Effect of short-term dark incubation with sulfate, chloride and selenate on the glutathione content of spinach leaf discs

A 24 h incubation of leaf discs of spinach ( Spinacia oleracea L. cv. Estivato) in darkness with 50 and 100 m M sulfate resulted in a two- to three-fold increase in the level of glutathione (GSH), a compound which may serve as storage of excess reduced sulfur in the plant. The accumulated GSH was a small fraction (around 1%) of the sulfate taken up in the spinach leaf discs. Incubation of spinach leaf discs with 50 and 100 m M chloride resulted in only a 30% increase of the water-soluble non-protein-SH; the uptake of electrolytes was comparable to that observed with sulfate. This indicated that the increase of the GSH level upon incubation with sulfate was rather specific and not due to salinity. Incubation with 50 m M Na₂SO₄ did not affect water-soluble protein-SH content after 24 h. Addition of 1 and 10 m M selenate, an inhibitor of sulfate reduction, strongly reduced sulfate-induced GSH accumulation in spinach leaf discs, both in light and darkness. It was concluded that the sulfate-induced SH accumulation was due to a substantial de novo reduction of sulfate in darkness and subsequent incorporation of the reduced sulfur into GSH. The role of the sulfate concentration at the reaction site of ATP sulfurylase in the regulation of sulfur assimilation in the plant is discussed with respect to the low affinity of the enzyme for sulfate.     

Freezing injury in spinach leaf tissue: Effects on water-soluble proteins, protein-sulfhydryl and water-soluble non-protein-sulfhydryl groups

Freezing of spinach leaf discs ( Spinacia aleracea L. cv. Estivato) resulted in an irreversible and parallel loss of protein-sulfhydryl (SH) and water-soluble protein. This decrease was inversely related to the increase in freezing injury as determined by the loss of electrolytes from the tissue after thawing. Loss of proteins and protein-SH occurred during freezing of the tissue and was not enhanced by thawing. The parallel decreases in content of soluble proteins and SH groups make it impossible to determine whether oxidation of protein-SH groups is the primary step in decline of protein content. During freezing the content of non-protein-SH compounds, mainly glutathione (GSH), was decreased to a lesser extent than that of protein-SH. Contrary to protein-SH, the levels of non-protein-SH declined substantially after thawing. The data indicate that GSH is not directly involved in protection of soluble proteins against freezing-induced denaturation.     

Effects of frost-hardening and salinity on glutathione and sulfhydryl levels and on glutathione reductase activity in spinach leaves

In frost-hardened spinach leaves ( Spinucea oleracea L. ev. Vroeg Reuzenblad ) an enhanced content of water-soluble non-protein sulfhydryl compounds was observed. The enhancement was due to higher levels of glutathione as well as to other non-protein-bound sulfhydryl compounds. In addition glutathione reductase activity was increased upon hardening. The affinity of the enzyme for oxidized glutathione was slightly lowered during hardening. The significance of glutathione accumulation during frost-hardening is discussed. Exposure of spinach to NaCl-stress did not affect the levels of glutathione and glutathione reductase of the leaves. In addition the kinetic properties of the enzyme remained unaltered by salinity. It is suggested that glutathione and glutathione reductase activity are not involved in adaptation of spinach to saline conditions.     

Exogenously supplied glycine betaine in spinach and rapeseed leaf discs: compatibility or non-compatibility?

CMS, cell membrane stability
GB, glycine betaine
PEG, polyethylene glycol
TTC, 2,3,5-triphenyltetrazolium chloride

