Specific role of phosphatidylglycerol and functional overlaps with other thylakoid lipids in Arabidopsis chloroplast biogenesis

Key message

With phosphate deficiency, the role of phosphatidylglycerol is compensated by increased glycolipid content in thylakoid membrane biogenesis but not photosynthetic electron transport in Arabidopsis chloroplasts.

Abstract

In plants and cyanobacteria, anionic phosphatidylglycerol (PG) is the only major phospholipid in thylakoid membranes, where neutral galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are predominant. In addition to provide a lipid bilayer matrix, PG plays a specific role in photosynthetic electron transport. Non-phosphorous sulfoquinovosyldiacylglycerol (SQDG) is another anionic lipid in thylakoids; it substitutes for PG under phosphate (Pi) deficiency to maintain proper balance of anionic charge in thylakoid membranes. Although the crucial role of PG in photosynthesis has been deeply analyzed in cyanobacteria, its physiological function in seed plants other than photosynthesis remains unclear. To reveal specific roles of PG and functional overlaps with other thylakoid lipids, we characterized a PG-deficient Arabidopsis mutant (pgp1-2) under Pi-controlled conditions. Under Pi-sufficient conditions, the proportion of PG and other thylakoid lipids was decreased in pgp1-2, which led to severe disruption of thylakoid membrane biogenesis. Under Pi-deficient conditions, the proportion of all glycolipids in the mutant was greatly increased, with that of PG further decreased. In Pi-deficient pgp1-2, thylakoid membranes remarkably developed, which was accompanied by a change in nucleoid morphology and restored expression of nuclear- and plastid-encoded photosynthesis genes. Increase in glycolipid content with Pi deficiency may compensate for the loss of PG in terms of thylakoid membrane biogenesis. Although Pi deficiency increased chlorophyll and photosynthesis protein content in pgp1-2, it critically decreased photochemical activity in PSII. Further deprivation of PG in photosynthesis complexes may abolish the PSII activity in Pi-deficient pgp1-2, which suggests that glycolipids cannot replace PG in photosynthesis.

     

The Involvement of the Complexes of Photosystems in the Organization of Chloroplast Ultrastructure in Pea Mutants

We studied the involvement of pigment-protein complexes of photosystems (PS) in the development and spatial arrangement of thylakoids in chloroplasts of pea (Pisum sativum L.) leaves. The initial line (cv. Torsdag) and its mutants, chlorotica 2004 displaying primary disturbances in the PSI reaction centers and chlorotica 2014 containing only 50% of chlorophyll and, as a sequence, the reduced amount of all pigment-protein complexes. A proportional decrease in the content of PSI and PSII complexes in the chlorotica 2014 mutant resulted in a partial reduction of the whole chloroplast membrane system, whereas grana and stroma thylakoid regions were well developed. In contrast, a loss of only 20% of chlorophyll and destruction of PSI complexes in the chlorotica 2004 mutant by 50% resulted in the destruction of stroma thylakoid regions and disturbed longitudinal thylakoid and grana orientation. It was concluded that protein-protein interactions in pigment-protein complexes played a key role in the structure of thylakoid membranes and their longitudinal orientation.     

Disturbance of chlorophyll biosynthesis at Mg branch affects the chloroplast ROS homeostasis and Ca<Superscript>2+</Superscript> signaling in <Emphasis Type="Italic">Pisum sativum</Emphasis>

Reactive oxygen species (ROS) and calcium (Ca²⁺), two crucial intracellular signaling molecules, have been reported to play important roles in chlorophyll biosynthesis. In this study, we aimed to investigate whether disturbance of chlorophyll synthesis affects chloroplast ROS and Ca²⁺ homeostases. Chlorophyll biosynthesis was inhibited at the Mg branch by virus-induced gene silencing (VIGS) of CHLI gene encoding the Mg chelatase CHLI subunit in pea (Pisum sativum). Subsequently, ROS and intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in these chlorophyll-deficient pea plants were evaluated by histochemical and fluorescent staining assays. The results showed that the superoxide anion and hydrogen peroxide were predominantly generated in chloroplasts of the yellow leaves of pea VIGS-CHLI plants. The expression of genes encoding chloroplast antioxidant enzymes (CuZn-superoxide dismutase, ascorbate peroxidase, glutathione reductase, phospholipid glutathione peroxidase, peroxiredoxin and thioredoxins) were also decreased in the leaves of VIGS-CHLI plants compared with the control plants. Additionally, the [Ca²⁺]_i were significantly reduced in the yellow leaves of VIGS-CHLI plants compared with the green leaves of VIGS-GFP control plants. The expression of genes encoding Ca²⁺ signaling related proteins (thylakoid Ca²⁺ transporter, calmodulins and calcineurin B-like protein) was down-regulated in yellow VIGS-CHLI leaves. These results indicate that inhibition of chlorophyll biosynthesis at the Mg branch by silencing CHLI affects chloroplast ROS homeostasis and Ca²⁺ signaling and down-regulates the expression of ROS scavenging genes and Ca²⁺ signaling related genes.     

