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Light-induced biogenesis of light-harvesting complex I (LHC I) during chloroplast development in barley (hordeum vulgare). Studies using cDNA clones of the 21- and 20-kilodalton LHC I apoproteins.

The light-harvesting complex (LHC) Ib pigment-proteins form the major component of the LHC I complex in higher plants. They comprise chlorophylls a and b, xanthophylls, and at least two polypeptide subunits of 21 and 20 kD in barley (Hordeum vulgare). We have identified two cDNA clones, LHC Ib-21 and LHC Ib-20, encoding the 21- and 20-kD LHC Ib apoproteins, respectively. N-terminal protein sequences of the purified LHC Ib polypeptides were used for the unequivocal correlation of these clones to their respective apoproteins. The cDNA clones encode two proteins that have strong sequence similarity to other LHC I and LHC II pigment-binding polypeptides of photosystems I and II. The 21-kD polypeptide contains 201 amino acid residues (22.14 kD), and the 20-kD polypeptide contains 200 amino acid residues (22.18 kD). The biogenesis of the LHC Ib apoproteins and the pigmented LHC I during the light-induced development of the chloroplast was studied. Accumulation of the two LHC Ib mRNAs is induced by light, and their amount is regulated by phytochrome. LHC Ib polypeptide accumulation in the thylakoid membrane temporally lags behind transcript accumulation. The rates of accumulation of LHC Ib transcripts and of their apoproteins lag behind those of the major LHC II component, LHC IIb. Complete assembly of the LHC Ib pigment-protein, as observed by low-temperature fluorescence spectroscopy, requires exposure of dark-grown seedlings to 72 h or more of light.

Amino Acid Sequence↗

Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate.

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/BgI2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/BgI2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.

Base Sequence↗

CHLOROPLAST BIOGENESIS genes act cell and noncell autonomously in early chloroplast development.

In order to identify nuclear genes required for early chloroplast development, a collection of photosynthetic pigment mutants of Arabidopsis was assembled and screened for lines with extremely low levels of chlorophyll. Nine chloroplast biogenesis (clb) mutants that affect proplastid growth and thylakoid membrane formation and result in an albino seedling phenotype were identified. These mutations identify six new genes as well as a novel allele of cla1. clb mutants have less than 2% of wild-type chlorophyll levels, and little or no expression of nuclear and plastid-encoded genes required for chloroplast development and function. In all but one mutant, proplastids do not differentiate enough to form elongated stroma thylakoid membranes. Analysis of mutants during embryogenesis allows differentiation between CLB genes that act noncell autonomously, where partial maternal complementation of chloroplast development is observed in embryos, and those that act cell autonomously, where complementation during embryogenesis is not observed. Molecular characterization of the noncell autonomous clb4 mutant established that the CLB4 gene encodes for hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the next to the last enzyme of the methylerythritol 4-phosphate (MEP) pathway for the synthesis of plastidic isoprenoids. The noncell autonomous nature of the clb4 mutant suggests that products of the MEP pathway can travel between tissues, and provides in vivo evidence that some movement of MEP intermediates exists from the cytoplasm to the plastid. The isolation and characterization of clb mutants represents the first systematic study of genes required for early chloroplast development in Arabidopsis.

Arabidopsis↗

Biogenesis and Ultrastructure of Carboxysomes from Wild Type and Mutants of Synechococcus sp. Strain PCC 7942.

Immature inclusions representing three progressive steps of carboxysome biogenesis have been identified in Synechococcus during the period of adaptation to low-CO2 conditions: (a) ring-shaped structures, (b) electron-translucent inclusions with the shape of a carboxysome and the internal orderly arrangement of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) molecules, and (c) carboxysomes with an internal electron-translucent area, which seem to be the penultimate stage of carboxysome maturation. The ability to build up normal carboxysomes is impaired in three (M3, EK6, and D4) of four high-carbon-requiring mutants studied in this work. M3 and EK6 exhibit abundant immature electron-translucent carboxysomes but no mature ones. This finding supports the contention that an open reading frame located 7.5 kb upstream of the gene encoding the large subunit of Rubisco (altered in M3) is involved in the carboxysome composition and confirms the structural role of the small subunit of Rubisco (slightly modified in EK6) in the assembly of these structures. D4 shows few typical carboxysomes and frequent immature types, its genetic lesion affecting the apparently unrelated gene encoding a subunit of phosphoribosyl aminoamidazole carboxylase of the purine biosynthesis pathway. Revertants EK20 (EK6) and RK13 (D4) have normal carboxysomes, which means that the restoration of the ability to grow under low CO2 coincides with the proper assembling of these structures. N5, a transport mutant due to the alteration of the gene encoding subunit 2 of NADH dehydrogenase, shows an increase in the number and size of carboxysomes and frequent bar-shaped ones.

