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Chloroplast Membrane Organization in Chilling Tolerant and Chilling-Sensitive Maize Seedlings
• • OUR.ALOF
j. Plant Physiol. Vol. 155. pp. 691-698 (1999) http://www.urbanfischer.de/journals/j pp
Plan. Ph,s.olo., © 1999 URBAN Ii! FISCHER
Chloroplast Membrane Organization in Chilling-Tolerant and Chilling-Sensitive Maize Seedlings 2 1 2 REENA GRITTLE PINHER0 , GOPINADHAN PALIYATH , RICKEYY. YADA , 1 DENNIS p. MURR 1
Department of Plant Agriculture, Division of Horticultural Science and
2
Department of Food Science, University of Guelph, Guelph, Ontario, Canada N I G 2W1
and
Accepted May 25, 1999
Summary
Resistance of maize (Zea mays L.) seedlings to chilling-induced damage and its potential bearing on chloroplast membrane organization have been investigated using a chilling-sensitive inbred CO 316 and a chilling-tolerant inbred CO 328. Paclobutrazol treatment of the chilling-sensitive CO 316 (CO 316P) induced chilling tolerance, also causing several morphological and physiological changes, and served as another means of exploring the relation between chloroplast membrane changes and chilling tolerance. Chilling treatment [6 'CI2 'C (day/night)] resulted in a reduction of Fv/Fm ratio, photosynthetic pigment levels and an increase in leakage of electrolytes. Within 24 h of exposure of CO 316 to chilling, the granal and stromal membrane showed extensive vesiculation and disruption of the granal array. By contrast, the granal and stromal organization of chilling-tolerant CO 328 and P-treated CO 316 remained nearly intact. Our studies suggest that paclobutrazol treatment can alter the membrane deterioration and disassembly processes of the thylakoid membranes of chilling-sensitive CO 316 to make them functionally similar to the chloroplast membrane of genetically chilling-tolerant CO 328.
Key words: Maize, Zea mays L., chilling stress, chilling tolerance, membrane, paclobutrazol, vesiculation. Abbreviations: P = paclobutrazol. Introduction
An important factor that dictates the geographic distribution of several plant species is their ability to withstand chilling stress (Oquist, 1983). Chilling injury occurs when crops indigenous to tropical and subtropical regions are exposed to low, non-freezing temperatures (0-12 'C, Lyons, 1973). The common symptoms of chilling injury include wilting, inhibition of metabolic processes, chlorosis, and changes in the molecular ordering or physical state of cell membranes that lead to increased permeability or leakage. The damaging effects ultimately result in reduced growth and yield (Saltveit and Morris, 1990; Rab and Saltveit, 1996). Chilling in the presence of light causes photo oxidation, bleaching of leaf pigments, chloroplast ultrastructural changes and cellular lipid degradation (Wise and Naylor, 1987). Maize, which ranks
among the top three food crops of the world, is chilling-sensitive. Chilling-sensitivity of several crop species varies depending on the stage of crop development and cultivar (Miedema et al., 1987; Janowiak and Markowski, 1994). Numerous attempts such as breeding for increased chilling tolerance, modifYing crop management practices and application of chemicals have been used to avoid chilling injury (Lee et al., 1985; Zhang et al., 1987). Paclobutrazol [(2RS,3RS)-I(4-chlorophenyl)-4,4-dimethyl-2-(I,2, 4-triazolyl)-pentan-301] is a triazole compound that exhibits varying degrees of plant growth regulatory and fungicidal activities due to its inhibition of cytochrome P4SO - dependent oxidation of entkaurene to GAlz-aldehyde, leading to the inhibition of gibber ellin and ergosterol biosynthesis in plants and fungi, respectively (Rademacher, 1992). Triazoles such as uniconazole, paclobutrazol and triadimefon protect plants from various 0176-1617/991155/691 $ 12.00/0
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REENA GRITTLE PINHERO, GOPINADHAN PALIYATH, RICKEY Y. YADA, and DENNIS P. MURR
environmental stresses (Davis and Curry, 1991; Kraus and Fletcher, 1994; Paliyath and Fletcher, 1995 a, 1995 b). Inhibition of gibberellin biosynthesis appear to alter several aspects of the regulation of the isoprenoid pathway leading to increased absciscic acid and cytokinin levels (Asare-Boamah et al., 1986; Grossman et aI., 1987), membrane properties, changes in the composition of antioxidants and antioxidant enzymes (Pinhero et aI., 1997) as well as several morphological parameters. The biochemical mechanisms resulting in the beneficial effects of triazole treatment need to be fully understood in order to develop transgenic stress-tolerant crop plants. The present study was undertaken to explore the biochemical adaptations to chilling tolerance exhibited by two maize inbreds, one of which is genetically chilling-tolerant (CO 328), whereas the other (CO 316) exhibits a low degree of chilling tolerance. However, chilling tolerance can be induced in this chilling-sensitive inbred by paclobutrazol treatment (Pinhero et aI., 1997). Since chilling injury is manifested at the membrane level (Lyons, 1973; Murata, 1990), we explored changes in thylakoid membrane organization during chilling as a potential indicator of the degree of chilling tolerance. Other parameters of monitoring chilling damage included measuring the variable (Fv) to maximal (Fm) fluorescence ratio, and membrane integrity in terms of leakage of electrolytes. Thus, changes in chloroplast membrane organization during chilling and a subsequent recovery period will indicate potential biochemical parameters that afford chilling tolerance to CO 328. Additionally by comparing the thylakoid membrane organization between CO 328 and paclobutrazol-treated CO 316, we delineated potential similarities and differences in membrane deterioration and disassembly that could be relevant to chilling tolerance mechanisms.
Materials and Methods Plant material and treatments Separate experiments were conducted to determine the extent of chilling injury in two maize (Zea mays L.) inbreds, CO 316 and CO 328, and to establish the effect of paclobutrazol in the alleviation of chilling injury. The CO lines of corn were developed at the Eastern Cereals and Oilseeds Research Centre, Agriculture and Agri-Food Canada, Ottawa. CO 316 was developed from the pedigree CO 266 x CO 273 / / CO 266. CO 273 and CO 266 were developed from the Pioneer hybrid 3990. CO 328 was developed from the ped igree CO 216 x CO 258 / / CO 216 BC2. CO 216 originated from the hybrid Pride 5 which is a four way or double cross hybrid. Thus CO 328 is believed to be completely different from CO 316. However, molecular or isozyme analysis have not been conducted to delineate differences (Robert Hamilton, personal communication). Seeds (50 g) were imbibed for 18 h in 100 mL of 0.17 mmol· L-1 paclobutrazol (P) or in water (controls) and air dried for 4 days. They were sown in a commercial potting medium (Promix PGX, Premier Brands, Red Hill, PAl in plastic cells (17.5 X 13.5 x 5.5 em). Seedlings were grown for 11 days in a growth cabinet, maintained at day/night temperatures of 23 ± 2 °C/19 ± 2°C, 50 to 70 % relative humidity, and a 16h photoperiod at 200 !lmol m -2 s-I,
Growth analyses Eleven days after sowing, plant height, fresh and dry weights of shoots, leaf area and photosynthetic pigment contents were determined. Leaf area was measured using a Li - Cor Li - 3100 area meter (Li - Cor Inc., USA). Dry weights of shoots were measured by drying the shoot in an oven at 70°C until consistently constant weights were obtained.
