Chilling root temperature causes rapid ultrastructural changes in cortical cells of cucumber...

DOI: 10.1093/jxb/erf071

Chilling root temperature causes rapid ultrastructuralchanges 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 Korea2 Department of Forest Products and Technology, College of Agriculture, Chonnam National University, Gwangju500-757, 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 peri-

ods 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, mito-

chondria, plastids, and ribosomes. The extent of

alterations varied greatly among cells, moderate to

severe alterations to cell components being observ-

able 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 bath-

ing 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 plantfor many parts of the world, Asian countries in particular.

However, this plant is sensitive to temperatures belowabout 15 °C, and massive economic losses can occur if thetemperature suddenly falls below this level during thegrowing season.

The effect of cold temperatures on the ultrastructureof plant cells has been studied for a long time, andrecently reviewed (Kratsch and Wise, 2000). Theextent of alterations in cell components appears to berelated to the severity of the cold temperatures and thelength of exposure. Generally, agricultural plants grownin tropical and sub-tropical regions are susceptible tochilling damage at temperatures ranging from 0±15 °C.Plant response to cold temperatures appears to be bothspecies- and tissue-speci®c. Ultrastructural studies sug-gest that cold-temperature-related changes involve awide range of cell components, and plastids are one ofthe most extensively investigated organelles. Plastidsand thylakoid membranes swell and become disorga-nized, thylakoids and peripheral reticulum vesiculate,plastoglobuli or lipid droplets accumulate, and eventu-ally the entire plastid becomes disorganized leading tothe disintegration of the envelope (Klein, 1960; Millerdet al., 1969; Taylor and Craig, 1971; Kimball andSalisbury, 1973; Forde et al., 1975; Moline, 1976; Nikiet al., 1978; Nesler and Wernsman, 1980; Murphy andWilson, 1981; Wise et al., 1983; Ishikawa, 1996).Signi®cant alterations have also been observed in othercell components. Mitochondria undergo distinct swell-ing (Ilker et al., 1976; Moline, 1976; Niki et al., 1978;Murphy and Wilson, 1981; Ishikawa, 1996), thepopulation of ribosomes decreases, endoplasmic reticu-lum (ER) dilates, cytoplasmic membranes vesiculate,

3 To whom correspondence should be addressed. Fax: +82 62 5300190. E-mail: [email protected]

Published by Oxford University Press

Journal of Experimental Botany, Vol. 53, No. 378, pp. 2225±2237, November 2002

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the nuclear chromatin condenses forming clumps,plasmalemma invaginates and there is an increase invacuolation and the number of membraneous vesicles(Ilker et al., 1976; Moline, 1976; Platt-Aloia andThomson, 1976; Niki et al., 1978; Murphy and Wilson,1981; Ishikawa, 1996).

Cucumber roots respond rapidly to 8 °C chillingtemperature, with a drastic reduction in water uptakewithin about 10 min of exposure to this temperature(SH Lee et al., unpublished observations), and thusprovide a valuable experimental system to studyvarious aspects of cell biology in relation to this andother factors. Using the root pressure probe formeasurements of root pressure developed in the rootsystem (Steudle, 2000), evidence is provided for asudden, steep drop in root pressure in this specieswithin minutes of exposure to chilling temperature. Theultrastructural observations reported here on the effectof 8 °C temperature on cucumber root tips are part ofa detailed study to investigate tissue organization andanatomical composition of various regions of cucumberroots, including the root-hair-producing region wheremuch of the water uptake is likely to occur. The aimis to understand the basis for the observed sudden dropin the root pressure in cucumber in response to chillingtemperature using a combined physiological, biochem-ical and anatomical approach in this study.

Materials and methods

Cucumber (Cucumis sativus L.) seeds were germinated on moistpaper towels in plastic trays, in the dark, in an incubator. Theseedlings were grown for 4 d in the dark and were then exposed to8 °C for periods ranging from 15 min to 96 h. About 1±2 mm longsegments were collected from the root tip region at 0 h (control) andafter 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 processedfor light (LM) and transmission electron microscopy (TEM) asdescribed 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 stereomicroscope, and ®xed overnight at 4 °C. After washing in the buffer,the samples were post-®xed in 2% osmium tetroxide (also preparedin 0.05 M sodium cacodylate buffer) and washed again in the buffer.They were dehydrated in a graded series of acetone, and in®ltratedand embedded in Spurr's low viscosity resin (Spurr, 1969).

