Effects of chilling temperature on photosynthetic rates, photosynthetic enzyme activities and metabolite levels in leaves of three sugarcane species
Akihiro Nose Fax, + 81 – 952 – 28 – 8737; e-mail: email@example.com
NADP-MDH, NADP-malate dehydrogenase
NADP-ME, NADP-malic enzyme
OAA, oxaloacetic acid
PEPcase, phosphoenolpyruvate carboxylase
PPDK, pyruvate orthophosphate dikinase
Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase
The aim of this study was to investigate the mechanism of photosynthetic changes in sugarcane leaves in response to chilling temperature by using three species (Saccharum sinense R. cv. Yomitanzan, Saccharum sp. cv. NiF4 and Saccharum officinarum L. cv. Badira) differing in origin and cold sensitivity. Yomitanzan is native to subtropical areas, Badira is native to tropical areas and NiF4 is a hybrid species containing genes of both tropical and subtropical species. At exposure to chilling temperature (10 °C), the photosynthetic rate in the leaves at either 10 °C or 30 °C showed a greater decrease in Badira than in NiF4 and Yomitanzan. After 28 h exposure of plants to the chilling temperature, the extractable activities of pyruvate, orthophosphate dikinase (PPDK) and NADP-malate dehydrogenase (NADP-MDH) increased or were relatively stable in the leaves of NiF4 and Yomitanzan, but decreased substantially in Badira. Correspondingly, there was a substantial accumulation of aspartate, and the level of alanine increased in Badira leaves during the chilling treatment. It is suggested that NADP-MDH and PPDK are key enzymes which may determine the cold sensitivity in photosynthesis of sugarcane.
Numerous studies have shown that temperature affects plant photosynthesis in various ways (Berry & Björkman 1980). When C3 plants are exposed to low temperature for a relatively long time, the activities of several photosynthetic enzymes have been found to increase significantly. For example, at exposure to low temperature for several days or months, the maximum activities of Rubisco, stromal and cytosolic fructose-1,6-bisphosphatase (FBPase), and sucrose-phosphate synthase in the leaves of several species increase markedly, and lead to significant increase in whole plant photosynthetic capacity (Holaday et al. 1992; Hurry et al. 1994, 1995a, b). Under short-term exposure to low temperature, changes in enzyme activation state seem to play a more important role in compensating plant photosynthesis (Holaday et al. 1992). However, species which do not tolerate low temperature may not have the same ability to acclimate by up regulation of enzymes (Bruggemann, van der Kooij & van Hasselt 1992; Holaday et al. 1992).
C4 plants differ from C3 plants; they are mainly distributed in tropical and subtropical areas, and most of them are viewed as cold-sensitive (Berry & Björkman 1980; Sugiyama et al. 1979). The temperature optima in C4 plants are usually higher than in C3 plants (Berry & Björkman 1980). It has been suggested that the poor growth of C4 species in cool areas could be attributed to various factors, such as a high temperature optimum for phosphoenolpyruvate carboxylase (PEPcase) (Treharne & Cooper 1969) and the high activation energy of PEPcase and pyruvate orthophosphate dikinase (PPDK) at temperatures below 12 °C (Edwards et al. 1985; Uedan & Sugiyama 1976). Within C4 species cold sensitivity differs among subtypes; most of the cold-sensitive species belong to the NADP-malic enzyme (NADP-ME) subtype (Edwards et al. 1985; Sugiyama et al. 1979). PPDK is an important C4 photosynthetic enzyme which is cold-labile, and has long been considered as a limiting enzyme in C4 photosynthesis at low temperature (Hatch 1979; Sugiyama et al. 1979; Potvin, Simon & Strain 1986; Burnell 1990; Potvin & Simon 1990; Usami et al. 1995). Recently, Kingston-Smith et al. (1997) have also shown that the production and carboxylation of phosphoenolpyruvate (PEP) are the most temperature-sensitive steps of photosynthesis in maize leaves. However, 14CO2-labelling studies show no signs that PPDK limits carbon flux in the C4 pathway in vivo in some plants when exposed to low temperature (Caldwell, Osmond & Nott 1977).