Imposition of chilling stress Eleven-day old seedlings were exposed for two days to a cold temperature regime of 6 °C/2 °C (day/night) and a photoperiod of 16 h at 450 !lmol m -2 s-l. After exposure to chilling stress, seedlings were returned to the previous growth conditions to assess the extent of chilling damage on chlorophyll fluorescence (Fv/Fm), electrolyte leakage, photosynthetic pigments, and thylakoid membrane organization.
Variable (Fv) to maximal (Fm) fluorescence ratio Fv/Fm ratio was measured in situ on the second true leaf with a portable Hansatech Plant Efficiency Analyzer (Hansatech Instrument Ltd., King's Lynn, UK) after dark-adapting the seedlings for 30 min as described previously (Pinhero and Fletcher, 1994). Fv/Fm was measured prior to chilling, immediately after 48 h of chilling (0 h post-chilling), 24 h, and 48 h of post-chilling periods.
Leakage ofelectrolytes Damage to membrane integrity was monitored by measuring the increase in leakage of ions from the second true leaf. The leaves were cut into 2 cm segments, rinsed in distilled water and placed in a test tube with 15 mL of distilled water at 24 °C for 24 h. Initial conductivity of the solution (spontaneous leakage of ions due to membrane damage) and total ionic conductivity after boiling the sample for 20 min was measured using a Model 32 conductance meter (Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio, USA). Percent leakage was measured as described previously (Pinhero and Fletcher, 1994) using the following formula 01.
,0
Ieakage 0 f'IOns = Initial - - conductivity ----'Total conductivity
X
100
Photosynthetic pigments Photosynthetic pigments were quantified two days after removal from chilling stress. For estimating the level of photosynthetic pigments, 2.5 cm long leaf segments were taken starting from the tip of the second true leaf. Chlorophyll a, b and carotenoid contents were determined after extracting the pigments in 80 % ethanol, as described earlier by Pinhero and Fletcher (1994).
Electron microscopy Three leaf pieces, each of 1 X 5 mm area were cut from the middle portion of the second true leaf from three different plants using a sharp razor blade. These pieces were fixed in cold 3 % glutaraldehyde (Sigma Chemical Co., St. Louis, MO) in 100 mmol. L-1 potas-
Chloroplast Membrane and Chilling Tolerance sium phosphate buffer, pH 7.2, vacuum-infiltrated for 10 min and left overnight at 4 'co The fixed material was washed with the same buffer and post-fixed in 1 % osmium tetroxide at 4 'C overnight. Post-fixed material was dehydrated in a graded series of ethanol (25 %, 50 % and 100 %). Dehydrated specimens were infiltrated, embedded in LR White contained in gelatin caps and allowed to polymerize at 60 'C for 1 h (Marty et aI., 1995). Sections were cut with a diamond knife and post-stained in 2 % aqueous uranyl acetate followed by Reynold's lead citrate. These sections were examined in a Philips EM 300 electron microscope set at 60 kV: Each figure is representative of 25-30 sections examined under the microscope.
693
ing of the potential mechanism of chilling tolerance in CO 328 and P-treated CO 316, the chloroplast membrane organization in these two sets of plants were investigated as a function of chilling duration. Since major differences were observed in the changes in Fv/Fm ratio as a function of chilling and post-chilling recovery in CO 328, CO 316 and P-treated CO 316, it was logical to assume that significant differences could exist in the chloroplast membrane organization and disassembly between plants in the three experimental systems studied.
Effect of chilling on maize inbreds and its protection by paclobutrazol
Statistical analysis ofdata The data were statistically analysed using analysis of variance in a completely randomized design and significant differences were determined using the protected LSD, at P=0.05.
Results
Since paclobutrazol at 0.17 mmol . L-1 did not alter the chilling tolerance of CO 328, only data from studies carried out with CO 328, CO 316 and P-treated CO 316 are presented. Initial values on growth parameters, Fv/Fm ratio, photosynthetic pigment contents and percent leakage of ions measured on ll-day-old seedlings are given in Table 1. Genetically chilling-tolerant CO 328 and the chilling-susceptible CO 316 possessed inherent differences in their morphology and growth parameters. Paclobutrazol treatment of chillingsusceptible CO 316 caused drastic morphological changes that included reduced height and increased root to shoot ratio that are characteristic to plants treated with triazole growth regulators (Pinhero and Fletcher, 1994). Even though CO 328 and P-treated CO 316 differed considerably in their morphology, the degree of chilling tolerance exhibited by these seedlings was nearly the same. These observations suggested that the underlying biochemical mechanisms of chilling tolerance exhibited by CO 328 and P-treated CO 316 may not be exactly identical. To obtain a better understand-
Table 1: Growth analyses of eleven-day old maize seedlings. Seedlings were grown for 11 days in growth cabinet maintained at 23 ± 2 'C I 19 ± 2 'C and measurements were taken as described in materials and methods. Parameters
CO 316
CO 316P
CO 328
Height (cm) Fresh wt. of shoots (g plant-I) Fresh wt. of roots (g plant -1) Root: shoot ratio (fresh wt.) Dry wt. of shoots ~ plant-I) Total leaf area (cm plant-I) Fv/Fm ratio Leakage of ions (%) Chlorophyll a (mg. g fresh wt. -1) Chlorophyll b (mg. g fresh wt. -\) Carotenoids (mg. g fresh wt. -\)
15.70a 0.78 a 0.37b 0.47 b 0.07" 2l.20a 0.75 b 3.20 b l.94a 0.82a 0.6 2 b
9.30 C O.71 a 0.46 a 0.65 a 0.06 a 20.20a
13.60 b 0.76 a 0.32b 0.43 b 0.07" 18.03 a 0.80 a 4.50 a l.61 b 0.62b 0.46 a
O.77b
4.30 a l.72 ab 0.69 ab 0.52a
a. b, ab denotes significant differences between values within a row at p ~ 0.05.
