Wei Chi

LOW PSII ACCUMULATION1 Is Involved in Efficient Assembly of Photosystem II in Arabidopsis thaliana

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.

A chloroplast envelope-bound PHD transcription factor mediates chloroplast signals to the nucleus

Chloroplast development, maintenance and function depend on the coordinated expression of chloroplast and nuclear genes. The retrograde chloroplast signals are essential in coordinating nuclear gene expression. Although the sources of signals in chloroplasts have been identified and the associated transcription factors in the nucleus extensively studied, the molecular mechanism that relays chloroplast signals to the nucleus remains a mystery. Here we show that PTM, a chloroplast envelope-bound plant homeodomain (PHD) transcription factor with transmembrane domains, functions in multiple retrograde signal pathways. The proteolytic cleavage of PTM occurs in response to retrograde signals and amino-terminal PTM accumulates in the nucleus, where it activates ABI4 transcription in a PHD-dependent manner associated with histone modifications. These results provide a molecular basis for the critical function of PTM in retrograde chloroplast signaling and shed new light on the mechanism whereby chloroplast signals are transmitted to the nucleus through the cytosol.

Formation of DEG5 and DEG8 Complexes and Their Involvement in the Degradation of Photodamaged Photosystem II Reaction Center D1 Protein in Arabidopsis

The widely distributed DEGP proteases play important roles in the degradation of damaged and misfolded proteins. Arabidopsis thaliana contains 16 DEGP-like proteases, four of which are located in the chloroplast. Here, we show that DEG5 and DEG8 form a hexamer in the thylakoid lumen and that recombinant DEG8 is proteolytically active toward both a model substrate (β-casein) and photodamaged D1 protein of photosystem II (PSII), producing 16-kD N-terminal and 18-kD C-terminal fragments. Inactivation of DEG5 and DEG8 resulted in increased sensitivity to photoinhibition. Turnover of newly synthesized D1 protein in the deg5 deg8 double mutant was impaired, and the degradation of D1 in the presence of the chloroplast protein synthesis inhibitor lincomycin under high-light treatment was slowed in the mutants. Thus, DEG5 and DEG8 are important for efficient turnover of the D1 protein and for protection against photoinhibition in vivo. The deg5 deg8 double mutant showed increased photosensitivity and reduced rates of D1 degradation compared with single mutants of deg5 and deg8. A 16-kD N-terminal degradation fragment of the D1 protein was detected in wild-type plants but not in the deg5 deg8 mutant following in vivo photoinhibition. Therefore, our results suggest that DEG5 and DEG8 have a synergistic function in the primary cleavage of the CD loop of the PSII reaction center protein D1.

Intracellular Signaling from Plastid to Nucleus

Intracellular signaling from plastids to the nucleus, called retrograde signaling, coordinates the expression of nuclear and plastid genes and is essential for plastid biogenesis and for maintaining plastid function at optimal levels. Recent identification of several components involved in plastid retrograde generation, transmission, and control of nuclear gene expression has provided significant insight into the regulatory network of plastid retrograde signaling. Here, we review the current knowledge of multiple plastid retrograde signaling pathways, which are derived from distinct sources, and of possible plastid signaling molecules. We describe the retrograde signaling-dependent regulation of nuclear gene expression, which involves multilayered transcriptional control, as well as the transcription factors involved. We also summarize recent advances in the identification of key components mediating signal transduction from plastids to the nucleus.

The Pentratricopeptide Repeat Protein DELAYED GREENING1 Is Involved in the Regulation of Early Chloroplast Development and Chloroplast Gene Expression in Arabidopsis

An Arabidopsis (Arabidopsis thaliana) mutant that exhibited a delayed greening phenotype (dg1) was isolated from a population of activation-tagged Arabidopsis lines. Young, inner leaves of dg1 mutants were initially very pale, but gradually greened and mature outer leaves, more than 3 weeks old, appeared similar to those of wild-type plants. Sequence and transcription analyses showed that DG1 encodes a chloroplast protein consisting of eight pentratricopeptide repeat domains and that its expression depends on both light and developmental status. In addition, analysis of the transcript profiles of chloroplast genes revealed that plastid-encoded polymerase-dependent transcript levels were markedly reduced, while nucleus-encoded polymerase-dependent transcript levels were increased, in dg1 mutants. Thus, DG1 is probably involved in the regulation of plastid-encoded polymerase-dependent chloroplast gene expression during early stages of chloroplast development.

The Function of RH22, a DEAD RNA Helicase, in the Biogenesis of the 50S Ribosomal Subunits of Arabidopsis Chloroplasts

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.

LPA19, a Psb27 homolog in Arabidopsis thaliana, facilitates D1 protein precursor processing during PSII biogenesis.

The biogenesis and assembly of photosystem II (PSII) are mainly regulated by the nuclear-encoded factors. To further identify the novel components involved in PSII biogenesis, we isolated and characterized a high chlorophyll fluorescence low psii accumulation19 (lpa19) mutant, which is defective in PSII biogenesis. LPA19 encodes a Psb27 homolog (At1g05385). Interestingly, another Psb27 homolog (At1g03600) in Arabidopsis was revealed to be required for the efficient repair of photodamaged PSII. These results suggest that the Psb27 homologs play distinct functions in PSII biogenesis and repair in Arabidopsis. Chloroplast protein labeling assays showed that the C-terminal processing of D1 in the lpa19 mutant was impaired. Protein overlay assays provided evidence that LPA19 interacts with D1, and coimmunoprecipitation analysis demonstrated that LPA19 interacts with mature D1 (mD1) and precursor D1 (pD1). Moreover, LPA19 protein was shown to specifically interact with the soluble C terminus present in the precursor and mature D1 through yeast two-hybrid analyses. Thus, these studies suggest that LPA19 is involved in facilitating the D1 precursor protein processing in Arabidopsis.

Interaction of the pentatricopeptide‐repeat protein DELAYED GREENING 1 with sigma factor SIG6 in the regulation of chloroplast gene expression in Arabidopsis cotyledons

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 roles of chloroplast proteases in the biogenesis and maintenance of photosystem II.

Photosystem II (PSII) catalyzes one of the key reactions of photosynthesis, the light-driven conversion of water into oxygen. Although the structure and function of PSII have been well documented, our understanding of the biogenesis and maintenance of PSII protein complexes is still limited. A considerable number of auxiliary and regulatory proteins have been identified to be involved in the regulation of this process. The carboxy-terminal processing protease CtpA, the serine-type protease DegP and the ATP-dependent thylakoid-bound metalloprotease FtsH are critical for the biogenesis and maintenance of PSII. Here, we summarize and discuss the structural and functional aspects of these chloroplast proteases in these processes. This article is part of a Special Issue entitled: SI: Photosystem II.

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway.

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.