Relationship between Thermal Transitions and Freezing Injury in Pea and Soybean Seeds

In an attempt to correlate freezable water with freezing injury, the thermal behavior of pea (Pisum sativum L.) and soybean (Glycine max L. Merr) seed parts at different moisture contents were compared with survival of the seeds when exposed to low temperatures. Thermal transitions between −150 and 10°C were studied using differential scanning calorimetry. In pea, reduction of germinability, after exposure of seeds to temperatures between − 18 and − 180°C, occurred at a constant moisture content (about 0.33 gram H₂O/gram dry weight) regardless of the temperature; this moisture level was above that at which freezable water was first detectable by differential scanning calorimetry (0.26 gram H₂O/gram dry weight). In contrast, damage to soybean seeds was observed at progressively lower moisture contents (from 0.33 to 0.20 gram H₂O/gram dry weight) when the temperature was decreased from −18°C to −50°C. At −18 and −30°C, moisture contents at which damage to soybean seeds was evident were above that at which freezable water was first detectable (0.23 gram H₂O/gram dry weight). However, at −50, −80, and −180°C, damage was evident even when freezable water was not detectable. The data suggest that, while the quantity of water is important in the expression of freezing injury, the presence of freezable water does not account for the damage.     

Different plant provenance same seed tolerance to abiotic stress: implications for <Emphasis Type="Italic">ex situ</Emphasis> germplasm conservation of a widely distributed coastal dune grass (<Emphasis Type="Italic">Uniola paniculata</Emphasis> L.)

Preservation of genetic diversity within germplasm repositories represents an important tool for plant conservation. However, seeds must tolerate extreme levels of post-harvest desiccation and cold to realize benefits of ex situ storage. Factors including local climate and habitat impact expression of desiccation and freezing tolerance especially for widely distributed species. Our aim here was to understand the influence of a latitudinal gradient on seed desiccation and cryo-freezing tolerance. We sampled mature U. paniculata seeds from two geographically and genetically distinct populations then examined seed-water relations and germination following desiccation via equilibrium drying assays (0.5 to 91% RH; ?797 to ?12.9 MPa). Germination ability after drying and subsequent cryo-freezing treatments (?196?°C, 1 to 1440 min) was also evaluated. Seeds of both populations displayed similar reverse sigmoid moisture sorption isotherms characteristic of desiccation tolerant tissues. Furthermore, initial seed water potential (?63 and ?90 MPa) was considerably lower than the lethal limit (?20 MPa) identified for desiccation sensitive tissues. Final germination (range 58–93%) and temporal patterns differed significantly between populations following desiccation and cryo-freezing stress, but these germination responses were similar to initial germination. A higher proportion of non-germinated, yet viable seeds remained for the northern compared to southern population. Location does influence germination response, but differential germination is related to seed dormancy rather than desiccation or cryo-freezing sensitivity. Ex situ conservation of U. paniculata is therefore feasible across the latitudinal gradient studied here.     

Effects of cooling rate on seeds exposed to liquid nitrogen temperatures

The effect of cooling rate on seeds was studied by hydrating pea (Pisum sativum), soybean (Glycine max), and sunflower (Helianthus annuus) seeds to different levels and then cooling them to − 190°C at rates ranging from 1°C/minute to 700°C/minute. When seeds were moist enough to have freezable water (> 0.25 gram H₂O/gram dry weight), rapid cooling rates were optimal for maintaining seed vigor. If the seeds were cooled while at intermediate moisture levels (0.12 to 0.20 gram H₂O per gram dry weight), there appeared to be no effect of cooling rate on seedling vigor. When seeds were very dry (< 0.08 gram H₂O per gram dry weight), cooling rate had no effect on pea, but rapid cooling rates had a marked detrimental effect on soybean and sunflower germination. Glass transitions, detected by differential scanning calorimetry, were observed at all moisture contents in sunflower and soybean cotyledons that were cooled rapidly. In pea, glasses were detectable when cotyledons with high moisture levels were cooled rapidly. The nature of the glasses changed with moisture content. It is suggested that, at high moisture contents, glasses were formed in the aqueous phase, as well as the lipid phase if tissues had high oil contents, and this had beneficial effects on the survival of seeds at low temperatures. At low moisture contents, glasses were observed to form in the lipid phase, and this was associated with detrimental effects on seed viability.     

