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Plants possess the most highly compartmentalized eukaryotic cells. To coordinate their intracellular functions, plastids and the mitochondria are dependent on the flow of information to and from the nuclei, known as retrograde and anterograde signals. One mobile retrograde signaling molecule is the monophosphate 3'-phosphoadenosine 5'-phosphate (PAP), which is mainly produced from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the cytosol and regulates the expression of a set of nuclear genes that modulate plant growth in response to biotic and abiotic stresses. The adenosine bisphosphate phosphatase enzyme SAL1 dephosphorylates PAP to AMP in plastids and the mitochondria, but can also rescue sal1 Arabidopsis phenotypes (PAP accumulation, leaf morphology, growth, etc.) when expressed in the cytosol and the nucleus. To understand better the roles of the SAL1 protein in chloroplasts, the mitochondria, nuclei, and the cytosol, we have attempted to complement the sal1 mutant by specifically cargoing the transgenic SAL1 protein to these four cell compartments. Overexpression of SAL1 protein targeted to the nucleus or the mitochondria alone, or co-targeted to chloroplasts and the mitochondria, complemented most aspects of the sal1 phenotypes. Notably, targeting SAL1 to chloroplasts or the cytosol did not effectively rescue the sal1 phenotypes as these transgenic lines accumulated very low levels of SAL1 protein despite overexpressing SAL1 mRNA, suggesting a possibly lower stability of the SAL1 protein in these compartments. The diverse transgenic SAL1 lines exhibited a range of PAP levels. The latter needs to reach certain thresholds in the cell for its impacts on different processes such as leaf growth, regulation of rosette morphology, sulfate homeostasis, and glucosinolate biosynthesis. Collectively, these findings provide an initial platform for further dissection of the role of the SAL1-PAP pathway in different cellular processes under stress conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ashykhmina, Chan, Frerigmann, Van Breusegem, Kopriva, Flügge and Gigolashvili.)

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

During their first year of growth, overwintering biennial plants transport Suc through the phloem from photosynthetic source tissues to storage tissues. In their second year, they mobilize carbon from these storage tissues to fuel new growth and reproduction. However, both the mechanisms driving this shift and the link to reproductive growth remain unclear. During vegetative growth, biennial sugar beet ( Beta vulgaris ) maintains a steep Suc concentration gradient between the shoot (source) and the taproot (sink). To shift from vegetative to generative growth, they require a chilling phase known as vernalization. We studied sugar beet sink-source dynamics upon vernalization and showed that before flowering, the taproot underwent a reversal from a sink to a source of carbohydrates. This transition was induced by transcriptomic and functional reprogramming of sugar beet tissue, resulting in a reversal of flux direction in the phloem. In this transition, the vacuolar Suc importers and exporters TONOPLAST SUGAR TRANSPORTER2;1 and SUCROSE TRANSPORTER4 were oppositely regulated, leading to the mobilization of sugars from taproot storage vacuoles. Concomitant changes in the expression of floral regulator genes suggest that these processes are a prerequisite for bolting. Our data will help both to dissect the metabolic and developmental triggers for bolting and to identify potential targets for genome editing and breeding., (© 2020 American Society of Plant Biologists. All rights reserved.)

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., (© 2018 American Society of Plant Biologists. All rights reserved.)

The xylulose 5-phosphate/phosphate translocator (PTs) (XPT) represents a link between the plastidial and extraplastidial branches of the oxidative pentose phosphate pathway. Its role is to retrieve pentose phosphates from the extraplastidial space and to make them available to the plastids. However, the XPT transports also triose phosphates and to a lesser extent phosphoenolpyruvate (PEP). Thus, it might support both the triose phosphate/PT (TPT) in the export of photoassimilates from illuminated chloroplasts and the PEP/PT (PPT) in the import of PEP into green or non-green plastids. In mutants defective in the day- and night-path of photoassimilate export from the chloroplasts (i.e., knockout of the TPT [ tpt-2 ] in a starch-free background [ adg1-1 ])the XPT provides a bypass for triose phosphate export and thereby guarantees survival of the adg1-1/tpt-2 double mutant. Here we show that the additional knockout of the XPT in adg1-1/tpt-2/xpt-1 triple mutants results in lethality when the plants were grown in soil. Thus the XPT can functionally support the TPT. The PEP transport capacity of the XPT has been revisited here with a protein heterologously expressed in yeast. PEP transport rates in the proteoliposome system were increased with decreasing pH-values below 7.0. Moreover, PEP transport determined in leaf extracts from wild-type plants showed a similar pH-response, suggesting that in both cases PEP 2- is the transported charge-species. Hence, PEP import into illuminated chloroplasts might be unidirectional because of the alkaline pH of the stroma. Here the consequence of a block in PEP transport across the envelope was analyzed in triple mutants defective in both PPTs and the XPT. PPT1 is knocked out in the cue1 mutant. For PPT2 two new mutant alleles were isolated and established as homozygous lines. In contrast to the strong phenotype of cue1 , both ppt2 alleles showed only slight growth retardation. As plastidial PEP is required e.g., for the shikimate pathway of aromatic amino acid synthesis, a block in PEP import should result in a lethal phenotype. However, the cue1-6/ppt2-1/ppt2-1 triple mutant was viable and even exhibited residual PEP transport capacity. Hence, alternative ways of PEP transport must exist and are discussed.

The xylulose 5-phosphate/phosphate translocator (XPT) represents the fourth functional member of the phosphate translocator (PT) family residing in the plastid inner envelope membrane. In contrast to the other three members, little is known on the physiological role of the XPT. Based on its major transport substrates (i.e., pentose phosphates) the XPT has been proposed to act as a link between the plastidial and extraplastidial branches of the oxidative pentose phosphate pathway (OPPP). As the XPT is also capable of transporting triose phosphates, it might as well support the triose phosphate PT (TPT) in exporting photoassimilates from the chloroplast in the light ('day path of carbon') and hence in supplying the whole plant with carbohydrates. Two independent knockout mutant alleles of the XPT ( xpt-1 and xpt-2 ) lacked any specific phenotype, suggesting that the XPT function is redundant. However, double mutants generated from crossings of xpt-1 to different mutant alleles of the TPT ( tpt-1 and tpt-2 ) were severely retarded in size, exhibited a high chlorophyll fluorescence phenotype, and impaired photosynthetic electron transport rates. In the double mutant the export of triose phosphates from the chloroplasts is completely blocked. Hence, precursors for sucrose biosynthesis derive entirely from starch turnover ('night path of carbon'), which was accompanied by a marked accumulation of maltose as a starch breakdown product. Moreover, pentose phosphates produced by the extraplastidial branch of the OPPP also accumulated in the double mutants. Thus, an active XPT indeed retrieves excessive pentose phosphates from the extra-plastidial space and makes them available to the plastids. Further metabolic profiling revealed that phosphorylated intermediates remained largely unaffected, whereas fumarate and glycine contents were diminished in the double mutants. The assessment of C/N-ratios suggested co-limitations of C- and N-metabolism as possible cause for growth retardation of the double mutants. Feeding of sucrose partially rescued the growth and photosynthesis phenotypes of the double mutants. Immunoblots of thylakoid proteins, spectroscopic determinations of photosynthesis complexes, and chlorophyll a fluorescence emission spectra at 77 Kelvin could only partially explain constrains in photosynthesis observed in the double mutants. The data are discussed together with aspects of the OPPP and central carbon metabolism.