Sugarcane is a typical NADP-ME subtype C4 plant. In this study we selected three sugarcane species, that differ in origin and cold sensitivity, and measured the changes of photosynthesis on exposure to chilling temperature in order to investigate the mechanism of cold sensitivity of photosynthesis in this NADP-ME subtype plant. Saccharum sinense R. cv. Yomitanzan is native to, and grows in subtropical areas, Saccharum officinarum L. cv. Badira is native to, and grows in tropical areas, and Saccharum sp. (S. officinarum×Saccharum spontaneum×Saccharum barberi) cv. NiF4 is a hybrid species containing genes of both tropical and subtropical species. Field observations show that these three species all grow well under higher temperatures. However, although Yomitanzan and NiF4 could germinate and grow well under lower temperatures, Badira could not. Previous studies have indicated that exposure of warm-grown maize and sorghum to 10 °C at a moderate light intensity for more than 4 d caused severe necrosis of exposed leaves (Taylor & Craig 1971), while within 3 d most photosynthetic enzymes in leaves remained constant (Taylor, Slack & McPherson 1974). Considering this, we measured the gas exchange, photosynthetic enzyme activities and metabolite levels in leaves of the three warm-grown sugarcane species in response to exposure to chilling temperature (10 °C) for a period of 52 h (less than 3 d).
MATERIALS AND METHODS
Sugarcane species, S. sinense R. cv. Yomitanzan, S. officinarum L. cv. Badira and S. sp. (S. officinarum×S. spontaneum× S. barberi) cv. NiF4, were germinated in vermiculite in the growth chamber (KG-50HLA, Koito In. Ltd, Tokyo, Japan) at day/night temperatures of 30/25 °C for about 2 weeks, and then the seedlings were transplanted to 4·5 L plastic pots containing about 2·5 kg soil in the growth chamber at day/night temperatures of 30/25 °C, and a photoperiod of 14 h. The relative humidity in the growth chamber was kept near 65%, light intensity at leaf height was about 700 μmol m–2 s–1 (400–700 nm), and CO2 was at ambient level. A 200 mL nutrient solution made from the commercial fertilizers (Otsuka 1 and Otsuka 2, Otsuka Chem. Co., Osaka, Japan) containing 260 p.p.m. N, 120 p.p.m. P, 405 p.p.m. K and necessary mineral elements was supplied to the plants twice a week. Tapwater was given as needed.
Chilling treatment, gas exchange and sampling for enzyme and metabolite determinations
After the sugarcane had been grown in the growth chamber at day/night temperatures of 30/25 °C for about 2 months, the plants were transferred to another growth chamber with the same photoperiod and light intensity but with a constant day/night temperature of 10 °C. The transfer was conducted from the start of the photoperiod. After the plants had been exposed to the chilling temperature for 4 h (at 1030 h on the first day of chilling treatment), 28 h (at 1030 h on the second day) and 52 h (at 1030 h on the third day), the uppermost fully expanded mature leaves were used for the measurements of gas exchange, enzyme assays and metabolite determinations. Gas exchange was measured with a Portable Photosynthesis System (LI-6400, Li-COR Inc., Lincoln, USA). Measurements were conducted within the growth chamber. At first, four separate plants in four pots were measured at a leaf temperature of 10 °C in the growth chamber at 10 °C. The photosynthetic rate at 10 °C for 0 h chilled leaves was measured after exposure of the leaves to the chilling temperature for about 10 min for all three species. After the gas exchange had been measured samples were taken immediately on the leaf that was used for gas exchange from two plants for measurements of enzymes and metabolites, and the other two plants were moved sequentially to another growth chamber with the same conditions except that the temperature was 30 °C. After being held in the growth chamber for 10 min for equilibrium of leaf temperature, the gas exchange was measured again at a leaf temperature of 30 °C. The light intensity in the leaf chamber was 1500 μmol m–2 s–1 supplied with a 6400–02 LED light source (Li-COR Inc.) and the leaf-to-air vapour pressure difference was maintained at 1·5 kPa when gas exchange was measured at a leaf temperature of 30 °C, and at around 1·0 kPa when measured at 10 °C (at 10 °C it is difficult to maintain the leaf-to-air vapour pressure difference at 1·5 kPa due to the extremely low flow rate of the system). The CO2 concentration was 350 μL L–1 supplied with a 6400–01 CO2 Injector System (Li-COR Inc.).
