Nucleolar vacuolation in soybean root meristematic cells during recovery after chilling

The nucleolar vacuole formation in soybean root meristematic cells from seedlings grown 3 d at temperature 25 °C (control), 3 d at temperature 25 °C and then transferred to 10 °C (chilling) for 4 d, and after recovery for 1.5, 3, 6, 12 and 24 h at 25 °C were observed on semi-thin sections. Simultaneously, autoradiographic studies with ³H-uridine on squashed preparations were carried out. During recovery of plants, the number of vacuolated nucleoli increased gradually from 24 % after 1.5 h up to 40 % after 24 h, while in the control there were 18 % of nucleoli with vacuoles and after 4-d chilling only 5 %. Labelling of cells during 20-min incubation in ³H-uridine and during 80-min post-incubation in non-radioactive medium was increased in recovered plants in comparison with the control and chilled plants. The conclusion has been drawn that nucleolar vacuoles in soybean plants are formed as a result of migration of granular component accumulated in nucleolus during 4-d chilling.     

Selenium Modifies the Effect of Short-Term Chilling Stress on Cucumber Plants

The objective of this study was to investigate the effect of selenium (Se) supply (0, control; 2.5, 5, 10, or 20 μM) on cucumber (Cucumis sativus L.) cv. Polan F1 plants grown under short-term low temperature stress. About 14–16 day-old seedlings, grown at an optimal temperature (25/20°C; day/night), were exposed to short-term chilling stress with a day/night temperature of 10°C/5°C for 24 h, for a further 24 h at 20°C/15°C, and then transferred to 25/20°C (re-warming) for 7 days. Se did not affect the fresh weight (FW) of plants at a concentration of 2.5–10 μM, but in the presence of 20 μM Se, the biomass of shoots significantly decreased. The contents of chlorophylls and carotenoids witnessed no significant change after Se supplementation. Compared with the control, the Se-treated plants showed an increase of proline content in leaves, once after chilling and again after 7 days of re-warming. However, proline levels were much higher immediately after chilling than after re-warming. The malondialdehyde (MDA) content in the root of plants treated with 2.5–10 μM Se decreased directly after stress. This was in comparison with the plants grown without Se, whereas it increased in roots and leaves of plants exposed to 20 μM Se. Seven days later, the MDA level in the root of plants grown in the presence of Se was still lower than those of plants not treated with Se and generally witnessed no significant change in leaves. Although Se at concentrations of 2.5–10 μM modified the physiological response of cucumber to short-term chilling stress, causing an increase in proline content in leaves and diminishing lipid peroxidation in roots, the resistance of plants to low temperature was not clearly enhanced, as concluded on the basis of FW and photosynthetic pigments accumulation.     

Cold Stress-Induced Acclimation in Rice is Mediated by Root-Specific Aquaporins

Cold acclimation process plays a vital role in the survival of chilling- and freezing-tolerant plants subjected to cold temperature stress. However, it remains elusive whether a cold acclimation process enhances root water uptake (a component of chilling tolerance) in chilling-sensitive crops such as rice. By analyzing the root hydraulic conductivity under cold stress for a prolonged time, we found that cold stress induced a gradual increase in root osmotic hydraulic conductivity [Lp(r(os))]. Compared with the control treatment (roots and shoots at 25°C), low root temperature (LRT) treatment (roots at 10°C; shoots at 25°C) dramatically reduced Lp(r(os)) within 1 h. However, Lp(r(os)) gradually increased during prolonged LRT treatment and it reached 10-fold higher values at day 5. Moreover, a coordinated up-regulation of root aquaporin gene expression, particularly OsPIP2;5, was observed during LRT treatment. Further, comparison of aquaporin gene expression under root-only chilling (LRT) and whole-plant chilling conditions, and in the roots of intact plants vs. shootless plants, suggests that a shoot to root signal is necessary for inducing the expression of aquaporin genes in the root. Collectively, these results demonstrate that a cold acclimation process for root water uptake functions in rice and is possibly regulated through aquaporins.     