Increased Water Activity Reduces the Thermal Resistance of Salmonella enterica in Peanut Butter

Increased water activity in peanut butter significantly (P < 0.05) reduced the heat resistance of desiccation-stressed Salmonella enterica serotypes treated at 90°C. The difference in thermal resistance was less notable when strains were treated at 126°C. Using scanning electron microscopy, we observed minor morphological changes of S. enterica cells resulting from desiccation and rehydration processes in peanut oil.     

Temperature-Induced Change in the Water Relations of Abies amabilis (Dougl.) Forbes

Water conductance through Abies amabilis seedlings was measured while the roots were exposed to temperatures from 15 to 0.25°C. Before conductance was measured, the seedlings were preconditioned for 3 months at either a high temperature (23°C) or a low temperature (3°C). For both groups of seedlings, conductance decreased as root temperature decreased. Conductance was lowest at 0.25°C. In addition, preconditioning at 3°C for 3 months significantly lowered conductance to water at all root temperatures. Under the same environmental conditions, seedlings preconditioned at 3°C had less than 25% of the transpirational water loss of seedlings preconditioned at high temperature. A decrease in leaf osmotic potential also resulted from low temperature preconditioning. In trees growing in the subalpine forest, which is the natural habitat of Abies amabilis, both decreased leaf conductance to water vapor and lower osmotic potentials were evident in winter. Since in winter the temperature of the soil in the subalpine zone remains less than 1°C for many months, lowered leaf conductance and decreased osmotic potentials appear to be mechanisms which aid in preventing desiccation damage.     

Influence of the Natural Microbial Flora on the Acid Tolerance Response of Listeria monocytogenes in a Model System of Fresh Meat Decontamination Fluids

Depending on its composition and metabolic activity, the natural flora that may be established in a meat plant environment can affect the survival, growth, and acid tolerance response (ATR) of bacterial pathogens present in the same niche. To investigate this hypothesis, changes in populations and ATR of inoculated (10⁵ CFU/ml) Listeria monocytogenes were evaluated at 35°C in water (10 or 85°C) or acidic (2% lactic or acetic acid) washings of beef with or without prior filter sterilization. The model experiments were performed at 35°C rather than lower (≤15°C) temperatures to maximize the response of inoculated L. monocytogenes in the washings with or without competitive flora. Acid solution washings were free (<1.0 log CFU/ml) of natural flora before inoculation (day 0), and no microbial growth occurred during storage (35°C, 8 days). Inoculated L. monocytogenes died off (negative enrichment) in acid washings within 24 h. In nonacid (water) washings, the pathogen increased (approximately 1.0 to 2.0 log CFU/ml), irrespective of natural flora, which, when present, predominated (>8.0 log CFU/ml) by day 1. The pH of inoculated water washings decreased or increased depending on absence or presence of natural flora, respectively. These microbial and pH changes modulated the ATR of L. monocytogenes at 35°C. In filter-sterilized water washings, inoculated L. monocytogenes increased its ATR by at least 1.0 log CFU/ml from days 1 to 8, while in unfiltered water washings the pathogen was acid tolerant at day 1 (0.3 to 1.4 log CFU/ml reduction) and became acid sensitive (3.0 to >5.0 log CFU/ml reduction) at day 8. These results suggest that the predominant gram-negative flora of an aerobic fresh meat plant environment may sensitize bacterial pathogens to acid.     

