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The short-term changes in metabolism that occurred after adding glucose or sucrose to freshly cut discs from growing potato (Solanum tuberosum L.) tubers were investigated. (i) When glucose was supplied, there was a marked increase in glycolytic metabolites, and respiration was stimulated. When sucrose was supplied, amounts of glycolytic metabolites including hexose phosphates and 3-phosphoglycerate (3PGA) were similar to or lower than in control discs incubated without sugars, and respiration did not rise initially above that in control discs. This different response to sucrose and glucose was found across the concentration range 5–200 mM. A larger proportion of the metabolised 14C was converted to starch when [14C] sucrose was supplied than when [14C] glucose was supplied. The different effect on metabolite levels, respiration and starch synthesis was largest after 20–30 min, and decreased in longer incubations. (ii) When 5 or 25 mM sucrose was added in the presence of [14C] glucose, it led to a decrease in hexose phosphates and 3PGA, and a small increase in the rate of starch synthesis compared to discs incubated with glucose in the absence of sucrose. These differences were seen in a 30-min pulse and a 2-h pulse. Whereas ADP-glucose levels after adding sucrose resembled those in control discs, glucose led to a decrease in ADP-glucose. This decrease did not occur when 5 or 25 mM sucrose was added with the glucose. (iii) To check the relevance of these experiments for intact tubers, water or 100 mM mannitol, sucrose or glucose were supplied through the stolon to intact tubers for 24 h. A 0.2 mM solution of [14C] glucose was then introduced into the tubers, and its metabolism investigated during the next 30 min. Labelling of starch was increased after preincubation with sucrose, and significantly inhibited after preincubation with glucose. (iv) It is concluded that glucose and sucrose have different effects on tuber metabolism. Whereas glucose leads to a preferential stimulation of respiration, sucrose preferentially stimulates starch synthesis via a novel mechanism that allows stimulation of ADP-glucose pyrophosphorylase even though the levels of hexose phosphates and the allosteric activator 3PGA decrease.

Summary Expression of the gene Nrt2Np, which encodes a putative high-affinity nitrate transporter of Nicotiana plumbaginifolia was studied under variable physiological conditions. Nrt2Np is rapidly induced by very low nitrate concentrations and repressed by reduced nitrogen metabolites. Furthermore, Nrt2Np is expressed in coordination with other genes involved in nitrate assimilation (Nia, Nii). A deficiency in nitrate reductase activity, which is accompanied by high internal nitrate concentration and low levels of nitrogen metabolites, e.g. glutamine, leads to an overexpression of Nrt2Np, showing that high nitrate concentration per se does not repress Nrt2Np expression. By investigating plants with altered nitrate uptake properties, we showed a correlation between Nrt2 mRNA accumulation and 15N nitrate influx rates, providing the first evidence that the expression of Nrt2 correlates with the rate of nitrate uptake. In situ hybridization revealed a tissue-specific expression pattern. Nrt2Np mRNA accumulation is localized throughout all layers of the root tip, being highest in epidermal and endodermal cells. However, in mature root tissue, Nrt2 expression was detected mainly in the lateral root primordia and in the epidermis.

Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa 1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571-581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa 1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa 1 tubers. Unexpectedly, the rate of degradation of [14C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20-30% in ppa 1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa 1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to three-fold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30-100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa 1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider-significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed.

The effect of glucose and glucose analogs on the amount of rbcS transcript and on metabolism was investigated in autotrophic cell-suspension cultures of Chenopodiumrubrum L. Within 10 h glucose led to a decrease of the transcript for rbcS, and an increase of phosphorylated metabolites and carbohydrates. The changes of transcript and metabolism showed the same concentration dependence, and were already marked at 1 mM glucose. Mannose at 10 mM led to a decrease of rbcS transcript, a decrease of phosphorylated intermediates, and an inhibition of photosynthesis. [14C] Mannose was slowly metabolised to anions and cations. Very low concentrations (0.2 mM) of 2-deoxyglucose (2DOG) led to a decrease of rbcS transcript within 6 h, a large depletion of phosphorylated intermediates, ATP and UDPglucose, and an inhibition of photosynthesis. The changes of metabolism and photosynthesis were not reversed by phosphate. There was a gradual recovery of rbcS transcript after 24–36 h, which occurred earlier and was larger than the recovery of metabolites or photosynthesis. When 14C-labelled 2DOG was supplied at 0.2 mM to investigate 2DOG uptake and metabolism during this transient repression of rbcS expression, net uptake and net phosphorylation of 2DOG stopped after 4 and 6 h, respectively, even though considerable amounts of 2DOG were still present in the medium and the cells. Of the 2DOG entering the cells, over 70% was metabolised in the first 6 h. Unexpectedly, only 20% remained in 2DOG-6-phosphate, and 30–35% was converted to 2-deoxysucrose. At later time points, label in 2DOG decreased, in parallel with a decrease of label in 2DOG-6-phosphate, and in 2DOG in the cells. Label accumulated in unidentified acidic compounds, in three further low-molecular-weight neutral compounds, and in insoluble compounds. To investigate why net uptake and phosphorylation of 2DOG was inhibited, cells were preincubated with 0.2 mM 2DOG for 24 h, and then supplied with [14C]2DOG or [14C]glucose. Preincubation with 2DOG led to a small (about 50%) inhibition of [14C]2DOG uptake and phosphorylation (indicating that there is recycling of 2DOG), and a strong inhibition of deoxysucrose (85%) and sucrose (50%) synthesis from [14C]2DOG and [14C]glucose, respectively. The implications of these results for the interpretation of experiments with 2DOG are discussed.

Tobacco seedlings were grown in nutrient agar at a range of ammonium nitrate concentrations either without added sucrose, or with 100 mol m -3 sucrose. In the absence of added sucrose, nitrogen-limited plants had increased levels of glucose, fructose and sucrose, decreased chlorophyll, decreased protein, and decreased Rubisco activity, but the level of the transcript for the small suhunit of Rubisco (RbcS) did not decrease compared with nitrogen-sufficient plants. When sucrose was added to nitrogen-sufficient seedlings, there was an increase of sucrose, glucose and fructose in the leaves, growth was increased, and the chlorophyll and protein content, Rubisco activity, and the RbcS transcript level did not change. When sucrose was added to nitrogen-limited seedlings, there was a further increase of sucrose, glucose and fructose, growth was not increased, and there was a further decrease of chlorophyll, protein and Rubisco activity, and a marked decrease of the RbcS transcript level. To check that the decrease of the RbcS transcript level was not an indirect effect due to changes of nitrogen metabolites after adding sugars, glucose was added to Chenopodium cells in the presence and absence of glutamine or azaserine. Changes of glutamine that suffice to increase and decrease the level of the transcript for nitrate reductase (Nia) do not affect the RbcS transcript concentration, and glucose addition still led to a decrease of the RbcS transcript level when the internal glutamine concentration was high. Tobacco seedlings were also grown in nutrient agar at a range of phosphate concentrations either without added sucrose, or with 100 mol m -3 sucrose. Phosphate-limited seedlings did not show a decrease of chlorophyll, protein, Rubisco activity, or the level of the RbcS transcript, compared with phosphate-sufficient seedlings. The addition of sucrose to phosphate-limited plants led to a similar increase of sugars to that seen after adding sucrose to nitrogen-limited seedlings, but did not alter chlorophyll, protein, Rubisco activity, or the level of the RbcS transcript. The addition of sucrose to phosphate-limited plants led to a slight increase of the level of the transcript for nitrate reductase (Nia), increased nitrate reductase activity, and a marked increase of the amino acid content. Phosphate limitation led to an increased level of the transcript for the regulatory subunit of ADP glucose pyrophosphorylase (AgpS2), and this response was strengthened when sucrose was added. The regulation of AgpS2 expression by phosphate and sucrose was further investigated by feeding sucrose and phosphate to detached source leaves via the transpiration stream. The level of the AgpS2 transcript decreased after feeding phosphate and increased after feeding sucrose, and the effect of sucrose was antagonised by phosphate. It is concluded that the response to sugar signalling is modulated by nitrogen and phosphate in a gene-specific manner. The significance of these results for understanding the visual phenotype of nitrogen- and phosphate-limited plants, and the response of photosynthesis and starch synthesis to the plant nutrient status is discussed.

