Plastidic Transporter Research Articles

Effects of Trophic Acclimation on Growth and Expression Profiles of Genes Encoding Enzymes of Primary Metabolism and Plastid Transporters of Chlamydomonas reinhardtii.

In this paper, the effect of prolonged trophic acclimation on the subsequent growth of Chlamydomonas reinhardtii batch cultures was studied. The mixotrophic (light + acetate) acclimation stimulated subsequent growth at both mixotrophy and autotrophy conditions and altered the expression profile of genes encoding enzymes of primary metabolism and plastid transporters. Besides the trophic effect, the influence of Chlamydomonas culture growth stage on gene expression was determined. Under mixotrophic conditions, this effect was most pronounced in the first half of the exponential growth with partial retention of the previous acclimation period traits. The autotrophy acclimation effect was more complex and its significance was enhanced at the end of the growth and in the stationary phase.

A metabolic, phylogenomic and environmental atlas of diatom plastid transporters from the model species Phaeodactylum.

Diatoms are an important group of algae, contributing nearly 40% of total marine photosynthetic activity. However, the specific molecular agents and transporters underpinning the metabolic efficiency of the diatom plastid remain to be revealed. We performed in silico analyses of 70 predicted plastid transporters identified by genome-wide searches of Phaeodactylum tricornutum. We considered similarity with Arabidopsis thaliana plastid transporters, transcriptional co-regulation with genes encoding core plastid metabolic pathways and with genes encoded in the mitochondrial genomes, inferred evolutionary histories using single-gene phylogeny, and environmental expression trends using Tara Oceans meta-transcriptomics and meta-genomes data. Our data reveal diatoms conserve some of the ion, nucleotide and sugar plastid transporters associated with plants, such as non-specific triose phosphate transporters implicated in the transport of phosphorylated sugars, NTP/NDP and cation exchange transporters. However, our data also highlight the presence of diatom-specific transporter functions, such as carbon and amino acid transporters implicated in intricate plastid-mitochondria crosstalk events. These confirm previous observations that substrate non-specific triose phosphate transporters (TPT) may exist as principal transporters of phosphorylated sugars into and out of the diatom plastid, alongside suggesting probable agents of NTP exchange. Carbon and amino acid transport may be related to intricate metabolic plastid-mitochondria crosstalk. We additionally provide evidence from environmental meta-transcriptomic/meta- genomic data that plastid transporters may underpin diatom sensitivity to ocean warming, and identify a diatom plastid transporter (J43171) whose expression may be positively correlated with temperature.

Divergence of plastid 2-oxoglutarate “only” transporters away from general transporters by using a cysteine-rich architecture

The common carbon and nitrogen currency, 2-oxoglutarate, could become a valuable resource for nitrogen assimilation and carbon centered biochemical fates. Here in this in silico study, a myriad of factors was used, namely phylogeny, sequence comparisons, and presence and location of clustered cysteines in specific plastid transporters of 2-oxoglutarate, to examine their evolution away from more generalized transporters. This transition would be to adopt the capability of internalizing 2-oxoglutarate alone or with superior specificities at the expense of malate. In phylogeny, the specific 2-oxoglutarate transporters (Cluster 1) are clustered in a separate clade away from 2 clades of general transporters (Cluster 2 and 3). The exclusivity (Cluster 1) and promiscuity of transporters (Cluster 2 and 3) compared to Arabidopsis counterparts characterized prior to this study, were used as a benchmark for my study. Within this mother clade of exclusive transporters, C4 and C3 2-oxoglutarate transporters once again form separate clusters of monophyly. Furthermore, a pattern of Cys –X-X-Cys-X(19)-Cys is conserved within the 2-oxo-glutarate only transporters that is missing in general transporters. Cysteines which are functionally key residues are inferred to be mediating intra- or inter-reactive disulfide bond formation or using a thiol (sulfhydryl) group for transport or to be forming a metal binding site. When a disulfide bond prediction tool was employed, it showed with negligible doubt that the Cys-X-X Cys-X(19) -Cys region was a strong contender for 2 separate disulfide bonds, although the middle cysteine was predicted to be involved in both. In addition, Cluster 2 general Zea mays C4 transporters are shown to be more recalcitrant to mutations of cysteines, compared to Panicum and Oryza counterparts. The study of 2-oxoglutarate and its availability in the chloroplast could play a two-prong role in C4 plants: to be a candidate for synthesis of bundle sheath cell Rubisco enzyme, which makes up ~50% of plant proteins, via ammonia assimilation, and even playing a role in carbon-centered biochemical pathways. This study could greatly facilitate choices in the tinkering of the right transporters for a future C4 rice in a climate change impacted world.

