Loss of Plastoglobule Kinases ABC1K1 and ABC1K3 Causes Conditional Degreening,Modified Prenyl-Lipids,and Recruitment of the Jasmonic Acid Pathway

Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen–derived carotenoid β-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyl-lipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wild-type PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.     

Biogenesis of photosynthetic complexes in the chloroplast of Chlamydomonas reinhardtii requires ARSA1, a homolog of prokaryotic arsenite transporter and eukaryotic TRC40 for guided entry of tail‐anchored proteins

as1, for antenna size mutant 1, was obtained by insertion mutagenesis of the unicellular green alga Chlamydomonas reinhardtii. This strain has a low chlorophyll content, 8% with respect to the wild type, and displays a general reduction in thylakoid polypeptides. The mutant was found to carry an insertion into a homologous gene, prokaryotic arsenite transporter (ARSA), whose yeast and mammal counterparts were found to be involved in the targeting of tail‐anchored (TA) proteins to cytosol‐exposed membranes, essential for several cellular functions. Here we present the characterization in a photosynthetic organism of an insertion mutant in an ARSA‐homolog gene. The ARSA1 protein was found to be localized in the cytosol, and yet its absence in as1 leads to a small chloroplast and a strongly decreased chlorophyll content per cell. ARSA1 appears to be required for optimal biogenesis of photosynthetic complexes because of its involvement in the accumulation of TOC34, an essential component of the outer chloroplast membrane translocon (TOC) complex, which, in turn, catalyzes the import of nucleus‐encoded precursor polypeptides into the chloroplast. Remarkably, the effect of the mutation appears to be restricted to biogenesis of chlorophyll‐binding polypeptides and is not compensated by the other ARSA homolog encoded by the C. reinhardtii genome, implying a non‐redundant function.     

ERIL1, the plant homologue of ERI‐1, is involved in the processing of chloroplastic rRNAs

Proteins belonging to the enhancer of RNA interference‐1 subfamily of 3′–5′ exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE‐1‐LIKE‐1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3′–5′ exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus‐encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.     

An allele of ZmPORB2 encoding a protochlorophyllide oxidoreductase promotes tocopherol accumulation in both leaves and kernels of maize

Phytol is one of the key precursors for tocopherol synthesis in plants, however, the underlying mechanisms concerning the accumulation of tocopherol remain poorly understood. In this study, qVE5, a major QTL affecting tocopherol accumulation in maize kernels was identified via a positional cloning approach. qVE5 encodes a protochlorophyllide oxidoreductase (ZmPORB2), which localizes to the chloroplast. Overexpression of ZmPORB2 increased tocopherol content in both leaves and kernels. Candidate gene association analysis identified a 5/8‐bp insertion/deletion (InDel058) in the 5′ untranslated region (UTR) as the causal polymorphism in affecting ZmPORB2 expression and being highly associated with tocopherol content. We showed that higher expression of ZmPORB2 correlated with more chlorophyll metabolites in the leaf following pollination. RNA‐sequencing and metabolic analysis in near isogenic lines (NILs) support that ZmPORB2 participates in chlorophyll metabolism enabling the production of phytol, an important precursor of tocopherol. We also found that the tocopherol content in the kernel is mainly determined by the maternal genotype, a fact that was further confirmed by in vitro culture experiments. Finally, a PCR‐based marker based on Indel058 was developed in order to facilitate the high tocopherol (vitamin E) maize breeding.     

Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue

In plants, lipids of the photosynthetic membrane are synthesized by parallel pathways associated with the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Lipids derived from the two pathways are distinguished by their acyl‐constituents. Following this plant paradigm, the prevalent acyl composition of chloroplast lipids suggests that Chlamydomonas reinhardtii (Chlamydomonas) does not use the ER pathway; however, the Chlamydomonas genome encodes presumed plant orthologues of a chloroplast lipid transporter consisting of TGD (TRIGALACTOSYLDIACYLGLYCEROL) proteins that are required for ER‐to‐chloroplast lipid trafficking in plants. To resolve this conundrum, we identified a mutant of Chlamydomonas deleted in the TGD2 gene and characterized the respective protein, CrTGD2. Notably, the viability of the mutant was reduced, showing the importance of CrTGD2. Galactoglycerolipid metabolism was altered in the tgd2 mutant with monogalactosyldiacylglycerol (MGDG) synthase activity being strongly stimulated. We hypothesize this to be a result of phosphatidic acid accumulation in the chloroplast outer envelope membrane, the location of MGDG synthase in Chlamydomonas. Concomitantly, increased conversion of MGDG into triacylglycerol (TAG) was observed. This TAG accumulated in lipid droplets in the tgd2 mutant under normal growth conditions. Labeling kinetics indicate that Chlamydomonas can import lipid precursors from the ER, a process that is impaired in the tgd2 mutant.     

Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1‐2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient‐sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi‐starved mgd1‐2 leaves, biogenesis of thylakoid‐like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress‐induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light‐harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1‐2 mutant. Moreover, the reduced expression of nuclear‐ and plastid‐encoded photosynthetic genes observed in the mgd1‐2 mutant under Pi‐sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid‐ and nuclear‐encoded photosynthetic genes, independently of photosynthesis.     

Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis

Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (E_GSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal E_GSH dynamics, we show that stromal E_GSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.     

Arabidopsis phosphatidylglycerophosphate phosphatase 1 involved in phosphatidylglycerol biosynthesis and photosynthetic function

Phosphatidylglycerol (PG) is an indispensable lipid constituent of photosynthetic membranes, whose function is essential in photosynthetic activity. In higher plants, the biological function of the last step of PG biosynthesis remains elusive because an enzyme catalyzing this reaction step, namely phosphatidylglycerophosphate phosphatase (PGPP), has been a missing piece in the entire glycerolipid metabolic map. Here, we report the identification and characterization of AtPGPP1 encoding a PGPP in Arabidopsis thaliana. Heterologous expression of AtPGPP1 in yeast Δgep4 complemented growth phenotype and PG‐producing activity, suggesting that AtPGPP1 encodes a functional PGPP. The GUS reporter assay showed that AtPGPP1 was preferentially expressed in hypocotyl, vasculatures, trichomes, guard cells, and stigmas. A subcellular localization study with GFP reporter indicated that AtPGPP1 is mainly localized at chloroplasts. A T‐DNA‐tagged knockout mutant of AtPGPP1, designated pgpp1‐1, showed pale green phenotype with reduced PG and chlorophyll contents but no defect in embryo development. In the pgpp1‐1 mutant, ultrastructure of plastids indicated defective development of chloroplasts and measurement of photosynthetic parameters showed impaired photosynthetic activity. These results suggest that AtPGPP1 is a primary plastidic PGPP required for PG biosynthesis and photosynthetic function in Arabidopsis.     

Molecular changes of Arabidopsis thaliana plastoglobules facilitate thylakoid membrane remodeling under high light stress

Plants require rapid responses to adapt to environmental stresses. This includes dramatic changes in the size and number of plastoglobule lipid droplets within chloroplasts. Although the morphological changes of plastoglobules are well documented, little is known about the corresponding molecular changes. To address this gap, we have compared the quantitative proteome, oligomeric state, prenyl-lipid content and kinase activities of Arabidopsis thaliana plastoglobules under unstressed and 5-day light-stressed conditions. Our results show a specific recruitment of proteins related to leaf senescence and jasmonic acid biosynthesis under light stress, and identify nearly half of the plastoglobule proteins in high native molecular weight masses. Additionally, a specific increase in plastoglobule carotenoid abundance under the light stress was consistent with enhanced thylakoid disassembly and leaf senescence, supporting a specific role for plastoglobules in senescence and thylakoid remodeling as an intermediate storage site for photosynthetic pigments. In vitro kinase assays of isolated plastoglobules demonstrated kinase activity towards multiple target proteins, which was more pronounced in the plastoglobules of unstressed than light-stressed leaf tissue, and which was diminished in plastoglobules of the abc1k1/abc1k3 double-mutant. These results strongly suggest that plastoglobule-localized ABC1 kinases hold endogenous kinase activity, as these were the only known or putative kinases identified in the isolated plastoglobules by deep bottom-up proteomics. Collectively, our study reveals targeted changes to the protein and prenyl-lipid composition of plastoglobules under light stress that present strategies by which plastoglobules appear to facilitate stress adaptation within chloroplasts.     