Bound Water in Durum Wheat under Drought Stress

To study drought stress effects on bound water, adsorption isotherms and pressure-volume curves were constructed for two durum wheat (Triticum durum Desf.) cultivars: Capeiti 8 (drought tolerant) and Creso (drought sensitive). Plants were grown under well-watered and water-stressed conditions in a controlled environment. Differential enthalpy (ΔH) was calculated through van't Hoff analysis of adsorption isotherms at 5 and 20°C, which allowed us to determine the strength of water binding. ΔH reached the most negative values at approximately 0.06 gram H₂O/gram dry weight and then increased rapidly for well-watered plants (until 0.10 gram H₂O/gram dry weight) or more slowly for drought-stressed plants (until 0.15-0.20 gram H₂O/gram dry weight). Bound water values from pressure-volume curves were greater for water-stressed (0.17 gram H₂O/gram dry weight) than for well-watered plants (0.09 gram H₂O/gram dry weight). They may be estimates of leaf moisture content where ΔH reaches the less negative values and hence some free water appears. With respect to the well-watered plants, tightly bound water tended to be less bound during drought, and more free water was observed in cv Creso compared to cv Capeiti 8 at moisture contents >0.10 gram H₂O/gram dry weight.     

Temperature Effects on Mitochondrial Respiration in Phaseolus acutifolius A. Gray and Phaseolus vulgaris L

Electron transport, using succinate as a substrate, was measured polarographically in mitochondria isolated from Phaseolus vulgaris and P. acutifolius plants at 25°C and 32°C. Mitochondria isolated from P. vulgaris plants grown at 32°C had reduced electron transport and were substantially uncoupled. Growth at 32°C had no effect on electron transport or oxidative phosphorylation in P. acutifolius compared to 25°C grown plants. Mitochondria isolated from 25°C grown P. vulgaris plants measured at 42°C were completely uncoupled. Similarly treated P. acutifolius mitochondria remained coupled. The uncoupling of P. vulgaris was due to increased proton permeability of inner mitochondrial membrane. The alternative pathway was more sensitive to heat than the regular cytochrome pathway. At 42°C, no alternative pathway activity was detected. The substantially greater heat tolerance of P. acutifollus compared to P. vulgaris mitochondrial electron transport suggests that mitochondrial sensitivity to elevated temperatures is a major limitation to growth of P. vulgaris at high temperatures and is an important characteristic conveying tolerance in P. acutifolius.     

Desiccation tolerance,longevity and seed-siring ability of entomophilous pollen from UK native orchid species

Background and Aims

Pollinator-limited seed-set in some terrestrial orchids is compensated for by the presence of long-lived flowers. This study tests the hypothesis that pollen from these insect-pollinated orchids should be desiccation tolerant and relatively long lived using four closely related UK terrestrial species; Anacamptis morio, Dactylorhiza fuchsii, D. maculata and Orchis mascula.

Methods

Pollen from the four species was harvested from inflorescences and germinated in vitro, both immediately and also after drying to simulate interflower transit. Their tolerance to desiccation and short-term survival was additionally assessed after 3 d equilibration at a range of relative humidities (RHs), and related to constructed sorption isotherms (RH vs. moisture content, MC). Ageing of D. fuchsii pollen was further tested over 2 months against temperature and RH, and the resultant survival curves were subjected to probit analysis, and the distribution of pollen death in time (σ) was determined. The viability and siring ability, following artificial pollinations, were determined in D. fuchsii pollen following storage for 6 years at –20 °C.

Key Results

The pollen from all four species exhibited systematic increases in germinability and desiccation tolerance as anthesis approached, and pollen from open flowers generally retained high germinability. Short-term storage revealed sensitivity to low RH, whilst optimum survival occurred at comparable RHs in all species. Similarly, estimated pollen life spans (σ) at differing temperatures were longest under the dry conditions. Despite a reduction in germination and seeds per capsule, long-term storage of D. fuchsii pollen did not impact on subsequent seed germination in vitro.