Extraction and assay of enzymes
After the gas exchange had been measured, leaf samples were taken and immersed in liquid nitrogen immediately and stored in liquid nitrogen until analysed. Leaf pieces (2 cm2) were extracted in 2 mL of extraction medium with 0·2 g sea sand and 20 mg of polyvinylpolypyrrolidone in a chilled mortar and pestle. The grinding medium contained 50 mM Tris-HCl pH 7·8, 8 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA)-NaOH pH 7·0, 5 mM dithiothreitol, 2·5 mM pyruvate and 0·2% (w/v) bovine serum albumin. The homogenate was filtered through one layer of Miracloth (Calbiochem-Novabiochem, La Jolla, CA, USA) and the filtrate was centrifuged at 12000 r.p.m. for 10 s (Diskboy Kurabo Fb4000, Kurabo Co., Ltd, Tokyo, Japan). The supernatant was immediately snap-frozen in liquid nitrogen separately in centrifuge tubes, and stored on liquid nitrogen until analysed. These extracts were used for assays of PEPcase, NADP-malate dehydrogenase (NADP-MDH), NADP-ME, PPDK and ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco). The PEPcase, NADP-ME, PPDK and Rubisco were measured as described previously (Du et al. 1996). The NADP-MDH initial activity assay medium contained 100 mM Hepes-NaOH pH 8·0, 0·5 mM EDTA-NaOH pH 7·0, 0·2 mM NADPH, 2 mM oxaloacetic acid (OAA) and 30 μL extract. For NADP-MDH total activity, 30 mM dithiothreitol was added to the extract and incubated for 30 min at room temperature, and then the activity was measured in the same assay mixture as for initial activity except that 20 mM dithiothreitol was included in the assay mixture (Holaday et al. 1992).
Stromal FBPase was extracted and assayed according to Holaday et al. (1992). Briefly, 2 cm2 of frozen leaf pieces was extracted in 2 mL of extraction medium with 0·2 g sea sand and 20 mg of polyvinylpolypyrrolidone in a chilled mortar and pestle. The extraction medium contained 50 mM Tris-HCl pH 8·0, 10 mM MgCl2, 1 mM EDTA-NaOH pH 7·0, 15 mM 2-mercaptoethanol, 1 mM fructose-1,6-bisphosphate and 0·1% (v/v) Triton X-100. The homogenate was centrifuged at 12000 r.p.m. for 10 s (Diskboy Kurabo Fb4000) and then the supernatant was used immediately for assay of stromal FBPase. The assay mixture (1 mL) contained 50 mM Tris-HCl pH 8·0, 10 mM MgCl2, 1 mM EDTA-NaOH pH 7·0, 0·5 mM NADP, 0·1 mM fructose-1,6-bisphosphate, 4 Units each of phosphoglucose isomerase and glucose-6-phosphate dehydrogenase, and 20 μL extract. The reaction was initiated by leaf extract. All enzymes activities were measured at 30 °C.
Extraction and determination of metabolites
Samples were taken and stored as for enzymes. The metabolites were extracted as described previously (Du et al. 1998) except that 5% HClO4 was employed to extract the metabolites instead of 3% in the previous study. The extract of metabolites at the final preparation was snap-frozen in liquid nitrogen separately in plastic centrifuge tubes and stored on liquid nitrogen until analysed.
Aspartate was measured spectrophotometrically in an assay mixture (1 mL) containing 60 mM phosphate buffer pH 7·2, 0·2 mM NADH, 3 mMα-ketoglutarate pH 7·0, 10 Units mL–1 malate dehydrogenase, 1 Unit mL–1 glutamate-oxaloacetate transaminase and 100μL extract. Alanine was measured in an assay mixture (1 mL) containing 100 mM Tris-HCl pH 7·6, 3 mM EDTA-NaOH pH 7·0, 6 mMα-ketoglutarate pH 7·0, 0·2 mM NADH, 7 Units mL–1 lactate dehydrogenase, 10 Units mL–1 glutamate-pyruvate transaminase and 100 μL extract. The concentrations of aspartate and alanine were estimated by the reduction of NADPH monitored at 340 nm. Other metabolites were measured as described previously (Du et al. 1998).