Changes in variable (Fv) to maximal (Fm) chlorophyll fluorescence has been widely used as a parameter in determining chilling injury before visible symptoms appeared (Wilson and Greaves, 1990). Immediately after chilling, Fvl Fm ratio decreased drastically in both inbreds (Fig. 1 A). However, the extent of reduction in Fv/Fm ratio was more in CO 316 than in CO 328. Paclobutrazol treatment prevented the extent of reduction in Fv/Fm ratio in CO 316. However, no marked change was observed in Fv/Fm ratio of CO 328 after paclobutrazol treatment. Photosynthetic efficiency as measured by Fv/Fm ratio recovered to 90 % of the initial values within 48 h of post-chilling period in CO 328 and in P-treated CO 316. However, the recovery was only 52 % in the chilling-sensitive CO 316 in the absence of paclobutrazol treatment (Fig. 1 A). Both CO 328 and P-treated CO 328 showed similar photosynthetic efficiency at all the time periods studied after chilling exposure. This result showed that paclobutrazol treatment was not giving any added protection to CO 328 from chilling damage. However, the sensitive inbred CO 316 exhibited protection from chilling after paclobutrazol treatment. Membrane damage as measured by leakage of ions was significantly higher in CO 316 when compared to that of CO 328 at all the time periods studied (Fig. 1 B). Paclobutrazol treatment reduced leakage of ions in CO 316 to a level exhibited by the chilling-tolerant CO 328. No added effect was noticed in CO 328 after paclobutrazol treatment. In parallel with the changes in Fv/Fm ratio, the decline in photosynthetic pigments (chlorophyll a, band carotenoids) was the highest in CO 316 (nearly 40 %), with only lO-20 % decline in CO 328 and P-treated CO 316 after 48 h of postchilling period (Fig. 2).
Changes in chloroplast membrane organization Examination of chloroplasts revealed that mesophyll chloroplasts of non-chilled CO 316 possessed granal and stromal thylakoids organized in typical fashion as in other graminaceous chloroplasts with abundant granal stacks and intervening stromal membranes (Fig. 3 A). However, within 24 h of chilling, swelling of thylakoids occurred in many grana, resulting in an increase in intrathylakoid space and formation of large vesicular structures (Fig. 3 B). After 48 h of chilling, granal stacks had undergone disorganization and dissolution forming large numbers of vesicular structures (Fig. 3C).
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Maize is a chilling-sensitive plant and sustains injury when exposed to low temperature (Miedema et al., 1987). Under field conditions, the seedlings are usually exposed to chilling temperatures and the damage is accentuated by simultaneous exposure to bright sunlight which leads to the production of active oxygen species due to the disruption in photosynthetic electron transport (Powels, 1984; Walker et aI., 1991). The damage to the cells has also been examined through ultrastructural studies (Wise and Naylor, 1987; van Hasselt, 1990). It has been reported that chilling in the presence of light caused severe chloroplast damage in the chilling-sensitive cucumber as compared with the chilling-tolerant pea (Wise and Naylor, 1987). In the present study, we have compared aspects of chillingtolerance in a genetically chilling-tolerant maize inbred CO 328 to a related inbred CO 316 which is chilling-sensitive. Chilling-tolerance could be induced in CO 316 by treating with the triazole plant growth regulator, paclobutrazol. However, after paclobutrazol treatment, the morphology of the plant changes altogether. Even though the resemblance to CO 328 is remote, P-treated CO 316 is equally chilling-tolerant as CO 328, perhaps suggesting that there could be different means of adaptation to attain chilling tolerance. Therefore, we have compared several physiological parameters between CO 316, CO 328 and P-treated CO 316 during chill-
o unchilled control
48 h chilling
24 h
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48 h postchilling
Fig. 1: Changes in Fv/Fm ratio expressed as a percent of the nonstressed control values obtained from plants (A). Eleven-day old seedlings were exposed for 48 h ro a chilling temperature of 6 'C/ 2 'C (day/ni~ht) and a photoperiod of 16 h at a light intensity of 450 Ilmol m- S-1 and subsequently grown in the growth cabinet for the periods indicated. (B). The leakage of electrolytes was monitored at the specified time intervals. Values are means of six (A) and five (B) replications. Error bars marked by different letters within each cluster denote significant difference at P:S;0.05.
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Chilling-tolerant CO 328 and P-treated CO 316 showed similarities in the organization of granal and stromal membranes in the chloroplast. Even though CO 328 possessed much larger granal stacks when compared to that in P-treated CO 316, both their stromal structures showed intermittent swollen nature (Figs. 4, 5). No major changes were observed in the thylakoid organization of CO 328 and P-treated CO 316 during chilling for 48 h (Figs. 4 C, 5 B). P-treated CO 316 showed very low dissolution of starch during chilling (Fig. 4 B, C) compared to CO 316 or CO 328 in which starch grains disappeared within 24 h of chilling (Fig. 3 B, data not shown for CO 328). In addition, chloroplasts of P-treated CO 316 showed large scale microvesiculation and bleb bing of membranes leading to the accumulation of vesicles in the cytoplasm (Fig. 4 B, C, V).
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Fig. 2: Changes in chlorophyll fl, chlorophyll band carotenoids of maize seedlings during two days of post-chilling period expressed as a percent of non-stressed values. For non-stressed values, see Table 1. Eleven-day old seedlings were exposed for 48 h to a chilling temperature of 6 'C/2 'C (day/night) and a photoperiod of 16 h at a light intensity of 450 Ilmol m- I s-I and subsequently grown in the growth cabinet. Values are means of six replications. Error bars marked by different letters within each cluster denote significant difference at P:S; 0.05.
Chloroplast Membrane and Chilling Tolerance
695
Fig. 3A: Electron micrographs of mesophyll chloroplasts from unchilled CO 316 seedlings. Magnification = 19,470 X; bar = 0.5 11m; S, stroma; G, granum; SG, starch granule.
Fig. 4 A: Chloroplast membrane structure and organization of CO 316 maize seedlings treated with paclobutrazol. Magnification = 24,977X; bar = 0.4 11m; S, stroma; G, granum.
Fig. 3 B, C: Changes in chloroplast membrane organization of CO 316 seedlings exposed to a chilling duration of 24 h (3 B) and 48 h (3 C). Magnification = 38,854 X; bar = 0.3 11m; S, stroma; G, granum; V, vesicles. Large scale vesiculation and membrane damage can be noticed during 48 h of chilling.
Fig. 4 B: Chloroplasts of paclobutrazol-treated CO 316 seedlings after exposure to 24 h of chilling. Magnification 0.4 /.lm; S, stroma; G, granum; V, vesiculation.
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696
REENA GRITTLE PINHERO, GOPINADHAN PALIYATH, RICKEY Y. YADA, and DENNIS P. MURR
Fig.4C: Chloroplasts of paclobutrazol-treated CO 316 seedlings after exposure to 48 h of chilling. Magnification = 38,854; bar = 0.26 11m; S, stroma; G, granum; V, vesiculation.
