Multi-omics data integration reveals link between epigenetic modifications and gene expression in sugar beet (Beta vulgaris subsp. vulgaris) in response to cold.

BACKGROUND: DNA methylation is thought to influence the expression of genes, especially in response to changing environmental conditions and developmental changes. Sugar beet (Beta vulgaris ssp. vulgaris), and other biennial or perennial plants are inevitably exposed to fluctuating temperatures throughout their lifecycle and might even require such stimulus to acquire floral competence. Therefore, plants such as beets, need to fine-tune their epigenetic makeup to ensure phenotypic plasticity towards changing environmental conditions while at the same time steering essential developmental processes. Different crop species may show opposing reactions towards the same abiotic stress, or, vice versa, identical species may respond differently depending on the specific kind of stress. RESULTS: In this study, we investigated common effects of cold treatment on genome-wide DNA methylation and gene expression of two Beta vulgaris accessions via multi-omics data analysis. Cold exposure resulted in a pronounced reduction of DNA methylation levels, which particularly affected methylation in CHH context (and to a lesser extent CHG) and was accompanied by transcriptional downregulation of the chromomethyltransferase CMT2 and strong upregulation of several genes mediating active DNA demethylation. CONCLUSION: Integration of methylomic and transcriptomic data revealed that, rather than methylation having directly influenced expression, epigenetic modifications correlated with changes in expression of known players involved in DNA (de)methylation. In particular, cold triggered upregulation of genes putatively contributing to DNA demethylation via the ROS1 pathway. Our observations suggest that these transcriptional responses precede the cold-induced global DNA-hypomethylation in non-CpG, preparing beets for additional transcriptional alterations necessary for adapting to upcoming environmental changes.

Cold-Triggered Induction of ROS- and Raffinose Metabolism in Freezing-Sensitive Taproot Tissue of Sugar Beet.

Sugar beet (Beta vulgaris subsp. vulgaris) is the exclusive source of sugar in the form of sucrose in temperate climate zones. Sugar beet is grown there as an annual crop from spring to autumn because of the damaging effect of freezing temperatures to taproot tissue. A collection of hybrid and non-hybrid sugar beet cultivars was tested for winter survival rates and freezing tolerance. Three genotypes with either low or high winter survival rates were selected for detailed study of their response to frost. These genotypes differed in the severity of frost injury in a defined inner region in the upper part of the taproot, the so-called pith. We aimed to elucidate genotype- and tissue-dependent molecular processes during freezing and combined analyses of sugar beet anatomy and physiology with transcriptomic and metabolite profiles of leaf and taproot tissues at low temperatures. Freezing temperatures induced strong downregulation of photosynthesis in leaves, generation of reactive oxygen species (ROS), and ROS-related gene expression in taproots. Simultaneously, expression of genes involved in raffinose metabolism, as well as concentrations of raffinose and its intermediates, increased markedly in both leaf and taproot tissue at low temperatures. The accumulation of raffinose in the pith tissue correlated with freezing tolerance of the three genotypes. We discuss a protective role for raffinose and its precursors against freezing damage of sugar beet taproot tissue.

Auxin signaling and vascular cambium formation enable storage metabolism in cassava tuberous roots.

Cassava storage roots are among the most important root crops worldwide, and represent one of the most consumed staple foods in sub-Saharan Africa. The vegetatively propagated tropical shrub can form many starchy tuberous roots from its stem. These storage roots are formed through the activation of secondary root growth processes. However, the underlying genetic regulation of storage root development is largely unknown. Here we report distinct structural and transcriptional changes occurring during the early phases of storage root development. A pronounced increase in auxin-related transcripts and the transcriptional activation of secondary growth factors, as well as a decrease in gibberellin-related transcripts were observed during the early stages of secondary root growth. This was accompanied by increased cell wall biosynthesis, most notably increased during the initial xylem expansion within the root vasculature. Starch storage metabolism was activated only after the formation of the vascular cambium. The formation of non-lignified xylem parenchyma cells and the activation of starch storage metabolism coincided with increased expression of the KNOX/BEL genes KNAT1, PENNYWISE, and POUND-FOOLISH, indicating their importance for proper xylem parenchyma function.