Semi-thick (2±3 mm) and ultra-thin sections (60±80 nm) were cutwith glass and diamond knives, respectively, on an RMC MT Xultramicrotome. Semi-thick sections were stained with 0.1% aque-ous toluidine blue and examined with a Zeiss Axiolab photomicro-scope. Ultra-thin sections were sequentially stained with uranylacetate and lead citrate and then examined with a JEOL 1010 TEM.

The root pressure of an excised cucumber root system wasmeasured by root pressure probe as previously described (Steudle,2000). Once the cut end of a root segment was connected tightly tothe probe, the root pressure developed due to active uptake ofnutrients into root, and the root pressure was measured continuouslyusing the probe.

Results

The ultrastructural observations presented are based on theconventional chemical ®xation of samples, which is knownto alter the morphology of some cell components. It is alsolikely that cells undergoing cold-induced changes mayreact somewhat differently to ®xatives compared touninjured cells. Therefore, the observations presentedhave to be viewed with these considerations in mind.

Observations were con®ned to examining cortical cellsfrom a region about 1 mm behind the root apex. Cells inthis region were undergoing expansion growth as indicatedby the presence of developing as well as fully developedintercellular spaces. Examination of semi-thick monitorsections with LM provided early indications that changesin root cortical cells due to cold treatment may be ratherrapid.

In roots receiving 15 min of cold exposure some areas ofthe cortex stained only lightly with toluidine blue. Cells insuch regions appeared abnormal; they had a highly reducedcytoplasmic density, and the presence of cell walls couldnot be con®rmed (Fig. 1B). However, the staining of alarge proportion of the cortex was similar to that of thecortex of control roots (Fig. 1A). Nuclei and cell wallswere easily recognized in these cells and they had a normalappearance, judging by comparisons with cortical cells ofcontrol roots.

With longer periods of cold exposure, greater areas ofthe cortex were affected (Fig. 1C). However, damagedareas did not appear to be proportional to the time ofexposure. Even after 96 h of exposure only about 25% ofthe cortex appeared to be severely affected. However, thisestimate is based on single sections only and not serialsections.

TEM observations of damaged and adjacent corticalareas con®rmed that cells in the lighter areas were severelyaffected, in addition to providing detailed information onthe nature and extent of alterations in various subcellularcomponents.

Control roots

Cortical cells from control roots (roots not exposed to cold)displayed features typically found in a metabolically activecell. Nuclei were large and spherical, containing fairlydisperse chromatin and a prominent nucleolus (Fig. 2A).Nuclei were more or less centrally located in the cells. Arelatively dense cytoplasm contained numerous mitochon-dria and variously shaped plastids, which had a stromadenser than that of mitochondria and were thus readilydistinguishable from them even at low magni®cations(Figs 2A, B, 3A). The mitochondria had a moderatelydense stroma, which contained ribosomes, DNA and well-developed cristae (Fig. 6A, B). The two membranes of themitochondrial envelope were parallel and evenly spaced(Fig. 6A, B). The plastids contained few internal mem-

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branes which were not readily distinguishable because ofthe high density of the stroma (Fig. 6A). Also, the plastidenvelope, which consisted of two parallel membranes, wasnot easily resolvable because of the high density of theplastid stroma (Fig. 6A, B) and the pleomorphic nature ofthe plastid form (Figs 2A, 3A). The cytoplasm alsocontained abundant cisternae of rough ER, which weredif®cult to discern at low magni®cations, mainly becauseof the high density of the cytoplasm (Fig. 2A, B). As seenat high magni®cations, ER cisternae were long, with auniform intracisternal spacing between the limiting mem-branes, and were studded with ribosomes (Fig. 6A).Polysomes were common also in other parts of thecytoplasm.

The plasmalemma appeared to be closely associatedwith the cell walls, which were thin and somewhatsinuous in the intercorner regions of the cells (Fig. 2A,B). The cell walls were thicker in the cell cornerregions, particularly in those cells which had developedintercellular cavities or were in the process of doing so(Figs 2B, 3A). Initially, vacuoles were small (Fig. 2A),but they progressively enlarged mainly through thecoalescence of adjoining vacuoles, and this processappeared to coincide with the development of inter-cellular cavities (Figs 2B, 3A). The intercellularcavities were formed mainly by the dissolution of theintercellular material in the cell corner regions, asshown by the presence of reticular, vacuolate anddense materials in these areas (Fig. 2B).