Fig. 5: Chloroplasts of CO 328 seedlings prior to chilling (A, 24,977x; bar = 0.4 11m;) and after 48 h (B, 30,528 X; bar = 0.3 11m;)
exposure to chilling. Note that the thylakoid and stroma are not disrupted. S, stroma; G, granum.
ing and post-chilling recovery, which will give us an indication regarding the potential biochemical parameters that could afford chilling-tolerance to maize plants. Our study shows that the parameters employed clearly differentiate the chilling-tolerant CO 328 and P-treated CO 316 from the chilling-sensitive CO 316. The decline in Fv/Fm ratio is lower in CO 328 and P-treated CO 316 as compared with CO 316 (Fig. 1 A). Various studies have used chlorophyll fluorescence ratio (Walker et aI., 1990; Wilson and Greaves, 1990) and electrolyte leakage (Janowiak and Markowski, 1994) as quantitative methods for differentiating chilling-tolerance of cultivars. Since the initial studies to screen the toler-
ance level of paclobutrazol-treated maize seedlings showed the ineffectiveness of paclobutrazol in augmenting the genetic chilling-tolerance in CO 328, further characterization of the chilling effect on maize seedlings was limited to CO 328, CO 316 and P-treated CO 316. Most of the higher altitude and latitude areas, where chilling-sensitive crops are grown, are characterized by large differences between day and night temperatures. For example, cold clear nights are likely to be followed by bright sunlight, early in the morning when the temperature is still low. Exposure to light during chilling can damage chilling-sensitive plants by causing light-induced degradation of photosynthetic pigments or photooxidation (Zhang et aI., 1987). It has been reported that photodegradation of leaf pigments in the chloroplast membrane can occur at temperatures below 10 °C (van Hasselt, 1990). Chloroplast membrane contains a remarkably high level of unsaturated lipids. Photoperoxidation of chloroplast lipids, concurrent with leaf pigment degradation was observed in cucumber leaf segments during chilling (Wise and Naylor, 1987). It has been reported that the use of electron transport inhibitors dichlorophenyl dimethyl urea and atrazine inhibited the photoperoxidation of lipids in cucumber leaf discs during chilling, indicating an involvement of superoxide in the damaging process (Wise and Naylor, 1987). The lower degree of damage noticed in thylakoid organization of P-treated CO 316 and CO 328 indicated that these plants possessed some protective mechanism against chilling injury, presumably at the level of preventing the formation or detoxification of active oxygen species. This contention is supported by our observations on changes in photosynthetic pigments, indicating a higher degree of protection in CO 328 and P-treated CO 316. Protection from photooxidation of chlorophyll has been obtained after treating bean plants with triadimefon (Asare-Boamah et aI., 1986) and corn seedlings with a combination of paclobutrazol and ancymidol (Pinhero and Fletcher, 1994). It has been reported that several environmental factors such as chilling and drought can impair the photosynthetic membrane's ability to process excitation energy, thereby resulting in oxidative degradation of thylakoid constituents (Powels, 1984). The dependency of photo oxidation on light and oxygen suggest that the light reactions of photosynthesis may be leaking energy to molecular oxygen, forming potentially toxic oxygen species such as Oz -', HzO z, 'OH, and 10 2 resulting in oxidative damage (Scandalios, 1993). Pigment bleaching, membrane damage and thylakoid membrane disorganization are symptoms of damage caused by active oxygen species (Wise and Naylor, 1987; Scandalios, 1993). The present study indicated that chilling protection was conferred in the sensitive inbred after paclobutrazol treatment, and involved a decrease in photo inhibition and photosynthetic pigment damage, as well as causing increased membrane integrity and chloroplast membrane stabilization. The reduction of chilling-related symptoms in CO 328 and P-treated CO 316 suggests that they experience less photooxidative damage. It has been reported that chilling-tolerance in maize (Anderson et al., 1995), cucumber (Upadhyaya et al., 1989) and tomato (Senaratna et al., 1988) seedlings are related to increased antioxidants and antioxidant enzymes. In another study with CO 328 and CO 316 inbreds, differential
Chloroplast Membrane and Chilling Tolerance
activities of antioxidant enzymes were observed (Pinhero et aI., 1997). CO 328 possessed higher activities of superoxide dis mutase and a new isozyme of glutathione reductase as compared to CO 316. Paclobutrazol treatment enhanced the activities of superoxide dismutase and ascorbate peroxidase in CO 316. The higher degree of protection of the chloroplast membrane observed in CO 328 and P-treated CO 316 during chilling could be related to increased antioxidant enzymes in these inbreds. Previous studies (Paliyath and Fletcher, 1995 a, 1995 b) have shown that maize coleoptiles derived from paclobutrazol-treated seeds sustain less heat damage as compared with the untreated coleoptiles. This protection appeared to result from efficient removal of damaged lipids/proteins in the form of microvesicles. Microvesiculation of membrane leading to the formation of deteriosomes has been proposed to be an efficient means of discarding degraded and peroxidized lipids thus stabilizing membrane structure (Yao et al., 1991 a; 1991 b; 1993; Paliyath and Droillard, 1992). Membrane lipid degradation is a multiple process and the accumulation of specific intermediates will alter the relative composition of lipids in the membrane, thus conferring specific structural features. For instance, the formation of hexagonal phase lipid (HII) during freezing (Gordon-Kamm and Steponkus, 1984) indicate that phospholipase D could be activated during freezing leading to the accumulation of phosphatidic acid in the membrane. Similarly, accumulation of diacylglycerol by increased phospholipase D and phosphatidate phosphatase activity can cause membrane microvesiculation (Paliyath and Thompson, 1987). Large numbers of vesicular structures were observed in the cytosol of P-treated CO 316 suggesting that this could be unique to paclobutrazol treatment. Therefore, protection from chilling could also involve modulation of membrane lipid degradation so as to discard/minimize damage to the membrane structure and function. Acknowledgements
This research was supported by a Canadian Commonwealth Scholarship to RGP and Research Grants from Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs. We thank Mr. Robert Harris, Electron Microscopy Centre, Dept. of Microbiology, University of Guelph, for helping with electron microscopy work. The authors also wish to thank Dr. Bob Hamilton of Eastern Cereals and Oilseeds Research Centre, Agriculture and Agri-Food Canada, Ottawa for kindly supplying the seeds of the two inbreds and Mr. Brad Smith, Zeneca Corp. for a generous supply of paclobutrazol for this study.