Hydrogen Sulfide: A Novel Gaseous Molecule for Plant Adaptation to Stress

Hydrogen sulfide (H2S) has emerged as a novel gaseous signal molecule with multifarious effects on seed germination, plant growth, development, and physiological processes. Due to its dominant role in plant stress tolerance and cross-adaptation, it is getting more attention nowadays, although it has been largely referred as toxic and environmental hazardous gas. In this review work, we are highlighting the importance of H2S as an essential gaseous molecule to help in signaling, metabolism, and stress tolerance in plants. Firstly, production of H2S from different natural and artificial sources were discussed with its transformation from sulfur (S) to sulfate (SO42−) and then to sulfite (SO32−). The importance of different kinds of transporters that helps to take SO42− from the soil solution was presented. Mainly, these transporters are SULTRs (H+/SO42− cotransporters) and multigene family encodes them. Furthermore, these SULTRs have LAST (Low affinity transport proteins), HAST (High affinity transport proteins), vacuole transporters, and plastid transporters. Since it is well known that there is strong relationship between SO42− and synthesis of hydrogen sulfide or dihydrogen sulfide or sulfane in plant cells. Thus, cysteine (Cys) metabolism through which H2S could be generated in plant cell with the role of different enzymes has been presented. Furthermore, H2S in interaction with other molecules could help to mitigate biotic and abiotic stress. Based on this review work, it can be concluded that H2S has potential to induce cross-adaptation to biotic and abiotic stress; thus, it is recommended that it should be considered in future studies to answer the questions like what are the receptors of H2S in plant cell, where in plants the physiological concentration of H2S is high in response to multiple stress and how it induces cross-adaptation by interaction with other signal molecules.

Plastidial transporters KEA1 and KEA2 at the inner envelope membrane adjust stromal pH in the dark.

Photosynthesis and carbon fixation depend critically on the regulation of pH in chloroplast compartments in the daylight and at night. While it is established that an alkaline stroma is required for carbon fixation, it is not known how alkaline stromal pH is formed, maintained or regulated. We tested whether two envelope transporters, AtKEA1 and AtKEA2, directly affected stromal pH in isolated Arabidopsis chloroplasts using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). External K+ -induced alkalinization of the stroma was observed in chloroplasts from wild-type (WT) plants but not from kea1kea2 mutants, suggesting that KEA1 and KEA2 mediate K+ uptake/H+ loss to modulate stromal pH. While light-stimulated alkalinization of the stroma was independent of KEA1 and KEA2, the rate of decay to neutral pH in the dark is delayed in kea1kea2 mutants. However, the dark-induced loss of a pH gradient across the thylakoid membrane was similar in WT and mutant chloroplasts. This indicates that proton influx from the cytosol mediated by envelope K+ /H+ antiporters contributes to adjustment of stromal pH upon light to dark transitions.