Antisense expression of a full length cDNA encoding plastid aldolase led to decreased expression of aldolase at the transcript and protein level in several 'antisense' potato transformants. To quantify the inhibition, activity was compared in corresponding leaves down a plant and in plants of different ages. Aldolase activity was decreased by 32-43%, 56-71%, 79-83% and 91-97% in A-70, A-3, A-51 and A-2. Separation on a Q-Sepharose-FF column showed the decrease was due to inhibition of plastid aldolase. The transformants showed a small increase of Rubisco activity, a small decrease of phosphoribulokinase activity, and larger but subproportional decreases of sedoheptulose-1,7-biphosphatase and plastid fructose-1,6-bisphosphatase activity. Ambient photosynthesis was inhibited by 10%, 40%, 66% and 85% in A-70, A-3, A-51 and A-2. The transformants contained increased triose phosphates, and very low ribulose-1,5-bisphosphate and glycerate-3-phosphate. Chlorophyll fluorescence indicated that photosystem II was more reduced and thylakoid energization was increased. Starch synthesis was decreased by 16% and 36% in A-70 and A-3, whereas sucrose synthesis was less strongly inhibited. Plant growth was not significantly altered in A-70, was decreased by 41% in A-3, and was severely inhibited in plants with under 20% of wild-type aldolase activity. Although plastid aldolase catalyses a readily reversible reaction, possesses no known regulatory properties, and would appear irrelevant for the control of metabolism and growth, small changes in its activity have marked consequences for photosynthesis, carbon partitioning and growth.

Transfer of potato tubers to low temperature leads after 2-4 d to a stimulation of sucrose synthesis, a decline of hexose-phosphates and a change in the kinetic properties, and the appearance of a new form of sucrose phosphate synthase (SPS). Antisense and co-suppression transformants with a 70-80% reduction in SPS expression have been used to analyse the contribution of SPS to the control of cold sweetening. The rate of sucrose synthesis in cold-stored tubers was investigated by measuring the accumulation of sugars, by injecting labelled glucose of high specific activity into intact tubers, and by providing 50 mol m -3 labelled glucose to fresh tuber slices from cold-stored tubers. A 70-80 % decrease of SPS expression resulted in a reproducible but non-proportional (10-40%) decrease of soluble sugars in cold-stored tubers, and a non-proportional (about 25%) inhibition of label incorporation into sucrose, increased labelling of respiratory intermediates and carbon dioxide, and increased labelling of glucans. The maximum activity of SPS is 50-fold higher than the net rate of sugar accumulation in wild-type tubers, and decreased expression of SPS in the transformants was partly compensated for increased levels of hexose-phosphates. It is concluded that SPS expression per se does not control sugar synthesis. Rather, a comparison of the in vitro properties of SPS with the estimated in vivo concentrations of effectors shows that SPS is strongly substrate limited in vivo. Alterations in the kinetic properties of SPS, such as occur in response to low temperature, will provide a more effective way to stimulate sucrose synthesis than changes of SPS expression.

The onset of sugar accumulation in cold-stored potato tubers coincides with an activation of sucrose phosphate synthase (SPS) and the appearance of a new form of amylase (Hill et al. 1996; Nielsen et al. 1997). To provide more evidence that these changes are involved in the regulation of cold-induced sugar accumulation we have compared the temperature dependence of sugar accumulation with the temperature dependence of changes in SPS and amylase activity. To do this, we investigated: sugars and metabolites; SPS activity, protein and transcript; invertase activity; and amylolytic activity and amylase forms, during the first 10 d after transferring tubers from room temperature to 3, 5, 7, 9 and 11 °C. After 10 d there was negligible accumulation of sugars at 11, 9 and 7 °C, and a large accumulation at 5 and 3 °C. Activation of SPS was assayed by comparing activity in an assay with limiting substrates and phosphate and an assay with saturating substrate concentrations. The activation state increased slightly at 7 °C, 3- to 4-fold at 5 °C and 5- to 6-fold at 3 °C. The cold-induced change in the kinetic properties of SPS was accompanied by the appearance of a new form of SPS with a slightly higher apparent molecular weight, and by an increase of the SPS transcript. The changes in SPS protein and transcript showed the same temperature dependence as the changes in the kinetic properties. Total starch hydrolysing enzyme activity was unaltered at 7°C, increased at 5°C and increased further (up to 7- to 8-fold) at 3 °C. The increase in amylolytic activity correlated with the appearance of a new amylase band on zymograms. Acid invertase activity showed a similar increase at 3, 5, and 7°C, and it did not correlate with the total sugar accumulation. The cold-induced accumulation of sugar can be reversed by transferring tubers back to warmer temperatures. We compared the decline of sugar levels with the changes of SPS, amylolytic activity and metabolites at various times (up to 14 d) after transfer of tubers from a 4 °C storage (for 14 d) back to 20°C. SPS activation state is reversed and the cold-induced form of SPS virtually disappears during the first 2–4 d at 20 °C. The cold-induced amylase activity also vanishes within 2–4 d.

Most previous analyses of shoot-root allocation have investigated correlations between changes in putative signals and shoot-root allocation. It is argued that studies of shoot-root allocation need to be extended to include investigations of mutants with specific lesions in nutrient metabolism, to identify the compounds that are sensed as indicators for the plant nutrient status and act as the starting point for specific transduction pathways. The mechanisms of nutrient sensing can then be investigated using molecular and genetic strategies analogous to those that have been successfully used to investigate other signal transduction events. Investigations of shoot-root allocation should also pay more attention to the way in which root architecture is modified in response to nutrient supply, and need to be designed and interpreted in the light of molecular and genetic analyses of root development.

