Plant Physiol. (1999) 119: 1387-1398

Acclimation of Arabidopsis Leaves Developing at Low Temperatures. Increasing Cytoplasmic Volume Accompanies Increased Activities of Enzymes in the Calvin Cycle and in the Sucrose-Biosynthesis Pathway¹

Åsa Strand^*, Vaughan Hurry, Stefan Henkes, Norman Huner, Petter Gustafsson, Per Gardeström, and Mark Stitt

Department of Plant Physiology, University of Umeå, S-901 87 Umeå, Sweden (Å.S., V.H., P. Gustafsson, P. Gardeström); Botanisches Institut, Universität Heidelberg, D-69120 Heidelberg, Germany (V.H., S.H., M.S.); and Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada (N.H.)

	ABSTRACT

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Photosynthetic and metabolic acclimation to low growth temperatures were studied in Arabidopsis (Heynh.). Plants were grown at 23°C and then shifted to 5°C. We compared the leaves shifted to 5°C for 10 d and the new leaves developed at 5°C with the control leaves on plants that had been left at 23°C. Leaf development at 5°C resulted in the recovery of photosynthesis to rates comparable with those achieved by control leaves at 23°C. There was a shift in the partitioning of carbon from starch and toward sucrose (Suc) in leaves that developed at 5°C. The recovery of photosynthetic capacity and the redirection of carbon to Suc in these leaves were associated with coordinated increases in the activity of several Calvin-cycle enzymes, even larger increases in the activity of key enzymes for Suc biosynthesis, and an increase in the phosphate available for metabolism. Development of leaves at 5°C also led to an increase in cytoplasmic volume and a decrease in vacuolar volume, which may provide an important mechanism for increasing the enzymes and metabolites in cold-acclimated leaves. Understanding the mechanisms underlying such structural changes during leaf development in the cold could result in novel approaches to increasing plant yield.

	INTRODUCTION

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A rapid shift from warm growth conditions to chilling temperatures (typically between 5°C and 10°C) leads to a pronounced inhibition of photosynthetic carbon fixation. In chilling-sensitive C₃ plants such as tomato and bean, this inhibition is associated with the inactivation of regulatory thioredoxin-activated enzymes of the Calvin cycle (Sassenrath and Ort, 1990; Sassenrath et al., 1991; Holaday et al., 1992; Brüggemann et al., 1994). Because the shift to low temperature is also associated with a strong activation of NADP-malate dehydrogenase in bean (Holaday et al., 1992), it is not due to a chill-induced lack of stromal reducing equivalents. In tomato the chill-induced reduction in photosynthesis is also associated with irreversible inactivation of Rubisco and loss of Rubisco protein (Brüggemann et al., 1992). Low temperature leads to a selective inhibition of end-product synthesis. This results in an accumulation of phosphorylated metabolites and a short-term phosphate limitation of photosynthesis (Leegood and Furbank, 1986; Sharkey et al., 1986) that may be partly due to the temperature-dependent changes in the affinity of cFBPase for its effectors, especially AMP (Stitt and Grosse, 1988).

Chilling inhibits phloem export, leading to a rapid accumulation of soluble sugars and repressing photosynthetic gene expression (Krapp and Stitt, 1995); Strand et al. (1997) showed this effect recently for Arabidopsis during short-term exposure to low temperatures. The inhibition of carbon fixation by low temperature may be followed by photoinhibition (Somersalo and Krause, 1989; Hurry and Huner, 1992). Chilling also interrupts the circadian-regulated transcription of several nuclear-encoded photosynthetic genes in both chilling-sensitive tomato (Martino-Catt and Ort, 1992) and chilling-tolerant Arabidopsis (Kreps and Simon, 1997). These data show that short-term exposure of chilling-sensitive and -tolerant species to low temperatures results in feedback-mediated down-regulation of photosynthesis and photosynthetic gene expression, perturbations to circadian-regulated processes, and, in some instances, irreversible damage to photosynthetic proteins.

