Characteristics of Cold Acclimation and Deacclimation in Tuber-bearing Solanum Species

The effect of temperatures on cold acclimation and deacclimation in foliage tissues was studied in Solanum commersonii (Oka 4583), a tuber-bearing potato. The threshold temperature for cold acclimation was about 12 C. In a temperature range of 2 to 12 C, the increase in hardiness was dependent on the acclimating temperature; the lower the acclimating temperature, the more hardiness achieved. A day/night temperature of 2 C, regardless of photoperiod, appeared to the optimum acclimating temperature for the Solanum species studied. A subfreezing temperature hardened plants less effectively. The maximum level of hardiness could be reached after 15 days of cold acclimation. However, it took only 1 day to deacclimate the hardened plants to a preacclimation level when plants were subjected to a warm regime from cold. The degree of deacclimation was dependent on the temperature of the warm regime.     

Genetic Analysis of Cold Tolerance at the Germination and Booting Stages in Rice by Association Mapping

Low temperature affects the rice plants at all stages of growth. It can cause severe seedling injury and male sterility resulting in severe yield losses. Using a mini core collection of 174 Chinese rice accessions and 273 SSR markers we investigated cold tolerance at the germination and booting stages, as well as the underlying genetic bases, by association mapping. Two distinct populations, corresponding to subspecies indica and japonica showed evident differences in cold tolerance and its genetic basis. Both subspecies were sensitive to cold stress at both growth stages. However, japonica was more tolerant than indica at all stages as measured by seedling survival and seed setting. There was a low correlation in cold tolerance between the germination and booting stages. Fifty one quantitative trait loci (QTLs) for cold tolerance were dispersed across all 12 chromosomes; 22 detected at the germination stage and 33 at the booting stage. Eight QTLs were identified by at least two of four measures. About 46% of the QTLs represented new loci. The only QTL shared between indica and japonica for the same measure was qLTSSvR6-2 for SSvR. This implied a complicated mechanism of old tolerance between the two subspecies. According to the relative genotypic effect (RGE) of each genotype for each QTL, we detected 18 positive genotypes and 21 negative genotypes in indica, and 19 positive genotypes and 24 negative genotypes in japonica. In general, the negative effects were much stronger than the positive effects in both subspecies. Markers for QTL with positive effects in one subspecies were shown to be effective for selection of cold tolerance in that subspecies, but not in the other subspecies. QTL with strong negative effects on cold tolerance should be avoided during MAS breeding so as to not cancel the effect of favorable QTL at other loci.     

Adaptive Changes in ATPase Activity in the Cells of Winter Wheat Seedlings during Cold Hardening

A cytochemical study of ATPase activity in the cells of cold hardened and nonhardened winter wheat (Triticum aestivum L. cv. Nongke No. 1) seedlings was carried out by electron microscopic observation of lead phosphate precipitation. ATPase activity associated with various cellular organelles was altered during cold hardening. (a) At 22°C, high plasmalemma ATPase activity was observed in both cold hardened and nonhardened tissues; at 5°C, high activity of plasmalemma ATPase was observed in hardened tissues, but not in unhardened tissues. (b) In nonhardened tissues, tonoplast and vacuoles did not exhibit high ATPase activity at either 22 or 5°C, while in hardened tissues high activity was observed at both temperatures. (c) At 5°C, ATPase activity of nucleoli and chromatin was decreased in hardened tissues, but not in nonhardened tissues. It is suggested that adaptive changes in ATPase activity associated with a particular cellular organelle or membrane may be associated with the development of frost resistance of winter wheat seedlings.     

Polysome Metabolism during Cold Acclimation in Wheat

The content, composition and biological activity of polysomesfrom three wheat genotypes were studied during cold acclimation.The structural integrity of the different polysome populationswas not affected by the hardening temperature. Polysomes werealso found to accumulate at higher level in cold hardened seedlingssuggesting a high protein synthesis capacity during the acclimationperiod. The in vitro translation of polysome-bound mRNAs inthe wheat germ cell-free system showed a high translation potentialof polysomes from cold hardened seedlings compared to that ofcontrol. The electrophoretic analysis of the translation productsby two-dimensional SDS-PAGE revealed the induction of severalnew mRNAs in cold hardened wheat seedlings. The presence ofthese new messengers in the polysomal fraction suggests thatnew messages have already been processed, transported and preferentiallyselected for translation by the ribosomes. The most importantchange was the induction and pronounced synthesis of four peptides[one high mol wt peptide of 200 kDa (pI 6.5) and three smallerones of 58 (pI 7.0), 48 (pI 7.1) and 48 (pI 7.2) kDa respectively]in the freezing tolerant cultivar Norstar. These specific polypeptideswere absent in the freezing sensitive cultivar Glenlea suggestingthat their induction and expression was associated with thefreezing tolerance capacity. (Received January 19, 1990; Accepted August 24, 1990)     