Cold-treated roots

Chilling injury to cortical cells of exposed roots wasapparent as early as 15 min after exposure (Fig. 1B). Injuryin these samples varied greatly in severity, ranging fromchanges in a few cell components to almost completedisintegration of the cytoplasm. Longer exposure periodscaused greater parts of the cortex to be affected, as seen inthe light micrograph illustrated (Fig. 1C). Therefore, thefollowing description is primarily based on the featuresconsidered to represent sequential injury, and not neces-sarily on the sequence of the exposure time. Also, rootsexposed to all periods had cells showing signs of injury aswell as those which appeared to have normal ultrastruc-ture. This feature is illustrated in Fig. 3B, where cells withearly injuries can be seen next to those without anyapparent injuries.

Early stages of chilling injury

A noticeable increase in the density of cytoplasm, anddistention of endoplasmic reticulum (ER) were earlysymptoms of injuries to cells (Figs 3B, 4A). At lowmagni®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 earlystages of cell injury because of the presence of wider,

Fig. 1. (A) Transverse section through a control root tip. Corticalcells show features typical of metabolically active cells. They have adense cytoplasm and large nuclei containing a prominent nucleolus.LM. Bar=100 mm. (B) Transverse section through a root tip exposedto cold temperature for 15 min. A small part of the cortex has stainedlightly, and shows signs of cell damage (arrow). LM. Bar=100 mm.(C) Transverse section through a root tip exposed to cold temperaturefor 48 h. A relatively large part of the cortex has stained lightly andshows signs of cell damage (arrows). LM. Bar=100 mm.

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variable intra-cisternal spaces in them (Figs 3B, 4A). Thestroma of both mitochondria and plastids became moretranslucent and granular, as viewed in low magni®cation

images (Fig. 4A). Both nuclei and nucleoli becameirregularly shaped (Fig. 4A). Cell walls were thinnerthan 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 thecisternae of ER (circles). The vacuoles (V) are relatively small. The nuclei (N) are centrally located and contain a large nucleolus (NU). Thechromatin is largely uncondensed. TEM. Bar=2 mm. (B) The cells shown here are similar in their ultrastructure to those in (A), except that cellcorners show signs of developing spaces. The cell corners are prominent and contain dense and vacuolate materials (arrowheads). The cytoplasmis dense and rich in mitochondria (M), plastids (P) and ER (circles). Some plastids contain starch grains. The vacuoles (V) are small, discrete andalso show signs of fusion. N, nucleus. TEM. Bar=2 mm.

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Intermediate stages of chilling injury

Cytoplasmic density was still very high and wascomparable to that in the cells showing signs ofearly injuries (Figs 4B, 7A, B). ER cisternae became

more distended and more irregular in their form (Figs4B, 8A). The cytoplasm also contained other unusualmembraneous formations (Fig. 8A). The nuclear envel-ope 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 inFig. 2B. The intercellular regions (IS) have developed cavities. The cell walls (CW) are thicker and less sinuous. The vacuoles are larger andshow signs of continued enlargement through fusion (arrowheads). The presence of starch in plastids is more common. The mitochondria (M) areabundant. 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 earlysigns 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 normalappearance. TEM. Bar=2 mm.



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Mitochondria and plastids became progressively moreswollen and translucent, losing much of their internal

membranes and ribosomes (Figs 4B, 7A, B). Somemitochondria 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 ofchilling injury. The cytoplasm is dense and ®lled with numerous distended membraneous structures, most of which are probably ER. Themitochondria (M) are abundant and their interior appears granular. The plastids (P) appear swollen, contain starch grains, and have a moderatelydense interior. The number and size of vacuoles (V) are highly variable. The nucleus (N) has an irregular shape and contains an irregularly shapednucleolus (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 positionedand 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 smallermitochondria (M). The interior of these bodies appears granular. The plasmalemma has pulled away from the cell wall in places (arrows). The starindicates a completely damaged cell, which is virtually empty. TEM. Bar=2 mm.