Shift in Expression of the Genes of Primary Metabolism and Chloroplast Transporters in Chlamydomonas reinhardtii under Different Trophic Conditions

Chlamydomonas reinhardtii P.A. Dangeard is a unicellular green alga capable to assimilate acetate. C. reinhardtii growth and metabolism distinctly depend on trophic conditions. Its influence on batch culture interferes with changes in the medium composition and metabolism of microalgae occurring during culture growth. The aim of this work was to estimate the effect of acetate on changes in the expression of 32 genes encoding enzymes of central metabolism and plastid transporters during growth of batch cultures of C. reinhardtii cc-124. In autotrophic conditions, transcription profiles considerably differed from the profiles in the presence of acetate. The strongest influence of trophic conditions was observed in the log phase of growth. In the presence of acetate, a more intense expression of gene ACS2 encoding plastid acetyl-CoA synthase and of the genes encoding subunits of enzymes directing acetyl groups to the synthesis of fatty acids was recorded. Elevated expression of genes PCK1 and PPT1 under mixotrophic conditions may be corresponded to the entry of acetate carbon to gluconeogenesis. In the presence of acetate, a high expression level of the starch metabolism genes was observed. Autotrophic conditions were notable for an elevated accumulation of transcripts of the genes encoding subunits of citrate lyase, which may be responsible for an outflow of acetyl groups from the Krebs cycle. Moreover, a higher level of expression was shown for genes of plastid transporters participating in the export of sugars from plastids, which is associated with the redistribution of reducing power in the cell. It was concluded that, on the level of transcription, similarity between mixotrophic and autotrophic cultures becomes more pronounced in the course of development.

Alteration in the Expression of Genes Encoding Primary Metabolism Enzymes and Plastid Transporters during the Culture Growth of Chlamydomonas reinhardtii

Alteration in the Expression of Genes Encoding Primary Metabolism Enzymes and Plastid Transporters during the Culture Growth of Chlamydomonas reinhardtii

Alteration in the Expression of Genes Encoding Primary Metabolism Enzymes and Plastid Transporters during the Culture Growth of Chlamydomonas reinhardtii

In a mixotrophic Chlamydomonas reinhardtii culture, the expression levels of genes encoding primary metabolic enzymes and chloroplast plastid transporters were analyzed. For the majority of the genes studied, their expression levels decreased as they approached the final stages of culture growth. During the period of exponential growth, the expression profiles changed more intensively than during the stationary phase. In the middle of exponential growth, significant changes of mRNA profiles reflected reorganization of metabolism, with an emphasis on the induction of lipid synthesis, accompanied by alterations in carbon fluxes through biochemical pathways and a shift in the energy balance between the plastid and cytosol.

Differential expression of genes involved in the response of Prunus spp. rootstocks under soil flooding

The State of Rio Grande do Sul is used to produce the largest volume of peaches in Brazil. However, productivity in the state is lower than elsewhere in the country. One of the challenges faced by producers in the area is the occurrence soil drainage problems. These problems are observed mainly in Pelotas and its surroundings, and depending on the time of year, may produce flooding issues that hamper both the development and productivity of cultures. In situations in which soil is flooded, oxygen levels are insufficient for root growth, can hinder cellular respiration and may lead to a cellular energy crisis. In addition, global climate change is affecting the climate, and such changes are expected to increase the frequency and severity of flood events in many the agricultural regions. In order to design studies that aim to enhance the selection of flood tolerant genotypes, it is essential to understand the physiological and molecular changes that occur in plants under conditions of oxygen deprivation. Since the production of peach trees in the region is accomplished using rootstocks, the aim of this study was to evaluate gas exchange parameters and gene expression changes involved in glycolytic, fermentative and ethylene metabolism in the peach cultivar NR0170401 and plum cultivars Marianna 2624 and Myrobalan 29C under flooded soil conditions. To this end, gas exchange parameters [net photosynthetic rate (A), and stomatal conductance (gs)] and the expression of nine genes [ACO1 (1-aminocyclopropane-1-carboxylic acid oxidase), ACS5 (1-aminoacyclopropane 1-carboxylic acid synthase), ADH1 (alcohol dehydrogenase 1), G6P (glucose-6-phosphate plastidic transporter), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), LDH1 (lactate dehydrogenase 1), PDC (Pyruvate decarboxylase), SAMs (SAM synthase), and UDPg (UDP-glucose pyrophosphorylase] were evaluated under conditions of flooding relative to controls. An evaluation of gas exchange showed that the plum cultivars are more tolerant to flooding than the peach cultivar studied. RT-qPCR results showed that the three evaluated rootstocks activated fermentative metabolism in roots in the first six hours post-flooding period, and root tissues were more highly affected than leaves. 'Marianna 2426' appears to have a more efficient gene response system compared to the other cultivars studied under conditions of oxygen deprivation. Although more studies are needed, such information has the potential to facilitate the selection of rootstocks used for peach production, for orchard management and for plant breeding programs aiming to produce new cultivars with increased flood tolerance.

