Effect of sugars on the development of oxidative stress induced by hypothermia in potato plants expressing yeast invertase gene

The influence of sugars on the development of oxidative stress induced by hypothermia was investigated in the leaves of two genotypes of potato (Solanum tuberosum L.) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. We used wild-type plants of potato, cv. Désirée, and potato plants expressing a yeast invertase gene under the control of the B33 class I patatin promoter and carrying a sequence of proteinase inhibitor II leader peptide for the apoplastic enzyme localization. At temperature of 22°C optimal for growth, expression of the yeast invertase gene in the leaves of transformed plants brought about a modification in the carbohydrate metabolism manifested in the activation of acid forms of invertase and accumulation of intracellular sugars (predominantly of sucrose because of its resynthesis). The exposure of plants to light under prolonged hypothermia (5°C, 6 days) activated all the forms of invertase (predominantly of acid invertase) and induced accumulation of sugars. In the leaves of potato expressing the yeast invertase gene, these processes were more intense. Under chilling, superoxide dismutase activity and the rate of lipid peroxidation in the leaves of investigated potato genotypes depended on the level of accumulated intracellular sugars. It was concluded that sugars play an important role as stabilizers of cellular membranes and scavengers of reactive oxygen species decelerating the processes of free radical oxidation of biomolecules upon the development of oxidative stress induced by hypothermia.     

CO<Subscript>2</Subscript> exchange and structural organization of chloroplasts under hypothermia in potato plants transformed with a gene for yeast invertase

Growth, CO₂ exchange, and the ultrastructure of chloroplasts were investigated in the leaves of potato plants (Solanum tuberosum L., cv. Désirée) of wild type and transformed with a gene for yeast invertase under the control of patatin class I B33 promoter (for apoplastic enzyme) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. At a temperature of 22°C optimal for growth, the transformed plants differed from the plants of wild type in retarded growth and a lower rate of photosynthesis as calculated per plant. On a leaf dry weight basis, photosynthesis of transformed plants was higher than in control plants. Under hypothermia (5°C), dark respiration and especially photosynthesis of transformed plants turned out to be more intense than in control material. After a prolonged exposure to low temperature (6 days at 5°C), in the plants of both genotypes, the ultrastructure of chloroplasts changed. Absolute areas of sections of chloroplasts and starch grains rose, and the area of plastoglobules decreased; in transformed plants, these changes were more pronounced. By some ultrastructural characteristics: a reduction in the cold of relative total area of sections of starch grains and plastoglobules (in percents of the chloroplast section area) and in the number of granal thylakoids (per a chloroplast section area), transformed plants turned out to be more cold resistant than wild-type plants. The obtained results are discussed in connection with changes in source-sink relations in transformed potato plants. These changes modify the balance between photosynthesis and retarded efflux of assimilates, causing an increase in the intracellular level of sugars and a rise in the tolerance to chilling.     

Chilling Tolerance of Potato Plants Transformed with a Yeast-Derived Invertase Gene under the Control of the B33 Patatin Promoter

Tolerance to chilling was compared under in vitro conditions in potato plants (Solanum tuberosum L., cv. Désirée) transformed with a yeast-derived invertase gene under the control of the B33 class 1 tuber-specific promoter (the B33-inv plants) and potato plants transformed only with a reporter gene (the control plants). The expression of the inserted yeast invertase gene was proved by following the acid and alkaline invertase activities and sugar contents in the leaves under the optimum temperature (22°C). The total activities of acid and alkaline invertases in the B33-inv plants exceeded those in the control plants by the factors of 2–3 and 1.3, respectively. In the B33-inv plants, the activity of acid invertase twice exceeded that of the alkaline invertase, whereas the difference equaled 12% in the control plants. The contents of sucrose and glucose increased in the B33-inv plants by 21 and 13%, respectively, as compared to the control. Chilling at +3 and –1°C for 1, 3, and 6 h did not affect the rate of lipid peroxidation, as measured by the content of malonic dialdehyde (MDA) in the leaves of the genotypes under study. Only the longer exposures (24 h at +3 and –1°C and 7 days at +5°C) produced a significant decline in the MDA content in the B33-invplants, as compared to the control. Following short freezing (20 min at –9°C), the content of MDA increased by 50% in the leaves of the control plants, while in the B33-inv plants, cold-treated and control plants did not differ in the MDA content. The authors presume that the potato plants transformed with the yeast invertase gene acquire a higher tolerance to low temperatures as compared to the control plants, apparently due to the changes in sugar ratio produced by the foreign invertase.     

