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.     

The Decreased Cold-Resistance of Chilling-Sensitive Plants Is Related to Suppressed CO2 Assimilation in Leaves and Sugar Accumulation in Roots

Tomato (Lycopersicon esculentum L., cv. Sibirskii skorospelyi) and cucumber (Cucumis sativus L., cv. Konkurent) plants were grown in a soil culture in a greenhouse at an average daily temperature of 20°C and ambient illumination until the development of five and eight true leaves, respectively. During the subsequent three days, some plants were kept in a climatic chamber at 6°C in the light, whereas other plants remained in a greenhouse (control). The cold-resistance of cucumber leaves and roots, as assayed from the electrolyte leakage, was reduced after cold exposure stronger than cold-resistance of tomato organs. The ratio photosynthesis/dark respiration was lower in cucumber than in tomato leaves at all measurement temperatures. The concentrations of sugars (sucrose + glucose + fructose) increased in chilled tomato roots but decreased in cucumber roots. Cold exposure changed the activities of various invertase forms (soluble and insoluble acidic and alkaline invertases). The total invertase activity and the ratio of mono- to disaccharides increased. The lower cucumber cold-resistance is related to the higher sensitivity of its photosynthetic apparatus to chilling and, as a consequence, insufficient root supply with sugars.     

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.     

Involvement of cell-wall invertase in low-temperature hardening of tobacco plants

Specific features of low-temperature hardening (6 days at 8°C) of cold-sensitive tobacco plants (Nicotiana tabacum, cv. Samsun) related to changes in the cell-wall invertase activity were studied. During cold hardening, oppositely directed changes in this enzyme activity occurred in tobacco leaves and roots. In the leaves, cell-wall invertase was activated (approximately by 30%), the content of sugars increased (approximately by 25%), and the content of sucrose, the main transport form of sugars, in the apoplast reduced by three times; all these changes indicate that assimilate outflow from leaves to roots was inhibited. In contrast, in the root system, enzyme activity was decreased almost twice and the content of sugars in them was essentially unchanged. It is suggested that a strategy of low-temperature adaptation of cold-sensitive tobacco plants aimed at creating the high cold tolerance of aboveground parts, even at the expense of the root system, which, under conditions of native vegetation, is not practically exposed to damaging low temperatures.     

Apoplastic sugars and cell-wall invertase are involved in formation of the tolerance of cold-resistant potato plants to hypothermia

We studied the involvement of apoplastic sugars (glucose, fructose, and sucrose) and the cell-wall invertase (CWI) in the formation of the tolerance of cold-resistant potato plants (Solanum tuberosum L., cv Désirée) to hypothermia. The activity of CW1 and the content in the cell and the apoplast substrate (sucrose) and the reaction products of this enzyme (glucose and fructose) have a significant influence on the formation of the tolerance of cold-resistant potato plants to hypothermia.     

Impact of soluble sugar concentrations on the acquisition of freezing tolerance in accessions of Arabidopsis thaliana with contrasting cold adaptation – evidence for a role of raffinose in cold acclimation

In the present study the cold acclimation potential of two accessions of Arabidopsis thaliana was investigated. Significant variation was found for basic tolerance as well as the capacity to acclimate to freezing temperatures. During cold acclimation, levels of soluble sugars increased in both genotypes, but raffinose accumulation discriminated the more tolerant accession Col‐0 from C24. Concentrations of other compatible solutes such as proline and glutamine were also higher in cold‐acclimated Col‐0 than C24 plants. Changes of invertase activity during cold exposure corresponded to changes in sucrose and fructose, but not glucose concentrations and were consistent with an initial chilling response and a later decline in hexose metabolization. When vacuolar invertase was suppressed by siRNA expression, reduced sucrolytic activity resulted in elevated leaf sucrose concentration, whereas the fructose content was strongly reduced. This led to elevated freezing tolerance in the cold‐tolerant genotype Col‐0, but not in C24. The most pronounced metabolic changes in invertase‐inhibited Col‐0 plants occurred for proline and glutamine concentrations, indicating indirect metabolic effects of altered sugar concentrations.     

