Antisense inhibition of the iron-sulphur subunit of succinate dehydrogenase enhances photosynthesis and growth in tomato via an organic acid-mediated effect on stomatal aperture

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the Sl SDH2-2 gene encoding the iron sulfur subunit of the succinate dehydrogenase protein complex in the antisense orientation under the control of the 35S promoter exhibit an enhanced rate of photosynthesis. The rate of the tricarboxylic acid (TCA) cycle was reduced in these transformants, and there were changes in the levels of metabolites associated with the TCA cycle. Furthermore, in comparison to wild-type plants, carbon dioxide assimilation was enhanced by up to 25% in the transgenic plants under ambient conditions, and mature plants were characterized by an increased biomass. Analysis of additional photosynthetic parameters revealed that the rate of transpiration and stomatal conductance were markedly elevated in the transgenic plants. The transformants displayed a strongly enhanced assimilation rate under both ambient and suboptimal environmental conditions, as well as an elevated maximal stomatal aperture. By contrast, when the Sl SDH2-2 gene was repressed by antisense RNA in a guard cell-specific manner, changes in neither stomatal aperture nor photosynthesis were observed. The data obtained are discussed in the context of the role of TCA cycle intermediates both generally with respect to photosynthetic metabolism and specifically with respect to their role in the regulation of stomatal aperture.     

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 (F _v/F _m), 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.     

Photosynthetic compensation by the reproductive structures in the spring ephemeral Gagea lutea

Growth and reproduction of spring ephemerals inhabiting deciduous forests progress simultaneously during a short period from snowmelt to canopy closure. To clarify the mechanism to mitigate the cost of reproduction, contributions of foliar and non-foliar photosynthetic products to seed production were examined in a spring ephemeral Gagea lutea. Leaf growth, foliar and non-foliar photosynthetic activities, and total assimilated products were compared among reproductive-intact, floral bud-removal, and vegetative plants. Translocation of current photosynthetic products to individual organs was quantified by ¹³CO₂-trace experiment. Bulb growth was compared between hand-pollination and floral bud-removal treatments. Finally, seed set was compared between intact, leaf-clipping, and bract-clipping treatments. Fruit-forming plants retained leaves longer than vegetative and floral bud-removal plants, but the assimilative contribution of extended leaf longevity was negligible. Carbon supply by bract photosynthesis was large enough for fruit development, while carbon supply by fruit photosynthesis was offset by the high respiration loss. Foliar photosynthetic products were largely transported to bulbs, while translocation to reproductive functions was negligible. Because the floral bud-removal increased the bulb growth, lack of reproduction could lead to more storage. The leaf-clipping had no effect on seed production, while the bract-clipping significantly reduced the seed production. Therefore, current photosynthesis of leafy bracts might be a major carbon source for fruit development. This self-compensative mechanism of reproductive structure enables the continuous reproductive activity in this species.     

Antisense acid invertase (TIV1) gene alters soluble sugar composition and size in transgenic tomato fruit.

Invertase (beta-fructosidase, EC 3.2.1.26) hydrolyzes sucrose to hexose sugars and thus plays a fundamental role in the energy requirements for plant growth and maintenance. Transgenic plants with altered extracellular acid invertase have highly disturbed growth habits. We investigated the role of intracellular soluble acid invertase in plant and fruit development. Transgenic tomato (Lycopersicon esculentum Mill.) plants expressing a constitutive antisense invertase transgene grew identically to wild-type plants. Several lines of transgenic fruit expressing a constitutive antisense invertase gene had increased sucrose and decreased hexose sugar concentrations. Each transgenic line with fruit that had increased sucrose concentrations also had greatly reduced levels of acid invertase in ripe fruit. Sucrose-accumulating fruit were approximately 30% smaller than control fruit, and this differential growth correlated with high rates of sugar accumulation during the last stage of development. These data suggest that soluble acid invertase controls sugar composition in tomato fruit and that this change in composition contributes to alterations in fruit size. In addition, sucrose-accumulating fruit have elevated rates of ethylene evolution relative to control fruit, perhaps as a result of the smaller fruit size of the sucrose-accumulating transgenic lines.     

