Influence of yeast-derived invertase gene expression in potato plants on membrane lipid peroxidation at low temperature

Tolerance to chilling was compared 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 and with the proteinase inhibitor II leader peptide sequence providing for the apoplastic enzyme localization (the B33-inv plants) and potato plants transformed only with a reporter gene (the control plants) under in vitro conditions. The total activities of acid and alkaline invertases and contents of sugars in the B33-inv plants exceeded those in the control plants. The exposures at low temperature produced a significant less lipid peroxidation activity in the B33-inv plants, as compared to the control. The authors presume that the B33-inv plants acquire a higher tolerance to low temperatures apparently due to the changes in sugar ratio produced by the yeast invertase.     

Effect of insertion of apoplastic invertase gene on photosynthesis of potato plants grown at various light intensities

We studied the influence of yeast invertase gene (inv), with the apoplastic localization of the enzyme, on photosynthesis of potato plants (Solanum tuberosum L., cv. Desiree) grown at various irradiances. Plants were raised in vitro, planted in soil in gauze-insulated stands, and grown at irradiances of 100, 200, and 380 W/m² of photosynthetically active radiation. Wild-type plants (WT) and the plants transformed with yeast invertase gene (B33-inv) were used. In the beginning of flowering stage, assimilation of ¹⁴CO₂ and ¹⁴C incorporation into photosynthates were measured. Irrespective of irradiance, the carbon assimilation was higher in WT plants, than in transformed B33-inv plants. In the plants studied, we observed divergent light dependences of ¹⁴C inclusion into sucrose: the highest labeling was observed at low irradiance in WT plants and at high irradiance in B33-inv transformed plants. The content of ¹⁴C incorporated into amino acids changed in the opposite direction compared to ¹⁴C incorporation into sucrose. Irrespective of the plant type, similar light dependences were observed for ¹⁴C content in the products of glycolate metabolism and in glycerate. At the intermediate irradiance, the patterns of ¹⁴C distribution among photosynthetic products showed minimal differences between the plants of two types. The role of apoplast invertase in sugar export from the leaf and the possible control of plant productivity through this enzyme activity are discussed.     

The changes in invertase activity and the content of sugars in the course of adaptation of potato plants to hypothermia

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.     

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.     

Contribution of Oxidative Stress to the Development of Cold-Induced Damage to Leaves of Chilling-Sensitive Plants: 3. Injury of Cell Membranes by Chilling Temperatures

Changes in permeability of cell membranes (judged from electrolyte leakage) were examined on leaves of 7- to 11-day-old seedlings of maize (Zea mays L.), cucumber (Cucumis sativus L.), millet (Panicum miliaceum L.), and on etiolated shoots of potato (Solanum tuberosum L.) immediately after cooling plants for 1–24 h at 2°C and one day after a 24-h chilling treatment. A gradually increasing leakage of ions from the cells was observed upon prolongation of chilling exposure, with the maximum attained by the end of 24-h chilling treatment. The leakage of electrolyte was slightly reduced in the post-treatment period but it was still higher than the electrolyte leakage from the control samples (untreated plants). The cold treatment of chilling-sensitive plants (but not of potato) revealed a positive correlation between the rates of lipid peroxidation, indicative of chilling injury, and the electrolyte efflux (r = 0.61–0.96). The evaluation of plant susceptibility to injury showed that millet and potato plants recovered from the chilling damage in 24 h after the treatment, whereas maize and cucumber plants did not show such a recovery.     

Effect of Low Temperature on the Protein Metabolism of Wheat Leaves

The effect of low temperature on the protein metabolism of wheat primary leaves was examined. In seedlings transferred from 25 to 5 °C, total soluble protein accumulation, in vivo protein synthesis and breakdown, in vitro protein breakdown, and SDS-PAGE profiles of proteinases in gelatine-containing gels were analysed. Leaf protein content increased within a 7-d period (70 % over the initial value) in plants exposed to 5 °C. The fast protein accumulation observed on days 0 – 2 was mainly attributed to a decreased breakdown. In further days, parallelly to a slowdown in the rate of protein accumulation, the leaf proteolytic activity increased. The incubation temperature also had an influence on the proteolytic activity: Q ₁₀ values for the 15 – 5 °C range were 80 – 200 % higher than those observed for the 25 – 15 °C range. On the other hand, the in vivo protein synthesis capacity, at either 25 or 55 °C, was not significantly modified in cold-treated plants. In addition to the enhanced activities of two serine-proteinases (previously found in control plants by SDS-PAGE analysis), cold-treated plants displayed a new proteinase, which had not been detected so far.     

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.     

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.     