In contrast, when leaves of cold-hardy herbaceous plants develop at low growth temperatures (5°C), they show a remarkable recovery of photosynthetic capacity. Even in the short term, low temperatures lead to an increase in the activation state of the Calvin-cycle enzymes in chilling-tolerant C₃ plants such as spinach, winter rye, and winter oilseed rape (Holaday et al., 1992; Hurry et al., 1994, 1995b), which is the opposite of the response in cold-sensitive species (see above). When Arabidopsis leaves develop at low temperature, they no longer show the suppression of transcript levels for photosynthetic proteins that is found after shifting warm-grown leaves to low temperatures (Strand et al., 1997). As a result, leaves that have developed at low temperature contain higher activities of selected enzymes of the photosynthetic carbon-reduction cycle (Hurry et al., 1994, 1995b). This reversal of the sink regulation of photosynthetic gene expression (Jang and Sheen, 1994; Jang et al., 1997) occurs even though these leaves produce and maintain large pools of soluble sugars.

Leaves of Arabidopsis that develop at 5°C greatly increase their expression of SPS and cFBPase in the Suc-synthesis pathway in the cytosol but not in the starch-synthesis pathway in the plastid (Strand et al., 1997). Marked increases in the SPS activity have also been reported for spinach (Guy et al., 1992; Holaday et al., 1992), for winter cultivars of oilseed rape and wheat (Hurry et al., 1995b), and rye (Hurry et al., 1994) after prolonged exposure to cold. The increased expression and activity of Suc-synthesis enzymes correlates with an accumulation of sugars in leaves at low temperature. It has been proposed that high sugar levels might be important for cryoprotection (Santarius, 1982; Anchordoguy et al., 1987; Carpenter and Crowe, 1988). However, there is no direct evidence that the increased sugar levels are due to increased synthesis and not just to a passive response to the inhibition of growth and phloem transport. It is also not clear whether the increased activities of the enzymes leading to Suc merely counteract the direct inhibitory effect of low temperature (see above) or whether they actually lead to increased Suc synthesis.

Another marked short-term effect found after shifting warm-grown plants to low temperatures is the accumulation of large pools of phosphorylated intermediates (Labate and Leegood, 1988; Hurry et al., 1994; Strand et al., 1997), thought to reflect the inhibition of end-product synthesis at low temperatures (see above). It has been suggested that this accumulation of phosphorylated intermediates contributes to the feedback down-regulation of photosynthesis, because the cytosolic and chloroplastic pools of Pi become depleted to the point where photophosphorylation is Pi limited (Herold, 1980; Mächler et al., 1984; Leegood and Furbank, 1986; Labate and Leegood, 1988; Stitt and Grosse, 1988). Although this may be true for leaves exposed to short-term (minutes to hours) drops in temperature, it is unclear whether Pi limitation persists in the long term (days) in plants acclimating to low temperature. The subcellular compartmentation of Pi is tightly regulated, and 85% to 95% of the Pi may be located in the vacuole in well-fertilized plants (Bieleski and Ferguson, 1983). Prolonged exposure to low-temperature-induced Pi deficiency might therefore be buffered by the release of Pi from the vacuole (Woodrow et al., 1984; Mimura et al., 1990), as occurs when choline feeding depletes Pi in the cytosol (Bligny et al., 1990). Reports that cold-acclimated plants show damping of oscillations in O₂ evolution under conditions of light- and CO₂-saturated photosynthesis (Hurry et al., 1993) indicate a repoising of the cytosolic Pi pool after cold development of leaves. This conclusion is supported by data showing an increase, rather than a decrease, in the ATP/ADP ratio in cold-developed leaves (Sobczyk et al., 1985; Hurry et al., 1995c; Strand et al., 1997). The role of Pi in acclimation to the cold and in the shift from starch to soluble carbohydrate accumulation therefore remains unclear.