Although nitrate reductase (NR, EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40–45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two instead of four functional nia genes compensate. (i) The nia transcript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3–4 h light in WT leaves, and then decreased by 50–60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40–60% less NR protein and NRA after 3–4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1–3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount of nia transcript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants, nia transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak of nia transcript, NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate, nia transcript and NRA 2–3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and weakening the feedback regulation, to allow assimilation to continue for a longer period each day.

Nia30(145) transformants with very low nitrate reductase activity provide an in vivo screen to identify processes that are regulated by nitrate. Nia30(145) resembles nitrate-limited wild-type plants with respect to growth rate and protein and amino acid content but accumulates large amounts of nitrate when it is grown on high nitrate. The transcripts for nitrate reductase (NR), nitrite reductase, cytosolic glutamine synthetase, and glutamate synthase increased; NR and nitrite reductase activity increased in leaves and roots; and glutamine synthetase activity increased in roots. The transcripts for phosphoenolpyruvate carboxylase, cytosolic pyruvate kinase, citrate synthase, and NADP-isocitrate dehydrogenase increased; phosphoenolpyruvate carboxylase activity increased; and malate, citrate, isocitrate, and [alpha]-oxoglutarate accumulated in leaves and roots. There was a decrease of the ADP-glucose pyrophosphorylase transcript and activity, and starch decreased in the leaves and roots. After adding 12 mM nitrate to nitrate-limited Nia30(145), the transcripts for NR and phosphoenolpyruvate carboxylase increased, and the transcripts for ADP-glucose pyrophosphorylase decreased within 2 and 4 hr, respectively. Starch was remobilized at almost the same rate as in wild-type plants, even though growth was not stimulated in Nia30(145). It is proposed that nitrate acts as a signal to initiate coordinated changes in carbon and nitrogen metabolism.

To investigate the effect of water stress on carbon metabolism in growing potato tubers (Solanum tuberosum L.), freshly cut and washed discs were incubated in a range of mannitol concentrations corresponding to external water potential between 0 and −1.2 MPa. (i) Incorporation of [14C]glucose into starch was inhibited in water-stressed discs, and labeling of sucrose was increased. High glucose overrode the changes at low water stress (up to −0.5 MPa) but not at high water stress. (ii) Although [14C]sucrose uptake increased in water-stressed discs, less of the absorbed [14C]sucrose was metabolised. (iii) Analysis of the sucrose content of the discs confirmed that increasing water deficit leads to a switch, from net sucrose degradation to net sucrose synthesis. (iv) In parallel incubations containing identical concentrations of sugars but differing in which sugar was labeled, degradation of [14C]sucrose and labeling of sucrose from [14C]glucose and fructose was found at each mannitol concentration. This shows that there is a cycle of sucrose degradation and resynthesis in these tuber discs. Increasing the extent of water stress changed the relation between sucrose breakdown and sucrose synthesis, in favour of synthesis. (v) Analysis of metabolites showed a biphasic response to increasing water deficit. Moderate water stress (0–200 mM mannitol) led to a decrease of the phosphorylated intermediates, especially 3-phosphoglycerate (3PGA). The decrease of metabolites at moderate water stress was not seen when high concentrations of glucose were supplied to the discs. More extreme water stress (300–500 mM mannitol) was accompanied by an accumulation of metabolites at low and high glucose. (vi) Moderate water stress led to an activation of sucrose phosphate synthase (SPS) in discs, and in intact tubers. The stimulation involved a change in the kinetic properties of SPS, and was blocked␣by protein phosphatase inhibitors. (vii) The amount of ADP-glucose (ADPGlc) decreased when discs were incubated on 100 or 200 mM mannitol. There was a strong correlation between the in vivo levels of ADPGlc and 3PGA when discs were subjected to moderate water stress, and when the sugar supply was varied. (viii) The level of ADPGlc increased and starch synthesis was further inhibited when discs were incubated in 300–500 mM mannitol. (ix) It is proposed that moderate water stress leads to an activation of SPS and stimulates sucrose synthesis. The resulting decline of 3PGA leads to a partial inhibition of ADP-glucose pyrophosphorylase and starch synthesis. More-extreme water stress leads to a further alteration of partitioning, because it inhibits the activities of one or more of the enzymes involved in the terminal reactions of starch synthesis.

Mutants and transformants of tobacco (Nicotiania tabacum L. cv Gatersleben 1) with decreased expression of nitrate reductase have been used to investigate whether nitrate accumulation in the shoot acts as a signal to alter allocation between shoot and root growth. (a) Transformants with very low (1–3% of wild-type levels) nitrate reductase activity had growth rates, and protein, amino acid and glutamine levels similar to or slightly lower than a nitrate-limited wild-type, but accumulated large amounts of nitrate. These plants should resemble a nitrate-limited wild-type, except in responses where nitrate acts as a signal. (b) Whereas the shoot:root ratio decreases from about 3.5 in a well-fertilized wild-type to about 2 in a nitrate-limited wild-type, the transformants had a very high shoot:root ratio (8–10) when they were grown on high nitrate. When they were grown on lower nitrate concentrations their shoot:root ratio declined progressively to a value similar to that in nitrate-limited wild-types. Mutants with a moderate (30–50%) decrease of nitrate reductase also had a small but highly significant increase of their shoot:root ratio, compared to the wild-type. The increased shoot:root ratio in the mutants and transformants was due to a stimulation of shoot growth and an inhibition of root growth. (c) There was a highly significant correlation between leaf nitrate content and the shoot:root ratio for eight genotypes growing at a wide range of nitrate supply. (d) A similar increase of the shoot:root ratio in nitrate reductase-deficient plants, and correlation between leaf nitrate content and the shoot:root ratio, was found in plants growing on ammonium nitrate. (f) Split-root experiments, in which the transformants were grown with part of their root system in high nitrate and the other part in low nitrate, showed that root growth is inhibited by the accumulation of nitrate in the shoot. High concentrations of nitrate in the rooting medium actually stimulate local root growth. (g) The inhibition of root growth in the transformants was relieved when the transformants were grown on limiting phosphate, even though the nitrate content of the root remained high. This shows that the nitrate-dependent changes in allocation can be overridden by other signals that increase allocation to root growth. (h) The reasons for the changed allocation were investigated in transformants growing normally, and in split-root culture. Accumulation of nitrate in the shoot did not lead to decreased levels of amino acids or protein in the roots. However, it did lead to a strong inhibition of starch synthesis and turnover in the leaves, and to decreased levels of sugars in the root. The rate of root growth was correlated with the root sugar content. It is concluded that these changes of carbon allocation could contribute to the changes in shoot and root growth.

