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Journal of Experimental Botany, Vol. 53, No. 378, pp. 2225±2237, November 2002 DOI: 10.1093/jxb/erf071 Chilling root temperature causes rapid ultrastructural changes in cortical cells of cucumber (Cucumis sativus L.) root tips Seong Hee Lee1, Adya P. Singh1,3, Gap Chae Chung1, Yoon Soo Kim2 and In Bae Kong1 1 Agricultural Plant Stress Research Centre (APSRC), Division of Applied Plant Science, College of Agriculture, Chonnam National University, Gwangju 500-757, South Korea 2 Department of Forest Products and Technology, College of Agriculture, Chonnam National University, Gwangju 500-757, South Korea Received 7 January 2002; Accepted 1 July 2002 Abstract Examination of root tips from cucumber (Cucumis sativus L.) seedlings grown at 8 °C for varying periods ranging from 15 min to 96 h, showed marked changes in the ultrastructure of cortical cells within only 15 min of exposure. Greater parts of the cortex were affected with longer periods of exposure, but the sequence of morphological changes in cell components was similar to that found for the roots exposed for 15 min. The effect of chilling injury included alterations in cell walls, nuclei, ER, mitochondria, plastids, and ribosomes. The extent of alterations varied greatly among cells, moderate to severe alterations to cell components being observable among adjoining cells. The measurements of root pressure using the root pressure probe showed a sudden, steep drop in the root pressure in response to lowering of the temperature of the bathing solution from 25 °C to 8 °C. These observations are discussed in the light of the information available on the ultrastructural and biochemical characteristics of the effect of cold exposure in chilling-sensitive plants. Keywords: Chilling injury, cortical cells, Cucumis sativus roots, transmission electron microscopy, root pressure. Introduction Cucumber (Cucumis sativus L.) is an important crop plant for many parts of the world, Asian countries in particular. 3 However, this plant is sensitive to temperatures below about 15 °C, and massive economic losses can occur if the temperature suddenly falls below this level during the growing season. The effect of cold temperatures on the ultrastructure of plant cells has been studied for a long time, and recently reviewed (Kratsch and Wise, 2000). The extent of alterations in cell components appears to be related to the severity of the cold temperatures and the length of exposure. Generally, agricultural plants grown in tropical and sub-tropical regions are susceptible to chilling damage at temperatures ranging from 0±15 °C. Plant response to cold temperatures appears to be both species- and tissue-speci®c. Ultrastructural studies suggest that cold-temperature-related changes involve a wide range of cell components, and plastids are one of the most extensively investigated organelles. Plastids and thylakoid membranes swell and become disorganized, thylakoids and peripheral reticulum vesiculate, plastoglobuli or lipid droplets accumulate, and eventually the entire plastid becomes disorganized leading to the disintegration of the envelope (Klein, 1960; Millerd et al., 1969; Taylor and Craig, 1971; Kimball and Salisbury, 1973; Forde et al., 1975; Moline, 1976; Niki et al., 1978; Nesler and Wernsman, 1980; Murphy and Wilson, 1981; Wise et al., 1983; Ishikawa, 1996). Signi®cant alterations have also been observed in other cell components. Mitochondria undergo distinct swelling (Ilker et al., 1976; Moline, 1976; Niki et al., 1978; Murphy and Wilson, 1981; Ishikawa, 1996), the population of ribosomes decreases, endoplasmic reticulum (ER) dilates, cytoplasmic membranes vesiculate, To whom correspondence should be addressed. Fax: +82 62 5300190. E-mail: [email protected] Published by Oxford University Press 2226 Lee et al. the nuclear chromatin condenses forming clumps, plasmalemma invaginates and there is an increase in vacuolation and the number of membraneous vesicles (Ilker et al., 1976; Moline, 1976; Platt-Aloia and Thomson, 1976; Niki et al., 1978; Murphy and Wilson, 1981; Ishikawa, 1996). Cucumber roots respond rapidly to 8 °C chilling temperature, with a drastic reduction in water uptake within about 10 min of exposure to this temperature (SH Lee et al., unpublished observations), and thus provide a valuable experimental system to study various aspects of cell biology in relation to this and other factors. Using the root pressure probe for measurements of root pressure developed in the root system (Steudle, 2000), evidence is provided for a sudden, steep drop in root pressure in this species within minutes of exposure to chilling temperature. The ultrastructural observations reported here on the effect of 8 °C temperature