PAPST2 Plays Critical Roles in Removing the Stress Signaling Molecule 3′-Phosphoadenosine 5′-Phosphate from the Cytosol and Its Subsequent Degradation in Plastids and Mitochondria

The compartmentalization of PAPS (the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate) synthesis (mainly in plastids), PAPS consumption (in the cytosol), and PAP (the stress signaling molecule 3'-phosphoadenosine 5'-phosphate) degradation (in plastids and mitochondria) requires organellar transport systems for both PAPS and PAP. The plastidial transporter PAPST1 (PAPS TRANSPORTER1) delivers newly synthesized PAPS from the stroma to the cytosol. We investigated the activity of PAPST2, the closest homolog of PAPST1, which unlike PAPST1 is targeted to both the plastids and mitochondria. Biochemical characterization in Arabidopsis thaliana revealed that PAPST2 mediates the antiport of PAP, PAPS, ATP, and ADP. Strongly increased cellular PAP levels negatively affect plant growth, as observed in the fry1 papst2 mutant, which lacks the PAP-catabolizing enzyme SALT TOLERANCE 1 and PAPST2. PAP levels were specifically elevated in the cytosol of papst2 and fiery1 papst2, but not in papst1 or fry1 papst1 PAPST1 failed to complement the papst2 mutant phenotype in mitochondria, because it likely removes PAPS from the cell, as demonstrated by the increased expression of phytosulfokine genes. Overexpression of SAL1 in mitochondria rescued the phenotype of fry1 but not fry1 papst2 Therefore, PAPST2 represents an important organellar importer of PAP, providing a piece of the puzzle in our understanding of the organelle-to-nucleus PAP retrograde signaling pathway.

Coordinated alterations in gene expression and metabolomic profiles of Chlamydomonas reinhardtii during batch autotrophic culturing

Chlamydomonas reinhardtii was grown under autotrophic batch culturing, which is known to be a widely applicable method for both fundamental research and applied purposes. This type of cultivation results in elevation of cell density and fast exhaustion of nutrient resources. We expected the revealed metabolic adaptation to be triggered at the transcriptional level. This investigation focuses on analyzing expression of the genes encoding enzymes involved in primary metabolism and plastid transporters during the exponential phase of C. reinhardtii autotrophic batch culture. About two-thirds of the tested genes demonstrated differential expression during algae growth. Patterns of expression were clustered into 5 groups. Most of the genes were gathered in two large clusters, characterized by peaks of expression at early or later exponential growth (EG). Genes which showed maximal expression in early EG were OMT1, HXK1, AMYB1, ACK1,2, CHLREDRAFT_123419, APE2, PCK1, CHLREDRAFT_195672, CIS2, TPT2 and ACLA1. Among the genes with maximal expression in later EG were SBE3, TPIC, CHLREDRAFT_137300, CHLREDRAFT_111372, PPT1 and CHLREDRAFT_122970. There were no genes detected with maximal expression at the cessation of proliferation. PCA showed that the expression profiles in the beginning EG were similar, and profiles changed drastically in the middle of exponential growth. PLS-DA revealed the difference between the beginning of EG and later periods linked to PC1 (44 %), between late EG and early stationary linked to PC2 (23 %) and finally between two points at the beginning of growth linked to PC3 (10 %). Mapping of genes and metabolites according to their correlation revealed a graph with two clusters. The first, smaller cluster contains genes that encode plastid exporters, enzymes of starch and carbohydrates metabolism. The expression level of these genes peaked later in EG. These genes are mainly associated with metabolites such as carbohydrates, acylglycerols and fatty acids metabolism. The second cluster is larger and more diverse. It combines genes with maximum expression in the beginning of EG. The core of this cluster is formed by genes encoding enzymes of fatty acids synthesis, energy and plastic pathways, and plastid transporters. This cluster included the majority of amino acids, carboxylic acids and many fatty acids.