When leaf discs of spinach ( Spinacia oleracea cv. Junius) and rapeseed ( Brassica napus var. oleifera cv. Samourai) were incubated in the light in the presence of glycine betaine (GB), they accumulated GB at a very high level. In comparison with the spinach leaf explants, the uptake of GB by rapeseed tissues was restricted, probably by the destabilizing effects exerted by GB in this plant material. In contrast, the viability of spinach leaf discs, as assessed by their capacity to reduce 2,3,5-triphenyltetrazolium chloride (TTC), was not affected, suggesting that the GB taken up was compatible in the leaf tissues of the GB accumulator. In rapeseed leaf discs treated with GB, chlorophyll loss as well as significant changes in polyamine content were induced, leading to a dramatic increase of the putrescine/(spermidine + spermine) ratio. In contrast, this ratio remained constant in the GB treated spinach explants, suggesting that spinach has the capacity to stabilize polyamine metabolism in the presence of high amounts of GB. The treatment of spinach leaf discs with GB prior to application of osmotic or salt shocks provided protection from stress. A weak capacity to accumulate proline under stress conditions was partially suppressed. The protein content decreased while the free amino acid level increased independently of the presence of GB. It is concluded that GB behaves as a true compatible solute in spinach, which is a typical GB accumulator, and that GB is damaging when loaded into the leaf tissues of rapeseed, which do not normally accumulate GB.     

Cysteine, γ-glutamyl-cysteine and glutathione contents of spinach leaves as affected by darkness and application of excess sulfur. II. Glutathione accumulation in detached leaves exposed to H2S in the absence of light is stimulated by the supply of glycine to the petiole

When illuminated leaf discs and detached leaves of spinach ( Spinacia oleracea L. cv. Estivato) were exposed to 0.4 and 0.25 μl 1^-1 H₂S, respectively, pool sizes of cysteine and glutathione increased. In the dark, apart from these compounds, the level of γ-glutamyl-cysteine also increased. Incubation of leaf discs with 1.0 m M buthionine sulfoximine (BSO) resulted in the accumulation of cysteine only, both in the light and in darkness. When glycine was supplied to the petioles of detached leaves exposed to H₂S in the dark, the accumulation of glutathione was stimulated, while γ-glutamyl-cysteine accumulation was prevented completely. Glycolate and glyoxylate, precursors of glycine in the glycolate pathway, had nearly the same effect as glycine. Although other amino acids were apparently taken up equally well as glycine when supplied to the petiole, they were much less effective, or not effective at all, in restoring glutathione synthesis in the dark. These results provide evidence, that H₂S-induced glutathione accumulation in spinach leaves in the dark is limited by the availability of glycine, giving rise to the accumulation of the metabolic precursor γ-glutamyl-cysteine.     

Cysteine, γ-glutamyl-cysteine and glutathione contents of spinach leaves as affected by darkness and application of excess sulfur

In the light, glutathione was the major water-soluble, non-protein, sulfhydryl compound in leaves of spinach ( Spinacia oleracea L. cv. Estivato). In the dark, another sulfhydryl compound accumulated, which proved to be γ-glutamyl-cysteine. In the light, exposure of leaves to excess sulfur in the form of atmospheric H₂S (0.25 μl l⁻¹) resulted in considerably increased levels of glutathione and cysteine. In the dark, in addition to these thiols, levels of γ-glutamyl-cysteine were also enhanced considerably. When leaves of plants exposed to H₂S in the dark were illuminated, the dipeptide rapidly disappeared. At the same time, glutathione contents increased by approximately the same amount, indicating a light-dependent conversion of γ-glutamyl-cysteine into glutathione. Possible mechanisms for these light-induced changes in thiol metabolism are discussed.     

Acyl carrier protein is conjugated to glutathione in spinach seed

Acyl carrier protein (ACP) contains an essential sulfhydryl group in its phosphopantetheine prosthetic group. We have investigated the state of this sulfhydryl in developing and mature spinach seed (Spinacia oleracea). Seed extracts were separated on sodium dodecyl sulfate or native polyacrylamide gels, blotted to nitrocellulose, and probed with antibodies raised against spinach ACP-I. In extracts of mature seeds prepared with reducing agents, ACP-II migrated as a single major band, whereas extracts prepared without reducing agents gave two major bands. The additional band was identified as a conjugate of ACP-II to glutathione (ACP-S-S-G) on the basis of its sensitivity to reducing agents and its comigration with standards in both native and sodium dodecyl sulfate gel electrophoresis. In developing spinach seeds ACP-II exists primarily in its free sulfhydryl form or as acyl derivatives, with essentially no ACP-S-S-G present. During later stages of seed development, as seed water content declines, ACP-S-S-G accumulates to approximately 50% of the total ACP. Seed imbibition results in a rapid decline in ACP-S-S-G levels. The ACP-S-S-G:ACP-SH ratio of seeds during storage was found to be a function of seed water content and this could be manipulated by controlling the relative humidity under which the seeds were stored. We speculate that conjugation of ACP to glutathione protects the ACP from sulfhydryl oxidative damage in dry seeds.     