Studies of Arabidopsis and other cold-tolerant herbaceous plants indicate that growth at low temperature leads to a developmental reprogramming of carbon metabolism that involves changes in gene expression and enzyme activity and possibly also changes of phosphate compartmentation. The resulting recovery of the photosynthetic rate and the preferential accumulation of soluble sugars could be an essential element for acclimation to low growth temperatures. Most previous studies have concentrated on a single or a small number of enzymes, however, and have not demonstrated that the changes in expression and activity actually lead to significant increases in the rate of photosynthesis and Suc synthesis. Furthermore, investigations of metabolism have not yet been integrated with other effects of low temperature on leaf development. Older studies of winter rye leaf development at low growth temperatures reported increases in cell size and cytoplasmic content, smaller vacuoles, and multivacuolated cells (Huner et al., 1981, 1984; Griffith et al., 1985). Ristic and Ashworth (1993) have shown that Arabidopsis parenchyma cells also undergo ultrastructural changes after transfer to low temperatures in continuous light, leading to multiple invaginations of the plasma membrane, and the accumulation of microvesicles associated with the plasma membrane, tonoplast, chloroplast envelope, and mitochondrial outer membrane.

In this study we investigated whether (a) the recovery of photosynthesis in the cold is associated with coordinate changes in the activity of several Calvin-cycle enzymes; (b) these changes plus the up-regulation of the cytosolic pathway for Suc synthesis reported previously for Arabidopsis after growth and development at low temperature (Strand et al., 1997) result in an increased flux into Suc; and (c) the Pi status in the cytoplasm is also adjusted during acclimation to low growth temperatures. Finally, we assessed whether there is a link between metabolic adaptations and anatomical changes after leaf development at low temperatures.

	MATERIALS AND METHODS

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Plant Material

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Chlorophyll and Protein Content and Specific Leaf Weight

Enzyme Activities

To assay the activity of the Calvin-cycle enzymes, 25 mg of frozen tissue was homogenized in 500 µL of extraction buffer (50 Hepes-KOH, pH 7.4, 12 mM MgCl₂, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM PMSF, 1 mM -aminocapronic acid, 10 µM leupeptin, 2 mM DTT, 0.1% [v/v] Triton X-100, 10 mg of polyvinylpolypyrrolidone, 5 mM KHCO₃ [for Rubisco only], 1 mM Fru-1,6-bisP [for stromal FBPase only], and 10% [v/v] glycerol) and then centrifuged at 11,000g for 5 min. We collected the supernatant and immediately assayed enzymatic activity (Haake et al., 1998).

Photosynthetic Carbon Flux

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Soluble Sugars and Starch

For starch extraction, we added 0.5 mL of distilled water to resuspend the pellet and autoclaved the samples for 3 h. For starch cleavage we added a 50-µL aliquot of the autoclaved suspension to 450 µL of 50 mM sodium-acetate incubation buffer, pH 4.8, containing 28 units of amyloglucosidase and 36 units of -amylase, and we incubated the samples at 37°C for 16 h. We then centrifuged the samples for 15 min at 11,000g and assayed the supernatant for Glc (Stitt et al., 1989).

Pi Determination

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Transmission Electron Microscopy

	RESULTS

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Calvin-Cycle Enzymes

View larger version (40K): [in this window] [in a new window]   Figure 1. Activity of the Calvin-cycle enzymes, Rubisco, PGK, GAPDH, aldolase, transketolase, stromal FBPase (sFBPase), and PRK, per unit of chlorophyll (Chl) in 23°C leaves (open bars), 23°C/5°C leaves (shaded bars), and 5°C leaves (black bars). Leaves were collected 2 h into the photoperiod and frozen at the temperature of liquid N2. Each value represents the mean ± SD of three different leaves from three different plants.