Low temperature acclimation of root fatty acid composition, leaf water potential, gas exchange and growth of soybean seedlings

Abstract Root fatty acid composition, photosynthesis, leaf water potentials, stomatal resistances, leaf specific weights, and root: shoot ratios of soybean were measured in two temperature regimes. Groups of soybean plants were grown in controlled chambers of the Duke University Phytotron under two thermoperiods. One group of the plants was grown from seed for 3 weeks in either 29/23°C or 17/11°C thermoperiods, and another group was grown for 2 weeks in 29/23°C and then transferred to the 17/11°C thermoperiod where it remained for 8 days. Broccoli was also grown in either 29/23°C or 17/11°C thermoperiods. Soybean roots contained more unsaturated fatty acids than broccoli roots, although broccoli roots showed a larger increase in unsaturation than soybean roots with decreased temperature. The fatty acid unsaturation in the roots of soybean began to increase rapidly after the temperature regime was changed. The increase was in the new roots produced in the cold regime rather than in the pre-existing roots. The soybean leaf water and osmotic potentials decreased about 0.4 MPa, beginning one day after the transfer from 29/23°C to 17/11°C, but recovered significantly after 8 d. Plants grown at 17/11 °C had lower rates of photosynthesis and adaxial stomatal resistances, but higher root: shoot ratios and specific leaf weights compared to plants grown at 29/23°C. Plants grown and maintained at 29/23°C showed a steady increase in photosynthetic rates over the 8-d experimental period, whether rates were measured in 1 mol m^?3 or 9 mol m^?3 oxygen. Plants transferred to 17/11°C however maintained constant rates of photosynthesis at 1 mol m^?3 O₂, whereas at 9 mol m^?3 rates declined for 2 d then were constant for the remaining 6 d of the experimental period. These results suggest that changes in membrane fatty acid unsaturation is an important aspect of plant acclimation to chilling temperatures in terms of maintaining root permeability and water uptake. However, the degree of unsaturation is not a good indicator of differences in chilling tolerance among species. The apparent acclimation of photorespiration to a constant percentage of photosynthesis suggests a role of photorespiration in the plant.     

Different chilling stresses stimulated the accumulation of different types of ginsenosides in <Emphasis Type="Italic">Panax ginseng</Emphasis> cells

Ginseng (Panax ginseng) is one of the most medically important plants in the world. Dammarane-type ginsenosides, which mainly include protopanaxatriol-type (PPT-type) and protopanaxadiol-type (PPD-type) ginsenosides, are the major pharmacologically relevant compounds that are produced by ginseng. Dammarenediol-II synthase (DDS) is the first committed enzyme in the ginsenoside biosynthetic pathway for dammarane-type ginsenosides, and PPD-type and PPT-type ginsenosides are catalyzed by protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), respectively. Ginseng cells are often used in stress studies. During their growth and development, ginseng plants are often exposed to cold stress. This study evaluated the effects of different chilling stresses on the accumulation of ginsenosides and the expressions of the DDS, PPDS and PPTS genes in ginseng cells. The results showed that continuous chilling (5 °C for 12 h) induced the PPT-type ginsenosides; whereas intermittent chilling (25 °C for 12 h and 5 °C for 12 h) stimulated the accumulation of PPD-type ginsenosides. The expression levels of DDS, PPDS and PPTS were clearly consistent with the accumulation pattern for PPT-type ginsenosides under continuous chilling stress or PPD-type ginsenosides under intermittent chilling stress, as was their order of involvement in the PPT-type or PPD-type biosynthetic pathway. These results indicate that different chilling treatments stimulated the accumulation of different types of ginsenosides, suggesting that cold stress may be one of the reasons for ginsenoside accumulation in ginseng cells.     

Phytophthora cryptogea root rot of tomato in rock woo] nutrient culture. II. Effect of root zone temperature on infection, sporulation and symptom development

The effect of root-zone temperature on Phytophthora cryptogea root rot was studied in tomato cv. Counter grown under winter and summer conditions in rockwool culture. A nutrient temperature of 25°C resulted in increased root initiation and growth, higher in winter-grown than in summer-grown plants. Rhizosphere zoospore populations were greatly reduced at 25°C and above. Growth of P. cryptogea in vitro was optimal between 20°C and 25°C and completely suppressed at 30°C. Encystment was enhanced by increased temperatures above 20°C. Zoospore release in vitro occurred in cultures maintained at constant temperatures in the absence of the normal chilling stimulus. Optimal release was at 10°C; no zoospores were released at 30°C. Inoculated, winter-grown tomato plants maintained at 15°C developed acute aerial symptoms and died after 21 days. Comparable plants grown at a root-zone temperature of 25°C remained symptomless for the 3-months duration of the experiment. Summer-grown infected plants at the higher root temperature wilted but did not die. Enhanced temperature was ineffective as a curative treatment in summer-grown plants with established infection. Aerial symptoms of Phytophthora infection are seen as a function of the net amount of available healthy root. With high root zone temperatures this is determined by new root production and decreased inoculum and infection.     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chlorophyll fluorescence and anthocyanin content in chilled maize plants after return to a non- chilling temperature under various irradiances