An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport

BackgroundThe preparation of functional thylakoid membranes from diatoms with a silica cell wall is still a largely unsolved challenge. Therefore, an optimized protocol for the isolation of oxygen evolving thylakoid membranes of the centric diatom Cyclotella meneghiniana has been developed. The buffer used for the disruption of the cells was supplemented with polyethylene glycol based on its stabilizing effect on plastidic membranes. Disruption of the silica cell walls was performed in a French Pressure cell and subsequent linear sorbitol density gradient centrifugation was used to isolate the thylakoid membrane fraction.ResultsSpectroscopic characterization of the thylakoids by absorption and 77 K fluorescence spectroscopy showed that the photosynthetic pigment protein complexes in the isolated thylakoid membranes were intact. This was supported by oxygen evolution measurements which demonstrated high electron transport rates in the presence of the artificial electron acceptor DCQB. High photosynthetic activity of photosystem II was corroborated by the results of fast fluorescence induction measurements. In addition to PSII and linear electron transport, indications for a chlororespiratory electron transport were observed in the isolated thylakoid membranes. Photosynthetic electron transport also resulted in the establishment of a proton gradient as evidenced by the quenching of 9-amino-acridine fluorescence. Because of their ability to build-up a light-driven proton gradient, de-epoxidation of diadinoxanthin to diatoxanthin and diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence could be observed for the first time in isolated thylakoid membranes of diatoms. However, the ∆pH, diadinoxanthin de-epoxidation and diatoxanthin-dependent NPQ were weak compared to intact diatom cells or isolated thylakoids of higher plants.ConclusionsThe present protocol resulted in thylakoids with a high electron transport capacity. These thylakoids can thus be used for experiments addressing various aspects of the photosynthetic electron transport by, e.g., employing artificial electron donors and acceptors which do not penetrate the diatom cell wall. In addition, the present isolation protocol yields diatom thylakoids with the potential for xanthophyll cycle and non-photochemical quenching measurements. However, the preparation has to be further refined before these important topics can be addressed systematically.

Identification and functional characterization of grapevine transporters that mediate glucose-6-phosphate uptake into plastids.