Of the seven Calvin-cycle enzymes measured, only Rubisco (Fig. 1A), GAPDH (Fig. 1C), aldolase (Fig. 1D), and possibly stromal FBPase (Fig. 1F) showed substantial increases in total extractable activity in the 23°C/5°C leaves. In contrast, 5°C leaves showed significant increases in the extractable activity of all seven enzymes, with Rubisco (2.5-fold), aldolase (2.1-fold), and, to a lesser degree, GAPDH (1.8-fold) and stromal FBPase (1.9-fold) showing the largest increases. The remaining enzymes, PGK (Fig. 1B), transketolase (Fig. 1E), and PRK (Fig. 1G), all showed approximately 1.5-fold increases in activity per unit of chlorophyll when 5°C leaves were compared with control 23°C leaves. Although increases in Rubisco and stromal FBPase activity at 5°C have been reported previously for spinach (Holaday et al., 1992), winter rye (Hurry et al., 1994), and winter oilseed rape (Hurry et al., 1995b), our data demonstrate that the activities of all of the Calvin-cycle enzymes, including nonregulated enzymes such as aldolase, increased significantly in the cold.

Cytosolic Enzyme Activities

View larger version (19K): [in this window] [in a new window]   Figure 2. Activity per unit of chlorophyll (Chl) of two cytosolic enzymes, SPS (A) and cFBPase (B), for 23°C leaves (open bars), 23°C/5°C leaves (shaded bars), and 5°C leaves (black bars). Leaves were collected 2 h into the photoperiod and frozen at the temperature of liquid N2. Each value represents the mean ± SD of three different leaves from three different plants.

Calvin-Cycle Enzyme Activities Increased Because of a General Increase of Protein

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We recalculated the enzyme activities shown in Figures 1 and 2 on a protein basis (Table II). There were only minor changes in the activities of Rubisco, PGK, GAPDH, aldolase, stromal FBPase, transketolase, and PRK among 23°C leaves, 23°C/5°C leaves, and 5°C leaves when the activity was related to total leaf protein. The increased activity of the Calvin-cycle enzymes on a chlorophyll, fresh-weight, or leaf-area basis was therefore mainly due to the general increase in leaf protein.

SPS activity still showed a 1.8-fold increase in activity on a protein basis, which was already evident in the 23°C/5°C leaves (Table II). cFBPase showed a 1.2-fold increase in the 23°C/5°C leaves, but the biggest increase was in the 5°C leaves (1.7-fold). The increased activity of the Suc-biosynthesis enzymes at low temperatures is therefore attributable to two processes: (a) a selective increase in the expression and activity of the enzymes for Suc synthesis that also occurs when preformed leaves are transferred to low temperature and (b) a further increase, linked to a general increase in protein, in the leaves that developed at low temperature.

Photosynthetic Carbon Flux

When photosynthesis was measured at 23°C, no differences were found in ¹⁴C incorporation between the control 23°C and the 23°C/5°C leaves; the 5°C leaves showed a 1.5-fold higher rate of ¹⁴CO₂ incorporation at 23°C (Fig. 3A). When similar measurements were made at 5°C, the control 23°C leaves had very low rates of C incorporation, the 23°C/5°C leaves showed a slight enhancement of ¹⁴C incorporation, and the 5°C leaves showed a dramatic 5-fold increase in photosynthetic carbon fixation (Fig. 3B). The rate of ¹⁴C incorporation in the 5°C leaves at 5°C was similar to that in the 23°C or the 23°C/5°C leaves at 23°C. These data demonstrate that the enhancements in enzymatic capacity translated into an almost complete recovery of photosynthetic carbon flux at the lower growth temperature.

View larger version (25K): [in this window] [in a new window]   Figure 3. Photosynthetic carbon flux measured as 14C incorporation after illumination in a leaf disc electrode for 20 and 40 min at 23°C (A) and 5°C (B), respectively. The carbon flux was measured for 23°C leaves (open bars), 23°C/5°C leaves (shaded bars), and 5°C leaves (black bars). Leaves were collected 2 h into the photoperiod. Each bar represents the mean ± SD of at least three different incubations. Insoluble, Starch; Neutral, sugars; Cationic, amino acids; Anionic, organic acids and phosphorylated intermediates.

