Lactate metabolism in potato tubers deficient in lactate dehydrogenase activity

The aim of this work was to investigate the effect of decreased activity of lactate dehydrogenase (EC 1.1.1.27; LDH) on lactate metabolism in potato tubers. By expressing a cDNA‐encoding potato tuber LDH in the antisense orientation, we generated transgenic potato plants with a preferential decrease in two of the five isozymes of LDH. Surprisingly, transgenic tubers grown under normoxic conditions did not contain less lactate, but rather instead contained approximately two‐fold more lactate than control tubers. This result is explicable if the decreased isozymes of LDH are responsible for the oxidation of lactate to pyruvate in vivo. This was confirmed by measurements of the rate of metabolism of lactate supplied to tuber discs: the rate in transgenic tubers was approximately half that of control tubers. The decrease in LDH activity had no measurable effect on the accumulation of lactate in cold‐stored tubers under anoxia, nor during the subsequent utilization of this lactate upon return to normoxia. In both control and transgenic tubers, the accumulation of lactate during anoxia was not accompanied by an induction of LDH activity or a change in isozyme distribution. In contrast, the metabolism of lactate after a period of anoxia was accompanied by a two‐fold increase in LDH activity and the induction of two isozymes that were distinct from those which had been decreased in the transgenic plants.     

Transgenic Potato with PVY Coat Protein Gene and Its Small-Scale Field Test

Potato virus Y (PVY) infection may cause a severe yield depression up to 80%. To develop the potato (Solanum tuberosum L. ) cultivars that resist PVY infection is very crucial in potato production. The authors have been cloned the coat protein gene of PVY from its Chinese isolate. A chimaeric gene containing the cauliflower mosaic virus 35S promoter and PVY coat protein coding region was introduced into the potato cultivars “Favorita”, “Tiger head” and “K4” via Agrobacterium tumefaciens. Results from PCR and Southern blot analysis confirmed that the foreign gene has integrated into the potato chromosomes. These transgenic potato plants were mechanically inoculated with PVY virus (20 mg/L). The presence of the virus in the potato plants was determined by ELISA and method of back inoculation into tobacco. The authors observed a drastic reduction in the accumulation of virus in some transgenic potato lines. Furthermore, some transgenic potato lines produced more tubers per plant than the untransformed potato did, and the average weight of these transgenic plant tubers was also increased. In the field test, the morphology and development of these transgenic potato plants were normal, 3 transgenic lines of “Favorita” exhibited a higher yield than the untrasformed virus-free potato with an increase ranged from 20% to 30%. From these transgenic lines, it will be very hopeful to develop a potato cultivar which not only has a significant resistance to PVY infection, but also a good harvest in potato production.     

The catecholamine potentiates starch mobilization in transgenic potato tubers.

In human and animal cells, the catecholamines are involved in glycogen mobilization. Since the compounds are found in a potato, their function in starch mobilization was hypothesized. In order to verify this hypothesis, the transgenic potato plants Solanum tuberosum L. cv. Desiree overexpressing tyrosine decarboxylase (TD EC 4.1.1.25) cDNA from parsley has been generated. The cDNA expression was judged by the northern blot analysis and the enzyme activity measurements. Four independent transgenic lines with the highest TD mRNA expression were selected and analyzed. The expected substantial decrease in tyrosine content was followed by significant increase in tyramine and dramatic enhancement of norepinephrine synthesis was detected. The level of L-3,4-dihydroxyphenylalanin (L-Dopa) was only slightly increased and dopamine significantly decreased in most cases in these plants. The increase in norepinephrine was accompanied by changes in carbohydrate metabolism. The significant increase in glucose and sucrose and the decrease in starch content were characteristic features of TD overexpressed transgenic potato tubers. The features mentioned above indicate that catecholamines potentiate starch mobilization in potato plants in common with animal cells. The decrease in tyrosine content in transgenic plants is also compensated by significant increase in chlorogenic acid synthesis thus potentially increasing the antioxidant capacity of transgenic tubers. The glycoalkaloids content is changed in the transformants. This may originate from glucose accumulation and glycolysis activation. The obtained transgenic potato provides material for further detailed studies of the physiological function of catecholamines in plants.     

Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes

Metabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography-mass spectrometry (GC-MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization-based GC-MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U-(13)C]glucose. The reproducibility of this method was similar to that found for other GC-MS-based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC-MS method has been applied to compare the metabolism of wild type and well-characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism.     

Control of potato tuber dormancy and sprouting by expression of sense and antisense genes of pyrophosphatase in potato

Inorganic pyrophosphate (PPi) is an enzyme involved in sugar metabolism in potato tubers. In our previous study, we isolated an inorganic pyrophosphatase (PPase) gene from potato and obtained the transgenic potato plants transformed with the sense and antisense PPase genes respectively. In the present experiment, the physiological indexes, tuber dormancy, and sprouting characteristics of the transgenic potatoes were analyzed and evaluated. The result showed that the PPase activity and the inorganic phosphate content of tubers were lower in the antisense transgenic plant lines but were higher in the sense transgenic plant lines, compared with wild-type tubers. Soluble sugars, such as glucose, fructose and sucrose increased in transgenic plants that had overexpression of the sense PPase gene, but decreased in the antisense transgenic plant lines, compared with wild-type tubers. Tuber sprouting time of the antisense transgenic plants were delayed for 2 and 3 weeks and reached the 100 % sprouting rate only after 14 and 16 weeks storage compared with the wild-type when tubers are stored under 25 and 4 °C, respectively. In contrast, tuber sprouting time of the sense transgenic plants was earlier by approximately 2 weeks than that of wild-type tubers under these storage temperatures.     

Activation of Host Defense Mechanisms by Elevated Production of H2O2 in Transgenic Plants

Active oxygen species have been postulated to perform multiple functions in plant defense, but their exact role in plant resistance to diseases is not fully understood. We have recently demonstrated H2O2-mediated disease resistance in transgenic potato (Solanum tuberosum) plants expressing a foreign gene encoding glucose oxidase. In this study we provide further evidence that the H2O2-mediated disease resistance in potato is effective against a broad range of plant pathogens. We have investigated mechanisms underlying the H2O2-mediated disease resistance in transgenic potato plants. The constitutively elevated levels of H2O2 induced the accumulation of total salicylic acid severalfold in the leaf tissue of transgenic plants, although no significant change was detected in the level of free salicylic acid. The mRNAs of two defense-related genes encoding the anionic peroxidase and acidic chitinase were also induced. In addition, an increased accumulation of several isoforms of extracellular peroxidase, including a newly induced one, was observed. This was accompanied by a significant increase in the lignin content of stem and root tissues of the transgenic plants. The results suggest that constitutively elevated sublethal levels of H2O2 are sufficient to activate an array of host defense mechanisms, and these defense mechanisms may be a major contributing factor to the H2O2-mediated disease resistance in transgenic plants.     

Superoxide stimulates a proton leak in potato mitochondria that is related to the activity of uncoupling protein

The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial. We have re-examined conditions that lead to uncoupling of mitochondria isolated from the tubers of potato (Solanum tuberosum). Specifically, we have investigated the effect of superoxide. In the absence of superoxide, linoleic acid stimulated a proton leak in mitochondria respiring NADH that was insensitive to GTP. However, when exogenous superoxide was generated by the addition of xanthine and xanthine oxidase, there was an additional linoleic acid-stimulated proton leak that was specifically inhibited by GTP. Under these conditions of assay (NADH as a respiratory substrate, in the presence of linoleic acid and xanthine/xanthine oxidase) there was a higher rate of proton conductance in mitochondria from transgenic potato tubers overexpressing the StUCP gene than those from wild type. The increased proton leak in the transgenic mitochondria was completely abolished by the addition of GTP. This suggests that superoxide and linoleic acid stimulate a proton leak in potato mitochondria that is related to the activity of uncoupling protein. Furthermore, it demonstrates that changes in the amount of StUCP can alter the rate of proton conductance of potato mitochondria.     