Two grapevine glucose-6-Pi plastidial transporters differently expressed in plant organs and in response to environmental and hormonal signals are characterized. They are involved in starch accumulation in berries and canes. In grapevine, starch accumulation in the trunk is important for winter storage of carbon and in the flower for reproductive development. Berries also accumulate starch in their plastids, which are also involved in the synthesis of aroma compounds important for fruit quality. The present work characterizes two glucose-phosphate translocators (VvGPT1, VvGPT2) that control the accumulation of starch in grape amyloplasts. Three different splicing variants identified for VvGPT2 (VvGPT2α, VvGPT2β and VvGPT2Ω) were more expressed in the leaves than in other organs. In contrast, VvGPT1 transcripts were more abundant in mature berries, canes and flowers than in the leaves. Expression of 35S-VvGPT1-GFP and 35S-VvGPT2Ω-GFP in tobacco leaf epidermal cells showed that the fusion proteins localized at the plastidial envelope. Complementation of the Arabidopsis pgi1-1 mutant impaired in leaf starch synthesis restored its ability to synthesize starch, demonstrating that VvGPT1 and VvGPT2Ω mediate the transport of glucose-6-Pi across the plastidial envelope. In grape cell suspensions, ABA, light and galactinol, together with sucrose and fructose, significantly increased the transcript abundance of VvGPT1, whereas VvGPT2Ω expression was affected only by sucrose. In addition, elicitation with methyl jasmonate strongly upregulated VvGPT1, VvGPT2Ω and VvPAL1, suggesting a role for GPTs in the production of secondary compounds in grapevine. Moreover, in grapevines cultivated in field conditions, VvGPT1 expression was higher in berries more exposed to the sun and subjected to higher temperatures. Although both VvGPT1 and VvGPT2 mediate the same function at the molecular level, they exhibit different expression levels and regulation in plant organs and in response to environmental and hormonal signals.

Plastidial transporters KEA1, -2, and -3 are essential for chloroplast osmoregulation, integrity, and pH regulation in Arabidopsis.

Multiple K(+) transporters and channels and the corresponding mutants have been described and studied in the plasma membrane and organelle membranes of plant cells. However, knowledge about the molecular identity of chloroplast K(+) transporters is limited. Potassium transport and a well-balanced K(+) homeostasis were suggested to play important roles in chloroplast function. Because no loss-of-function mutants have been identified, the importance of K(+) transporters for chloroplast function and photosynthesis remains to be determined. Here, we report single and higher-order loss-of-function mutants in members of the cation/proton antiporters-2 antiporter superfamily KEA1, KEA2, and KEA3. KEA1 and KEA2 proteins are targeted to the inner envelope membrane of chloroplasts, whereas KEA3 is targeted to the thylakoid membrane. Higher-order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in the kea1kea2 mutants, but increased in the kea3 mutant, indicating an altered chloroplast pH homeostasis. Electron microscopy of kea1kea2 leaf cells revealed dramatically swollen chloroplasts with disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed the observed phenotypes. Furthermore, the kea1kea2 background enables genetic analyses of the functional significance of other chloroplast transporters as exemplified here in kea1kea2Na(+)/H(+) antiporter1 (nhd1) triple mutants. Taken together, the presented data demonstrate a fundamental role of inner envelope KEA1 and KEA2 and thylakoid KEA3 transporters in chloroplast osmoregulation, integrity, and ion and pH homeostasis.

Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning

Oil palm can accumulate up to 90% oil in its mesocarp, the highest level observed in the plant kingdom. In contrast, the closely related date palm accumulates almost exclusively sugars. To gain insight into the mechanisms that lead to such an extreme difference in carbon partitioning, the transcriptome and metabolite content of oil palm and date palm were compared during mesocarp development. Compared with date palm, the high oil content in oil palm was associated with much higher transcript levels for all fatty acid synthesis enzymes, specific plastid transporters, and key enzymes of plastidial carbon metabolism, including phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase. Transcripts representing an ortholog of the WRI1 transcription factor were 57-fold higher in oil palm relative to date palm and displayed a temporal pattern similar to its target genes. Unexpectedly, despite more than a 100-fold difference in flux to lipids, most enzymes of triacylglycerol assembly were expressed at similar levels in oil palm and date palm. Similarly, transcript levels for all but one cytosolic enzyme of glycolysis were comparable in both species. Together, these data point to synthesis of fatty acids and supply of pyruvate in the plastid, rather than acyl assembly into triacylglycerol, as a major control over the storage of oil in the mesocarp of oil palm. In addition to greatly increasing molecular resources devoted to oil palm and date palm, the combination of temporal and comparative studies illustrates how deep sequencing can provide insights into gene expression patterns of two species that lack genome sequence information.