on cucumber root tips are part of a detailed study to investigate tissue organization and anatomical composition of various regions of cucumber roots, including the root-hair-producing region where much of the water uptake is likely to occur. The aim is to understand the basis for the observed sudden drop in the root pressure in cucumber in response to chilling temperature using a combined physiological, biochemical and anatomical approach in this study. Materials and methods Cucumber (Cucumis sativus L.) seeds were germinated on moist paper towels in plastic trays, in the dark, in an incubator. The seedlings were grown for 4 d in the dark and were then exposed to 8 °C for periods ranging from 15 min to 96 h. About 1±2 mm long segments were collected from the root tip region at 0 h (control) and after the following periods of exposure to 8 °C: 15 min, 30 min, 1 h, 4 h, 8 h, 16 h, 24 h, 48 h, 72 h, and 96 h. The samples were processed for light (LM) and transmission electron microscopy (TEM) as described below. Root tip segments were transferred to vials containing 3% glutaraldehyde (prepared in 0.05 M sodium cacodylate buffer) immediately after cutting with a sharp razor blade under a stereo microscope, and ®xed overnight at 4 °C. After washing in the buffer, the samples were post-®xed in 2% osmium tetroxide (also prepared in 0.05 M sodium cacodylate buffer) and washed again in the buffer. They were dehydrated in a graded series of acetone, and in®ltrated and embedded in Spurr's low viscosity resin (Spurr, 1969). Semi-thick (2±3 mm) and ultra-thin sections (60±80 nm) were cut with glass and diamond knives, respectively, on an RMC MT X ultramicrotome. Semi-thick sections were stained with 0.1% aqueous toluidine blue and examined with a Zeiss Axiolab photomicroscope. Ultra-thin sections were sequentially stained with uranyl acetate and lead citrate and then examined with a JEOL 1010 TEM. The root pressure of an excised cucumber root system was measured by root pressure probe as previously described (Steudle, 2000). Once the cut end of a root segment was connected tightly to the probe, the root pressure developed due to active uptake of nutrients into root, and the root pressure was measured continuously using the probe. Results The ultrastructural observations presented are based on the conventional chemical ®xation of samples, which is known to alter the morphology of some cell components. It is also likely that cells undergoing cold-induced changes may react somewhat differently to ®xatives compared to uninjured cells. Therefore, the observations presented have to be viewed with these considerations in mind. Observations were con®ned to examining cortical cells from a region about 1 mm behind the root apex. Cells in this region were undergoing expansion growth as indicated by the presence of developing as well as fully developed intercellular spaces. Examination of semi-thick monitor sections with LM provided early indications that changes in root cortical cells due to cold treatment may be rather rapid. In roots receiving 15 min of cold exposure some areas of the cortex stained only lightly with toluidine blue. Cells in such regions appeared abnormal; they had a highly reduced cytoplasmic density, and the presence of cell walls could not be con®rmed (Fig. 1B). However, the staining of a large proportion of the cortex was similar to that of the cortex of control roots (Fig. 1A). Nuclei and cell walls were easily recognized in these cells and they had a normal appearance, judging by comparisons with cortical cells of control roots. With longer periods of cold exposure, greater areas of the cortex were affected (Fig. 1C). However, damaged areas did not appear to be proportional to the time of exposure. Even after 96 h of exposure only about 25% of the cortex appeared to be severely affected. However, this estimate is based on single sections only and not serial sections. TEM observations of damaged and adjacent cortical areas con®rmed that cells in the lighter areas were severely affected, in addition to providing detailed information on the nature and extent of alterations in various subcellular components. Control roots Cortical cells from control roots (roots not exposed to cold) displayed features typically found in a metabolically active cell. Nuclei were large and spherical, containing fairly disperse chromatin and a prominent nucleolus (Fig. 2A). Nuclei were more or less centrally located in the cells. A relatively dense cytoplasm contained numerous mitochondria and variously shaped plastids, which had a stroma denser than that of mitochondria and were thus readily distinguishable from them even at low magni®cations (Figs 2A, B, 3A). The mitochondria had a moderately dense stroma, which