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SCANDALIOS, ]. G.: Oxygen stress and superoxide dismutases. Plant Physiol. 101,7-12 (1993). SENARATNA, T., c. E. MACAY, B. D. MCKERSIE, and R. A. FLETCHER: Uniconazol-induced chilling tolerance in tomato and its relationship to antioxidant content. J. Plant Physiol. 133, 56-61 (1988). UPADHYAYA, A., T. D. DAVIS, R. H. WALSER, A. B. GALBRAITH, and N. SANKHLA: Uniconazole - induced alleviation of low temperature damage in relation to antioxidant activity. HortScience 24, 955-957 (1989). VAN HASSELT, P. R.: Light-induced damage during chilling. In: WANG, C. Y. (ed.): Chilling injuty of horticultural crops, pp. 113-128. CRC Press, Boca Raton, Florida (1990). WALKER, M. A., B. D. MCKERSIE, and K. P. PAULS: Effects of chilling on the biochemical and functional properties of thylakoid membranes. Plant Physiol. 97, 663-669 (1991). WALKER, M. A., D. M. SMITH, K. P. PAULS, and B. D. MCKERSIE: A chlorophyll fluorescence screening test to evaluate chilling tolerance in tomato. HortScience 25, 334-339 (1990). WILSON, J. M. and]. A. GREAVES: Assessment of chilling sensitivity by chlorophyll fluorescence analysis. In: WANG, C. Y. (ed.): Chill-
ing injuty of horticultural crops, pp.129-141. CRC Press, Boca Raton, Florida (1990). WISE, R. R. and A. NAYLOR: Chillipg - enhanced photooxidation. The peroxidative destruction of lipids during chilling injuty to photosynthesis and ultrastructure. Plant Physiol. 83, 272-277 (1987). YAO, K., G. PALIYATH, R. W. HUMPH RAY, F. R. HALLET, and ]. E. THOMPSON: Identification and characterization of non-sedimentable lipid-protein microvesicles. Proc. Nat!. Acad. Sci. USA 88, 2269-2273 (1991 a). YAO, K., G. PALIYATH, and]. E. THOMPSON: Non-sedimentable microvesicles from senescing bean cotyledons contain gel phaseforming phospholipid degradation products. Plant Physiol. 97, 502-508 (1991 b). - - - Localization of peroxidized lipids in non-sedimentable microvesicles of senescing bean cotyledons. ]. Exp. Bot. 44, 12671274 (1993). ZHANG, L. X. P. H. LI, and M. J. TSENG: Amelioration of chilling injuty in rice seedlings by mefluidide. Crop Sci. 27, 531-534 (1987).
j. Plant Physiol. Vol. 155. pp. 691-698 (1999) http://www.urbanfischer.de/journals/j pp
Plan. Ph,s.olo., © 1999 URBAN Ii! FISCHER
Chloroplast Membrane Organization in Chilling-Tolerant and Chilling-Sensitive Maize Seedlings 2 1 2 REENA GRITTLE PINHER0 , GOPINADHAN PALIYATH , RICKEYY. YADA , 1 DENNIS p. MURR 1
Department of Plant Agriculture, Division of Horticultural Science and
2
Department of Food Science, University of Guelph, Guelph, Ontario, Canada N I G 2W1
and
Accepted May 25, 1999
Summary
Resistance of maize (Zea mays L.) seedlings to chilling-induced damage and its potential bearing on chloroplast membrane organization have been investigated using a chilling-sensitive inbred CO 316 and a chilling-tolerant inbred CO 328. Paclobutrazol treatment of the chilling-sensitive CO 316 (CO 316P) induced chilling tolerance, also causing several morphological and physiological changes, and served as another means of exploring the relation between chloroplast membrane changes and chilling tolerance. Chilling treatment [6 'CI2 'C (day/night)] resulted in a reduction of Fv/Fm ratio, photosynthetic pigment levels and an increase in leakage of electrolytes. Within 24 h of exposure of CO 316 to chilling, the granal and stromal membrane showed extensive vesiculation and disruption of the granal array. By contrast, the granal and stromal organization of chilling-tolerant CO 328 and P-treated CO 316 remained nearly intact. Our studies suggest that paclobutrazol treatment can alter the membrane deterioration and disassembly processes of the thylakoid membranes of chilling-sensitive CO 316 to make them functionally similar to the chloroplast membrane of genetically chilling-tolerant CO 328.
Key words: Maize, Zea mays L., chilling stress, chilling tolerance, membrane, paclobutrazol, vesiculation. Abbreviations: P = paclobutrazol. Introduction
An important factor that dictates the geographic distribution of several plant species is their ability to withstand chilling stress (Oquist, 1983). Chilling injury occurs when crops indigenous to tropical and subtropical regions are exposed to low, non-freezing temperatures (0-12 'C, Lyons, 1973). The common symptoms of chilling injury include wilting, inhibition of metabolic processes, chlorosis, and changes in the molecular ordering or physical state of cell membranes that lead to increased permeability or leakage. The damaging effects ultimately result in reduced growth and yield (Saltveit and Morris, 1990; Rab and Saltveit, 1996). Chilling in the presence of light causes photo oxidation, bleaching of leaf pigments, chloroplast ultrastructural changes and cellular lipid degradation (Wise and Naylor, 1987). Maize, which ranks
among the top three food crops of the world, is chilling-sensitive. Chilling-sensitivity of several crop species varies depending on the stage of crop development and cultivar (Miedema et al., 1987; Janowiak and Markowski, 1994). Numerous attempts such as breeding for increased chilling tolerance, modifYing crop management practices and application of chemicals have been used to avoid chilling injury (Lee et al., 1985; Zhang et al., 1987). Paclobutrazol [(2RS,3RS)-I(4-chlorophenyl)-4,4-dimethyl-2-(I,2, 4-triazolyl)-pentan-301] is a triazole compound that exhibits varying degrees of plant growth regulatory and fungicidal activities due to its inhibition of cytochrome P4SO - dependent oxidation of entkaurene to GAlz-aldehyde, leading to the inhibition of gibber ellin and ergosterol biosynthesis in plants and fungi, respectively (Rademacher, 1992). Triazoles such as uniconazole, paclobutrazol and triadimefon protect plants from various 0176-1617/991155/691 $ 12.00/0
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REENA GRITTLE PINHERO, GOPINADHAN PALIYATH, RICKEY Y. YADA, and DENNIS P. MURR
environmental stresses (Davis and Curry, 1991; Kraus and Fletcher, 1994; Paliyath and Fletcher, 1995 a, 1995 b). Inhibition of gibberellin biosynthesis appear to alter several aspects of the regulation of the isoprenoid pathway leading to increased absciscic acid and cytokinin levels (Asare-Boamah et al., 1986; Grossman et aI., 1987), membrane properties, changes in the composition of antioxidants and antioxidant enzymes (Pinhero et aI., 1997) as well as several morphological parameters. The biochemical mechanisms resulting in the beneficial effects of triazole treatment need to be fully understood in order to develop transgenic stress-tolerant crop plants. The present study was undertaken to explore the biochemical adaptations to chilling tolerance exhibited by two maize inbreds, one of which is genetically chilling-tolerant (CO 328), whereas the other (CO 316) exhibits a low degree of chilling tolerance. However, chilling tolerance can be induced in this chilling-sensitive inbred by paclobutrazol treatment (Pinhero et aI., 1997). Since chilling injury is manifested at the membrane level (Lyons, 1973; Murata, 1990), we explored changes in thylakoid membrane organization during chilling as a potential indicator of the degree of chilling tolerance. Other parameters of monitoring chilling damage included measuring the variable (Fv) to maximal (Fm) fluorescence ratio, and membrane integrity in terms of leakage of electrolytes. Thus, changes in chloroplast membrane organization during chilling and a subsequent recovery period will indicate potential biochemical parameters that afford chilling tolerance to CO 328. Additionally by comparing the thylakoid membrane organization between CO 328 and paclobutrazol-treated CO 316, we delineated potential similarities and differences in membrane deterioration and disassembly that could be relevant to chilling tolerance mechanisms.