Membrane proteins are estimated to constitute a quarter of all proteins encoded in plant genomes, yet only a limited number have been experimentally characterized. This is mainly due to the large variation in particular physical properties coupled with purification difficulties. Computational methods are therefore very helpful for the initial characterization of a candidate membrane protein. Individual prediction tools can, with varying levels of success, predict the occurrence of transmembrane spans, the subcellular location, and lipid posttranslational modifications. Since it can be tedious to consult each prediction tool separately, ARAMEMNON has been designed to compile various computational predictions for plant membrane proteins and to present the results via a user-friendly web interface. This protocol describes how to use ARAMEMNON to identify and characterize plant membrane proteins.

Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the β-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks., (© 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Photosynthesis is central to all life on earth, providing not only oxygen but also organic compounds that are synthesized from atmospheric CO 2 and water using light energy as the driving force. The still-increasing world population poses a serious challenge to further enhance biomass production of crop plants. Crop yield is determined by various parameters, inter alia by the light energy conversion efficiency of the photosynthetic machinery. Photosynthesis can be looked at from different perspectives: (i) light reactions and carbon assimilation, (ii) leaves and canopy structure, and (ii) source-sink relationships. In this review, we discuss opportunities and prospects to increase photosynthetic performance at the different layers, taking into account the recent progress made in the respective fields., Competing Interests: The authors declare that they have no competing interests. No competing interests were disclosed. No competing interests were disclosed. No competing interests were disclosed.

A rapid accumulation of γ-aminobutyric acid (GABA) during biotic and abiotic stresses is well documented. However, the specificity of the response and the primary role of GABA under such stress conditions are hardly understood. To address these questions, we investigated the response of the GABA-depleted gad1/2 mutant to drought stress. GABA is primarily synthesized from the decarboxylation of glutamate by glutamate decarboxylase (GAD) which exists in five copies in the genome of Arabidopsis thaliana. However, only GAD1 and GAD2 are abundantly expressed, and knockout of these two copies dramatically reduced the GABA content. Phenotypic analysis revealed a reduced shoot growth of the gad1/2 mutant. Furthermore, the gad1/2 mutant was wilted earlier than the wild type following a prolonged drought stress treatment. The early-wilting phenotype was due to an increase in stomata aperture and a defect in stomata closure. The increase in stomata aperture contributed to higher stomatal conductance. The drought oversensitive phenotype of the gad1/2 mutant was reversed by functional complementation that increases GABA level in leaves. The functionally complemented gad1/2 x pop2 triple mutant contained more GABA than the wild type. Our findings suggest that GABA accumulation during drought is a stress-specific response and its accumulation induces the regulation of stomatal opening thereby prevents loss of water., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance., (© 2016 American Society of Plant Biologists. All rights reserved.)

Sugar beet provides around one third of the sugar consumed worldwide and serves as a significant source of bioenergy in the form of ethanol. Sucrose accounts for up to 18% of plant fresh weight in sugar beet. Most of the sucrose is concentrated in the taproot, where it accumulates in the vacuoles. Despite 30 years of intensive research, the transporter that facilitates taproot sucrose accumulation has escaped identification. Here, we combine proteomic analyses of the taproot vacuolar membrane, the tonoplast, with electrophysiological analyses to show that the transporter BvTST2.1 is responsible for vacuolar sucrose uptake in sugar beet taproots. We show that BvTST2.1 is a sucrose-specific transporter, and present evidence to suggest that it operates as a proton antiporter, coupling the import of sucrose into the vacuole to the export of protons. BvTST2.1 exhibits a high amino acid sequence similarity to members of the tonoplast monosaccharide transporter family in Arabidopsis, prompting us to rename this group of proteins 'tonoplast sugar transporters'. The identification of BvTST2.1 could help to increase sugar yields from sugar beet and other sugar-storing plants in future breeding programs.

Carbohydrate metabolism in plants is tightly linked to photosynthesis and is essential for energy and carbon skeleton supply of the entire organism. Thus, the hexose phosphate pools of the cytosol and the chloroplast represent important metabolic resources that are maintained through action of phosphoglucose isomerase (PGI) and phosphoglucose mutase interconverting glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. Here, we investigated the impact of disrupted cytosolic PGI (cPGI) function on plant viability and metabolism. Overexpressing an artificial microRNA targeted against cPGI (amiR-cpgi) resulted in adult plants with vegetative tissue essentially free of cPGI activity. These plants displayed diminished growth compared with the wild type and accumulated excess starch in chloroplasts but maintained low sucrose content in leaves at the end of the night. Moreover, amiR-cpgi plants exhibited increased nonphotochemical chlorophyll a quenching during photosynthesis. In contrast to amiR-cpgi plants, viable transfer DNA insertion mutants disrupted in cPGI function could only be identified as heterozygous individuals. However, homozygous transfer DNA insertion mutants could be isolated among plants ectopically expressing cPGI. Intriguingly, these plants were only fertile when expression was driven by the ubiquitin10 promoter but sterile when the seed-specific unknown seed protein promoter or the Cauliflower mosaic virus 35S promoter were employed. These data show that metabolism is apparently able to compensate for missing cPGI activity in adult amiR-cpgi plants and indicate an essential function for cPGI in plant reproduction. Moreover, our data suggest a feedback regulation in amiR-cpgi plants that fine-tunes cytosolic sucrose metabolism with plastidic starch turnover., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

The concept of retrograde control of nuclear gene expression assumes the generation of signals inside the chloroplasts, which are either released from or sensed inside of the organelle. In both cases, downstream signaling pathways lead eventually to a differential regulation of nuclear gene expression and the production of proteins required in the chloroplast. This concept appears reasonable as the majority of the over 3000 predicted plastidial proteins are encoded by nuclear genes. Hence, the nucleus needs information on the status of the chloroplasts, such as during acclimation responses, which trigger massive changes in the protein composition of the thylakoid membrane and in the stroma. Here, we propose an additional control mechanism of nuclear- and plastome-encoded photosynthesis genes, taking advantage of pathways involved in sugar- or hormonal signaling. Sugars are major end products of photosynthesis and their contents respond very sensitively to changes in light intensities. Based on recent findings, we ask the question as to whether the carbohydrate status outside the chloroplast can be directly sensed within the chloroplast stroma. Sugars might synchronize the responsiveness of both genomes and thereby help to coordinate the expression of plastome- and nuclear-encoded photosynthesis genes in concert with other, more specific retrograde signals., (© The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.)

Phosphoenolpyruvate (PEP) serves not only as a high energy carbon compound in glycolysis, but it acts also as precursor for plastidial anabolic sequences like the shikimate pathway, which produces aromatic amino acids (AAA) and subsequently secondary plant products. After conversion to pyruvate, PEP can also enter de novo fatty acid biosynthesis, the synthesis of branched-chain amino acids, and the non-mevalonate way of isoprenoid production. As PEP cannot be generated by glycolysis in chloroplasts and a variety of non-green plastids, it has to be imported from the cytosol by a phosphate translocator (PT) specific for PEP (PPT). A loss of function of PPT1 in Arabidopsis thaliana results in the chlorophyll a/b binding protein underexpressed1 (cue1) mutant, which is characterized by reticulate leaves and stunted roots. Here we dissect the shoot- and root phenotypes, and also address the question whether or not long distance signaling by metabolites is involved in the perturbed mesophyll development of cue1. Reverse grafting experiments showed that the shoot- and root phenotypes develop independently from each other, ruling out long distance metabolite signaling. The leaf phenotype could be transiently modified even in mature leaves, e.g. by an inducible PPT1RNAi approach or by feeding AAA, the cytokinin trans-zeatin (tZ), or the putative signaling molecule dehydrodiconiferyl alcohol glucoside (DCG). Hormones, such as auxins, abscisic acid, gibberellic acid, ethylene, methyl jasmonate, and salicylic acid did not rescue the cue1 leaf phenotype. The low cell density1 (lcd1) mutant shares the reticulate leaf-, but not the stunted root phenotype with cue1. It could neither be rescued by AAA nor by tZ. In contrast, tZ and AAA further inhibited root growth both in cue1 and wild-type plants. Based on our results, we propose a model that PPT1 acts as a net importer of PEP into chloroplast, but as an overflow valve and hence exporter in root plastids.