转ipt和反义ACO基因番茄的叶片衰老相关特性

以ipt和反义ACO转化的两类转基因番茄纯系为材料,研究在植株不同生长发育阶段,不同叶位中,与叶片衰老相关的生理生化指标.结果表明:两类基因导入番茄后,均可增强内源iPA和IAA表达水平,增加或保持番茄叶片的叶绿素含量、提高光合效率,进而明显地延缓植株的叶片衰老,提高单株果实产量.但它们调控叶片衰老的途径不同,ipt主要通过提高CTK的水平延缓叶片衰老,而反义ACO则主要是通过抑制乙烯生成,间接提高IAA的水平来实现.     

Improved growth,productivity and quality of tomato (Solanum lycopersicum L.) plants through application of shikimic acid

A field experiment was conducted to investigate the effect of seed presoaking of shikimic acid (30, 60 and 120 ppm) on growth parameters, fruit productivity and quality, transpiration rate, photosynthetic pigments and some mineral nutrition contents of tomato plants. Shikimic acid at all concentrations significantly increased fresh and dry weights, fruit number, average fresh and dry fruit yield, vitamin C, lycopene, carotenoid contents, total acidity and fruit total soluble sugars of tomato plants when compared to control plants. Seed pretreatment with shikimic acid at various doses induces a significant increase in total leaf conductivity, transpiration rate and photosynthetic pigments (Chl. a, chl. b and carotenoids) of tomato plants. Furthermore, shikimic acid at various doses applied significantly increased the concentration of nitrogen, phosphorus and potassium in tomato leaves as compared to control non-treated tomato plants. Among all doses of shikimic acid treatment, it was found that 60 ppm treatment caused a marked increase in growth, fruit productivity and quality and most studied parameters of tomato plants when compared to other treatments. On the other hand, no significant differences were observed in total photosynthetic pigments, concentrations of nitrogen and potassium in leaves of tomato plants treated with 30 ppm of shikimic acid and control plants. According to these results, it could be suggested that shikimic acid used for seed soaking could be used for increasing growth, fruit productivity and quality of tomato plants growing under field conditions.     

The influence of cytosolic phosphoglucomutase on photosynthetic carbohydrate metabolism

The aim of this work was to examine the role of cytosolic phosphoglucomutase (cPGM; EC 5.4.2.2) in photosynthetic carbon partitioning. We have previously described the generation and characterisation of the tuber metabolism of transgenic potato ( Solanum tuberosum cv. Desiree) lines expressing the StcPGM gene in the antisense orientation under the control of the 35S promoter. Here we extend the characterisation of leaf metabolism within these lines, examining properties of gas exchange, carbon partitioning, and the effect of the genetic manipulation on a wide range of metabolites including metabolites of the sucrose-starch transition, glycolysis, the Krebs cycle and amino acid metabolism. The data acquired in the present study surprisingly reveal that the photosynthetic sucrose synthetic capacity of the leaves is largely unaltered but that these plants display a reduced rate of photosynthesis, a dramatic reduction in nucleotide levels, and a general decline of biosynthesis. We conclude that these lines exhibit only moderate changes in sucrose synthesis but more complex changes on a range of diverse metabolic pathways.     

A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit

The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild‐type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose : sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species.     

Sex-specific physiology and source-sink relations in the dioecious plant Silene latifolia

Differences in reproductive demands between the sexes of dioecious plants could cause divergence in physiology between the sexes. We found that the reproductive effort of female Silene latifolia plants increased to more than twice that of male plants or female plants that were prevented from setting fruit by lack of pollination after 4 weeks of flowering. Whole-plant source/sink ratios of pollinated females were significantly lower than those of males or unpollinated females because of investment in fruit. We hypothesized that these differences in source/sink ratio between the sexes and within females, depending on pollination, would lead to differences in leaf photosynthetic rates. Within females, we found that photosynthetic capacity was consistent with measurement of whole-plant source/sink ratio. Females that were setting fruit had 30% higher light-saturated photosynthetic rates by 28 days after flowering than females that were not setting fruit. Males, however, had consistently higher photosynthetic rates than females from 10 days after flowering onwards. Males also had approximately twice the dark respiration rates of fruiting females. We found that female reproductive structures are longer-lived and contribute more carbon to their own support than male reproductive structures. Despite the higher rates of leaf dark respiration and lower calyx photosynthetic rates, males fix more carbon than do females. We conclude that females have a sink-regulated mechanism of photosynthesis that allows them to respond to variations in fruit set. This mechanism is not, however, sufficient to explain why male S. latifolia plants have higher rates of photosynthesis, higher source/sink ratios, and lower reproductive allocation, but fail to grow larger than female plants.     