contained ribosomes, DNA and welldeveloped cristae (Fig. 6A, B). The two membranes of the mitochondrial envelope were parallel and evenly spaced (Fig. 6A, B). The plastids contained few internal mem- Chilling injury in cucumber root 2227 branes which were not readily distinguishable because of the high density of the stroma (Fig. 6A). Also, the plastid envelope, which consisted of two parallel membranes, was not easily resolvable because of the high density of the plastid stroma (Fig. 6A, B) and the pleomorphic nature of the plastid form (Figs 2A, 3A). The cytoplasm also contained abundant cisternae of rough ER, which were dif®cult to discern at low magni®cations, mainly because of the high density of the cytoplasm (Fig. 2A, B). As seen at high magni®cations, ER cisternae were long, with a uniform intracisternal spacing between the limiting membranes, and were studded with ribosomes (Fig. 6A). Polysomes were common also in other parts of the cytoplasm. The plasmalemma appeared to be closely associated with the cell walls, which were thin and somewhat sinuous in the intercorner regions of the cells (Fig. 2A, B). The cell walls were thicker in the cell corner regions, particularly in those cells which had developed intercellular cavities or were in the process of doing so (Figs 2B, 3A). Initially, vacuoles were small (Fig. 2A), but they progressively enlarged mainly through the coalescence of adjoining vacuoles, and this process appeared to coincide with the development of intercellular cavities (Figs 2B, 3A). The intercellular cavities were formed mainly by the dissolution of the intercellular material in the cell corner regions, as shown by the presence of reticular, vacuolate and dense materials in these areas (Fig. 2B). Cold-treated roots Chilling injury to cortical cells of exposed roots was apparent as early as 15 min after exposure (Fig. 1B). Injury in these samples varied greatly in severity, ranging from changes in a few cell components to almost complete disintegration of the cytoplasm. Longer exposure periods caused greater parts of the cortex to be affected, as seen in the light micrograph illustrated (Fig. 1C). Therefore, the following description is primarily based on the features considered to represent sequential injury, and not necessarily on the sequence of the exposure time. Also, roots exposed to all periods had cells showing signs of injury as well as those which appeared to have normal ultrastructure. This feature is illustrated in Fig. 3B, where cells with early injuries can be seen next to those without any apparent injuries. Fig. 1. (A) Transverse section through a control root tip. Cortical cells show features typical of metabolically active cells. They have a dense cytoplasm and large nuclei containing a prominent nucleolus. LM. Bar=100 mm. (B) Transverse section through a root tip exposed to cold temperature for 15 min. A small part of the cortex has stained lightly, and shows signs of cell damage (arrow). LM. Bar=100 mm. (C) Transverse section through a root tip exposed to cold temperature for 48 h. A relatively large part of the cortex has stained lightly and shows signs of cell damage (arrows). LM. Bar=100 mm. Early stages of chilling injury A noticeable increase in the density of cytoplasm, and distention of endoplasmic reticulum (ER) were early symptoms of injuries to cells (Figs 3B, 4A). At low magni®cations ER cisternae were recognized with dif®culty in the cortical cells of control roots (Figs 2A, B, 3A), whereas they were easily recognized even at the early stages of cell injury because of the presence of wider, 2228 Lee et al. variable intra-cisternal spaces in them (Figs 3B, 4A). The stroma of both mitochondria and plastids became more translucent and granular, as viewed in low magni®cation images (Fig. 4A). Both nuclei and nucleoli became irregularly shaped (Fig. 4A). Cell walls were thinner than in the control material and were distorted (Fig. 4A). Fig. 2. Cortical cells from control root tips. (A) The cell corners lack intercellular spaces. The cell walls (CW) are thin and variable in thickness. The thinnest regions of cell walls are sinuous (arrowhead). The cytoplasm is dense and contains numerous mitochondria (M), plastids (P) and the cisternae of ER (circles). The vacuoles (V) are relatively small. The nuclei (N) are centrally located and contain a large nucleolus (NU). The chromatin is largely uncondensed. TEM. Bar=2 mm. (B) The cells shown here are similar in their ultrastructure to those in (A), except that cell corners show signs of developing spaces. The cell corners are prominent and contain dense and vacuolate materials (arrowheads). The cytoplasm is dense and rich in mitochondria (M), plastids (P) and ER (circles). Some plastids contain starch grains. The