Changes in photosynthetic rate and stomatal conductance in the leaves of three sugarcane species in response to chilling temperature are shown in Table 1. The changes in photosynthetic rate exhibited the following two features. (1) After chilling treatment, the photosynthetic rates at leaf temperature of 30 °C in leaves of NiF4 and Yomitanzan did not show a large decrease in comparison with that for Badira. For example, after 52 h exposure to the chilling treatment the photosynthetic rates in leaves of NiF4 and Yomitanzan remained at 70 to 75% of the rates in their unstressed controls, but the photosynthetic rate in Badira was only about 20% of the control rate. (2) After 52 h exposure to the chilling temperature, the photosynthetic rates at a leaf temperature of 10 °C in the leaves of NiF4 and Yomitanzan increased by 20 to 30% relative to the unstressed plants. In contrast, the photosynthetic rate in leaves of Badira decreased by more than 50% under the same conditions. The results in (1) and (2) suggest that the photosynthetic capacity of leaves of Badira is seriously damaged by the chilling temperature, but not that of NiF4 and Yomitanzan.
Table 1. .
Effects of chilling temperature on photosynthetic rate and stomatal conductance in leaves of three sugarcane species. Values at 0 h represent unchilled-stressed control plants. Values in parentheses show the percentage of the photosynthetic rate in chilling-stressed plants of that in control plants. Data for photosynthetic rate and stomatal conductance are means ± SE of four measurements from two (at 30 °C) or four (at 10 °C) separate plants
Changes in stomatal conductance in leaves of the three species in response to chilling showed that (1) stomatal conductance at 30 °C exhibited a similar pattern in photosynthetic rate in relation to chilling temperature. After 52 h exposure to the chilling temperature the stomatal conductance in leaves of NiF4 and Yomitanzan remained at 65 to 70% of the conductance in their unstressed controls, compared with about 20% in Badira; (2) The stomatal conductance measured at 10 °C in leaves of NiF4 was only reduced by 15 to 25% relative to that measured at 30 °C. In Yomitanzan, the stomatal conductance at 10 °C in leaves of unstressed plants was reduced markedly, to 27% of that measured at 30 °C. However, after chilling treatment, the stomatal conductance at 10 °C recovered to approximately 60 to 70% of the conductance measured at 30 °C. In Badira, the stomatal conductance at 10 °C was reduced markedly relative to that measured at 30 °C; after 4 h exposure to the chilling temperature the stomata were almost completely closed.
As the reductions in stomatal conductance in leaves of the three species coincided with reductions in photosynthetic rate at exposure to the chilling temperature, we measured the photosynthetic rate in leaves at doubled CO2 concentration in order to determine whether the stomata limited the photosynthetic rate at low temperature. The results showed that photosynthesis in all three species did not exhibit any evident responses to the enhanced CO2 concentration at chilling temperature, although the intercellular partial pressure of CO2 in the leaves of the three species all increased markedly at the enhanced CO2 concentration. For example, after 52 h exposure of plants to the chilling temperature, when the external CO2 partial pressure was changed from 350 μL L–1 to 700 μL L–1, the intercellular partial pressure of CO2 in leaves of NiF4, Yomitanzan and Badira increased greatly, from 83, 91 and 130 μL L–1 to 219, 266 and 338 μL L–1, respectively. However, the photosynthetic rates at 30 °C in leaves of the three species changed very little, namely from 25·3, 20·6 and 6·1 μmol m–2 s–1 to 26·4, 21·1 and 6·7 μmol m–2 s–1, respectively. These results suggest that the reduction in stomatal conductance at chilling temperature was not responsible for the reduction in photosynthetic rate in the three species. This conclusion is consistent with the previous findings in sorghum (Taylor et al. 1974) and other species (Leegood & Edwards 1996)
Photosynthetic enzyme activities