Vernalization Alters Sink and Source Identities and Reverses Phloem Translocation from Taproots to Shoots in Sugar Beet.

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.

The Cassava Source-Sink project: opportunities and challenges for crop improvement by metabolic engineering.

Cassava (Manihot esculenta Crantz) is one of the important staple foods in Sub-Saharan Africa. It produces starchy storage roots that provide food and income for several hundred million people, mainly in tropical agriculture zones. Increasing cassava storage root and starch yield is one of the major breeding targets with respect to securing the future food supply for the growing population of Sub-Saharan Africa. The Cassava Source-Sink (CASS) project aims to increase cassava storage root and starch yield by strategically integrating approaches from different disciplines. We present our perspective and progress on cassava as an applied research organism and provide insight into the CASS strategy, which can serve as a blueprint for the improvement of other root and tuber crops. Extensive profiling of different field-grown cassava genotypes generates information for leaf, phloem, and root metabolic and physiological processes that are relevant for biotechnological improvements. A multi-national pipeline for genetic engineering of cassava plants covers all steps from gene discovery, cloning, transformation, molecular and biochemical characterization, confined field trials, and phenotyping of the seasonal dynamics of shoot traits under field conditions. Together, the CASS project generates comprehensive data to facilitate conventional breeding strategies for high-yielding cassava genotypes. It also builds the foundation for genome-scale metabolic modelling aiming to predict targets and bottlenecks in metabolic pathways. This information is used to engineer cassava genotypes with improved source-sink relations and increased yield potential.

Metabolic profiles of six African cultivars of cassava (Manihot esculenta Crantz) highlight bottlenecks of root yield.

Cassava is an important staple crop in sub-Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenolpyruvate carboxylase activity support a C3 mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin-Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse-grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.

Cassava Metabolomics and Starch Quality.

Cassava plays an important role as a staple food for more than 800 million people in the world due to its ability to maintain relatively high productivity even in nutrient-depleted soils. Even though cassava has been the focus of several breeding programs and has become a strong focus of research in the last few years, relatively little is currently known about its metabolism and metabolic composition in different tissues. In this article, the absolute content of sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, carotenoids, chlorophylls, tocopherols, and total protein as well as starch quality is described based on multiple analytical techniques, with protocols specifically adjusted for material from different cassava tissues. Moreover, quantification of secondary metabolites relative to internal standards is presented using both non-targeted and targeted metabolomics approaches. The protocols have also been adjusted to apply to freeze-dried material in order to allow processing of field harvest samples that typically will require long-distance transport. © 2019 The Authors. Basic Protocol 1: Metabolic profiling by gas chromatography-mass spectrometry (GC-MS) Support Protocol 1: Preparation of freeze-dried cassava material Support Protocol 2: Preparation of standard compound mixtures for absolute quantification of metabolites by GC-MS Support Protocol 3: Preparation of retention-time standard mixture Basic Protocol 2: Determination of organic acids and phosphorylated intermediates by ion chromatography-mass spectrometry (IC-MS) Support Protocol 4: Preparation of standards and recovery experimental procedure Basic Protocol 3: Determination of soluble sugars, starch, and free amino acids Alternate Protocol: Determination of soluble sugars and starch Basic Protocol 4: Determination of anions Basic Protocol 5: Determination of elements Basic Protocol 6: Determination of total protein Basic Protocol 7: Determination of non-targeted and targeted secondary metabolites Basic Protocol 8: Determination of carotenoids, chlorophylls, and tocopherol Basic Protocol 9: Determination of starch quality.

Symplasmic phloem unloading and radial post-phloem transport via vascular rays in tuberous roots of Manihot esculenta.

