Chloroplast Localization Research Articles

Bioinformatic analysis of maize gene encoding starch branching enzyme SBEIIb.

Purpose. Investigation of maize ae1 gene polymorphism by bioinformatic methods. Methods. Global and local alignment of the nucleotide and amino acid sequences, in silico translation and transcription, translates modeling, pri­mers design, phylogenetic analysis. Results. 255 nucleotide sequences of maize аe1 gene, 500 amino acid sequences of homology translates of maize ae1 gene (SBEIIb enzyme homologs) and 100 mRNA expressed from the maize ae1 gene were analyzed to establish phylogenetic relationships. Polymorphism of maize ae1 gene different regions was investigated by bioinformatic methods. Modeling of the maize enzyme SBEIIb was performed. Conclusions. According to the results of amino acid sequences of SBEIIb enzyme homologs alignment, it was found that ae1 gene orthologs are pre­sent only in monocots, paralogs – in monocots, dicots, and other taxa, including algae and animals. Based on the results of alignment of plants mRNA from which enzyme SBEIIb is translated, maize ae1 gene orthologs and the nearest paralogs encoding starch branching enzymes with chloroplast localization were defined; this suggests a possible origin of ae1 gene due to duplication of the gene encoding the 1,4-alpha-glucan-branching enzyme 2 with chloroplast or amyloplast localization. In the maize ae1 gene structure, regions were found that include polymorphic sites not defined previously. For the polymorphic sites design primers were developed that allowed to differentiate the maize lines. It was determined that the detection of polymorphism in theory can influence the enzyme function and, as a result, change the concentration of amylopectin in maize grain.

TaTypA, a Ribosome-Binding GTPase Protein, Positively Regulates Wheat Resistance to the Stripe Rust Fungus.

Tyrosine phosphorylation protein A (TypA/BipA) belongs to the ribosome-binding GTPase superfamily. In many bacterial species, TypA acts as a global stress and virulence regulator and also mediates resistance to the antimicrobial peptide bactericidal permeability-increasing protein. However, the function of TypA in plants under biotic stresses is not known. In this study, we isolated and functionally characterized a stress-responsive TypA gene (TaTypA) from wheat, with three copies located on chromosomes 6A, 6B, and 6D, respectively. Transient expression assays indicated chloroplast localization of TaTypA. The transcript levels of TaTypA were up-regulated in response to treatment with methyl viologen, which induces reactive oxygen species (ROS) in chloroplasts through photoreaction, cold stress, and infection by an avirulent strain of the stripe rust pathogen. Knock down of the expression of TaTypA through virus-induced gene silencing decreased the resistance of wheat to stripe rust accompanied by weakened ROS accumulation and hypersensitive response, an increase in TaCAT and TaSOD expression, and an increase in pathogen hyphal growth and branching. Our findings suggest that TaTypA contributes to resistance in an ROS-dependent manner.

Molecular characterization of XopAG effector AvrGf2 from Xanthomonas fuscans ssp. aurantifolii in grapefruit.

Xanthomonas fuscans ssp. aurantifolii group C strains exhibit host specificity on different citrus species. The strains possess a type III effector, AvrGf2, belonging to the XopAG effector gene family, which restricts host range on citrus. We dissected the modular nature and mode of action of AvrGf2 in grapefruit resistance. XopAG effectors possess characteristic features, such as a chloroplast localization signal, a cyclophilin-binding domain characteristic amino acid sequence motif (GPLL) and a C-terminal domain-containing CLNAxYD. Mutation of GPLL to AASL in AvrGf2 abolished the elicitation of the hypersensitive response (HR), whereas mutation of only the first amino acid to SPLL delayed the HR in grapefruit. Yeast two-hybrid experiments showed strong interaction of AvrGf2 with grapefruit cyclophilin (GfCyp), whereas AvrGf2-SPLL and AvrGf2-AASL mutants showed weak and no interaction, respectively. Molecular modelling and insilico docking studies for the cyclophilin-AvrGf2 interaction predicted the binding of citrus cyclophilins (CsCyp, GfCyp) to hexameric peptides spanning the cyclophilin-binding domain of AvrGf2 and AvrGf2 mutants (VAGPLL, VASPLL and VAAASL) with affinities equivalent to or better than a positive control peptide (YSPSA) previously demonstrated to bind CsCyp. In addition, the C-terminal domain of XopAG family effectors contains a highly conserved motif, CLNAxYD, which was identified to be crucial for the induction of HR based on site-directed mutagenesis (CLNAxYD to CASAxYD). Our results suggest a model in which grapefruit cyclophilin promotes a conformational change in AvrGf2, thereby triggering the resistance response.

Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis.

Plants have varying abilities to tolerate chilling (low but not freezing temperatures), and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance.

Rice OsVAMP714, a membrane-trafficking protein localized to the chloroplast and vacuolar membrane, is involved in resistance to rice blast disease.

Membrane trafficking plays pivotal roles in many cellular processes including plant immunity. Here, we report the characterization of OsVAMP714, an intracellular SNARE protein, focusing on its role in resistance to rice blast disease caused by the fungal pathogen Magnaporthe oryzae. Disease resistance tests using OsVAMP714 knockdown and overexpressing rice plants demonstrated the involvement of OsVAMP714 in blast resistance. The overexpression of OsVAMP7111, whose product is highly homologous to OsVAMP714, did not enhance blast resistance to rice, implying a potential specificity of OsVAMP714 to blast resistance. OsVAMP714 was localized to the chloroplast in mesophyll cells and to the cellular periphery in epidermal cells of transgenic rice plant leaves. We showed that chloroplast localization is critical for the normal OsVAMP714 functioning in blast resistance by analyzing the rice plants overexpressing OsVAMP714 mutants whose products did not localize in the chloroplast. We also found that OsVAMP714 was located in the vacuolar membrane surrounding the invasive hyphae of M. oryzae. Furthermore, we showed that OsVAMP714 overexpression promotes leaf sheath elongation and that the first 19 amino acids, which are highly conserved between animal and plant VAMP7 proteins, are crucial for normal rice plant growths. Our studies imply that the OsVAMP714-mediated trafficking pathway plays an important role in rice blast resistance as well as in the vegetative growth of rice.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

Molecular characterization of lipoxygenase genes and their expression analysis against biotic and abiotic stresses in Panax ginseng

Lipoxygenase (LOX) belongs to a family of non-heme-iron-containing fatty acid dioxygenases that are widely distributed in plants and animals. LOX involved in the synthesis of jasmonic acid and six-carbon (C6) volatiles which is necessary for plant growth and responses to a wide range of biotic and abiotic stresses. We have isolated and characterized LOX cDNA clones from Panax ginseng Meyer. From their deduced amino acid sequences, two diverse classes of 9-LOX (LOX1, LOX2, and LOX3) and 13-LOX (LOX4, LOX5) are defined in P. ginseng. A GenBank Blast X search revealed that the deduced amino acid of PgLOXs share a high degree of homology with LOX from other plants and mammals especially in three distinct motifs; motif1 harboring iron binding regions, motif2 and motif3. Chloroplast localization was predicted for PgLOX5. PgLOXs displayed organ-specific expression, highly expressed in aerial parts of the plant such as 3-year old flower, stem and leaf tissues. PgLOXs mRNAs were elevated strongly by bacterial infection. Expression of PgLOXs was differentially induced in ginseng not only by mechanical damage and methyl jasmonate but also after exposure to withholding water. Ginseng 13-LOXs positively respond to wounding that may involve in production of C6 volatiles and jasmonic acid at the wounded sites. However, the higher expression of PgLOX3 by water deficit and 82 % of the nucleotide sequence identity with the EST from severe drought-stressed leaves of Populus (CU229089.1) at +6371 bp downstream of PgLOX3 genomic DNA structure can suggest drought tolerance role for PgLOX3. Ginseng LOX genes have different expression pattern which may suggest different specific function against various environmental stresses.

The chloroplastic DEVH-box RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 involved in plasmodesmata regulation is required for group II intron splicing.

INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) encodes a putative DEVH-box RNA helicase originally identified through a genetic screening for Arabidopsis mutants altered in plasmodesmata (PD) aperture. Depletion of ISE2 also affects chloroplasts activity, decreases accumulation of photosynthetic pigments and alters expression of photosynthetic genes. In this work, we show the chloroplast localization of ISE2 and decipher its role in plastidic RNA processing and, consequently, PD function. Group II intron-containing RNAs from chloroplasts exhibit defective splicing in ise2 mutants and ISE2-silenced plants, compromising plastid viability. Furthermore, RNA immunoprecipitation suggests that ISE2 binds in vivo to several splicing-regulated RNAs. Finally, we show that the chloroplast clpr2 mutant (defective in a subunit of a plastidic Clp protease) also exhibits abnormal PD function during embryogenesis, supporting the idea that chloroplast RNA processing is required to regulate cell-cell communication in plants.

A proteome map of a quadruple photoreceptor mutant sustains its severe photosynthetic deficient phenotype

Light is the environmental factor that most affects plant growth and development through its impact on photomorphogenesis and photosynthesis. A quadruple photoreceptor mutant lacking four of the most important photoreceptors in plants, phytochromes A and B (phyA, phyB) and cryptochromes 1 and 2 (cry1, cry2), is severely affected in terms of growth and development. Previous studies have suggested that in addition to a photomorphogenic disorder, the phyA phyB cry1 cry2 quadruple mutant might have severe alterations in photosynthetic ability. Here, we investigated the photosynthetic processes altered in the quadruple mutant and performed a proteomic profiling approach to identify some of the proteins involved. The phyA phyB cry1 cry2 quadruple mutant showed reduced leaf area and total chlorophyll content. Photosynthetic rates at high irradiances were reduced approximately 65% compared to the wild type (WT). Light-saturated photosynthesis and the response of net CO2 exchange to low and high internal CO2 concentrations suggest that the levels or activity of the components of the Calvin cycle and electron transport might be reduced in the quadruple mutant. Most of the under-expressed proteins in the phyA phyB cry1 cry2 quadruple mutant consistently showed a chloroplastic localization, whereas components of the Calvin cycle and light reaction centers were overrepresented. Additionally, Rubisco expression was reduced threefold in the phyA phyB cry1 cry2 quadruple mutant.Together, these results highlight the importance of the phytochrome and cryptochrome families in proper autotrophy establishment in plants. They also suggest that an overall limitation in the chlorophyll levels, expression of Rubisco, and enzymes of the Calvin Cycle and electron transport that affect ribulose-1,5-biphosphate (RuBP) regeneration reduced photosynthetic capacity in the phyA phyB cry1 cry2 quadruple mutant.

Molecular identification and functional characterization of the first Nα-acetyltransferase in plastids by global acetylome profiling.

Protein Nα-terminal acetylation represents one of the most abundant protein modifications of higher eukaryotes. In humans, six Nα-acetyltransferases (Nats) are responsible for the acetylation of approximately 80% of the cytosolic proteins. N-terminal protein acetylation has not been evidenced in organelles of metazoans, but in higher plants is a widespread modification not only in the cytosol but also in the chloroplast. In this study, we identify and characterize the first organellar-localized Nat in eukaryotes. A primary sequence-based search in Arabidopsis thaliana revealed seven putatively plastid-localized Nats of which AT2G39000 (AtNAA70) showed the highest conservation of the acetyl-CoA binding pocket. The chloroplastic localization of AtNAA70 was demonstrated by transient expression of AtNAA70:YFP in Arabidopsis mesophyll protoplasts. Homology modeling uncovered a significant conservation of tertiary structural elements between human HsNAA50 and AtNAA70. The in vivo acetylation activity of AtNAA70 was demonstrated on a number of distinct protein Nα-termini with a newly established global acetylome profiling test after expression of AtNAA70 in E. coli. AtNAA70 predominately acetylated proteins starting with M, A, S and T, providing an explanation for most protein N-termini acetylation events found in chloroplasts. Like HsNAA50, AtNAA70 displays Nε-acetyltransferase activity on three internal lysine residues. All MS data have been deposited in the ProteomeXchange with identifier PXD001947 (http://proteomecentral.proteomexchange.org/dataset/PXD001947).