Antibodies raised against a peptide fragment (residues 60-456) of potato sucrose phosphate synthase (SPS) were used to investigate whether potato plants contain multiple forms of SPS. When a partially purified preparation of SPS from cold-stored potato tubers was separated on 5% polyacrylamide gel electrophoresis (PAGE), four immunopositive bands were found with estimated molecular weights of 125, 127, 135 and 145 kDa. These bands were also found in rapidly prepared extracts and were termed SPS-la, SPS-lb, SPS-2 and SPS-3, respectively. Direct evidence that SPS-1a and SPS-lb represent active SPS was provided by the finding that both are greatly reduced in plants expressing an antisense sequence derived from the potato leaf SPS gene. SPS-2 was not decreased in the antisense plants, indicating that it has a significantly different sequence. Evidence that SPS-2 represents active SPS was obtained by showing that the amount of SPS-1a and SPS-1b protein remaining in the leaves and tubers of antisense potato plants was too low to account for the remaining SPS activity. The four immunopositive SPS forms had different tissue distributions. SPS-1a was the major form in all tissues except petals, sepals and stamens. SPS-1b and SPS-2 were absent in very young growing tissues but were present as minor forms in source leaves and sprouting tubers. The SPS-1b level was especially high in petals and sepals, and the SPS-2 level was especially high in the stamens. SPS-3 was only detected in very young tissues. The four forms also showed different responses to low temperature. Transfer of tubers to 4 °C led to a specific and reversible increase of SPS-1b during the next 4 d. The appearance of SPS-1b correlated with a change in the kinetic properties of SPS that has recently been shown (Hill et al. 1996) to play a key role in triggering the accumulation of sugars in cold-stored tubers. The appearance of SPS-lb protein at low temperature was accompanied by an increase of SPS transcript. Incubation of tuber slices with calyculin A and okadaic acid to alter the phosphorylation state of SPS did not lead to appearance or disappearance of SPS-1b. It is concluded that potato plants contain several forms of SPS that have different functions in growing and mature tissues, in flower parts, and in acclimation to low temperature.

Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by beta-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 µM in the cytosol, 0.2 to 0.5 µM in the chloroplasts, and 0.05 µM in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night. Fil: Mattos Martins, Marina Camara. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Hejazi, Mahdi. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Fettke, Joerg. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Steup, Martin. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Feil, Regina. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Krause, Ursula. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Arrivault, Stéphanie. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Vosloh, Daniel. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Ivakov, Alexander. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Yadav, Umesh Prasad. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Piques, Maria. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Metzner, Daniela. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Stitt, Mark. Max Planck Institute of Molecular Plant Physiology; Alemania Fil: Lunn, John Edward. Max Planck Institute of Molecular Plant Physiology; Alemania

Next-generation technologies generate an overwhelming amount of gene sequence data. Efficient annotation tools are required to make these data amenable to functional genomics analyses. The Mercator pipeline automatically assigns functional terms to protein or nucleotide sequences. It uses the MapMan 'BIN' ontology, which is tailored for functional annotation of plant 'omics' data. The classification procedure performs parallel sequence searches against reference databases, compiles the results and computes the most likely MapMan BINs for each query. In the current version, the pipeline relies on manually curated reference classifications originating from the three reference organisms (Arabidopsis, Chlamydomonas, rice), various other plant species that have a reviewed SwissProt annotation, and more than 2000 protein domain and family profiles at InterPro, CDD and KOG. Functional annotations predicted by Mercator achieve accuracies above 90% when benchmarked against manual annotation. In addition to mapping files for direct use in the visualization software MapMan, Mercator provides graphical overview charts, detailed annotation information in a convenient web browser interface and a MapMan-to-GO translation table to export results as GO terms. Mercator is available free of charge via http://mapman.gabipd.org/web/guest/app/Mercator.

Sweet Enough to Flower In making the developmental switch from vegetative growth to flowering, plants integrate diverse information, including photoperiod, hormone signals, and carbohydrate status. Wahl et al. (p. 704 ; see the Perspective by Danielson and Frommer ) analyzed the physiology of the signaling sugar trehalose-6-phosphate (T6P) in Arabidopsis . Quantities of T6P cycle in daily rhythms that peak toward the end of the day. T6P levels in the shoot apical meristem mirrored sucrose levels. Disruption of T6P production also disrupted expression of the FLOWERING LOCUS T gene, which responds in leaves to day length and generates signals that direct the meristem to initiate flowering programs. T6P production also affected the signaling pathway that links the age of the plant to flowering. By incorporating requirements for T6P signaling in the flowering induction pathways, the plant ensures that adequate carbohydrate reserves have been accumulated. Thus, T6P regulates the shift to flowering by linking carbohydrate status to day length in the leaves and to developmental age in the shoot apical meristem.

Trehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity., A kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ≥88%. A. thaliana Col-0 rosettes contain about 20-30 nmol g-1FW of trehalose, increasing to about 50-60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively., The fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.

Natural genetic diversity provides a powerful tool to study the complex interrelationship between metabolism and growth. Profiling of metabolic traits combined with network-based and statistical analyses allow the comparison of conditions and identification of sets of traits that predict biomass. However, it often remains unclear why a particular set of metabolites is linked with biomass and to what extent the predictive model is applicable beyond a particular growth condition. A panel of 97 genetically diverse Arabidopsis (Arabidopsis thaliana) accessions was grown in near-optimal carbon and nitrogen supply, restricted carbon supply, and restricted nitrogen supply and analyzed for biomass and 54 metabolic traits. Correlation-based metabolic networks were generated from the genotype-dependent variation in each condition to reveal sets of metabolites that show coordinated changes across accessions. The networks were largely specific for a single growth condition. Partial least squares regression from metabolic traits allowed prediction of biomass within and, slightly more weakly, across conditions (cross-validated Pearson correlations in the range of 0.27–0.58 and 0.21–0.51 and P values in the range of

These experiments investigate events involved in triggering sugar accumulation in the cold in tubers of Solanum tuberosum L. cv. Desiree. Sugar content, 14 C-glucose metabolism, metabolite levels and activities of sucrose phosphate synthase (SPS) and starch-degrading enzymes were followed after transfer to 4 °C. (i) Net sucrose accumulation began between 2 and 4 d. By 10 d, reducing sugars were also increasing. From 20 d onwards, sugar accumulation slowed. Sucrose fell, but reducing sugars continued to increase. (ii) To measure unidirectional sucrose synthesis, U-[ 14 C]glucose was injected into tubers after various times at 4 °C. The tubers were then incubated for 6 h. After 1 d at 4 °C, both the absolute and the relative (expressed as a percentage of the metabolized label) rates of sucrose synthesis decreased compared to those at 20 °C. Between 2 and 4 d at 4 °C, labelling of sucrose increased 3-fold, to over 60% of the metabolized label. This high rate was maintained for up to 50 d in cold storage. When tissue slices were incubated with 2.5 mol m -3 U-[ 14 C]glucose, the rate of labelling of sucrose in slices from 6 d cold-stored material was higher than in slices from warm-stored material, irrespective of whether the incubation occurred at 4 °C or at 20 °C. (iii) Hexose-phosphates increased during the first day after transfer to 4 °C. Their levels fell during the next 3 d, as sucrose synthesis increased. They then rose (until 20 d) and fell, in parallel with the rise and decline of sucrose levels. UDPglucose remained unaltered during the first 4 d, and then increased and decreased in parallel with sucrose. (iv) SPS activity assayed in optimal conditions and the total amount of SPS protein did not change. However, when assayed in the presence of phosphate and limiting substrate concentrations, activity rose 3-5-fold between 2 and 4 d. (v) Amylases and phosphorylases were investigated using zymograms to separate isoforms. Phosphorylases did not change. Between 2 and 4 d at 4 °C, a new amylolytic activity appeared. (vi) Estimates of the specific activity of the phosphorylated intermediates and the absolute rate of sucrose synthesis (calculated from the 14 C-labelling data and metabolite analysis) showed that changed kinetic properties of SPS and decreased levels of hexose-phosphate are accompanied by a 6-8-fold stimulation of sucrose synthesis. They also show that the final level of sugar is partly determined by a cycle of sugar synthesis and degradation. (vii) It is concluded that the onset of sugar accumulation in cold-stored tubers is initiated by a change in the kinetic properties of SPS and the appearance of a new amylolytic activity. It is discussed how other factors, including hexose-phosphate levels and subcellular compartmentalization, could also influence the final levels of sugars by altering the balance of sugar synthesis and remobilization.