Exploration of biochemical and molecular diversity in chickpea seeds to categorize cold stress-tolerant and susceptible genotypes

Chickpea (Cicer arietinum L.) genotypes are sensitive to low temperature (<10°C) during its reproductive stage suffer from abortion of flowers, infertile pods and small shriveled seeds that resulted in a significant decrease in crop yield. In the present investigation seeds of a number of cold stress-tolerant and susceptible genotypes were evaluated for biochemical and molecular diversity with the purpose to categorize them. The activities of various antioxidative enzymes (superoxide dismutase, glutathione reductase, ascorbate peroxidase and catalase), content of H₂O₂ and malondialdehyde, enzymes involved in phosphate metabolism (acid and alkaline phosphatases), and content of phytic acid and proline were determined in seeds of 20 cold stress tolerant and seven cold stress susceptible genotypes. Higher activities of superoxide dismutase, ascorbate peroxidase, catalase and acid phosphatase and low content of malondialdehyde and phytic acid were observed in cold stress-tolerant genotypes as compared to cold stress susceptible genotypes. Seventeen chickpea genotypes comprising both cold stress-tolerant and susceptible ones were evaluated through 20 randomly amplified polymorphic DNA (RAPD) primers. The results of cluster analysis revealed two major groups. In the first group five tolerant (group 1a) and six susceptible genotypes (group 1b) clustered together whereas in second group all the tolerant genotypes clustered together (group 2). Out of 20 RAPD primers, 4 primers (Opa-13, Opa-14, Opa-15 and Opa-16) have been identified as markers for cold stress tolerance. In general high SOD activity, and H₂O₂ content and low MDA and phytic acid content are related with cold stress tolerance. The status of these markers was more pronounced in genotypes clustered in group 2 after RAPD analysis than in group 1a of cold stress-tolerant genotypes as compared to susceptible genotypes. The observed biochemical and molecular diversity could be useful for identifying and developing cold stress-tolerant genotypes of chickpea.     

Change in citrus leaf lipids during freeze-thaw stress

Fatty acids in the leaves of the citrus hybrid B5-9-68, a BC₁ progeny [(Citrus paradisi x Poncirus trifoliata) x C. sinensis], were compared with those in P. trifoliata and C. sinensis. Total lipid and triacylglycerol fatty acid profiles of the cold hardened hybrid were similar to the profiles of the hardy P. trifoliata and different from the profiles of the less hardy C. sinensis. When subjected to a freeze regime, the unhardened hybrid lost 22% leaf fatty acids during the freeze stage and 13% during the thaw stage. Linolenic acid accounted for 98% of the fatty acid decrease. Degradation was greatest in phosphatidylcholine (89%), phosphatidylglycerol (83%), monogalactosyldiglyceride (79%) and digalactosyldiglyceride (50%). Phosphatidic acid increased 4-fold over the two stress stages. Total leaf fatty acids during the freeze-thaw regime increased 12% in the cold hardened hybrid. Three molecular species of triacylglycerol which were rich in linolenic acid increased in the hardened hybrid during the freeze-thaw regime. The increase in highly unsaturated triacylglycerol species under freeze-thaw stress suggests that triacylglycerol has a role in maintaining the fluidity of biomembranes during freezing conditions.     

