Ameliorative effect of melatonin on meristematic cells of chilled and re-warmed Vigna radiata roots

Changes in ultrastructure of meristematic cells as well as growth and lipid peroxidation in roots of 3-d-old seedlings obtained from control (C), hydroprimed (H) and hydroprimed with melatonin (H-MEL) seeds after 2 d of incubation at 25 or 5 °C and 2 d of re-warming after chilling were investigated. Under 25 °C hydropriming (H and H-MEL) inhibited root growth, but after chilling and re-warming a positive MEL effect on root elongation was observed. The results show decreased lipid peroxidation in H-MEL roots already after chilling, but the significant extent of MEL impact was seen after re-warming. Similarly at the ultrastructural level, the protective effect of MEL at chilling was also visible, especially in plastids, and this effect maintained also after re-warming.     

Involvement of melatonin applied to Vigna radiata L. seeds in plant response to chilling stress

The impact of melatonin (50 µM L^?1) applied to Vigna radiata seeds by hydro-priming on phenolic content, L-phenylalanine ammonialyase (PAL) activity, MEL level, antioxidant properties of ethanol extracts as well as electrolyte leakage from chilled and re-warmed Vigna radiata roots of seedlings were examined. Seedlings obtained from non-primed seeds, hydro-primed and hydro-primed with MEL were investigated after 2 days of chilling and 2 days of re-warming. At 25°C, the level of MEL in roots derived from seeds hydro-primed with MEL was 7-fold higher than in roots derived from non-primed seeds. However, the content of MEL significantly decreased in all variants investigated after re-warming, in contrast to PAL activity and phenolic levels, which reached the highest values. The antioxidant capacity of ethanol extracts from chilled and re-warmed roots, determined by ABTS+· assay, was correlated with phenolic content while the reducing ability of these extracts, determined by the FRAP method, correlated with PAL activity. However, both were the highest in rewarmed roots with applied MEL, which was accompanied by a significant decline in electrolyte leakage. Taken together, results may indicate that MEL can play a positive role in plant acclimation to stressful conditions and activation of phenolic pathway by this molecule.     

Effects of seed hydropriming in presence of exogenous proline on chilling injury limitation in <Emphasis Type="Italic">Vigna radiata</Emphasis> L. seedlings

Three-day-old seedlings (t ₀ stage) of Vigna radiata (L.) Wilczek obtained from seeds hydroprimed (H) and hydroprimed with proline (HPro) were examined. H and HPro slightly improved mung bean seed germination and seedlings growth at 5°C. The best growth was observed in the seedlings obtain from HPro5 (5 mM) seeds in comparison with the seedlings obtained from the control-non-primed seeds and H seeds. Exposure of mung bean seedlings grown from non-primed seeds to chilling for 4 days induced chilling injury: membrane lipid peroxidation, decrease in endogenous proline level and inhibition of growth of roots and hypocotyls. The seedlings obtain from HPro seeds grew better during the time of chilling and after rewarming at 25°C. The possible role of HPro in chilling injury limitation is discussed.     

Induction of phenolic compounds in two dark-grown lentil cultivars with different tolerance to copper ions

The influence of a high copper sulphate concentration on growth, Cu accumulation, lipid peroxidation as well as on the contents of total phenolic compounds (PhC) and UV-absorbing compounds (UVAC) in roots of lentil (Lens culinars Medic.) cvs. Krak and Tina was investigated. The plants were subjected to 0.5 mM Cu²⁺ for 3 and 5 days in darkness. Growth inhibition and increased lipid peroxidation in the roots of both cultivars, especially in cv. Tina which accumulated more Cu, were observed. Cu²⁺ treatment caused greater PhC and UVAC accumulation in cv. Krak; however, constitutive levels of these compounds were higher in cv. Tina. The maximum absorption peak of UVAC was determined at 270 nm. HPLC analyses of these compounds revealed the presence of two main derivatives of the soluble (aglycone and ester-bound) fraction of the hydroxycinnamic acids, ferulic (FA) and p-coumaric (p-CA) acids and the flavonol, kaempferol (Kam). Greater changes in the content of phenolic acids than of Kam may suggest that the former play a more important role in protecting lentil roots against high Cu²⁺ concentration. Thus, while the lower PhC levels at a higher Cu content in the roots of cv. Tina were probably due to stress, their higher levels in cv. Krak could have been a response to ROS signaling. However, though the high concentration of Cu²⁺ stimulated PhC in cv. Krak, it was not sufficient to counteract the amount of ROS generated by metal presence. These observations may suggest that ROS can serve as a common signal for acclimation to Cu²⁺ stress and cause PhC accumulation in dark-grown roots. The role of PhC in lentil tolerance to Cu²⁺ stress is discussed.     