Cassava (Manihot esculenta) is one of the most important staple food crops worldwide. Its starchy tuberous roots supply over 800 million people with carbohydrates. Yet, surprisingly little is known about the processes involved in filling of those vital storage organs. A better understanding of cassava carbohydrate allocation and starch storage is key to improving storage root yield. Here, we studied cassava morphology and phloem sap flow from source to sink using transgenic pAtSUC2::GFP plants, the phloem tracers esculin and 5(6)-carboxyfluorescein diacetate, as well as several staining techniques. We show that cassava performs apoplasmic phloem loading in source leaves and symplasmic unloading into phloem parenchyma cells of tuberous roots. We demonstrate that vascular rays play an important role in radial transport from the phloem to xylem parenchyma cells in tuberous roots. Furthermore, enzymatic and proteomic measurements of storage root tissues confirmed high abundance and activity of enzymes involved in the sucrose synthase-mediated pathway and indicated that starch is stored most efficiently in the outer xylem layers of tuberous roots. Our findings form the basis for biotechnological approaches aimed at improved phloem loading and enhanced carbohydrate allocation and storage in order to increase tuberous root yield of cassava.

In Concert: Orchestrated Changes in Carbohydrate Homeostasis Are Critical for Plant Abiotic Stress Tolerance.

The sessile lifestyle of higher plants is accompanied by their remarkable ability to tolerate unfavorable environmental conditions. This is because, during evolution, plants developed a sophisticated repertoire of molecular and metabolic reactions to cope with changing biotic and abiotic challenges. In particular, the abiotic factors light intensity and ambient temperature are characterized by altering their amplitude within comparably short periods of time and are causative for onset of dynamic plant responses. These rapid responses in plants are also classified as 'acclimation reactions' which differ, due to their reversibility and duration, from non-reversible 'adaptation reactions'. In this review, we demonstrate the remarkable importance of stress-induced changes in carbohydrate homeostasis of plants exposed to high light or low temperatures. These changes represent a co-ordinated process comprising modifications of (i) the concentrations of selected sugars; (ii) starch turnover; (iii) intracellular sugar compartmentation; and (iv) corresponding gene expression patterns. The critical importance of these individual processes has been underlined in the recent past by the analyses of a large number of mutant plants. The outcome of these analyses raised our understanding of acclimation processes in plants per se but might even become instrumental to develop new concepts for directed breeding approaches with the aim to increase abiotic stress tolerance of crop species, which in most cases have high stress sensitivity. The latter direction of plant research is of special importance since abiotic stress stimuli strongly impact on crop productivity and are expected to become even more pronounced because of human activities which alter environmental conditions rapidly.

Evidence for GABA-Induced Systemic GABA Accumulation in Arabidopsis upon Wounding.

The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is present in all plant species analyzed so far. Its synthesis is stimulated by either acidic conditions occurring after tissue disruption or higher cytosolic calcium level. In mammals, GABA acts as inhibitory neurotransmitter but its function in plants is still not well understood. Besides its involvement in abiotic stress resistance, GABA has a role in the jasmonate-independent defense against invertebrate pests. While the biochemical basis for GABA accumulation in wounded leaves is obvious, the underlying mechanisms for wounding-induced GABA accumulation in systemic leaves remained unclear. Here, the Arabidopsis thaliana knock-out mutant lines pop2-5, unable to degrade GABA, and tpc1-2, lacking a wounding-induced systemic cytosolic calcium elevation, were employed for a comprehensive investigation of systemic GABA accumulation. A wounding-induced systemic GABA accumulation was detected in tpc1-2 plants demonstrating that an increased calcium level was not involved. Similarly, after both mechanical wounding and Spodoptera littoralis feeding, GABA accumulation in pop2-5 plants was significantly higher in local and systemic leaves, compared to wild-type plants. Consequently, larvae feeding on these GABA-enriched mutant plants grew significantly less. Upon exogenous application of a D2-labeled GABA to wounded leaves of pop2-5 plants, its uptake but no translocation to unwounded leaves was detected. In contrast, an accumulation of endogenous GABA was observed in vascular connected systemic leaves. These results suggest that the systemic accumulation of GABA upon wounding does not depend on the translocation of GABA or on an increase in cytosolic calcium.