A TIR–NBS protein encoded by Arabidopsis Chilling Sensitive 1 (CHS1) limits chloroplast damage and cell death at low temperature

Survival of plants at low temperature depends on mechanisms for limiting physiological damage and maintaining growth. We mapped the chs1‐1 (chilling sensitive1‐1) mutation in Arabidopsis accession Columbia to the TIR‐NBS gene At1g17610. In chs1‐1, a single amino acid exchange at the CHS1 N‐terminus close to the conserved TIR domain creates a stable mutant protein that fails to protect leaves against chilling stress. The sequence of another TIR‐NBS gene (At5g40090) named CHL1 (CHS1‐like 1) is related to that of CHS1. Over‐expression of CHS1 or CHL1 alleviates chilling damage and enhances plant growth at moderate (24°C) and chilling (13°C) temperatures, suggesting a role for both proteins in growth homeostasis. chs1‐1 mutants show induced salicylic acid production and defense gene expression at 13°C, indicative of autoimmunity. Genetic analysis of chs1‐1 in combination with defense pathway mutants shows that chs1‐1 chilling sensitivity requires the TIR‐NBS‐LRR and basal resistance regulators encoded by EDS1 and PAD4 but not salicylic acid. By following the timing of metabolic, physiological and chloroplast ultrastructural changes in chs1‐1 leaves during chilling, we have established that alterations in photosynthetic complexes and thylakoid membrane integrity precede leaf cell death measured by ion leakage. At 24°C, the chs1‐1 mutant appears normal but produces a massive necrotic response to virulent Pseudomonas syringae pv. tomato infection, although this does not affect bacterial proliferation. Our results suggest that CHS1 acts at an intersection between temperature sensing and biotic stress pathway activation to maintain plant performance over a range of conditions.     

Hunting the main player enabling Chlamydomonas reinhardtii growth under fluctuating light

Photosynthetic organisms have evolved numerous photoprotective mechanisms and alternative electron sinks/pathways to fine‐tune the photosynthetic apparatus under dynamic environmental conditions, such as varying carbon supply or fluctuations in light intensity. In cyanobacteria flavodiiron proteins (FDPs) protect the photosynthetic apparatus from photodamage under fluctuating light (FL). In Arabidopsis thaliana, which does not possess FDPs, the PGR5‐related pathway enables FL photoprotection. The direct comparison of the pgr5, pgrl1 and flv knockout mutants of Chlamydomonas reinhardtii grown under ambient air demonstrates that all three proteins contribute to the survival of cells under FL, but to varying extents. The FDPs are crucial in providing a rapid electron sink, with flv mutant lines unable to survive even mild FL conditions. In contrast, the PGRL1 and PGR5‐related pathways operate over relatively slower and longer time‐scales. Whilst deletion of PGR5 inhibits growth under mild FL, the pgrl1 mutant line is only impacted under severe FL conditions. This suggests distinct roles, yet a close relationship, between the function of PGR5, PGRL1 and FDP proteins in photoprotection.     

SULTR3;1 is a chloroplast‐localized sulfate transporter in Arabidopsis thaliana

Plants play a prominent role as sulfur reducers in the global sulfur cycle. Sulfate, the major form of inorganic sulfur utilized by plants, is absorbed and transported by specific sulfate transporters into plastids, especially chloroplasts, where it is reduced and assimilated into cysteine before entering other metabolic processes. How sulfate is transported into the chloroplast, however, remains unresolved; no plastid‐localized sulfate transporters have been previously identified in higher plants. Here we report that SULTR3;1 is localized in the chloroplast, which was demonstrated by SULTR3;1‐GFP localization, Western blot analysis, protein import as well as comparative analysis of sulfate uptake by chloroplasts between knockout mutants, complemented transgenic plants, and the wild type. Loss of SULTR3;1 significantly decreases the sulfate uptake of the chloroplast. Complementation of the sultr3;1 mutant phenotypes by expression of a 35S‐SULTR3;1 construct further confirms that SULTR3;1 is one of the transporters responsible for sulfate transport into chloroplasts.     