Sucrose metabolism in spring and winter wheat in response to high irradiance, cold stress and cold acclimation

Effects of low‐temperature stress, cold acclimation and growth at high irradiance in a spring (Triticum aestivum L. cv. Katepwa) and a winter wheat (Triticum aestivum L. cv. Monopol) were examined in leaves and crowns with respect to the sucrose utilisation and carbon allocation. Light‐saturated and carbon dioxide (CO₂)‐saturated rates of CO₂ assimilation were decreased by 50% in cold‐stressed spring and winter wheat cultivars. Cold‐ or high light‐acclimated Katepwa spring wheat maintained light‐saturated rates of CO₂ assimilation comparable to those of control spring wheat. In contrast, cold‐ or high light‐acclimated winter wheat maintained higher light and CO₂‐saturated rates of CO₂ assimilation than non‐acclimated controls. In leaves, during either cold stress, cold acclimation or acclimation to high irradiance, the sucrose/starch ratio increased by 5‐ to 10‐fold and neutral invertase activity increased by 2‐ to 2.5‐fold in both the spring and the winter wheat. In contrast, Monopol winter wheat, but not Katepwa spring wheat, exhibited a 3‐fold increase in leaf sucrose phosphate synthase (SPS) activity, a 4‐fold increase in sucrose:sucrose fructosyl transferase activity and a 6.6‐fold increase in acid invertase upon cold acclimation. Although leaves of cold‐stressed and high light‐grown spring and winter wheat showed 2.3‐ to 7‐fold higher sucrose levels than controls, these plants exhibited a limited capacity to adjust either sucrose phosphate synthase or sucrose synthase activity (SS[s]). In addition, the acclimation to high light resulted in a 23–31% lower starch abundance and no changes at the level of fructan accumulation in leaves of either winter or spring wheat when compared with controls. However, high light‐acclimated winter wheat exhibited a 1.8‐fold higher neutral invertase activity and high light‐acclimated spring wheat exhibited an induction of SS(d) activity when compared with controls. Crowns of Monopol showed higher fructan accumulation than Katepwa upon cold and high light acclimation. We suggest that the differential adjustment of CO₂‐saturated rates of CO₂ assimilation upon cold acclimation in Monopol winter wheat, as compared with Katepwa spring wheat, is associated with the increased capacity of Monopol for sucrose utilisation through the biosynthesis of fructans in the leaves and subsequent export to the crowns. In contrast, the differential adjustment of CO₂‐saturated rates of CO₂ assimilation upon high light acclimation of Monopol appears to be associated with both increased fructan and starch accumulation in the crowns.     

Sucrose metabolism of perennial ryegrass in relation to cold acclimation

Sugar metabolism is one of the important factors involved in winter hardiness and since the discovery of sucrose biosynthesis, considerable advances have been made in understanding its regulation and crucial role. This investigation examined the changes in activities of sucrose metabolizing enzymes and sugar content during cold hardening of perennial ryegrass (Lolium perenne L.). Changes in acid invertase (AI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) along with all the three soluble sugars glucose, fructose and sucrose were measured in leaves and stem base tissue during cold acclimation. Although fructans were the predominant carbohydrate the changes in glucose, fructose and sucrose were significant. All the three soluble sugars in both leaf and stem tissues started to decrease from the first day and continued up to day 7 and thereafter started to increase until day 28. AI in the soluble fraction showed a higher activity than that in the cell wall bound fraction. In both the leaf and stem bases soluble AI activity increased during the first week and after that it started to decrease gradually. On the other hand both the SS and SPS increased gradually throughout the acclimation period. Sucrose content was negatively correlated with AI and positively correlated with SS and SPS accounting well for the relation between the substrate and enzyme activity. These results suggest that AI, SS and SPS in ryegrass are regulated by cold acclimation and play an important role in sugar accumulation and acquisition of freezing tolerance.     

Activities of Sucrose Synthase and Acid Invertase in Pea Seedling Organs

The organ topography of sucrose synthase and soluble acid invertase in pea seedlings at heterotrophic stage (3–14 days) was studied. Sucrose synthase was most active in the roots, with the highest activity on the 6–8th days. In the leaves, its activity decreased from day 3 to day 14. In the stems, sucrose synthase activity was at an invariantly low level. The patterns of sucrose synthase activity in etiolated and green plants were similar. As distinct from sucrose synthase, invertase activity was the highest in the stem, especially in etiolated plants. The peak of its activity was observed on the 6-8th days. In the leaves, invertase activity was lower but its pattern was the same. In the roots, acid invertase activity decreased from the 3rd day and did not depend on illumination. The conclusion is that differences in sucrose synthase and acid invertase activities in roots, leaves, and stem are determined by differences in the import of hydrolytic products of stored compound from the cotyledons as well as by different demands of these organs for these products for the processes of organ expansion and for the maintenance of organ metabolism.     