These experiments also provided an opportunity to assess whether leaf development at 5°C alters the partitioning of newly fixed carbon. When photosynthesis was carried out at 23°C, the control 23°C leaves allocated slightly more than 50% of their newly fixed carbon into starch (Fig. 3A). This proportion remained at a similarly high level (approximately 45%) in the 23°C/5°C leaves but decreased to less than 30% in the 5°C leaves (Fig. 3B). This was not simply an effect of treatment temperature because the reduced partitioning into starch on a percentage basis was similar irrespective of when the 5°C-labeling experiment was carried out. Of the three soluble fractions, the newly fixed carbon was primarily partitioned into the neutral fraction, representing soluble carbohydrates. In the control (23°C) leaves, approximately 30% of the labeled carbon went into the neutral fraction, whereas in the 5°C leaves it increased to 40% (Fig. 3). There was also a small relative increase in partitioning into the cationic (amino acid) fraction in the 5°C leaves (18% versus 11% for the 23°C leaves) and a relatively larger increase of flux into the anionic fraction (phosphorylated intermediates and organic acids) in the 5°C leaves (14% versus 5% for the 23°C leaves). Therefore, the primary effect of leaf development at 5°C is to restore a high overall rate of photosynthesis, but there is also a shift of emphasis in partitioning, away from starch and toward soluble compounds including sugars.

Starch and Soluble Sugar Content

View larger version (34K): [in this window] [in a new window]   Figure 4. Carbohydrate content for 23°C leaves (), 23°C/5°C leaves (), and 5°C leaves (). Samples were frozen in the light at various times during the photoperiod at the temperature of liquid N2. Each point represents the mean ± SD of three different leaves from three different plants. The bar at the top of the graphs indicates the times when the lights were off (black bars) or on (white bars). Chl, Chlorophyll; hex, hexose.

In contrast, the 5°C leaves contained a large Suc pool that showed marked diurnal changes. Large amounts of Suc accumulated throughout the day and a substantial amount was mobilized after the end of the day. A significant pool was nevertheless maintained throughout the dark period. The cold-developed 5°C leaves also contained substantial pools of reducing sugars, although it is noteworthy that they were smaller than in the 23°C/5°C leaves and that they were relatively stable throughout the photoperiod. The starch pool in the 5°C leaves built up during the day, although to a lesser extent than in the control 23°C leaves, and was remobilized during the dark period. These data support the conclusions drawn from Figure 3 that development at 5°C enabled Arabidopsis leaves to recover significant photosynthetic capacity and simultaneously allowed a marked shift toward partitioning into a large and active Suc pool.

Total Pi Pools and Available Pi

View larger version (22K): [in this window] [in a new window]   Figure 5. Total Pi content (A) and available Pi pools (B) in Arabidopsis leaves. Leaf samples for the determination of total Pi were collected 2 h into the photoperiod by freezing in light at the temperature of liquid N2. To determine available Pi, 200 mM Glc was fed through the petiole in ambient light for 30 min. The increase in hexose-P was then determined. The figures show the results for 23°C leaves (open bars, ), 23°C/5°C leaves (shaded bars, ), and 5°C leaves (black bars, ). Each value represents the mean ± SD of at least three different leaves. Chl, Chlorophyll.

Leaf Anatomy

View larger version (74K): [in this window] [in a new window]   Figure 6. Electron micrographs of Arabidopsis mesophyll cells. A and B, 23°C leaves; C and D, 23°C/5°C leaves; E and F, 5°C leaves. Bars in A, C, and E = 1 µm; bars in B, D, and F = 0.1 µm. T, Tonoplast membrane.

This interpretation of the electron micrographs is supported by the large decrease in the water content of the leaves and by the observation that this decrease was accompanied by a marked increase of protein (Table I) but not of starch (Fig. 4).

