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Abstract

Accumulation of non-structural carbohydrate in leaves represses photosynthesis. However, the extent of repression should be different between sink leaves (sugar consumers) and source leaves (sugar exporters). We investigated the effects of carbohydrate accumulation on photosynthesis in the primary leaves of bean (Phaseolus vulgaris L.) during leaf expansion. To increase the carbohydrate content of the leaves, we supplied 20 mM sucrose solution to the roots for 5 d (sugar treatment). Plants supplied only with water and nutrients were used as controls. The carbohydrate contents, which are the sum of glucose, sucrose and starch, of the sugar-treated leaves were 1.5–3 times of those of the control leaves at all developmental stages. In the young sink leaves, the photosynthetic rate at saturating light and at an ambient CO2 concentration (A360) did not differ between the sugar-treated and control leaves. The A360 of sugar-treated source leaves gradually decreased relative to the control source leaves with leaf expansion. The initial slope of the A–Ci (CO2 concentration in the intercellular space) curve, and the Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) content per leaf area showed trends similar to that of A360. Differences in Amax between the treatments were slightly smaller than those in A360. These results indicate that the effect of carbohydrate accumulation on photosynthesis is significant in the source leaves, but not in the young sink leaves, and that the decrease in Rubisco content was the main cause of the carbohydrate repression of photosynthesis.

Introduction

Accumulation of non-structural carbohydrates in leaves often represses photosynthesis (Krapp et al. 1991, Krapp and Stitt 1995, Jeannette et al. 2000). For the carbohydrate repression of photosynthesis, three mechanisms have been proposed.

Accumulation of carbohydrate in leaves often causes feedback inhibition of sucrose synthesis and accumulation of sugar phosphates in the cytosol. The accumulation of sugar phosphates decreases the orthophosphate concentration in the cytosol and thereby suppresses the antiport of triose phosphate/orthophosphate across the chloroplast envelope (Stitt and Quick 1989). Low availability of orthophosphate in the stroma suppresses ATP synthesis and reduction of PGA (phosphoglyceric acid) to triose phosphate, and thereby photosynthesis (Stitt 1986, Sharkey and Vanderveer 1989). The phosphate-limited photosynthesis is most likely to occur at saturating CO2 concentrations (Sharkey 1985). Low orthophosphate concentrations in the stroma may also lower the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Sawada et al. 1992). However, some mutants of Arabidopsis thaliana with impaired sucrose synthesis did not show signs of reduction of photosynthesis due to orthophosphate depletion, although they accumulated the phosphorylated intermediates (Strand et al. 2000, Chen et al. 2005). Thus, the phosphate limitation does not always occur in response to inhibition of sucrose synthesis.

Nafziger and Koller (1976) claimed that starch accumulation in chloroplasts causes deformation of the chloroplasts and thereby decreases the conductance for CO2 diffusion from the intercellular spaces to the catalytic site of Rubisco (gi). Nakano et al. (2000) also suggested that starch accumulation itself, not the decreased amounts of photosynthetic enzymes, caused the repression of photosynthesis in the leaves of bean plants with their pods removed.

Expression of photosynthetic genes is suppressed by soluble sugars, which would eventually repress photosynthesis (Sheen 1990, Pego et al. 2000). Long-term cultivation of plants at elevated CO2 increases the carbohydrate level in the leaves of many plants, and it has often been reported that expression of photosynthetic genes is repressed (Nie et al. 1995, Miller et al. 1997, Chang et al. 1998).

The carbohydrate repression of photosynthesis, however, has rarely been examined in relation to leaf developmental stages, although the metabolic roles of carbohydrates dramatically change depending on the leaf developmental stages. Young sink leaves import carbohydrates to construct their photosynthetic systems, while mature source leaves with high photosynthetic activities export photosynthates to sink organs. In one of such studies, Krapp et al. (1991) fed a 50 mM glucose solution to sink and source spinach leaves from their petioles via the transpiration stream. The photosynthetic rate per leaf area of the glucose-fed source leaves was 30% of that of the control source leaves, while that of the glucose-fed sink leaves was not different from that of the control sink leaves. The results indicate that the effect of carbohydrate accumulation on photosynthesis differs between the sink and source leaves.

In the present study, we investigated the effects of carbohydrate accumulation on photosynthesis in leaves of bean (Phaseolus vulgaris L.) at various developmental stages during the leaf expansion. The sink–source transition in the primary leaves of bean was examined previously (Miyazawa et al. 2003) and the transition occurred when the leaf area was about 40% of the fully expanded leaf area. We fed sucrose solution to the pot-grown plants with the primary leaves at various developmental stages to increase the carbohydrate contents in the leaves, and compared the photosynthetic properties of sugar-treated and control leaves. The effects on the ribulose-1,5-bisphosphate (RuBP) regeneration capacity and on the RuBP carboxylation capacity were separately evaluated. To identify mechanisms responsible for the decrease in these capacities, we examined photosynthetic parameters including the orthophosphate content, gi and Rubisco content in relation to the three potential mechanisms mentioned above. Based on these results, the significance of the developmental stage-dependent repression of photosynthesis is discussed.