Changes in glutathione content during cold acclimation in Cornus sericea and Citrus sinensis

Glutathione content was evaluated in relation to freezing tolerance in red osier dogwood stems and Valencia orange leaves. Exposure of dogwood and citrus to cold-acclimating conditions in controlled environments led to increases in reduced glutathione (GSH) content which were correlated with freezing tolerance. GSH did not accumulate in field-grown dogwood stems during cold acclimation in fall, but did increase in content prior to deacclimation in late winter. Further studies showed that accumulation of GSH in dogwood at low temperatures is dependent on adequate levels of sulfate in the soil. In citrus, modulation of GSH content by infiltration of leaf tissue with various compounds including GSH did not alter freezing tolerance. Root treatment with N,N-diallyl-2,2-dichloroacetamide (R-25788) increased leaf GSH content, but not hardiness. Evidence presented indicates that glutathione accumulates in plant tissues exposed to low temperatures, but that GSH accumulation is not associated with freezing tolerance.     

The mechanism of photosynthetic sulfate reduction by isolated chloroplasts

The reduction of sulfate by isolated spinach chloroplasts was studied. A reconstituted system of broken chloroplasts and of chloroplast extract reduced sulfate to sulfite in the light when ADP, NADP⁺, ferredoxin and glutathione were added. The chloroplast extract reduced sulfate to sulfite in the dark if supplemented with ATP and with reduced glutathione. Neither ferredoxin nor NADPH were needed for this reduction in the dark.

A sulfite reductase was purified from spinach leaves. Broken chloroplasts and sulfite reductase reduced sulfite to sulfide in the light when ferredoxin was added. NADP⁺ was not required for this reduction.

The results suggest that in chloroplasts a sulfate activated by ATP (phosphoadenosine phosphosulfate) is reduced to sulfite by a sulfhydryl compound and that sulfite is reduced to sulfide by a ferredoxin-dependent sulfite reductase.     

Interaction between exogenous glycine betaine and the photorespiratory pathway in canola leaf discs

The uptake and accumulation of exogenously supplied glycine betaine (GB) by canola (which never accumulates GB in response to stress) leaf discs has been found to induce damage to some of their structural and functional components. As a consequence some free amino acids were accumulated, particularly glutamine and glycine. Similar results were obtained with leaf discs of Arabidopsis thaliana i.e. another cruciferous plant that does not naturally produce significant amounts of GB. In contrast no changes in glutamine and glycine contents were observed in response to the GB treatment in leaf discs of spinach, a natural producer of GB. The change in glutamine content might be related to the senescing effects caused by the GB treatment. Glycine accumulation in response to GB has been more thoroughly studied with canola leaf discs. It only occurred under light conditions and was suppressed under non-photorespiratory conditions. The accumulation of glycine in canola leaf discs in response to GB was either restricted when GB was added in the presence of aminooxyacetate (an inhibitor of transaminases) or enhanced when added in the presence of aminoacetonitrile (an inhibitor of glycine decarboxylation by mitochondria). Both compounds are known to block the glycolate pathway. Glycine accumulation was not found in leaf discs of Zea mays treated in the light in the presence of GB. These results suggest that the absorbed GB could exert destabilizing effects on the photorespiration of the C₃ cruciferous plants canola and Arabidopsis via competitive effects between GB and glycine at the mitochondrial step of the glycolate pathway. The mechanism of the GB effect remains to be elucidated as well as that of its apparent compatibility in spinach, the well known natural producer of GB.     