Journal Article↗

Heterogeneity of mitochondrial protein biogenesis during primary leaf development in barley

The natural developmental gradient of light-grown primary leaves of barley (Hordeum vulgare L.) was used to analyze the biogenesis of mitochondrial proteins in relation to the age and physiological changes within the leaf. The data indicate that the protein composition of mitochondria changes markedly during leaf development. Three distinct patterns of protein development were noted: group A proteins, consisting of the E1 beta-subunit of the pyruvate dehydrogenase complex, ORF156, ORF577, alternative oxidase, RPS12, cytochrome oxidase subunits II and III, malic enzyme, and the alpha- and beta-subunits of F1-ATPase; group B proteins, consisting of the E1 alpha-subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, HSP70A, cpn60C, and cpn60B; and group C proteins, consisting of the four subunits of the glycine decarboxylase complex (P, H, T, and L proteins), fumarase, and formate dehydrogenase. All of the proteins increased in concentration from the basal meristem to the end of the elongation zone (20.0 mm from the leaf base), whereupon group A proteins decreased, group B proteins increased to a maximum at 50 mm from the leaf base, and group C proteins increased to a maximum at the leaf tip. This study provides evidence of a marked heterogeneity of mitochondrial protein composition, reflecting a changing function as leaf cells develop photosynthetic and photorespiratory capacity.

Journal Article↗

Mitochondrial biogenesis during germination in maize embryos.

Mitochondrial biogenesis and metabolism were investigated during maize (Zea mays) seed germination. Mitochondria from dry and imbibed seed exhibited NADH-dependent O(2) uptake that was completely inhibited by KCN and antimycin A. Mitochondria in the dry seed had a lower rate of succinate-dependent O(2) uptake relative to that measured in imbibed and germinated seed. The activities of the tricarboxylic acid (TCA) cycle enzymes, pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex, NAD-malic enzyme, and citrate synthase, are similarly low in mitochondria from dry seed and this correlates with a lower relative abundance of the mitochondrial matrix-located citrate synthase and pyruvate dehydrogenase complex E1alpha-subunit polypeptides. Electron microscopy revealed that mitochondria in the dry seed have a poorly developed internal membrane structure with few cristae; following 24 h of germination the mitochondria developed a more normal structure with more developed cristae. The mitochondria from maize embryos could be fractionated into two subpopulations by Suc density gradient centrifugation: one subpopulation of buoyant density equivalent to 22% to 28% (w/w) Suc; the other equivalent to 37% to 42% (w/w) Suc. These two subpopulations had different activities of specific mitochondrial enzymes and contained different amounts of specific mitochondrial proteins as revealed by western-blot analysis. Both subpopulations from the dry embryo were comprised of poorly developed mitochondria. However, during imbibition mitochondria in the heavy fraction (37%-42% [w/w] Suc) progressively acquired characteristics of fully functional mitochondria found in the germinated seedling in terms of structure, enzymic activity, and protein complement. In contrast, mitochondria in the light fraction (22% to 28% [w/w] Suc) show no significant structural change during imbibition and the amounts of specific mitochondrial proteins decreased significantly during germination.

Gene Expression Regulation, Developmental↗

Biogenesis of Mitochondria in Germinating Peanut Cotyledons II. Changes in Cytochromes and Mitochondrial DNA.

Biogenesis of mitochondria occurs in the germinating cotyledons of peanuts. This process was demonstrated by measuring both constitutive and enzymatic properties of mitochondria as a function of germination time. Direct counting by phase contrast microscopy of sucrose density gradient preparations showed that the number of mitochondria increased markedly during germination. DNA with a buoyant density distinct from the major cellular DNA was associated with these mitochondrial preparations. During germination the amount of this DNA in mitochondrial pellets increased. This increase closely paralleled the increase in number of mitochondria.Succinoxidase and succinic dehydrogenase increased during germination. Both activities were confined to the mitochondrial fraction. The rate of increase of succinoxidase activity was significantly greater than the rate of increase of succinic dehydrogenase and both increased at least initially at a greater rate than the amount of mitochondrial DNA or numbers of mitochondria.The amounts of cytochromes present in mitochondrial preparations were measured spectrophotometrically. All of the cytochromes increased in amount during germination. The rate of increase of cytochrome a - a(3) very close to the rate of increase in succinoxidase activity.