Results

We designated the day when we started imbibition of the bean seeds as day 0 (Fig. 1). The juvenile bean plants were planted in the vermiculite on day 4 and watered every day. Half-strength Hoagland solution was supplied every day from day 7. The primary leaves emerged on day 7–9 and were fully expanded on day 23. According to Miyazawa et al. (2003), the sink–source transition of the primary leaves of the same bean plants occurred when the leaf area attained 40% of the fully expanded area. In the present study, the leaf area reached 40% on around day 10. The daily net photosynthesis calculated from the dark respiration rate and the photosynthetic rate at growth photosynthetic photon flux density (PPFD) of 300 µmol m–2 s–1 and CO2 concentration of 360 µmol mol–1 increased gradually with leaf development and turned to be positive around day 10 (data not shown). We fed 50 ml of 20 mM sucrose solution to the pot every day for 5 d before the measurement of photosynthesis and sampling. We call this treatment ‘sugar treatment’. The leaves collected on day 12 were treated with sucrose solution for 5 d when the leaves were mainly in the sink phase. With leaf expansion, the sugar-treated period was shifted from the sink to source phases, and we were able to see the effect of sugar treatment on the photosynthetic properties of the leaves at various stages in terms of the sink–source transition.

Carbohydrates contents

Changes in carbohydrate contents per leaf area with leaf development are shown in Fig. 2. We sampled the leaves towards the end of the day. Thus, the carbohydrate contents in Fig. 2 would be regarded the as maximum values within the diurnal cycle. Glucose contents in the control leaves were high when the leaves were young, and gradually decreased with leaf development (Fig. 2A). In contrast, the glucose contents of the sugar-treated leaves decreased only slightly. The glucose contents of the sugar-treated leaves were higher than those of the control leaves, but the differences on day 12 were small. The differences between the treatments on days 14 and 16 were statistically significant.

The sucrose contents of the sugar-treated leaves were higher than those of the control ones at any stage (Fig. 2B). The difference in the sucrose content between the control and sugar-treated leaves was relatively small on day 12, and increased with leaf development.

The starch contents were relatively low on days 12 and 14 in both sugar-treated and control leaves, and increased with leaf expansion (Fig. 2C). The starch contents of the sugar-treated leaves were 1.5–3 times higher than those of the control leaves. Total carbohydrate content, the sum of the glucose, sucrose and starch contents, also showed trends similar to the starch contents (Fig. 2D).

Leaf mass per area (LMA)

In the control leaves, the LMA was almost constant throughout the experimental periods (Table 1). The LMA did not differ between the treatments on day 12 or 14. The LMA of the sugar-treated leaves was slightly higher than that of the control leaves on day 16, 18 and 20. The increase in the LMA in the sugar-treated leaves was mostly attributed to the increase in the carbohydrate contents (Fig. 2D).

Photosynthesis

Changes in the photosynthetic rate on a unit leaf area basis at a PPFD of 1,000 µmol m–2 s–1 and at an ambient CO2 concentration (Ca) of 360 µmol mol–1 (A360) are shown in Fig. 3A. The A360 gradually decreased with the developmental stage. The A360 was not different between the sugar-treated and control leaves on day 12, but was lower in the sugar-treated leaves than in the control leaves on other days. The difference in A360 between the treatments gradually increased with leaf expansion, and the differences were statistically significant on days 18 and 20. The initial slope of the A–Ci (CO2 concentration in the intercellular space) curve, the indicator of the Rubisco carboxylation capacity in vivo, showed a trend similar to that of A360 (Fig. 3B).

As the indicator of RuBP regeneration capacity in vivo, we measured the rate of photosynthesis at 1,000 µmol photon m–2 s–1 and at a saturating Ca of 1,500 µmol mol–1 (Amax) (Fig. 3C). The Amax of the control leaves was almost constant after day 14. In contrast, the Amax of sugar-treated leaves gradually decreased, and the Amax values of sugar-treated leaves were significantly lower than those of the control leaves on days 18 and 20.

Chlorophyll content and chlorophyll fluorescence

Chl contents per leaf area decreased with leaf development, and Chl a/b ratios did not change during the experimental periods (Table 1). There was no difference in the Chl contents and a/b ratio between the treatments, except for the Chl a/b ratio on day 20. The maximum quantum yield of PSII (Fv/Fm), measured just after the dark pre-treatment for 20 min at the Ca of 360 µmol mol–1, was unchanged during the leaf development (Table 1). Though the Fv/Fm values in the sugar-treated leaves were slightly higher than those of the control leaves, they did not statistically differ between the treatments.