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modi®ed ribosomes (Fig. 7A, B). However, there didnot appear to be a close correlation between the loss inribosomes and other changes taking place in mitochon-

dria. For example, there were examples of highlyswollen mitochondria, which had only a few internalmembranes, 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. Cellwalls can not be recognized and may have disintegrated. The nuclei (N) are highly irregular and have almost completely lost the integrity of theirenvelope. 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 havevesiculated greatly and can not be recognized. The cytoplasmic density is greatly reduced. TEM. Bar=2 mm. (B) Cortical cells from a root tipexposed to cold temperature for 16 h. The cell marked by an asterisk shows signs of an intermediate stage of chilling injury. The other cells aregreatly transformed and show signs of the ®nal stages of disintegration. The cytoplasm in these cells is sparsely granular and contains scatteredvesicles of varying sizes (arrowheads). The cell walls are extremely thin and show sign of discontinuity (arrow). TEM. Bar=2 mm.



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ribosomes (Fig. 7A). In some instances the remaininginternal membranes in mitochondria had vesiculated(Fig. 7A). The cell walls were extremely thin and theplasmalemma had pulled away from the cell wall inplaces (Fig. 4B).

Advanced stages of chilling injury

Cytoplasmic density progressively declined (Figs 5A,8B, 9A, B). In the ®nal stages, the cytoplasm appearedvery thin and electron-lucent, containing sparselydistributed 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.TheER cisternae are long with a uniform intracisternal spacing between the limiting membranes. The plastids (P) are dense and occasionally containstarch 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 membraneousenvelope; the membranes are parallel with a uniform spacing (circles). N, nucleus; NP, nuclear pore. TEM. Bar=500 nm.

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ribosomes (Fig. 5B). Cell walls were extremely thin,wavy and disrupted in places (Fig. 5B). In some casescell walls had disappeared completely, and thuspartitioning between the nuclei of neighbouring cells

was lost (Fig. 5A). The nuclei had lost the integrity oftheir envelope, which appeared highly irregular anddisrupted in places (Fig. 5A). Fragmented membranesof 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 greatlyswollen. They have lost much of their internal membranes, although the envelope is still present. Ribosomes are still in abundance in themitochondrial stroma, which also contains some vesiculate, membraneous structures (arrowheads). Membraneous vesicles are also common in thecytoplasm (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 coldtemperature for 16 h. The cytoplasm is highly dense. Mitochondria (M) appear swollen. The mitochondrial stroma has lost many of its ribosomesand thus appears considerably more lucent than the mitochondria of control root tips. A few cristae and the limiting envelope are still present insome mitochondria. The large vacuolate body (P) with sparse granular inclusions may be a highly swollen plastid. TEM. Bar=500 nm.



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to parent nuclei (Fig. 9A). ER and other cytoplasmicmembranes were highly modi®ed (Fig. 8B) formingvesicles of varying shapes and sizes (Figs 5A, 9B).The envelopes of both mitochondria and plastids weredisrupted (Fig. 5A). The cytoplasm also containedspherical, electron dense bodies (Figs 8B, 9B).Eventually, the enucleate cell appeared very electron

transparent and sparsely granular, containing vesicles ofvarying shapes and sizes (Fig. 5B).

Root pressure measurement

Root pressure in excised cucumber root system meas-ured with the root pressure probe was about 0.15±0.20MPa 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 ERappears distended and has also vesiculated. The cytoplasm also contains some highly dense deposits (arrowheads). TEM. Bar=500 nm.

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However, when the temperature was gradually loweredfrom 25 °C to 8 °C, the root pressure decreasedrapidly, reaching zero MPa within 15±20 min (Fig. 10).

This steep drop in the root pressure is a clearindication that the cucumber root system is verysensitive 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 brokendown, but the nucleolus (NU) is still present. The membranes (arrowheads) present near the nucleolus may have been part of the nuclearenvelope. 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. Thecytoplasmic density is greatly reduced as compared to the cells in Fig. 7. The mitochondrion (M) present contains only a few cristae andaggregates of ribosomes and appears highly translucent. Membraneous vesicles (arrowheads) are common in the cytoplasm. The cytoplasm alsocontains some dense deposits (arrows). TEM. Bar=500 nm.