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

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。     

Characterization of the early events of potato tuber development

The early events of potato ( Solanum tuberosum L. cv. Superior) tuberization were examined by using a model system of axillary bud tuber development from petiole-leaf single-node cuttings. Both fresh weight and starch accumulation were monitored to establish a developmental framework for morphological changes. Fresh weight and starch content began to increase in axillary buds after 2 days. Visible changes in bud morphology could be detected 4 days after the start of incubation. Substantial increases in both total protein and total RNA were observed at the onset of tuber morphology. Immunoblot analysis showed that the major tuber protein, patatin, could be initially detected in day 4 buds and that a 22-kDa proteinase inhibitor could be initially detected at day 8. Northern blot analysis corroborated this pattern of accumulation at the RNA level for both protein types. Substantial accumulation of the two proteinase inhibitor mRNAs occurred later than patatin mRNA accumulation. The results of this study showed that there is considerable accumulation of both protein and mRNA occurring during the early stages of tuber development prior to the substantial accumulation of the major tuber storage proteins.     

Water deficit enhancement of proline and α-amino nitrogen accumulation in potato plants and its association with susceptibility to drought

Potato plants ( Solanum tuberosum L. cvs 'Up-to-Date', 'Desiree', 'Alpha', 'Spunta', 'Elvira' and 'Troubadour') were exposed to cycles of water stress and relief during growth. Severe water deficit induced increased proline content 6- to 7-fold in nonturgid leaves which just started to wilt, and 8- to 27-fold in fully wilted leaves of potatoes. However, proline content was not affected during the early stages of stress development over a range of osmotic potentials in the leaves. The rising proline content was related to turgor loss of leaves independent of changes in the osmotic potentials, which indicates that proline involvement in osmoregulation of potato leaves is unlikely.
Repeated cycles of water stress and relief resulted in increased proline and α-amino nitrogen content in the tuber tissue of some of the cultivars. The smallest increase in proline content was obtained in 'Alpha' tubers and the content of α-amino nitrogen remained unaffected by the water stress. Concomitantly, 'Alpha' was the most drought-tolerant cultivar, as determined by its capacity to accumulate dry matter in tubers under stress conditions. On the other hand, in tubers of cultivars which were more susceptible to drought, a marked increase in proline and α-amino nitrogen was observed in response to water stress. The possible association of these findings with tolerance of potatoes to repeated short periods of drought is discussed.     

马铃薯HMGR基因的克隆,序列分析及其表达特征

采用ＲＴ－ＰＣＲ技术,从马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏｓｕｍ Ｌ．）幼叶中克隆了一个约１．０ｋｂ的ｃＤＮＡ片段,序列分析结果表明,该ｃＤＮＡ与忆报道的马铃薯ＨＭＧＲ基因家族的三种类型基因具有较高核酸序列同源性,与ＨＭＧＲⅠ基因同源性为７７．０％,ＨＭＧＲⅡ基因为９３．２％,ＨＭＧＲⅢ基因为７７．１％。其中３’端非翻译区序列与ＨＭＧＲⅠ、ＨＭＧＲⅡ、ＨＭＧＲⅢ三种基因同源性分别为５０．２％,８     

不同生育期马铃薯根系构型及生理对干旱胁迫的响应

【目的】明确马铃薯各生育期根系对干旱胁迫的响应特征,初步解析根系的抗旱机制,为干旱胁迫下马铃薯的科学管理提供理论依据。【方法】以‘冀张薯12号’马铃薯为材料,采用室内盆栽对比试验,设置重度干旱[土壤相对含水量45%]和正常浇水[土壤相对含水量75%]处理,在4个生育期观测马铃薯生长、根系构型和根系生理指标。【结果】干旱胁迫组马铃薯的株高、茎粗、总根长、总表面积和根总体积在整个生育期都显著低于对照组;干旱胁迫组单株产量、单株结薯数和淀粉显著低于对照组,干旱胁迫组还原糖显著高于对照组;其根系活力、MDA含量、脯氨酸含量、可溶性糖含量也显著高于对照组,并随着干旱时间的延长呈上升趋势;干旱胁迫组马铃薯根系SOD、POD活性在后期也显著高于对照组,且SOD活性在前期响应较慢,POD活性则响应较快。【结论】马铃薯的生长、根系发育和产量受到干旱胁迫的显著抑制,其根系的抗氧化酶活性、渗透调节物质含量均能迅速做出响应,缓解干旱胁迫带来的伤害,表现出一定的抗旱性。     

