Mild Reductions in Mitochondrial NAD-Dependent Isocitrate Dehydrogenase Activity Result in Altered Nitrate Assimilation and Pigmentation But Do Not Impact Growth

Transgenic tomato (Solanum lycopersicum) plants were generated expressing a fragment of the mitochondrial NAD-dependent isocitrate dehydrogenase gene (SlIDH1) in the antisense orientation. The transgenic plants displayed a mild reduction in the activity of the target enzyme in the leaves but essentially no visible alteration in growth from the wild-type. Fruit size and yield were, however, reduced. These plants were characterized by relatively few changes in photosynthetic parameters, but they displayed a minor decrease in maximum photosynthetic efficiency (Fv/Fm). Furthermore, a clear reduction in flux through the tricarboxylic acid (TCA) cycle was observed in the transformants. Additionally, biochemical analyses revealed that the transgenic lines exhibited considerably altered metabolism, being characterized by slight decreases in the levels of amino acids, intermediates of the TCA cycle, photosynthetic pigments, starch, and NAD(P)H levels, but increased levels of nitrate and protein. Results from these studies show that even small changes in mitochondrial NAD-dependent isocitrate dehydrogenase activity lead to noticeable alterations in nitrate assimilation and suggest the presence of different strategies by which metabolism is reprogrammed to compensate for this deficiency

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (Fv/Fm), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism

Simultaneous antisense inhibition of two starch-synthase isoforms in potato tubers leads to accumulation of grossly modified amylopectin.

A chimaeric antisense construct was used to reduce the activities of the two major starch-synthase isoforms in potato tubers simultaneously. A range of reductions in total starch-synthase activities were found in the resulting transgenic plants, up to a maximum of 90% inhibition. The reduction in starch-synthase activity had a profound effect on the starch granules, which became extremely distorted in appearance compared with the control lines. Analysis of the starch indicated that the amounts produced in the tubers, and the amylose content of the starch, were not affected by the reduction in activity. In order to understand why the starch granules were distorted, amylopectin was isolated and the constituent chain lengths analysed. This indicated that the amylopectin was very different to that of the control. It contained more chains of fewer than 15 glucose units in length, and fewer of between 15 and 80 glucose units. In addition, the amylopectin contained more very long chains. Amylopectin from plants repressed in just one of the activities of the two starch-synthase isoforms, which we have reported upon previously, were also analysed. Using a technique different to that used previously we show that both isoforms also affect the amylopectin, but in a way that is different to when both isoforms are repressed together

Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers.

The tricarboxylic acid (TCA) cycle constitutes a major component of the mitochondrial metabolism of eucaryotes, including higher plants. To analyze the importance of this pathway, we down-regulated mitochondrial citrate synthase (mCS; EC 4.1.3.7), the first enzyme of the TCA cycle, in transgenic potato plants using an antisense RNA approach. Several transformants were identified with reduced citrate synthase activity (down to approximately 6% of wild-type activity). These plants were indistinguishable from wild-type plants in the greenhouse during vegetative growth. A major change, however, was seen upon initiation of the generative phase (flower formation). In the case of transgenic plants with a strong reduction in citrate synthase activity (< 30% of wild-type levels), flower buds formed > 2 weeks later as compared with wild-type plants. Furthermore, flower buds from these plants did not develop into mature flowers but rather were aborted at an early stage of development. Microscopic analysis showed that in these cases ovaries disintegrated during flower development. We conclude that the TCA cycle is of major importance during the transition from the vegetative to the generative phase

A double-blind, placebo-controlled, cross-over study to establish the bifidogenic effect of a very-long-chain inulin extracted from globe artichoke (Cynara scolymus) in healthy human subjects

There is growing interest in the use of inulins as substrates for the selective growth of beneficial gut bacteria such as bifidobacteria and lactobacilli because recent studies have established that their prebiotic effect is linked to several health benefits. In the present study, the impact of a very-long-chain inulin (VLCI), derived from globe artichoke (Cynara scolymus), on the human intestinal microbiota compared with maltodextrin was determined. A double-blind, cross-over study was carried out in thirty-two healthy adults who were randomised into two groups and consumed 10 g/d of either VLCI or maltodextrin, for two 3-week study periods, separated by a 3-week washout period. Numbers of faecal bifidobacteria and lactobacilli were significantly higher upon VLCI ingestion compared with the placebo. Additionally, levels of Atopobium group significantly increased, while Bacteroides–Prevotella numbers were significantly reduced. No significant changes in faecal SCFA concentrations were observed. There were no adverse gastrointestinal symptoms apart from a significant increase in mild and moderate bloating upon VLCI ingestion. These observations were also confirmed by in vitro gas production measurements. In conclusion, daily consumption of VLCI extracted from globe artichoke exerted a pronounced prebiotic effect on the human faecal microbiota composition and was well tolerated by all volunteers