Arabidopsis casein kinase 2 α4 subunit regulates various developmental pathways in a functionally overlapping manner

Casein kinase 2 (CK2) is an essential and well-conserved Ser/Thr kinase that regulates proteins in a posttranslational manner. CK2 has been shown to affect a large number of developmental processes across eukaryotes. It is a tetrameric protein composed of a dimer of alpha (catalytic) and beta (regulatory) subunit each. In our previous study we showed that three of the four CK2 α subunits in Arabidopsis act in a functionally redundant manner to regulate various developmental pathways. In this study we constructed two independent CK2 α4 RNAi lines in the CK2 alpha triple mutant background. Through functional characterization of these RNAi lines we show that the fourth α subunit in Arabidopsis also functions redundantly in regulating ABA response, lateral root formation and flowering time. CK2 α4-GFP localizes to the chloroplast in transgenic Arabidopsis seedlings, consistent with the presence of a chloroplast localization signal at the amino-terminus of CK2 α4 subunit. Taken together, our results suggest a functionally overlapping role for the CK2 α4 subunit in regulating various developmental processes in plants.

Polyphenol oxidase in leaves: is there any significance to the chloroplastic localization?

Polyphenol oxidase (PPO) catalyses the oxidation of monophenols and/or o-diphenols to o-quinones with the concomitant reduction of oxygen to water which results in protein complexing and the formation of brown melanin pigments. The most frequently suggested role for PPO in plants has been in defence against herbivores and pathogens, based on the physical separation of the chloroplast-localized enzyme from the vacuole-localized substrates. The o-quinone-protein complexes, formed as a consequence of cell damage, may reduce the nutritional value of the tissue and thereby reduce predation but can also participate in the formation of structural barriers against invading pathogens. However, since a sufficient level of compartmentation-based regulation could be accomplished if PPO was targeted to the cytosol, the benefit derived by some plant species in having PPO present in the chloroplast lumen remains an intriguing question. So is there more to the chloroplastic location of PPO? An interaction between PPO activity and photosynthesis has been proposed on more than one occasion but, to date, evidence either for or against direct involvement has been equivocal, and the lack of identified chloroplastic substrates remains an issue. Similarly, PPO has been suggested to have both pro- and anti-oxidant functions. Nevertheless, several independent lines of evidence suggest that PPO responds to environmental conditions and could be involved in the response of plants to abiotic stress. This review highlights our current understanding of the in vivo functions of PPO and considers the potential opportunities it presents for exploitation to increase stress tolerance in food crops.

Chloroplast localization of Cry1Ac and Cry2A protein--an alternative way of insect control in cotton.

BackgroundInsects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants.ResultsSuccessful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of Cry1Ac and 0.568 μg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein.ConclusionLocating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Identification and characterization of chloroplast casein kinase II from Oryza sativa (rice).

Plastid casein kinase II is an important regulator of transcription, posttranscriptional processes, and, most likely, different metabolic functions in dicotyledonous species. Here we report the identification and characterization of pCKII from the monocotyledonous species Oryza sativa. OspCKII activity was enriched from isolated rice chloroplasts using heparin-Sepharose chromatography, in which it co-elutes with the transcriptionally active chromosome (TAC) and several ribosomal proteins. Inclusion mass scanning of the kinase-active fraction identified the gene model for OspCKII. Transient expression of GFP fused to the 184 N-terminal amino acids of the OspCKII sequence in rice confirmed the chloroplastic localization of the kinase. OspCKII activity shows the characteristic features of casein kinase II, such as the utilization of GTP as phosphate donor, inhibition by low concentrations of heparin and poly-lysine, and utilization of the canonical pCKII motif E-S-E-G-E in the model substrate RNP29. Phosphoproteome analysis of a protein extract from rice leaves combined with a meta-analysis with published phosphoproteomics data revealed differences in the target protein spectrum between rice and Arabidopsis. Consistently, several pCKII phosphorylation sites in dicotyledonous plants are not conserved in monocots and algae, suggesting that details of pCKII regulation in plastids have changed during evolution.