Journal Article↗

Auxin biogenesis: subcellular compartmentation of indoleacetaldehyde reductases in cucumber seedlings.

Subcellular fractionation of cucumber (Cucumis sativus L.) seedlings was achieved, and two of the enzymes in the auxin biosynthetic pathway were localized. NADH-specific indoleacetaldehyde reductase activity was observed only in the cytosol fractions obtained from separated hypocotyl and cotyledon tissue. In contrast, a portion of the NADPH-specific indoleacetaldehyde reductase activity was associated with a microsomal fraction derived from these tissues. The NADPH-specific indoleacetaldehyde reductase was consistently found to be more firmly associated with the microsomal fraction derived from hypocotyls than with that from the cotyledons. These results indicate a division of the terminal steps of auxin biogenesis into at least two subcellular compartments.

Journal Article↗

Biogenesis of Mitochondria in Imbibed Peanut Cotyledons : II. DEVELOPMENT OF LIGHT AND HEAVY MITOCHONDRIA.

THERE ARE TWO TYPES OF MITOCHONDRIA PRESENT IN IMBIBED PEANUT COTYLEDONS: a light type (density 1.182 grams per cubic centimeter) and a heavy type (density 1.205 grams per cubic centimeter). The membrane fractions from these two types can be distinguished using sucrose density gradient analysis, and differences in membrane density between the light and heavy types are reflected in differences in their protein N and phospholipid P composition. With increasing time after imbibition, there is a substantial increase in the amount and activity of the light type of mitochondria due to their de novo synthesis. The membrane density of the light mitochondrial fraction declines over 5 days after the start of imbibition as the phospholipid P to protein N ratio increases. The heavy mitochondrial fraction declines during the first 3 days after the start of imbibition, and then it remains at a low, but constant, level thereafter. Even during the decline, however, there is synthesis of proteins comparable to that into light mitochondria. The mitochondrial biogenesis that has been observed in peanut cotyledons is of the light type, the function and physiological importance of the minor heavy type is not known.

Journal Article↗

A role for membrane lipid polyunsaturation in chloroplast biogenesis at low temperature.

Two different mutants of Arabidopsis thaliana deficient in chloroplast membrane lipid polyunsaturation were indistinguishable in appearance from the wild-type when grown at 22 degrees C. By contrast, leaf tissues of the mutants that developed during growth at 5 degrees C were chlorotic, whereas the wild type was not. This is the first direct evidence that chloroplast lipid polyunsaturation contributes to low-temperature fitness. Chloroplasts from mutant lines grown at 5 degrees C were much smaller than those of the wild-type, and the thylakoid membrane content was reduced by up to 70%. However, there was no discernible effect of low temperature on chloroplasts that developed prior to exposure to low temperatures. These and related observations suggest that the high degree of chloroplast membrane lipid polyunsaturation is required for some aspect of chloroplast biogenesis.

Journal Article↗

Regulation of Mitochondrial Function and Biogenesis in Cucumber (Cucumis sativus L.) Cotyledons during Early Seedling Growth.

The aim of this work was to characterize the respiratory metabolism of the greening cotyledons of cucumber (Cucumis sativus L.) during early seedling growth and to investigate how this is integrated with changes in mitochondrial biogenesis and function. In light-grown cotyledons, lipid mobilization extended from germination to 6 days postimbibition, reaching a maximum at 3 to 4 days postimbibition. The rate of dark oxygen uptake reached a maximum at 2 days postimbibition in dark-grown and 3 days postimbibition in light-grown cotyledons. Development of photosynthetic capacity occurred from 4 to 7 days postimbibition. In dark-grown cotyledons, lipid mobilization extended beyond 7 days postimbibition, and there was no greening or acquisition of photosynthetic competence. Measurements of mitochondrial function indicated that the respiratory capacity of the tissue changed such that during lipid mobilization there was a much greater capacity for the operation of the nondecarboxylating portion of the citric acid cycle (succinate to oxaloacetate), whereas during the development of photosynthetic function the activity of the remainder of the cycle (oxaloacetate to succinate) was induced. Comparison of the maximum capacities for mitochondrial substrate oxidations in vitro with the rates of in vivo substrate oxidations, predicted from the rate of lipid breakdown, indicated that mitochondria in this tissue operate at or below state 4 rates, suggesting limitation by both availability of ADP and substrate.