Cold hardening of raspberry plants in vitro is enhanced by increasing sucrose in the culture medium

The influence of exogenously applied sucrose on cold hardening of raspberry ( Rubus idaeus L.) in vitro was examined. Raspberry plants (cv. Preussen) were cultured on Murashige-Skoog (MS) media with different levels (1, 3, 5 and 7%) of sucrose and subjected to low-temperature acclimation (3/−3°C day/night temperature, 8-h photoperiod) for 14 days. Cold hardiness (LT₅₀ in controlled freezing), shoot moisture content, osmolality and the amounts of sucrose, glucose and fructose were determined. Exogenously applied sucrose was taken up by plants, but the uptake corresponded to less than 10% of total sugar reserves in the culture. Cold hardiness was primarily affected by acclimation treatment, but sucrose increased cold hardiness of nonacclimated plants and significantly enhanced the effect of acclimation treatment, 5% sucrose in the culture medium being optimal for cold hardening. LT₅₀ values ranged between −4.1 and −7.1°C for nonacclimated, and between −14.2 and −20.7°C for cold-acclimated shoots. Shoot moisture content was inversely related to medium sucrose level and declined only slightly during cold acclimation. After cold acclimation, plant osmolality predicted hardiness better than shoot moisture content. Plant osmolality and sugar content were increased by increasing the medium sucrose level and, to a greater extent, by cold acclimation. Sucrose, glucose and fructose accumulated during hardening. Sucrose was the predominant sugar, and the rate of sucrose accumulation during cold acclimation was independent of the medium sucrose level or the initial plant sucrose content. A close correlation between cold hardiness and total sugars, sucrose, glucose and fructose was established. These results suggest that sugars have more than a purely osmotic effect in protecting acclimated raspberry plants from cold.     

蔗糖脂肪酸脂对离体和活体大豆蔗糖酶活力和构象的影响

研究了蔗糖脂肪酸脂(SFE)对离体和活体大豆蔗糖酶活力和构象的影响。当SFE的浓度大于1.0mmol/L后,开始激活离体蔗糖酶的活力。在大豆的开花期和结荚期,SFE可以增加蔗糖酶的活力。荧光实验结果表明,在小于2mmol/L时,SFE不改变蔗糖酶的荧光最大发射峰峰位和峰高。施用SFE后,大豆叶片中果糖、葡萄糖和蔗糖酶的含量,分别是对照的170%±10%,190%±10%,和260%±20%;而蔗糖的含量几乎不变。对蔗糖酶 的SDS-凝胶电泳图进行扫描分析的结果表明,经SFE处理后的蔗糖酶含量比对照高两倍左右。这些结果说明SFE可以显著增加活体蔗糖酶的活力,但活力的增加既不是因为蔗糖酶构象的改变,也不是蔗糖(作为底物)诱导所致,而是SFE增加了大豆叶片中蔗糖酶的含量引起的。     

Soluble acid invertase activity in leaves is independent of species differences in leaf carbohydrates, diurnal sugar profiles and paths of phloem loading

Leaf sucrose, starch, hexose and maximum extractable soluble acid invertase activity were compared throughout the day in source leaves of 13 plant species chosen for their putative phloem-loading type (apoplastic or symplastic). Four species which represent the different phloem-loading types (tomato, barley, maize and Fuchsia ) were studied in detail. Using this information we wished to determine whether a positive correlation between foliar carbohydrates and acid invertase activity exists in leaves from different species and, furthermore, whether this relationship is determined by phloem-loading type. Acid invertase activity was relatively constant throughout the day in all species. The extent of sucrose, hexose and starch accumulation and the sucrose: starch ratio measured at a given time were species-dependent. No correlations were found between foliar soluble acid invertase activity and the hexose, sucrose or starch content of the leaves in any of the species, regardless of phloem-loading type. The species examined could be divided into three distinct groups: (1) high sucrose, low invertase; (2) low sucrose, low invertase; and (3) low sucrose, high invertase. The absence of an inverse relationship between leaf sucrose, hexose or starch contents and endogenous soluble acid invertase suggests that this enzyme is not directly involved in carbon partitioning in leaves but serves an auxiliary function.     