Retrograde signals from chloroplasts are thought to control the expression of nuclear genes associated with plastidial processes such as acclimation to varying light conditions. Arabidopsis mutants altered in the day and night path of photoassimilate export from the chloroplasts served as tools to study the involvement of carbohydrates in high light (HL) acclimation. A double mutant impaired in the triose phosphate/phosphate translocator (TPT) and ADP-glucose pyrophosphorylase (AGPase) (adg1-1/tpt-2) exhibits a HL-dependent depletion in endogenous carbohydrates combined with a severe growth and photosynthesis phenotype. The acclimation response of mutant and wild-type plants has been assessed in time series after transfer from low light (LL) to HL by analysing photosynthetic performance, carbohydrates, MgProtoIX (a chlorophyll precursor), and the ascorbate/glutathione redox system, combined with microarray-based transcriptomic and GC-MS-based metabolomic approaches. The data indicate that the accumulation of soluble carbohydrates (predominantly glucose) acts as a short-term response to HL exposure in both mutant and wild-type plants. Only if carbohydrates are depleted in the long term (e.g. after 2 d) is the acclimation response impaired, as observed in the adg1-1/tpt-2 double mutant. Furthermore, meta-analyses conducted with in-house and publicly available microarray data suggest that, in the long term, reactive oxygen species such as H₂O₂ can replace carbohydrates as signals. Moreover, a cross-talk exists between genes associated with the regulation of starch and lipid metabolism. The involvement of genes responding to phytohormones in HL acclimation appears to be less likely. Various candidate genes involved in retrograde control of nuclear gene expression emerged from the analyses of global gene expression.

Assembly of photosystem II (PSII) occurs sequentially and requires several auxiliary proteins, such as ALB3 (ALBINO3). Here, we describe the role of the Arabidopsis thaliana thylakoid membrane protein Tellurite resistance C (AtTerC) in this process. Knockout of AtTerC was previously shown to be seedling-lethal. This phenotype was rescued by expressing TerC fused C-terminally to GFP in the terc-1 background, and the resulting terc-1TerC- GFP line and an artificial miRNA-based knockdown allele (amiR-TerC) were used to analyze the TerC function. The alterations in chlorophyll fluorescence and thylakoid ultrastructure observed in amiR-TerC plants and terc-1TerC- GFP were attributed to defects in PSII. We show that this phenotype resulted from a reduction in the rate of de novo synthesis of PSII core proteins, but later steps in PSII biogenesis appeared to be less affected. Yeast two-hybrid assays showed that TerC interacts with PSII proteins. In particular, its interaction with the PSII assembly factor ALB3 has been demonstrated by co-immunoprecipitation. ALB3 is thought to assist in incorporation of CP43 into PSII via interaction with Low PSII Accumulation2 (LPA2) Low PSII Accumulation3 (LPA3). Homozygous lpa2 mutants expressing amiR-TerC displayed markedly exacerbated phenotypes, leading to seedling lethality, indicating an additive effect. We propose a model in which TerC, together with ALB3, facilitates de novo synthesis of thylakoid membrane proteins, for instance CP43, at the membrane insertion step., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

In plants, two independent serine biosynthetic pathways, the photorespiratory and glycolytic phosphoserine (PS) pathways, have been postulated. Although the photorespiratory pathway is well characterized, little information is available on the function of the PS pathway in plants. Here, we present a detailed characterization of phosphoglycerate dehydrogenases (PGDHs) as components of the PS pathway in Arabidopsis thaliana. All PGDHs localize to plastids and possess similar kinetic properties, but they differ with respect to their sensitivity to serine feedback inhibition. Furthermore, analysis of pgdh1 and phosphoserine phosphatase mutants revealed an embryo-lethal phenotype and PGDH1-silenced lines were inhibited in growth. Metabolic analyses of PGDH1-silenced lines grown under ambient and high CO2 conditions indicate a direct link between PS biosynthesis and ammonium assimilation. In addition, we obtained several lines of evidence for an interconnection between PS and tryptophan biosynthesis, because the expression of PGDH1 and phosphoserine aminotransferase1 is regulated by MYB51 and MYB34, two activators of tryptophan biosynthesis. Moreover, the concentration of tryptophan-derived glucosinolates and auxin were reduced in PGDH1-silenced plants. In essence, our results provide evidence for a vital function of PS biosynthesis for plant development and metabolism.

Plants assimilate carbon dioxide during photosynthesis in chloroplasts. Assimilated carbon is subsequently allocated throughout the plant. Generally, two types of organs can be distinguished, mature green source leaves as net photoassimilate exporters, and net importers, the sinks, e.g., roots, flowers, small leaves, and storage organs like tubers. Within these organs, different tissue types developed according to their respective function, and cells of either tissue type are highly compartmentalized. Photoassimilates are allocated to distinct compartments of these tissues in all organs, requiring a set of metabolite transporters mediating this intercompartmental transfer. The general route of photoassimilates can be briefly described as follows. Upon fixation of carbon dioxide in chloroplasts of mesophyll cells, triose phosphates either enter the cytosol for mainly sucrose formation or remain in the stroma to form transiently stored starch which is degraded during the night and enters the cytosol as maltose or glucose to be further metabolized to sucrose. In both cases, sucrose enters the phloem for long distance transport or is transiently stored in the vacuole, or can be degraded to hexoses which also can be stored in the vacuole. In the majority of plant species, sucrose is actively loaded into the phloem via the apoplast. Following long distance transport, it is released into sink organs, where it enters cells as source of carbon and energy. In storage organs, sucrose can be stored, or carbon derived from sucrose can be stored as starch in plastids, or as oil in oil bodies, or - in combination with nitrogen - as protein in protein storage vacuoles and protein bodies. Here, we focus on transport proteins known for either of these steps, and discuss the implications for yield increase in plants upon genetic engineering of respective transporters.