Rubisco large subunit (LSU) and light-harvesting Chl binding protein of PSII (LHCII) content

Changes in Rubisco LSU and the LHCII contents per leaf area are shown in Fig. 4. The Rubisco LSU contents decreased with leaf development (Fig. 4A). The decrease in Rubisco LSU content showed a trend similar to those of the A360 and the initial slope of the A–Ci curve (Fig. 3A, B), though the decrease in Rubisco LSU contents was slightly faster than the decreases in A360 and the initial slope. While the Rubisco LSU content did not differ between the sugar-treated and control leaves on day 12, the Rubisco LSU contents of the sugar-treated leaves were lower than those of the control leaves after day 14. The difference between the treatments gradually increased with leaf age. The LHCII content also decreased with leaf development (Fig. 4B). There was no difference in LHCII content between the treatments.

Nitrogen and carbon content

The nitrogen content of the sugar-treated leaves was slightly lower than that of the control leaves on day 12, and the difference between the treatments increased with leaf development (Table 1). The carbon content showed a trend similar to that of the LMA (data not shown). The nitrogen/carbon (N/C) ratio of the sugar-treated leaves was always lower than that of the control leaves, and the differences between the treatments were greater at the later stages of leaf development.

Orthophosphate content

The orthophosphate contents gradually increased with leaf development (Fig. 5A). On day 12, the orthophosphate contents did not differ between the treatments. On the contrary, the orthophosphate content of the sugar-treated leaves were slightly lower than that of the control leaves after day 14, and the difference between the treatments gradually increased with leaf development.

Internal conductance for CO2 diffusion

Changes in the internal conductance for CO2 diffusion from the intercellular space to the Rubisco catalytic site, gi, with leaf development are shown in Fig. 5B. gi of the control leaves gradually deceased with leaf development. In contrast, gi of the sugar-treated leaves on day 12 was slightly higher than those on other days. From day 14, the gi of the sugar-treated leaves was almost constant.

Discussion

In the present study, we divide the leaf expansion into three stages. The leaves examined on day 12 were treated with sugar when they were mainly in the sink phase, and thus are called ‘sink leaves’ (see the first paragraph of the Results; see also Miyazawa et al. 2003). The leaves examined in day 14 were treated when they were in the sink–source transition and thus are called ‘transitional leaves’. The leaves examined after day 16 are called ‘source leaves’.

Although the carbohydrate contents of the sugar-treated sink leaves examined on day 12 were about twice of that in the control sink leaves (Fig. 2D), the photosynthetic rates were not affected by the sugar treatment (Fig. 3). However, the carbohydrate contents, even in the sugar-treated leaves, were lower than the levels in the transitional and source leaves. Thus, it is probable that the carbohydrate levels in the sink leaves were lower than the threshold that induces the sugar effect on photosynthesis. The low photosynthetic rates and high growth respiration rate in the sink leaves, especially before day 10 (data not shown), would keep the carbohydrate content low in the primary leaves. In separate experiments, we fed the sucrose solution at 60 or 120 mM to the pots. However, the total carbohydrate contents of such ‘sink’ primary leaves were not significantly different from that of the plant fed 20 mM sucrose solution. The feeding of higher concentrations of sucrose solution also caused shrinkage of the primary leaves. Thus, we used the 20 mM solution. In the study of Krapp et al. (1991), the sugar-fed sink leaves also contained lower amounts of carbohydrates than the sugar-fed source leaves. Except for an extreme supplement of soluble sugars, which was used in some studies (Jang and Sheen 1997), the carbohydrate would not accumulate to levels that are enough to induce the sugar repression of photosynthesis in the sink leaves.

In the sink leaves, however, glucose levels, which repress photosynthetic genes (Jang and Sheen 1997), were highest in the experimental periods. Thus, it is also probable that the glucose repression of photosynthetic genes does not occur in sink leaves.

In the transitional leaves, the soluble sugar contents were enhanced by the sugar treatment, although the starch content of the leaves did not increase markedly. The A360 of the sugar-treated transitional leaves was slightly lower than that of the control transitional leaves (Fig. 3A). The initial slope of the A–Ci curve was also decreased in the sugar-treated leaves, but Amax was not affected by the sugar treatment (Fig. 3B, C). Thus, the decrease in A360 was attributed to the decrease in the initial slope of the A–Ci curve, which reflects the RuBP carboxylation capacity of the leaf, but not to that in Amax.

The initial slope of A–Ci curves, which reflects the RuBP carboxylation capacity of Rubisco (Farquhar et al. 1980), is mainly determined by the content and the properties of Rubisco and gi. However, in the transitional leaves, the starch accumulation, which is thought to cause the decrease in gi (Nafziger and Koller 1976), was not marked. Moreover, gi of the sugar-treated transitional leaves was even slightly greater than that of the control leaves (Fig. 5B). Thus, the decrease in gi with starch accumulation did not occur in the transitional leaves.

