Engineering ketocarotenoid biosynthesis in potato tubers

Consumption of astaxanthin is increasingly associated with a range of health benefits. Attempts to engineer ketocarotenoid biosynthesis in plants have been successful although there are no reports of nutritionally significant levels of astaxanthin in plant storage organs. Thus, in this study, ketocarotenoid biosynthesis was engineered in potato tubers. Both Solanum tuberosum and Solanum phureja transgenic lines were produced that expressed an algal bkt1 gene, encoding a beta-ketolase, and accumulated ketocarotenoids. Two major ketocarotenoids were detected, ketolutein and astaxanthin. The level of unesterified astaxanthin reached ca. 14 microg g(-1) DW in some bkt1 expressing lines of S. phureja but was much lower in the S. tuberosum background. Co-transformation of S. tuberosum with crtB, encoding phytoene synthase, and the bkt1 gene was achieved in order to determine whether this would enhance the levels of S. tuberosum ketocarotenoid.     

Regulatory role of polyamine in the acid phosphatase from potato tubers.

Effects of polyamine and metal ions on the new type of acid phosphatase purified from potato (Solanum tuberosum L. Irish Cobbler) tubers were analyzed. The enzyme belongs to nonspecific acid phosphatase family (EC 3.1.3.2), which hydrolyzes various phosphorylated substrates. The enzyme hydrolyzed inorganic pyrophosphate as a preferred substrate, and exhibited the hyperbolic kinetics with respect to the substrate, inorganic pyrophosphate in the absence of metal cations. Polyamine activated the enzyme effectively by lowering the K(m) value without appreciable changes in the maximal velocity. The most effective polyamines as activators were spermine and spermidine. Mg(2+) ion increased the K(m) value without affecting the maximal velocity of the enzyme, but Ca(2+) ion decreased both the K(m) and V(max) values. Increasing concentrations of spermine also decreased the K(m) value irrespective of Mg(2+) ion included, but gave a constant K(m) and V(max) values in the absence and presence of Ca(2+) ion. Action of spermine and metal ions can be explained by the complex formation with the substrate pyrophosphate. The acid phosphatase from potato can utilize the pyrophosphate-spermine or pyrophosphate-Ca(2+) complex as the preferred substrates. However, the enzyme can use the pyrophosphate-Mg complex with a weak affinity for the active site. Polyamine activates acid phosphatase in the absence and presence of metal cations, and activation by polyamine of the enzyme may contribute to the stimulation of starch biosynthesis and the control of glycolysis/gluconeogenesis by regulating PPi levels in growing potato tubers.     

Changes in cytokinins during initiation and development of potato tubers

Cytokinin-like activity was assayed in stolons and tubers of Solanum tuberosum L. ssp. andigena (Juz. et Buk.) Hawkes cv. 165 grown in pots under controlled environment conditions. The plants were allowed to tuberise without the application of environmental or other external stimuli. The soluble sugar and starch contents of stolon tips and tubers were measured. Starch accumulation was a precise indicator of tuber initiation. Cytokinin-like activity began to increase in tubers with a diameter greater than 7.5 mm and, as assessed on a per tuber basis, was greatest in the largest size-category analysed. However, expressed as a function of fresh and dry weight, activity was greatest in tubers of 15–20 mm in diameter. Increases in cytokinin-like activity occurred subsequent to tuber formation, indicating that the tuberisation stimulus is unlikely to be cytokinin-like in nature.     

Changes in the levels of free IAA and cytokinins in potato tubers during dormancy and sprouting

Changes in the levels of free indol-3-ylacetic acid (IAA) and free cytokinins were determined in the course of dormancy and sprouting period in potato tubers(Solanum tuberosum L., cv. Nevskii) stored at 4 °C. The same analyses were performed in potato tubers after Ethrel application, which prolongs dormancy. No significant changes were found in free IAA level during dormancy followed by a rapid decrease during sprouting. After Ethrel application a significant lower IAA level was found 3 weeks after application, but further on no changes in free IAA level between treated and non-treated tubers were detected. Cytokinin level was relatively low and constant till sprouting and increases then by about 55 %, mainly due to an increase in the level of zeatin riboside and isopentenyladenosine. Ethrel application decreased cytokinin level during dormancy slightly, but postpones the increase coupled with sprouting by about 1 month. Thus, IAA does not seem to have a significant effect on tuber dormancy, while cytokinins are probably necessary for sprouting initiation.     