Heat Tolerance of Cold Acclimated Puma Winter Rye Seedlings and the Effect of a Heat Shock on Freezing Tolerance

An increase in tolerance to one form of abiotic stress oftenresults in an increase in tolerance to another stress. The heattolerance of Puma rye (Secale cereale) was determined for seedlingseither not cold hardened or hardened under either controlledenvironmental or natural conditions. The heat tolerance wasdetermined either as a function of time at 42°C or the abilityto tolerate a maximum temperature. The seedlings were eithernot heat preconditioned or heat preconditioned before the heatstress. In all cases cold hardened seedlings were more heattolerant than non or partially cold hardened seedlings. Heatpreconditioning had no effect on the heat tolerance of naturallycold hardened seedlings. In contrast, seedlings cold hardenedin a controlled environment chamber, then heat preconditioned,were more heat tolerant than non preconditioned seedlings. Aheat shock of 36°C for 2 h increased the freezing toleranceof non hardened seedlings from –2.5°C to –4.5°C.Analysis of heat shock protein 70 (HSP70) gene expression indicatedthat the HSP70 gene was not induced by cold acclimation andtherefore not directly involved in the increased thermo toleranceobserved. A number of heat stable proteins, simple sugars andlong chain carbohydrate polymers accumulated during the coldacclimation process and may have a role in increasing heat toleranceas well as freezing tolerance. These data suggest cold hardeningincreases heat tolerance, however, a heat shock to non acclimatedseedlings only marginally increased the freezing tolerance ofPuma rye seedlings. ³Present address: Agriculture and Agri-Food Canada, 107 SciencePlace, Saskatoon SK S7N 0X2, Canada.     

Constitutive and cold-induced resistance of rye and wheat seedlings to oxidative stress

The influence of cold hardening of rye (Secale cereale L.) and wheat (Triticum aestivum L.) seedlings on their resistance to the oxidative stress (OS) agents, namely, 50 mM hydrogen peroxide or 5 mM iron (II) sulfate was studied. Unhardened rye seedlings were more resistant to hydrogen peroxide than those of wheat, since their growth was less inhibited, and they accumulated lesser amounts of lipid peroxidation products after a treatment with H₂O₂. The interspecific differences in responses to FeSO₄ were less significant. The unhardened seedlings of rye, in comparison with those of wheat, possessed more active guaiacol peroxidase (GPO) and more levels of anthocyanins and proline. In response to the OS agents, the unhardened rye seedlings enhanced activities of superoxide dismutase and catalase, whereas the wheat seedlings enhanced GPO activity and proline content. The cold hardening (6 days at 2°C) increased activities of antioxidant (AO) enzymes, contents of proline, sugars, and anthocyanins in seedlings of both species, and made the seedlings more resistant to the OS agents. After the cold hardening, rye seedlings were more resistant to OS than wheat seedlings. The hardened seedlings of both species activated the AO enzymes in response to H₂O₂ or FeSO₄ greater than the unhardened ones. However, the hardened wheat seedlings, in contrast to the unhardened ones, did not augment the proline content in contact with the OS agents. The conclusion was drawn on different contributions of AO enzymes and low-molecular weight compounds to the basal and induced by the cold—hardening resistances of rye and wheat seedlings to OS.     

The impact of cold on photosynthesis in genotypes of Coffea spp.--photosystem sensitivity, photoprotective mechanisms and gene expression

Environmental constraints disturb plant metabolism and are often associated with photosynthetic impairments and yield reductions. Among them, low positive temperatures are of up most importance in tropical plant species, namely in Coffea spp. in which some acclimation ability has been reported. To further explain cold tolerance, the impacts on photosynthetic functioning and the expression of photosynthetic-related genes were analyzed. The experiments were carried out along a period of slow cold imposition (to allow acclimation), after chilling (4 °C) exposure and in the following rewarming period, using 1.5-year-old coffee seedlings of 5 genotypes with different cold sensitivity: Coffea canephora cv. Apoatã, Coffea arabica cv. Catuaí, Coffea dewevrei and 2 hybrids, Icatu (C. arabica × C. canephora) and Piatã (C. dewevrei × C. arabica). All genotypes suffered a significant leaf area loss only after chilling exposure, with Icatu showing the lowest impact, a first indication of a higher cold tolerance, contrasting with Apoatã and C. dewevrei. During cold exposure, net photosynthesis and Chl a fluorescence parameters were strongly affected in all genotypes, but stomatal limitations were not detected. However, the extent of mesophyll limitation, reflecting regulatory mechanisms and/or damage, was genotype dependent. Overnight retention of zeaxanthin was common to Coffea genotypes, but the accumulation of photoprotective pigments was highest in Icatu. That down-regulated photochemical events but efficiently protected the photosynthetic structures, as shown, e.g., by the lowest impacts on A_max and PSI activity and the strongest reinforcement of PSII activity, the latter possibly reflecting the presence of a photoprotective cycle around PSII in Icatu (and Catuaí). Concomitant to these protection mechanisms, Icatu was the sole genotype to present simultaneous upregulation of caCP22, caPI and caCytf, related to, respectively, PSII, PSI and to the complex Cytb₆/f, which could promote better repair ability, contributing to the maintenance of efficient thylakoid functioning. We conclude that Icatu showed the best acclimation ability among the studied genotypes, mostly due to a better upregulation of photoprotection and repair mechanisms. We confirmed the presence of important variability in Coffea spp. that could be exploited in breeding programs, which should be assisted by useful markers of cold tolerance, namely the upregulation of antioxidative molecules, the expression of selected genes and PSI sensitivity.     