Conclusions

Substantial pollen desiccation tolerance and life span of the four entomophilous orchids reflects a resilient survival strategy in response to unpredictable pollinator visitation, and presents an alternative approach to germplasm conservation.     

Evaluation of water binding, seed coat permeability and germination characteristics of wheat seeds equilibrated at different relative humidities

The relative binding of seed water and seed coat membrane stability were measured in two contrasting wheat (Triticum aestivum L) varieties, HDR 77 (drought-tolerant) and HD 2009 (susceptible) using seed water sorption isotherms, electrical conductivity (EC) of leachates and desorption-absorption isotherms. Analysis of sorption isotherm at 25 degrees C showed that the seeds of HDR 77 had significantly higher number of strong binding sites, with correspondingly greater amount of seed water as strongly bound water, as compared to HD 2009. Total number of binding sites was also higher in HDR 77 than HD 2009, which explained the better desiccation tolerance and higher capacity to bind water in seeds of HDR 77. EC of seed leachate in both varieties did not change with respect to change in equilibrium relative humidity (RII), indicating the general seed coat membrane stability of wheat seeds. However, absolute conductivity values were higher for HD 2009. showing its relatively porous seed coat membrane. Significantly lower area enclosed by the desorption-absorption isotherm loop in HDR 77, as compared to HD 2009 also indicated the greater membrane integrity of HDR 77. Germination and seedling vigour of HD 2009 were reduced when equilibrated over very low and very high RH. In contrast, germination and vigour in HDR 77 were maintained high, except at very high RH, indicating again its desiccation tolerance. Thus, the study demonstrated the relative drought tolerance of HDR 77, on the basis of seed water-binding characteristics and seed membrane stability. Seed membrane stability as measured by seed leachate conductivity or as area under dehydration-rehydration loop may be used as a preliminary screening test for drought tolerance in wheat.     

Influence of Temperature Stress on in Vitro Fertilization and Heat Shock Protein Synthesis in Maize (Zea mays L.) Reproductive Tissues

This study was conducted to investigate the response of maize (Zea mays) male and female mature reproductive tissues to temperature stress. We have tested the fertilization abilities of the stressed spikelets and pollen using in vitro pollination-fertilization to determine their respective tolerance to stress. The synthesis of heat shock proteins (HSPs) was also analyzed in male and female tissues using electrophoresis of ³⁵S-labeled proteins and fluorography, to establish a relationship between the physiological and molecular responses. Pollen, spikelets, and pollinated spikelets were exposed to selected temperatures (4, 28, 32, 36, or 40°C) and tested using an in vitro fertilization system. The fertilization rate is highly reduced when pollinated spikelets are exposed to temperatures over 36°C. When pollen and spikelets are exposed separately to temperature stress, the female tissues appear resistant to 4 hours of cold stress (4°C) or heat stress (40°C). Under heat shock conditions, the synthesis of a typical set of HSPs is induced in the female tissues. In contrast, the mature pollen is sensitive to heat stress and is responsible for the failure of fertilization at high temperatures. At the molecular level, no heat shock response is detected in the mature pollen.     

Thermotolerance of isolated mitochondria associated with heat shock proteins

Mitochondria isolated from 2-day-old etiolated soybean (Glycine max) seedlings which had been subjected to various heat shock treatments, i.e. (A) 28°C (2 h), (B) 38°C (2 h), (C) 38°C (2 h)-42.5°C (0.5 h), and (D) 38°C (2 h)-42.5°C (0.5 h)-28°C (4 h), were monitored for O₂ uptake using an oxygen electrode. Mitochondria isolated after all four heat shock treatments were active in O₂ consumption at 28°C in response to succinate and ADP (derived P/O ratios were 1.6, 1.7, 1.3, and 1.3, respectively.) The mitochondria from all four treatments were also active in O₂ uptake at 42.5°C. However, only mitochondria isolated after treatment (C) were tightly coupling at 42.5°C (derived ADP/O ratio was about 1.4). Combined with our earlier findings on the subcellular localization of heat shock proteins, our present data demonstrate that association of heat shock proteins with mitochondria by treatment (C) enables them to phosphorylate at 42.5°C (i.e. they become thermotolerant). Isolated mitochondria from treatment (C) and treatment (A) were compared by electron microscopy. They appeared to be very similar and no significant ultrastructural differences were noted.     