Starch biosynthesis from triose-phosphate in transgenic potato tubers expressing plastidic fructose-1,6-bisphosphatase

A full length cDNA clone encoding plastidic fructose-1,6-bisphosphatase (cp-FBPase), together with a transit peptide, was isolated from a potato (Solanum tuberosum L.) leaf cDNA library. Potato plants were transformed with the isolated cp-FBPase sequence behind a patatin class I promoter to ensure tuber-specific expression of the enzyme. Plant lines were selected which expressed up to 250 mU (g FW)-1 in the developing tubers, which is 10- to 20-fold the activity found in wild-type tubers. Intact amyloplasts were isolated from in vitro-grown minitubers developed in darkness. Comparison with marker enzymes showed that cp-FBPase activity in transgenic tubers, as well as the low FBPase activity in the wild-type tubers, was localised inside the amyloplasts. The intact amyloplasts isolated from both wild-type and transgenic tubers synthesised starch from [U-14C] glucose-6-phosphate. Conversely, only the transgenic tubers expressing cp-FBPase showed appreciable synthesis of starch from [U-14C] dihydroxyacetone phosphate, and this synthesis rate was correlated to the activity of cp-FBPase. Thus, the expression of cp-FBPase in tubers allows for a new route of starch biosynthesis from triose-phosphates imported from the cytosol. The transgenic tubers did not differ from wild-type tubers with respect to starch content, or the levels of neutral sugars and phosphorylated hexoses.     

Tissue distribution and change in potato starch phosphorylase mRNA levels in wounded tissue and sprouting tubers.

Starch phosphorylase has been cloned from a lambda gt10 cDNA library of potato tuber mRNA. Selected recombinants have been used to demonstrate that phosphorylase mRNA is most abundant in tubers but is also detectable in stolon, root, stem and leaf tissue. The level of phosphorylase mRNA was greatly reduced in wounded stem and tuber tissue. The wounding-induced decrease in phosphorylase mRNA levels is not reversed in the presence of sucrose or mannitol. Regional differences are described in the levels of phosphorylase and patatin mRNA in different parts of the tuber and in the shoot of sprouting potatoes.     

萌芽菊芋块茎对盐碱土壤胁迫的生理响应

土壤盐碱化是影响全球农业生产和生态环境的重要问题。在农田、轻度盐碱草地和重度盐碱草地设置样地以块茎种植菊芋,次年5月块茎萌发阶段取块茎样品测定丙二醛、游离脯氨酸、可溶性糖含量以及抗氧化酶活性并进行蛋白质组学分析,分析了萌芽菊芋块茎对盐碱土壤胁迫的生理响应。0—20 cm土层的电导率(表征土壤可溶盐含量)表明从农田到轻度、重度盐碱草地土壤盐碱胁迫逐渐增强,丙二醛含量变化反映出菊芋块茎受害程度逐渐增加,并且基于游离脯氨酸的渗透调节能力也在逐渐增强。蛋白质组学分析结果显示与遗传信息加工相关的差异蛋白数量最多(占28.75%)且多为表达上调,意味着DNA复制和转录、蛋白质合成和折叠的相关蛋白在响应盐碱胁迫中发挥关键作用。碳水化合物及多糖代谢(占15%)、氨基酸代谢(占11.25%)以及能量代谢(占7.5%)相关的差异蛋白数量也较多,说明调节物质代谢平衡在萌芽菊芋块茎应对盐碱土壤胁迫过程中有重要作用。这些结果为揭示萌芽菊芋块茎适应盐胁迫的生理机制奠定了基础。     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

Application of enzyme-linked immunosorbent assay to the detection of potato leafroll virus in potato tubers