Materials and Methods Plant material and treatments Separate experiments were conducted to determine the extent of chilling injury in two maize (Zea mays L.) inbreds, CO 316 and CO 328, and to establish the effect of paclobutrazol in the alleviation of chilling injury. The CO lines of corn were developed at the Eastern Cereals and Oilseeds Research Centre, Agriculture and Agri-Food Canada, Ottawa. CO 316 was developed from the pedigree CO 266 x CO 273 / / CO 266. CO 273 and CO 266 were developed from the Pioneer hybrid 3990. CO 328 was developed from the ped igree CO 216 x CO 258 / / CO 216 BC2. CO 216 originated from the hybrid Pride 5 which is a four way or double cross hybrid. Thus CO 328 is believed to be completely different from CO 316. However, molecular or isozyme analysis have not been conducted to delineate differences (Robert Hamilton, personal communication). Seeds (50 g) were imbibed for 18 h in 100 mL of 0.17 mmol· L-1 paclobutrazol (P) or in water (controls) and air dried for 4 days. They were sown in a commercial potting medium (Promix PGX, Premier Brands, Red Hill, PAl in plastic cells (17.5 X 13.5 x 5.5 em). Seedlings were grown for 11 days in a growth cabinet, maintained at day/night temperatures of 23 ± 2 °C/19 ± 2°C, 50 to 70 % relative humidity, and a 16h photoperiod at 200 !lmol m -2 s-I,
Growth analyses Eleven days after sowing, plant height, fresh and dry weights of shoots, leaf area and photosynthetic pigment contents were determined. Leaf area was measured using a Li - Cor Li - 3100 area meter (Li - Cor Inc., USA). Dry weights of shoots were measured by drying the shoot in an oven at 70°C until consistently constant weights were obtained.
Imposition of chilling stress Eleven-day old seedlings were exposed for two days to a cold temperature regime of 6 °C/2 °C (day/night) and a photoperiod of 16 h at 450 !lmol m -2 s-l. After exposure to chilling stress, seedlings were returned to the previous growth conditions to assess the extent of chilling damage on chlorophyll fluorescence (Fv/Fm), electrolyte leakage, photosynthetic pigments, and thylakoid membrane organization.
Variable (Fv) to maximal (Fm) fluorescence ratio Fv/Fm ratio was measured in situ on the second true leaf with a portable Hansatech Plant Efficiency Analyzer (Hansatech Instrument Ltd., King's Lynn, UK) after dark-adapting the seedlings for 30 min as described previously (Pinhero and Fletcher, 1994). Fv/Fm was measured prior to chilling, immediately after 48 h of chilling (0 h post-chilling), 24 h, and 48 h of post-chilling periods.
Leakage ofelectrolytes Damage to membrane integrity was monitored by measuring the increase in leakage of ions from the second true leaf. The leaves were cut into 2 cm segments, rinsed in distilled water and placed in a test tube with 15 mL of distilled water at 24 °C for 24 h. Initial conductivity of the solution (spontaneous leakage of ions due to membrane damage) and total ionic conductivity after boiling the sample for 20 min was measured using a Model 32 conductance meter (Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio, USA). Percent leakage was measured as described previously (Pinhero and Fletcher, 1994) using the following formula 01.
,0
Ieakage 0 f'IOns = Initial - - conductivity ----'Total conductivity
X
100
Photosynthetic pigments Photosynthetic pigments were quantified two days after removal from chilling stress. For estimating the level of photosynthetic pigments, 2.5 cm long leaf segments were taken starting from the tip of the second true leaf. Chlorophyll a, b and carotenoid contents were determined after extracting the pigments in 80 % ethanol, as described earlier by Pinhero and Fletcher (1994).
Electron microscopy Three leaf pieces, each of 1 X 5 mm area were cut from the middle portion of the second true leaf from three different plants using a sharp razor blade. These pieces were fixed in cold 3 % glutaraldehyde (Sigma Chemical Co., St. Louis, MO) in 100 mmol. L-1 potas-
Chloroplast Membrane and Chilling Tolerance sium phosphate buffer, pH 7.2, vacuum-infiltrated for 10 min and left overnight at 4 'co The fixed material was washed with the same buffer and post-fixed in 1 % osmium tetroxide at 4 'C overnight. Post-fixed material was dehydrated in a graded series of ethanol (25 %, 50 % and 100 %). Dehydrated specimens were infiltrated, embedded in LR White contained in gelatin caps and allowed to polymerize at 60 'C for 1 h (Marty et aI., 1995). Sections were cut with a diamond knife and post-stained in 2 % aqueous uranyl acetate followed by Reynold's lead citrate. These sections were examined in a Philips EM 300 electron microscope set at 60 kV: Each figure is representative of 25-30 sections examined under the microscope.
693
ing of the potential mechanism of chilling tolerance in CO 328 and P-treated CO 316, the chloroplast membrane organization in these two sets of plants were investigated as a function of chilling duration. Since major differences were observed in the changes in Fv/Fm ratio as a function of chilling and post-chilling recovery in CO 328, CO 316 and P-treated CO 316, it was logical to assume that significant differences could exist in the chloroplast membrane organization and disassembly between plants in the three experimental systems studied.
Effect of chilling on maize inbreds and its protection by paclobutrazol
Statistical analysis ofdata The data were statistically analysed using analysis of variance in a completely randomized design and significant differences were determined using the protected LSD, at P=0.05.
Results
Since paclobutrazol at 0.17 mmol . L-1 did not alter the chilling tolerance of CO 328, only data from studies carried out with CO 328, CO 316 and P-treated CO 316 are presented. Initial values on growth parameters, Fv/Fm ratio, photosynthetic pigment contents and percent leakage of ions measured on ll-day-old seedlings are given in Table 1. Genetically chilling-tolerant CO 328 and the chilling-susceptible CO 316 possessed inherent differences in their morphology and growth parameters. Paclobutrazol treatment of chillingsusceptible CO 316 caused drastic morphological changes that included reduced height and increased root to shoot ratio that are characteristic to plants treated with triazole growth regulators (Pinhero and Fletcher, 1994). Even though CO 328 and P-treated CO 316 differed considerably in their morphology, the degree of chilling tolerance exhibited by these seedlings was nearly the same. These observations suggested that the underlying biochemical mechanisms of chilling tolerance exhibited by CO 328 and P-treated CO 316 may not be exactly identical. To obtain a better understand-
Table 1: Growth analyses of eleven-day old maize seedlings. Seedlings were grown for 11 days in growth cabinet maintained at 23 ± 2 'C I 19 ± 2 'C and measurements were taken as described in materials and methods. Parameters
CO 316
CO 316P
CO 328
Height (cm) Fresh wt. of shoots (g plant-I) Fresh wt. of roots (g plant -1) Root: shoot ratio (fresh wt.) Dry wt. of shoots ~ plant-I) Total leaf area (cm plant-I) Fv/Fm ratio Leakage of ions (%) Chlorophyll a (mg. g fresh wt. -1) Chlorophyll b (mg. g fresh wt. -\) Carotenoids (mg. g fresh wt. -\)
15.70a 0.78 a 0.37b 0.47 b 0.07" 2l.20a 0.75 b 3.20 b l.94a 0.82a 0.6 2 b
9.30 C O.71 a 0.46 a 0.65 a 0.06 a 20.20a
13.60 b 0.76 a 0.32b 0.43 b 0.07" 18.03 a 0.80 a 4.50 a l.61 b 0.62b 0.46 a
O.77b
4.30 a l.72 ab 0.69 ab 0.52a
a. b, ab denotes significant differences between values within a row at p ~ 0.05.