Disturbed structure and function of chloroplasts during blocked biosynthesis of 5-aminolevulinic acid in the light

Xantha-702 mutant of cotton (Gossypium hirsutum L.) proved to have blocked synthesis of 5-aminolevulinic acid in the light. Accordingly, mutant leaves accumulated 2–5% chlorophyll of baseline. Mutant plants demonstrated disturbed production of pigment-protein complexes of photosystems I (PSI) and II (PSII) and generation of the chloroplast membrane system blocked at the early stages, largely, at the stages of vesicles and single short thylakoid. The functional activity of the PSI and PSII reaction centers was close to zero. Only the chlorophyll a/b light-harvesting complexes of PSI and PSII with the chlorophyll fluorescence peaks at 728 and 681 nm, respectively, were produced in the xantha-702 mutant. We propose that the genetic block of 5-aminolevunilic acid biosynthesis in the light in the xantha-702 mutant disturbs the formation and activity of the complexes of the reaction centers of PS-I and PS-II and inhibits the development of the whole membrane system of chloroplasts.     

Functional characterization of chloroplast-targeted RbgA GTPase in higher plants

Key message

Plant RbgA GTPase is targeted to chloroplasts and co-fractionated with chloroplast ribosomes, and plays a role in chloroplast rRNA processing and/or ribosome biogenesis.

Abstract

Ribosome Biogenesis GTPase A (RbgA) homologs are evolutionarily conserved GTPases that are widely distributed in both prokaryotes and eukaryotes. In this study, we investigated functions of chloroplast-targeted RbgA. Nicotiana benthamiana RbgA (NbRbgA) and Arabidopsis thaliana RbgA (AtRbgA) contained a conserved GTP-binding domain and a plant-specific C-terminal domain. NbRbgA and AtRbgA were mainly localized in chloroplasts, and possessed GTPase activity. Since Arabidopsis rbgA null mutants exhibited an embryonic lethal phenotype, virus-induced gene silencing (VIGS) of NbRbgA was performed in N. benthamiana. NbRbgA VIGS resulted in a leaf-yellowing phenotype caused by disrupted chloroplast development. NbRbgA was mainly co-fractionated with 50S/70S ribosomes and interacted with the chloroplast ribosomal proteins cpRPL6 and cpRPL35. NbRbgA deficiency lowered the levels of mature 23S and 16S rRNAs in chloroplasts and caused processing defects. Sucrose density gradient sedimentation revealed that NbRbgA-deficient chloroplasts contained reduced levels of mature 23S and 16S rRNAs and diverse plastid-encoded mRNAs in the polysomal fractions, suggesting decreased protein translation activity in the chloroplasts. Interestingly, NbRbgA protein was highly unstable under high light stress, suggesting its possible involvement in the control of chloroplast ribosome biogenesis under environmental stresses. Collectively, these results suggest a role for RbgA GTPase in chloroplast rRNA processing/ribosome biogenesis, affecting chloroplast protein translation in higher plants.