Cosuppression of the chloroplast localized molecular chaperone HSP90.5 impairs plant development and chloroplast biogenesis in Arabidopsis.

BackgroundHSP90.5 is a chloroplast localized HSP90 family molecular chaperone in Arabidopsis, and it has been implicated in plant abiotic stress resistance, photomorphogenesis and nuclear-encoded protein import into the chloroplast. However, how these processes are controlled by HSP90 is not well understood. To understand the role of HSP90.5 in chloroplast function and biogenesis, in this study, we generated transgenic Arabidopsis plants that overexpress a C-terminally FLAG-tagged HSP90.5. By characterizing three HSP90.5 cosuppression lines, we demonstrated the essential role of HSP90.5 in plant growth and chloroplast biogenesis.ResultsImmunoblotting and quantitative PCR analyses revealed three independent HSP90.5 cosuppressing transgenic lines. All three cosuppression lines displayed a certain degree of variegated phenotype in photosynthetic tissues, and the cosuppression did not affect the expression of cytosolic HSP90 isoforms. HSP90.5 cosuppression was shown to be developmentally regulated and occurred mostly at late developmental stage in adult leaves and inflorescence tissues. HSP90.5 cosuppression also caused significantly reduced rosette leaf growth, transient starch storage, but did not affect rosette leaf initiation or inflorescence production, although the fertility was reduced. Isolation of chloroplasts and size exclusion chromatography analysis indicated that the FLAG at the HSP90.5 C-terminus does not affect its proper chloroplast localization and dimerization. Finally, transmission electron microscopy indicated that chloroplast development in HSP90.5 cosuppression leaves was significantly impaired and the integrity of chloroplast is highly correlated to the expression level of HSP90.5.ConclusionWe thoroughly characterized three HSP90.5 cosuppression lines, and demonstrated that properly controlled expression of HSP90.5 is required for plant growth and development in many tissues, and especially essential for chloroplast thylakoid formation. Since the homozygote of HSP90.5 knockout mutant is embryonically lethal, this study provides transgenic lines that mimic the conditional knockout line or siRNA line of the essential HSP90.5 gene in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-0500-7-643) contains supplementary material, which is available to authorized users.

Functional screening of a novel Δ15 fatty acid desaturase from the coccolithophorid Emiliania huxleyi

The coccolithophorid Emiliania huxleyi is a bloom-forming marine phytoplankton thought to play a key role as a biological pump that transfers carbon from the surface to the bottom of the ocean, thus contributing to the global carbon cycle. This alga is also known to accumulate a variety of polyunsaturated fatty acids. At 25°C, E. huxleyi produces mainly 14:0, 18:4n−3, 18:5n−3 and 22:6n−3. When the cells were transferred from 25°C to 15°C, the amount of unsaturated fatty acids, i.e. 18:1n−9, 18:3n−3 and 18:5n−3, gradually increased. Among the predicted desaturase genes whose expression levels were up-regulated at low temperature, we identified a gene encoding novel ∆15 fatty acid desaturase, EhDES15, involved in the production of n−3 polyunsaturated fatty acids in E. huxleyi. This desaturase contains a putative transit sequence for localization in chloroplasts and a ∆6 desaturase-like domain, but it does not contain a cytochrome b5 domain nor typical His-boxes found in ∆15 desaturases. Heterologous expression of EhDES15 cDNA in cyanobacterium Synechocystis sp. PCC 6803 cells increased the level of n−3 fatty acid species, which are produced at low levels in wild-type cells grown at 30°C. The orthologous genes are only conserved in the genomes of prasinophytes and cryptophytes. The His-boxes conserved in orthologues varied from that of the canonical ∆15 desaturases. These results suggested the gene encodes a novel ∆15 desaturase responsible for the synthesis of 18:3n−3 from 18:2n−6 in E. huxleyi.