The effect of irradiance on the ratio of variable to maximum fluorescence (F_v/F_m) and on the anthocyanin content of chilled (0.5 °C) young maize plants was investigated after returning the plants to a non-chilling temperature (25 °C). Compared to control plants grown throughout at 25 °C in the light, the F_v/F_m hardly changed during chilling or when returned to a non-chilling temperature in the dark, but there was a decrease in this parameter if the plants were shifted to the light after the cold treatment. Similarly, compared to the control plants there was no change in the anthocyanin content either at low temperature or after transfer to 25 °C in the dark. However, there was a sudden increase in the anthocyanin level after returning the plants from dark cold conditions to a non-chilling temperature in the light.     

Antioxidant enzymes and isoflavonoids in chilled soybean (Glycine max (L.) Merr.) seedlings

Changes of activity antioxidant enzymes and of levels of isoflavonoids were studied in the roots and hypocotyls of the etiolated soybean (Glycine max (L.) Merr. var. Essor) seedlings, submitted to cold. Prolonged exposure to 1 degrees C inhibited hypocotyl and root elongation and limited their growth after seedlings were transferred to 25 degrees C. Roots were more sensitive to chilling than hypocotyls. At 1 degrees C a gradual increase in MDA concentration in roots but not in hypocotyls was observed. An increase in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activity in hypocotyls was observed both at 1 degrees C and after transfer of plants to 25 degrees C. In roots, CAT activity increased after 4 days of chilling, while SOD activity only after rewarming. L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity decreased in roots of chilled seedlings, but did not change in hypocotyls until activity increased after transfer to 25 degrees C. The content of genistein and daidzein increased after 24 h of treatment by low temperature and then decreased with prolonged chilling in hypocotyls and remained high in roots. However, it should be noted that genistin level (genistein glucoside) in chilled hypocotyls is 10 times higher than in roots, despite falling tendency. The role of antioxidant enzymes and isoflavonoids in preventing chilling injury in hypocotyls and roots of soybean seedlings is discussed.     

Chilling stress and oxygen metabolizing enzymes in Zea mays and Zea diploperennis*

Abstract. The activities of five active-oxygen scavenging enzymes were compared for cold-lability and three were compared for chilling induction in two Zea genotypes of contrasting susceptibility to photoinhibition during chilling. Activities of superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GTR, EC 1.6.4.2) in leaf extracts from plants grown without chilling stress were assayed at 19°C and 5°C. Enzymes from the chilling-susceptible Z. Mays cv. LG11 had lower specific activities at 5°C than did enzymes from the chilling-tolerant Z. diploperennis, except for MDHAR where no significant differences were observed. The activities of SOD and APX from Z. diploperennis were double those of Z. mays at both assay temperatures. Monodehydroa-scrobate reductase and glutathione reductase activities in both species were reduced by 63–78% at a 5°C assay temperature. The dehydroascorbate reductase (DHAR) showed the greatest low-temperature lability losing 96% (Z. diploperennis) and 100% (Z. mays) of its activity at 5°C. To examine possible chilling-induced changes in levels of enzyme activity, plants of both Zea genotypes were transferred to growth chambers at 10°C at moderate light intensities. Glutathione reductase activity was found to increase within 24h in Z. diploperennis, but it decreased slightly in Z. mays. MDHAR activity decreased by 50% in Z. diploperennis but showed only a transient increase in activity in Z. mays.     

Ultrastructure and functional activity of chloroplasts in wheat leaves under root chilling

The effects of root chilling (2 °C; during 1, 5 h, 1, 2, 4 and 7 days) on the ultrastructure, functional activity of chloroplasts and cold tolerance of leaf cells of wheat (Triticum aestivum L.) were studied. Results indicated that the area of the chloroplasts increased and the number of grana in the chloroplast decreased already within first hours of the experiment. On the 2nd–7th day of the cold treatment, the length of photosynthetic membranes in the chloroplasts increased owing to the membranes of thylakoids in grana. The number of chloroplasts per cell was increased by the end of the experiment. Reduction of electron transport rate and intensification of non-photochemical quenching of chlorophyll fluorescence were observed in the first hours of root chilling. The growth of the leaves slowed in the first day of the treatment and resumed on the second day. Leaf area in the root-chilled plants by the end of the experiment exceeded the initial values by 60 %. The significant rise in cold tolerance of leaf cells was detected after 24 h of root chilling. After 48 h of the treatment, the cold tolerance reached a maximum, and did not change thereafter. It is assumed that most of the observed structural and functional changes are adaptive, and meant to support the photosynthetic function and promote the cold tolerance of the plants.     