An important goal in biotechnological research is to improve the yield of crop plants. Here, we genetically modified simultaneously source and sink capacities in potato (Solanum tuberosum cv. Desirée) plants to improve starch yield. Source capacity was increased by mesophyll-specific overexpression of a pyrophosphatase or, alternatively, by antisense expression of the ADP-glucose pyrophosphorylase in leaves. Both approaches make use of re-routing photoassimilates to sink organs at the expense of leaf starch accumulation. Simultaneous increase in sink capacity was accomplished by overexpression of two plastidic metabolite translocators, that is, a glucose 6-phosphate/phosphate translocator and an adenylate translocator in tubers. Employing such a 'pull' approach, we have previously shown that potato starch content and yield can be increased when sink strength is elevated. In the current biotechnological approach, we successfully enhanced source and sink capacities by a combination of 'pull' and 'push' approaches using two different attempts. A doubling in tuber starch yield was achieved. This successful approach might be transferable to other crop plants in the future., (© 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

An Arabidopsis thaliana double mutant (adg1-1/tpt-2) defective in the day- and night-path of photoassimilate export from the chloroplast due to a knockout in the triose phosphate/phosphate translocator (TPT; tpt-2) and a lack of starch [mutation in ADP glucose pyrophosphorylase (AGPase); adg1-1] exhibits severe growth retardation, a decrease in the photosynthetic capacity, and a high chlorophyll fluorescence (HCF) phenotype under high light conditions. These phenotypes could be rescued when the plants were grown on sucrose (Suc) or glucose (Glc). Here we address the question whether Glc-sensing hexokinase1 (HXK1) defective in the Glc insensitive 2 (gin2-1) mutant is involved in the sugar-dependent rescue of adg1-1/tpt-2. Triple mutants defective in the TPT, AGPase, and HXK1 (adg1-1/tpt-2/gin2-1) were established as homozygous lines and grown together with Col-0 and Landsberg erecta (Ler) wild-type plants, gin2-1, the adg1-1/tpt-2 double mutant, and the adg1-1/tpt-2/gpt2-1 triple mutant [additionally defective in the glucose 6-phosphate/phosphate translocator 2 (GPT2)] on agar in the presence or absence of 50 mM of each Glc, Suc, or fructose (Fru). The growth phenotype of the double mutant and both triple mutants could be rescued to a similar extent only by Glc and Suc, but not by Fru. All three sugars were capable of rescuing the HCF and photosynthesis phenotype, irrespectively of the presence or absence of HXK1. Quantitative RT-PCR analyses of sugar-responsive genes revealed that plastidial HXK (pHXK) was up-regulated in adg1-1/tpt-2 plants grown on sugars, but showed no response in adg1-1/tpt-2/gin2-1. It appears likely that soluble sugars are directly taken up by the chloroplasts and enter further metabolism, which consumes ATP and NADPH from the photosynthetic light reaction and thereby rescues the photosynthesis phenotype of the double mutant. The implication of sugar turnover and probably signaling inside the chloroplasts for the concept of retrograde signaling is discussed.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the high-energy sulfate donor for sulfation reactions. Plants produce some PAPS in the cytosol, but it is predominantly produced in plastids. Accordingly, PAPS has to be provided by plastids to serve as a substrate for sulfotransferase reactions in the cytosol and the Golgi apparatus. We present several lines of evidence that the recently described Arabidopsis thaliana thylakoid ADP/ATP carrier TAAC transports PAPS across the plastid envelope and thus fulfills an additional function of high physiological relevance. Transport studies using the recombinant protein revealed that it favors PAPS, 3'-phosphoadenosine 5'-phosphate, and ATP as substrates; thus, we named it PAPST1. The protein could be detected both in the plastid envelope membrane and in thylakoids, and it is present in plastids of autotrophic and heterotrophic tissues. TAAC/PAPST1 belongs to the mitochondrial carrier family in contrast with the known animal PAPS transporters, which are members of the nucleotide-sugar transporter family. The expression of the PAPST1 gene is regulated by the same MYB transcription factors also regulating the biosynthesis of sulfated secondary metabolites, glucosinolates. Molecular and physiological analyses of papst1 mutant plants indicate that PAPST1 is involved in several aspects of sulfur metabolism, including the biosynthesis of thiols, glucosinolates, and phytosulfokines.

The transfer of fatty acids across biological membranes is a largely uncharacterized process, although it is essential at membranes of several higher plant organelles like chloroplasts, peroxisomes, or the endoplasmic reticulum. Here, we analyzed loss-of-function mutants of the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a model system to circumvent redundancy problems encountered in eukaryotic organisms. Cells deficient in the only cytoplasmic Synechocystis acyl-acyl carrier protein synthetase (SynAas) were highly resistant to externally provided α-linolenic acid, whereas wild-type cells bleached upon this treatment. Bleaching of wild-type cells was accompanied by a continuous increase of α-linolenic acid in total lipids, whereas no such accumulation could be observed in SynAas-deficient cells (Δsynaas). When SynAas was disrupted in the tocopherol-deficient, α-linolenic acid-hypersensitive Synechocystis mutant Δslr1736, double mutant cells displayed the same resistance phenotype as Δsynaas. Moreover, heterologous expression of SynAas in yeast (Saccharomyces cerevisiae) mutants lacking the major yeast fatty acid import protein Fat1p (Δfat1) led to the restoration of wild-type sensitivity against exogenous α-linolenic acid of the otherwise resistant Δfat1 mutant, indicating that SynAas is functionally equivalent to Fat1p. In addition, liposome assays provided direct evidence for the ability of purified SynAas protein to mediate α-[(14)C]linolenic acid retrieval from preloaded liposome membranes via the synthesis of [(14)C]linolenoyl-acyl carrier protein. Taken together, our data show that an acyl-activating enzyme like SynAas is necessary and sufficient to mediate the transfer of fatty acids across a biological membrane.

The photorespiratory pathway helps illuminated C(3)-plants under conditions of limited CO(2) availability by effectively exporting reducing equivalents in form of glycolate out of the chloroplast and regenerating glycerate-3-P as substrate for RubisCO. On the other hand, this pathway is considered as probably futile because previously assimilated CO(2) is released in mitochondria. Consequently, a lot of effort has been made to reduce this CO(2) loss either by reducing fluxes via engineering RubisCO or circumventing mitochondrial CO(2) release by the introduction of new enzyme activities. Here we present an approach following the latter route, introducing a complete glycolate catabolic cycle in chloroplasts of Arabidopsis thaliana comprising glycolate oxidase (GO), malate synthase (MS), and catalase (CAT). Results from plants bearing both GO and MS activities have already been reported (Fahnenstich et al., 2008). This previous work showed that the H(2)O(2) produced by GO had strongly negative effects. These effects can be prevented by introducing a plastidial catalase activity, as reported here. Transgenic lines bearing all three transgenic enzyme activities were identified and some with higher CAT activity showed higher dry weight, higher photosynthetic rates, and changes in glycine/serine ratio compared to the wild type. This indicates that the fine-tuning of transgenic enzyme activities in the chloroplasts seems crucial and strongly suggests that the approach is valid and that it is possible to improve the growth of A. thaliana by introducing a synthetic glycolate oxidative cycle into chloroplasts.

