108 transcript was greatly reduced in hfp108-1, so hfp108-1 showed a weak phenotype, and null alleles of HFP108 (hfp108-2) were embryonic lethal. Observations with cleared seeds in the same silique showed that homozygous hfp108-2 seeds were blocked at the heart stage and did not develop further. Thus, these results suggest that AtcpRRF is essential for embryogenesis and chloroplast biogenesis.
[Show abstract][Hide abstract] ABSTRACT: Photosystem II (PSII) is a multisubunit membrane protein complex that is assembled in a sequence of steps. However, the molecular mechanisms responsible for the assembly of the individual subunits into functional PSII complexes are still largely unknown. Here, we report the identification of a chloroplast protein, Low PSII Accumulation3 (LPA3), which is required for the assembly of the CP43 subunit in PSII complexes in Arabidopsis (Arabidopsis thaliana). LPA3 interacts with LPA2, a previously identified PSII CP43 assembly factor, and a double mutation of LPA2 and LPA3 is more deleterious for assembly than either single mutation, resulting in a seedling-lethal phenotype. Our results indicate that LPA3 and LPA2 have overlapping functions in assisting CP43 assembly and that cooperation between LPA2 and LPA3 is essential for PSII assembly. In addition, we provide evidence that LPA2 and LPA3 interact with Albino3 (Alb3), which is essential for thylakoid protein biogenesis. Thus, the function of Alb3 in some PSII assembly processes is probably mediated through interactions with LPA2 and LPA3.
[Show abstract][Hide abstract] ABSTRACT: Light is the ultimate source of energy for photosynthesis; however, excessive light leads to photooxidative damage and hence reduced photosynthetic efficiency, especially when combined with other abiotic stresses. Although the photosystem II (PSII) reaction center D1 protein is the primary target of photooxidative damage, other PSII core proteins are also damaged and degraded. However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases. Here, we show that Deg7 is peripherally associated with the stromal side of the thylakoid membranes and that Deg7 interacts directly with PSII. Our results show that Deg7 is involved in the primary cleavage of photodamaged D1, D2, CP47, and CP43 and that this activity is essential for its function in PSII repair. The double mutants deg5 deg7 and deg8 deg7 showed no obvious phenotypic differences under normal growth conditions, but additive effects were observed under high light. These results suggest that Deg proteases on both the stromal and luminal sides of the thylakoid membranes are important for the efficient PSII repair in Arabidopsis (Arabidopsis thaliana).
[Show abstract][Hide abstract] ABSTRACT: To gain insight into the molecular mechanism of RNA editing, we have characterized the low psii accumulation66 (lpa66) Arabidopsis (Arabidopsis thaliana) mutant, which displays a high chlorophyll fluorescence phenotype. Its perturbed chlorophyll fluorescence is reflected in reduced levels of photosystem II (PSII) proteins. In vivo protein labeling showed that synthesis rates of the PSII reaction center protein D1/D2 were lower, and turnover rates of PSII core proteins higher, than in wild-type counterparts. The assembly of newly synthesized proteins into PSII occurs in the lpa66 mutant but with reduced efficiency compared with the wild type. LPA66 encodes a chloroplast protein of the pentatricopeptide repeat family. In lpa66 mutants, editing of psbF that converts serine to phenylalanine is specifically impaired. Thus, LPA66 is specifically required for editing the psbF transcripts in Arabidopsis, and the amino acid alternation due to lack of editing strongly affects the efficiency of the assembly of PSII complexes.
[Show abstract][Hide abstract] ABSTRACT: The phytohormone abscisic acid (ABA) is involved in the adaptive stress response and regulates expression of many stress-responsive genes, including some transcriptional factors. A bZIP transcription factor, OsABI5, was isolated from the panicle of Oryza sativa L. Expression of the OsABI5 gene was induced by abscisic acid (ABA) and high salinity, and down-regulated by drought and cold (4 degrees C) in seedlings. The OsABI5 protein was localized in the nucleus and has trans-activation activity. The N-terminal of the protein is necessary for its activity. OsABI5 could bind to a G-box element and trans-activate reporter gene expression. Complementation analysis revealed that the expression of OsABI5 driven by the 35S promoter could rescue ABA-insensitivity of abi5-1 during seed germination and result in hypersensitivity to ABA. Over-expression of OsABI5 in rice conferred high sensitivity to salt stress. Repression of OsABI5 promoted stress tolerance and resulted in low fertility of rice. These results suggested that OsABI5 could regulate the adaptive stress response and plant fertility of rice as a transcription factor.
[Show abstract][Hide abstract] ABSTRACT: Alternative splicing allows many gene products to alter their biological functions. A bZIP-type transcription factor, OsABI5, undergoes alternative splicing. Two OsABI5 splicing variants were identified, designated OsABI5-1, and OsABI5-2 and their different expression patterns in tissues were analyzed. Despite a completely identical functional domain, OsABI5-2 could specifically bind to G-box element, but OsABI5-1 could not; the transactivation activity of OsABI5-1 was higher than that of OsABI5-2; the interaction strength of OsABI5-2 and OsVP1 was stronger than that of OsABI5-1 and OsVP1; indicating a different function in the regulation of downstream target genes. Complementation tests and ABA (abscisic acid) hypersensitivity of Arabidopsis transgenic lines revealed the redundant function of OsABI5 splicing variants in ABA signaling. The interaction between OsABI5-1 and OsABI5-2 was also confirmed. These results suggest that OsABI5 variants may have overlapping and distinct functions to fine tune gene expression in ABA signaling as transcription factors together with OsVP1.
Biochemical and Biophysical Research Communications 09/2007; 360(2):307-13. DOI:10.1016/j.bbrc.2007.05.226 · 2.28 Impact Factor