Effect of Light and Chilling Temperatures on Chilling-sensitive and Chilling-resistant Plants. Pretreatment of Cucumber and Spinach Thylakoids in Vivo and in Vitro

The effects of chilling temperatures, in light or dark, on the isolated thylakoids and leaf discs of cucumber (Cucumis sativa L. “Marketer”) and spinach (Spinacia oleracea L. “Bloomsdale”) were studied. The pretreatment of isolated thylakoids and leaf discs at 4 C in the dark did not affect the phenazine methosulfate-dependent phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, or chlorophyll content. Exposure of cucumber cotyledon discs and isolated thylakoids of cucumber and spinach to 4 C in light resulted in a rapid inactivation of the thylakoids. The sequence of activities or components lost during inactivation (starting with the most sensitive) are: phenazine methosulfate-dependent cyclic phosphorylation, proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity, and chlorophyll. The rate of loss of proton uptake, osmotic response to sucrose, Ca²⁺-dependent ATPase activity and chlorophyll is similar for isolated cucumber and spinach thylakoids, whereas spinach thylakoids are more resistant to the loss of phenazine methosulfate-dependent phosphorylation. The thylakoids of spinach leaf discs were unaffected by exposure to 4 C in light. The results question whether the extreme resistance of spinach thylakoids treated in vivo is solely a function of the chloroplast thylakoid membranes and establish the validity of using in vitro results to make inferences about cucumber thylakoids treated in vivo at 4 C in light.     

Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter, Plant Physiol [1991] 97: 175-181). The objective of this study was to determine whether the LT₅₀ (lethal temperature: the freezing temperature at which 50% of the tissue is killed) of spinach leaf tissue can be changed by diminishing the extent of microtubule depolymerization in response to freezing. Also examined was how tolerance to the components of extracellular freezing, low temperature and dehydration, is affected by microtubule stabilization. Leaf sections of nonacclimated and cold-acclimated spinach were treated with 20 micromolar taxol, a microtubule-stabilizing compound, prior to freezing, supercooling, or dehydration. Taxol stabilized microtubules against depolymerization in cells subjected to these stresses. When pretreated with taxol both nonacclimated and cold-acclimated cells exhibited increased injury during freezing and dehydration. In contrast, supercooling did not injure cells with taxol-stabilized microtubules. Electrolyte leakage, visual appearance of the cells, or a microtubule repolymerization assay were used to assess injury. As leaves were cold-acclimated beyond the normal period of 2 weeks taxol had less of an effect on cell survival during freezing. In leaves acclimated for up to 2 weeks, stabilizing microtubules with taxol resulted in death at a higher freezing temperature. At certain stages of cold acclimation, it appears that if microtubule depolymerization does not occur during a freeze-thaw cycle the plant cell will be killed at a higher temperature than if microtubule depolymerization proceeds normally. An alternative explanation of these results is that taxol may generate abnormal microtubules, and connections between microtubules and the plasma membrane, such that normal cellular responses to freeze-induced dehydration and subsequent rehydration are blocked, with resultant enhanced freezing injury.     

Choline Synthesis in Spinach in Relation to Salt Stress

Choline metabolism was examined in spinach (Spinacia oleracea L.) plants growing under nonsaline and saline conditions. In spinach, choline is required for phosphatidylcholine synthesis and as a precursor for the compatible osmolyte glycine betaine (betaine). When control (nonsalinized) leaf discs were incubated for up to 2 h with [1,2-14C]ethanolamine, label appeared in the N-methylated derivatives of phosphoethanolamine including phosphomono-, phosphodi-, and phosphotri- (i.e. phosphocholine) methyl-ethanolamine, as well as in choline and betaine, whereas no radioactivity could be detected in the mono- and dimethylated derivatives of the free base ethanolamine. Leaf discs from salinized plants showed the same pattern of labeling, although the proportion of label that accumulated in betaine was almost 3-fold higher in the salinized leaf discs. Enzymes involved in choline metabolism were assayed in crude leaf extracts of plants. The activites of ethanolamine kinase and of the three S-adenosylmethionine:phospho-base N-methyltransferase enzymes responsible for N-methylating phosphoethanolamine to phosphocholine were all higher in extracts of plants salinized step-wise to 100, 200, or 300 mM NaCI compared with controls. In contrast, choline kinase, phosphocholine phosphatase, and cytidine 5[prime]-triphosphate: phosphocholine cytidylyltransferase activities showed little variation with salt stress. Thus, the increased diversion of choline to betaine in salt-stressed spinach appears to be mediated by the increased activity of several key enzymes involved in choline biosynthesis.     

Cryopreservation alters membrane sulfhydryl status of bull spermatozoa: protection by oxidized glutathione.

