Rubisco Accumulation Factor1-like (RAFL) interacts with RAF1 to mediate Rubisco assembly in Arabidopsis.

Rubisco catalysis a rate-limiting step in photosynthesis. It is a complex of eight large (RbcL) and eight small (RbcS) subunits. The biogenesis of Rubisco requires assembly chaperones. One of the key Rubisco assembly chaperones, Rubisco accumulation factor1 (RAF1), assembled as a dimer, acts downstream of chaperonin-assisted RbcL folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes. In maize, lacking RAF1 causes Rubisco deficient and seedling lethal. A RAF1 homologue, RAF1-like (RAFL), has been detected in Arabidopsis. We found RAFL shares 61.98 % sequence similarity with RAF1. They have similar conserved domains, predicted 3D structures and gene expression pattern. Phylogenetic tree analysis showed that RAFL and RAF1 only present in analyzed dicots, while only one copy of RAF presented in monocots, mosses and green algae. Combined analysis by three different protein-protein interaction methods showed that RAFL interacts with RAF1 both in vivo and in vitro. Taken together, we conclude that RAFL and RAF1 are close paralogous genes, and they can form heterodimer and/or homodimers to mediate Rubisco assembly in Arabidopsis.

Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism.

Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START-dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid- and phosphate (Pi) starvation-related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression.

The photosystem-II repair cycle: updates and open questions.

MAIN CONCLUSION: The photosystem-II (PSII) repair cycle is essential for the maintenance of photosynthesis in plants. A number of novel findings have illuminated the regulatory mechanisms of the PSII repair cycle. Photosystem II (PSII) is a large pigment-protein complex embedded in the thylakoid membrane. It plays a vital role in photosynthesis by absorbing light energy, splitting water, releasing molecular oxygen, and transferring electrons for plastoquinone reduction. However, PSII, especially the PsbA (D1) core subunit, is highly susceptible to oxidative damage. To prevent irreversible damage, plants have developed a repair cycle. The main objective of the PSII repair cycle is the degradation of photodamaged D1 and insertion of newly synthesized D1 into the PSII complex. While many factors are known to be involved in PSII repair, the exact mechanism is still under investigation. In this review, we discuss the primary steps of PSII repair, focusing on the proteolytic degradation of photodamaged D1 and the factors involved.

Research progress on the biosynthesis and delivery of iron-sulfur clusters in the plastid.

Iron-sulfur (Fe-S) clusters are ancient protein cofactors ubiquitously exist in organisms. They are involved in many important life processes. Plastids are semi-autonomous organelles with a double membrane and it is believed to originate from a cyanobacterial endosymbiont. By learning form the research in cyanobacteria, a Fe-S cluster biosynthesis and delivery pathway has been proposed and partly demonstrated in plastids, including iron uptake, sulfur mobilization, Fe-S cluster assembly and delivery. Fe-S clusters are essential for the downstream Fe-S proteins to perform their normal biological functions. Because of the importance of Fe-S proteins in plastid, researchers have made a lot of research progress on this pathway in recent years. This review summarizes the detail research progress made in recent years. In addition, the scientific problems remained in this pathway are also discussed.

Effect of the Enhanced Production of Chlorophyll b on the Light Acclimation of Tomato.