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Discussion

It is apparent that some of the cortical cells of cucumberroots had undergone dramatic ultrastructural changes afteronly 15 min of exposure to 8 °C. In addition, all stages ofchilling injury, including those showing disruption andbreakdown of cell walls, nuclei and cytoplasmiccomponents, were observable after this very short periodof cold exposure. These events are undoubtedly inresponse to early changes in the cell components associ-ated with vital cell activities. An early increase incytoplasmic density appears to be due to an increase inthe population of free ribosomes, apparently resulting fromthe dissociation of polysomes. This means that a loss in thecapacity of cells to synthesize the new proteins that arerequired for normal growth and development, occurs veryearly on. There are no indications from earlier studies thatthe response to cold even among the chilling-sensitiveplants is so rapid, particularly in terms of ultrastructuralmodi®cation of cell components leading to cell destruc-tion.

Many of the earlier ultrastructural studies on chillingstress to plants have been focused on aerial parts; fewerstudies have examined roots. Accumulation of lipids anddense deposits in response to cold stress has been observedin various cell components, including plastids, cytoplasm,regions between the plasmalemma and the cell wall, and inintercellular 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 dramaticchanges. The swelling of plastids and mitochondria, anddilation, disorganization and disruption of their mem-branes, disruption and vesiculation of ER and othercytoplasmic membranes appear to be common features

among the ultrastructural changes associated with chilling-induced 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; Nesslerand Wernsman, 1980; Murphy and Wilson, 1981; Wiseet al., 1983; Ishikawa, 1996). The observations presentedhere are consistent with the results of earlier studies and, inaddition, provide substantial new information on these andother changes related to cytoplasmic, nuclear and cell wallmodi®cations.

Recent biochemical studies on plant response to chillingstress provide the basis for understanding the rapidultrastructural changes observed in cucumber roots inresponse to cold exposure. While some studies have shownno noticeable effect of cold temperatures on mitochondrialultrastructure (Nessler and Wernsman, 1980; Wise andNaylor, 1987), cell membranes and mitochondria generallyappear to be among the main components affected duringcold-induced loss of cell integrity and also during coldacclimation. It is well known that cold stress affects fattyacid composition and the ¯uidity of cellular membranes,proteins and metabolic processes (Graham and Patterson,1982; Nishida and Murata, 1996). Mitochondria appear tobe one of the main cellular components to be affected (DeSantis et al., 1999), and chilling-acclimation-responsivenuclear genes, including the catalase gene (Prasad, 1997),have been isolated. Within a metabolically active plant cellthe involvement of oxygen in the respiratory andphotosynthetic processes leads to the generation ofhydroxyl radicals and other free radicals which candamage DNA, proteins and lipids (Bowler et al., 1992).However, plants are equipped with enzymes such ascatalases, peroxidases and superoxide dismutase and freeradical scavengers to protect cell components fromoxidative stress.

The degree of protection against environmental stressesvaries among species, and there are indications thatchilling-sensitive plants either do not produce theseenzymes to the level required for protection from oxidativestress, or the level of these enzymes goes down in responseto exposure of plants to chilling temperatures. The drasticultrastructural changes observed in mitochondria suggestthat the oxidative stress may also be the main cause of thisrapid response in cucumber roots. Monitoring the level ofcatalases, peroxidases and superoxide dismutase in cucum-ber root tips during the periods of cold exposure will benecessary to con®rm this.

Cold sensing of plants is mediated by an early event inthe signalling process, involving transient in¯ux ofcalcium into the cytosol (Knight et al., 1991; Monroyand Dhindsa, 1995). Oxidative signals also cause atransient increase in cytosolic calcium (Price et al.,1994). These studies point to a link between cold sensing,calcium and oxidative stress, and studies related tocytochemical localization of calcium in cucumber root

Fig. 10. The effect of low root temperature on cucumber root pressure(n=6). A steep drop in the root pressure occurred when thetemperature was lowered from 25 °C to 8 °C.

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tips after varying periods of cold exposure are presentlybeing undertaken by the authors.

The observation of a sudden, steep drop in root pressurewhen the temperature of the bathing solution was loweredto a chilling temperature provides further support for thehigh sensitivity of cucumber roots to low temperature. Thechallenge now is to understand the biochemical andstructural basis for the rapid response of cucumber tochilling temperature in its root-water relations.

Acknowledgements

This work was supported by grants from the Korea Science andEngineering Foundation (KOSEF) to the Agricultural Plant StressResearch Centre (APSRC, R11-2001-09201004-0) of ChonnamNational University.

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