On the other hand, the Rubisco LSU contents of the sugar-treated transitional leaves slightly decreased (Fig. 4A). Thus, the decrease in the initial slope of the A–Ci curve in the sugar-treated transitional leaves was mainly attributed to the smaller amount of Rubisco. The decrease in Rubisco content was probably due to the sugar repression of expression of Rubisco genes (Koch 1996). In rice, Rubisco protein is mainly synthesized during leaf expansion (Mae et al. 1983). Thus, the decrease in the Rubisco content in sugar-treated transitional leaves is probably caused by the decrease in de novo synthesis of Rubisco. The nitrogen content of the sugar-treated leaves also decreased slightly in the transitional stage (Table 1). This decrease in nitrogen content would be partly due to the decrease in Rubisco content (Fig. 4A). Because the Chl content, Chl a/b ratio or LHCII content were not different between the treatments (Table 1, Fig. 4B), the contents of Chl proteins probably did not respond to the sugar treatment. Thus, the decrease in nitrogen content in the sugar-treated leaves did not cause a general decrease in the protein contents.

In the source leaves, both the soluble sugar and starch content were increased by the sugar treatment (Fig. 2). These increases also affect the LMA of the sugar-treated leaves (Table 1). The A360 of the sugar-treated source leaves was lower than that of control leaves (Fig. 3A). The decrease in A360 would be due to the decrease in both the RuBP carboxylation and regeneration capacity, because both the Amax, the indicator of RuBP regeneration capacity and the initial slope of the A–Ci curve decreased in the sugar-treated leaves (Fig. 3B, C). Also, A–Ci analysis suggested that the A360 was co-limited by the RuBP carboxylation and regeneration capacities (data not shown). The Rubisco contents of the source leaves were decreased by the sugar treatment (Fig. 4A). In contrast, gi of the source leaves was not affected by the sugar treatment (Fig. 5B), although the starch accumulated markedly in the sugar-treated leaves. Thus, as was the case with the transitional leaves, the decrease in the RuBP carboxylation capacity would be the result of the sugar repression of photosynthetic genes due to the soluble sugar accumulation. The decrease in gi with starch accumulation was not apparent in this study.

Amax was decreased by the sugar treatment only in the source leaves (Fig. 3C). Also, the marked and significant starch accumulation was observed only in the source leaves (Fig. 2C). These results suggest the possibility that the starch accumulation decreases the Amax, although starch per se is solid and biochemically inactive. The relationship between the starch accumulation and the decrease in Amax is unclear, but the glucose, which affects photosynthetic gene expression through hexokinase (Jang and Sheen 1997), is generated by the starch degradation in the night. This glucose generation by starch degradation is suggested to affect the photosynthetic gene expression level (Chang et al. 1998). Also, the components of the photosynthetic electron transport chain, which affect the Amax, are more stable than Rubisco in the senesced leaves (Hidema et al. 1991, Mae et al. 1993). The stability partially explains the fact that significant decreases in Amax occur only in source leaves.

Amax is thought to be limited by the photosynthetic electron transport rate. The Chl content, Chl a/b ratio and LHCII were not influenced by the sugar treatment of the source leaves (Table 1, Fig. 4B). Thus, the Chl protein contents would be unaffected by the carbohydrate accumulation. Fv/Fm, which represents the activity of PSII, also did not change with sugar treatment. These results suggest that the decreases in the amounts or the activities of other electron transport chain components such as plastocyanin (Dijkwel et al. 1996) and H+-ATPase (Krapp et al. 1993) are caused by the carbohydrate accumulation. The gene for LHCII (cab) is known to be down-regulated by soluble sugars (Dijkwel et al. 1996), but the effect was not observed in the present study. Some studies suggested that the LHCII protein content was little affected by the mRNA level of the cab gene (Flachmann and Kühlbrandt 1995).

The orthophosphate contents of the leaves, which also affect the photosynthetic rate at saturated CO2 concentration, slightly decreased due to the sugar treatment (Fig. 5A). However, the orthophosphate content of the leaves increased with leaf development. Thus, the orthophosphate would not be depleted, at least in source leaves, and the low level of orthophosphate content in the sugar-treated source leaves would not totally explain the decrease in Amax. In addition, because the orthophosphate is released in starch synthesis (Stitt et al. 1990), the extreme starch accumulation due to the sugar treatment (Fig. 2C) might cause considerable release of orthophosphate. Therefore, it is most likely that the orthophosphate content would not be depleted in the chloroplasts in the present experiment.

In the present study, we demonstrated that the sucrose supply to the bean roots influenced the photosynthesis in the source leaves, but not in the sink leaves. It is most probable that the accumulation of soluble sugars in the sugar-treated leaves at the transitional and source stage caused the decrease in Rubisco content of the leaves.

