Frank Ludewig

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.

High CO2-mediated down-regulation of photosynthetic gene transcripts is caused by accelerated leaf senescence rather than sugar accumulation.

The influence of elevated atmospheric CO2 on transcript levels of photosynthetic genes was investigated in leaves of Nicotiana tabacum cv. SamsunNN and cv. Wisconsin38 plants. Plants were grown under ambient (400 ppm) and elevated (800/1000 ppm) atmospheric CO2, and transcript levels were determined in leaves of different age. Down‐regulation of photosynthetic gene transcripts was apparent in senescing leaves only. A correlation between transcript levels and leaf contents of soluble sugars could not be found. To investigate whether a shift in leaf ontogeny would be involved in the regulation of photosynthetic genes transgenic tobacco plants expressing either the gus or ipt gene under control of the senescence‐specific SAG‐12 promoter [Gan, S. and Amasino, R.M. (1995) Science 270, 1986–1988] were included in our studies. As expected SAG‐12‐driven GUS activity increased with leaf age. This increase of GUS activity was stimulated by elevated atmospheric CO2, accompanied by a loss of chlorophyll and the down‐regulation of photosynthetic genes, verifying that high CO2 accelerates leaf ontogeny. Senescence as well as down‐regulation of photosynthetic genes could be delayed by ipt expression. Levels of soluble sugars were indistinguishable from wild type or even slightly elevated in ipt transgenic plants. Therefore, sugar accumulation as a cause for down‐regulation of photosynthetic genes under high CO2 can be excluded. It appears more likely that the high CO2‐mediated decline in photosynthetic gene transcripts is due to a temporal shift in leaf ontogeny.

The role of transient starch in acclimation to elevated atmospheric CO2

Although increased concentrations of CO2 stimulate photosynthesis, this stimulation is often lost during prolonged exposure to elevated carbon dioxide, leading to an attenuation of the potential gain in yield. Under these conditions, a wide variety of species accumulates non‐structural carbohydrates in leaves. It has been proposed that starch accumulation directly inhibits photosynthesis, that the rate of sucrose and starch synthesis limits photosynthesis, or that accumulation of sugars triggers changes in gene expression resulting in lower activities of Rubisco and inhibition of photosynthesis. To distinguish these explanations, transgenic plants unable to accumulate transient starch due to leaf mesophyll‐specific antisense expression of AGP B were grown at ambient and elevated carbon dioxide. There was a positive correlation between the capacity for starch synthesis and the rate of photosynthesis at elevated CO2 concentrations, showing that the capability to synthesize leaf starch is essential for photosynthesis in elevated carbon dioxide. The results show that in elevated carbon dioxide, photosynthesis is restricted by the rate of end product synthesis. Accumulation of starch is not responsible for inhibition of photosynthesis. Although transgenic plants contained increased levels of hexoses, transcripts of photosynthetic genes were not downregulated and Rubisco activity was not decreased arguing against a role of sugar sensing in acclimation to high CO2.

Overriding the co-limiting import of carbon and energy into tuber amyloplasts increases the starch content and yield of transgenic potato plants

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.

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.

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.

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.

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.

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.