Relationships of leaf Fatty acids to cold hardening of citrus seedlings

Three cultivars of citrus with different sensitivities to freezing temperatures (citron, Citrus medica L.; rough lemon, C. limon Burm. F; sour orange, C. aurantium L.) were cold hardened for 4 weeks. Lipids from leaves of hardened and control seedlings were fractionated and analyzed for fatty acids. The absolute amount of triglycerides and phospholipids increased in the leaves upon hardening. With hardening, total linoleic acid also increased 141% in citron, 210% in rough lemon, and 233% in sour orange. Specific increases in linoleic acid were found in triglycerides, in the four phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol), and in neutral lipids more polar than triglycerides. Trans-3-hexadecenoic acid was found only in phosphatidylglycerol.     

β-1,3-glucanase and chitinase activities in winter triticales during cold hardening and subsequent infection by Microdochium nivale

The accumulation of pathogenesis-related proteins such as β-1,3-glucanases and chitinases was studied in cold induced snow mould resistance in two Polish cultivars of winter triticale, cv. Hewo and cv. Magnat that substantially differ in resistance to Microdochium nivale. The plants were pre-hardened at 12°C for 10 days and hardened at 4°C for 28 days. Subsequently, cold hardened plants were inoculated with fungal mycelium (M. nivale) and incubated at 4°C for 7 days in dark. Cold acclimatisation resulted in suppression of the total glucanase and chitinases activities in the resistant Hewo as well as sensitive Magnat cultivars that possibly coincides with altered metabolism. However, upon infection with M. nivale the chitinases were markedly induced in the cv. Hewo. At the same time, total β-1,3 glucanases activities did not seem to be affected by fungus in any of the tested triticale cultivars. The pattern and/or the activity of chitinases in plants might be indicative for the resistance/susceptibility against M. nivale.     

Biochemical Changes in Tuber-bearing Solanum Species in Relation to Frost Hardiness during Cold Acclimation

Biochemical changes in potato leaves during cold acclimation have been examined and compared between a frost-tolerant S. acaule and a frost-susceptible S. tuberosum species. Changes were also examined in S. tuberosum, S. acaule, and S. commersonii species when they were hardened at different temperatures to varying hardiness levels.     

The effect of CCC on the nitrogen compounds content in rape plants and their frost hardiness. Relation to the conditions of day-length and temperature

Both CCC and cold (5°C) treatment gave rise to an increased content of the water-soluble proteins in leaf tissue of the winter rape, irrespective of the day length. This effect was accompanied by a decrease of the insoluble nitrogen compounds content, mainly under theLD (the 16 hour day). The applied retardant also stimulated the consumption of the structural compounds induced by low temperature. Low temperature treatment hardened plants more distinctly than the CCC application. The frost hardening effect of CCC andSD (the 8 hour day) was manifested only at 20°C and it disappeared at 5°C. Changes in frost hardiness were not correlated with the changes in the reducing sugar content and in the reducing ability of the studied tissue. The coincident effect of CCC and cold on the reducing sugar content was observed underSD conditions.     

Changes in fatty acid composition of lipids in chloroplast membranes of tobacco plants during cold hardening

Changes in fatty acid composition of chloroplast membrane lipids were investigated using tobacco (Nicotiana tabacum L., cv. Samsun) plants subjected to cold hardening for 6 days at 8°C. Under optimal growing temperature (22°C), the lipids of thylakoid membranes were characterized by elevated content of 16:3n-3 and 18:3n-3 fatty acids (FA). Compared to the lipids of chloroplast envelope membranes, the thylakoid lipids were less rich in the content of saturated, mono- and diunsaturated FA. The relative content of unsaturated FA in chloroplast membranes increased substantially during cold hardening, which was mainly due to the accumulation of 18:3n-3 FA. It is concluded that the observed changes in FA composition of chloroplast lipids during cold hardening adjust the fluidity of these membranes to the level sufficient for functioning of tobacco photosynthetic apparatus, which is a prerequisite for accumulation of assimilates and allows the hardened tobacco plants to survive under conditions of hypothermia.     