     

Genetic analysis and fine-mapping of a new rice mutant, <Emphasis Type="Italic">white and lesion mimic leaf1</Emphasis>

Rice (Oryza sativa L.) leaf color mutants are excellent models for studying chlorophyll biosynthesis and chloroplast development. In this study, we isolated a stable genetic white and lesion mimic leaf1 (wlml1) mutant from an ethyl methanesulfonate (EMS)-mutagenized population of the indica cultivar TN1. Compared with wild-type TN1, the wlml1 mutant had lower contents of chlorophyll and carotenoids, altered chloroplast ultrastructure, and altered regulation of genes associated with chlorophyll metabolism and chloroplast development. In addition, lesions formed on the leaves of wlml1 plants grown at 20 °C and genes related to disease resistance and antioxidant functions were up-regulated; by contrast, the mutant phenotype was partially suppressed at 28 °C. These findings indicated that WLML1 might play a role in chlorophyll metabolism and chloroplast development, as well as in biotic and abiotic stress responses. Genetic analysis showed that WLML1 was controlled by a recessive nuclear gene, and map-based cloning delimited WLML1 to a 159.7-kb region on chromosome 4 that includes 30 putative open reading frames. Based on these findings, the wlml1 mutant will be a good genetic material for further studies on chlorophyll metabolism and stress responses in rice.     

A putative plastidial adenine nucleotide transporter,BRITTLE1-3, plays an essential role in regulating chloroplast development in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Differentiation from proplastids into chloroplasts is a light- and energy-dependent process. How this process is regulated is still poorly understood at the molecular level. We herein report a new putative plastidial adenine nucleotide transporter, BRITTLE1-3 (referred to as OsBT1-3), encoded by the rice (Oryza sativa) White Stripe Leaf 2 (WSL2) gene. Loss of OsBT1-3 function results in defective chloroplast biogenesis, severely reduced photosynthetic efficiency, and finally a white stripe leaf phenotype in the first four leaves. The expression levels of genes related to chlorophyll biosynthesis and photosynthesis are drastically reduced, accompanied with over accumulation of reactive oxygen species (ROS) in the wsl2 mutant. OsBT1-3 is targeted to the chloroplasts and it expresses in almost all tissues in plants, especially in young leaves. OsBT1-3 consists of 419 amino acids and exhibits features of all mitochondrial carrier proteins, including a typical transmembrane-spanning domain and a highly conserved sequence motif designated as the ‘mitochondrial energy transfer signatures’. Phylogenetic analysis shows that OsBT1-3 is a putative plastidial adenine nucleotide transporter and is most closely related to ZmBT1-2. Together, these observations suggest that the new putative adenine nucleotide transporter, OsBT1-3, plays an essential role in regulating chloroplast biogenesis and maintenance of ROS homeostasis during rice seedling de-etiolation.     

A single nucleotide mutation of <Emphasis Type="Italic">IspF</Emphasis> gene involved in the MEP pathway for isoprenoid biosynthesis causes yellow-green leaf phenotype in rice

Key message

We identified IspF gene through yellow-green leaf mutant 505ys in rice. OsIspF was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. On expression levels of genes in this mutant, OsIspF itself and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase were all up-regulated, however, among eight genes associated with photosynthesis, only psaA, psaN and psbA genes for three reaction center subunits of photosystem obviously changed.

Abstract

Isoprenoids are the most abundant natural compounds in all organisms, which originate from the basic five-carbon units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, IPP and DMAPP are synthesized through two independent pathways, the mevalonic acid pathway in cytoplasm and the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids. The MEP pathway comprises seven enzymatic steps, in which IspF is the fifth enzyme. So far, no IspF gene has been identified in monocotyledonous plants. In this study, we isolated a leaf-color mutant, 505ys, in rice (Oryza sativa). The mutant displayed yellow-green leaf phenotype, reduced level of photosynthetic pigments, and arrested development of chloroplasts. By map-based cloning of this mutant, we identified OsIspF gene (LOC_Os02g45660) showing significant similarity to IspF gene of Arabidopsis, in which a missense mutation occurred in the mutant, resulting in an amino acid change in the encoded protein. OsIspF gene was expressed in all tissues detected, and its encoded protein was targeted to the chloroplast. Further, the mutant phenotype of 505ys was complemented by transformation with the wild-type OsIspF gene. Therefore, we successfully identified an IspF gene in monocotyledonous plants. In addition, real-time quantitative RT-PCR implied that a positive regulation could exist between the OsIspF gene and the genes encoding other enzymes of the MEP pathway and chlorophyll synthase. At the same time, it also implied that the individual genes involved in the MEP pathway might differentially regulated expression levels of the genes associated with photosynthesis.

