A Cytoplasmic Regulatory Mutant of Euglena: Constitutivity for the Light-Inducible Chloroplast Transfer RNAs

An ultraviolet-induced cytoplasmic mutant (G₁BU) of Euglena gracilis Klebs var. bacillaris Pringsheim is described. G₁BU, in addition to being golden in color and containing smaller amounts of chlorophyll than wild type when grown in the light, is a regulatory constitutive mutant for the light-inducible chloroplast isoleucine and methionine tRNAs; i.e., these two tRNAs are present in dark-grown G₁BU cells at approximately the levels present in light-grown wild-type cells. Six other mutants were also examined for normal control of plastid tRNA biosynthesis; all four of the aplastidic mutants (lacking appreciable plastid structures and detectable plastid DNA) are incapable of chloroplast tRNA biosynthesis, whereas two other color mutants that do contain plastids and chloroplast DNA have normal plastid tRNA regulation.     

Nonvesicular phospholipid transfer between peroxisomes and the endoplasmic reticulum

The endoplasmic reticulum (ER) plays an important role in peroxisome biogenesis; some peroxisomal membrane proteins are inserted into the ER and trafficked to peroxisomes in vesicles. These vesicles could also provide the phospholipids required for the growth of peroxisomal membranes, because peroxisomes lack phospholipid biosynthesis enzymes. To test this, we established a novel assay to monitor phospholipid transfer between the ER and peroxisomes and found that phospholipids are rapidly trafficked between these compartments. This transport is not blocked in mutants with conditional defects in Sec proteins required for vesicular trafficking from the ER or in Pex3p, a protein required for peroxisome membrane biogenesis. ER to peroxisome lipid transport was reconstituted in vitro and does not require cytosolic factors or ATP. Our findings indicate that lipids are directly transferred from the ER to peroxisomes by a nonvesicular pathway and suggest that ER to peroxisome vesicular transport is not required to provide lipids for peroxisomal growth.     

Plastid protein synthesis is required for plant development in tobacco

Chloroplasts fulfill important functions in cellular metabolism. The majority of plastid genome-encoded genes is involved in either photosynthesis or chloroplast gene expression. Whether or not plastid genes also can determine extraplastidic functions has remained controversial. We demonstrate here an essential role of plastid protein synthesis in tobacco leaf development. By using chloroplast transformation, we have developed an experimental system that produces recombination-based knockouts of chloroplast translation in a cell-line-specific manner. The resulting plants are chimeric and, in the presence of translational inhibitors, exhibit severe developmental abnormalities. In the absence of active plastid protein synthesis, leaf blade development is abolished because of an apparent arrest of cell division. This effect appears to be cell-autonomous in that adjacent sectors of cells with translating plastids are phenotypically normal but cannot complement for the absence of plastid translation in mutant sectors. Developmental abnormalities also are seen in flower morphology, indicating that the defects are not caused by inhibited expression of plastid photosynthesis genes. Taken together, our data point to an unexpected essential role of plastid genes and gene expression in plant development and cell division.     

From the Cover: Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival

The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period.     

Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea.

This communication demonstrates that all de novo fatty acid biosynthesis in spinach leaf cells requires acyl carrier protein (ACP) and occurs specifically in the chloroplasts. Antibodies raised to purified spinach ACP inhibited at least 98% of malonyl CoA-dependent fatty acid synthesis by spinach leaf homogenates. Therefore, the presence of ACP in a compartment of the spinach leaf cell would serve as a marker for de novo fatty acid biosynthesis. A radioimmunoassay capable of detecting 10(15) mol (10(-11) g) of spinach ACP was developed to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach leaf protoplasts. All of the ACP of the leaf cell could be attributed to the chloroplast. Less than 1% of the ACP associated with chloroplasts resulted from binding of free ACP to chloroplasts. Of interest, ACP from Escherichia coli, soybean, and sunflower showed only partial crossreactivity with spinach ACP by the radioimmunoassay. These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the de novo synthesis of C16 and C18 fatty acids. These fatty acids are then transported into the cytoplasm for further modification and are either inserted into extrachloroplastic membrane lipids or returned to the chloroplast for insertion into lamellar membrane lipids.     

Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts

Tocopherols are nonpolar compounds synthesized and localized in plastids but whose genetic elimination specifically impacts fatty acid desaturation in the endoplasmic reticulum (ER), suggesting a direct interaction with ER-resident enzymes. To functionally probe for such interactions, we developed transorganellar complementation, where mutated pathway activities in one organelle are experimentally tested for substrate accessibility and complementation by active enzymes retargeted to a companion organelle. Mutations disrupting three plastid-resident activities in tocopherol and carotenoid synthesis were complemented from the ER in this fashion, demonstrating transorganellar access to at least seven nonpolar, plastid envelope-localized substrates from the lumen of the ER, likely through plastid:ER membrane interaction domains. The ability of enzymes in either organelle to access shared, nonpolar plastid metabolite pools redefines our understanding of the biochemical continuity of the ER and chloroplast with profound implications for the integration and regulation of organelle-spanning pathways that synthesize nonpolar metabolites in plants.     

Cytoplasmic and chloroplast synthesis of phycobilisome polypeptides

In vivo labeling of eukaryotic phycobilisomes in the presence of inhibitors of translation on 70S and 80S ribosomes demonstrates that some of the polypeptides of this light-harvesting complex are synthesized in the cytoplasm while others are synthesized in the chloroplast. The major pigmented polypeptides, the α and β subunits of the biliproteins (molecular weights between 15,000 and 20,000) and the anchor protein (molecular weight about 90,000) are translated on 70S ribosomes. This suggests that these polypeptides are made within the algal chloroplast. Because the α and β subunits comprise a group of closely related polypeptides, the genes encoding these polypeptides may reside in the plastid genome as a multigene family. Other prominent phycobilisome polypeptides, including a nonpigmented polypeptide that may be involved in maintaining the structural integrity of the complex, are synthesized on cytoplasmic ribosomes. Because the synthesis of phycobilisomes appears to require the expression of genes in two subcellular compartments, this system may be an excellent model for: (i) examining interaction between nuclear and plastid genomes: (ii) elucidating the molecular processes involved in the evolution of plastid genes: (iii) clarifying the events in the synthesis and assembly of macromolecular complexes in the chloroplast.     

Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii

The polypeptide subunits of the photosynthetic electron transport complexes in plants and algae are encoded by two genomes. Nuclear genome-encoded subunits are synthesized in the cytoplasm by 80S ribosomes, imported across the chloroplast envelope, and assembled with the subunits that are encoded by the plastid genome. Plastid genome-encoded subunits are synthesized by 70S chloroplast ribosomes directly into membranes that are widely believed to belong to the photosynthetic thylakoid vesicles. However, in situ evidence suggested that subunits of photosystem II are synthesized in specific regions within the chloroplast and cytoplasm of Chlamydomonas. Our results provide biochemical and in situ evidence of biogenic membranes that are localized to these translation zones. A “chloroplast translation membrane” is bound by the translation machinery and appears to be privileged for the synthesis of polypeptides encoded by the plastid genome. Membrane domains of the chloroplast envelope are located adjacent to the cytoplasmic translation zone and enriched in the translocons of the outer and inner chloroplast envelope membranes protein import complexes, suggesting a coordination of protein synthesis and import. Our findings contribute to a current realization that biogenic processes are compartmentalized within organelles and bacteria.     

TIC236 gain-of-function mutations unveil the link between plastid division and plastid protein import

TIC236 is an essential component of the translocon for protein import into chloroplasts, as evidenced by the embryonic lethality of the knockout mutant. Here, we unveil a TIC236-allied component, the chloroplast outer membrane protein CRUMPLED LEAF (CRL), absence of which impairs plastid division and induces autoimmune responses in Arabidopsis thaliana. A forward genetic screen exploring CRL function found multiple dominant TIC236 gain-of-function (tic236-gf) mutations that abolished crl-induced phenotypes. Moreover, CRL associates with TIC236, and a tic236-knockdown mutant exhibited multiple lesions similar to the crl mutant, supporting their shared functionality. Consistent with the defective plastid division phenotype of crl, CRL interacts with the transit peptides of proteins essential in plastid division, with tic236-gf mutations reinforcing their import via increased TIC236 stability. Ensuing reverse genetic analyses further revealed genetic interaction between CRL and SP1, a RING-type ubiquitin E3 ligase, as well as with the plastid protease FTSH11, which function in TOC and TIC protein turnover, respectively. Loss of either SP1 or FTSH11 rescued crl mutant phenotypes to varying degrees due to increased translocon levels. Collectively, our data shed light on the links between plastid protein import, plastid division, and plant stress responses.