Demand for food as driver for plant sink development.

Starting with the first humans settling down to build their lives on agriculture and stock breeding, the development of food and feed became tremendously important. With increasing population, in particular boosted by industrialization, the need for more food rose further. One way to cope with the needs of people was to open up new and optimize already existing resources like the introduction of potato into the European population's diet and the development of grasses to high-yielding cereals, respectively. The process of plant improvement is still ongoing. Nowadays, yield enhancement is still an important breeding aim for several plant species as world population further increases, especially in less developed regions. However, in addition to quantity improvement, food quality is in the focus to prevent human malnutrition and resulting diseases or early death. In this review we will give a brief historical overview on how plants were developed to nourish the population and will discuss more recent approaches to secure sufficient food production.

Phenotypic and chemotypic studies using Arabidopsis and yeast reveal that GHB converts to SSA and induce toxicity.

γ-Hydroxybutyric acid (GHB) is a naturally occurring compound. It is detected in organisms such as yeasts, plants and mammals. GHB is produced from the reduction of succinic semialdehyde (SSA) by the activity of GHB dehydrogenase. Arabidopsis genome contains two GHB dehydrogenase encoding genes. The accumulation of GHB in ssadh mutants led to the speculation that GHB is the cause of aberrant phenotypes. Conversely, the accumulation of GHB in Arabidopsis plants subjected to abiotic stresses was described as a way of avoiding SSA induced damage. To resolve these contrasting views on GHB, we examined the effect of exogenous GHB and SSA on the growth of yeast and Arabidopsis plants. GHB concentrations up to 1.5 mM didn't affect shoots of Arabidopsis plants; however, root growth was inhibited. In contrast, 0.3 mM SSA has severely affected the growth of plants. Treatment of yeast wild-type strain with 10 mM SSA and 10 mM GHB didn't affect the growth. However, the growth of yeast uga2 mutant was greatly inhibited by the same concentration of SSA, but not GHB. Metabolic analysis and enzyme activity assay on native gel showed that Arabidopsis, but not yeast, possesses a GHB dehydrogenase activity that converts GHB back to SSA. The enzymatic assay has also indicated the existence of an additional GHB dehydrogenase encoding gene(s) in Arabidopsis genome. Taken together, we conclude that GHB is less toxic than SSA. Its accumulation in ssadh mutants and during abiotic stresses is a response to avoid the SSA induced damage.

Gamma-aminobutyric acid depletion affects stomata closure and drought tolerance of Arabidopsis thaliana.

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.

Identification of the transporter responsible for sucrose accumulation in sugar beet taproots.

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.

Insect Herbivory-Elicited GABA Accumulation in Plants is a Wound-Induced, Direct, Systemic, and Jasmonate-Independent Defense Response.

The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is present in all organisms analyzed so far. In invertebrates GABA acts as a neurotransmitter; in plants different functions are under discussion. Among others, its involvement in abiotic stress reactions and as a defensive compound against feeding insects is suggested. GABA is synthesized from glutamate by glutamate decarboxylases and degraded by GABA-transaminases. Here, in Arabidopsis thaliana, gad1/2 double mutants showing reduced GABA concentrations as well as GABA-enriched triple mutants (gad1/2 x pop2-5) were generated and employed for a systematic study of GABA induction, accumulation and related effects in Arabidopsis leaves upon herbivory. The results demonstrate that GABA accumulation is stimulated by insect feeding-like wounding by a robotic caterpillar, MecWorm, as well as by real insect (Spodoptera littoralis) herbivory. Higher GABA levels in both plant tissue and artificial dietary supplements in turn affect the performance of feeding larvae. GABA enrichment occurs not only in the challenged but also in adjacent leaf. This induced response is neither dependent on herbivore defense-related phytohormones, jasmonates, nor is jasmonate induction dependent on the presence of GABA. Thus, in Arabidopsis the rapid accumulation of GABA very likely represents a general, direct and systemic defense reaction against insect herbivores.