vacuoles (V) are small, discrete and also show signs of fusion. N, nucleus. TEM. Bar=2 mm. Chilling injury in cucumber root 2229 Intermediate stages of chilling injury Cytoplasmic density was still very high and was comparable to that in the cells showing signs of early injuries (Figs 4B, 7A, B). ER cisternae became more distended and more irregular in their form (Figs 4B, 8A). The cytoplasm also contained other unusual membraneous formations (Fig. 8A). The nuclear envelope was also markedly distended (Fig. 4B). Fig. 3. (A) Parts of adjoining cortical cells from a control root tip. The cells are at a slightly more advanced stage of differentiation than those in Fig. 2B. The intercellular regions (IS) have developed cavities. The cell walls (CW) are thicker and less sinuous. The vacuoles are larger and show signs of continued enlargement through fusion (arrowheads). The presence of starch in plastids is more common. The mitochondria (M) are abundant. N, nucleus. TEM. Bar=2 mm. (B) A group of cortical cells from a root tip exposed to cold temperature for 1 h. Several cells show early signs of chilling injury. The ER is distended. In addition to prominent vacuoles (V) the cytoplasm contains smaller vacuolate structures (arrowheads). The nuclei (N) are not centrally located and nucleoli (NU) are irregular. The cells marked by unlabelled arrows have a normal appearance. TEM. Bar=2 mm. 2230 Lee et al. Mitochondria and plastids became progressively more swollen and translucent, losing much of their internal membranes and ribosomes (Figs 4B, 7A, B). Some mitochondria showed irregular aggregates of remaining/ Fig. 4. (A) Cortical cells from a root tip exposed to cold temperature for 15 min. The cells show signs of both early and intermediate stages of chilling injury. The cytoplasm is dense and ®lled with numerous distended membraneous structures, most of which are probably ER. The mitochondria (M) are abundant and their interior appears granular. The plastids (P) appear swollen, contain starch grains, and have a moderately dense interior. The number and size of vacuoles (V) are highly variable. The nucleus (N) has an irregular shape and contains an irregularly shaped nucleolus (NU). The cell walls are thin and irregular. TEM. Bar=2 mm. (B) Cortical cells from a root tip exposed to cold temperature for 48 h. Some cells show intermediate stages of chilling injury. The nucleus (N) is irregular in shape and the nucleolus (NU) is asymmetrically positioned and corresponds to the nucleus in its shape. The nuclear envelope is grossly distended in places (arrowheads). The ER is also highly distended. The cytoplasm is highly dense and contains many spherical to oval-shaped bodies, the larger of which may be plastids (P) and the smaller mitochondria (M). The interior of these bodies appears granular. The plasmalemma has pulled away from the cell wall in places (arrows). The star indicates a completely damaged cell, which is virtually empty. TEM. Bar=2 mm. Chilling injury in cucumber root 2231 modi®ed ribosomes (Fig. 7A, B). However, there did not appear to be a close correlation between the loss in ribosomes and other changes taking place in mitochon- dria. For example, there were examples of highly swollen mitochondria, which had only a few internal membranes, but contained abundant, apparently normal Fig. 5. (A) Cortical cells from a root tip exposed to cold temperature for 15 min. Cells show signs of advanced stages of chilling injury. Cell walls can not be recognized and may have disintegrated. The nuclei (N) are highly irregular and have almost completely lost the integrity of their envelope. Nucleoli (NU) are still present but are irregular. The interior of the mitochondria (M) is greatly altered and appears translucent. Also, the envelope appears to have been damaged in some mitochondria. The plastid (P) envelope also appears to be damaged. The ER may have vesiculated greatly and can not be recognized. The cytoplasmic density is greatly reduced. TEM. Bar=2 mm. (B) Cortical cells from a root tip exposed to cold temperature for 16 h. The cell marked by an asterisk shows signs of an intermediate stage of chilling injury. The other cells are greatly transformed and show signs of the ®nal stages of disintegration. The cytoplasm in these cells is sparsely granular and contains scattered vesicles of varying sizes (arrowheads). The cell walls are extremely thin and show sign of discontinuity (arrow). TEM. Bar=2 mm. 2232 Lee et al. ribosomes (Fig. 7A). In some instances the remaining internal membranes in mitochondria had vesiculated (Fig. 7A). The cell walls were extremely thin and the plasmalemma had pulled away from the cell wall in places (Fig. 4B). Advanced stages of chilling injury Cytoplasmic density progressively