马铃薯块SGAs合成途径关键基因表达量的GGE双标图分析

糖苷生物碱(steroidal glycoalkaloid,SGAs)含量密切关系马铃薯块茎的食用品质和加工品质。本文利用GGE-biplot双标图分析了红光处理6、12、24 h后,5个马铃薯品种块茎中调控糖苷生物碱合成途径7个主要基因的表达情况。pvs1、sgt1和sgt3这3个基因的表达量均远高于其他4个基因的表达。在所测基因型中,sgt3的表达量不仅高而且还相对于pvs1和sgt1来说比较稳定,而在不同处理时间之间差异较大,稳定性较差。在12与24 h处理后所有基因表达的趋势比较接近,但是除了pvs1基因的表达量,其他6个基因的表达量均低于6 h处理的。从基因型的角度分析,所有基因在野生种HA和当地栽培种ZH-3中表达量较高而且表达趋势比较一致。通过比较调控糖苷生物碱合成途径的不同阶段的基因的表达量,发现不同阶段关键基因的表达量在基因型之间存在显著差异。所以,通过GGE-biplot分析,结果展示了7个基因在不同基因型马铃薯块茎的表达趋势,而且也根据基因表达量直观的展示了处理时间与各基因型之间的聚类与关系,为将来进一步分析不同光质在基因水平调控马铃薯糖苷生物碱的生物合成提供了重要参考。     

Three genes encode 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hevea brasiliensis: hmg1 and hmg3 are differentially expressed

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyses an important step in isoprenoid biosynthesis in plants. In Hevea brasiliensis, HMGR is encoded by a small gene family comprised of three members, hmg1, hmg2 and hmg3. We have previously described hmg1 and hmg2 (Plant Mol Biol 16: 567–577, 1991). Here we report the isolation and characterization of hmg3 genomic and cDNA clones. In comparison to hmg1 which is more highly expressed in laticifers than in leaves, the level of hmg3 mRNA level is equally abundant in laticifers and leaves. In situ hybridization experiments showed that the expression of hmg3 is not cell-type specific while hmg1 is expressed predominantly in the laticifers. Primer-extension experiments using laticifer RNA showed that hmg1 is induced by ethylene while hmg3 expression remains constitutive. The hmg3 promoter, like the promoters of most house-keeping genes, lacks a TATA box. Our results suggest that hmg1 is likely to encode the enzyme involved in rubber biosynthesis while hmg3 is possibly involved in isoprenoid biosynthesis of a housekeeping nature.     

Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase 1 in Tobacco Results in Sterol Overproduction

A genomic fragment encoding one (HMGR1) of the three 3-hydroxy-3-methylglutaryl coenzyme A reductases (HMGRs) from Hevea brasiliensis (H.B.K.) Mull. Arg. (M.-L. Chye, C.-T. Tan, N.-H. Chua [1992] Plant Mol Biol 19: 473-484) was introduced into Nicotiana tabacum L. cv xanthi via Agrobacterium transformation to study the influence of the hmg1 gene product on plant isoprenoid biosynthesis. Transgenic plants were morphologically indistinguishable from control wild-type plants and displayed the same developmental pattern. Transgenic lines showed an increase in the level of total sterols up to 6-fold, probably because of an increased expression level of hmg1 mRNA and a corresponding increased enzymatic activity for HMGR, when compared with the level of total sterols from control lines not expressing the hmg1 transgene. In addition to the pathway end products, campesterol, sitosterol, and stigmasterol, some biosynthetic intermediates such as cycloartenol also accumulated in transgenic tissues. Most of the overproduced sterols were detected as steryl-esters and were likely to be stored in cytoplasmic lipid bodies. These data strongly support the conclusion that plant HMGR is a key limiting enzyme in phytosterol biosynthesis.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes

Quality traits such as flavour and texture are assuming a greater importance in crop breeding programmes. This study takes advantage of potato germplasm differentiated in tuber flavour and texture traits. A recently developed 44,000-element potato microarray was used to identify tuber gene expression profiles that correspond to differences in tuber flavour and texture as well as carotenoid content and dormancy characteristics. Gene expression was compared in two Solanum tuberosum group Phureja cultivars and two S. tuberosum group Tuberosum cultivars; 309 genes were significantly and consistently up-regulated in Phureja, whereas 555 genes were down-regulated. Approximately 46% of the genes in these lists can be identified from their annotation and amongst these are candidates that may underpin the Phureja/Tuberosum trait differences. For example, a clear difference in the cooked tuber volatile profile is the higher level of the sesquiterpene alpha-copaene in Phureja compared with Tuberosum. A sesquiterpene synthase gene was identified as being more highly expressed in Phureja tubers and its corresponding full-length cDNA was demonstrated to encode alpha-copaene synthase. Other potential 'flavour genes', identified from their differential expression profiles, include those encoding branched-chain amino acid aminotransferase and a ribonuclease suggesting a mechanism for 5'-ribonucleotide formation in potato tubers on cooking. Major differences in the expression levels of genes involved in cell wall biosynthesis (and potentially texture) were also identified, including genes encoding pectin acetylesterase, xyloglucan endotransglycosylase and pectin methylesterase. Other gene expression differences that may impact tuber carotenoid content and tuber life-cycle phenotypes are discussed.     

Involvement of de Novo Protein Synthesis,Protein Kinase,Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.)

A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested.     

A Genetic and Pharmacological Analysis of Isoprenoid Pathway by LC-MS/MS in Fission Yeast

Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level.     

Effects of postharvest storage and dormancy status on ABA content, metabolism, and expression of genes involved in ABA biosynthesis and metabolism in potato tuber tissues

At harvest, and for an indeterminate period thereafter, potato tubers will not sprout and are physiologically dormant. Abscisic acid (ABA) has been shown to play a critical role in tuber dormancy control but the mechanisms controlling ABA content during dormancy as well as the sites of ABA synthesis and catabolism are unknown. As a first step in defining the sites of synthesis and cognate processes regulating ABA turnover during storage and dormancy progression, gene sequences encoding the ABA biosynthetic enzymes zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED) and three catabolism-related genes were used to quantify changes in their relative mRNA abundances in three specific tuber tissues (meristems, their surrounding periderm and underlying cortex) by qRT-PCR. During storage, StZEP expression was relatively constant in meristems, exhibited a biphasic pattern in periderm with transient increases during early and mid-to-late-storage, and peaked during mid-storage in cortex. Expression of two members of the potato NCED gene family was found to correlate with changes in ABA content in meristems (StNCED2) and cortex (StNCED1). Conversely, expression patterns of three putative ABA-8′-hydroxylase (CYP707A) genes during storage varied in a tissue-specific manner with expression of two of these genes rising in meristems and periderm and declining in cortex during storage. These results suggest that ABA synthesis and metabolism occur in all tuber tissues examined and that tuber ABA content during dormancy is the result of a balance of synthesis and metabolism that increasingly favors catabolism as dormancy ends and may be controlled at the level of StNCED and StCYP707A gene activities Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Potato Glycoalkaloids: A Burden or a Blessing?

Steroidal glycoalkaloids (SGAs) are produced following the general steroid biosynthesis pathway, starting from acetyl-coenzyme A and followed by the intermediates mevalonic acid, squalene, cycloartenol, and cholesterol. α-Chaconine and α-solanine are the main SGAs of the cultivated potato (Solanum tuberosum), whereas many other SGAs are known in the wild potato species. Low concentrations of SGAs improve the taste of potato, but concentrations greater than 200 mg/kg can have toxic effects on animals and humans. SGAs have antimicrobial activity and confer resistance to some insects, but many such pests of potato are not greatly affected. Certain environmental conditions and wounding enhance SGA accumulation in tubers in the field and storage. Low production of SGAs is a dominant character inherited in a relatively simple manner and can be selected for in potato-breeding programs, whereas the use of wild potato germplasm tends to increase the SGA accumulation in the breeding lines. Further efforts are likely to be directed toward the reduction of the SGA content in the edible potato products through breeding and biotechnological methodologies, whereas potato genotypes with high SGA production may be developed for use in the pharmaceutical industry.     

Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step in the cytosolic isoprenoid biosynthesis pathway in higher plants. To understand the contribution of HMGR to plant development, we isolated T-DNA insertion mutants for HMG1 and HMG2. The hmg1 and hmg2 mutants were both more sensitive than the wild type (WT) to lovastatin, an inhibitor of HMGR. The hmg2 mutant showed no visible phenotype under normal growth conditions. In contrast, the hmg1 mutant exhibited dwarfing, early senescence, and sterility. Expression of senescence-associated genes 12 (SAG12), a marker gene for senescence, was induced in the hmg1 mutant at an earlier stage than in the WT. Levels of trans-cytokinins--hormones known to inhibit senescence--were not lower in hmg1. The mutant did not have the typical appearance of brassinosteroid (BR)-deficient mutants, except for a dwarf phenotype, because of the suppression of cell elongation. The expression of several genes involved in cell elongation was suppressed in hmg1. WT plants treated exogenously with inhibitors of sterol biosynthesis had similar gene expression and sterility characteristics as the hmg1 mutants. Pleiotropic phenotypes were rescued by feeding with squalene, the precursor of sterols and triterpenoids. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 plays a critical role in triterpene biosynthesis, and that sterols and/or triterpenoids contribute to cell elongation, senescence, and fertility.     

Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene: increase of the antisense effect during tuber growth

Transgenic plants of a tetraploid potato cultivar were obtained in which the amylose content of tuber starch was reduced via antisense RNA-mediated inhibition of the expression of the gene encoding granule-bound starch synthase (GBSS). GBSS is one of the key enzymes in the biosynthesis of starch and catalyses the formation of amylose. The antisense GBSS genes, based on the full-length GBSS cDNA driven by the 35S CaMV promoter or the potato GBSS promoter, were introduced into the potato genome by Agrobacterium tumefaciens-mediated transformation. Expression of each of these genes resulted in the complete inhibition of GBSS gene expression, and thus in the production of amylose-free tuber starch, in mature field-grown plants originating from rooted in vitro plantlets of 4 out of 66 transgenic clones. Clones in which the GBSS gene expression was incompletely inhibited showed an increase of the extent of inhibition during tuber growth. This is likely to be due to the increase of starch granule size during tuber growth and the specific distribution pattern of starch components in granules of clones with reduced GBSS activity. Expression of the antisense GBSS gene from the GBSS promoter resulted in a higher stability of inhibition in tubers of field-grown plants as compared to expression from the 35S CaMV promoter. Field analysis of the transgenic clones indicated that inhibition of GBSS gene expression could be achieved without significantly affecting the starch and sugar content of transgenic tubers, the expression level of other genes involved in starch and tuber metabolism and agronomic characteristics such as yield and dry matter content.     

Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose-6-phosphate

The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole-plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose-6-phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.     

Evidence against sink limitation by the sucrose-to-starch route in potato plants expressing fructosyltransferases

To investigate whether the route from sucrose to starch limits sink strength of potato tubers, we established an additional storage carbohydrate pool and analyzed allocation of imported assimilates to the different pools. Tuber specific expression of the fructan biosynthetic enzymes of globe artichoke resulted in accumulation of fructans to about 5% of the starch level, but did not increase tuber dry weight per plant. While partial repression of starch synthesis caused yield reduction in wild-type plants, it stimulated fructan accumulation, and yield losses were ameliorated in tubers expressing fructosyltransferases. However, a nearly complete block of the starch pathway by inhibition of sucrose synthase could not be compensated by the fructan pathway. Although fructan concentrations rose, yield reduction was even enhanced, probably because of a futile cycle of fructan synthesis and degradation by invertase, which is induced when sucrose synthase is knocked out. The data do not support a limitation of sink strength by enzyme activities of the starch pathway but point to an energy limitation of storage carbohydrate formation in potato tubers.     

Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.