Phylogenetic and Biochemical Evidence Supports the Recruitment of an ADP-Glucose Translocator for the Export of Photosynthate during Plastid Endosymbiosis

The acquisition of photosynthesis by eukaryotic cells through enslavement of a cyanobacterium represents one of the most remarkable turning points in the history of life on Earth. In addition to endosymbiotic gene transfer, the acquisition of a protein import apparatus and the coordination of gene expression between host and endosymbiont genomes, the establishment of a metabolic connection was crucial for a functional endosymbiosis. It was previously hypothesized that the first metabolic connection between both partners of endosymbiosis was achieved through insertion of a host-derived metabolite transporter into the cyanobacterial plasma membrane. Reconstruction of starch metabolism in the common ancestor of photosynthetic eukaryotes suggested that adenosine diphosphoglucose (ADP-Glc), a bacterial-specific metabolite, was likely to be the photosynthate, which was exported from the early cyanobiont. However, extant plastid transporters that have evolved from host-derived endomembrane transporters do not transport ADP-Glc but simple phosphorylated sugars in exchange for orthophosphate. We now show that those eukaryotic nucleotide sugar transporters, which define the closest relatives to the common ancestor of extant plastid envelope carbon translocators, possess an innate ability for transporting ADP-Glc. Such an unexpected ability would have been required to establish plastid endosymbiosis.

Disruption of Nap14, a plastid‐localized non‐intrinsic ABC protein in Arabidopsis thaliana results in the over‐accumulation of transition metals and in aberrant chloroplast structures

Chloroplasts are the major sink for Fe in shoot tissues because of the requirements of the photosynthetic process and to storage in ferritins. Such requirements are common both to plastids and to their evolutionary progenitors, the cyanobacteria. Here, we examined whether iron transport mechanisms were conserved throughout the evolution of photosynthetic organisms. Comparison of the sequences of putative plastid transporters from Arabidopsis thaliana with those involved in cyanobacterial Fe transport identified two orthologs of the FutC protein, AtNAP11 and AtNAP14. To study their function, we analysed insertional mutants in the genes coding for these proteins. Both nap11/nap11 and nap14/nap14 plants exhibited severe growth defects. Significant changes in transition metal homeostasis were detected only in nap14/nap14. This mutant was found to contain approximately 18 times more Fe in the shoot tissue than in wild-type plants. The increased shoot transition metal content was accompanied by a specific loss of chloroplast structures and by a reduction in transcript levels of Fe homeostasis-related genes. Based on these results, we propose that AtNAP14 plays an important role in plastid transition metal homeostasis. One possibility is that AtNAP14 is part of a chloroplast transporter complex. Alternatively, AtNAP14 function may be in regulating transition metal homeostasis.

The Plastidic Bile Acid Transporter 5 Is Required for the Biosynthesis of Methionine-Derived Glucosinolates inArabidopsis thaliana

Aliphatic glucosinolate biosynthesis is highly compartmentalized, requiring import of 2-keto acids or amino acids into chloroplasts for side chain elongation and export of the resulting compounds into the cytosol for conversion into glucosinolate. Aliphatic glucosinolate biosynthesis in Arabidopsis thaliana is regulated by three R2R3-MYB transcription factors, the major player being High Aliphatic Glucosinolate 1 (HAG1/MYB28). Here, we show that BAT5, which belongs to the putative bile acid transporter family, is the only member of this family that is transactivated by HAG1/MYB28, HAG2/MYB76, and HAG3/MYB29. Furthermore, two isopropylmalate isomerases genes, IPMI1 and IPMI2, and the isopropylmalate dehydrogenase gene, IPMDH1, were identified as targets of HAG1/MYB28 and the corresponding proteins localized to plastids, suggesting a role in plastidic chain elongation reactions. The BAT proteins also localized to plastids; however, only mutants defective in BAT5 function contained strongly reduced levels of aliphatic glucosinolates. The bat5 mutant chemotype was rescued by induced overexpression of BAT5. Feeding experiments using 2-keto acids and amino acids of different chain length suggest that BAT5 is a plastidic transporter of (chain-elongated) 2-keto acids. Mechanical stimuli and methyl jasmonate transiently induced BAT5 expression in inflorescences and leaves. Thus, BAT5 was identified as the first transporter component of the aliphatic glucosinolate biosynthetic pathway.