Tomato (Solanum lycopersicum Mill.) is one of the widely cultured vegetables under protected cultivation, in which insufficient light is one of the major factors that limit its growth, yield, and quality. Chlorophyll b (Chl b) is exclusively present in the light-harvesting complex (LHC) of photosystems, while its synthesis is strictly regulated in response to light conditions in order to control the antenna size. Chlorophyllide a oxygenase (CAO) is the sole enzyme that converts Chl a to Chl b for Chl b biosynthesis. Previous studies have shown that overexpressing CAO without the regulating domain (A domain) in Arabidopsis overproduced Chl b. However, the growth characteristics of the Chl b overproduced plants under different light environmental conditions are not well studied. Considering tomatoes are light-loving plants and sensitive to low light stress, this study aimed to uncover the growth character of tomatoes with enhanced production of Chl b. The A domain deleted Arabidopsis CAO fused with the FLAG tag (BCF) was overexpressed in tomatoes. The BCF overexpressed plants accumulated a significantly higher Chl b content, resulting in a significantly lower Chl a/b ratio than WT. Additionally, BCF plants possessed a lower maximal photochemical efficiency of photosystem II (Fv/Fm) and anthocyanin content than WT plants. The growth rate of BCF plants was significantly faster than WT plants under low-light (LL) conditions with light intensity at 50-70 µmol photons m-2 s-1, while BCF plants grew slower than WT plants under high-light (HL) conditions. Our results revealed that Chl b overproduced tomato plants could better adapt to LL conditions by absorbing more light for photosynthesis but adapt poorly to excess light conditions by accumulating more ROS and fewer anthocyanins. Enhanced production of Chl b is able to improve the growth rate of tomatoes that are grown under LL conditions, indicating the prospect of employing Chl b overproduced light-loving crops and ornamental plants for protected or indoor cultivation.

Updated Progress on Group II Intron Splicing Factors in Plant Chloroplasts.

Group II introns are large catalytic RNAs (ribozymes) in the bacteria and organelle genomes of several lower eukaryotes. Many critical photosynthesis-related genes in the plant chloroplast genome also contain group II introns, and their splicing is critical for chloroplast biogenesis and photosynthesis processes. The structure of chloroplast group II introns was altered during evolution, resulting in the loss of intron self-splicing. Therefore, the assistance of protein factors was required for their splicing processes. As an increasing number of studies focus on the mechanism of chloroplast intron splicing; many new nuclear-encoded splicing factors that are involved in the chloroplast intron splicing process have been reported. This report reviewed the research progress of the updated splicing factors found to be involved in the splicing of chloroplast group II introns. We discuss the main problems that remain in this research field and suggest future research directions.

DJC78 is a cochaperone that interacts with cpHsc70-1 in the chloroplasts.

Heat shock proteins 70 (HSP70s) could cooperate with structurally diverse HSP40s (J proteins) to generate diverse chaperone networks in various cellular compartments, performing multiple housekeeping and stress-related functions in the organisms. There are two kinds of chloroplast heat shock protein 70 (cpHsc70-1, cpHsc70-2) and multiple J proteins in the Arabidopsis chloroplasts, while the interaction between cpHsc70s and J proteins and the function of most J proteins are largely unknown. In the present study, we found that AtDJC78 interacts with cpHsc70-1 through its C terminal, according to the results of yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). Bioinformatics analysis showed that DJC78 is one of the widespread and highly conserved J proteins in plants, AtDJC78 could be transported into chloroplasts, and the expression of AtDJC78 was significantly up-regulated under heat stress. Furthermore, we found that AtDJC78 may be associated with regulating hydrogen peroxide levels under heat stress in plants. These findings suggest that AtDJC78 is a new cochaperone interacting with cpHsc70-1 in the chloroplasts.

Progress on Understanding Transcriptional Regulation of Chloroplast Development in Fleshy Fruit.

Edible fleshy fruits are important food sources in the human diet. Their yield and nutritional quality have long been considered as breeding targets for improvement. Various developing fleshy fruits with functional chloroplasts are capable of photosynthesis and contribute to fruit photosynthate, leading to the accumulation of metabolites associated with nutritional quality in ripe fruit. Although tomato high-pigment mutants with dark-green fruits have been isolated for more than 100 years, our understanding of the mechanism of chloroplast development in fleshy fruit remain poor. During the past few years, several transcription factors that regulate chloroplast development in fleshy fruit were identified through map-based cloning. In addition, substantial progress has been made in elucidating the mechanisms that how these transcription factors regulate chloroplast development. This review provides a summary and update on this progress, with a framework for further investigations of the multifaceted and hierarchical regulation of chloroplast development in fleshy fruit.

The SUFBC2 D complex is required for the biogenesis of all major classes of plastid Fe-S proteins.

