Changes in carbohydrate metabolism and assimilate export in starch-excess mutants of Arabidopsis

The aim of this work was to investigate the effects on carbohydrate metabolism of a reduction in the capacity to degrade leaf starch in Arabidopsis. The major roles of leaf starch are to provide carbon for sucrose synthesis, respiration and, in developing leaves, for biosynthesis and growth. Wild-type plants were compared with plants of a starch-excess mutant line (sex4) deficient in a chloroplastic isoform of endoamylase. This mutant has a reduced capacity for starch degradation, leading to an imbalance between starch synthesis and degradation and the gradual accretion of starch as the leaves age. During the night the conversion of starch into sucrose in the mutant is impaired; the leaves of the mutant contained less sucrose than those of the wild type and there was less movement of ¹⁴C-label from starch to sucrose in radio-labelling experiments. Furthermore, the rate of assimilate export to the roots during the night was reduced in the mutant compared with the wild type. During the day however, photosynthetic partitioning was altered in the mutant, with less photosynthate partitioned into starch and more into sugars. Although the sucrose content of the leaves of the mutant was similar to the wild type during the day, the rate of export of sucrose to the roots was increased more than two-fold. The changes in carbohydrate metabolism in the mutant leaves during the day compensate partly for its reduced capacity to synthesize sucrose from starch during the night.     

Altering flux through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana modifies photosynthetic acclimation at low temperatures and the development of freezing tolerance

To test the hypothesis that the up‐regulation of sucrose biosynthesis during cold acclimation is essential for the development of freezing tolerance, the acclimation responses of wild‐type (WT) Arabidopsis thaliana (Heynh.) were compared with transgenic plants over‐expressing sucrose phosphate synthase (over‐sps) or with antisense repression of either cytosolic fructose‐1,6‐bisphosphatase (antifbp) or sucrose phosphate synthase (antisps). Plants were grown at 23 °C and then shifted to 5 °C. The leaves shifted to 5 °C for 10 d and the new leaves that developed at 5 °C were compared with control leaves on plants at 23 °C. Plants over‐expressing sucrose phosphate synthase showed improved photosynthesis and increased flux of fixed carbon into sucrose when shifted to 5 °C, whereas both antisense lines showed reduced flux into soluble sugars relative to WT. The improved photosynthetic performance by the over‐sps plants shifted to 5 °C was associated with an increase in freezing tolerance relative to WT (?9.1 and ?7.2 °C, respectively). In contrast, both antisense lines showed impaired development of freezing tolerance (? 5.2 and ?5.8 °C for antifbp and antisps, respectively) when shifted to 5 °C. In the new leaves developed at 5 °C the recovery of photosynthesis as typically seen in WT was strongly inhibited in both antisense lines and this inhibition was associated with a further failure of both antisense lines to cold acclimate. Thus, functional sucrose biosynthesis at low temperature in the over‐sps plants reduced the inhibition of photosynthesis, maintained the mobilization of carbohydrates from source leaves to sinks and increased the rate at which freezing tolerance developed. Modification of sucrose metabolism therefore represents an additional approach that will have benefits both for the development of freezing tolerance and over‐wintering, and for the supply of exportable carbohydrate to support growth at low temperatures.     

Growth and reproduction of Arabidopsis thaliana in relation to storage of starch and nitrate in the wild-type and in starch-deficient and nitrate-uptake-deficient mutants