Thermal Tolerance of Zymomonas mobilis: Temperature-Induced Changes in Membrane Composition

The membrane composition of Zymomonas mobilis changed dramatically in response to growth temperature. With increasing temperature, the proportion of vaccenic acid declined with an increase in myristic acid, the proportion of phosphatidylcholine and cardiolipin increased with decreases in phosphatidylethanolamine and phosphatidylglycerol, and the phospholipid/protein ratio of the membrane declined. These changes in membrane composition were correlated with changes in thermal tolerance and with changes in membrane fluidity. Cells grown at 20°C were more sensitive to inactivation at 45°C than were cells grown at 30°C, as expected. However, cells grown at 41°C (near the maximal growth temperature for Z. mobilis) were hypersensitive to thermal inactivation, suggesting that cells may be damaged during growth at this temperature. When cells were held at 45°C, soluble proteins from cells grown at 41°C were rapidly lost into the surrounding buffer in contrast to cells grown at lower temperatures. The synthesis of phospholipid-deficient membranes during growth at 41°C was proposed as being responsible for this increased thermal sensitivity.     

Photosynthetic Response of Soybean to Microclimate in 26-Year-Old Tree-Based Intercropping Systems in Southern Ontario,Canada

In order to study the effect of light competition and microclimatic modifications on the net assimilation (NA), growth and yield of soybean (Glycine max L.) as an understory crop, three 26-year-old soybean-tree (Acer saccharinum Marsh., Populus deltoides X nigra, Juglans nigra L.) intercropping systems were examined. Tree competition reduced photosynthetically active radiation (PAR) incident on soybeans and reduced net assimilation, growth and yield of soybean. Soil moisture of 20 cm depth close (< 3 m) to the tree rows was also reduced. Correlation analysis showed that NA and soil water content were highly correlated with growth and yield of soybean. When compared with the monoculture soybean system, the relative humidity (RH) of the poplar-soybean, silver maple-soybean, and black walnut-soybean intercropped systems was increased by 7.1%, 8.0% and 5.9%, soil water content was reduced by 37.8%, 26.3% and 30.9%, ambient temperature was reduced by 1.3°C, 1.4°C and 1.0°C, PAR was reduced by 53.6%, 57.9% and 39.9%, and air CO₂ concentration was reduced by 3.7μmol·mol^-1, 4.2μmol·mol^-1 and 2.8μmol·mol^-1, respectively. Compared to the monoculture, the average NA of soybean in poplar, maple and walnut treatments was also reduced by 53.1%, 67.5% and 46.5%, respectively. Multivariate stepwise regression analysis showed that PAR, ambient temperature and CO₂ concentration were the dominant factors influencing net photosynthetic rate.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

Increased rate of drying reduces metabolic inequity and critical water content in radicles of Cicer arietinum L.

Orthodox seed serves as easily accessible model to study desiccation-sensitivity in plant tissues because once they undergo germination, they become sensitive to desiccation imposed injuries. In the proposed study, effects of rate of drying on the viability, electrolyte leakage, superoxide accumulation, lipid-protein oxidation and antioxidant enzymes were explored in excised radicles of Cicer arietinum L. under dehydration and wet storage. For both the drying conditions, desiccation could be explained by exponential and inverse functions. Under rapid drying tissue viability as scored by germination efficiency and tetrazolium staining remained 100 % all through the analysis (24 h) but declined remarkably after 0.30 g g⁻¹ fresh mass water content (4 days) under slow drying. Moreover, precipitous fall in tissue viability was observed after 2 weeks of wet storage. Rapid drying was also accompanied with limited amounts of electrolyte leakage, superoxide radical, malondialdehyde and protein hydroperoxide, together with enhanced level of protein. Additionally, activities of both superoxide dismutase and ascorbate peroxidase were increased in rapidly dried radicles, but guaiacol peroxidase was declined. In contrary, above referred biomarkers were observed to perform either inversely or poorly during slow drying and wet storage suggesting that above documented alterations might be the resultant of ageing and not desiccation. Gathered data demonstrated that increased drying lowers the critical water content for tissue survival and also reduces the risk of damage resulting from aqueous-based deleterious reactions. Additionally, it also showed that growing radicles are a popular model to explore desiccation-sensitivity in plant tissues and/or seeds.     