Tuber Formation and Growth of in vitro Cultivated Transgenic Potato Plants Overproducing Phytochrome B

We studied the effect of the ectopic expression of the Arabidopsis PHYB gene, which encodes the phytochrome B (phyB) apoprotein, under the control of cauliflower mosaic virus 35S promoter on the photoperiodic response of tuberization and growth of potato (Solanum tuberosum L., cv. Désirée) transformed lines. Stem cuttings of transformed and control plants were cultured on Murashige and Skoog nutrient medium containing 5 or 8% sucrose in the phytotron chambers at 20°C under conditions of a long day (16 h), a short day (10 h), or in darkness. We showed that the overexpression of the PHYB gene enhanced the inhibitory effect of the long day on tuberization. In addition, tuber initiation in these transformed plants occurred at a higher sucrose concentration. The insertion of the PHYB gene decreased plant and tuber weights and shortened stems and internodes. Thus, we demonstrated the complex result of the PHYB gene insertion: it affected the photoperiodic response of tuberization, the control of tuber initiation by sucrose, and the growth of potato vegetative organs.     

The effect of light intensity on the assay of the low temperature limit of photosynthesis using msec delayed light emission

Steady state millisecond delayed fluorescence (DLE) of intact leaves and cyanobacterial cells was measured continuously with a Becquerel-type phosphoroscope while cooling from the growth temperature to near 0°C or heating from the low to high temperature at about 1°C/min. The temperature of maximum DLE depended upon light intensity. In Anacystis grown at 28 and 38°C DLE maximum occurred near 15 and 23°C, respectively, which are the temperatures where thylakoid membrane lipids have been shown to pass from the liquid crystalline to the mixed solid-liquid crystalline state in these cyanobacteria. In some plants such as field mallow DLE increased continuously as the temperature decreased, whereas in others it rose to a maximum, then decreased. Chilling-sensitive plants such as tomato, sweet potato and Trichospermum, showed DLE maxima around 10–14°C while the chilling-resistant plant, oat, had a maximum near 4°C and field mallow had no maximum above 0°C.Abbreviations DLE delayed light emission CIW-DPB Publ. No. 1022.     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Cis regulatory elements directing tuber-specific and sucrose-inducible expression of a chimeric class I patatin promoter/GUS-gene fusion

Summary The 5-upstream region of the class I patatin gene B33 directs strong expression of the -glucuronidase (GUS) reporter gene in potato tubers and in leaves treated with sucrose. Cis-acting elements affecting specificity and level of expression were identified by deletion analysis in transgenic potato plants. A putative tuber-specific element is located downstream from position –195. Nuclear proteins present in leaf and tuber extracts bind specifically to a conserved AT rich motif within this region. A DNA fragment between –183 and –143, including the binding site is, however, not able to enhance the expression of a truncated 35S promoter from cauliflower mosaic virus. Independent positive elements contributing to a 100-fold increase relative to the basic tuber-specific element are located between –228 and –195; –736 and –509, –930 and –736 and –1512 and –951. Sucrose inducibility is controlled by sequences downstream of position –228, indicating that the tuber-specific and sucrose-inducible elements are in close proximity.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Assessment of the tolerance of Lactococcus lactis cells at elevated temperatures

When Lactococcus lactis strains were exposed directly to the lethal temperature of 50 C for 30 ;min, 0.1–31% of the cells survived. However, when pre-exposed to 40 °C, prior to exposure at 50 °C, 4–61% of the cells survived. A plasmid carrying a unique heat shock gene from the thermophile Streptococcus thermophilus was cloned into L. ;lactis. When the transformed cells were cultivated at 30 °C the introduction of the plasmid had no obvious effect on the growth of L. ;lactis. However, when the temperature was abruptly shifted from 30 °C to 42 °C at mid-growth phase the growth decreased by 50%.     

Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. ssp. Oleifera

Summary Conditions favourable to embryogenesis from isolated microspores of Brassica rapa L. ssp. oleifera (canola quality) were identified. A population with enhanced responsiveness for microspore embryogenesis (C200) was synthesized by crossing individual plants showing microspore embryogenic potential. For optimal microspore embryogenesis, buds (2–3mm in length, containing mid-late uninucieate microspores) were collected from older plants (2 months old) and microspores isolated and washed in iron-free B5 medium. NLN medium with its iron content reduced to half was beneficial for initial microspore culture. An elevated temperature(33–35°C) during the first day of culture, followed by maintenance at 25°C resulted in dozens of embryos from each isolation (about 100 buds). Seeds were obtained from plants regenerated from microsporederived embryos after colchicine treatment.     