Cryopreservation induces extensive biophysical and biochemical changes in the membrane of spermatozoa that ultimately decrease the fertility potential of the cells. Sulfhydryl groups of sperm proteins regulate a number of activities of the cells. Qualitative and quantitative analyses of sulfhydryl groups in the sperm membrane were performed by fluorescence microscopy, fluorimetry and electrophoresis. Fluorimetric analysis using 5-iodoacetamidofluoresceine indicated a two-fold increase in the content of sulfhydryl groups in sperm membrane after a freezing/thawing cycle. Electrophoresis of Triton-soluble sperm proteins after labeling with 3-(N-maleimidylpropionyl) biocytin indicated that proteins of 40-65 and 34 kDa range expose more sulfhydryl groups after cooling at 4 degrees C and freezing/thawing. Cryopreservation of spermatozoa changed the distribution pattern of sulfhydryl groups on sperm surface measured with fluorescence microscopy using 5-iodoacetamidofluoresceine. The percentage of spermatozoa labeled at the level of the mid-piece decreased by 50 and 90% after cooling and freezing/thawing, respectively. Spin labeling studies showed a 15% faster rotational diffusion (mobility) of sulfhydryl containing proteins in the membrane of frozen/thawed spermatozoa as compared to that of fresh spermatozoa. Addition of glutathione, reduced (GSH) or oxidized (GSSG), to the cryoprotectant partially prevented the effects of freezing/thawing, such as higher exposure of sulfhydryl groups, changes in the cellular distribution, and enhanced rotational diffusion of sulfhydryl containing proteins of sperm membrane. Addition of GSSG to the cryoprotectant reduced by 35% the loss of motility of spermatozoa undergoing a freezing/thawing cycle. We concluded that cryopreservation perturbs sperm membrane sulfhydryl containing proteins and that these modifications could be partially prevented by the addition of GSSG to the cryopreservation medium.     

Synthesis of Glutathione in Leaves of Transgenic Poplar Overexpressing [gamma]-Glutamylcysteine Synthetase

Internode stem fragments of the poplar hybrid Populus tremula x Populus alba were transformed with a bacterial gene (gshl) for [gamma]-glutamylcysteine synthetase ([gamma]-ECS) targeted to the cytosol. Lines overexpressing [gamma]-ECS were identified by northern analysis, and the transformant with the highest enzyme activity was used to investigate the control of glutathione synthesis. Whereas foliar [gamma]-ECS activity was below the limit of detection in untransformed plants, activities of up to 8.7 nmol mg-1 protein min-1 were found in the transformant, in which the foliar contents of [gamma]-glutamylcysteine ([gamma]-EC) and glutathione were increased approximately 10- and 3-fold, respectively, without affecting either the reduction state of the glutathione pool or the foliar cysteine content. A supply of exogenous cysteine to leaf discs increased the glutathione content from both transformed and untransformed poplars, and caused the [gamma]-EC content of the transformant discs to increase still further. The following conclusions are drawn: (a) the native [gamma]-ECS of untransformed poplars exists in quantities that are limiting for foliar glutathione synthesis; (b) foliar glutathione synthesis in untransformed poplars is limited by cysteine availability; (c) in the transformant interactions between glutathione synthesis and cysteine synthesis operate to sustain the increased formation of [gamma]-EC and glutathione; and (d) the foliar glutathione content of the transformant is restricted by cysteine availability and by the activity of glutathione synthetase.     

Chloroplast and Nuclear DNA Content of Cultured Spinach Leaf Discs

The amounts of chloroplast DNA and nuclear DNA in the cellsof spinach leaf discs cultutred under different light regimeshave been measured. The cellular level of ctDNA increased 10-foldin discs cultured in white light and was accompanied by a 2-foldincrease in the cellular level of nuclear DNA. In low intensitygreen light the cellular level of ctDNA increased 6-fold butwas not accompanied by an increase in the level of nuclear DNA.No net DNA synthesis on a per cell basis occurred in discs culturedin darkness. Chloroplasts of uncultured leaf discs containedan average of 83 plastome copies. The number of plastome copiesper chloroplast after 6 days culture decreased to 36 copiesin darkness, remained almost constant at 73 copies in whitelight and increased to 215 copies in low intensity green light. These results suggest that ctDNA replication can be independentof cellular levels of nuclear DNA and chloroplast division.     