declined (Figs 5A, 8B, 9A, B). In the ®nal stages, the cytoplasm appeared very thin and electron-lucent, containing sparsely distributed particles, many of which may be degraded Fig. 6. A high magni®cation view of cortical cells from control root tips. (A, B) The cytoplasm is dense and contains numerous polysomes.The ER cisternae are long with a uniform intracisternal spacing between the limiting membranes. The plastids (P) are dense and occasionally contain starch grains (S). The internal plastid membranes are few. The mitochondria (M) have a moderately dense stroma, which contains ribosomes, DNA (arrowheads) and numerous infoldings (cristae) of the inner envelope membrane. The mitochondria have a typical double membraneous envelope; the membranes are parallel with a uniform spacing (circles). N, nucleus; NP, nuclear pore. TEM. Bar=500 nm. Chilling injury in cucumber root 2233 ribosomes (Fig. 5B). Cell walls were extremely thin, wavy and disrupted in places (Fig. 5B). In some cases cell walls had disappeared completely, and thus partitioning between the nuclei of neighbouring cells was lost (Fig. 5A). The nuclei had lost the integrity of their envelope, which appeared highly irregular and disrupted in places (Fig. 5A). Fragmented membranes of the nuclear envelope were often seen in proximity Fig. 7. (A) High magni®cation view of a cortical cell from a root tip exposed to cold temperature for 48 h. The mitochondria (M) are greatly swollen. They have lost much of their internal membranes, although the envelope is still present. Ribosomes are still in abundance in the mitochondrial stroma, which also contains some vesiculate, membraneous structures (arrowheads). Membraneous vesicles are also common in the cytoplasm (arrows), which is highly dense. TEM. Bar=500 nm. (B) A high magni®cation view of a cortical cell from a root tip exposed to cold temperature for 16 h. The cytoplasm is highly dense. Mitochondria (M) appear swollen. The mitochondrial stroma has lost many of its ribosomes and thus appears considerably more lucent than the mitochondria of control root tips. A few cristae and the limiting envelope are still present in some mitochondria. The large vacuolate body (P) with sparse granular inclusions may be a highly swollen plastid. TEM. Bar=500 nm. 2234 Lee et al. to parent nuclei (Fig. 9A). ER and other cytoplasmic membranes were highly modi®ed (Fig. 8B) forming vesicles of varying shapes and sizes (Figs 5A, 9B). The envelopes of both mitochondria and plastids were disrupted (Fig. 5A). The cytoplasm also contained spherical, electron dense bodies (Figs 8B, 9B). Eventually, the enucleate cell appeared very electron transparent and sparsely granular, containing vesicles of varying shapes and sizes (Fig. 5B). Root pressure measurement Root pressure in excised cucumber root system measured with the root pressure probe was about 0.15±0.20 MPa when root temperature was kept at 25 °C. Fig. 8. High magni®cation views of modi®ed cytoplasmic membranes in cortical cells from root tips exposed to cold temperature for 15 min. (A) The ER is distended. Also present in the cytoplasm are some unusual membraneous formations (arrowheads), TEM. Bar=500 nm. (B) The ER appears distended and has also vesiculated. The cytoplasm also contains some highly dense deposits (arrowheads). TEM. Bar=500 nm. Chilling injury in cucumber root 2235 However, when the temperature was gradually lowered from 25 °C to 8 °C, the root pressure decreased rapidly, reaching zero MPa within 15±20 min (Fig. 10). This steep drop in the root pressure is a clear indication that the cucumber root system is very sensitive to low root temperature. Fig. 9. (A) A high magni®cation view of a cortical cell from a root tip exposed to cold temperature for 15 min. The nuclear envelope had broken down, but the nucleolus (NU) is still present. The membranes (arrowheads) present near the nucleolus may have been part of the nuclear envelope. TEM. Bar=500 nm. (B) A high magni®cation view of a cortical cell from a root tip exposed to cold temperature for 16 h. The cytoplasmic density is greatly reduced as compared to the cells in Fig. 7. The mitochondrion (M) present contains only a few cristae and aggregates of ribosomes and appears highly translucent. Membraneous vesicles (arrowheads) are common in the cytoplasm. The cytoplasm also contains some dense deposits (arrows). TEM. Bar=500 nm. 2236 Lee et al. Fig. 10. The effect of low root temperature on cucumber root pressure (n=6). A steep drop in the root pressure occurred when the temperature was lowered from 25 °C to 8 °C. Discussion It is apparent that some of the cortical cells of cucumber roots had undergone dramatic ultrastructural changes after only 15 min of exposure to 8 °C. In addition, all stages of