Nucleotide metabolism operates in all living organisms, embodies an evolutionarily ancient and indispensable complex of metabolic pathways and is of utmost importance for plant metabolism and development. In plants, nucleotides can be synthesized de novo from 5-phosphoribosyl-1-pyrophosphate and simple molecules (e.g., CO(2), amino acids, and tetrahydrofolate), or be derived from preformed nucleosides and nucleobases via salvage reactions. Nucleotides are degraded to simple metabolites, and this process permits the recycling of phosphate, nitrogen, and carbon into central metabolic pools. Despite extensive biochemical knowledge about purine and pyrimidine metabolism, comprehensive studies of the regulation of this metabolism in plants are only starting to emerge. Here we review progress in molecular aspects and recent studies on the regulation and manipulation of nucleotide metabolism in plants.

SummaryThe fungal pathogen Ustilago maydis establishes a biotrophic relationship with its host plant maize (Zea mays). Hallmarks of the disease are large plant tumours in which fungal proliferation occurs. Previous studies suggested that classical defence pathways are not activated. Confocal microscopy, global expression profiling and metabolic profiling now shows that U. maydis is recognized early and triggers defence responses. Many of these early response genes are downregulated at later time points, whereas several genes associated with suppression of cell death are induced. The interplay between fungus and host involves changes in hormone signalling, induction of antioxidant and secondary metabolism, as well as the prevention of source leaf establishment. Our data provide novel insights into the complexity of a biotrophic interaction.

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Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promoter was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.

Stomata are pores on the leaf surface, bounded by two guard cells, which control the uptake of CO(2) for photosynthesis and the concomitant loss of water vapor. In 1898, Francis Darwin showed that stomata close in response to reduced atmospheric relative humidity (rh); however, our understanding of the signaling pathway responsible for coupling changes in rh to alterations in stomatal aperture is fragmentary. The results presented here highlight the primacy of abscisic acid (ABA) in the stomatal response to drying air. We show that guard cells possess the entire ABA biosynthesis pathway and that it appears upregulated by positive feedback by ABA. When wild-type Arabidopsis and the ABA-deficient mutant aba3-1 were exposed to reductions in rh, the aba3-1 mutant wilted, whereas the wild-type did not. However, when aba3-1 plants, in which ABA synthesis had been specifically rescued in guard cells, were challenged with dry air, they did not wilt. These data indicate that guard cell-autonomous ABA synthesis is required for and is sufficient for stomatal closure in response to low rh. Guard cell-autonomous ABA synthesis allows the plant to tailor leaf gas exchange exquisitely to suit the prevailing environmental conditions.

Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5′‐upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber‐specific gene activity which was on average 100‐ to 1000‐fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the β‐glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.

Chimeric genes consisting of the coding sequence of the yeast invertase gene suc 2 and different N‐terminal portions of the potato‐derived vacuolar protein proteinase inhibitor II fused to the 35S CaMV promoter and the poly‐A site of the octopine synthase gene were transferred into tobacco and Arabidopsis thaliana plants using Agrobacterium based systems. Regenerated transgenic plants display a 50‐ to 500‐fold higher invertase activity compared to non‐transformed control plants. This invertase is N‐glycosylated and efficiently secreted from the plant cell leading to its apoplastic location. Whereas expression of the invertase does not lead to drastic changes in transgenic Arabidopsis thaliana plants, transgenic tobacco plants show dramatic changes with respect to development and phenotype. Expression of the invertase leads to stunted growth due to reduction of internodal distances, to development of bleached and/or necrotic regions in older leaves and to suppressed root formation. In mature leaves, high levels of soluble sugars and starch accumulate. These carbohydrates do not show a diurnal turnover. The accumulation of carbohydrate is accompanied by an inhibition of photosynthesis, and in tobacco, by an increase in the rate of respiration. Measurements in bleached versus green areas of the same leaf show that the bleached section contains high levels of carbohydrates and has lower photosynthesis and higher respiration than green sections. It is concluded that expression of invertase in the cell wall interrupts export and leads to an accumulation of carbohydrates and inhibition of photosynthesis.

SummaryThe original aim of this work was to increase starch accumulation in potato tubers by enhancing their capacity to metabolise sucrose. We previously reported that specific expression of a yeast invertase in the cytosol of tubers led to a 95% reduction in sucrose content, but that this was accompanied by a larger accumulation of glucose and a reduction in starch. In the present paper we introduced a bacterial glucokinase from Zymomonas mobilis into an invertase-expressing transgenic line, with the intention of bringing the glucose into metabolism. Transgenic lines were obtained with up to threefold more glucokinase activity than in the parent invertase line and which did not accumulate glucose. Unexpectedly, there was a further dramatic reduction in starch content, down to 35% of wild-type levels. Biochemical analysis of growing tuber tissue revealed large increases in the metabolic intermediates of glycolysis, organic acids and amino acids, two-to threefold increases in the maximum catalytic activities of key enzymes in the respiratory pathways, and three-to fivefold increases in carbon dioxide production. These changes occur in the lines expressing invertase, and are accentuated following introduction of the second transgene, glucokinase. We conclude that the expression of invertase in potato tubers leads to an increased flux through the glycolytic pathway at the expense of starch synthesis and that heterologous overexpression of glucokinase enhances this change in partitioning.