As reported in a previous paper [Lerchl et al. (1995) Plant Cell, 7, 259–270], expression of Escherichia coli inorganic pyrophosphatase in the cytosol under the control of the phloem-specific rolC promoter from, Agobacterium rhizogenes results in decreased growth of transgenic tobacco plants. In this paper we investigate the effect of the phloem-specific expression of pyrophosphatase on phloem metabolism, and on plant growth and allocation. A small decrease in the hexose phosphate/UDP-glucose ratio, the ATP/ADP ratio and the respiration rate in the midribs of the transformants provides evidence Hint mobilization of sucrose via pyrophosphate-dependent reactions is necessary for phloem energy metabolism. The source leaves of the transformants had higher levels of carbohydrates and amino acids and a much higher glutamine/glutamate ratio than the wild type, showing that export was inhibited and that the growth inhibition was not due to a lack of photoas-similates or organic nitrogen in the leaves. The accumulation of photoassimilates was paralleled by a decrease in photosynthesis, chlorophyll content and ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity, a small increase in hexose phosphates and triose phosphates and a decrease in glycerate 3-phosphate in the source leaves. There was a decrease of soluble sugars and amino acids in sink leaves of the transformants. In sink leaves amino acids decreased more than carbohydrates and a decrease in the glutamine/ glutamate ratio was observed. This was accompanied by a large decrease of nitrate. Sugars and amino acids were also reduced in the root tips of the transformants. The carbohydrate /amino acid ratio decreased 5-fold in the root tips, indicating a particularly smile shortage of carbohydrates. Relatively high levels of sugars and amino acids in the basal regions of the root and the increase in sugars in the midrib indicate that there is also increased leakage of assimilates out of the phloem during long-distance transport. Metabolism is required to maintain phloem function along the transport route, as well as for the initial step of loading. The transformants showed decreased stem and root growth. The growth inhibition was largest in conditions allowing rapid growth of the wild type (high light and nitrogen supply).

This review discusses how genetically manipulated plants are being used to study the regulation of metabolism in plants, using carbohydrate metabolism as an example. The molecular tools required are introduced, including the history of Agrobacterium tumefaciens-mediated gene transfer and other transformation techniques, the availability of promoters to achieve a specific or induced expression, strategies to target proteins to subcellular compartments of the cell, and the use of antisense or cosuppression to inhibit expression of endogenous genes. A discussion then follows of how such plants can be used in biochemical and physiological experiments to identify and quantify the importance of enzymes and processes that control metabolic fluxes, storage, and growth. These results are leading to a reassessment of ideas about metabolic regulation and have consequences for design of bioengineering strategies in plants. Emerging commercial applications are also surveyed.

The shx mutant of barley (Hordeum vulgare L. cv. Bomi) produces grains greatly decreased in starch content and containing smaller A-starch granules together with normal B-granules. Soluble starch synthase (SSS) and ADP-glucose pyrophosphorylase (AGP) are reduced to about 20% normal activity. To help determine the position of the block to starch synthesis in shx, we measured enzyme activities and metabolite levels in developing grains of normal and mutant genotypes in a cv. Bomi background. We demonstrate that sucrose, free hexose, hexose phosphates and, critically, ADP-Glc accumulate as a result of the mutation. In addition to AGP and SSS, several other enzyme activities are affected in the shx mutant, most of them showing activities 50-80% of normal. Northern blots showed that transcripts for the AGP small subunit are much less abundant, but of normal size, in shx. These and earlier results together indicate that the metabolic block is at the end of the starch synthetic pathway, with a primary effect on a type I, primer-independent soluble starch synthase.

Plants often respond to environmental changes by reprogramming metabolic and stress-associated pathways. Homeostatic integration of signaling is a central requirement for ensuring metabolic stability in living organisms. Under diurnal conditions, properly timed rhythmic metabolism provides fitness benefits to plants. TIME FOR COFFEE (TIC) is a circadian regulator known to be involved in clock resetting at dawn. Here we explored the mechanism of influence of TIC in plant growth and development, as initiated by a microarray analysis. This global profiling showed that a loss of TIC function causes a major reprogramming of gene expression that predicts numerous developmental, metabolic, and stress-related phenotypes. This led us to demonstrate that this mutant exhibits late flowering, a plastochron defect, and diverse anatomical phenotypes. We further observed a starch-excess phenotype and altered soluble carbohydrate levels. tic exhibited hypersensitivity to oxidative stress and abscisic acid, and this was associated with a striking resistance to drought. These phenotypes were connected to an increase in total glutathione levels that correlated with a readjustment of amino acids and polyamine pools. By comparatively analyzing our transcriptomic and metabolomic data, we concluded that TIC is a central element in plant homeostasis that integrates and coordinates developmental, metabolic, and environmental signals.

The maximum yield of crop plants depends on the efficiency of conversion of sunlight into biomass. This review summarises recent models that estimate energy conversion efficiency for successive steps in photosynthesis and metabolism. Photorespiration was identified as a major reason for energy loss during photosynthesis and strategies to modify or suppress photorespiration are presented. Energy loss during the conversion of photosynthate to biomass is also large but cannot be modelled as precisely due to incomplete knowledge about pathways and turnover and maintenance costs. Recent research on pathways involved in metabolite transport and interconversion in different organs, and recent insights into energy requirements linked to the production, maintenance and turnover of the apparatus for cellular growth and repair processes are discussed.

Summary Proteaceae species in south-western Australia occur on severely phosphorus (P)-impoverished soils. They have very low leaf P concentrations, but relatively fast rates of photosynthesis, thus exhibiting extremely high photosynthetic phosphorus-use-efficiency (PPUE). Although the mechanisms underpinning their high PPUE remain unknown, one possibility is that these species may be able to replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. For six Proteaceae species, we measured soil and leaf P concentrations and rates of photosynthesis of both young expanding and mature leaves. We also assessed the investment in galactolipids, sulfolipids and phospholipids in young and mature leaves, and compared these results with those on Arabidopsis thaliana, grown under both P-sufficient and P-deficient conditions. In all Proteaceae species, phospholipid levels strongly decreased during leaf development, whereas those of galactolipids and sulfolipids strongly increased. Photosynthetic rates increased from young to mature leaves. This shows that these species extensively replace phospholipids with nonphospholipids during leaf development, without compromising photosynthesis. A considerably less pronounced shift was observed in A. thaliana. Our results clearly show that a low investment in phospholipids, relative to nonphospholipids, offers a partial explanation for a high photosynthetic rate per unit leaf P in Proteaceae adapted to P-impoverished soils.