A Plastidic Transporter Involved in Aliphatic Glucosinolate Biosynthesis

The distinctive flavors and anticarcinogenic properties of cruciferous vegetables are largely due to the presence of glucosinolates. These sulfur-rich metabolites are derived from one of several amino acids via three stages: (1) elongation of the amino acid side chain; (2) formation of the core glucosinolate skeleton; and (3) secondary modification of the side chain (reviewed in Grubb and Abel, 2006). Met-derived aliphatic glucosinolates constitute the majority of glucosinolates in many Brassicaceae species, including Arabidopsis (Kliebenstein et al., 2001). Biosynthesis of these glucosinolates is highly compartmentalized, with preliminary reactions and final stages occurring in the cytosol and side chain elongation in the chloroplast. Prompted by the discovery that the R2R3-MYB transcription factor High Aliphatic Glucosinolate 1 (HAG1/MYB28) is a key regulator of Met-derived glucosinolate biosynthesis in Arabidopsis (Gigolashvili et al., 2007; Hirai et al., 2007), Gigolashvili et al. (pages 1813–1829) set out to identify novel targets of HAG1. Using transcriptome coexpression analysis, they found that BAT5, which encodes a putative bile acid transporter with a hitherto uncharacterized role in plants, is coexpressed with HAG1. Cotransformation assays using a HAG1 effector and a ProBAT5-GUS reporter showed that HAG1 transactivates the BAT5 promoter, confirming that BAT5 is indeed a target of HAG1. Furthermore, the authors established that full-length BAT5-GFP localizes to chloroplasts in transiently transformed Arabidopsis leaves (see figure). Figure 1 BAT5-GFP localizes to chloroplasts. BAT5-GFP fluorescence (A) and chloroplast autofluorescence (B) in transgenic Arabidopsis leaves and the overlay (C) of (A) and (B) showing colocalization (yellow) of BAT5-GFP and chloroplasts. The authors tested whether BAT5 has a role in aliphatic glucosinolate biosynthesis by examining the effect of downregulation of BAT5 on glucosinolate content. Although a homozygous bat5 mutant with vastly reduced steady state levels of BAT5 mRNA appeared to be normal under standard conditions, metabolic analysis revealed that it had significantly reduced levels of aliphatic glucosinolates. Complementation of the bat5 mutant with an estradiol-inducible BAT5 vector rescued the glucosinolate composition to almost wild-type levels, and the degree of induced gene expression correlated with the level of aliphatic glucosinolates produced. The effect on aliphatic glucosinolate content was even more pronounced in BAT5 microRNA overexpressing lines, which have undetectable levels of BAT5 mRNA. The observation that wounding and methyl jasmonate, which are known to induce glucosinolate biosynthesis, upregulate the expression of BAT5, provides further evidence that BAT5 is involved in glucosinolate biosynthesis. Finally, the authors sought to pinpoint the function of BAT5 in aliphatic glucosinolate biosynthesis by feeding bat5 and wild-type Arabidopsis plants with various intermediates of the Met-derived glucosinolate pathway and then analyzing the glucosinolate content. The results suggest that BAT5 transports Met derivatives into the chloroplast for side chain elongation and exports elongated products back into the cytosol for conversion into glucosinolates. Thus, BAT5 plays an integral role in Met-derived glucosinolate biosynthesis in Arabidopsis.

Molecular and functional characterization of the plastid-localized Phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana

The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.