Uptake and Growth-promoting Activity of Indoleacetic Acid in Segments of Cold-hardened Wheat Coleoptiles

Seedings of winter wheat (Triticum aestivum L. cv. Kharkov MC 22) were grown at 24 C (unhardened) and 4 C (hardened). Indoleacetic acid (IAA) was added to excised coleoptile segments after lengthy incubation and their responses were determined by photometric auxanometry at both 25 C and 5 C. The segments' rates of uptake of ¹⁴CIAA were also compared at both temperatures. Cold hardening had no significant effect on the rates of elongation and uptake in a saturating concentration of IAA (2 to 10 μM) at either temperature. Elongation was more sensitive to temperature of measurement than was uptake. At suboptimal concentrations of IAA and 25 C, hardened coleoptiles took up [2-¹⁴C]-IAA twice as fast but elongated half as fast as unhardened coleoptiles. This and the lack of effect of cold hardening on apparent uptake of [1-¹⁴C]-IAA raised the possibility that a higher rate of IAA-decarboxylation was coupled with the higher rate of uptake of IAA by hardened coleoptiles. Homeostatic hormonal regulation was also evident in the same endogenous rates of elongation of segments of cold-hardened and unhardened coleoptiles.     

Cold Acclimation in Genetically Related (Sibling) Deciduous and Evergreen Peach (Prunus persica [L.] Batsch): I. Seasonal Changes in Cold Hardiness and Polypeptides of Bark and Xylem Tissues

Seasonal patterns of proteins and of cold hardiness were characterized in bark and xylem tissues of genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). In contrast with deciduous trees, which entered endodormancy and abscised leaves in the fall, evergreen trees retained their leaves and exhibited shoot elongation under favorable environmental conditions. A successive increase in the cold hardiness of bark and xylem was observed during the fall in both genotypes. This was followed by a subsequent decrease from midwinter to spring. Xylem tissue in both genotypes exhibited deep supercooling and a significant correlation (r = 0.99) between the midpoint of the low-temperature exotherm and the subzero temperature at which 50% injury occurred (assessed by electrolyte leakage) was noted. The maximum hardiness level attained in deciduous trees was more than twofold that of evergreens. Seasonal pattern of proteins from bark and xylem of the sibling genotypes was characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among other qualitative and quantitative changes, accumulation of a 19-kilodalton polypeptide in the bark of both genotypes was observed during fall followed by a decrease in spring. This polypeptide accumulated to higher levels in the deciduous peach compared with the evergreen. Additionally, a 16-kilodalton protein exhibited the same pattern in deciduous trees but not in the evergreen trees. Both the 19- and a 16-kilodalton bark proteins conform to the criteria of a bark storage protein. The relationship of seasonal changes in protein to cold hardiness and dormancy in these genetically related peach genotypes is discussed.     

Microbial Diversity and PAH Catabolic Genes Tracking Spatial Heterogeneity of PAH Concentrations

We analyzed the within-site spatial heterogeneity of microbial community diversity, polyaromatic hydrocarbon (PAH) catabolic genotypes, and physiochemical soil properties at a creosote contaminated site. Genetic diversity and community structure were evaluated from an analysis of denaturant gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified sequences of 16S rRNA gene. The potential PAH degradation capability was determined from PCR amplification of a suit of aromatic dioxygenase genes. Microbial diversity, evenness, and PAH genotypes were patchily distributed, and hot and cold spots of their distribution coincided with hot and cold spots of the PAH distribution. The analyses revealed a positive covariation between microbial diversity, biomass, evenness, and PAH concentration, implying that the creosote contamination at this site promotes diversity and abundance. Three patchily distributed PAH-degrading genotypes, NAH, phnA, and pdo1, were identified, and their abundances were positively correlated with the PAH concentration and the fraction of soil organic carbon. The covariation of the PAH concentration with the number and spatial distribution of catabolic genotypes suggests that a field site capacity to degrade PAHs may vary with the extent of contamination.