Factors affecting the detection of potato leafroll virus (PLRV) by enzyme-linked immunosorbent assay (ELISA) in tubers of field-grown potato plants with primary or secondary infection were studied. The reactions of extracts of virus-free potato tubers were minimised by pre-incubating the extracts at room temperature and by careful choice of the dilution of enzyme-conjugated globulin. PLRV was reliably detected in tubers produced by secondarily infected plants of all six cultivars tested. PLRV concentration was greater in heel-end than in rose-end vascular tissue of recently harvested tubers but increased in rose-end tissue when tubers stored at 4°C for at least 5 months were placed at 15–24°C for 2 wk. PLRV occurred at greater concentration in tubers from plants of cv. Maris Piper with natural or experimentally induced primary infection than in tubers from secondarily infected plants; again PLRV concentration was greater in heel-end than in rose-end vascular tissue. Plants whose shoots were infected earliest in the growing season were invaded systemically and produced the greatest proportion of infected tubers; plants infected late in the season also produced infected tubers but PLRV was not detected in their shoot tops. PLRV concentration in tubers from the earliest-infected plants was less than in tubers from later-infected plants. PLRV was detected reliably by ELISA in tubers from progenies that were totally infected but was not detected in all infected tubers from partially infected progenies. ELISA is suitable as a routine method of indexing tubers for PLRV, although the virus will not be detected in all infected tubers produced by plants to which it is transmitted late in the growing season.     

Effects of gibberellin and auxin on the synthesis of abscisic acid and ethylene in buds of dormant and sprouting potato tubers

Gibberellic and β-indolylacetic acids at concentrations of 10⁻⁷-10⁻⁵ M were shown to change the hormonal status and duration of true dormancy in potato tubers. Gibberellic acid shortened the true dormancy and decreased the contents of abscisic acid and ethylene in the apical meristem. β-Indolylacetic acid elongated the true dormancy and decreased abscisic acid production, but caused a more than tenfold increase in the production of ethylene by apical tissues. The data suggest that β-indolylacetic acid and ethylene, as well as gibberellic and abscisic acids, are involved in the regulation of true dormancy in potato tubers.     

Evolutionary diversification in polyamine biosynthesis

Polyamine biosynthesis is an ancient metabolic pathway present in all organisms. Aminopropyltransferases are key enzymes that mediate the synthesis of spermidine, spermine, and thermospermine. The relatively high sequence similarity between aminopropyltransferases and their similarity with putrescine N-methyltransferases (PMT) raises the question of whether they share a common ancestor or have evolved by convergence. Here we show that aminopropyltransferases and PMT are phylogenetically interconnected, and the different activities have been generated by unusually frequent events of diversification of existing functions. Although all spermidine synthases (SPDSs) derive from a common ancestor preceding the separation between prokaryotes and eukaryotes, they have been the origin of a variety of new activities. Among those, spermine synthases (SPMSs) represent a novelty independently arisen at least 3 times, in animals, fungi, and plants. The most parsimonious mechanism would involve the duplication and change of function of preexisting SPDS genes in each phylum. Although spermine is not essential for life, the repeated invention of SPMS and its conservation strongly argues for an evolutionary advantage derived from its presence. Moreover, the appearance of thermospermine synthase (tSPMS) in several genera of Archaea and Bacteria was accompanied by a loss of SPDS, suggesting that the new activity originated as a change of function of this enzyme. Surprisingly, tSPMS was later acquired by plants at an early stage of evolution by horizontal gene transfer and has proven to be essential for vascular development in tracheophytes. Finally, the synthesis of nicotine and tropane alkaloids in Solanales was favored by the origination of a new activity, PMT, as a duplication and change of function from SPDS.     

Effect of vitamins on sprouting of purple nutsedge tubers

Vitamins of the B group and vitamin C were applied to purple nutsedge (Cyperus rotundus L.) tubers to study their effect on the sprouting rate, initiation and establishment of sprouts, growth of plantlets and development of orthotropic rhizomes in comparison with the corresponding effects of kinetin. Ascorbic acid up to 100 mg I^?1 hastened sprouting, whereas vitamins of the B group and kinetin retarded sprouting; 100% tubers sprouted in all treatments within 10 days. Unlike kinetin, none of the vitamins resulted in the establishment of more than one sprout per tuber. Riboflavin and pyridoxine promoted root and shoot growth of the plantlets, whereas kinetin produced short, thick shoots and inhibited root growth, with increasing concentration. Ascorbic acid was only second to kinetin in the induction of orthotropic rhizomes, but the former resulted in an increase in rhizome length.