Understanding molecular factors determining local adaptation is a key challenge, particularly relevant for plants, which are sessile organisms coping with a continuously fluctuating environment. Here we introduce a rigorous network-based approach for investigating the relation between geographic location of accessions and heterogeneous molecular phenotypes. We demonstrate for Arabidopsis accessions that not only genotypic variability but also flowering and metabolic phenotypes show a robust pattern of isolation-by-distance. Our approach opens new avenues to investigate relations between geographic origin and heterogeneous molecular phenotypes, like metabolite profiles, which can easily be obtained in species where genome data is not yet available.

The diversity of metabolites found in plants is by far greater than in most other organisms. Metabolic profiling techniques, which measure many of these compounds simultaneously, enabled investigating the regulation of metabolic networks and proved to be useful for predicting important agronomic traits. However, little is known about the genetic basis of metabolites in crops such as maize. Here, a set of 289 diverse maize inbred lines was genotyped with 56,110 SNPs and assayed for 118 biochemical compounds in the leaves of young plants, as well as for agronomic traits of mature plants in field trials. Metabolite concentrations had on average a repeatability of 0.73 and showed a correlation pattern that largely reflected their functional grouping. Genome-wide association mapping with correction for population structure and cryptic relatedness identified for 26 distinct metabolites strong associations with SNPs, explaining up to 32.0% of the observed genetic variance. On nine chromosomes, we detected 15 distinct SNP–metabolite associations, each of which explained more then 15% of the genetic variance. For lignin precursors, including p -coumaric acid and caffeic acid, we found strong associations ( P values to ) with a region on chromosome 9 harboring cinnamoyl-CoA reductase, a key enzyme in monolignol synthesis and a target for improving the quality of lignocellulosic biomass by genetic engineering approaches. Moreover, lignin precursors correlated significantly with lignin content, plant height, and dry matter yield, suggesting that metabolites represent promising connecting links for narrowing the genotype–phenotype gap of complex agronomic traits.

Sucrose is the main transported form of carbon in several plant species, including Populus species. Sucrose metabolism in developing wood has therefore a central role in carbon partitioning to stem biomass. Half of the sucrose-derived carbon is in the form of fructose, but metabolism of fructose has received little attention as a factor in carbon partitioning to walls of wood cells. We show that RNAi-mediated reduction of FRK2 activity in developing wood of hybrid aspen (Populus tremula × tremuloides) led to the accumulation of soluble neutral sugars and a decrease in hexose phosphates and UDP-glucose, indicating that carbon flux to cell-wall polysaccharide precursors is decreased. Reduced FRK2 activity also led to thinner fiber cell walls with a reduction in the proportion of cellulose. No pleiotropic effects on stem height or diameter were observed. The results establish a central role for FRK2 activity in carbon flux to wood cellulose.

We suspect that many biologists who have been following the development of functional ’omics and systems biology are wondering why, compared with the success in integrating information from large-scale studies of transcripts and, more recently, proteins, there is less success in integrating

Recent rapid advances in next generation RNA sequencing (RNA-Seq)-based provide researchers with unprecedentedly large data sets and open new perspectives in transcriptomics. Furthermore, RNA-Seq-based transcript profiling can be applied to non-model and newly discovered organisms because it does not require a predefined measuring platform (like e.g. microarrays). However, these novel technologies pose new challenges: the raw data need to be rigorously quality checked and filtered prior to analysis, and proper statistical methods have to be applied to extract biologically relevant information. Given the sheer volume of data, this is no trivial task and requires a combination of considerable technical resources along with bioinformatics expertise. To aid the individual researcher, we have developed RobiNA as an integrated solution that consolidates all steps of RNA-Seq-based differential gene-expression analysis in one user-friendly cross-platform application featuring a rich graphical user interface. RobiNA accepts raw FastQ files, SAM/BAM alignment files and counts tables as input. It supports quality checking, flexible filtering and statistical analysis of differential gene expression based on state-of-the art biostatistical methods developed in the R/Bioconductor projects. In-line help and a step-by-step manual guide users through the analysis. Installer packages for Mac OS X, Windows and Linux are available under the LGPL licence from http://mapman.gabipd.org/web/guest/ robin.

Deep profiling of the transcriptome and proteome during leaf development reveals unexpected responses to water deficit, as well as a surprising lack of protein-level fluctuations during the day–night cycle, despite clear changes at the transcript level., Transcript and protein variation patterns reflect the functional stages of the leaf. Protein and transcript levels correlate well during leaf development, with some notable exceptions. Diurnal transcript-level fluctuations are not matched by corresponding diurnal fluctuations in the detected proteome. Continuous reduced soil water content results in reduced leaf growth, but the plant adapts at molecular levels without showing a typical drought response., Leaves have a central role in plant energy capture and carbon conversion and therefore must continuously adapt their development to prevailing environmental conditions. To reveal the dynamic systems behaviour of leaf development, we profiled Arabidopsis leaf number six in depth at four different growth stages, at both the end-of-day and end-of-night, in plants growing in two controlled experimental conditions: short-day conditions with optimal soil water content and constant reduced soil water conditions. We found that the lower soil water potential led to reduced, but prolonged, growth and an adaptation at the molecular level without a drought stress response. Clustering of the protein and transcript data using a decision tree revealed different patterns in abundance changes across the growth stages and between end-of-day and end-of-night that are linked to specific biological functions. Correlations between protein and transcript levels depend on the time-of-day and also on protein localisation and function. Surprisingly, only very few of >1700 quantified proteins showed diurnal abundance fluctuations, despite strong fluctuations at the transcript level.

Diurnal cycles provide a tractable system to study the response of metabolism and growth to fluctuating temperatures. We reasoned that the response to daytime and night temperature may vary; while daytime temperature affects photosynthesis, night temperature affects use of carbon that was accumulated in the light. Three Arabidopsis thaliana accessions were grown in thermocycles under carbon-limiting conditions with different daytime or night temperatures (12 to 24 degrees C) and analyzed for biomass, photosynthesis, respiration, enzyme activities, protein levels, and metabolite levels. The data were used to model carbon allocation and growth rates in the light and dark. Low daytime temperature led to an inhibition of photosynthesis and an even larger inhibition of growth. The inhibition of photosynthesis was partly ameliorated by a general increase in protein content. Low night temperature had no effect on protein content, starch turnover, or growth. In a warm night, there is excess capacity for carbon use. We propose that use of this capacity is restricted by feedback inhibition, which is relaxed at lower night temperature, thus buffering growth against fluctuations in night temperature. As examples, the rate of starch degradation is completely temperature compensated against even sudden changes in temperature, and polysome loading increases when the night temperature is decreased.