Sulla carnosa modulates root invertase activity in response to the inhibition of long‐distance sucrose transport under magnesium deficiency

• Being the principal product of photosynthesis, sucrose is involved in many metabolic processes in plants. As magnesium (Mg) is phloem mobile, an inverse relationship between Mg shortage and sugar accumulation in leaves is often observed.
• Mg deficiency effects on carbohydrate contents and invertase activities were determined in Sulla carnosa Desf. Plants were grown hydroponically at different Mg concentrations (0.00, 0.01, 0.05 and 1.50 mM Mg) for one month.
• Mineral analysis showed that Mg contents were drastically diminished in shoots and roots mainly at 0.01 and 0.00 mM Mg. This decline was adversely associated with a significant increase of sucrose, fructose and mainly glucose in shoots of plants exposed to severe deficiency. By contrast, sugar contents were severely reduced in roots of these plants indicating an alteration of carbohydrate partitioning between shoots and roots of Mg‐deficient plants. Cell wall invertase activity was highly enhanced in roots of Mg‐deficient plants, while the vacuolar invertase activity was reduced at 0.00 mM Mg. This decrease of vacuolar invertase activity may indicate the sensibility of roots to Mg starvation resulting from sucrose transport inhibition. ¹⁴CO₂ labeling experiments were in accordance with these findings showing an inhibition of sucrose transport from source leaves to sink tissues (roots) under Mg depletion.
• The obtained results confirm previous findings about Mg involvement in photosynthate loading into phloem and add new insights into mechanisms evolved by S. carnosa to cope with Mg shortage in particular the increase of the activity of cell wall invertase.

     

Acid invertase (sucrose hydrolysis) is not required for sucrose mobilization from the vacuole

The possible involvement of acid invertase (sucrose hydrolysis) as a prerequisite for sucrose mobilization from the vacuole of storage cells was investigated. Sugarcane ( Saccharum officinarum ) stalks, carrot ( Daucus carota ) roots and red beet ( Beta vulgaris ) hypocotyls were planted under greenhouse conditions and allowed to resume growth. The plants, however, were not permitted to become photosynthetically autotrophic by removing the new expanded leaves. Sucrose levels declined significantly in all three tissues without the development of acid invertase (EC 3.2.1.26) during the 21‐day experimental period. Acid invertase and thus sucrose hydrolysis within the vacuole was, therefore, not required for sucrose mobilization.     

Post‐translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold‐induced sweetening of potato tubers

Cold‐induced sweetening (CIS) is a serious post‐harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark‐coloured and bitter‐tasting product and generating the probable carcinogen acrylamide as a by‐product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS‐susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS‐resistant line increased susceptibility to CIS. The results show that post‐translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS.     

Biochemical basis for effects of k-deficiency on assimilate export rate and accumulation of soluble sugars in soybean leaves

The effects of K-deficiency on carbon exchange rates (CER), photosynthate partitioning, export rate, and activities of key enzymes involved in sucrose metabolism were studied in soybean (Glycine max [L.] Merr.) leaves. The different parameters were monitored in mature leaves that had expanded prior to, or during, imposition of a complete K-deficiency (plants received K-free nutrition solution). In general, recently expanded leaves had the highest concentration of K, and imposition of K-stress at any stage of leaf expansion resulted in decreased K concentrations relative to control plants (10 millimolar K). A reduction in CER, relative to control plants, was only observed in leaves that expanded during the K-stress. Stomatal conductance also declined, but this was not the primary cause of the decrease in carbon fixation because internal CO₂ concentration was unaffected by K-stress. Assimilate export rate from K-deficient leaves was reduced but relative export, calculated as a percentage of CER, was similar to control leaves. Over all the data, export rate was correlated positively with both CER and activity of sucrose phosphate synthase in leaf extracts. K-deficient leaves had higher concentrations of sucrose and hexose sugars. Accumulation of hexose sugars was associated with increased activities of acid invertase. Neutral invertase activity was low and unaffected by K-nutrition. It is concluded that decreased rates of assimilate export are associated with decreased activities of sucrose phosphate synthase, a key enzyme involved in sucrose formation, and that accumulation of hexose sugars may occur because of increased hydrolysis of sucrose in K-deficient leaves.