Effects of Temperature Acclimation Pretreatment on the Ultrastructure of Mesophyll Cells in Young Grape Plants （Vitis vinifera L. cv. Jingxiu） Under Cross-Temperature Stresses

Leaves from annual young grape plants （Vitis vinifera L. cv. Jingxiu） were used as experimental materials. The ultrastructural characteristics of mesophyll cells in chilling-treated plants after heat acclimation （HA） and in heat-treated plants after cold acclimation （CA） were observed and compared using transmission electron microscopy. The results showed that slight injury appeared in the ultrastructure of mesophyll cells after either HA （38℃ for 10 h） or CA （8℃ for 2.5 d）, but the tolerance to subsequent extreme temperature stress was remarkably improved by HA or CA pretreatment. The increases in membrane permeability and malondialdehyde concentration under chilling （0℃） or heat （45℃） stress were markedly inhibited by HA or CA pretreatment. The mesophyll cells of plants not pretreated with HA were markedly damaged following chilling stress. The chloroplasts appeared irregular in shape, the arrangement of the stroma lamellae was disordered, and no starch granules were present. The cristae of the mitochondria were disrupted and became empty. The nucleus became irregular in shape and the nuclear membrane was digested. In contrast, the mesophyll cells of HA-pretreated plants maintained an intact ultrastructure under chilling stress. The mesophyll cells of control plants were also severely damaged under heat stress. The chloroplast became round in shape, the stroma lamellae became swollen, and the contents of vacuoles formed clumps. In the case of mitochondria of control plants subjected to heat stress, the outer envelope was digested and the cristae were disrupted and became many small vesicles. Compared with cellular organelles in control plants, those in CA plant cells always maintained an integrated state during whole heat stress, except for the chloroplasts, which became round in shape after 10 h heat stress. From these data, we suggest that the stability of mesophyll cells under chilling stress can be increased by HA pretreatment. Similarly, CA pretreatment can protect chloroplasts, mitochondria, and the nucleus against subsequent heat stress; thus, the thermoresistance of grape seedlings was improved. The results obtained in the present study are the first, to our knowledge, to offered cytological evidence of cross-adaptation to temperature stresses in grape plants.     

Effects of long-term chilling on growth and photosynthesis of the C4 gramineae Paspalum dilatatum

Dallis grass (Paspalum dilatatum Poir.) is a C₄/NADP‐ME gramineae, previously classified as semi‐tolerant to cold, although a complete study on this species acclimation process under a long‐term chilling and controlled environmental conditions has never been conducted. In the present work, plants of the variety Raki maintained at 25/18°C (day/night) (control) were compared with plants under a long‐term chilling at 10/8°C (day/night) (cold‐acclimated) in order to investigate how growth and carbon assimilation mechanisms are engaged in P. dilatatum chilling tolerance. Although whole plant mean relative growth rate (mean RGR) and leaf growth were significantly decreased by cold exposure, chilling did not impair plant development nor favour the investment in biomass below ground. Cold‐acclimated P. dilatatum cv. Raki had a lower leaf chlorophyll content, but a higher photosynthetic capacity at optimal temperatures, its range being shifted to lower values. Associated with this higher capacity to use the reducing power in CO₂ assimilation, cold‐acclimated plants further showed a higher capacity to oxidize the primary stable quinone electron acceptor of PSII, Q_A. The activity and activation of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were not significantly affected by the long‐term chilling. Cold‐acclimated P. dilatatum cv. Raki apparently showed a lower transfer of excitation energy from the light‐harvesting complex of photosystem II to the respective reaction centre and enhancement of radiationless energy‐dissipating mechanisms at suboptimal temperatures. Overall, long‐term chilling resulted in several effects that comprise responses with an intermediate character of both chilling‐tolerant and –sensitive plants, which seem to play a significant role in the survival and acclimation of P. dilatatum cv. Raki at low temperature.     