Changes in variable (Fv) to maximal (Fm) chlorophyll fluorescence has been widely used as a parameter in determining chilling injury before visible symptoms appeared (Wilson and Greaves, 1990). Immediately after chilling, Fvl Fm ratio decreased drastically in both inbreds (Fig. 1 A). However, the extent of reduction in Fv/Fm ratio was more in CO 316 than in CO 328. Paclobutrazol treatment prevented the extent of reduction in Fv/Fm ratio in CO 316. However, no marked change was observed in Fv/Fm ratio of CO 328 after paclobutrazol treatment. Photosynthetic efficiency as measured by Fv/Fm ratio recovered to 90 % of the initial values within 48 h of post-chilling period in CO 328 and in P-treated CO 316. However, the recovery was only 52 % in the chilling-sensitive CO 316 in the absence of paclobutrazol treatment (Fig. 1 A). Both CO 328 and P-treated CO 328 showed similar photosynthetic efficiency at all the time periods studied after chilling exposure. This result showed that paclobutrazol treatment was not giving any added protection to CO 328 from chilling damage. However, the sensitive inbred CO 316 exhibited protection from chilling after paclobutrazol treatment. Membrane damage as measured by leakage of ions was significantly higher in CO 316 when compared to that of CO 328 at all the time periods studied (Fig. 1 B). Paclobutrazol treatment reduced leakage of ions in CO 316 to a level exhibited by the chilling-tolerant CO 328. No added effect was noticed in CO 328 after paclobutrazol treatment. In parallel with the changes in Fv/Fm ratio, the decline in photosynthetic pigments (chlorophyll a, band carotenoids) was the highest in CO 316 (nearly 40 %), with only lO-20 % decline in CO 328 and P-treated CO 316 after 48 h of postchilling period (Fig. 2).
Changes in chloroplast membrane organization Examination of chloroplasts revealed that mesophyll chloroplasts of non-chilled CO 316 possessed granal and stromal thylakoids organized in typical fashion as in other graminaceous chloroplasts with abundant granal stacks and intervening stromal membranes (Fig. 3 A). However, within 24 h of chilling, swelling of thylakoids occurred in many grana, resulting in an increase in intrathylakoid space and formation of large vesicular structures (Fig. 3 B). After 48 h of chilling, granal stacks had undergone disorganization and dissolution forming large numbers of vesicular structures (Fig. 3C).
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Maize is a chilling-sensitive plant and sustains injury when exposed to low temperature (Miedema et al., 1987). Under field conditions, the seedlings are usually exposed to chilling temperatures and the damage is accentuated by simultaneous exposure to bright sunlight which leads to the production of active oxygen species due to the disruption in photosynthetic electron transport (Powels, 1984; Walker et aI., 1991). The damage to the cells has also been examined through ultrastructural studies (Wise and Naylor, 1987; van Hasselt, 1990). It has been reported that chilling in the presence of light caused severe chloroplast damage in the chilling-sensitive cucumber as compared with the chilling-tolerant pea (Wise and Naylor, 1987). In the present study, we have compared aspects of chillingtolerance in a genetically chilling-tolerant maize inbred CO 328 to a related inbred CO 316 which is chilling-sensitive. Chilling-tolerance could be induced in CO 316 by treating with the triazole plant growth regulator, paclobutrazol. However, after paclobutrazol treatment, the morphology of the plant changes altogether. Even though the resemblance to CO 328 is remote, P-treated CO 316 is equally chilling-tolerant as CO 328, perhaps suggesting that there could be different means of adaptation to attain chilling tolerance. Therefore, we have compared several physiological parameters between CO 316, CO 328 and P-treated CO 316 during chill-
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48 h postchilling
Fig. 1: Changes in Fv/Fm ratio expressed as a percent of the nonstressed control values obtained from plants (A). Eleven-day old seedlings were exposed for 48 h ro a chilling temperature of 6 'C/ 2 'C (day/ni~ht) and a photoperiod of 16 h at a light intensity of 450 Ilmol m- S-1 and subsequently grown in the growth cabinet for the periods indicated. (B). The leakage of electrolytes was monitored at the specified time intervals. Values are means of six (A) and five (B) replications. Error bars marked by different letters within each cluster denote significant difference at P:S;0.05.
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Chilling-tolerant CO 328 and P-treated CO 316 showed similarities in the organization of granal and stromal membranes in the chloroplast. Even though CO 328 possessed much larger granal stacks when compared to that in P-treated CO 316, both their stromal structures showed intermittent swollen nature (Figs. 4, 5). No major changes were observed in the thylakoid organization of CO 328 and P-treated CO 316 during chilling for 48 h (Figs. 4 C, 5 B). P-treated CO 316 showed very low dissolution of starch during chilling (Fig. 4 B, C) compared to CO 316 or CO 328 in which starch grains disappeared within 24 h of chilling (Fig. 3 B, data not shown for CO 328). In addition, chloroplasts of P-treated CO 316 showed large scale microvesiculation and bleb bing of membranes leading to the accumulation of vesicles in the cytoplasm (Fig. 4 B, C, V).
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Fig. 2: Changes in chlorophyll fl, chlorophyll band carotenoids of maize seedlings during two days of post-chilling period expressed as a percent of non-stressed values. For non-stressed values, see Table 1. Eleven-day old seedlings were exposed for 48 h to a chilling temperature of 6 'C/2 'C (day/night) and a photoperiod of 16 h at a light intensity of 450 Ilmol m- I s-I and subsequently grown in the growth cabinet. Values are means of six replications. Error bars marked by different letters within each cluster denote significant difference at P:S; 0.05.
Chloroplast Membrane and Chilling Tolerance
695
Fig. 3A: Electron micrographs of mesophyll chloroplasts from unchilled CO 316 seedlings. Magnification = 19,470 X; bar = 0.5 11m; S, stroma; G, granum; SG, starch granule.
Fig. 4 A: Chloroplast membrane structure and organization of CO 316 maize seedlings treated with paclobutrazol. Magnification = 24,977X; bar = 0.4 11m; S, stroma; G, granum.
Fig. 3 B, C: Changes in chloroplast membrane organization of CO 316 seedlings exposed to a chilling duration of 24 h (3 B) and 48 h (3 C). Magnification = 38,854 X; bar = 0.3 11m; S, stroma; G, granum; V, vesicles. Large scale vesiculation and membrane damage can be noticed during 48 h of chilling.