International audience; Many plants, including Arabidopsis thaliana, retain a substantial portion of their photosynthate in leaves in the form of starch, which is remobilized to support metabolism and growth at night. ADP-glucose pyrophosphorylase (AGPase) catalyses the first committed step in the pathway of starch synthesis, the production of ADP-glucose. The enzyme is redox-activated in the light and in response to sucrose accumulation, via reversible breakage of an intermolecular cysteine bridge between the two small (APS1) subunits. The biological function of this regulatory mechanism was investigated by complementing an aps1 null mutant (adg1) with a series of constructs containing a full-length APS1 gene encoding either the wild-type APS1 protein or mutated forms in which one of the five cysteine residues was replaced by serine. Substitution of Cys81 by serine prevented APS1 dimerization, whereas mutation of the other cysteines had no effect. Thus, Cys81 is both necessary and sufficient for dimerization of APS1. Compared to control plants, the adg1/APS1C81S lines had higher levels of ADP-glucose and maltose, and either increased rates of starch synthesis or a starch-excess phenotype, depending on the daylength. APS1 protein levels were five- to tenfold lower in adg1/APS1C81S lines than in control plants. These results show that redox modulation of AGPase contributes to the diurnal regulation of starch turnover, with inappropriate regulation of the enzyme having an unexpected impact on starch breakdown, and that Cys81 may play an important role in the regulation of AGPase turnover.

International audience; Every year thousands of experiments are conducted using plants grown under more-or-less controlled environmental conditions. The aim of many such experiments is to compare the phenotype of different species or genotypes in a specific environment, or to study plant performance under a range of suboptimal conditions. Our paper aims to bring together the minimum knowledge necessary for a plant biologist to set up such experiments and apply the environmental conditions that are appropriate to answer the questions of interest. We first focus on the basic choices that have to be made with regard to the experimental setup (e.g. where are the plants grown; what rooting medium; what pot size). Second, we present practical considerations concerning the number of plants that have to be analysed considering the variability in plant material and the required precision. Third, we discuss eight of the most important environmental factors for plant growth (light quantity, light quality, CO2, nutrients, air humidity, water, temperature and salinity); what critical issues should be taken into account to ensure proper growth conditions in controlled environments and which specific aspects need attention if plants are challenged with a certain a-biotic stress factor. Finally, we propose a simple checklist that could be used for tracking and reporting experimental conditions.

Wild-type and antisense rbcS tobacco (Nicotiana tabacum) plants were grown in a glasshouse in midsummer in Portugal with an irradiance of 1500–2000 μmol m−2s−1 and daytime temperatures of 30–35 °C. The Rubisco content of the transformants was lower by 35, 80 and over 90% than that of the wild-type. Gas exchange was measured over three separate days. There was a near-linear relation between Rubisco content and photosynthetic rate during the period of high irradiance, allowing a flux control coefficient of 0.83–0.89 to be estimated. The relation deviated slightly from linearity, because the internal CO2 concentration (c;) was higher in the transformants than in the wild-type (190 and 275 μmol mol−1 in plants with 35 and 80% less Rubisco, respectively, compared with 175 μmol mol−1 for wild-type), compensating to some extent for the decreased Rubisco content. This increase in ci occurred because the stomatal conductance (g) remained unaltered or was even higher in plants with decreased Rubisco, despite the lower rate of CO2 assimilation. As a consequence, water use efficiency declined. The decreased rate of photosynthesis was not accompanied by a stoichiometric decrease in apparent growth rate. These results are discussed in relation to earlier studies of the plant set in growth cabinets. It is concluded that tobacco can adjust over a wide range of growth conditions to avoid a onesided limitation by Rubisco, but that in extreme environmental conditions this capacity to adapt is exhausted.

Labelling experiments in which high-specific-activity [U-14C]sucrose or [U-14C]hexoses were injected into potato (Solanum tuberosum L. cv. Desiree) tubers showed that within 1 d of detaching growing tubers from their mother plant, there is an inhibition of starch synthesis, a stimulation of the synthesis of other major cell components, and rapid resynthesis of sucrose. This is accompanied by a general increase in phosphorylated intermediates, an increase in UDP-glucose, and a dramatic decrease of ADP-glucose. No significant decline in the extracted activity of enzymes for sucrose degradation or synthesis, or starch synthesis is seen within 1 d, nor is there a significant decrease in sucrose, amino acids, or fresh weight. Over the next 7 d, soluble carbohydrates decline. This is accompanied by a decline in sucrose-synthase activity, hexose-phosphate levels, and the synthesis of structural cell components. It is argued that a previously unknown mechanism acting at ADP-glucose pyrophosphorylase allows sucrose-starch interconversions to be regulated independently of the use of sucrose for cell growth.

The reverse genetics approach is allowing the definition of specific reaction steps in plant carbohydrate metabolism. This is enabling the design of studies to alter sucrose and starch partitioning in the leaf, to inhibit phloem transport, or to increase/inhibit the rate of sucrose import into storage organs and its conversion to starch. These studies provide information about what kind of change should be made to biosynthetic pathways to achieve a desired aim. They also highlight the flexibility of plant primary metabolism and reveal a multi-faceted interaction between carbohydrate metabolism and many other aspects of plant growth and development.

Biological systems contain an immense number of individual components, which undergo dynamic and highly interactive responses in time and space. Analysis of these responses will provide a key to unlock the information encrypted in the genome sequences that are accumulating around the world. This task is being driven by powerful methods that allow comprehensive of gene expression, protein localization and protein–protein interactions. Emerging technologies that allow a comprehensive analysis of metabolites (1) will also make a vital contribution, by uncovering many of the phenotypic changes that result from alterations of the genotype, or that accompany changes in gene expression. High throughput profiling technologies suffer, however, from a serious blind spot. Understanding of biological function also requires spatial resolution, at the cellular and subcellular level. For several decades, this sort of information was obtained in a painstaking way, for example, by isolating cell types or organelles and investigating what proteins, enzyme activities, and metabolites they contained (2), or by producing tissue sections for in situ hybridization or immunolocalization of transcripts and proteins. Molecular cell biology now uses generic methods to investigate the tissue and cell-specific localization of transcripts and the cellular and subcellular distribution of proteins, for example reporter genes or GFP fusion proteins. Analogous techniques are urgently needed to measure metabolite levels in situ. Metabolites change even more dynamically than transcripts or proteins, but only a minute fraction of metabolites possess spectral properties that allow them to be directly visualized and imaging by NMR is restricted to metabolites that are present at relatively high concentrations (3). The development of generic techniques to monitor in situ metabolite levels is an enormous technical challenge, because of their immense chemical heterogeneity.