Effect of Low Temperature and Rh izospheric Application of Naringenin on Pea-Rhizobium leguminosarum biovar viciae Symbiosis

The production of Tsr factor by Rhizobium leguminosarum bv. viciae was influenced by low temperature (10°C) In the presence of seed exudate collected at 10°C and 25°C or naringenin (10fuM). Root exudate collected at 25°C and naringenin induced Tsr factor in R. leguminosarum causing thick and short root phenotype and root hair curling and deformation of host root. Root exudate collected at 10°C also induced root hair curling but Tsr activity was low. low temperature grown plants had poor nodulation, nitrogen fixation, nitrogen content and total blomass as compared to plants grown at 25°C. Rhizospheric application of naringenin partially alleviated the deleterious effect of low temperature on nodulation status and nodule efficiency.     

Differential gene expression in cucumber plants in response to brief daily cold treatments

The mechanisms of plant responses to short-term cold treatments applied daily in the period of active growth remain unknown. Cucumber (Cucumis sativus L.) plants were subjected to brief drops of temperature (2 h, 12°C) at the end of each night over a 6-day period (DROP treatment) and to prolonged (6 days) cooling at 12°C (permanent low-temperature treatment, PLT). The plants exposed to cold treatments and control plants grown at 20°C were compared in terms of cold resistance and changes in gene expression. Cold resistance of plants was determined on the basis of LT₅₀ temperature. The response of cucumber genetic machinery was assessed by means of a differential display method based on polymerase chain reaction (PCR). The changes in mRNA pool in cells of cucumber plants subjected to permanent and periodic chilling were assessed after comparing the populations of PCR fragments of cDNA. In both types of chilling protocols, the cold resistance started to increase from the 2nd day of low temperature treatment. At the end of the experiment (on the 6th day), the increment in cold resistance was three times larger for DROP compared to PLT treatment. Analysis of mRNA pool showed that the numbers of amplified fragments were nearly identical in both types of low-temperature treatment. The higher level of cold resistance under DROP conditions was assumed to depend on features of metabolism.     

Role of phytochrome B in the development of cold tolerance in cucumber plants under light and in darkness

Cold tolerance of cucumber (Cucumis sativus L.) seedlings was investigated using wild-type plants and the phytochrome B-deficient mutant (lh-mutant). Plants were subjected for 6 days to intermittent short-term cooling (12°C for 2 h per day) and to continuous chilling under conditions of 16-h photoperiod (day/night = 16/8 h) and permanent illumination. “Dehardening” process was initiated by the transfer of plants to either light or dark conditions at 23°C. It was concluded that phytochrome B participates in the development of cold tolerance in cucumber plants under stress conditions, i.e., under short-term intermittent chilling at nights and during dehardening in continuous darkness.     

Changes in soluble carbohydrates in polar Caryophyllaceae and Poaceae plants in response to chilling

Four species of flowering plants comprising Arctic populations of Cerastium alpinum and Poa arctica var. vivipara and indigenous Antarctic species Colobanthus quitensis and Deschampsia antarctica were investigated. Plants derived from natural origins were grown in an experimental greenhouse in Poland (53°47′N and 20°30′E latitude). Plants for experiment were collected during spring of 2010. Soluble carbohydrates in the intact shoots of C. alpinum and C. quitensis, polar plants of the family Caryophyllaceae, and D. antarctica and P. arctica var. vivipara, representatives of the family Poaceae, were analyzed by gas chromatography, and their involvement in the plants’ response to chilling stress was examined. Plant tissues of the examined families growing in a greenhouse conditions (18–20 °C, short day 10/14 h light/darkness) differed in the content and composition of soluble carbohydrates. In addition to common monosaccharides, myo-inositol and sucrose, Caryophyllaceae plants contained raffinose family oligosaccharides (RFOs), d-pinitol and mono-galactosyl pinitols. RFOs and d-pinitol were not detected in plants of the family Poaceae which contain 1-kestose, a specific tri-saccharide. The accumulation of significant quantities of sucrose in all investigated plants, RFOs in Caryophyllaceae plants and 1-kestose in Poaceae plants in response to chilling stress (4 °C for 48 h with a long day photoperiod, 20/4 h) indicates that those compounds participate in the stress response. The common sugar accumulating in cold stress response and probably most important for chilling tolerance of four investigated plants species seems to be sucrose. On the other hand, the accumulation of above-mentioned carbohydrates during chilling stress can be a return to sugars metabolism, occurring in natural environmental conditions. No changes in d-pinitol concentrations were observed in the tissues of C. alpinum and C. quitensis plants subjected to both low and elevated temperatures, which probably rules out the protective effects of d-pinitol in response to cold stress.