	DISCUSSION

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Increased Photosynthetic Capacity and Reprogramming of Allocation in Cold-Acclimated Leaves

Arabidopsis leaves that developed at 5°C showed a 5-fold increase in carbon fixation compared with the control 23°C leaves measured at 5°C. When the fluxes were compared at warm temperature (23°C), the 5°C leaves also possessed a higher capacity for photosynthetic carbon fixation than did the control leaves. Indeed, the fully acclimated 5°C leaves were able to maintain a flux of fixed carbon at 5°C that was similar to that maintained by the control leaves at 23°C. This increased capacity for photosynthetic carbon flux required a reprogramming of carbon metabolism (Strand et al., 1997). As a result, the Arabidopsis 5°C leaves were able to produce and accumulate large pools of soluble sugars without any associated suppression of photosynthetic gene expression or metabolism. As we discuss below, the metabolic changes resulting from leaf development at 5°C included changes in Pi allocation and in the activities of Calvin-cycle enzymes and enzymes for Suc synthesis.

Coordinate Increases in the Activities of Regulated and Nonregulated Calvin-Cycle Enzymes Stimulated Photosynthetic Carbon Fixation

There were differences in the extent to which individual Calvin-cycle enzyme activities increased following acclimation to a lower growth temperature. Whether these differential responses resulted from variable sensitivities of the enzymes to low temperature or from other factors remains to be determined. The most pronounced increases in activity in the 5°C leaves were found for Rubisco (2.5-fold) and for the "nonregulated" enzyme, aldolase (2.1-fold). Rubisco activity has frequently been investigated and shown to increase in the cold in several species of monocots and dicots (Treharne and Eagles, 1970; Chabot et al., 1972; Holaday et al., 1992; Hurry et al., 1994, 1995b). Nonregulated enzymes such as aldolase, on the other hand, have been considered to be expressed in excess and to be unimportant for regulation processes such as acclimation. This view clearly requires re-evaluation; Haake et al. (1998) showed that antisense down-regulation of aldolase in tobacco leaves resulted in a pronounced reduction in carbon fluxes in the Calvin cycle.

Coordinate Increase of the Activities of Enzymes for Suc Synthesis and Changes to Pi Compartmentation Stimulated Suc Synthesis

It has been suggested that the low rates of Pi release in end-product synthesis limit photophosphorylation at low temperatures (Leegood and Furbank, 1986; Sharkey et al., 1986). The increased activities of these cytosolic enzymes involved in Suc biosynthesis could counteract this limitation. Our results also indicate that changes in Pi compartmentation occurred during low-temperature acclimation. Although the total pool of Pi in the leaf cells did not change after cold exposure, the availability of Pi increased, indicating that more Pi was available for metabolism in the cytoplasm, especially in leaves that developed at low temperature. This may explain why Pi did not become limiting for photophosphorylation and photosynthesis, even though phosphorylated intermediates were present at much higher levels in the cold. This conclusion is supported by earlier studies of the effect of leaf development at low temperature on ATP-to-ADP ratios and oscillations in CO₂-saturated O₂ evolution and room-temperature chlorophyll fluorescence (Hurry et al., 1993, 1995c). Further studies may reveal how low temperatures trigger the release of Pi from the vacuole.

Previous investigators reported a significant increase in the soluble sugar-to-starch ratio in several species after leaf development in the cold (Hurry et al., 1995b; Strand et al., 1997). However, analyses of carbohydrate pools at a single time do not reveal whether the changes were due to increased synthesis or to inhibition of export. In this report we show that cold development of leaves led to a shift in carbon partitioning toward soluble sugar synthesis. Whereas in the control 23°C leaves, 50% of the incorporated [¹⁴C]CO₂ was incorporated into starch and only 30% into sugars; in the 5°C leaves, only 30% of the labeled carbon was found in starch and as much as 40% was converted to sugars. The diurnal changes in leaf carbohydrate levels provided independent evidence for a change in allocation. The 5°C leaves accumulated less starch during the photoperiod than did the control 23°C leaves and instead maintained a large Suc pool that showed significant turnover during the diurnal period. Suc was not fully mobilized during the night, which is consistent with the hypothesis that, in addition to being important for the export of photosynthate, Suc may play an osmoregulatory or cryoprotective role in leaves in the cold (Anchordoguy et al., 1987; Carpenter and Crowe, 1988). In contrast, fully developed source leaves that had been shifted to 5°C (the 23°C/5°C leaves) accumulated large pools of soluble carbohydrates, including very large pools of free hexoses. These leaves also contained a large amount of starch that was not remobilized during the night. The accumulation of carbohydrates in the 23°C/5°C leaves therefore appears to be due at least partly to limited export and mobilization.