Background: We have studied the impact of carbohydrate-starvation on the acclimation response to high light using Arabidopsis thaliana double mutants strongly impaired in the day- and night path of photoassimilate export from the chloroplast. A complete knock-out mutant of the triose phosphate/phosphate translocator (TPT; tpt-2 mutant) was crossed to mutants defective in (i) starch biosynthesis (adg1-1, pgm1 and pgi1-1; knock-outs of ADP-glucose pyrophosphorylase, plastidial phosphoglucomutase and phosphoglucose isomerase) or (ii) starch mobilization (sex1-3, knock-out of glucan water dikinase) as well as in (iii) maltose export from the chloroplast (mex1-2)., Results: All double mutants were viable and indistinguishable from the wild type when grown under low light conditions, but--except for sex1-3/tpt-2--developed a high chlorophyll fluorescence (HCF) phenotype and growth retardation when grown in high light. Immunoblots of thylakoid proteins, Blue-Native gel electrophoresis and chlorophyll fluorescence emission analyses at 77 Kelvin with the adg1-1/tpt-2 double mutant revealed that HCF was linked to a specific decrease in plastome-encoded core proteins of both photosystems (with the exception of the PSII component cytochrome b559), whereas nuclear-encoded antennae (LHCs) accumulated normally, but were predominantly not attached to their photosystems. Uncoupled antennae are the major cause for HCF of dark-adapted plants. Feeding of sucrose or glucose to high light-grown adg1-1/tpt-2 plants rescued the HCF- and growth phenotypes. Elevated sugar levels induce the expression of the glucose-6-phosphate/phosphate translocator2 (GPT2), which in principle could compensate for the deficiency in the TPT. A triple mutant with an additional defect in GPT2 (adg1-1/tpt-2/gpt2-1) exhibited an identical rescue of the HCF- and growth phenotype in response to sugar feeding as the adg1-1/tpt-2 double mutant, indicating that this rescue is independent from the sugar-triggered induction of GPT2., Conclusions: We propose that cytosolic carbohydrate availability modulates acclimation to high light in A. thaliana. It is conceivable that the strong relationship between the chloroplast and nucleus with respect to a co-ordinated expression of photosynthesis genes is modified in carbohydrate-starved plants. Hence carbohydrates may be considered as a novel component involved in chloroplast-to-nucleus retrograde signaling, an aspect that will be addressed in future studies.

D-Lactate negatively affects Arabidopsis thaliana seedling development in a concentration-dependent manner. At media D-lactate concentrations greater than 5-10mM the development of wild-type plants is arrested shortly after germination whereas plants overexpressing the endogenous D-lactate dehydrogenase (D-LDH) detoxify D-lactate to pyruvate and survive. When the transgenic plants are further transferred to normal growth conditions they develop indistinguishably from the wild type. Thus, D-LDH was successfully established as a new marker in A. thaliana allowing selecting transgenic plants shortly after germination. The selection on D-lactate containing media adds a new optional marker system, which is especially useful if the simultaneous selection of multiple constructs is desired., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

With the development of high-throughput metabolic technologies, a plethora of primary and secondary compounds have been detected in the plant cell. However, there are still major gaps in our understanding of the plant metabolome. This is especially true with regards to the compartmental localization of these identified metabolites. Non-aqueous fractionation (NAF) is a powerful technique for the determination of subcellular metabolite distributions in eukaryotic cells, and it has become the method of choice to analyze the distribution of a large number of metabolites concurrently. However, the NAF technique produces a continuous gradient of metabolite distributions, not discrete assignments. Resolution of these distributions requires computational analyses based on marker molecules to resolve compartmental localizations. In this article we focus on expanding the computational analysis of data derived from NAF. Along with an experimental workflow, we describe the critical steps in NAF experiments and how computational approaches can aid in assessing the quality and robustness of the derived data. For this, we have developed and provide a new version (v1.2) of the BestFit command line tool for calculation and evaluation of subcellular metabolite distributions. Furthermore, using both simulated and experimental data we show the influence on estimated subcellular distributions by modulating important parameters, such as the number of fractions taken or which marker molecule is selected. Finally, we discuss caveats and benefits of NAF analysis in the context of the compartmentalized metabolome.

The energy status of plant cells strongly depends on the energy metabolism in chloroplasts and mitochondria, which are capable of generating ATP either by photosynthetic or oxidative phosphorylation, respectively. Another energy-rich metabolite inside plastids is the glycolytic intermediate phosphoenolpyruvate (PEP). However, chloroplasts and most non-green plastids lack the ability to generate PEP via a complete glycolytic pathway. Hence, PEP import mediated by the plastidic PEP/phosphate translocator or PEP provided by the plastidic enolase are vital for plant growth and development. In contrast to chloroplasts, metabolism in non-green plastids (amyloplasts) of starch-storing tissues strongly depends on both the import of ATP mediated by the plastidic nucleotide transporter NTT and of carbon (glucose 6-phosphate, Glc6P) mediated by the plastidic Glc6P/phosphate translocator (GPT). Both transporters have been shown to co-limit starch biosynthesis in potato plants. In addition, non-photosynthetic plastids as well as chloroplasts during the night rely on the import of energy in the form of ATP via the NTT. During energy starvation such as prolonged darkness, chloroplasts strongly depend on the supply of ATP which can be provided by lipid respiration, a process involving chloroplasts, peroxisomes, and mitochondria and the transport of intermediates, i.e. fatty acids, ATP, citrate, and oxaloacetate across their membranes. The role of transporters involved in the provision of energy-rich metabolites and in pathways supplying plastids with metabolic energy is summarized here.

Background: The extensive subcellular compartmentalization of metabolites and metabolism in eukaryotic cells is widely acknowledged and represents a key factor of metabolic activity and functionality. In striking contrast, the knowledge of actual compartmental distribution of metabolites from experimental studies is surprisingly low. However, a precise knowledge of, possibly all, metabolites and their subcellular distributions remains a key prerequisite for the understanding of any cellular function., Methodology/principal Findings: Here we describe results for the subcellular distribution of 1,117 polar and 2,804 lipophilic mass spectrometric features associated to known and unknown compounds from leaves of the model plant Arabidopsis thaliana. Using an optimized non-aqueous fractionation protocol in conjunction with GC/MS- and LC/MS-based metabolite profiling, 81.5% of the metabolic data could be associated to one of three subcellular compartments: the cytosol (including the mitochondria), vacuole, or plastids. Statistical analysis using a marker-'free' approach revealed that 18.5% of these metabolites show intermediate distributions, which can either be explained by transport processes or by additional subcellular compartments., Conclusion/significance: Next to a functional and conceptual workflow for the efficient, highly resolved metabolite analysis of the fractionated Arabidopsis thaliana leaf metabolome, a detailed survey of the subcellular distribution of several compounds, in the graphical format of a topological map, is provided. This complex data set therefore does not only contain a rich repository of metabolic information, but due to thorough validation and testing by statistical methods, represents an initial step in the analysis of metabolite dynamics and fluxes within and between subcellular compartments.

Arabidopsis thaliana mutants impaired in starch biosynthesis due to defects in either ADP glucose pyrophosphorylase (adg1-1), plastidic phosphoglucose mutase (pgm) or a new allele of plastidic phosphoglucose isomerase (pgi1-2) exhibit substantial activity of glucose-6-phosphate (Glc6P) transport in leaves that is mediated by a Glc6P/phosphate translocator (GPT) of the inner plastid envelope membrane. In contrast to the wild type, GPT2, one of two functional GPT genes of A. thaliana, is strongly induced in these mutants during the light period. The proposed function of the GPT in plastids of non-green tissues is the provision of Glc6P for starch biosynthesis and/or the oxidative pentose phosphate pathway. The function of GPT in photosynthetic tissues, however, remains obscure. The adg1-1 and pgi1-2 mutants were crossed with the gpt2-1 mutant defective in GPT2. Whereas adg1-1/gpt2-1 was starch-free, residual starch could be detected in pgi1-2/gpt2-1 and was confined to stomatal guard cells, bundle sheath cells and root tips, which parallels the reported spatial expression profile of AtGPT1. Glucose content in the cytosolic heteroglycan increased substantially in adg1-1 but decreased in pgi1-2, suggesting that the plastidic Glc6P pool contributes to its biosynthesis. The abundance of GPT2 mRNA correlates with increased levels of soluble sugars, in particular of glucose in leaves, suggesting induction by the sugar-sensing pathway. The possible function of GPT2 in starch-free mutants is discussed in the background of carbon requirement in leaves during the light-dark cycle.