Photosynthetic enzyme activities in leaves of the three species showed different responses to chilling temperature (Table 2). In NiF4, almost all photosynthetic enzymes activities measured, including PEPcase, NADP-MDH, NADP-ME and PPDK in the C4 pathway, and Rubisco and stromal FBPase in the Calvin cycle, increased at exposure to the chilling temperature (except for NADP-MDH total activity). In particular, PPDK and stromal FBPase activities, and NADP-MDH in vivo activity (initial activity, measured immediately after extraction and without the presence of dithiothreitol) showed large increases. The NADP-MDH total activity (measured after in vitro activation and with the presence of dithiothreitol) showed no substantial changes before and after chilling treatment, leading to a substantial increase in the NADP-MDH activation state (initial activity/total activity), from 19 to 45%. Both Rubisco initial (measured immediately after extraction) and total activities (measured after being activated with CO2 and Mg2+in vitro) increased at the chilling temperature, although, the activation state was not affected by the chilling treatment. These results are evidently different from the findings in C3 plants in which both Rubisco initial activity and activation state are found to increase at exposure to chilling temperature in several species (Holaday et al. 1992; Hurry et al. 1994, 1995a, 1995b). In Yomitanzan, the NADP-MDH activity showed similar changes to those of NiF4; the activation state of NADP-MDH increased from 17 to 31%. PPDK activity showed no evident changes at exposure to the chilling temperature. Other enzymes activities were reduced slightly at short-term chilling (4 h and 28 h) and then increased after further chilling treatment (Table 2).
Table 2. .
Effects of chilling temperature on photosynthetic enzyme activities in leaves of three sugarcane species. Values in parentheses show the percentage of the enzyme activity in chilling-stressed plants of that in control plants. Data for enzyme activity are means ± SE of four determinations from two separate plants
In Badira, the enzyme activities all fell with exposure to the chilling temperature with the exception of stromal FBPase activity which was not evidently affected by exposure to the chilling temperature for 28 and 52 h, and NADP-MDH initial activity which showed an increase at 4 h exposure to the chilling temperature. In particular, PPDK activity and NADP-MDH in vivo activity showed marked reductions after 28 h exposure to the chilling temperature, which was consistent with the results obtained in sorghum (Taylor et al. 1974). The NADP-MDH activation state in leaves also decreased, from 17 to 5%. It should be noted that in comparison with the relative constant levels in NADP-MDH total activity and the increases in Rubisco total activity in leaves of NiF4 and Yomitanzan, the NADP-MDH and Rubisco total activities in Badira decreased at exposure to the chilling temperature, reflecting a reduction in amounts of photosynthetic enzyme protein in Badira leaves, but not in NiF4 and Yomitanzan (Table 2). The significant decrease of Rubisco activity at low temperature has also been observed in chill-sensitive tomato (Sassenrath & Ort 1990).
In NiF4, the malate and OAA levels were relatively constant during chilling treatment and remained at 40 to 55% of their unstressed controls (Table 3). The PEP level was high relative to other metabolites at chilling temperature, and was maintained at about 80 to 95% of that in the unstressed control. Pyruvate and aspartate levels showed a trend of initially decreasing and then increasing during chilling treatment. However, viewed in total, there were no abrupt changes in any metabolite level during chilling treatment, all metabolite pool sizes except for PEP were reduced to roughly half of their unstressed controls. In Yomitanzan, the PEP and OAA levels increased after 4 and 28 h exposure to the chilling temperature, but both levels fell gradually during chilling treatment. Aspartate, alanine and pyruvate levels fell at 4 h exposure to the chilling temperature, and rose rapidly after further chilling treatment, especially the aspartate and alanine. Malate level, as for NiF4, was relatively constant during the chilling period, remaining at approximately 50% of its unstressed control.
Table 3. .