Materials and Methods

Plant material

Seeds of P. vulgaris L. cv. Yamashiro-Kurosando were imbibed on wet paper for 4 d before sowing. The first day when the seeds were imbibed was called day 0. On day 4, germinated seedlings were planted in vermiculite in 12.7 cm diameter pots (six seedlings per pot) and watered every day. On day 10, five of them were thinned. The plants were grown in a light-controlled room with fluorescent light (FPL36EX-W, Matsushita Electric Industrial Co. Ltd, Kadoma, Japan) at a PPFD of 300 µmol m–2 s–1 during the light period of 14 h (6:00–20:00 h), a relative humidity of 50–70% and an air temperature of 25°C. From day 7, the plants were fertilized every day with 50 ml of half-strength Hoagland solution containing 2 mM KNO3, 2 mM Ca(NO3)2, 0.75 mM MgSO4, 0.665 mM NaHPO4, 25 µM Fe-EDTA, 5 µM ZnSO4, 0.5 µM CuSO4, 0.25 µM NaMoO4, 50 µM NaCl and 0.1 µM CoSO4.

Sugar treatment

To increase the carbohydrate content of the primary leaf, we supplied 50 ml of 20 mM sucrose solution to the bean roots every day for 5 d from days 7, 9, 11, 13 and 15 (Fig. 1). Sucrose solution was supplied between 15:00 and 17:00 h. Water and the nutrient solution were given at the same time. We call this treatment ‘sugar treatment’. Bean plants that were supplied with only water and the nutrient solution were used as controls. After the sugar treatment for 5 d, we measured various photosynthetic properties of the primary leaves: the measurements were thus conducted on days 12, 14, 16, 18 and 20.

Measurement of leaf nitrogen and carbon content

Nitrogen and carbon contents of dried leaf discs were analyzed with an NC analyzer (vario EL III, Elementar Analysensysteme GmbH, Hanau, Germany).

Measurements of photosynthesis

Rates of photosynthesis were measured with a portable infrared CO2 gas analyzer (LI-6400, Li-Cor, Lincoln, NE, USA). The primary leaf was enclosed in the assimilation chamber in which the Ca was kept at 360 µmol mol–1. The leaf temperature was 25°C. The leaf was illuminated at a PPFD of 1,000 µmol m–2 s–1 for 20 min, and the rates of photosynthesis were measured at various Cas ranging from 50 to 1,500 µmol mol–1. The Ci was calculated according to von Caemmerer and Farquhar (1981). The slope of the regression line of the A–Ci curve for Ci below 150 µ mol mol–1 was calculated by the least squares method and was regarded as the initial slope of the A–Ci curve.

Chlorophyll fluorescence and internal conductance for CO2 diffusion (gi)

Chl fluorescence was measured with a pulse amplitude modulation fluorometer (PAM-101, Waltz, Effeltrich, Germany). The leaf was enclosed in the assimilation chamber of the LI-6400 at an air temperature of 25°C and a Ca of 360 µmol mol–1. After keeping the leaf in the dark for 20 min, the Fv/Fm was measured.

We calculated the gi according to the method of Terashima and Ono (2002). We measured the A–Ci curve and Chl fluorescence at a PPFD of 500 µmol m–2 s–1. Leaf absorbance was assumed to be 85%.

After the measurements of the photosynthetic rate and Chl fluorescence, leaf discs of 1 cm in diameter were sampled and quickly frozen in liquid N2 at 17:00–20:00 h for the subsequent analyses.

Measurement of chlorophyll content

The frozen leaf disc was well ground with a mortar and a pestle in liquid N2. The Chl was extracted with 80% acetone and centrifuged at 15,000×g. The absorbance of the supernatant was measured according to Porra et al. (1989).

Non-structural carbohydrate contents

Contents of glucose, sucrose and starch in the leaf were measured as described by Ono et al. (1996). Frozen leaf discs (1.57 cm2) were ground in liquid N2 to powder, and carbohydrates were extracted with 80% ethanol. The suspension was incubated at 80°C for 1 h and centrifuged at 15,000×g for 10 min. The precipitations of these extracts were used for the estimation of starch. The supernatant was evaporated to remove ethanol with a centrifugal concentrator (CC-105, Tomy Seiko, Tokyo, Japan). The same volumes of distilled water and chloroform were added to the concentrated supernatant and mixed well. The mixture was centrifuged at 15,000×g and the upper clear phase was used for the estimation of glucose and sucrose.

Orthophosphate content

The orthophosphate concentration was measured as described by Sawada et al. (1992) and Saheki et al. (1985). The frozen leaf disc (0.785 cm2) was ground in liquid N2 and suspended with 1 ml of 1% perchloric acid. The extract was centrifuged at 750×g and the supernatant was diluted 10 times with 1% perchloric acid. A 400 µl of this diluted extract was mixed with 400 µl of molybdate reagent containing 15 mM ammonium molybdate, 100 mM zinc acetate (pH 5.0), 0.25% (w/v) SDS and 2.5% (w/v) ascorbic acid (pH 5.0). After incubation for 15 min at 30°C, A850 was measured. KH2PO4 solution was used as the standard.