Restriction of phosphoenolpyruvate (PEP) supply to plastids causes lethality of female and male gametophytes in Arabidopsis thaliana defective in both a phosphoenolpyruvate/phosphate translocator (PPT) of the inner envelope membrane and the plastid-localized enolase (ENO1) involved in glycolytic PEP provision. Homozygous double mutants of cue1 (defective in PPT1) and eno1 could not be obtained, and homozygous cue1 heterozygous eno1 mutants [cue1/eno1(+/-)] exhibited retarded vegetative growth, disturbed flower development, and up to 80% seed abortion. The phenotypes of diminished oil in seeds, reduced flavonoids and aromatic amino acids in flowers, compromised lignin biosynthesis in stems, and aberrant exine formation in pollen indicate that cue1/eno1(+/-) disrupts multiple pathways. While diminished fatty acid biosynthesis from PEP via plastidial pyruvate kinase appears to affect seed abortion, a restriction in the shikimate pathway affects formation of sporopollonin in the tapetum and lignin in the stem. Vegetative parts of cue1/eno1(+/-) contained increased free amino acids and jasmonic acid but had normal wax biosynthesis. ENO1 overexpression in cue1 rescued the leaf and root phenotypes, restored photosynthetic capacity, and improved seed yield and oil contents. In chloroplasts, ENO1 might be the only enzyme missing for a complete plastidic glycolysis.

All photosynthetic organisms face the difficulty of maintaining cellular metabolism in the absence of photosynthetic active radiation during the night. Although many consuming metabolic pathways (e.g., fatty acid synthesis) are only active in the light, plant cells still require basic levels of metabolic energy and reductive power during the night for sustained growth and development.

Glucosinolates are plant secondary metabolites involved in responses to biotic stress. The final step of their synthesis is the transfer of a sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) onto a desulfo precursor. Thus, glucosinolate synthesis is linked to sulfate assimilation. The sulfate donor for this reaction is synthesized from sulfate in two steps catalyzed by ATP sulfurylase (ATPS) and adenosine 5'-phosphosulfate kinase (APK). Here we demonstrate that R2R3-MYB transcription factors, which are known to regulate both aliphatic and indolic glucosinolate biosynthesis in Arabidopsis thaliana, also control genes of primary sulfate metabolism. Using trans-activation assays we found that two isoforms of APK, APK1, and APK2, are regulated by both classes of glucosinolate MYB transcription factors; whereas two ATPS genes, ATPS1 and ATPS3, are differentially regulated by these two groups of MYB factors. In addition, we show that the adenosine 5'-phosphosulfate reductases APR1, APR2, and APR3, which participate in primary sulfate reduction, are also activated by the MYB factors. These observations were confirmed by analysis of transgenic lines with modulated expression levels of the glucosinolate MYB factors. The changes in transcript levels also affected enzyme activities, the thiol content and the sulfate reduction rate in some of the transgenic plants. Altogether the data revealed that the MYB transcription factors regulate genes of primary sulfate metabolism and that the genes involved in the synthesis of activated sulfate are part of the glucosinolate biosynthesis network.

The Arabidopsis thaliana locus At5g06580 encodes an ortholog to Saccharomyces cerevisiae d-lactate dehydrogenase (AtD-LDH). The recombinant protein is a homodimer of 59-kDa subunits with one FAD per monomer. A substrate screen indicated that AtD-LDH catalyzes the oxidation of d- and l-lactate, d-2-hydroxybutyrate, glycerate, and glycolate using cytochrome c as an electron acceptor. AtD-LDH shows a clear preference for d-lactate, with a catalytic efficiency 200- and 2000-fold higher than that for l-lactate and glycolate, respectively, and a K(m) value for d-lactate of approximately 160 microm. Knock-out mutants showed impaired growth in the presence of d-lactate or methylglyoxal. Collectively, the data indicated that the protein is a d-LDH that participates in planta in the methylglyoxal pathway. Web-based bioinformatic tools revealed the existence of a paralogous protein encoded by locus At4g36400. The recombinant protein is a homodimer of 61-kDa subunits with one FAD per monomer. A substrate screening revealed highly specific d-2-hydroxyglutarate (d-2HG) conversion in the presence of an organic cofactor with a K(m) value of approximately 580 microm. Thus, the enzyme was characterized as a d-2HG dehydrogenase (AtD-2HGDH). Analysis of knock-out mutants demonstrated that AtD-2HGDH is responsible for the total d-2HGDH activity present in A. thaliana. Gene coexpression analysis indicated that AtD-2HGDH is in the same network as several genes involved in beta-oxidation and degradation of branched-chain amino acids and chlorophyll. It is proposed that AtD-2HGDH participates in the catabolism of d-2HG most probably during the mobilization of alternative substrates from proteolysis and/or lipid degradation.

Fatty acid beta-oxidation is essential for seedling establishment of oilseed plants, but little is known about its role in leaf metabolism of adult plants. Arabidopsis thaliana plants with loss-of-function mutations in the peroxisomal ABC-transporter1 (PXA1) or the core beta-oxidation enzyme keto-acyl-thiolase 2 (KAT2) have impaired peroxisomal beta-oxidation. pxa1 and kat2 plants developed severe leaf necrosis, bleached rapidly when returned to light, and died after extended dark treatment, whereas the wild type was unaffected. Dark-treated pxa1 plants showed a decrease in photosystem II efficiency early on and accumulation of free fatty acids, mostly alpha-linolenic acid [18:3(n-3)] and pheophorbide a, a phototoxic chlorophyll catabolite causing the rapid bleaching. Isolated wild-type and pxa1 chloroplasts challenged with comparable alpha-linolenic acid concentrations both showed an 80% reduction in photosynthetic electron transport, whereas intact pxa1 plants were more susceptible to the toxic effects of alpha-linolenic acid than the wild type. Furthermore, starch-free mutants with impaired PXA1 function showed the phenotype more quickly, indicating a link between energy metabolism and beta-oxidation. We conclude that the accumulation of free polyunsaturated fatty acids causes membrane damage in pxa1 and kat2 plants and propose a model in which fatty acid respiration via peroxisomal beta-oxidation plays a major role in dark-treated plants after depletion of starch reserves.

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.

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.

Plants can metabolize sulfate by two pathways, which branch at the level of adenosine 5'-phosphosulfate (APS). APS can be reduced to sulfide and incorporated into Cys in the primary sulfate assimilation pathway or phosphorylated by APS kinase to 3'-phosphoadenosine 5'-phosphosulfate, which is the activated sulfate form for sulfation reactions. To assess to what extent APS kinase regulates accumulation of sulfated compounds, we analyzed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, and the sulfated 12-hydroxyjasmonate were reduced approximately fivefold in apk1 apk2 plants. Although auxin levels were increased in the apk1 apk2 mutants, as is the case for most plants with compromised glucosinolate synthesis, typical high auxin phenotypes were not observed. The reduction in glucosinolates resulted in increased transcript levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. It also led to great alterations in sulfur metabolism: the levels of sulfate and thiols increased in the apk1 apk2 plants. The data indicate that the APK1 and APK2 isoforms of APS kinase play a major role in the synthesis of secondary sulfated metabolites and are required for normal growth rates.