Background 14-3-3 proteins are considered master regulators of many signal transduction cascades in eukaryotes. In plants, 14-3-3 proteins have major roles as regulators of nitrogen and carbon metabolism, conclusions based on the studies of a few specific 14-3-3 targets. Results In this study, extensive novel roles of 14-3-3 proteins in plant metabolism were determined through combining the parallel analyses of metabolites and enzyme activities in 14-3-3 overexpression and knockout plants with studies of protein-protein interactions. Decreases in the levels of sugars and nitrogen-containing-compounds and in the activities of known 14-3-3-interacting-enzymes were observed in 14-3-3 overexpression plants. Plants overexpressing 14-3-3 proteins also contained decreased levels of malate and citrate, which are intermediate compounds of the tricarboxylic acid (TCA) cycle. These modifications were related to the reduced activities of isocitrate dehydrogenase and malate dehydrogenase, which are key enzymes of TCA cycle. In addition, we demonstrated that 14-3-3 proteins interacted with one isocitrate dehydrogenase and two malate dehydrogenases. There were also changes in the levels of aromatic compounds and the activities of shikimate dehydrogenase, which participates in the biosynthesis of aromatic compounds. Conclusion Taken together, our findings indicate that 14-3-3 proteins play roles as crucial tuners of multiple primary metabolic processes including TCA cycle and the shikimate pathway.

Fruit of tomato (Solanum lycopersicum), like those from many species, have been characterized to undergo a shift from partially photosynthetic to truly heterotrophic metabolism. While there is plentiful evidence for functional photosynthesis in young tomato fruit, the rates of carbon assimilation rarely exceed those of carbon dioxide release, raising the question of its role in this tissue. Here, we describe the generation and characterization of lines exhibiting a fruit-specific reduction in the expression of glutamate 1-semialdehyde aminotransferase (GSA). Despite the fact that these plants contained less GSA protein and lowered chlorophyll levels and photosynthetic activity, they were characterized by few other differences. Indeed, they displayed almost no differences in fruit size, weight, or ripening capacity and furthermore displayed few alterations in other primary or intermediary metabolites. Although GSA antisense lines were characterized by significant alterations in the expression of genes associated with photosynthesis, as well as with cell wall and amino acid metabolism, these changes were not manifested at the phenotypic level. One striking feature of the antisense plants was their seed phenotype: the transformants displayed a reduced seed set and altered morphology and metabolism at early stages of fruit development, although these differences did not affect the final seed number or fecundity. Taken together, these results suggest that fruit photosynthesis is, at least under ambient conditions, not necessary for fruit energy metabolism or development but is essential for properly timed seed development and therefore may confer an advantage under conditions of stress.

The last decade has witnessed dramatic advances in our ability to determine the levels of an ever broadening range of metabolites. That said, while transcriptomic approaches provide almost complete coverage ([Gonzalez-Ballester et al., 2010][1]) and proteomics approaches are now capable of

Plants grow in a light/dark cycle. We have investigated how growth is buffered against the resulting changes in the carbon supply. Growth of primary roots of Arabidopsis seedlings was monitored using time-resolved video imaging. The average daily rate of growth is increased in longer light periods or by addition of sugars. It responds slowly over days when the conditions are changed. The momentary rate of growth exhibits a robust diel oscillation with a minimum 8-9 h after dawn and a maximum towards the end of the night. Analyses with starch metabolism mutants show that starch turnover is required to maintain growth at night. A carbon shortfall leads to an inhibition of growth, which is not immediately reversed when carbon becomes available again. The diel oscillation persists in continuous light and is strongly modified in clock mutants. Central clock functions that depend on CCA1/LHY are required to set an appropriate rate of starch degradation and maintain a supply of carbon to support growth through to dawn, whereas ELF3 acts to decrease growth in the light period and promote growth in the night. Thus, while the overall growth rate depends on the carbon supply, the clock orchestrates diurnal carbon allocation and growth.

The data explosion in the biological sciences has led to many novel challenges for the individual researcher. One of these is to interpret the sheer mass of data at hand. Typical high-throughput data sets from transcriptomic data can easily comprise hundred thousand data points. It is thus necessary to provide tools to interactively visualize these data sets in a way that aids in their interpretation. Thus we have developed the MAPMAN application. This application renders individual data points from different domains as different glyphs that are color coded to reflect underlying changes in the magnitude/abundance of the underlying data. In order to augment the human comprehensibility of the biologist domain experts these data are organized on meaningful pathway diagrams that the biologist has encountered numerous times. Using these representations together with a high level organization thus helps to quickly realize the main outcome of such a high throughput study and to further decide on additional tasks that should be performed to explore the data.

Climate-driven vegetation mortality is occurring globally and is predicted to increase in the near future. The expected climate feedbacks of regional-scale mortality events have intensified the need to improve the simple mortality algorithms used for future predictions, but uncertainty regarding mortality processes precludes mechanistic modeling. By integrating new evidence from a wide range of fields, we conclude that hydraulic function and carbohydrate and defense metabolism have numerous potential failure points, and that these processes are strongly interdependent, both with each other and with destructive pathogen and insect populations. Crucially, most of these mechanisms and their interdependencies are likely to become amplified under a warmer, drier climate. Here, we outline the observations and experiments needed to test this interdependence and to improve simulations of this emergent global phenomenon.

Despite the fact that the organic acid content of a fruit is regarded as one of its most commercially important quality traits when assessed by the consumer, relatively little is known concerning the physiological importance of organic acid metabolism for the fruit itself. Here, we evaluate the effect of modifying malate metabolism in a fruit-specific manner, by reduction of the activities of either mitochondrial malate dehydrogenase or fumarase, via targeted antisense approaches in tomato (Solanum lycopersicum). While these genetic perturbations had relatively little effect on the total fruit yield, they had dramatic consequences for fruit metabolism, as well as unanticipated changes in postharvest shelf life and susceptibility to bacterial infection. Detailed characterization suggested that the rate of ripening was essentially unaltered but that lines containing higher malate were characterized by lower levels of transitory starch and a lower soluble sugars content at harvest, whereas those with lower malate contained higher levels of these carbohydrates. Analysis of the activation state of ADP-glucose pyrophosphorylase revealed that it correlated with the accumulation of transitory starch. Taken together with the altered activation state of the plastidial malate dehydrogenase and the modified pigment biosynthesis of the transgenic lines, these results suggest that the phenotypes are due to an altered cellular redox status. The combined data reveal the importance of malate metabolism in tomato fruit metabolism and development and confirm the importance of transitory starch in the determination of agronomic yield in this species.

International audience; ADP-glucose pyrophosphorylase (AGPase) catalyses the synthesis of ADP-glucose, and is a highly regulated enzyme in the pathway of starch synthesis. In Arabidopsis thaliana, the enzyme is a heterotetramer, containing two small subunits encoded by the APS1 gene and two large subunits encoded by the APL1-4 genes. TILLING (Targeting Induced Local Lesions IN Genomes) of a chemically mutagenised population of A. thaliana plants identified 33 novel mutations in the APS1 gene, including 21 missense mutations in the protein coding region. High throughput measurements using a robotised cycling assay showed that maximal AGPase activity in the aps1 mutants varied from