Effects of chilling temperature on metabolite levels in leaves of three sugarcane species. Values in parentheses show the percentage of the metabolite content in chilling-stressed plants of that in control plants. Data for metabolite content are means ± SE of four determinations from two separate plants
In Badira, the malate and pyruvate pool sizes were reduced to only 20 to 25% of their unstressed controls, much lower than in NiF4 and Yomitanzan. The PEP level decreased gradually with chilling time. The OAA levels fluctuated during the chilling period, but remained high. The most striking changes occurred in aspartate, in which the level fell sharply to about 10% of its control at 4 h exposure to the chilling temperature, and then increased strikingly with further chilling treatment. After exposure to the chilling temperature for 52 h, aspartate levels increased nearly six-fold. Alanine was almost depleted in leaves after 4 h exposure to the chilling temperature (reduced to 2% of its control), but thereafter the level increased gradually upon further chilling.
Primarily due to the striking increase in aspartate level, the total level of C4 pathway intermediates (malate, OAA, pyruvate, PEP, aspartate and alanine) at 52 h exposure to the chilling temperature in Badira leaves accounted for 67% of that in unstressed controls, in comparison with 48 and 65% in leaves of NiF4 and Yomitanzan, respectively, in which metabolites other than aspartate accounted for the main portion (Table 3). If aspartate and alanine were excluded from the total C4 pathway intermediates, the above percentages for leaves of NiF4, Yomitanzan and Badira would be 49, 57 and 28%, respectively, indicating that the total level of C4 pathway intermediates, not including aspartate and alanine, in the leaves of Badira were reduced markedly at chilling temperature.
Gas exchange, enzyme activities and metabolite contents were also monitored in control plants kept at day/night temperatures of 30/25 °C, but no significant changes were found for any parameter during the 52 h experiment (data not shown).
Three sugarcane species that differ in origin exhibited large differences in photosynthesis in relation to chilling temperature, both in gas exchange and photosynthetic biochemical reactions. Photosynthetic capacity in leaves of the hybrid species NiF4 was not damaged by chilling temperature. This was reflected by the fact that almost all enzyme activities measured did not decrease but instead they increased at exposure to the chilling temperature (Table 2) and the photosynthetic rate at 10 °C increased by 20 to 30% (Table 1).
In Badira, after 52 h exposure to the chilling temperature, photosynthetic rate in leaves decreased by more than 50%; almost all of the enzyme activities measured decreased; in particular PPDK activity and NADP-MDH initial activity decreased by about 70% (Table 2). In corresponding to the marked fall of NADP-MDH in vivo activity, the aspartate level in leaves increased nearly six-fold, while the malate level decreased to 23% of its unstressed control, in comparison with 43% in NiF4, at 52 h exposure to the chilling temperature (Table 3). The PEP levels in Badira leaves decreased gradually with chilling time, which was a sign that the Calvin cycle was in an environment of progressively insufficient CO2 (Leegood & von Caemmerer 1989). These results suggest that NADP-MDH must strongly limit the carbon flux in the C4 pathway in leaves of Badira at chilling temperature; that is, the carbon flux in the C4 pathway was blocked by the reaction catalysed by NADP-MDH and the carbon was channelled into aspartate at chilling temperature. Brooking & Taylor (1973) have reported that after exposure to chilling temperature, 80% of the 14CO2-labelled carbon in leaves of sorghum, a chilling-sensitive C4 species, accumulated in aspartate. Our results are consistent with their findings. However, as chilling extended, with the rapid decrease in PPDK activity (Table 2) the alanine level began to increase in the leaves (Table 3), indicating that PPDK gradually came into play to limit or co-limit the carbon flux at chilling temperature. However, compared with NADP-MDH, PPDK seemed to play a relatively less important role. In contrast to NADP-MDH and PPDK, the two Calvin cycle enzymes, Rubisco and stromal FBPase, were not severely affected by this chilling treatment (Table 2). Therefore, the data suggest that the marked decreases in NADP-MDH and PPDK activities are responsible for the significant decrease in photosynthetic rate in Badira leaves at exposure to the chilling temperature (Table 1).
