Organ-specificity and inducibility of patatin class I promoter from potato in transgenic arabidopsis plants

Patatin class I promoter (B33 promoter) is a tissue-specific potato (Solanum tuberosum L.) promoter expressing the patatin gene mainly in tubers. However, it can be induced in other organs by sucrose or light. We compared the activity of this promoter fused with the reporter gene during heterological expression in B33::GUS transgenic arabidopsis (Arabidopsis thaliana L.) plants and homological expression of the same DNA construct in potato. Promoter activity was estimated from quantification of β-glucuronidase (GUS) activity. It was shown that, during heterological expression in arabidopsis seedlings, B33 promoter manifested a tissue-specificity and inducibility, although in a different manner than during homological expression in potato. In noninduced arabidopsis seedlings, B33 promoter was most active in the roots, whereas, after induction with sucrose treatment, it became most active in cotyledons. 10 mM sucrose was sufficient for a manifold activation of B33 promoter in intact seedlings. The degree of B33 promoter induction by sucrose in arabidopsis seedlings was strictly organ-specific and increased in the following sequence: root < hypocotyl < cotyledons. 150–200 mM sucrose enhanced B33 promoter activity in cotyledons by 200 to 300 times, i.e., much stronger than in potato organs. Glucose and fructose were less efficient than sucrose. Phytohormones affecting tuber formation in potato (gibberellins, auxins, and cytokinins) did not affect significantly B33 promoter activity in arabidopsis. A lag period of approximately 6 h preceded sucrose-induced B33 promoter activation. This indicates that the patatin promoter is not the primary target for the sucrose signal. The quantitative examination of heterological expression of patatin class I promoter further clarifies its basic functional characteristics and permits a better prognosis of its behavior after transferring into other plant species.     

Class I patatin基因5‘侧翼区驱动的GUS基因在马铃薯块茎中专一性表达

Binary vectors pPATIs (with partial signal sequence) and pPATI (without signal sequence) were constructed by fusing 1.4 kb 5' flanking regions of Class I patatin gene with GUS. Transient GUS expression was observed in in vitro tuber slices bombarded with pPATI. These constructs were then introduced into potato (cv. Desiree) via Agrobacterium tumefaciens transformation. Transgenic potato plants were confirmed by X-Gluc staining and PCR. Using in vitro tuberization system, GUS activities were assayed by fluorescence. It was shown that, in plants transformed with PATI-GUS, GUS specific activities were about 10-20 fold higher in tubers than in stems. Increased sucrose concentration could not induce PATI-GUS expression, but light enhanced PATI-GUS expression in cultured shoots.     

Sucrose-regulated expression of a chimeric potato tuber gene in leaves of transgenic tobacco plants

Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato tubers. Class-I patatin genes encode 98 to 99% of the patatin mRNA in tubers, but are not normally expressed in other tissues. They are not totally tuber-specific; however, since they can be induced to express at high levels in other tissues under conditions of sink limitation or in explants cultured on medium containing elevated levels of sucrose. To examine the evolution of the mechanisms that regulate patatin gene expression, we introduced a chimeric patatin--glucuronidase (GUS) gene containing 2.5 kb of 5 flanking sequence from the Class-I potato patatin gene PS20 into tobacco plants. The construct was not expressed at significant levels in leaves of juvenile plants or plantlets cultured in vitro, but was expressed at high levels in explants cultured on medium containing 0.3 to 0.4 M sucrose. While there were differences in the expression of the chimeric gene between transgenic tobacco and potato plants, the pattern of sucrose induction was very similar. These results suggest that the mechanism that controls patatin gene expression in potato tubers evolved from a widely distributed mechanism in which gene expression is regulated by the level of available photosynthate.     

Class I patatin基因5′侧翼区驱动的GUS基因在马铃薯块茎中专一性表达

将1.4kb Class I patatin基因的5′侧翼区与GUS基因融合,构建了双元表达载体pPATIs(含patatin部分信号顺序)和pPATI(不含patatin部分信号顺序)。pPATI通过基因枪介导在块茎切片中获得了瞬间表达。以上建构物通过农杆菌介导转入了马铃薯品种Desiree。X-Gluc染色(PATIs不能染色)及PCR结果证实已获得转基因植株。利用离体块茎诱导系统,GUS的表达进一步用荧光进行定量检测,结果显示,PATI-GUS的转基因植株中GUS比活性均以块茎明显高于茎段,达10-20倍。蔗糖浓度的升高,PATI-GUS植株中的GUS比活性无明显变化,与前人报道有不同。此外,光照促进PATI-GUS的表达。     