Developmental changes in carbohydrate content and sucrose degrading enzymes in tuberising stolons of potato (Solanum tuberosum)

Tuberising stolon tips of potato ( Solanum tuberosum L. cv. Record) accumulate starch and sucrose but the hexose content, particularly fructose, declines rapidly. Similar changes occur in the region 2 cm behind the swelling apex but the decline in glucose is far more pronounced than in the developing tuber. Tuberisation is characterised by an apparent switch from an invertase-dominated sucrolytic system (both acid and alkaline invertases [EC 3.2.1.26] are present) to one dominated by sucrose synthase (EC 2.4.1.13). Sucrose synthase and fructokinase (EC 2.7.1.4) activities were, at a maximum, ca 10- and 5-fold higher, respectively in the swelling stolon tip compared with the non-tuberising region. At the highest starch contents attained, the starch level in the young developing tuber was approximately double that in the adjacent non-tuberising stolon region. Immunoblots revealed that developmental changes in sucrose synthase. fructokinase and alkaline invertase polypeptides corresponded with enzyme activities. Antibodies raised against the N-terminal amino acid sequence of a soluble invertase purified from mature tubers did not detect significant quantities of a polypeptide in stolons and young, developing tubers. Antibodies raised against an in vitro expression product of an apoplastic invertase cloned from a leaf cDNA library detected a polypeptide in developing tubers but not in mature ones. However, expression of the protein did not correlate well with acid invertase activity during early tuber formation.     

An elicitor-and pathogen-induced cdna from potato encodes a stress-responsive cyclophilin

Differential mRNA display was used to identify pathogen-responsive, stress-related genes in potato cell suspensions treated with salicylic acid and a cell wall-derived elicitor from Phytophthora infestans. Among the positive clones identified, one was found to be expressed at a significantly higher level in elicited cells than in control cells. DNA sequencing of this amplicon revealed high homology and identified it as a potato cyclophilin cDNA. The maximum amount of the cyclophilin mRNA was found 9 to 12 h after elicitation. Cyclophilin (CyP) mRNA synthesis was also up-regulated from 12 to 24 h in potato leaves locally infected with zoospores from Phytophthora infestans. However, untreated leaves responding systemically to the pathogen showed only a weak, delayed response at 24 h post infection. The observed accumulation of potato CyP mRNA in response to salicylic acid, P. infestans elicitor and P. infestans infection, suggest that CyPs play an important role in plant stress responses.     

Lipid fatty acid composition of potato plants transformed with the Δ12-desaturase gene from cyanobacterium

Potato (Solanum tuberosum L.) plants were transformed with the desA gene encoding Δ12 acyl-lipid desaturase in the cyanobacterium Synechocystis sp. PCC 6803. To evaluate the efficiency of this gene expression in the plant, its sequence was translationally fused with the sequence of the reporter gene encoding thermostable lichenase. A comparison of native and hybrid gene expression showed that lichenase retained its activity and thermostability within the hybrid protein, whereas desaturase retained its capability of inserting the double bond in fatty acid (FA) chains and, thus, to modify their composition in membrane lipids. In most transformed plants, shoots contained higher amounts of polyunsaturated FAs, linoleic and linolenic (by 39–73 and 12–41%, respectively). The total absolute content of unsaturated FAs was also higher in transformants by 20–42% as compared to wild-type plants. When transformed plants were severely cooled (to ?7°C), the rate of their membrane lipid peroxidation was not enhanced, whereas in wild-type plants, it increased substantially (by 25%) under such conditions. These results could indicate a higher tolerance of transformed plants to low temperatures and the oxidative stress induced by hypothermia.