Iron-sulfur (Fe-S) proteins play crucial roles in plastids, participating in photosynthesis and other metabolic pathways. Fe-S clusters are thought to be assembled on a scaffold complex composed of SUFB, SUFC and SUFD proteins. However, several additional proteins provide putative scaffold functions in plastids, and, therefore, the contribution of SUFB, C and D proteins to overall Fe-S assembly still remains unclear. In order to gain insights regarding Fe-S cluster biosynthesis in plastids, we analyzed the complex composed of SUFB, C and D in Arabidopsis by blue native-polyacrylamide gel electrophoresis. Using this approach, a major complex of 170 kDa containing all subunits was detected, indicating that these proteins constitute a SUFBC2 D complex similar to their well characterized bacterial counterparts. The functional effects of SUFB, SUFC or SUFD depletion were analyzed using an inducible RNAi silencing system to specifically target the aforementioned components; resulting in a decrease of various plastidic Fe-S proteins including the PsaA/B and PsaC subunits of photosystem I, ferredoxin and glutamine oxoglutarate aminotransferase. In contrast, the knockout of potential Fe-S scaffold proteins, NFU2 and HCF101, resulted in a specific decrease in the PsaA/B and PsaC levels. These results indicate that the functions of SUFB, SUFC and SUFD for Fe-S cluster biosynthesis cannot be replaced by other scaffold proteins and that SUFBC2 D, NFU2 and HCF101 are involved in the same pathway for the biogenesis of PSI. Taken together, our results provide in vivo evidence supporting the hypothesis that SUFBC2 D is the major, and possibly sole scaffold in plastids.

The iron-sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis, but not phytochrome signaling.

Proteins that contain iron-sulfur (Fe-S) clusters play pivotal roles in various metabolic processes such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of Fe-S clusters in plants, the SUFB subunit of the SUFBCD complex appears to be unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome-mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNA interference (RNAi) lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate Mg-protoporphyrin IX monomethylester (Mg-proto MME), consistent with the impairment of Mg-proto MME cyclase activity. Both SUFC- and SUFD-deficient RNAi lines accumulated the same intermediate, suggesting that inhibition of Fe-S cluster synthesis is the primary cause of this impairment. Dark-grown laf6 seedlings also showed an increase in protoporphyrin IX (Proto IX), Mg-proto, Mg-proto MME and 3,8-divinyl protochlorophyllide a (DV-Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far-red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale-green SUFB-deficient phenotype, indicating it is not caused by mutation at the SUFB locus. These results demonstrate that biosynthesis of Fe-S clusters is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation.

Accumulation of the NON-YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in Arabidopsis thaliana.

Chlorophyll a and chlorophyll b are interconverted in the chlorophyll cycle. The initial step in the conversion of chlorophyll b to chlorophyll a is catalyzed by the chlorophyll b reductases NON-YELLOW COLORING 1 (NYC1) and NYC1-like (NOL), which convert chlorophyll b to 7-hydroxymethyl chlorophyll a. This step is also the first stage in the degradation of the light-harvesting chlorophyll a/b protein complex (LHC). In this study, we examined the effect of chlorophyll b on the level of NYC1. NYC1 mRNA and NYC1 protein were in low abundance in green leaves, but their levels increased in response to dark-induced senescence. When the level of chlorophyll b was enhanced by the introduction of a truncated chlorophyllide a oxygenase gene and the leaves were incubated in the dark, the amount of NYC1 was greatly increased compared with that of the wild type; however, the amount of NYC1 mRNA was the same as in the wild type. In contrast, NYC1 did not accumulate in the mutant without chlorophyll b, even though the NYC1 mRNA level was high after incubation in the dark. Quantification of the LHC protein showed no strong correlation between the levels of NYC1 and LHC proteins. However, the level of chlorophyll fluorescence of the dark adapted plant (Fo ) was closely related to the accumulation of NYC1, suggesting that the NYC1 level is related to the energetically uncoupled LHC. These results and previous reports on the degradation of chlorophyllide a oxygenase suggest that the a feedforward and feedback network is included in chlorophyll cycle.