We have investigated the interactions between resource assimilation and storage in rosette leaves, and their impact on the growth and reproduction of the annual species Arabidopsis thaliana. The resource balance was experimentally perturbed by changing (i) the external nutrition, by varying the nitrogen supply; (ii) the assimilation and reallocation of resources from rosette leaves to reproductive organs, by cutting or covering rosette leaves at the time of early flower bud formation, and (iii) the internal carbon and nitrogen balance of the plants, by using isogenic mutants either lacking starch formation (PGM mutant) or with reduced nitrate uptake (NU mutant). When plants were grown on high nitrogen, they had higher concentrations of carbohydrates and nitrate in their leaves during the rosette phase than during flowering. However, these storage pools did not significantly contribute to the bulk flow of resources to seeds. The pool size of stored resources in rosette leaves at the onset of seed filling was very low compared to the total amount of carbon and nitrogen needed for seed formation. Instead, the rosette leaves had an important function in the continued assimilation of resources during seed ripening, as shown by the low seed yield of plants whose leaves were covered or cut off. When a key resource became limiting, such as nitrogen in the NU mutants and in plants grown on a low nitrogen supply, stored resources in the rosette leaves (e.g. nitrogen) were remobilized, and made a larger contribution to seed biomass. A change in nutrition resulted in a complete reversal of the plant response: plants shifted from high to low nutrition exhibited a seed yield similar to that of plants grown continuously on a low nitrogen supply, and vice versa. This demonstrates that resource assimilation during the reproductive phase determines seed production. The PGM mutant had a reduced growth rate and a smaller biomass during the rosette phase as a result of changes in respiration caused by a high turnover of soluble sugars ( Caspar et al. 1986 ; W. Schulze et al. 1991 ). During flowering, however, the vegetative growth rate in the PGM mutant increased, and exceeded that of the wild-type. By the end of the flowering stage, the biomass of the PGM mutant did not differ from that of the wild-type. However, in contrast to the wild-type, the PGM mutant maintained a high vegetative growth rate during seed formation, but had a low rate of seed production. These differences in allocation in the PGM mutant result in a significantly lower seed yield in the starchless mutants. This indicates that starch formation is not only an important factor during growth in the rosette phase, but is also important for whole plant allocation during seed formation. The NU mutant resembled the wild-type grown on a low nitrogen supply, except that it unexpectedly showed symptoms of carbohydrate shortage as well as nitrogen deficiency. In all genotypes and treatments, there was a striking correlation between the concentrations of nitrate and organic nitrogen and shoot growth on the one hand, and sucrose concentration and root growth on the other. In addition, nitrate reductase activity (NRA) was correlated with the total carbohydrate concentration: low carbohydrate levels in starchless mutants led to low NRA even at high nitrate supply. Thus the concentrations of stored carbohydrates and nitrate are directly or indirectly involved in regulating allocation.     

Inactivation of thioredoxin f1 leads to decreased light activation of ADP‐glucose pyrophosphorylase and altered diurnal starch turnover in leaves of Arabidopsis plants

Chloroplast thioredoxin f (Trx f) is an important regulator of primary metabolic enzymes. However, genetic evidence for its physiological importance is largely lacking. To test the functional significance of Trx f in vivo, Arabidopsis mutants with insertions in the trx f1 gene were studied, showing a drastic decrease in Trx f leaf content. Knockout of Trx f1 led to strong attenuation in reductive light activation of ADP‐glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis, in leaves during the day and in isolated chloroplasts, while sucrose‐dependent redox activation of AGPase in darkened leaves was not affected. The decrease in light‐activation of AGPase in leaves was accompanied by a decrease in starch accumulation, an increase in sucrose levels and a decrease in starch‐to‐sucrose ratio. Analysis of metabolite levels at the end of day shows that inhibition of starch synthesis was unlikely due to shortage of substrates or changes in allosteric effectors. Metabolite profiling by gas chromatography–mass spectrometry pinpoints only a small number of metabolites affected, including sugars, organic acids and ethanolamine. Interestingly, metabolite data indicate carbon shortage in trx f1 mutant leaves at the end of night. Overall, results provide in planta evidence for the role played by Trx f in the light activation of AGPase and photosynthetic carbon partitioning in plants.     

Regulation of sucrose and starch metabolism in potato tubers in response to short-term water deficit

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. Received: 26 August 1996 / Accepted: 5 November 1996