Immobilized, thermostable S- and F-forms of the extracellular invertase from Candida utilis can hydrolyse sucrose up to 100 °C

When grown on a sucrose-containing medium, Candida utilis synthesizes and secretes two invertases: one of molecular size of 280 kDa (the S-form – Slow-migrating) and a new form of Mr of 62 kDa (the F-form – Fast-migrating). Prior to immobilization, purification of S- and F-forms of invertase increased the immobilization yield to 89–100%, in comparison with that of crude invertase preparation (52%). The immobilized purified S- and F-form of invertase remained partially active after 15 min at 100 °C; the F-form retained almost 30% of its maximum activity. The immobilized S-form or F-form of invertase almost completely inverted (95% hydrolysis) 60% (w/v) sucrose over 5 h continuous reaction at 80 °C. Moreover, at 90 °C the immobilized F-form hydrolysed 70% of 60% (w/v) sucrose over 5 h, while the capability of the immobilized S-form of inverting sucrose over 5 h reaction decreased from 80% to 45%.     

Contribution of Oxidative Stress to the Development of Cold-Induced Damage to Leaves of Chilling-Sensitive Plants: 1. Reactive Oxygen Species Formation during Plant Chilling

Changes in the levels of superoxide anion radical and total peroxides were studied immediately after the chilling of 7–11-day-old seedlings of maize (Zea mays L.), cucumber (Cucumis sativus L.), millet (Panicum miliaceum L.), and etiolated potato (Solanum tuberosum L.) shoots at 2°C for 1–24 h and one day after 24-h chilling. A short-term (1 h) chilling of chilling-sensitive plants resulted in the 2.4–7.5-fold acceleration of the O^– ₂ generation. A longer chilling period reduced somewhat the rate of O^– ₂ generation, but this rate did not achieve the control level. The highest level of H₂O₂ was observed after 2-h chilling with its subsequent lowering. In the cold-tolerant potato, the levels of O^– ₂ and peroxides reduced after chilling. The rate of lipid peroxidation (an index characterizing cold-induced membrane damage) increased gradually with the lengthening of the chilling period. Reactive oxygen species are supposed to be involved in the induction of the oxidative stress during chilling of chilling-sensitive plants and in the triggering of cold-induced damage.     

A culture unit system for the study of responses of mycorrhizal and non-mycorrhizal seedling to treatments

The time-dependence of Mn accumulation was confirmed in potato foliage (Solanum tuberosum. L.cv. Norland) grown in solution culture. Older leaves grown at 0.61 mM Mn had substantially higher Mn concentrations than younger leaves and stem samples. Levels of Mn in older leaves increased steadily from 4000 µg g^–1 at one week to 8–10,000 µg g^–1 at 6 weeks, but were relatively constant in the emerging leaves. Even foliage grown at low Mn levels (0.01 mM Mn) had 4 fold gradients in Mn concentration from younger (40 µg g^–1) to older leaves (180 µg g^–1).At 0.61 mM Mn, concentrations of 3–4000 µg g^–1 in the youngest fully-developed leaves did not bring about any decline in yield, and levels of up to 5000 µg g^–1 occurred in individual potato leaves before Mn toxicity symptoms were observed. Potato foliage grown at the high Mn had similar leaf numbers, but showed an increased stem length and smaller leaves than foliage grown at 0.01 mM Mn. In particular, the leaf area of the middle and lower leaf fractions were affected by the high Mn level.The ability of rapidly growing plants to withstand high concentrations of Mn is discussed in relation to the pattern of dry matter and Mn accumulation shown by potato foliage.     

Enhancement of frost tolerance in olive shoots in vitro by cold acclimation and sucrose increase in the culture medium

The evaluation of frost tolerance in olive shoots in vitro has been successfully accomplished. The behavior of in vitro shoots at freezing temperatures was comparable to that of intact plants. Cold acclimation was found to increase frost tolerance in cv. Moraiolo and the LT₅₀ was about 4 °C lower compared to nonacclimated shoots. Damage in acclimated shoots occurred at –15 °C, whereas control shoots were damaged at –10 °C. Olive shoots were unable to withstand freezing temperatures of –20 °C, even when acclimated. The effects of sucrose were also determined. 6% (w/v) sucrose in the medium conferred the highest frost tolerance in both acclimated and nonacclimated plants.     

Effect of High Temperature on Protein Expression in Strawberry Plants

Strawberry plants (Fragaria×ananassa Duch.) cvs. Nyoho and Toyonoka were exposed to temperatures of 20, 33, and 42 °C for 4 h, and protein patterns in leaves and flowers was analyzed by 2-dimensional polyacrylamide gel electrophoresis and immunoblotting. In leaves and flowers of both cultivars, the content of most proteins decreased, but a few new proteins appeared in response to heat stress. These heat shock proteins (Hsps) were detected in the range of 19 – 29 kDa in leaves, and 16 – 26 kDa in flowers. The intensity of a 43 kDa protein spot increased in response to heat stress in Nyoho flowers, but not in Toyonoka flowers. The peaHsp17.7 antibody recognized one band at approximately 26 kDa in leaves, and two bands at approximately 16 and 17 kDa in flowers of both cultivars. These results show that the effects of heat stress on Hsp synthesis in strawberry plants differ between plant organs and between cultivars.