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.     

Sucrose transporter LeSUT1 and LeSUT2 inhibition affects tomato fruit development in different ways

Sucrose transporters of higher plants belong to a large gene family. At least four different sucrose transporters are known in Solanaceous plants, although their function remains to be elucidated in detail. The isolation of LeSUT1 and LeSUT2 from Lycopersicon esculentum has been described earlier. Whereas SUT1 is supposed to be the main phloem loader of sucrose in Solanaceae , the role of SUT2 remains a matter of debate. A transgenic approach was taken to evaluate the potential functions of SUT2/SUC3 proteins in sucrose transport or sensing. Expression of LeSUT1 and LeSUT2 was inhibited independently in transgenic tomato plants, using the antisense technique, in order to analyse their specific functions. Although the phloem-specific inhibition of LeSUT1 antisense plants showed a phenotype consistent with an essential role in phloem loading, constitutive LeSUT2 antisense inhibition exclusively affected tomato fruit and seed development. Neither LeSUT1 , nor the LeSUT2 antisense plants were able to produce normal tomato fruits; however, it is likely that independent mechanisms underlie these phenomena. While phloem loading was blocked in LeSUT1 antisense plants, the fertility of fruits was reduced in LeSUT2 antisense plants. A detailed physiological analysis of these plants established a role for SUT2 in pollen tube growth and thus assigned a physiological role for SUT2.     

Deficiency of mitochondrial fumarase activity in tomato plants impairs photosynthesis via an effect on stomatal function

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of a fumarate hydratase (fumarase) gene in the antisense orientation and exhibiting considerable reductions in the mitochondrial activity of this enzyme show impaired photosynthesis. The rate of the tricarboxylic acid cycle was reduced in the transformants relative to the other major pathways of carbohydrate oxidation and the plants were characterized by a restricted rate of dark respiration. However, biochemical analyses revealed relatively little alteration in leaf metabolism as a consequence of reducing the fumarase activity. That said, in comparison to wild-type plants, CO(2) assimilation was reduced by up to 50% under atmospheric conditions and plants were characterized by a reduced biomass on a whole plant basis. Analysis of further photosynthetic parameters revealed that there was little difference in pigment content in the transformants but that the rate of transpiration and stomatal conductance was markedly reduced. Analysis of the response of the rate of photosynthesis to variation in the concentration of CO(2) confirmed that this restriction was due to a deficiency in stomatal function.     

Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening

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.     

Metabolic profiling of a mapping population exposes new insights in the regulation of seed metabolism and seed, fruit, and plant relations

To investigate the regulation of seed metabolism and to estimate the degree of metabolic natural variability, metabolite profiling and network analysis were applied to a collection of 76 different homozygous tomato introgression lines (ILs) grown in the field in two consecutive harvest seasons. Factorial ANOVA confirmed the presence of 30 metabolite quantitative trait loci (mQTL). Amino acid contents displayed a high degree of variability across the population, with similar patterns across the two seasons, while sugars exhibited significant seasonal fluctuations. Upon integration of data for tomato pericarp metabolite profiling, factorial ANOVA identified the main factor for metabolic polymorphism to be the genotypic background rather than the environment or the tissue. Analysis of the coefficient of variance indicated greater phenotypic plasticity in the ILs than in the M82 tomato cultivar. Broad-sense estimate of heritability suggested that the mode of inheritance of metabolite traits in the seed differed from that in the fruit. Correlation-based metabolic network analysis comparing metabolite data for the seed with that for the pericarp showed that the seed network displayed tighter interdependence of metabolic processes than the fruit. Amino acids in the seed metabolic network were shown to play a central hub-like role in the topology of the network, maintaining high interactions with other metabolite categories, i.e., sugars and organic acids. Network analysis identified six exceptionally highly co-regulated amino acids, Gly, Ser, Thr, Ile, Val, and Pro. The strong interdependence of this group was confirmed by the mQTL mapping. Taken together these results (i) reflect the extensive redundancy of the regulation underlying seed metabolism, (ii) demonstrate the tight co-ordination of seed metabolism with respect to fruit metabolism, and (iii) emphasize the centrality of the amino acid module in the seed metabolic network. Finally, the study highlights the added value of integrating metabolic network analysis with mQTL mapping.