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.     

Inhibition of glutathione synthesis reduces chilling tolerance in maize

 The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine (γEC) synthetase, the first enzyme of GSH synthesis. At 25 °C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 °C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or γEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity. Received: 9 November 1999 / Accepted: 17 February 2000     

Chilling injury and changes in adenosine triphosphate of cotton seedlings

Young Gossypium hirsutum L. seedlings chilled at 5° showed a continual decrease in ATP concentration with time of chilling. Chilled plants returned to optimum conditions were able to restore the initial ATP concentration when chilled only 1 day, but not when chilled 2 days. The decrease in ATP with chilling was prevented by hardening the seedlings at 15° for 2 days (14-hr-day-length) immediately before chilling. The ATP level of hardened plants was higher than of unhardened plants. When hardened plants were chilled at 5°, the ATP level increased in the leaves but decreased in the roots.     

Chilling-induced oxidative stress in young sal (Shorea robusta) seedlings

Aerial parts of the chilling-sensitive young sal seedlings showed overproduction of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) in response to constant chilling exposure during November to March (9–14.1 °C) in field conditions. Almost 4–6 fold increase in ROS was observed in aerial parts of chilling exposed seedlings than the control seedlings (maintained in greenhouse). Increased formation of ROS was found to be closely associated with the rise in TBARS in leaf (5.8 fold) and shoot (4.8 fold) tissues. On the contrary the leaf and shoot of control seedling and root of both control and chilling exposed seedlings exhibited relatively very low levels of superoxide and TBARS. The chilling exposed seedlings also showed striking weakening in the free radical processing enzyme systems. The low temperatures during November to March resulted in reduced activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POX) and ascorbate peroxidase (APX) almost by 49, 26, 7 and 78 % in leaves and 65, 46, 9 and 85% in shoots respectively compared to leaves and shoots of control seedlings. Our results indicated that, substantially higher rates of liberation of superoxide and TBARS along with drastic failure of antioxidant enzyme system in chilling sensitive sal seedlings leads to oxidative bursts terminating into irreversible injury in leaves and shoot of these seedlings.     

Chilling-induced ultrastructural changes to mesophyll cells of Arabidopsis grown under short days are almost completely reversible by plant re-warming

Exposure of plants to chilling (low temperatures above freezing) limits growth and development in all environments outside the lowest latitudes. Cell ultrastructure and morphometric studies may allow associations to be made between chilling-induced changes at the ultrastructural level, molecular events and their physiological consequences. We examined changes in the shape, size and membrane organization of the organelles of mesophyll cells in Arabidopsis thaliana (Col 0), a cold-resistant species, after subjecting 6-week-old plants grown at normal growth temperatures to chilling (2.5–4°C; 14-h dark/10-h light cycle) for 6, 24 and 72 h and after a re-warming period of 50 h. No ultrastructural differences were seen in the first 6 h of chilling but after 24 h we observed swollen and rounded chloroplasts with larger starch grains and dilated thylakoids compared to control plants. By 72 h, chilling had resulted in a large accumulation of starch in chloroplasts, an apparent crowding of the cytosol and a lower abundance of peripheral reticulum than in the controls. The average area per chloroplast in cell sections increased after 72-h chilling while the number of chloroplasts remained the same. Ring-shaped and other morphologically aberrant mitochondria were present in significantly higher abundance in plants given 72 h chilling than in the controls. Plant re-warming for 50 h reduced chloroplast size to those of the controls and returned mitochondria to standard morphology, but peripheral reticulum remained less abundant than in plants never given a cold treatment. The near full return to normal ultrastructure upon plant re-warming indicates that the morphological changes may be part of acclimation to cold.     