Amino acids--a life between metabolism and signaling.

Amino acids serve as constituents of proteins, precursors for anabolism, and, in some cases, as signaling molecules in mammalians and plants. This review is focused on new insights, or speculations, on signaling functions of serine, γ-aminobutyric acid (GABA) and phenylalanine-derived phenylpropanoids. Serine acts as signal in brain tissue and mammalian cancer cells. In plants, de novo serine biosynthesis is also highly active in fast growing tissues such as meristems, suggesting a similar role of serine as in mammalians. GABA functions as inhibitory neurotransmitter in the brain. In plants, GABA is also abundant and seems to be involved in sexual reproduction, cell elongation, patterning and cell identity. The aromatic amino acids phenylalanine, tyrosine, and tryptophan are precursors for the production of secondary plant products. Besides their pharmaceutical value, lignans, neolignans and hydroxycinnamic acid amides (HCAA) deriving from phenylpropanoid metabolism and, in the case of HCAA, also from arginine have been shown to fulfill signaling functions or are involved in the response to biotic and abiotic stress. Although some basics on phenylpropanoid-derived signaling have been described, little is known on recognition- or signal transduction mechanisms. In general, mutant- and transgenic approaches will be helpful to elucidate the mechanistic basis of metabolite signaling.

Role of metabolite transporters in source-sink carbon allocation.

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.

Simultaneous boosting of source and sink capacities doubles tuber starch yield of potato plants.

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.

The role of transporters in supplying energy to plant plastids.

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.

Mutants of GABA transaminase (POP2) suppress the severe phenotype of succinic semialdehyde dehydrogenase (ssadh) mutants in Arabidopsis.

BACKGROUND: The gamma-aminubutyrate (GABA) shunt bypasses two steps of the tricarboxylic acid cycle, and is present in both prokaryotes and eukaryotes. In plants, the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase (GAD), the mitochondrial enzymes GABA transaminase (GABA-T; POP2) and succinic semialdehyde dehydrogenase (SSADH). We have previously shown that compromising the function of the GABA-shunt, by disrupting the SSADH gene of Arabidopsis, causes enhanced accumulation of reactive oxygen intermediates (ROIs) and cell death in response to light and heat stress. However, to date, genetic investigations of the relationships between enzymes of the GABA shunt have not been reported. PRINCIPAL FINDINGS: To elucidate the role of succinic semialdehyde (SSA), gamma-hydroxybutyrate (GHB) and GABA in the accumulation of ROIs, we combined two genetic approaches to suppress the severe phenotype of ssadh mutants. Analysis of double pop2 ssadh mutants revealed that pop2 is epistatic to ssadh. Moreover, we isolated EMS-generated mutants suppressing the phenotype of ssadh revealing two new pop2 alleles. By measuring thermoluminescence at high temperature, the peroxide contents of ssadh and pop2 mutants were evaluated, showing that only ssadh plants accumulate peroxides. In addition, pop2 ssadh seedlings are more sensitive to exogenous SSA or GHB relative to wild type, because GHB and/or SSA accumulate in these plants. SIGNIFICANCE: We conclude that the lack of supply of succinate and NADH to the TCA cycle is not responsible for the oxidative stress and growth retardations of ssadh mutants. Rather, we suggest that the accumulation of SSA, GHB, or both, produced downstream of the GABA-T transamination step, is toxic to the plants, resulting in high ROI levels and impaired development.