chilling injury, including those showing disruption and breakdown of cell walls, nuclei and cytoplasmic components, were observable after this very short period of cold exposure. These events are undoubtedly in response to early changes in the cell components associated with vital cell activities. An early increase in cytoplasmic density appears to be due to an increase in the population of free ribosomes, apparently resulting from the dissociation of polysomes. This means that a loss in the capacity of cells to synthesize the new proteins that are required for normal growth and development, occurs very early on. There are no indications from earlier studies that the response to cold even among the chilling-sensitive plants is so rapid, particularly in terms of ultrastructural modi®cation of cell components leading to cell destruction. Many of the earlier ultrastructural studies on chilling stress to plants have been focused on aerial parts; fewer studies have examined roots. Accumulation of lipids and dense deposits in response to cold stress has been observed in various cell components, including plastids, cytoplasm, regions between the plasmalemma and the cell wall, and in intercellular spaces (Slack et al., 1974; Ilker et al., 1976; Platt-Aloia and Thomson, 1976; Wise et al., 1983). Chloroplasts, mitochondria and cytoplasmic membranes, including ER, have been observed to undergo dramatic changes. The swelling of plastids and mitochondria, and dilation, disorganization and disruption of their membranes, disruption and vesiculation of ER and other cytoplasmic membranes appear to be common features among the ultrastructural changes associated with chillinginduced injuries (Millerd et al., 1969; Taylor and Craig, 1971; Forde et al., 1975; Kimball and Salisbury, 1973; Ilker et al., 1976; Moline, 1976; Niki et al., 1978; Nessler and Wernsman, 1980; Murphy and Wilson, 1981; Wise et al., 1983; Ishikawa, 1996). The observations presented here are consistent with the results of earlier studies and, in addition, provide substantial new information on these and other changes related to cytoplasmic, nuclear and cell wall modi®cations. Recent biochemical studies on plant response to chilling stress provide the basis for understanding the rapid ultrastructural changes observed in cucumber roots in response to cold exposure. While some studies have shown no noticeable effect of cold temperatures on mitochondrial ultrastructure (Nessler and Wernsman, 1980; Wise and Naylor, 1987), cell membranes and mitochondria generally appear to be among the main components affected during cold-induced loss of cell integrity and also during cold acclimation. It is well known that cold stress affects fatty acid composition and the ¯uidity of cellular membranes, proteins and metabolic processes (Graham and Patterson, 1982; Nishida and Murata, 1996). Mitochondria appear to be one of the main cellular components to be affected (De Santis et al., 1999), and chilling-acclimation-responsive nuclear genes, including the catalase gene (Prasad, 1997), have been isolated. Within a metabolically active plant cell the involvement of oxygen in the respiratory and photosynthetic processes leads to the generation of hydroxyl radicals and other free radicals which can damage DNA, proteins and lipids (Bowler et al., 1992). However, plants are equipped with enzymes such as catalases, peroxidases and superoxide dismutase and free radical scavengers to protect cell components from oxidative stress. The degree of protection against environmental stresses varies among species, and there are indications that chilling-sensitive plants either do not produce these enzymes to the level required for protection from oxidative stress, or the level of these enzymes goes down in response to exposure of plants to chilling temperatures. The drastic ultrastructural changes observed in mitochondria suggest that the oxidative stress may also be the main cause of this rapid response in cucumber roots. Monitoring the level of catalases, peroxidases and superoxide dismutase in cucumber root tips during the periods of cold exposure will be necessary to con®rm this. Cold sensing of plants is mediated by an early event in the signalling process, involving transient in¯ux of calcium into the cytosol (Knight et al., 1991; Monroy and Dhindsa, 1995). Oxidative signals also cause a transient increase in cytosolic calcium (Price et al., 1994). These studies point to a link between cold sensing, calcium and oxidative stress, and studies related to cytochemical localization of calcium in cucumber root Chilling injury in cucumber root 2237 tips after varying periods of cold exposure are presently being undertaken by the authors. 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