Reexamination of chlorophyllase function implies its involvement in defense against chewing herbivores.

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.

Research Progress in J-Proteins in the Chloroplast.

The J-proteins, also called DNAJ-proteins or heat shock protein 40 (HSP40), are one of the famous molecular chaperones. J-proteins, HSP70s and other chaperones work together as constitute ubiquitous types of molecular chaperone complex, which function in a wide variety of physiological processes. J-proteins are widely distributed in major cellular compartments. In the chloroplast of higher plants, around 18 J-proteins and multiple J-like proteins are present; however, the functions of most of them remain unclear. During the last few years, important progress has been made in the research on their roles in plants. There is increasing evidence that the chloroplast J-proteins play essential roles in chloroplast development, photosynthesis, seed germination and stress response. Here, we summarize recent research advances on the roles of J-proteins in the chloroplast, and discuss the open questions that remain in this field.

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants.

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Chlorophyllase, a Common Plant Hydrolase Enzyme with a Long History, Is Still a Puzzle.

Chlorophyllase (Chlase, CLH) is one of the earliest discovered enzymes present in plants and green algae. It was long considered to be the first enzyme involved in chlorophyll (Chl) degradation, while strong evidence showed that it is not involved in Chl breakdown during leaf senescence. On the other hand, it is possible that CLH is involved in Chl breakdown during fruit ripening. Recently, it was discovered that Arabidopsis CLH1 is located in developing chloroplasts but not in mature chloroplasts, and it plays a role in protecting young leaves from long-term photodamage by catalysing Chl turnover in the photosystem II (PSII) repair cycle. However, there remain other important questions related to CLH. In this article, we briefly reviewed the research progress on CLH and listed the main unanswered questions related to CLH for further study.

Research Progress in the Interconversion, Turnover and Degradation of Chlorophyll.

Chlorophylls (Chls, Chl a and Chl b) are tetrapyrrole molecules essential for photosynthetic light harvesting and energy transduction in plants. Once formed, Chls are noncovalently bound to photosynthetic proteins on the thylakoid membrane. In contrast, they are dismantled from photosystems in response to environmental changes or developmental processes; thus, they undergo interconversion, turnover, and degradation. In the last twenty years, fruitful research progress has been achieved on these Chl metabolic processes. The discovery of new metabolic pathways has been accompanied by the identification of enzymes associated with biochemical steps. This article reviews recent progress in the analysis of the Chl cycle, turnover and degradation pathways and the involved enzymes. In addition, open questions regarding these pathways that require further investigation are also suggested.

Progress in Research on the Mechanisms Underlying Chloroplast-Involved Heat Tolerance in Plants.

Global warming is a serious challenge plant production has to face. Heat stress not only affects plant growth and development but also reduces crop yield and quality. Studying the response mechanisms of plants to heat stress will help humans use these mechanisms to improve the heat tolerance of plants, thereby reducing the harm of global warming to plant production. Research on plant heat tolerance has gradually become a hotspot in plant molecular biology research in recent years. In view of the special role of chloroplasts in the response to heat stress in plants, this review is focusing on three perspectives related to chloroplasts and their function in the response of heat stress in plants: the role of chloroplasts in sensing high temperatures, the transmission of heat signals, and the improvement of heat tolerance in plants. We also present our views on the future direction of research on chloroplast related heat tolerance in plants.

Overexpressing 7-Hydroxymethyl Chlorophyll a Reductase Alleviates Non-Programmed Cell Death during Dark-Induced Senescence in Intact Arabidopsis Plants.