Journal Article↗

Oxidative remodeling of chromoplast carotenoids: identification of the carotenoid dioxygenase CsCCD and CsZCD genes involved in Crocus secondary metabolite biogenesis.

The accumulation of three major carotenoid derivatives-crocetin glycosides, picrocrocin, and safranal-is in large part responsible for the color, bitter taste, and aroma of saffron, which is obtained from the dried styles of Crocus. We have identified and functionally characterized the Crocus zeaxanthin 7,8(7',8')-cleavage dioxygenase gene (CsZCD), which codes for a chromoplast enzyme that initiates the biogenesis of these derivatives. The Crocus carotenoid 9,10(9',10')-cleavage dioxygenase gene (CsCCD) also has been cloned, and the comparison of substrate specificities between these two enzymes has shown that the CsCCD enzyme acts on a broader range of precursors. CsZCD expression is restricted to the style branch tissues and is enhanced under dehydration stress, whereas CsCCD is expressed constitutively in flower and leaf tissues irrespective of dehydration stress. Electron microscopy revealed that the accumulation of saffron metabolites is accompanied by the differentiation of amyloplasts and chromoplasts and by interactions between chromoplasts and the vacuole. Our data suggest that a stepwise sequence exists that involves the oxidative cleavage of zeaxanthin in chromoplasts followed by the sequestration of modified water-soluble derivatives into the central vacuole.

Amino Acid Sequence↗

HCF164 encodes a thioredoxin-like protein involved in the biogenesis of the cytochrome b(6)f complex in Arabidopsis.

To understand the biogenesis of the plastid cytochrome b(6)f complex and to identify the underlying auxiliary factors, we have characterized the nuclear mutant hcf164 of Arabidopsis and isolated the affected gene. The mutant shows a high chlorophyll fluorescence phenotype and is severely deficient in the accumulation of the cytochrome b(6)f complex subunits. In vivo protein labeling experiments indicated that the mutation acts post-translationally by interfering with the assembly of the complex. Because of its T-DNA tag, the corresponding gene was cloned and its identity confirmed by complementation of homozygous mutant plants. HCF164 encodes a thioredoxin-like protein that possesses disulfide reductase activity. The protein was found in the chloroplast, where it is anchored to the thylakoid membrane at its lumenal side. HCF164 is closely related to the thioredoxin-like protein TxlA of Synechocystis sp PCC6803, most probably reflecting its evolutionary origin. The protein also shows a limited similarity to the eubacterial CcsX and CcmG proteins, which are required for the maturation of periplasmic c-type cytochromes. The putative roles of HCF164 for the assembly of the cytochrome b(6)f complex are discussed.

Amino Acid Sequence↗

Maize mutants lacking chloroplast FtsY exhibit pleiotropic defects in the biogenesis of thylakoid membranes.

A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion in bacteria and eukaryotic cells is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membranes. A conserved component of the SRP mechanism is a membrane-bound SRP receptor, denoted FtsY in bacteria. Plant genomes encode FtsY homologs that are targeted to the chloroplast (cpFtsY). To investigate the in vivo roles of cpFtsY, we characterized maize cpFtsY and maize mutants having a Mu transposon insertion in the corresponding gene (chloroplast SRP receptor1, or csr1). Maize cpFtsY accumulates to much higher levels in leaf tissue than in roots and stems. Interestingly, it is present at similar levels in etiolated and green leaf tissue and was found to bind the prolamellar bodies of etioplasts. A null cpFtsY mutant, csr1-1, showed a substantial loss of leaf chlorophyll, whereas a "leaky" allele, csr1-3, conditioned a more moderate chlorophyll deficiency. Both alleles caused the loss of various LHCPs and the thylakoid-bound photosynthetic enzyme complexes and were seedling lethal. By contrast, levels of the membrane-bound components of the thylakoid protein transport machineries were not altered. The thylakoid membranes in csr1-1 chloroplasts were unstacked and reduced in abundance, but the prolamellar bodies in mutant etioplasts appeared normal. These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus.

Amino Acid Sequence↗

The Arabidopsis chloroplastic NifU-like protein CnfU, which can act as an iron-sulfur cluster scaffold protein, is required for biogenesis of ferredoxin and photosystem I.