Chlorophyll fluorescence characteristics associated with hydration level in pea cotyledons

In order to study the effects of desiccation on a photosynthetic system, light harvesting and light-induced electron transport processes were examined in pea cotyledons at various moisture levels, using in vivo fluorescence excitation spectra and fluorescence induction kinetics. Water sorption isotherms yielded thermodynamic data that suggested very strong water binding between 4 to 11% water, intermediate sorption between water contents of 13 to 22%, and very weak binding at moisture contents between 24 to 32%. The fluorescence properties of the tissue changed with the moisture contents, and these changes correlated generally with the three regions of water binding. Peak fluorescence and fluorescence yield remained at low levels when water content was limited to the tightly bound regions, below 12%. Several new peaks appeared in the chlorophyll a excitation spectrum and both peak fluorescence and fluorescence yield increased at intermediate water-binding levels (12-22%). At moisture contents where water is weakly bound (>24%), peak fluorescence and fluorescence yield were maximum and the fluorescence excitation spectrum was unchanging with further increases in water content.

The state of water is an important component in the energy transfer and electron transport system. At hydration levels where water is most tightly bound, energy transfer from pigments is limited and electron transport is blocked. At intermediate water binding levels, energy transfer and electron transport increase and, in the region of weak water binding, energy transfer and electron transport are maximized.

     

The role of antioxidant defense in freezing tolerance of resurrection plant Haberlea rhodopensis

Haberlea rhodopensis Friv. is unique with its ability to survive two extreme environmental stresses—desiccation to air-dry state and subzero temperatures. In contrast to desiccation tolerance, the mechanisms of freezing tolerance of resurrection plants are scarcely investigated. In the present study, the role of antioxidant defense in the acquisition of cold acclimation and freezing tolerance in this resurrection plant was investigated comparing the results of two sets of experiments—short term freezing stress after cold acclimation in controlled conditions and long term freezing stress as a part of seasonal temperature fluctuations in an outdoor ex situ experiment. Significant enhancement in flavonoids and anthocyanin content was observed only as a result of freezing-induced desiccation. The total amount of polyphenols increased upon cold acclimation and it was similar to the control in post freezing stress and freezing-induced desiccation. The main role of phenylethanoid glucoside, myconoside and hispidulin 8-C-(2-O-syringoyl-b-glucopyranoside) in cold acclimation and freezing tolerance was elucidated. The treatments under controlled conditions in a growth chamber showed enhancement in antioxidant enzymes activity upon cold acclimation but it declined after subsequent exposure to −10 °C. Although it varied under ex situ conditions, the activity of antioxidant enzymes was high, indicating their important role in overcoming oxidative stress under all treatments. In addition, the activity of specific isoenzymes was upregulated as compared to the control plants, which could be more useful for stress counteraction compared to changes in the total enzyme activity, due to the action of these isoforms in the specific cellular compartments.Supplementary informationThe online version contains supplementary material available at 10.1007/s12298-021-00998-0.     

Enhancement of Chilling Resistance of Inoculated Grapevine Plantlets with a Plant Growth-Promoting Rhizobacterium, Burkholderia phytofirmans Strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26°C) and low (4°C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26°C and 4°C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26°C and 4°C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO₂ fixation and O₂ evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.