Development under low-temperature growth conditions allows plants to acclimate carbon metabolism by shifting partitioning toward soluble-sugar synthesis. Up-regulation of the cytosolic pathway for Suc biosynthesis could be of great importance for the recovery of photosynthesis and the maintenance of the flux of carbon at low temperatures, as well as for the capacity to grow and develop new leaves in the cold. The cFBPase and SPS enzymes play key roles in the cold-acclimation process and molecular modifications of these enzymes will be of great interest in the future.

Changes in Cytoplasmic and Vacuolar Volume Underlay the 2- to 3-Fold Increases in Protein and Enzyme Activity in Cold-Acclimated Leaves

A marked decrease of water in leaves that developed at low temperatures accompanied the changes in enzyme activities and protein. Electron micrographs showed that these changes were due to an increase in the relative volume of the cytoplasm and a decrease in the relative volume of the vacuole. In the 5°C leaves the cytoplasm occupied a larger volume of the leaf cell and appeared denser than in the 23°C leaves. The vacuoles were clearly smaller in the 23°C/5°C leaves and in the 5°C leaves, but these structural changes were most pronounced in the 5°C leaves. Similar increases in cytoplasmic volume have been reported previously for tree species (Siminovitch et al., 1967) and winter rye (Huner et al., 1981; Griffith et al., 1985), indicating that the changes observed for Arabidopsis in the present study represent a typical response to cold acclimation. However, the functional significance of these changes in cell structure has not previously been recognized.

The anatomical changes observed in cold-developed leaves interact with their metabolic adaptation. On one hand, it appears likely that the increase in the cytoplasmic volume allows the cells to accommodate the 2- to 3-fold increase in leaf protein and therefore plays an integral role in the acclimation of metabolism to low temperature, leading to an increased capacity for photosynthesis and sugar synthesis. But on the other hand, the increased cytoplasm may give rise to an increased need for solutes, such as Suc, that are typically restricted to the cytoplasm. Higher levels of sugars, especially Suc, may be necessary in the cytoplasm not only to maintain export and provide cryoprotection but possibly also to help mechanize a selective increase in the cytoplasmic volume. Investigations of plants with altered levels of sugars will test these ideas. Insight into the underlying mechanisms of these structural changes in the cold could result in novel approaches to increasing plant yield.

In conclusion, we have shown that leaf development at 5°C results in the recovery of photosynthetic carbon fixation and a shift in the partitioning of carbon away from starch and toward Suc. This photosynthetic recovery is supported by coordinated increases in the activity of several Calvin-cycle enzymes and by marked increases in the activity of key cytosolic enzymes for Suc synthesis. Furthermore, despite the large accumulation of hexose phosphates in the cold, Pi is not limited and probably even increases in the cytosol. Finally, we show a link between anatomical changes in leaf development at 5°C and metabolic adjustments to the cold. An increase in the volume of the cytoplasm may provide an important mechanism for increasing the enzymes and metabolites in cold-acclimated leaves. It correlates with an increased flux through the cytosol and Suc synthesis and possibly with an increased need for solutes in the cytosol.

	FOOTNOTES

   Received September 9, 1998; accepted December 28, 1998.

	ABBREVIATIONS

Abbreviations: cFBPase, cytosolic Fru-1,6-bisphosphatase. GAPDH, NADP-glyceraldehyde-P dehydrogenase. PGK, phosphoglycerate kinase. PRK, phosphoribulokinase. SPS, Suc-6-P synthase.

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