A sucrose non‐fermenting‐1‐related protein kinase‐1 gene,IbSnRK1, improves starch content,composition, granule size,degree of crystallinity and gelatinization in transgenic sweet potato

Sucrose non‐fermenting‐1‐related protein kinase‐1 (SnRK1) is an essential energy‐sensing regulator and plays a key role in the global control of carbohydrate metabolism. The SnRK1 gene has been found to increase starch accumulation in several plant species. However, its roles in improving starch quality have not been reported to date. In this study, we found that the IbSnRK1 gene was highly expressed in the storage roots of sweet potato and strongly induced by exogenous sucrose. Its expression followed the circandian rhythm. Its overexpression not only increased starch content, but also decreased proportion of amylose, enlarged granule size and improved degree of crystallinity and gelatinization in transgenic sweet potato, which revealed, for the first time, the important roles of SnRK1 in improving starch quality of plants. The genes involved in starch biosynthesis pathway were systematically up‐regulated, and the content of ADP‐glucose as an important precursor for starch biosynthesis and the activities of key enzymes were significantly increased in transgenic sweet potato. These findings indicate that IbSnRK1 improves starch content and quality through systematical up‐regulation of the genes and the increase in key enzyme activities involved in starch biosynthesis pathway in transgenic sweet potato. This gene has the potential to improve starch content and quality in sweet potato and other plants.     

Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.     

Effect of ambiol on stem growth in regenerants of source and transgenic potato plants

The effects of ambiol, a new growth regulator, on stem growth and morphological features of stem development have been compared in regenerants of potato (Solanum tuberosum L., var. Desire) plants transgenic for a defensin gene and in original potato plants. The original and transgenic plants exhibited differences in shoot development, which were observed both in control settings (no ambiol) and in the presence of various ambiol concentrations. In addition to normal plants of both forms, plant regenerants with morphological deviations were present in ambiol-treated groups. It is suggested that the abnormal shoot development observed in original and transgenic potato plants treated with ambiol is associated with (a) hormonal changes caused by expression of the defensin gene in the transgenic plants and (b) effects of ambiol on the hormonal balance of the plants.     

Effect of Ambiol on Stem Growth in Regenerants of Source and Transgenic Potato Plants

The effects of ambiol, a new growth regulator, on stem growth and morphological features of stem development have been compared in regenerants of potato (Solanum tuberosum L., var. Desire) plants transgenic for a defensin gene and in original potato plants. The original and transgenic plants exhibited differences in shoot development, which were observed both in control settings (no ambiol) and in the presence of various ambiol concentrations. In addition to normal plants of both forms, plant regenerants with morphological deviations were present in ambiol-treated groups. It is suggested that the abnormal shoot development observed in original and transgenic potato plants treated with ambiol is associated with (a) hormonal changes caused by expression of the defensin gene in the transgenic plants and (b) effects of ambiol on the hormonal balance of the plants.     

The control of source to sink carbon flux during tuber development in potato

We have used top-down metabolic control analysis to investigate the control of carbon flux through potato (Solanum tuberosum) plants during tuberisation. The metabolism of the potato plant was divided into two blocks of reactions (the source and sink blocks) that communicate through the leaf apoplastic sucrose pool. Flux was measured as the transfer of ¹⁴C from CO₂ to the tuber. Flux and apoplastic sucrose concentration were varied either by changing the light intensity or using transgenic manipulations that specifically affect the source or sink blocks, and elasticity coefficients were measured. We have provided evidence in support of our assumption that apoplastic sucrose is the only communicating metabolite between the source and sink blocks. The elasticity coefficients were used to calculate the flux control coefficients of the source and sink blocks, which were 0.8 and 0.2, respectively. This work suggests that the best strategy for the manipulation of tuber yield in potato will involve increases in photosynthetic capacity, rather than sink metabolism.     

过表达MtVP1对马铃薯表型及糖代谢的影响

I型H⁺-PPase参与糖异生和蔗糖分解代谢,利用不同的糖(蔗糖、葡萄糖和果糖)饲喂拟南芥(Arabidopsis thaliana)Ⅰ型H⁺-PPase基因不同类型的突变体,产生的表型不一致,因此,推测Ⅰ型H⁺-PPase可能存在其它影响糖代谢的机制。为进一步明确该酶对糖代谢的影响,以过表达MtVP1的马铃薯(Solanum tuberosum)渭薯4号为研究对象,观察不同培养条件下的表型,监测糖含量变化,并利用转录组测序分析转录谱。结果表明,过表达MtVP1马铃薯表现出红色茎、紫色花和表皮毛更发达,单株块茎数减少,块茎变大,块茎皱缩速度加快;转基因马铃薯块茎中淀粉、葡萄糖和果糖含量显著下降,芽中葡萄糖和果糖含量也显著下降。果糖饲喂导致转基因马铃薯花青素含量显著降低;转基因马铃薯体内果糖-1,6-二磷酸酶和果糖-2,6-二磷酸酶基因表达上调3–7倍。研究结果为进一步从糖代谢角度探究Ⅰ型H⁺-PPase的生理功能提供参考。     