Chloroplasts evolved from a gram-negative cyanobacterial endosymbiont, with most cyanobacterial genes having been transferred to the host plant genome. Therefore, thousands of nuclear-encoded chloroplast proteins are posttranslationally imported into chloroplasts, orchestrated by outer and inner envelope membrane (OEM and IEM) translocons, respectively termed TOC and TIC. Although an array of translocon proteins has been identified (1, 2), it has been unclear how TOC and TIC are linked across the two envelope membranes separated by an intermembrane space. The recent discovery of the TIC236 protein solved this long-standing question (3). TIC236 is an integral IEM protein associated with TIC components. Its C-terminal domain, located in the intermembrane space, directly interacts with the N-terminal polypeptide transport-associated (POTRA) domains of TOC75-III (hereafter TOC75), the channel protein in the TOC complex. TOC75 and TIC236 are homologs of bacterial TamA (TRANSLOCON ASSEMBLY MODULE A) and TamB, respectively, which together function in bacterial outer membrane protein biogenesis and protein export (3–5).Plastid division occurs in developing cells to ensure an optimal number of plastids is in place before cell division, requiring the import of a suite of plastid-division machinery (PDM) proteins. The loss of any vital PDM components results in gigantic plastids and a drastically reduced plastid number per cell (6). Surprisingly, several Arabidopsis mutants deficient in plastid division—including crumpled leaf (crl), accumulation and replication of chloroplasts6 (arc6), plastid division2 (pdv2), and filamenting temperature-sensitive z1 (ftsz1)—develop foliar cell death (7), resembling lesion-mimicking mutants (LMM) that exhibit a light-dependent hypersensitive response–like cell death (8, 9). Like LMM, crl and other plastid division mutants constantly up-regulate immune-related genes (7, 10). The gigantic chloroplasts of crl mutants also induce an abnormal cell cycle, with increased endoreduplication activity leading to stunted growth (11). Previous studies have indicated that autoimmune responses, abnormal cell cycle, and growth inhibition are likely mediated by a process called retrograde signaling [i.e., signaling from the gigantic chloroplasts back to the nucleus (7, 10, 11)].CRL is a nuclear-encoded chloroplast OEM protein. Its short N-terminal region resides in the intermembrane space, followed by a transmembrane domain and a cytosolic chromophore lyase CpcT/CpeT domain characterized from a cyanobacterial CpcT bilin lyase (12). Although the lyase domain retains phycocyanobilin-binding aptitude, there is no apparent correlation between phycocyanobilin-binding ability and crl-induced lesions in Arabidopsis (13), indicating that CRL has gained a divergent function.     

Single-stranded DNA from abutilon mosaic virus is present in the plastids of infected Abutilon sellovianum

The leaf mosaic of Abutilon sellovianum is caused by abutilon mosaic virus (AbMV), a geminivirus that has a circular single-stranded DNA genome. DNA was isolated from intact plastids of AbMV-infected and noninfected plants. Plastids from infected plants were shown to contain the single-stranded AbMV DNA by Southern-blot hybridization experiments that used a probe made from highly purified AbMV DNA. The possibility of adsorption of AbMV virions or viral DNA onto the plastid envelope was ruled out by several in vitro experiments with DNase I and protease. Furthermore, the lamellar system of plastids from AbMV-infected plants was degenerated and substituted by amorphous electron-dense material. The transport of AbMV DNA across the plastid envelope has implications for the development of a chloroplast transformation vector.     

Origin of a chloroplast protein importer

During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system. A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares ≈22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the Synechocystis PCC6803 genome. Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of Synechocystis PCC6803 and that SynToc75 forms a voltage-gated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes. These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane. Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.     

The steady-state level of Mg-protoporphyrin IX is not a determinant of plastid-to-nucleus signaling in Arabidopsis

The plastid plays a vital role in various cellular activities within plant cells including photosynthesis and other metabolic pathways. It is believed that the functional status of the plastid is somehow monitored by the nucleus to optimize the expression of genes encoding plastid proteins. The currently dominant model for plastid-derived signaling (“plastid signaling”) proposes that Mg-protoporphyrin IX (MgProto) is a negative signal that represses the expression of a wide range of nuclear genes encoding plastid-localized proteins when plastid development is inhibited. In this study, we have re-evaluated this hypothesis by quantifying the steady-state levels of MgProto (as well as its neighboring intermediates protoporphyrin IX and Mg-Proto monomethyl ester [MgProtoMe]) in Arabidopsis plants with altered plastid signaling responses as monitored by expression of the Lhcb1, RBCS, HEMA1, BAM3 and CA1 genes. In addition, we have examined the correlation between gene expression and MgProto (MgProtoMe) in a range of mutants and conditions in which the steady-state levels of MgProto (MgProtoMe) have been modified. Overall we found that there was no correlation between the steady-state levels of MgProto (MgProtoMe) and Lhcb1 expression or with any of the other genes tested. Taking these results together, we propose that the current model on plastid signaling must be revised.     