Formation of methanethiol from methionine by leaf tissue

Leaf discs, but not detached leaves, exposed to L-methionine or S-methyl-L-cysteine emitted a volatile sulphur compound identified as methanethiol by different trapping systems and by GC. Methanethiol emission was analyzed using pumpkin (Cucurbita pepo) leaf discs. Emission was observed in darkness or light, however methanethiol emission was greately stimulated by light. Light-dependent emission started after a lag-time of 5–6 hr with an emission peak after 36–40 hr. Maximum rates obtained were in the range of 200 pmol methanethiol/min/cm² leaf area. After a period of 42 hr about 60–80% of total methionine sulphur added was released as methanethiol. Addition of chloramphenicol did not alter the induction period nor the maximum emission rate of methanethiol in response to L-methionine. Emission was also observed in response to S-methyl-L-cysteine; however, the shorter lag-period for methanethiol formation suggests metabolism via a different enzyme system. In a cell-free system of pumpkin leaves methanethiol formation occured in response to L-methionine. Feeding experiments with L-[³⁵S]methionine to leaf discs showed that more than 80% of methanethiol emitted was derived from the labelled methionine fed. These findings suggest that plants have the capacity to degrade L-methionine to methanethiol. Whole leaves fed L-methionine by the petiole system do not emit methanethiol, but this compound is formed and transported into the feeding solution. Thus, methanethiol is also produced by the intact leaf, but, in contrast to sulphide, is not released into the atmosphere. It is suggested that translocation of methanethiol may function as a signal for the regulation of sulphate uptake.     

Role of silicon in enhancing resistance to freezing stress in two contrasting winter wheat cultivars

The main objective of this study was to elucidate the roles of silicon (Si) in enhancing tolerance to freezing stress (?5 °C) in two contrasting wheat (Triticum aestivum L.) cultivars: i.e. cv. Yangmai No. 5, a freezing-susceptible cultivar and cv. Linmai No. 2, a freezing-tolerant cultivar. Shoot dry weight of the freezing-susceptible wheat was significantly lower under freezing stress than in controls, but increased significantly with Si amendment. The freezing treatment did not affect shoot dry weight of the freezing-tolerant cultivar. The leaf water content was considerably decreased by freezing stress in the freezing-susceptible cultivar, but was significantly increased by Si amendment. In contrast, freezing treatment did not significantly reduce leaf water content in the freezing-tolerant cultivar and Si played no role in water retention in this cultivar. The concentrations of H₂O₂ and free proline along with malondialdehyde (MDA) were progressively enhanced by freezing stress in the two wheat cultivars used, but were significantly suppressed by amendment with Si. The major antioxidant enzyme activities and non-enzymatic antioxidants (i.e. glutathione and ascorbic acid) in the leaves of freezing-stressed plants were decreased, but were stimulated significantly by the exogenous Si. The possible mechanisms for Si-enhanced freezing stress may be attributed to the higher antioxidant defense activity and lower lipid peroxidation through water retention in leaf tissues.     

Metabolism of hydroxylamine by spinach leaf discs and its relationship to nitrate reduction

Nitrate reduction in vivo by spinach leaf discs was shown to be inhibited by hydroxylamine when this was included in the nitrate reductase assay solutions or introduced to the tissue during a preincubation period. The sensitivity of nitrate reduction to hydroxylamine was not sufficient to suggest a natural process, considering the small endogenous concentrations of hydroxylamine in the leaves. Inhibition of nitrate reduction in vivo could be approximately related to rates of in vitro inhibition of nitrate reductase by this compound. There was no need to suppose conversion of hydroxylamine to cyanide to inhibit nitrate reduction. Some of the in vivo and in vitro characteristics of hydroxylamine inhibition of nitrate reductase are described. Hydroxylamine was metabolised by discs at rates comparable to nitrate reduction. Rates of metabolism of hydroxylamine, and its accumulation in the tissues from an external solution were both enhanced by light but little affected by anaerobiosis.Abbreviations NR nitrate reductase     

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.