Inducible Positive Mutant Screening System to Unveil the Signaling Pathway of Late Blight Resistance

Late blight is the most devastating potato disease and it also causes serious yield loss in tomato.Several disease resistance genes (R genes) to late blight have been cloned from potato in the past decade.However,the resistance mechanisms remain elusive.Tomato and potato belong to the botanical family Solanaceace and share remarkably conserved genome structure.Since tomato is a model system in genetic and plant pathology research,we used tomato to develop a powerful mutant screening system that will greatly facilitate the analysis of the signaling pathway of resistance to Phytophthora infestans.First we proved that the R3a transgenic tomatoes developed specific hypersensitive cell death response (HR) to P.infestans strains carrying the corresponding avirulence gene Avr3a,indicating that the signaling pathway from the R3a-Avr3a recognition to HR is conserved between potato and tomato.Second,we generated transgenic tomatoes carrying both R3a and Avr3a genes,with the latter under the control of a glucocorticiod-inducible promoter.Dexamethasone induced expression of Avr3a and resulted in localized HR.This versatile system can be used to construct a mutant library to screen surviving mutants whose resistance signal transduction was interrupted,providing the basis to identify key genes involved in the resistance to late blight in Solanaceae.     

Response of transgenic potato seedlings to allelopathic pressure and the effect of nutrients in the culture medium

Chinese farmers frequently use a wheat–potato cropping system. The land area planted to transgenic potatoes is increasing because transgenic potatoes have greater resistance to pests and diseases. However, little is known about the bio-compatibility of transgenic potatoes with wheat straw. The objective of this tissue culture study was to determine the allelopathic effects of wheat straw on transgenic potato seedlings. Seedlings were cultured on normal MS medium (normal treatment) and nutrient-deficient MS medium (acclimated treatment) and then transferred to MS medium, which contained wheat straw powder. Wheat straw powder inhibited potato seedling growth in both treatments. Among the parameters analyzed in this study, inhibition was greatest for plant fresh weight and least for plant height. The inhibitive effects of wheat straw were greater for seedling roots compared to shoots. Resistance to allelopathic pressure from wheat straw was greater in acclimated seedlings compared to normal seedlings. This suggested that previous pressure may have induced tolerance in the transgenic potato seedlings. Furthermore, growth inhibition of potato seedlings from the normal treatment increased as the amount of wheat straw powder in the culture medium increased. Calculations indicated that the presence of wheat straw would lead to a 55% reduction in the total biomass of normal potato seedlings compared to a 39% reduction for acclimated seedlings. Parameters such as net photosynthesis rate (Pn) and quantum yield (Y(II)) changed as the nutrient content of the culture medium increased or decreased, but the changes in the parameters were smaller for acclimated seedlings compared to normal seedlings. This suggests that nutrient status during the culture period could help transgenic potato seedlings adapt and compensate for energy loss from seedlings in defending against allelopathic pressure. In summary, the results show that previous exposure to pressures such as nutrient deficiency may increase the allelopathic pressure resistance of transgenic potato seedlings.     

Low levels of pyrophosphate in transgenic potato plants expressing E. coli pyrophosphatase lead to decreased vitality under oxygen deficiency