Contents of LSU and LHCII

The frozen leaf discs (2.355 cm2) were ground in liquid N2. The soluble protein was extracted with 300 µl of extraction buffer containing 100 mM Na-phosphate buffer (pH 7.5), 50 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), one tablet/50 ml complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), 1% (w/v) polyvinylpyrrolidone, 0.7% (w/v) polyethylene glycol and 5% (w/v) Triton X-100. The extract was centrifuged at 750×g and the supernatant was used for analyses by SDS–PAGE.

LSU and LHCII contents were measured according to Makino et al. (1986) with some modifications. The gel was stained with Coomassie brilliant blue R-250. The 47 kDa band of LSU and the 27 kDa band of LHCII were cut out, and the Coomassie brilliant blue R-250 of these blocks was eluted with formamide at 55°C for 5 h. The A595 of these elutes were measured. Bovine serum albumin was used as the standard.

Statistical analyses

The difference between the treatments was analyzed with Student’s t-test.

Acknowledgments

We thank Drs. T. Kakimoto, K. Ono and S.-I. Miyazawa for endless helpful advice. We also thank Dr. A. Makino for providing primary antibody against LSU.

Fig. 1 Schedule of sugar treatment. Day 0 was the first day when the bean seeds were imbibed. The bean plants were planted at day 4 and watered every day. Six plants were planted in one pot, and they were thinned at day 10. A 50 ml of half-strength Hoagland solution was supplied every day from day 7. The black box shows the periods when 50 ml of 20 mM sucrose solution was supplied to the bean roots. Day 10, which is drafted with a solid line, is the sink–source transition point forecasted from Miyazawa et al. (2003) and the daily photosynthetic rate.

Fig. 1 Schedule of sugar treatment. Day 0 was the first day when the bean seeds were imbibed. The bean plants were planted at day 4 and watered every day. Six plants were planted in one pot, and they were thinned at day 10. A 50 ml of half-strength Hoagland solution was supplied every day from day 7. The black box shows the periods when 50 ml of 20 mM sucrose solution was supplied to the bean roots. Day 10, which is drafted with a solid line, is the sink–source transition point forecasted from Miyazawa et al. (2003) and the daily photosynthetic rate.

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Fig. 2 Changes in carbohydrate contents on a leaf area basis in the primary leaves of bean. (A) Glucose, (B) sucrose, (C) starch and (D) total carbohydrate. Total carbohydrate is the sum of the glucose, sucrose and starch contents. Filled circles, control leaves; open circles, sugar-treated leaves. Error bars denote the SE (n = 3–6). * and ** indicate a significant difference at P < 0.05 and P < 0.01, respectively, with the Student’s t-test.

Fig. 2 Changes in carbohydrate contents on a leaf area basis in the primary leaves of bean. (A) Glucose, (B) sucrose, (C) starch and (D) total carbohydrate. Total carbohydrate is the sum of the glucose, sucrose and starch contents. Filled circles, control leaves; open circles, sugar-treated leaves. Error bars denote the SE (n = 3–6). * and ** indicate a significant difference at P < 0.05 and P < 0.01, respectively, with the Student’s t-test.

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Fig. 3 Changes in the photosynthetic rate on a leaf area basis in the primary leaves of bean. (A) Photosynthetic rate at a PPFD of 1,000 µmol m–2 s–1 and CO2 concentration of 360 µmol mol–1 (A360). (B) A slope of the regression line of the A–Ci curve for the range of Ci below 150 µmol mol–1 measured at a PPFD of 1,000 µmol m–2 s–1. (C) Photosynthetic rate at a PPFD of 1,000 µmol m–2 s–1 and CO2 concentration of 1,500 µmol mol–1 (Amax). Filled circles, control leaves; open circles, sugar-treated leaves. Error bars denote the SE (n = 3–6). * , ** and *** indicate significant differences at P < 0.05, P < 0.01 and P < 0.001, respectively, with the Student’s t-test.

Fig. 3 Changes in the photosynthetic rate on a leaf area basis in the primary leaves of bean. (A) Photosynthetic rate at a PPFD of 1,000 µmol m–2 s–1 and CO2 concentration of 360 µmol mol–1 (A360). (B) A slope of the regression line of the A–Ci curve for the range of Ci below 150 µmol mol–1 measured at a PPFD of 1,000 µmol m–2 s–1. (C) Photosynthetic rate at a PPFD of 1,000 µmol m–2 s–1 and CO2 concentration of 1,500 µmol mol–1 (Amax). Filled circles, control leaves; open circles, sugar-treated leaves. Error bars denote the SE (n = 3–6). * , ** and *** indicate significant differences at P < 0.05, P < 0.01 and P < 0.001, respectively, with the Student’s t-test.

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Fig. 4 Changes in Rubisco large subunit content (A) and light-harvesting chl binding protein of PSII content (B) of the primary leaves of bean. These contents are shown on a leaf area basis. Filled circles, control leaves; open circles, sugar-treated leaves. Error bars denote the SE (n = 3–6). * indicates a significant difference at P < 0.05 with the Student’s t-test.