An Arabidopsis (Arabidopsis thaliana) double mutant impaired in starch biosynthesis and the triose phosphate/phosphate translocator (adg1-1/tpt-1) is characterized by a diminished utilization of photoassimilates and the concomitant consumption of reducing power and energy produced in the photosynthetic light reaction. In order to guarantee survival, the double mutant responds to this metabolic challenge with growth retardation, an 80% decline in photosynthetic electron transport, diminished chlorophyll contents, an enhanced reduction state of plastoquinone in the dark (up to 50%), a perturbation of the redox poise in leaves (increased NADPH/NADP ratios and decreased ascorbate/dehydroascorbate ratios), hyperstacking of grana thylakoids, and an increased number of plastoglobules. Enhanced oxygen consumption and applications of inhibitors of alternative mitochondrial and chloroplast oxidases (AOX and PTOX) suggest that chlororespiration as well as mitochondrial respiration are involved in the enhanced plastoquinone reduction state in the dark. Transcript amounts of PTOX and AOX were diminished and nucleus-encoded components related to plastidic NADH reductase (NDH1) were increased in adg1-1/tpt-1 compared with the wild type. Cytochrome b559, proposed to be involved in the reoxidation of photosystem II, was not regulated at the transcriptional level. The hyperstacking of grana thylakoids mimics adaptation to low light, and increased plastoglobule numbers suggest a response to enhanced oxidative stress. Altered chloroplast organization combined with perturbations in the redox poise suggests that adg1-1/tpt-1 could be a tool for the in vivo study of retrograde signaling mechanisms controlling the coordinated expression of nucleus- and plastome-encoded photosynthetic genes.

Reactive oxygen species (ROS) represent both toxic by-products of aerobic metabolism as well as signaling molecules in processes like growth regulation and defense pathways. The study of signaling and oxidative-damage effects can be separated in plants expressing glycolate oxidase in the plastids (GO plants), where the production of H(2)O(2) in the chloroplasts is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions. Thus, GO plants represent an ideal non-invasive model to study events related to the perception and responses to H(2)O(2) accumulation. Metabolic profiling of GO plants indicated that under high light a sustained production of H(2)O(2) imposes coordinate changes on central metabolic pathways. The overall metabolic scenario is consistent with decreased carbon assimilation, which results in lower abundance of glycolytic and tricarboxylic acid cycle intermediates, while simultaneously amino acid metabolism routes are specifically modulated. The GO plants, although retarded in growth and flowering, can complete their life cycle indicating that the reconfiguration of the central metabolic pathways is part of a response to survive and thus, to adapt to stress conditions imposed by the accumulation of H(2)O(2) during the light period.

Reactive oxygen species (ROS) are continuously produced in several organelles during aerobic metabolism. Furthermore, a wide range of environmental stresses such as chilling, salinity, drought and high light, lead to an elevated production of ROS. ROS can react with biomolecules and cause oxidative damage and even necrosis. Antioxidants and antioxidant-enzymes function to interrupt the cascades of uncontrolled oxidation. On the other hand, ROS influence the expression of genes playing a central role in many signaling pathways. Tools like the exogenous application of oxidative stress-causing agents and the in planta production of ROS in mutants altered in ROS metabolism are increasingly used to assess specific and common responses toward different types of ROS signals. The major challenge is the identification of ROS sensors and signaling components to finally elucidate the molecular mechanisms of oxidative stress response in plants.

Arabidopsis (Arabidopsis thaliana) overexpressing glycolate oxidase (GO) in chloroplasts accumulates both hydrogen peroxide (H(2)O(2)) and glyoxylate. GO-overexpressing lines (GO plants) grown at 75 micromol quanta m(-2) s(-1) show retarded development, yellowish rosettes, and impaired photosynthetic performance, while at 30 micromol quanta m(-2) s(-1), this phenotype virtually disappears. The GO plants develop oxidative stress lesions under photorespiratory conditions but grow like wild-type plants under nonphotorespiratory conditions. GO plants coexpressing enzymes that further metabolize glyoxylate but still accumulate H(2)O(2) show all features of the GO phenotype, indicating that H(2)O(2) is responsible for the GO phenotype. The GO plants can complete their life cycle, showing that they are able to adapt to the stress conditions imposed by the accumulation of H(2)O(2) during the light period. Moreover, the data demonstrate that a response to oxidative stress is installed, with increased expression and/or activity of known oxidative stress-responsive components. Hence, the GO plants are an ideal noninvasive model system in which to study the effects of H(2)O(2) directly in the chloroplasts, because H(2)O(2) accumulation is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions.

Transgenic potato (Solanum tuberosum) plants simultaneously over-expressing a pea (Pisum sativum) glucose-6-phosphate/phosphate translocator (GPT) and an Arabidopsis thaliana adenylate translocator (NTT1) in tubers were generated. Double transformants exhibited an enhanced tuber yield of up to 19%, concomitant with an additional increased starch content of up to 28%, compared with control plants. The total starch content produced in tubers per plant was calculated to be increased by up to 44% in double transformants relative to the wild-type. Single over-expression of either gene had no effect on tuber starch content or tuber yield, suggesting that starch formation within amyloplasts is co-limited by the import of energy and the supply of carbon skeletons. As total adenosine diphosphate-glucose pyrophosphorylase and starch synthase activities remained unchanged in double transformants relative to the wild-type, they cannot account for the increased starch content found in tubers of double transformants. Rather, an optimized supply of amyloplasts with adenosine triphosphate and glucose-6-phosphate seems to favour increased starch synthesis, resulting in plants with increased starch content and yield of tubers.

Although the nonphotosynthetic NAD-malic enzyme (NAD-ME) was assumed to play a central role in the metabolite flux through the tricarboxylic acid cycle, the knowledge on this enzyme is still limited. Here, we report on the identification and characterization of two genes encoding mitochondrial NAD-MEs from Arabidopsis (Arabidopsis thaliana), AtNAD-ME1 and AtNAD-ME2. The encoded proteins can be grouped into the two clades found in the plant NAD-ME phylogenetic tree. AtNAD-ME1 belongs to the clade that includes known alpha-subunits with molecular masses of approximately 65 kD, while AtNAD-ME2 clusters with the known beta-subunits with molecular masses of approximately 58 kD. The separated recombinant proteins showed NAD-ME activity, presented comparable kinetic properties, and are dimers in their active conformation. Native electrophoresis coupled to denaturing electrophoresis revealed that in vivo AtNAD-ME forms a dimer of nonidentical subunits in Arabidopsis. Further support for this conclusion was obtained by reconstitution of the active heterodimer in vitro. The characterization of loss-of-function mutants for both AtNAD-MEs indicated that both proteins also exhibit enzymatic activity in vivo. Neither the single nor the double mutants showed a growth or developmental phenotype, suggesting that NAD-ME activity is not essential for normal autotrophic development. Nevertheless, metabolic profiling of plants completely lacking NAD-ME activity revealed differential patterns of modifications in light and dark periods and indicates a major role for NAD-MEs during nocturnal metabolism.

