Involvement of apoplastic invertase in the formation of resistance of cold-tolerant plants to hypothermia was established for Solanum tuberosum L. cv. Désirée and a transformant of the same cultivar expressing the yeast suc2 gene encoding apoplastic invertase. The dependence of the formation of increased constitutive and stress-induced cold-tolerance on the activity of apoplastic invertase and, consequently, on the level of intracellular sugars in the roots and leaves was demonstrated.

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.

Some properties and activity of extracellular invertase in the Saccharomyces cerevisiae yeasts encoded by the suc2 gene in heterologous expression were described. It was shown that the target suc2 gene is actively expressed in the genome of the transformed potato plants and S. cerevisiae invertase synthesized by this gene is transported into the apoplast due to the signal peptide of the proteinase II inhibitor. This enzyme is present in the apoplast in a soluble form and absorbed into the cell wall.

Охарактеризованы некоторые свойства и активность внеклеточной инвертазы дрожжей Saccharomyces cerevisiae, кодируемой геном suc2 в условиях гетерологичной экспрессии. Показано, что целевой ген suc2 активно экспрессируется в геноме трансформированных растений картофеля, а синтезируемый им белок инвертазы S. cerevisiae благодаря наличию сигнального пептида ингибитора протеиназы II транспортируется в апопласт и присутствует в этом компартменте в растворимой форме, слабо адсорбируясь на клеточной стенке.

When following low-temperature acclimation (5 days at 2°C) of cold-resistant plants of Arabidopsis (Arabidopsis thaliana Heynh. (L.), ecotype Columbia) in relation to the changes in chloroplast ultrastructure, we registered the high efficiency of hardening and the ability of hardened plants to lower a threshold of frost damage by about 3°C. During hardening, the area of grana in the chloroplasts more than doubled, with considerably increased numbers of thylakoids per granum and thylakoids per chloroplast. The rate of apparent photosynthesis decreased to lesser extent than the rate of dark respiration, as a result the content of soluble sugars increased fourfold, ensuring an adaptive reorganization of metabolism, which enabled the hardened plants to survive even at below-zero temperatures (up to −7°C). The authors conclude that a considerable increase in the number of thylakoids in the chloroplasts helps maintain photosynthesis at low above-zero temperatures and is a prerequisite for the accumulation of soluble sugars in Arabidopsis leaves.

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.

Our study was carried out with potato plants (Solanum tuberosun L.,cv. Désirée) transformed with the yeast invertase gene under the control of the B33 class I patatin promoter and with the proteinase inhibitor II leader peptide sequence providing for the apoplastic enzyme localization (B33-inv plants) and with the plants transformed with the reporter gene encoding bb-glucuronidase under the control of the 35S CaMV promoter (control plants). Exposure to 5°C during 6 days caused an increase in invertase activity and sugar content in B33-inv leaves in comparison with the control plants. Cell membranes of B33-inv plant cells showed greater cold tolerance under low temperature conditions than control plants that was recorded by electrolyte release. We supposed that higher cold tolerance of B33-inv plants was caused by stabilizing effect of sugar on the membranes, because B33-inv plants differ from the control plants in higher invertase activity, induced by expression of yeast invertase gene, and high content of sugars.

The source-sink relations (SSR) in a plant under cold exposure to frost was studied in the plants of winter wheat (Triticum aestivum L.) Mironovskaya 808 cultivar and its allocytoplasmic hybrid containing alien cytoplasm of goat grass (Aegilops ovata L.) and grown in a water culture until phas of two leaves. The alteration of SSR was processed by treatment of plant with solutions of the photosynthesis inhibitors diuron and paraquat, keeping plants in the dark, changing from mixotrophic to autotrophic nutrient, and also through the exchange of nutrient solution to distillate water.

На полученных в культуре in vitro растениях картофеля (Solanum tuberosum L., сорт Дезире) исследовали динамику изменения в листьях активности разных форм инвертаз (кислой растворимой, щелочной, кислой нерастворимой) и содержания сахаров (глюкозы, фруктозы, сахарозы) при адаптации растений к длительной (6 суток) гипотермии (5°C). Использовали растения дикого типа и трансформанты с измененным углеводным метаболизмом, вызванным введением гена дрожжевой инвертазы (апопластный вариант локализации фермента). Выявлено, что при адаптации к гипотермии в листьях обоих генотипов повышалась активность кислых форм инвертаз и увеличивалось содержание сахарозы и глюкозы. Максимальная активность кислых инвертаз к третьим суткам холодовой адаптации соответствовала наибольшему содержанию сахаров, при этом трансформанты по этим показателям превосходили контрольные растения. Трансформанты проявили более высокую холодостойкость, по сравнению с растениями дикого типа, о чем свидетельствовало отсутствие нарушений в ионной проницаемости их мембран. Сделано заключение, что благодаря накоплению в период адаптации к гипотермии низкомолекулярных углеводов за счет повышения активности кислых форм инвертаз холодостойкие растения лучше приспосабливаются к перепаду температур.