BACKGROUND AND AIMS: The aim of this study was to investigate the importance of pyrophosphate (PPi) for plant metabolism and survival under low oxygen stress. Responses of roots of wild-type potato plants were compared with roots of transgenic plants containing decreased amounts of PPi as a result of the constitutive expression of Escherichia coli pyrophosphatase in the cytosol. METHODS: For the experiments, roots of young wild-type and transgenic potato plants growing in nutrient solution were flushed for 4 d with nitrogen, and subsequently metabolite contents as well as enzyme activities of the glycolytic pathway were determined. KEY RESULTS AND CONCLUSIONS: In roots of transgenic plants containing 40% less PPi, UDPglucose accumulated while the concentrations of hexose-6-phosphate, other glycolytic intermediates and ATP were decreased, leading to a growth retardation in aerated conditions. Apart from metabolic alterations, the activity of sucrose synthase was increased to a lower extent in the transgenic line than in wild type during hypoxia. These data suggest that sucrose cleavage was inhibited due to PPi deficiency already under aerated conditions, which has severe consequences for plant vitality under low oxygen. This is indicated by a reduction in the glycolytic activity, lower ATP levels and an impaired ability to resume growth after 4 d of hypoxia. Interestingly, the phosphorylation of fructose-6-phosphate via PPi-dependent phosphofructokinase was not altered in roots of transgenic plants. Nevertheless, our data provide some evidence for the importance of PPi to maintain plant growth and metabolism under oxygen deprivation.     

Effect of water stress on proline accumulation of genetically modified potatoes (Solanum tuberosum L.) generating fructans

This study aimed to investigate the effect of water stress on the accumulation of proline in the leaves of transgenic potato lines generating fructans. The results of the series of bifactorial experiments in 2002 and 2003 indicated an increase of the proline level in leaves of all potato lines examined under water deficit. In addition, an increase in proline concentration during plant development was observed. The proline content was related to leaf water potential and relative water content (RWC), which indicates that proline could be involved in osmoregulation of potato plants under the experimental conditions. Surprisingly, under water deficit, the proline level was lowest in most of the transgenic SST/FFT-lines, which generate fructan molecules with a high degree of polymerization. Therefore, a pleiotropic effect can not be excluded as the reason for the divergence in behavior of these transgenic lines. The present results suggest that the modification of carbohydrate metabolism, especially the high content of soluble carbohydrates, may affect water stress-induced proline accumulation.     

Characterization of the ethanol-inducible alc gene expression system in tomato

The efficacy of the ethanol-inducible alc transgene expression system, derived from the filamentous fungus Aspergillus nidulans, has been demonstrated in transgenic tomato. Two direct comparisons have been made. First, this study has utilized two transgenic lines carrying distinct reporter genes (chloramphenicol acetyltransferase and beta-glucuronidase) to distinguish aspects of induction determined by the nature of the gene/gene product rather than that of the plant. Second, comparisons have been made to data generated in other species in order to identify any species-specific effects. The induction profiles for different genes in different species have shown remarkable similarity indicating the broad applicability of this gene switch. While there are minor differences observed between species, these probably arise from diversity in their metabolism. A series of potential alternative inducers have also been tested, revealing that ethanol (through metabolism to acetaldehyde) is better than other alcohols and ketones included in this study. Expression driven by alc was demonstrated to vary spatially, the upper younger leaves having higher activity than the lower older leaves; this will be important for some applications, and for experimental design. The highest levels of activity from ethanol-inducible transgene expression were determined to be the equivalent of those from the constitutive Cauliflower Mosaic Virus 35S promoter. This suggests that the alc system could be an important tool for plant functional genomics.     

Starch self-processing in transgenic sweet potato roots expressing a hyperthermophilic α-amylase

Sweet potato is a major crop in the southeastern United States, which requires few inputs and grows well on marginal land. It accumulates large quantities of starch in the storage roots and has been shown to give comparable or superior ethanol yields to corn per cultivated acre in the southeast. Starch conversion to fermentable sugars (i.e., for ethanol production) is carried out at high temperatures and requires the action of thermostable and thermoactive amylolytic enzymes. These enzymes are added to the starch mixture impacting overall process economics. To address this shortcoming, the gene encoding a hyperthermophilic α-amylase from Thermotoga maritima was cloned and expressed in transgenic sweet potato, generated by Agrobacterium tumefaciens-mediated transformation, to create a plant with the ability to self-process starch. No significant enzyme activity could be detected below 40°C, but starch in the transgenic sweet potato storage roots was readily hydrolyzed at 80°C. The transgene did not affect normal storage root formation. The results presented here demonstrate that engineering plants with hyperthermophilic glycoside hydrolases can facilitate cost effective starch conversion to fermentable sugars. Furthermore, the use of sweet potato as an alternative near-term energy crop should be considered.