The biosynthesis of iron-sulfur clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of iron-sulfur clusters. Here, we report the identification of two chloroplast-localized NifU-like proteins, AtCnfU-V and AtCnfU-IVb, from Arabidopsis (Arabidopsis thaliana) with high sequence similarity to a cyanobacterial NifU-like protein that was proposed to serve as a molecular scaffold. AtCnfU-V is constitutively expressed in several tissues of Arabidopsis, whereas the expression of AtCnfU-IVb is prominent in the aerial parts. Mutant Arabidopsis lacking AtCnfU-V exhibited a dwarf phenotype with faint pale-green leaves and had drastically impaired photosystem I accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of ferredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vitro activity of iron-sulfur cluster insertion into apo-ferredoxin. When expressed in Escherichia coli cells, AtCnfU-V formed a homodimer carrying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-ferredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis in chloroplasts and thereby is required for biogenesis of ferredoxin and photosystem I.

Amino Acid Sequence↗

SLOW WALKER1, essential for gametogenesis in Arabidopsis, encodes a WD40 protein involved in 18S ribosomal RNA biogenesis.

The progression of mitotic division cycles and synchronous development between and within the male and female reproductive organs are essential for plant sexual reproduction. Little is known about the genetic control of the progression of mitotic cycles of the haploid genome during gametogenesis in higher plants. Here, we report the phenotypic and molecular characterization of an Arabidopsis thaliana mutant, slow walker1 (swa1), in which the progression of the mitotic division cycles of the female gametophyte was disrupted. Confocal microscopy revealed that megagametophyte development was asynchronous in swa1, causing embryo sacs to arrest at two-, four-, or eight-nucleate stages within the same pistil. A delayed pollination experiment showed that a small fraction of the swa1 embryo sacs were able to develop into functional female gametophytes. The swa1 mutation also showed a slight reduction in penetrance through the male gametophyte, although the pollen grains were morphologically normal. Molecular analysis indicates that SWA1 encodes a protein with six WD40 repeats that is localized in the nucleolus in interphase cells. The SWA1 gene is expressed in cells undergoing active cell divisions, including functional megaspores and the female gametophytic cells. RNA interference results indicated that knockout of SWA1 inhibited root growth significantly and led to the accumulation of unprocessed 18S pre-rRNA. These data suggest that SWA1 most likely plays a role in rRNA biogenesis that is essential for the progression of the mitotic division cycles during gametogenesis in plants.

Amino Acid Sequence↗

Chloroplast biogenesis of photosystem II cores involves a series of assembly-controlled steps that regulate translation.

The biogenesis of photosystem II, one of the major photosynthetic protein complexes, involves a cascade of assembly-governed regulation of translation of its major chloroplast-encoded subunits. In Chlamydomonas reinhardtii, the presence of the reaction center subunit D2 is required for the expression of the other reaction center subunit D1, while the presence of D1 is required for the expression of the core antenna subunit apoCP47. Using chimeric genes expressed in the chloroplast, we demonstrate that the decreased synthesis of D1 or apoCP47 in the absence of protein assembly is due to a genuine downregulation of translation. This regulation is mediated by the 5' untranslated region of the corresponding mRNA and originates from negative feedback exerted by the unassembled D1 or apoCP47 polypeptide. However, autoregulation of translation of subunit D1 is not implicated in the recovery from photoinhibition, which involves an increased translation of psbA mRNA in response to the degradation of photodamaged D1. De novo synthesis and repair of photosystem II complexes are independently controlled.

5' Untranslated Regions↗

Arabidopsis SAMT1 defines a plastid transporter regulating plastid biogenesis and plant development.

S-Adenosylmethionine (SAM) is formed exclusively in the cytosol but plays a major role in plastids; SAM can either act as a methyl donor for the biogenesis of small molecules such as prenyllipids and macromolecules or as a regulator of the synthesis of aspartate-derived amino acids. Because the biosynthesis of SAM is restricted to the cytosol, plastids require a SAM importer. However, this transporter has not yet been identified. Here, we report the molecular and functional characterization of an Arabidopsis thaliana gene designated SAM TRANSPORTER1 (SAMT1), which encodes a plastid metabolite transporter required for the import of SAM from the cytosol. Recombinant SAMT1 produced in yeast cells, when reconstituted into liposomes, mediated the counter-exchange of SAM with SAM and with S-adenosylhomocysteine, the by-product and inhibitor of transmethylation reactions using SAM. Insertional mutation in SAMT1 and virus-induced gene silencing of SAMT1 in Nicotiana benthamiana caused severe growth retardation in mutant plants. Impaired function of SAMT1 led to decreased accumulation of prenyllipids and mainly affected the chlorophyll pathway. Biochemical analysis suggests that the latter effect represents one prominent example of the multiple events triggered by undermethylation, when there is decreased SAM flux into plastids.

Anion Transport Proteins↗