     

A rice <Emphasis Type="Italic">White-stripe leaf3</Emphasis> (<Emphasis Type="Italic">wsl3</Emphasis>) mutant lacking an HD domain-containing protein affects chlorophyll biosynthesis and chloroplast development

Leaf-color mutants are ideal genetic materials for understanding the mechanism of chloroplast development and chlorophyll (Chl) biosynthesis. Here we isolated and identified a new leaf-color mutant of rice, named white-stripe leaf3 (wsl3), from a ⁶⁰Co-irradiated mutant pool. The wsl3 mutant displayed a visible white-stripe leaf in both young seedlings and flag leaves of mature plant. Chl content in homozygous wsl3 mutant was approximately 47% of that in the wild type. Besides, chloroplast development in the mutant was severely arrested. By a map-based cloning strategy, the wsl3 gene was finely confined to a 50.8 kb region on chromosome 1. Moreover, a 9-bp deletion was identified in the genomic region of LOC_Os01g01920, which encodes an HD (histidine and aspartic acid) domaincontaining protein. Genetic complementation confirmed that LOC_Os01g01920 could recover the lesion of wsl3 mutation. Real-time PCR analyses showed that the expression levels of WSL3 were the highest in young and flag leaves among various tissues, and most of the genes associated with Chl biosynthesis were significantly down-regulated in the wsl3 mutant. Meanwhile, in contrast to many nuclear gene-encoded phage-type RNA polymerase(s) (NEP) transcribed genes were up-regulated, most of plastid-encoded bacterialtype RNA polymerase (PEP) transcribed genes were downregulated. These results demonstrated that the WSL3 gene, as an HD domain-containing protein, is involved in chl biosynthesis and chloroplast development in rice.     

Cloning and functional characterization of the <Emphasis Type="Italic">SmNCED3</Emphasis> in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

As one of the most important phytohormones, the abscisic acid (ABA) is often used to breed stress-tolerant crop lines with both higher yields and active ingredient contents. In higher plants, the 9-cis-epoxycarotenoid dioxygenase (NCED) has been found to be a regulatory enzyme involved in ABA biosynthesis. In research, the novel gene SmNCED3 was isolated from S. miltiorrhiza. The open reading frame of SmNCED3 was 1725-bp, and it was encoding 574 amino acids with a calculated molecular mass of 63,822 kDa, which was verified by the expression of SmNCED3 in E. coli. The deduced SmNCED3 amino acid sequence had high sequence homology with NCED sequences from other plants and contained a putative chloroplast transit targeting signal peptide at its N terminus. Phylogenetic analysis demonstrated that SmNCED3 had a closer affinity to NCED3 in Arabidopsis thaliana (AtNCED3). The 1732-bp 5′ flanking sequence of SmNCED3 was also cloned. It contained several phytohormone response elements, biotic or abiotic stress-related elements, and plant development-related elements. Real-time PCR revealed that SmNCED3 was highly expressed in leaves, and was strongly induced by exogenous ABA. A subcellular localization experiment indicated that SmNCED3 was located in chloroplast stroma, chloroplast membranes, and thylakoid membranes. The overexpression of SmNCED3 promoted ABA accumulation. These results indicated that SmNCED3 might be a rate-limiting gene regulating ABA biosynthesis, and improving abiotic stresses tolerance and active ingredient contents in plants.     

Biogenesis of chlorophyll-binding proteins under iron stress in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The biogenesis of chlorophyll-binding proteins under iron stress has been investigated in vivo in a chlN deletion mutant of Synechocystis sp. PCC 6803. The chlN gene encodes one subunit of the light-independent protochlorophyllide reductase. The mutant is unable to synthesize chlorophyll in darkness, causing chlorophyll biosynthesis to become light dependent. When the mutant was propagated in darkness, essentially no chlorophyll and photosystems were detected. Upon return of the chlN deletion mutant to light, 77 K fluorescence emission spectra and oxygen evolution of greening cells under iron-sufficient or-deficient conditions were measured. The gradual blue shift of the photosystem I (PS I) peak upon greening under iron stress suggested the structural alteration of newly synthesized PS I. Furthermore, the rate of biogenesis of PS II was delayed under iron stress, which might be due to the presence of IsiA.