Effect of suppression of the source activity on some physiological characteristics of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) was studied on plants grown in water culture. The plants were examined at the mixotrophic stage of growth period, during their transition from vegetative state to relative dormancy in autumn. The average temperature over 10 days of the experiment was 6°C at 9-h photoperiod and illuminance of 8–20 klx. The source strength was suppressed successively with a series of treatments: intact control plants (V1); plants with the seed endosperm removed (V2); plants with photosynthesis inhibited (V3); plants with the seed endosperm removed and photosynthesis inhibited (V4); plants with the seed endosperm removed, photosynthesis inhibited, and the root nutrient medium replaced with distilled water (V5). On the 6th–10th day of the experiment, the relative growth rate (RGR) was determined from dry weight increments. At the same time, the distribution of biomass among organs, the CO2 exchange rates (photosynthesis and dark respiration), the content and proportions of sugars (sucrose, glucose, and fructose), the total content of phenolic compounds and flavonoids, the index of membrane stability (IMS) in leaves, and frost hardiness of plants were measured. Frost hardiness of vegetating plants was shown to be inversely related to RGR (R = −0.906), dark respiration rate (R = −0.789), the percentage of sucrose in total sugar content (R = −0.737), leaf IMS (R = −0.390), and the rate of apparent photosynthesis (R = −0.288); it was directly proportional to the content of flavonoids (R =0.973), total phenols (R = 0.743), and sugars (R = 0.385). The role of modified source-sink relations in frost hardiness of vegetating plants at the stage of their transition to cold hardening is discussed. The differences between plants undergoing this transition and cold-hardened plants are considered, as well as the importance of phenolic compounds for the development of frost hardiness.

The pattern of changes in the activity of various forms of invertase (acid soluble, alkaline, and acid insoluble) and the content of sugars (glucose, fructose, and sucrose) in the course of plant adaptation to prolonged (6 days) hypothermia (5°C) was investigated in the leaves of potato plants ( Solanum tuberosum L., cv. Desiree) produced in vitro. We used the wild-type plants as a control and transformed plants with carbohydrate metabolism modified by inserting the yeast gene for invertase (apoplastic enzyme). In the course of adaptation to hypothermia, the activity of acid invertase was shown to rise and the content of sucrose and glucose to increase in the leaves of both genotypes. The greatest activity of acid invertases by the third day of cold acclimation corresponded to the peak level of sugars; in transformed plants, these characteristics exceeded those in the control plants. The transformed plants were more cold resistant than the control plants as suggested by the lack of disturbance of ion permeability of their membranes. It was concluded that owing to accumulation of low-molecular carbohydrates in the course of cold acclimation caused by activation of acid invertase cold resistant plants better adapt to temperature drop.

We studied the influence of alien cytoplasm of spring goatgrass Aegilops ovata L. on some physiological parameters in winter wheat (Triticum aestivum L.), Mironovskaya 808, under normal conditions and in the case of modified source-sink relations. Measurements of relative rates of plant dry matter growth and its distribution among organs, CO2 exchange (photosynthesis upon light saturation and dark respiration), content of sugars (sucrose + glucose + fructose) and their ratio in leaves, frost hardiness, and indices of membrane stability and damage of leaves by frost have shown that, on average, alloplasmic hybrid differed from the initial cultivar by almost all parameters. Reduced frost hardiness, increased index of leaf damage by frost, lowered leaf content of sugars, and reduced sucrose/(glucose + fructose) ratio in the alloplasmic hybrid were combined with higher roots/leaves ratio, relative rate of dry matter growth, and photosynthesis and respiration rates. The alloplasmic hybrid was more tolerant to decreased source strength in source-sink relations as compared to the initial cultivar.

Изучали влияние чужеродной цитоплазмы ярового эгилопса (Aegilops ovata L.) на проявление ряда физиологических свойств у растений озимой пшеницы (Triticum aestivum L.) сорта Мироновская 808 в норме и при измененных донорно-акцепторных отношениях (ДАО) в растении. Измерения относительных скоростей роста (ОСР) сухой массы растений и ее распределения по органам, СО2-обмена растения (фотосинтеза при световом насыщении и темнового дыхания), содержания сахаров (сахароза + глюкоза + фруктоза) и их соотношения в листьях, морозостойкости растений, индексов мембранной стабильности и холодового повреждения листьев показали, что в среднем аллоцитоплазматический гибрид (АЦПГ) отличался от исходного сорта озимой пшеницы почти по всем показателям. Сниженная морозостойкость, повышенный индекс повреждения листьев морозом, сниженное содержание сахаров в листьях и пониженное отношение сахароза/(глюкоза + фруктоза) у АЦПГ сочетались с более высокими отношением корни/листья, ОСР сухой массы, скоростями фотосинтеза и дыхания. АЦПГ более толерантен к снижению донорной функции в ДАО по сравнению с исходным сортом.

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.