This conclusion is supported by the changes in the total level of C4 pathway intermediates. As explained in the Results section, the amount of the carbon flowing in the C4 pathway (malate, OAA, pyruvate and PEP) in Badira leaves at chilling temperature was much reduced due to the flow of the carbon into aspartate and alanine. The total level of the C4 pathway intermediates has been found to increase during induction of photosynthesis (Usuda 1985a) and to increase with increasing photosynthetic rate as CO2 concentration increased in maize leaves (Usuda 1985b), suggesting that the total level of the C4 intermediates has a closely positive relationship with the photosynthetic rate in maize leaves under normal conditions. The agreement between the marked reduction of the total level of the C4 intermediates in Badira leaves and the large decrease of its photosynthetic rate at exposure to the chilling temperature suggests that the reduction of PPDK and NADP-MDH activities at chilling temperature may be the primary cause of the reduction of photosynthetic rate in sugarcane leaves at chilling temperature.
Yomitanzan showed a intermediate response between NiF4 and Badira to chilling temperature. After 52 h exposure to the chilling temperature, some enzyme activities increased; for example, Rubisco, stromal FBPase activities and NADP-MDH initial activity, but other enzymes activities decreased slightly after the initial chilling, and then recovered or increased after the extended chilling treatment (Table 2). Metabolite levels in leaves showed mixed responses to chilling temperature. Aspartate and alanine levels in Yomitanzan leaves increased clearly after 28 h exposure to the chilling temperature, and OAA levels exhibited substantial rise at 4 and 28 h exposure to chilling, indicating that NADP-MDH and PPDK activities were limiting the carbon flux at chilling temperature. However, this limitation apparently is much less than in Badira.
In this study, NiF4 exhibited the strongest cold-tolerance among the three species tested. NiF4 is a hybrid species bred by crossing the hybrid derivatives of the subtropical species S. barberi and the tropical species S. officinarum with the hybrid derivatives involving S. spontaneum. The strong cold-tolerance of NiF4 might be due to the hybrid vigour of the species or due to the contributions from S. spontaneum. During chilling treatment, aspartate and pyruvate levels in NiF4 leaves only showed trends of slightly increasing with chilling time, suggesting that NADP-MDH and PPDK did not evidently limit the carbon flux. We think that if chilling time was extended or chilling temperature was lowered, NADP-MDH and PPDK activities would also gradually come into play to limit the photosynthesis in NiF4.
It has been clear that photo-inhibition and photo-oxidation usually happen when plants are exposed to low temperature in light. Short-term chilling treatment can result in photo-inhibition, which leads to inhibition of light-saturated photosynthesis, photon yield and electron transport; and prolonged treatment results in photo-oxidation, which leads to disruption of photosynthetic apparatus and bleaching of leaves in some plants (Powles 1984). In this experiment, the light-saturated photosynthetic rate in Badira leaves decreased markedly at exposure to chilling temperature (Table 1); furthermore, we observed some spots of bleaching in Badira leaves after 3 d exposure to chilling temperature, suggesting that severe photo-inhibition and photo-oxidation occurred in Badira. It has been known that both photochemistry and carbon metabolism in plant leaves can be effected by chilling injury, but impairments of photosynthesis in some species by chilling are due to effects on carbon metabolism rather than due to photo-inhibition or damage to photochemistry (Stitt & Grosse 1988; Labate et al. 1990; Leegood & Edwards 1996). It has also been shown that the maintenance of certain levels of photosynthetic carbon metabolism is necessary to prevent or minimize photo-inhibition (Powles 1984). NADP-MDH and PPDK are two enzymes located in the chloroplasts of mesophyll cells and the activation states of NADP-MDH and PPDK in leaves have been shown to be closely related to photosynthetic electron transport and adenosine diphosphate (ADP)-mediated phosphorylation (Edwards et al. 1985). Therefore, the results in this study support the proposal that the marked reductions in NADP-MDH and PPDK activities in Badira leaves at exposure to chilling temperature limited the CO2 assimilation and caused photo-inhibition and damage to the capacity for photosynthetic electron transport and ADP-mediated phosphorylation.
We thank Professor Gerald. E. Edwards (Washington State University) for informative and helpful comments and Mr Eric Haas (University of Nebraska-Lincoln) for reading through the manuscript. We also thank Professor Y. Uchida (Saga University) and Professor S. Murayama (University of the Ryukyus) for supporting this study, and Mr K. Oshima for assistance in some aspects of this study.
Present address: Department of Biochemistry, University of Nebraska, Lincoln, NE 68588–0664, USA