Fig. 4 B: Chloroplasts of paclobutrazol-treated CO 316 seedlings after exposure to 24 h of chilling. Magnification 0.4 /.lm; S, stroma; G, granum; V, vesiculation.
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696
REENA GRITTLE PINHERO, GOPINADHAN PALIYATH, RICKEY Y. YADA, and DENNIS P. MURR
Fig.4C: Chloroplasts of paclobutrazol-treated CO 316 seedlings after exposure to 48 h of chilling. Magnification = 38,854; bar = 0.26 11m; S, stroma; G, granum; V, vesiculation.
Fig. 5: Chloroplasts of CO 328 seedlings prior to chilling (A, 24,977x; bar = 0.4 11m;) and after 48 h (B, 30,528 X; bar = 0.3 11m;)
exposure to chilling. Note that the thylakoid and stroma are not disrupted. S, stroma; G, granum.
ing and post-chilling recovery, which will give us an indication regarding the potential biochemical parameters that could afford chilling-tolerance to maize plants. Our study shows that the parameters employed clearly differentiate the chilling-tolerant CO 328 and P-treated CO 316 from the chilling-sensitive CO 316. The decline in Fv/Fm ratio is lower in CO 328 and P-treated CO 316 as compared with CO 316 (Fig. 1 A). Various studies have used chlorophyll fluorescence ratio (Walker et aI., 1990; Wilson and Greaves, 1990) and electrolyte leakage (Janowiak and Markowski, 1994) as quantitative methods for differentiating chilling-tolerance of cultivars. Since the initial studies to screen the toler-
ance level of paclobutrazol-treated maize seedlings showed the ineffectiveness of paclobutrazol in augmenting the genetic chilling-tolerance in CO 328, further characterization of the chilling effect on maize seedlings was limited to CO 328, CO 316 and P-treated CO 316. Most of the higher altitude and latitude areas, where chilling-sensitive crops are grown, are characterized by large differences between day and night temperatures. For example, cold clear nights are likely to be followed by bright sunlight, early in the morning when the temperature is still low. Exposure to light during chilling can damage chilling-sensitive plants by causing light-induced degradation of photosynthetic pigments or photooxidation (Zhang et aI., 1987). It has been reported that photodegradation of leaf pigments in the chloroplast membrane can occur at temperatures below 10 °C (van Hasselt, 1990). Chloroplast membrane contains a remarkably high level of unsaturated lipids. Photoperoxidation of chloroplast lipids, concurrent with leaf pigment degradation was observed in cucumber leaf segments during chilling (Wise and Naylor, 1987). It has been reported that the use of electron transport inhibitors dichlorophenyl dimethyl urea and atrazine inhibited the photoperoxidation of lipids in cucumber leaf discs during chilling, indicating an involvement of superoxide in the damaging process (Wise and Naylor, 1987). The lower degree of damage noticed in thylakoid organization of P-treated CO 316 and CO 328 indicated that these plants possessed some protective mechanism against chilling injury, presumably at the level of preventing the formation or detoxification of active oxygen species. This contention is supported by our observations on changes in photosynthetic pigments, indicating a higher degree of protection in CO 328 and P-treated CO 316. Protection from photooxidation of chlorophyll has been obtained after treating bean plants with triadimefon (Asare-Boamah et aI., 1986) and corn seedlings with a combination of paclobutrazol and ancymidol (Pinhero and Fletcher, 1994). It has been reported that several environmental factors such as chilling and drought can impair the photosynthetic membrane's ability to process excitation energy, thereby resulting in oxidative degradation of thylakoid constituents (Powels, 1984). The dependency of photo oxidation on light and oxygen suggest that the light reactions of photosynthesis may be leaking energy to molecular oxygen, forming potentially toxic oxygen species such as Oz -', HzO z, 'OH, and 10 2 resulting in oxidative damage (Scandalios, 1993). Pigment bleaching, membrane damage and thylakoid membrane disorganization are symptoms of damage caused by active oxygen species (Wise and Naylor, 1987; Scandalios, 1993). The present study indicated that chilling protection was conferred in the sensitive inbred after paclobutrazol treatment, and involved a decrease in photo inhibition and photosynthetic pigment damage, as well as causing increased membrane integrity and chloroplast membrane stabilization. The reduction of chilling-related symptoms in CO 328 and P-treated CO 316 suggests that they experience less photooxidative damage. It has been reported that chilling-tolerance in maize (Anderson et al., 1995), cucumber (Upadhyaya et al., 1989) and tomato (Senaratna et al., 1988) seedlings are related to increased antioxidants and antioxidant enzymes. In another study with CO 328 and CO 316 inbreds, differential
Chloroplast Membrane and Chilling Tolerance
activities of antioxidant enzymes were observed (Pinhero et aI., 1997). CO 328 possessed higher activities of superoxide dis mutase and a new isozyme of glutathione reductase as compared to CO 316. Paclobutrazol treatment enhanced the activities of superoxide dismutase and ascorbate peroxidase in CO 316. The higher degree of protection of the chloroplast membrane observed in CO 328 and P-treated CO 316 during chilling could be related to increased antioxidant enzymes in these inbreds. Previous studies (Paliyath and Fletcher, 1995 a, 1995 b) have shown that maize coleoptiles derived from paclobutrazol-treated seeds sustain less heat damage as compared with the untreated coleoptiles. This protection appeared to result from efficient removal of damaged lipids/proteins in the form of microvesicles. Microvesiculation of membrane leading to the formation of deteriosomes has been proposed to be an efficient means of discarding degraded and peroxidized lipids thus stabilizing membrane structure (Yao et al., 1991 a; 1991 b; 1993; Paliyath and Droillard, 1992). Membrane lipid degradation is a multiple process and the accumulation of specific intermediates will alter the relative composition of lipids in the membrane, thus conferring specific structural features. For instance, the formation of hexagonal phase lipid (HII) during freezing (Gordon-Kamm and Steponkus, 1984) indicate that phospholipase D could be activated during freezing leading to the accumulation of phosphatidic acid in the membrane. Similarly, accumulation of diacylglycerol by increased phospholipase D and phosphatidate phosphatase activity can cause membrane microvesiculation (Paliyath and Thompson, 1987). Large numbers of vesicular structures were observed in the cytosol of P-treated CO 316 suggesting that this could be unique to paclobutrazol treatment. Therefore, protection from chilling could also involve modulation of membrane lipid degradation so as to discard/minimize damage to the membrane structure and function. Acknowledgements
This research was supported by a Canadian Commonwealth Scholarship to RGP and Research Grants from Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs. We thank Mr. Robert Harris, Electron Microscopy Centre, Dept. of Microbiology, University of Guelph, for helping with electron microscopy work. The authors also wish to thank Dr. Bob Hamilton of Eastern Cereals and Oilseeds Research Centre, Agriculture and Agri-Food Canada, Ottawa for kindly supplying the seeds of the two inbreds and Mr. Brad Smith, Zeneca Corp. for a generous supply of paclobutrazol for this study.
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