FtsHi1/ARC1 is an essential gene in Arabidopsis that links chloroplast biogenesis and division

The Arabidopsis arc1 (accumulation and replication of chloroplasts 1) mutant has pale seedlings and smaller, more numerous chloroplasts than the wild type. Previous work has suggested that arc1 affects the timing of chloroplast division but does not function directly in the division process. We isolated ARC1 by map‐based cloning and discovered it encodes FtsHi1 (At4g23940), one of several FtsHi proteins in Arabidopsis. These poorly studied proteins resemble FtsH metalloproteases important for organelle biogenesis and protein quality control but are presumed to be proteolytically inactive. FtsHi1 bears a predicted chloroplast transit peptide and localizes to the chloroplast envelope membrane. Phenotypic studies showed that arc1 (hereafter ftsHi1‐1), which bears a missense mutation, is a weak allele of FtsHi1 that disrupts thylakoid development and reduces de‐etiolation efficiency in seedlings, suggesting that FtsHi1 is important for chloroplast biogenesis. Consistent with this finding, transgenic plants suppressed for accumulation of an FtsHi1 fusion protein were often variegated. A strong T‐DNA insertion allele, ftsHi1‐2, caused embryo‐lethality, indicating that FtsHi1 is an essential gene product. A wild‐type FtsHi1 transgene rescued both the chloroplast division and pale phenotypes of ftsHi1‐1 and the embryo‐lethal phenotype of ftsHi1‐2. FtsHi1 overexpression produced a subtle increase in chloroplast size and decrease in chloroplast number in wild‐type plants while suppression led to increased numbers of small chloroplasts, providing new evidence that FtsHi1 negatively influences chloroplast division. Taken together, our analyses reveal that FtsHi1 functions in an essential, envelope‐associated process that may couple plastid development with division.     

Arabidopsis inositol phosphate kinases IPK1 and ITPK1 constitute a metabolic pathway in maintaining phosphate homeostasis

Emerging studies have suggested that there is a close link between inositol phosphate (InsP) metabolism and cellular phosphate (P_i) homeostasis in eukaryotes; however, whether a common InsP species is deployed as an evolutionarily conserved metabolic messenger to mediate P_i signaling remains unknown. Here, using genetics and InsP profiling combined with P_i‐starvation response (PSR) analysis in Arabidopsis thaliana, we showed that the kinase activity of inositol pentakisphosphate 2‐kinase (IPK1), an enzyme required for phytate (inositol hexakisphosphate; InsP₆) synthesis, is indispensable for maintaining P_i homeostasis under P_i‐replete conditions, and inositol 1,3,4‐trisphosphate 5/6‐kinase 1 (ITPK1) plays an equivalent role. Although both ipk1‐1 and itpk1 mutants exhibited decreased levels of InsP₆ and diphosphoinositol pentakisphosphate (PP‐InsP₅; InsP₇), disruption of another ITPK family enzyme, ITPK4, which correspondingly caused depletion of InsP₆ and InsP₇, did not display similar P_i‐related phenotypes, which precludes these InsP species from being effectors. Notably, the level of d /l ‐Ins(3,4,5,6)P₄ was concurrently elevated in both ipk1‐1 and itpk1 mutants, which showed a specific correlation with the misregulated P_i phenotypes. However, the level of d /l ‐Ins(3,4,5,6)P₄ is not responsive to P_i starvation that instead manifests a shoot‐specific increase in the InsP₇ level. This study demonstrates a more nuanced picture of the intersection of InsP metabolism and P_i homeostasis and PSRs than has previously been elaborated, and additionally establishes intermediate steps to phytate biosynthesis in plant vegetative tissues.