In a previous transactivation screen, two Arabidopsis thaliana R2R3-MYB transcription factors, HAG2/MYB76 and HAG3/MYB29, along with the already characterized HAG1/MYB28, were identified as putative regulators of aliphatic glucosinolate biosynthesis. Molecular and biochemical characterization of HAG2/MYB76 and HAG3/MYB29 functions was performed using transformants with increased or repressed transcript levels. Real-time PCR assays, cotransformation assays and measurements of glucosinolate contents were used to assess the impact of both MYB factors on the steady-state level of glucosinolate biosynthetic genes and accumulation of aliphatic glucosinolates. Both HAG2/MYB76 and HAG3/MYB29 were shown to be positive regulators of aliphatic glucosinolate biosynthesis. Expression of promoter-beta-glucuronidase (GUS) fusions indicated GUS activities in both vegetative and generative organs, with distinct characteristics for each MYB factor. HAG1/MYB28, HAG2/MYB76 and HAG3/MYB29 reciprocally transactivated each other in the control of aliphatic glucosinolate biosynthesis and downregulated the expression of genes involved in the control of indolic glucosinolate biosynthesis, pointing to a reciprocal negative regulation of these two pathways. All three HAG transcription factors exert a coordinated control on aliphatic glucosinolate biosynthesis.

The full-length cDNA encoding the maize (Zea mays) C(4) NADP-malic enzyme was expressed in Arabidopsis (Arabidopsis thaliana) under the control of the cauliflower mosaic virus 35S promoter. Homozygous transgenic plants (MEm) were isolated with activities ranging from 6- to 33-fold of those found in the wild type. The transformants did not show any differences in morphology and development when grown in long days; however, dark-induced senescence progressed more rapidly in MEm plants compared to the wild type. Interestingly, senescence could be retarded in the transgenic lines by exogenously supplying glucose, sucrose, or malate, suggesting that the lack of a readily mobilized carbon source is likely to be the initial factor leading to the premature induction of senescence in MEm plants. A comprehensive metabolic profiling on whole rosettes allowed determination of approximately 80 metabolites during a diurnal cycle as well as following dark-induced senescence and during metabolic complementation assays. MEm plants showed no differences in the accumulation and degradation of carbohydrates with respect to the wild type in all conditions tested, but accumulated lower levels of intermediates used as respiratory substrates, prominently malate and fumarate. The data indicated that extremely low levels of malate and fumarate are responsible for the accelerated dark-induced senescence encountered in MEm plants. Thus, in prolonged darkness these metabolites are consumed faster than in the wild type and, as a consequence, MEm plants enter irreversible senescence more rapidly. In addition, the data revealed that both malate and fumarate are important forms of fixed carbon that can be rapidly metabolized under stress conditions in Arabidopsis.

Methionine-derived glucosinolates belong to a class of plant secondary metabolites that serve as chemoprotective compounds in plant biotic defense reactions and also exhibit strong anticancerogenic properties beneficial to human health. In a screen for the trans-activation potential of various transcription factors toward glucosinolate biosynthetic genes, we could identify the HAG1 (HIGH ALIPHATIC GLUCOSINOLATE 1, also referred to as MYB28) gene as a positive regulator of aliphatic methionine-derived glucosinolates. The content of aliphatic glucosinolates as well as transcript levels of aliphatic glucosinolate biosynthetic genes were elevated in gain-of-function mutants and decreased in HAG1 RNAi knock-down mutants. Pro(HAG1):GUS expression analysis revealed strong HAG1 promoter activity in generative organs and mature leaves of A. thaliana plants, the main sites of accumulation of aliphatic glucosinolates. Mechanical stimuli such as touch or wounding transiently induced HAG1/MYB28 expression in inflorescences of flowering plants, and HAG1/MYB28 over-expression reduced insect performance as revealed by weight gain assays with the generalist lepidopteran herbivore Spodoptera exigua. Expression of HAG1/MYB28 was significantly induced by glucose, indicating a novel transcriptional regulatory mechanism for the integration of carbohydrate availability upon biotic challenge. We hypothesize that HAG1/MYB28 is a novel regulator of aliphatic glucosinolate biosynthesis that controls the response to biotic challenges.

Glucosinolates are a class of plant secondary metabolites that serve as antiherbivore compounds in plant defence. A previously identified Arabidopsis thaliana activation-tagged line, displaying altered levels of secondary metabolites, was shown here to be affected in the content of indolic and aliphatic glucosinolates. The observed chemotype was caused by activation of the R2R3-MYB transcription factor gene HIG1 (HIGH INDOLIC GLUCOSINOLATE 1, also referred to as MYB51). HIG1/MYB51 was shown to activate promoters of indolic glucosinolate biosynthetic genes leading to increased accumulation of indolic glucosinolates. The corresponding loss-of-function mutant hig1-1 contained low levels of glucosinolates. Overexpression of the related transcription factor ATR1/MYB34, which had previously been described as a regulator of indolic glucosinolate and indole-3-acetic acid homeostasis, in the hig1-1 mutant background led to a partial rescue of the mutant chemotype along with a severe high-auxin growth phenotype. Overexpression of MYB122, another close homologue of HIG1/MYB51, did not rescue the hig1-1 chemotype, but caused a high-auxin phenotype and increased levels of indolic glucosinolates in the wild-type. By contrast, overexpression of HIG1/MYB51 resulted in the specific accumulation of indolic glucosinolates without affecting auxin metabolism and plant morphology. Mechanical stimuli such as touch or wounding transiently induced the expression of HIG1/MYB51 but not of ATR1/MYB34, and HIG1/MYB51 overexpression reduced insect herbivory as revealed by dual-choice assays with the generalist lepidopteran herbivore, Spodoptera exigua. We hypothesize that HIG1/MYB51 is a regulator of indolic glucosinolate biosynthesis that also controls responses to biotic challenges.

Transient expression systems are intensively used to study the transactivation potential of transcription factors and to confirm target promoters. Here we present a novel system based on the high-efficiency transformation of cultured Arabidopsis thaliana cells by agrobacteria. To demonstrate the potential of this system, we compared it with a commonly used protoplast transfection assay, and studied the regulation of phenylpropanoid biosynthetic pathway genes by various transcription factors. Both systems led to comparable results on the regulation of the promoters tested. However, the agrobacterium-mediated co-transformation assay needs significantly less time, requires only mixing of cultured plant cells with agrobacteria, is less labour-intensive and allows handling of multiple assays in parallel, making it suitable for medium- to high-throughput analyses. In addition, the binary vectors used are the same for both cell-based assays and stable plant transformations.

By screening genome databases, 12 genes encoding membrane proteins homologous to nucleobase-ascorbate transporters (NATs) were identified in Arabidopsis thaliana. A similar number of genes was found in the rice genome. The plant NAT proteins split into five clades (I-V) based on protein multisequence alignments. This classification nicely correlates with the patterns of organ- and tissue-specific expression during the whole life cycle of A. thaliana. Interestingly, expression of two members of clade III, AtNAT7 and AtNAT8, was found to be up-regulated in undifferentiated tissues such as callus or tumors produced by Agrobacterium tumefaciens. Clade V comprises AtNAT12 possessing a hydrophilic N-terminal extension. Transient expression of green fluorescent protein (GFP) fusions in different systems showed that AtNAT12 along with AtNAT7 and -8 are located in the plasma membrane. Mutations in any of the AtNAT genes do not induce phenotypic alterations. The absence of obvious mutant phenotypes in single but also in double and triple mutants suggests a high degree of functional redundancy between AtNAT genes, but might also point to redundant functions provided by genes or pathways unrelated to the AtNATs.