Phosphorus deficiency enhances aluminum tolerance of rice (Oryza sativa) by changing the physicochemical characteristics of root plasma membranes and cell walls

The negative charge at the root surface is mainly derived from the phosphate group of phospholipids in plasma membranes (PMs) and the carboxyl group of pectins in cell walls, which are usually neutralized by calcium (Ca) ions contributing to maintain the root integrity. The major toxic effect of aluminum (Al) in plants is the inhibition of root elongation due to Al binding tightly to these negative sites in exchange for Ca. Because phospholipid and pectin concentrations decrease in roots of some plant species under phosphorus (P)-limiting conditions, we hypothesized that rice (Oryza sativa L.) seedlings grown under P-limiting conditions would demonstrate enhanced Al tolerance because of their fewer sites on their roots. For pretreatment, rice seedlings were grown in a culture solution with (+P) or without (−P) P. Thereafter, the seedlings were transferred to a solution with or without Al, and the lipid, pectin, hemicellulose, and mineral concentrations as well as Al tolerance were then determined. Furthermore, the low-Ca tolerance of P-pretreated seedlings was investigated under different pH conditions. The concentrations of phospholipids and pectins in the roots of rice receiving −P pretreatment were lower than those receiving +P pretreatment. As expected, seedlings receiving the −P pretreatment showed enhanced Al tolerance, accompanied by the decrease in Al accumulation in their roots and shoots. This low P-induced enhanced Al tolerance was not explained by enhanced antioxidant activities or organic acid secretion from roots but by the decrease in phospholipid and pectin concentrations in the roots. In addition, low-Ca tolerance of the roots was enhanced by the −P pretreatment under low pH conditions. This low P-induced enhancement of low-Ca tolerance may be related to the lower Ca requirement to maintain PM and cell wall structures in roots of rice with fewer phospholipids and pectins.     

Application of phytohormones during seed hydropriming and heat shock treatment on sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) chilling resistance and changes in soluble carbohydrates

The objective of this study was to analyze the mechanism of some physiological processes accompanying acquisition of sunflower (Helianthus annuus L.) chilling resistance due to seeds hydropriming in the presence of salicylic acid, jasmonic acid, 24-epibrassinolide followed exposition of seeds to short-term heat shock treatment. The seeds were hydroprimed at 25 °C in limited amounts of water or solution of salicylic or jasmonic acid at 10^?2, 10^?3 and 10^?4 M concentration, 24-epibrassinolide at 10^?6, 10^?8 and 10^?10 M concentration. The seeds were incubated for 2 days, subjected to short-term heat shock (45 °C, 2 h) and chilled for 21 days at 0 °C. Sunflower chilling susceptibility and physiological responses were evaluated according to the inhibition of radicle growth, the inhibition of the number of lateral roots formation, the activity of catalase and changes in soluble carbohydrates in seedlings developing for 72 h at 25 °C. Hydropriming and short-term heat shock application explicitly reduced inhibition of roots as well as lateral roots development by allowing the germinating seeds to recover from the growth-inhibiting effects of chilling. Seeds hydropriming in solutions containing salicylic acid, jasmonic acid and 24-epibrassinolide followed heat shock treatment additionally promoted the activity of catalase and sugars metabolism, which stimulated seedlings development and alleviated the decrease of F _v/F _m caused by chilling conditions. These beneficial effects contributed to increased resistance of sunflower seedlings to chilling stress. The present study demonstrated that the most profitable effect on reducing negative effect of chilling may be achieved by short-term heat shock applied during hydropriming in water supplemented with 24-epiBL (10^?8 and 10^?10 M) or salicylic acid (10^?3 and 10^?4 M).     