The plastid ndh genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes

The plastid genomes of several plants contain ndh genes—homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria. From sequence similarities with these genes, the ndh gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established. Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant Pisum sativum. The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step. The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS/PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting. The Ndh complex showed an NADH- and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors. This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase. Our results are compatible with a dual role for the Ndh complex in the chlororespiratory and cyclic photophosphorylation pathways.     

Lipid in the livers of adolescents with nonalcoholic steatohepatitis: combined effects of pathways on steatosis

Fatty liver is a prerequisite for the development of nonalcoholic steatohepatitis (NASH). The homeostasis of hepatic lipid is determined by the dynamic balance of multiple pathways introducing lipids into or removing lipids from hepatocytes. We aim to study the different contributions of major lipid pathways to fat deposition in NASH livers. Expression of the lipid metabolism-related genes was analyzed by microarray and quantitative real-time polymerase chain reaction analysis. The expression levels of genes responsible for the rate-limiting steps of fatty acid uptake (CD36, FABPpm, SLC27A2, and SLC27A5), de novo synthesis (ACACB), oxidation (CPT-1), and very low-density lipoprotein (VLDL) secretion (ApoB) were used to evaluate the relative activity of each pathway. The expression levels for CD36 and CPT-1 were confirmed by Western blot analysis. Fatty acid uptake pathways were up-regulated to a higher degree than other pathways. The de novo synthesis pathway was also up-regulated more than both VLDL secretion and fatty acid oxidation pathways. In contrast to other NASH livers, one NASH liver exhibited lower ApoB and CPT-1 expression levels than normal controls. The increased fatty acid uptake and de novo synthesis were the most common causes for steatosis in NASH patients. In a rare case, impaired VLDL secretion and fatty acid oxidation contributed to the development of steatosis. Our study promises a simple method for the determination of why hepatic steatosis occurs in individual patients. This method may allow specific targeting of therapeutic treatments in individual patients.     

Galactolipid synthesis in chloroplast inner envelope is essential for proper thylakoid biogenesis, photosynthesis, and embryogenesis

The biogenesis of thylakoid membranes, an indispensable event for the photoautotrophic growth of plants, requires a significant increase in the level of the unique thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG), which constitutes the bulk of membrane lipids in chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. Here we report the identification and characterization of an Arabidopsis mutant showing a complete defect in MGDG synthase 1. The mutant seeds germinated as small albinos only in the presence of sucrose. The seedlings lacked galactolipids and had disrupted photosynthetic membranes, leading to the complete impairment of photosynthetic ability and photoautotrophic growth. Moreover, invagination of the inner envelope, which is not seen in mature WT chloroplasts, was observed in the mutant, supporting an old hypothesis that envelope invagination is a major event in early chloroplast biogenesis. In addition to the defective seedling phenotype, embryo development was arrested in the mutant, although seeds with impaired embryos could germinate heterotrophically. These results demonstrate the importance of galactolipids not only in photosynthetic growth but also in embryogenesis.     

PIF3 is a repressor of chloroplast development

The phytochrome-interacting factor PIF3 has been proposed to act as a positive regulator of chloroplast development. Here, we show that the pif3 mutant has a phenotype that is similar to the pif1 mutant, lacking the repressor of chloroplast development PIF1, and that a pif1pif3 double mutant has an additive phenotype in all respects. The pif mutants showed elevated protochlorophyllide levels in the dark, and etioplasts of pif mutants contained smaller prolamellar bodies and more prothylakoid membranes than corresponding wild-type seedlings, similar to previous reports of constitutive photomorphogenic mutants. Consistent with this observation, pif1, pif3, and pif1pif3 showed reduced hypocotyl elongation and increased cotyledon opening in the dark. Transfer of 4-d-old dark-grown seedlings to white light resulted in more chlorophyll synthesis in pif mutants over the first 2 h, and analysis of gene expression in dark-grown pif mutants indicated that key tetrapyrrole regulatory genes such as HEMA1 encoding the rate-limiting step in tetrapyrrole synthesis were already elevated 2 d after germination. Circadian regulation of HEMA1 in the dark also showed reduced amplitude and a shorter, variable period in the pif mutants, whereas expression of the core clock components TOC1, CCA1, and LHY was largely unaffected. Expression of both PIF1 and PIF3 was circadian regulated in dark-grown seedlings. PIF1 and PIF3 are proposed to be negative regulators that function to integrate light and circadian control in the regulation of chloroplast development.