Genome-wide identification and bioinformatic analysis of PPR gene family in tomato

Pentatricopeptide repeats (PPRs) genes constitute one of the largest gene families in plants, which play a broad and essential role in plant growth and development. In this study, the protein sequences annotated by the tomato (S. lycopersicum L.) genome project were screened with the Pfam PPR sequences. A total of 471 putative PPR-encoding genes were identified. Based on the motifs defined in A. thaliana L., protein structure and conserved sequences for each tomato motif were analyzed. We also analyzed phylogenetic relationship, subcellular localization, expression and GO analysis of the identified gene sequences. Our results demonstrate that tomato PPR gene family contains two subfamilies, P and PLS, each accounting for half of the family. PLS subfamily can be divided into four subclasses i.e., PLS, E, E+ and DYW. Each subclass of sequences forms a clade in the phylogenetic tree. The PPR motifs were found highly conserved among plants. The tomato PPR genes were distributed over 12 chromosomes and most of them lack introns. The majority of PPR proteins harbor mitochondrial or chloroplast localization sequences, whereas GO analysis showed that most PPR proteins participate in RNA-related biological processes.

Subcellular localization of dinoflagellate polyketide synthases and fatty acid synthase activity.

Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & Ø. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K.brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [(3) H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K.brevis expressed sequence tag database have similarity (BLASTx e-value <10(-10) ) to PKSs, no transcripts for either Type I (cytosolic) or Type II (chloroplast) FAS are present. Further characterization of the FAS complexes may help to elucidate the functions of the PKS enzymes identified in dinoflagellates.

Identification and characterization of a novel chloroplast/mitochondria co-localized glutathione reductase 3 involved in salt stress response in rice

Glutathione reductases (GRs) are important components of the antioxidant machinery that plants use to respond against abiotic stresses. In rice, one cytosolic and two chloroplastic GR isoforms have been identified. In this work, we describe the cloning and characterization of the full-length cDNA encoding OsGR3, a chloroplast-localized GR that up to now was considered as a non-functional enzyme because of assumed lack of N-terminal conserved domains. The expression of OsGR3 in E. coli validated that it can be translated as a protein with GR activity. OsGR3 shows 76 and 53% identity with OsGR1 (chloroplastic) and OsGR2 (cytosolic), respectively. Phylogenetic analysis revealed 2 chloroplastic GRs in Poaceae species, including rice, sorghum and brachypodium, but only one chloroplastic GR in dicots. A plastid transit peptide is located at the N terminus of OsGR3, and genetic transformation of rice with a GR3-GFP fusion construct further confirmed its localization in chloroplasts. Furthermore, OsGR1 and OsGR3 are also targeted to mitochondria, which suggest a combined antioxidant mechanism in both chloroplasts and mitochondria. However, both isoforms showed a distinct response to salinity: the expression of OsGR3 but not OsGR1 was induced by salt stress. In addition, the transcript level of OsGR3 was greatly increased with salicylic acid treatment but was not significantly affected by methyl jasmonate, dehydration or heat shockstress. Our results provide new clues about the possible roles of functional OsGR3 in salt stress and biotic stress tolerance.

Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents

To understand the photosynthetic basis in a single seed descent line 10 (SSDL10) of wheat contained high ATP in leaves, the chloroplast proteome was compared to SSDL10 and its parents using a combination of 2-DE and MALDI-TOF MS and MS/MS. More than 300 protein spots could be reproducibly detected in the 2D gel. 18 spots were differentially expressed between SSDL10 and the parents, 16 of which were identified by MS with the localization in chloroplasts. These proteins are grouped into diverse functional categories, including Calvin cycle and electron transport in photosynthesis, redox homeostasis, metabolism, and regulation. In addition to Rubisco large subunit, the content of photosynthetic electron transfers such as chlorophyll a-b binding protein, ATP synthase δ subunit, ferredoxin-NADP+ oxidoreductase (FNR) was higher in SSDL10 than in its parents. Furthermore, cyclic electron transfer around photosystem I (CET) was faster in SSDL10 than in the parents. Analysis of NADPH-NBT oxidoreductase activity combined with immuno-detection further revealed that, the activity of two high molecular mass protein complexes containing FNR probably involved, the CET appeared higher in SSDL10 than in the parents. The possible mechanism for the regulative role of CET in photosynthesis in SSDL10 is discussed.