Alterations in phenylpropanoid content in soybean roots during low temperature acclimation

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, growth and phenolic acid content during low temperature acclimation in soybean (Glycine max. (L.) Merr.) roots were investigated. Elongation of soybean roots was inhibited after the transfer of 3-d-old seedlings grown at 25 to 10 °C. Extractable PAL activity as well as the total amount of phenolics increased 24 h after plant transfer to low temperature. The high pressure liquid chromatography analyses revealed the presence of six phenolic acids in soybean roots: p-hydroxybenzoic, vanillic, syringic, anisic, p-coumaric and ferulic. Analyses of different fractions of phenolic acids showed that during 24 h of low temperature exposure, an increase in the relative level of ester-bound-soluble phenolic acids occurred. The highest increase in this fraction was observed for ferulic acid (26 %). At the same time, a decrease in phenolic glycosides took place. The amount of phenolic acids released after alkaline treatment of the cell wall material was strongly inhibited (3-fold), which may suggest an alteration of the physical properties of the wall in acclimation to low temperature. The possible role of phenolics in acclimation to low temperature in roots is discussed.     

Mechanism of the Decline in Vacuolar H -ATPase Activity in Mung Bean Hypocotyls during Chilling

The mechanism responsible for the decrease in the activity of vacuolar H⁺ -ATPase during chilling was investigated in seedlings of mung bean (Vigna radiata). After chilling at 0°C for 3 d, the activity of vacuolar H⁺ -ATPase, calculated on the basis of membrane protein, decreased to 47% of the original value. Of the nine subunits of the ATPase, the specific contents of at least six subunits, of 68, 57, 44, 38, 37, and 32 kD, decreased in vacuolar membranes after chilling, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These subunits were released by treatment with chaotropic anions such as thiocyanate. The level of the 16-kD subunit did not change. Immunoblot analyses showed the decrease in the levels of the subunits of 68, 57, and 32 kD. Furthermore, the specific activity of the ATPase purified from chilled hypocotyls was two-thirds of that of the enzyme from nonchilled seedlings, and the enzyme from chilled tissue retained only a small amount of the 32-kD subunit. These results suggest that a selective release of the peripheral subunits of the ATPase from the membrane and a partial degradation of the ATPase complex may occur in vivo during chilling.     

Role of Calcium on Phenolic Compounds and Enzymes Related to Lignification in Soybean (Glycine max L.) Root Growth

Changes in soluble and cell wall bound peroxidases activities, phenylalanine ammonia-lyase activity and phenolic compounds and lignin contents in roots of calcium-treated soybean (Glycine max (L.) Merr.) seedlings and their relationships with root growth were investigated. Three-day-old soybean seedlings were cultivated in nutrient solution with or without 0.025–5.0 mM calcium for 24 h. In general, length and fresh and dry weights of roots increased, while activities of enzymes (soluble and cell-wall peroxidases and phenylalanine ammonia-lyase) and phenolic compounds and lignin contents decreased against calcium concentrations. In the absence of calcium, phenylalanine ammonia-lyase and peroxidases activities increased by accumulating phenolic compounds and lignin due to restricted growth of roots. Enhanced calcium supply reduced the production of phenolic compounds and lignification due to low phenylalanine ammonia-lyase and peroxidases activities, reinforcing the essential role of calcium to improve the soybean root growth.     

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.     

Differential responses of antioxidative system to chilling and drought in four rice cultivars differing in sensitivity