Use of an in vitro tuberization system to study tuber protein gene expression

Summary Nodal cuttings from micropropagated potato plantlets give rise to microtubers when placed on Murashige and Skoog medium containing 6% sucrose and 2.5 mg/liter kinetin and incubated in the dark at 19°C. Microtubers produced from the cultivar Superior were shown to contain the same characteristic group of proteins as field-grown tubers. As with field-grown tubers, the 40 000-dalton major tuber glycoprotein, patatin, accumulated to high levels in microtubers, reaching 3.7±0.2 mg/g fresh weight after 90 d. Also in agreement with field-grown plants, stems and leaves of micropropagated plantlets did not contain detectable levels of patatin, but small amounts of an electrophoretically distinct form accumulated transiently in roots. Patatin mRNA is readily detectable in developing microtubers 15 d after transfer of the cuttings to inductive medium. Patatin mRNA was also present in roots, but as with field-grown plants, was 50- to 100-fold less abundant and could be distinguished from that in tubers by primer extension. Microtuber development and patatin accumulation were inhibited by gibberellic acid. This work was supported by grants 83-CRCR-1-1348 and 85-CRCR-I-1792 from the U.S. Department of Agriculture Competitive Grants program and with funds from the Texas Agricultural Experiment Station.     

Storekeeper defines a new class of plant-specific DNA-binding proteins and is a putative regulator of patatin expression

The expression of class I patatin genes is restricted to potato tubers but can be induced in other tissues by exogenous sucrose. Here we show that tuber-specific and sucrose-inducible gene expression is reduced in transgenic potato plants by mutations in a conserved 10 base pair motif within the B-box of the patatin promoter. In a southwestern screen, we have isolated a novel DNA-binding protein designated Storekeeper (STK) that specifically recognises the B-box motif in vitro. Gel shift experiments with an STK-specific antibody suggest that STK is the B-box binding protein found in tuber nuclei. We propose that STK, the defining member of a new class of DNA binding proteins, regulates patatin expression in potato tubers via the B-box motif.     

Both developmental and metabolic signals activate the promoter of a class I patatin gene

Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5′-upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber-specific gene activity which was on average 100- to 1000-fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the β-glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.     

Immunocytochemical localization of patatin,the major glycoprotein in potato (Solanum tuberosum L.) tubers

Patatin is a family of glycoproteins with an apparent molecular weight of 40 kDa. The protein is synthesized as a pre-protein with a hydrophobic signal sequence of 23 amino acids. Using different immunocytochemical methods we determined the tissue-specific as well as subcellular localization of the patatin protein. Since antibodies raised against patatin showed crossreactivity with glycans of other glycoproteins, antibodies specific for the protein portion of the glycoprotein were purified. Using these antibodies for electron-microscopical immunocytochemistry, the protein was found to be localized mainly in the vacuoles of both tubers and leaves of potatoes (Solanum tuberosum L.) induced for patatin expression. Neither cell walls nor the intercellular space contained detectable levels of patatin protein. Concerning the tissue specificity, patatin was mainly found in parenchyma cells of potato tubers. The same distribution was observed for the esterase activity in potato tubers.Abbreviations PHA phytohemagglutinin - TFMS trifluoromethanesulfonic acid     

Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings

Summary The objective of this study was to separate and determine effects on the field performance of transgenic potatoes that originate from the tissue culture process of transformation and from the genes inserted. The constructs introduced contained the reporter gene for betaglucuronidase (GUS) under the control of the patatin promoter (four different constructs) and the neomycin phosphotransferase gene under the control of the nopaline synthase promoter. Both genes might be expected to have a neutral effect on plant phenotype. The field performance of transgenic plants (70 independent transformants) was compared with non-transgenic plants regenerated from tuber discs by adventitious shoot formation and from shoot cultures established from tuber nodal cuttings. Plants from all three treatments were grown in a field trial from previously field-grown tubers, and plant performance was measured in terms of plant height at flowering, weight of tubers, number of tubers, weight of large tubers and number of large tubers. There was evidence of somaclonal variation among the transgenic plants; mean values for all characters were significantly lower and variances generally higher than from plants derived from nodal shoot cultures. A similar change in means and variances was observed for the non-transgenic tuber-disc regenerants when compared with shoot culture plants. Plant height, tuber weight and tuber number were, however, significantly lower in transgenic plants than in tuber-disc regenerants, suggesting an effect on plant performance either of the tissue culture process used for transformation or of the genes inserted. There were significant differences between constructs for all five plant characters. The construct with the smallest segment of patatin promoter and the lowest level of tuber specificity for GUS expression had the lowest values for all five characters. It is proposed that the nature of GUS expression is influencing plant performance. There was no indication that the NPTII gene, used widely in plant transformation, has any substantial effect on plant performance in the field.     