Fig. 4 Changes in Rubisco large subunit content (A) and light-harvesting chl binding protein of PSII content (B) of the primary leaves of bean. These contents are shown on a leaf area basis. Filled circles, control leaves; open circles, sugar-treated leaves. Error bars denote the SE (n = 3–6). * indicates a significant difference at P < 0.05 with the Student’s t-test.

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Fig. 5 Changes in the orthophosphate content (A) and internal conductance (gi; B) on a leaf area basis of the leaves. Filled circles, control leaves; open circles, sugar-treated leaves. Error bars denote the SE [n = 3–6 except for gi of the control leaves at day 20 (n = 2)]. ** indicates a significant difference at P < 0.01 with the Student’s t-test.

Fig. 5 Changes in the orthophosphate content (A) and internal conductance (gi; B) on a leaf area basis of the leaves. Filled circles, control leaves; open circles, sugar-treated leaves. Error bars denote the SE [n = 3–6 except for gi of the control leaves at day 20 (n = 2)]. ** indicates a significant difference at P < 0.01 with the Student’s t-test.

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Table 1
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LMA, nitrogen content per leaf area, nitrogen/carbon (N/C) ratio, Chl content, Chl a/b ratio and Fv/Fm of the primary leaves of bean

1214161820
LMA (g m–2)Control24.0 ± 1.5724.3 ± 1.4524.3 ± 1.13**28.9 ± 1.6325.8 ± 3.12
Suc26.5 ± 0.6525.5 ± 1.9632.1 ± 1.7433.9 ± 2.5930.8 ± 2.88
Nitrogen content (g m–2)Control1.74 ± 0.111.34 ± 0.091.23 ± 0.081.17 ± 0.03**1.35 ± 0.09
Suc1.68 ± 0.081.23 ± 0.131.07 ± 0.090.90 ± 0.050.89 ± 0.13
N/C ratioControl0.150 ± 0.0060.121 ± 0.0110.116 ± 0.011*0.097 ± 0.006*0.125 ± 0.010**
Suc0.131 ± 0.0060.107 ± 0.0170.076 ± 0.0100.061 ± 0.0080.065 ± 0.004
Chl a + b (mmol m–2)Control0.45 ± 0.010.38 ± 0.040.33 ± 0.020.30 ± 0.010.23 ± 0.01
Suc0.44 ± 0.030.40 ± 0.010.34 ± 0.020.34 ± 0.030.21 ± 0.02
Chl a/b (mol mol–1)Control3.29 ± 0.083.55 ± 0.083.41 ± 0.033.47 ± 0.013.22 ± 0.05*
Suc3.45 ± 0.053.49 ± 0.043.36 ± 0.063.50 ± 0.093.34 ± 0.02
Fv/FmControl0.66 ± 0.010.73 ± 0.020.68 ± 0.020.71 ± 0.020.70 ± 0.03
Suc0.74 ± 0.020.77 ± 0.010.72 ± 0.010.70 ± 0.060.72 ± 0.02
1214161820
LMA (g m–2)Control24.0 ± 1.5724.3 ± 1.4524.3 ± 1.13**28.9 ± 1.6325.8 ± 3.12
Suc26.5 ± 0.6525.5 ± 1.9632.1 ± 1.7433.9 ± 2.5930.8 ± 2.88
Nitrogen content (g m–2)Control1.74 ± 0.111.34 ± 0.091.23 ± 0.081.17 ± 0.03**1.35 ± 0.09
Suc1.68 ± 0.081.23 ± 0.131.07 ± 0.090.90 ± 0.050.89 ± 0.13
N/C ratioControl0.150 ± 0.0060.121 ± 0.0110.116 ± 0.011*0.097 ± 0.006*0.125 ± 0.010**
Suc0.131 ± 0.0060.107 ± 0.0170.076 ± 0.0100.061 ± 0.0080.065 ± 0.004
Chl a + b (mmol m–2)Control0.45 ± 0.010.38 ± 0.040.33 ± 0.020.30 ± 0.010.23 ± 0.01
Suc0.44 ± 0.030.40 ± 0.010.34 ± 0.020.34 ± 0.030.21 ± 0.02
Chl a/b (mol mol–1)Control3.29 ± 0.083.55 ± 0.083.41 ± 0.033.47 ± 0.013.22 ± 0.05*
Suc3.45 ± 0.053.49 ± 0.043.36 ± 0.063.50 ± 0.093.34 ± 0.02
Fv/FmControl0.66 ± 0.010.73 ± 0.020.68 ± 0.020.71 ± 0.020.70 ± 0.03
Suc0.74 ± 0.020.77 ± 0.010.72 ± 0.010.70 ± 0.060.72 ± 0.02

Control and suc denote control and sugar-treated leaves, respectively. Means ± SE, n = 3–6.

* and ** indicate the statistical significance at P < 0.05 and P < 0.01, respectively, with Student’s t-test in each developmental stage.