Responses of antioxidative defense systems to chilling and drought stresses were comparatively studied in four cultivars of rice (Oryza sativa L.) differing in sensitivity, two of them (Xiangnuo no. 1 and Zimanuo) are tolerant to chilling but sensitive to drought and the other two (Xiangzhongxian no. 2 and IR50) are tolerant to drought but sensitive to chilling. The seedlings of rice were transferred into growth chamber for 5 d at 8 degrees C as chilling treatment, or at 28 degrees C as control, or at 28 degrees C but cultured in 23% PEG-6000 solution as drought stress treatment. Under drought stress the elevated levels of electrolyte leakage, contents of H(2)O(2) and total thiobarbituric acid-reacting substances (TBARS) in Xiangzhongxian no. 2 and IR50 are lower than those in Xiangnuo no. 1 and Zimanuo. On the contrary, Xiangnuo no. 1 and Zimanuo have much lower level of electrolyte leakage, H(2)O(2) and TBARS than Xiangzhongxian no. 2 and IR50 under chilling stress. Activities of antioxidant enzymes (superoxide dismutase (SOD), catalase, and ascorbate-peroxidase (APX)) and contents of antioxidants (ascorbaic acid and reduced glutathione) were measured during the stress treatments. All of them were enhanced greatly until 3 d after drought stress in the two drought-tolerant cultivars, or after chilling stress in the two chilling-tolerant cultivars. They all were decreased at 5 d after stress treatments. On the other hand, activities of antioxidant enzymes and contents of antioxidants were decreased greatly in the drought-sensitive cultivars after drought stress, or in the chilling-sensitive cultivars after chilling stress. The results indicated that tolerance to drought or chilling in rice is well associated with the enhanced capacity of antioxidative system under drought or chilling condition, and that the sensitivity of rice to drought or chilling is linear correlated to the decreased capacity of antioxidative system.     

Effect of LAB 173 711, an ABA analogue,on low-temperature resistance of mung bean seedlings

The effect of LAB 173 711, a synthetic analogue of abscisic acid, has been evaluated on chilling-sensitive mung bean (Vigna radiata L. cv. Local V.) seedlings. Electrical conductivity was used for assessing the degree of chilling injury. Exposure of 8-day-old mung bean seedlings to 4°C for 35 h resulted in a 50% electrolyte leakage and induced irreversible chilling injury. The seedlings gained the best protection against chilling injury by pretreatment with LAB 173 711 (5 × 10^–4 M) for 3 days. The protection effect could be sustained for 4 days. The LAB 173 711 pretreatment at 28°C did not cause a significant difference in the electrolyte leakage over the ambient temperature (28°C) control. Application of LAB 173 711 at 28°C reduced visible injury and the treated seedlings had higher ethylene production and respiration rate over the untreated control. LAB 173 711 helped maintain the integrity of the cell membrane and thus reduced the leakage of soluble sugar and amino acids. These combined effects led to a higher chilling tolerance in the mung bean seedlings.     

A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 °C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

Role of abscisic acid in chilling tolerance of rice (Oryza sativa L.) seedlings. I. Endogenous abscisic acid levels

Changes of ABA levels in chilled rice (Oryza sativa L.) seedlings of two varieties were determined. On exposure to chilling, ABA concentration rapidly increased in the chilling-tolerant cultivar (cv. Tainung 67, TNG.67) but not in the chilling-sensitive cultivar (cv. Taichung Native 1, TN.1). Both detached shoots and roots of TNG.67 seedlings showed a significant ABA increase after exposure to chilling. TN.1 seedlings could not accumulate ABA under low temperature but well-watered status. Exogenous application of the ABA biosynthetic inhibitor, fluridone, reduced ABA accumulation, as well as survival ratio of chilled TNG.67 seedlings. Electrolyte leakage and leaf conductance were also increased by the inhibitor and the effects could be reversed by exogenously applied ABA. ABA concentrations in xylem sap of TNG.67 seedlings increased within 4 h after chilling, and this was temporally coincident with the reduction of leaf conductance. The roles of endogenous ABA in the tolerance of rice seedlings to chilling on a whole plant basis are discussed and suggested.     

Chilling injury and nucleotide changes in young cotton plants

The effects of chilling at 3 to 5 C on the nucleotide composition of roots and leaves of cotton (Gossypium hirsutum L.) seedlings were determined. Chilling decreased the concentration of nucleotides, especially di- and triphosphates, in both leaves and roots. Chilling also caused an increase in free nucleosides. The results are interpreted to mean that general phosphorolytic activity is associated with chilling injury rather than damage to the phosphorylating mechanisms alone. Hardening at 10 to 20 C prior to chilling protected the seedlings against subsequent chilling injury and prevented nucleotide losses.