Leaf senescence, the last stage of leaf development, is a well-regulated and complex process for investigation. For simplification, dark-induced leaf senescence has frequently been used to mimic the natural senescence of leaves because many typical senescence symptoms, such as chlorophyll (Chl) and protein degradation, also occur under darkness. In this study, we compared the phenotypes of leaf senescence that occurred when detached leaves or intact plants were incubated in darkness to induce senescence. We found that the symptoms of non-programmed cell death (non-PCD) with remaining green coloration occurred more heavily in the senescent leaves of whole plants than in the detached leaves. The pheophorbide a (Pheide a) content was also shown to be much higher in senescent leaves when whole plants were incubated in darkness by analyses of leaf Chl and its metabolic intermediates. In addition, more serious non-PCD occurred and more Pheide a accumulated in senescent leaves during dark incubation if the soil used for plant growth contained more water. Under similar conditions, the non-PCD phenotype was alleviated and the accumulation of Pheide a was reduced by overexpressing 7-hydroxymethyl Chl a (HMChl a) reductase (HCAR). Taken together, we conclude that a high soil water content induced non-PCD by decreasing HCAR activity when whole plants were incubated in darkness to induce senescence; thus, the investigation of the fundamental aspects of biochemistry and the regulation of leaf senescence are affected by using dark-induced leaf senescence.

HCAR Is a Limitation Factor for Chlorophyll Cycle and Chlorophyll b Degradation in Chlorophyll-b-Overproducing Plants.

The chlorophyll (Chl) cycle is the metabolic pathway for Chl a and Chl b inter-conversion. In this pathway, Chl b is synthesized from Chl a by the catalyzing action of chlorophyllide a oxygenase (CAO). In contrast, Chl b is firstly reduced to produce 7-hydroxymethyl Chl (HMChl) a, which is catalyzed by two isozymes of Chl b reductase (CBR), non-yellow coloring 1 (NYC1) and NYC1-like (NOL). Subsequently, HMChl a is reduced to Chl a by HMChl a reductase (HCAR). CAO plays a pivotal role in Chl a/b ratio regulation and plants over-accumulate Chl b in CAO-overexpressing plants. NYC1 is more accumulated in Chl-b-overproducing plants, while HCAR is not changed. To investigate the role of HCAR in Chl cycle regulation, the Chl metabolites of Chl-b-overproducing plants were analyzed. The results showed that HMChl a accumulated in these plants, and it decreased and the Chl a/b ratio increased by overexpressing HCAR, implying HCAR is insufficient for Chl cycle in Chl-b-overproducing plants. Furthermore, during dark-induced senescence, the non-programmed cell death symptoms (leaves dehydrated with green color retained) of Chl-b-overproducing plants were obviously alleviated, and the content of HM pheophorbide (HMPheide) a and Pheide b were sharply decreased by overexpressing HCAR. These results imply that HCAR is also insufficient for Chl degradation in Chl-b-overproducing plants during senescence, thus causing the accumulation of Chl metabolites and non-programmed cell death of leaves. With these results taken together, we conclude that HCAR is not well regulated and it is a limiting factor for Chl cycle and Chl b degradation in Chl-b-overproducing plants.

Subcellular localization of chlorophyllase2 reveals it is not involved in chlorophyll degradation during senescence in Arabidopsis thaliana.

Chlorophyllase (CLH), which catalyzes the release of the phytol chain from chlorophyll (Chl), has been long considered to catalyze the first step of Chl degradation. Arabidopsis contains two isoforms of CLH (CLH1 and CLH2), and CLH1 was previously demonstrated to be localized in tonoplast and endoplasmic reticulum, and not be involved in Chl degradation. In contrast, CLH2 possesses a predicted signal-peptide for chloroplast localization, and phylogenetic analysis of CLHs in Arabidopsis and other species also indicate that CLH2 forms a different clade than CLH1. Therefore, the possibility remains that CLH2 is involved in the breakdown of Chl. In the current study, clh mutants lacking CLH2 or both CLH isoforms were analyzed after the induction of senescence. Results indicated that the clh knockout lines were still able to degrade Chl at the same rate as wild-type plants. Transgenic Arabidopsis plants were generated that constitutively expressed either CLH2 or CLH2 fused to a yellow fluorescent protein (YFP). Observations made using confocal microscopy indicated that CLH2-YFP was located external to chloroplasts. Additionally, in overexpression plants, CLH2 was enriched in tonoplast and endoplasmic reticulum fractions following membrane fractionation. Based on the collective data, we conclude that CLH2 is not involved in Chl breakdown during senescence in Arabidopsis.