Table 1
Open in new tab

LMA, nitrogen content per leaf area, nitrogen/carbon (N/C) ratio, Chl content, Chl a/b ratio and Fv/Fm of the primary leaves of bean

1214161820
LMA (g m–2)Control24.0 ± 1.5724.3 ± 1.4524.3 ± 1.13**28.9 ± 1.6325.8 ± 3.12
Suc26.5 ± 0.6525.5 ± 1.9632.1 ± 1.7433.9 ± 2.5930.8 ± 2.88
Nitrogen content (g m–2)Control1.74 ± 0.111.34 ± 0.091.23 ± 0.081.17 ± 0.03**1.35 ± 0.09
Suc1.68 ± 0.081.23 ± 0.131.07 ± 0.090.90 ± 0.050.89 ± 0.13
N/C ratioControl0.150 ± 0.0060.121 ± 0.0110.116 ± 0.011*0.097 ± 0.006*0.125 ± 0.010**
Suc0.131 ± 0.0060.107 ± 0.0170.076 ± 0.0100.061 ± 0.0080.065 ± 0.004
Chl a + b (mmol m–2)Control0.45 ± 0.010.38 ± 0.040.33 ± 0.020.30 ± 0.010.23 ± 0.01
Suc0.44 ± 0.030.40 ± 0.010.34 ± 0.020.34 ± 0.030.21 ± 0.02
Chl a/b (mol mol–1)Control3.29 ± 0.083.55 ± 0.083.41 ± 0.033.47 ± 0.013.22 ± 0.05*
Suc3.45 ± 0.053.49 ± 0.043.36 ± 0.063.50 ± 0.093.34 ± 0.02
Fv/FmControl0.66 ± 0.010.73 ± 0.020.68 ± 0.020.71 ± 0.020.70 ± 0.03
Suc0.74 ± 0.020.77 ± 0.010.72 ± 0.010.70 ± 0.060.72 ± 0.02
1214161820
LMA (g m–2)Control24.0 ± 1.5724.3 ± 1.4524.3 ± 1.13**28.9 ± 1.6325.8 ± 3.12
Suc26.5 ± 0.6525.5 ± 1.9632.1 ± 1.7433.9 ± 2.5930.8 ± 2.88
Nitrogen content (g m–2)Control1.74 ± 0.111.34 ± 0.091.23 ± 0.081.17 ± 0.03**1.35 ± 0.09
Suc1.68 ± 0.081.23 ± 0.131.07 ± 0.090.90 ± 0.050.89 ± 0.13
N/C ratioControl0.150 ± 0.0060.121 ± 0.0110.116 ± 0.011*0.097 ± 0.006*0.125 ± 0.010**
Suc0.131 ± 0.0060.107 ± 0.0170.076 ± 0.0100.061 ± 0.0080.065 ± 0.004
Chl a + b (mmol m–2)Control0.45 ± 0.010.38 ± 0.040.33 ± 0.020.30 ± 0.010.23 ± 0.01
Suc0.44 ± 0.030.40 ± 0.010.34 ± 0.020.34 ± 0.030.21 ± 0.02
Chl a/b (mol mol–1)Control3.29 ± 0.083.55 ± 0.083.41 ± 0.033.47 ± 0.013.22 ± 0.05*
Suc3.45 ± 0.053.49 ± 0.043.36 ± 0.063.50 ± 0.093.34 ± 0.02
Fv/FmControl0.66 ± 0.010.73 ± 0.020.68 ± 0.020.71 ± 0.020.70 ± 0.03
Suc0.74 ± 0.020.77 ± 0.010.72 ± 0.010.70 ± 0.060.72 ± 0.02

Control and suc denote control and sugar-treated leaves, respectively. Means ± SE, n = 3–6.

* and ** indicate the statistical significance at P < 0.05 and P < 0.01, respectively, with Student’s t-test in each developmental stage.

Abbreviations

     
  • A360

    photosynthetic rate at a PPFD of 1,000 µmol m–2 s–1 and CO2 concentration of 360 µmol mol–1

    •  
  • Amax

    photosynthetic rate at a PPFD of 1,000 µmol m–2 s–1 and CO2 concentration of 1,500 µmol mol–1

    •  
  • Ca

    ambient CO2 concentration

    •  
  • Ci

    CO2 concentration in the intercellular space

    •  
  • Fv/Fm

    maximum quantum yield of PSII

    •  
  • gi

    CO2 conductance between the intercellular space and the catalytic site of Rubisco

    •  
  • LHCII

    light-harvesting chlorophyll binding protein of PSII

    •  
  • LMA

    leaf mass per area

    •  
  • LSU

    large subunit of Rubisco

    •  
  • PPFD

    photosynthetic photon flux density

    •  
  • Rubisco

    ribulose-1,5-bisphosphate carboxylase/oxygenase

    •  
  • RuBP

    ribulose-1,5-bisphosphate

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