Factors affecting gene expression of patatin and proteinase-inhibitor-II gene families in detached potato leaves:

In whole intact potato (Solanum tuberosum L.) plants, the gene families of class-I patatin and proteinase inhibitor II (Pin 2) are constitutively expressed in the tubers. However, they are also induced in detached potato leaves in the presence of light. To further characterize this light action, the detached leaves were subjected to monochromatic light of different wavelengths and to darkness in the presence of metabolites and inhibitors. Patatin genes could be induced by the simultaneous application of sucrose (sugars) and glutamine in darkness. Neither of these metabolites was active when supplied alone. When photosynthesis was blocked by 3-(3,4-Di-chlorophenyl)-1, 1-dimethylurea (DCMU) in the light, patatin genes were not expressed; however, the inhibition was overcome in the presence of sucrose. This indicates that besides its role in photosynthetic carbohydrate production, light may be essential for the supply of amino acids (or reduced nitrogen). Unlike patatin, Pin 2 genes were, to a small extent, also active in darkness, and sucrose weakly enhanced this expression. However, DCMU did not affect Pin 2 expression in the light. Both abscisic acid and methyl jasmonate strongly promoted the accumulation of Pin 2 mRNA independent of the light conditions, indicating that the gene family is probably under hormonal control. The phytohormones did not affect patatin gene expression. Inhibitors of cytosolic (cycloheximide) and organellar (chloramphenicol) translation had opposite effects on the two gene families. Careful evaluation of the inhibitors' action indicates that protein synthesis (cytosol) is required for the expression of Pin 2 genes but not for the patatin genes. These results clearly demonstrate that, although in situ both gene families are constitutively expressed in the same plant organ (tuber) in intact plants, their expression is mediated by different factors.Abbreviations ABA cis-abscisic acid - DCMU 3-(3,4-dichlorphenyl)-1,1-dimethylurea - GUS -glucuronidase activity - MeJA methyl jasmonate - Pin 2 proteinase inhibitor II We thank Beate Küsgen and Regina Breitfeld for the greenhouse work. This work was supported by a grant from the Bundesministerium für Forschung und Technologie.     

Expression of a Patatin-like Protein in the Anthers of Potato and Sweet Pepper Flowers

Patatin, the major glycoprotein in potato tubers, is encoded by a multigene family. RNA and protein analyses reveal that a homologous mRNA and an immunologically cross-reacting protein can be found in potato flowers, which is similar to patatin in that it displays a lipid acyl hydrolase activity. The patatin-like protein found in flowers has a higher molecular weight than the authentic tuber patatin. Deglycosylation experiments show that this is not due to differences in the glycosylation pattern. Immunocytochemical analysis shows the patatin-like protein to be present only in the epidermal cell layer of the anther, the exothecium, and in petals of potato flowers. Furthermore, the fact that a patatin-like protein can be detected in a similar tissue in sweet pepper, another solanaceous plant, could give a clue concerning the evolutionary origin of patatin.     

Evaluation of potato tuber moth (Lepidoptera: Gelechiidae) resistance in tubers of Bt-cry5 transgenic potato lines

The potato tuber moth, Phthorimaea operculella (Zeller), in tropical and subtropical countries, is the most destructive pest of potato, Solanum tuberosum L. The larvae attack foliage and tubers in the field and in storage. The purpose of this study was to evaluate the efficacy of a Bt-cry5 transgene to control the potato tuber moth in tuber tissues. Tuber bioassays using stored (11-12 mo old) and newly harvested tubers of Bt-cry5-Lemhi Russet and Bt-cry5-Atlantic potato lines showed up to 100% mortality of 1st instars. Mortality was lowest in the newly harvested tubers of Bt-cry5-Atlantic lines (47.1-67.6%). Potato tuber moth mortality was 100% in the Bt-cry5-Spunta lines that were transformed with Bt-cry5 gene controlled by the CaMV 35S promoter (pBIML5 vector) and in 2 of 3 lines transformed with Bt-cry5 gene controlled by the Gelvin super promoter (pBIML1 vector). The transgenic Spunta lines expressing Bt-cry5 controlled by the patatin promoter (pBMIL2 vector) showed the lowest tuber moth mortality (25.6 and 31.1%). The Bt-cry5 transgenic lines with high tuber expression of B. thuringiensis have value in an integrated pest management system to control potato tuber moth.     

A transgenic study on affecting potato tuber yield by expressing the rice sucrose transporter genes OsSUT5Z and OsSUT2M

In many plants, sucrose transporters are essential for both sucrose exports from sources and imports into sinks, indicating a function in assimilate partitioning. To investigate whether sucrose transporters can improve the yield of starch plant, potato plants (Solanum tuberosum L. cv. Désirée) were transformed with cDNAs of the rice sucrose transporter genes OsSUT5Z and OsSUT2M under the control of a tuber-specific, class-I patatin promoter. Compared to the controls, the average fructose content of OsSUT5Z transgenic tubers significantly increased. However, the content of the sugars and starch in the OsSUT2M transgenic potato tubers showed no obvious difference. Correspondingly, the average tuber yield, average number of tubers per plant and average weight of single tuber showed no significant difference in OsSUT2M transgenic tubers with controls. In the OsSUT5Z transgenic lines, the average tuber yield per plant was 1.9-fold higher than the controls, and the average number of tubers per plant increased by more than 10 tubers on average, whereas the average weight of a single tuber did not increase significantly. These results suggested that the average number of tubers per plant showed more contribution than the average weight of a single tuber to the tuber yield per plant.