**4. Discussion**

The present study was conducted to examine the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity in intact soybean plants. The experimental construction of excessive photosynthetic source capacity was conducted by treating the plants with continuous light for 3 days. The data show that the treatment of continuous exposure to light for intact soybean plants decreased significantly the leaf photosynthetic rate (Fig. 1). Since the light treatment also decreased the leaf stomatal conductance in soybean plants (see Fig. 2), it is thought that the decrease in leaf photosynthetic rate caused by treatment of continuous exposure to light might have resulted from stomatal limitation of CO2 diffusion. However, the treatment of continuous exposure to light did not affect significantly leaf intercellular CO2 concentration (see Fig. 2), implicating that the light treatment decreased CO2 incorporation by leaf photosynthetic cells, as it affected leaf stomatal conductance. In addition, the light treatment decreased activation ratio of Rubisco in leaf extract and did not affect significantly leaf Rubisco content (see Fig. 3 and 6). Furthermore, the light treatment increased the amount of protein-bound RuBP in leaf extract (see Fig. 4). The decrease in activation ratio of Rubisco and increase in the amount of protein-bound RuBP in leaf extract (Brooks & Portis, 1988) strongly suggest an increase in the amount of uncarbamylated inactive Rubisco in leaf. Therefore, it is suggested that the decrease in leaf photosynthetic rate caused by treatment of continuous exposure to light is likely to be due to deactivation of Rubisco in leaf. Treatment of continuous exposure to light for intact soybean plants also increased significantly both the contents of sucrose and starch, which are the major photosynthetic carbohydrates, in leaf (see Fig. 5), indicating that the light treatment could result in an excessive photosynthetic source capacity in the plants. The present study also shows that analyzed leaf chlorophyll, total protein and water contents were not affected significantly by the treatment of continuous exposure to light (see Fig. 6 and 7). Therefore, results obtained in the present study strongly suggest that the decrease in leaf photosynthetic rate in intact soybean plants caused by treatment of continuous exposure to light is unlikely to be due to simple damages such as the breakdown of cellular compartments, but is likely to be due to deactivation of Rubisco, which is associated with accumulation of photosynthetic carbohydrates (sucrose and starch) in leaf under excessive photosynthetic source capacity.

As described in the Introduction, single-rooted soybean leaves have quite been helpful to study the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity, since the plants have simple source-sink organization and have excellent characteristics [growing only the sink organs (roots) without growing source organ (leaf)], which have not been found in other plants (Sawada et al., 1986, 2003). However, as already mentioned, as the plant leaf is constituted from only the primary leaf in intact soybean plants, there is the possibility that properties of single-rooted soybean leaves may not reflect those of the original intact soybean plants or the other intact plants. However, results obtained in the present study of the changes in leaf photosynthetic rate, initial activity and activation ratio of Rubisco in leaf extract, and contents of major photosynthetic carbohydrates (sucrose and starch) and chlorophyll in leaf caused by treatment of continuous exposure to light corresponded with results from studies that have performed similar experiments of continuous exposure to light using single-rooted soybean leaves (Sawada et al., 1986, 1990, 1992). Leaf intercellular CO2 concentration, amount of proteinbound RuBP in leaf extract and leaf Rubisco content have not been analyzed in the singlerooted soybean leaves. As already mentioned, the present study used the original intact soybean plants from which single-rooted soybean leaves can be made. Therefore, the correspondence of data from original intact soybean plants and those from single-rooted soybean leaves highlights that properties of single-rooted soybean leaves and those of original intact soybean plants are very similar, thus suggesting that properties of single-

continuous exposure to light for intact soybean plants decreased significantly the leaf photosynthetic rate (Fig. 1). Since the light treatment also decreased the leaf stomatal conductance in soybean plants (see Fig. 2), it is thought that the decrease in leaf photosynthetic rate caused by treatment of continuous exposure to light might have resulted from stomatal limitation of CO2 diffusion. However, the treatment of continuous exposure to light did not affect significantly leaf intercellular CO2 concentration (see Fig. 2), implicating that the light treatment decreased CO2 incorporation by leaf photosynthetic cells, as it affected leaf stomatal conductance. In addition, the light treatment decreased activation ratio of Rubisco in leaf extract and did not affect significantly leaf Rubisco content (see Fig. 3 and 6). Furthermore, the light treatment increased the amount of protein-bound RuBP in leaf extract (see Fig. 4). The decrease in activation ratio of Rubisco and increase in the amount of protein-bound RuBP in leaf extract (Brooks & Portis, 1988) strongly suggest an increase in the amount of uncarbamylated inactive Rubisco in leaf. Therefore, it is suggested that the decrease in leaf photosynthetic rate caused by treatment of continuous exposure to light is likely to be due to deactivation of Rubisco in leaf. Treatment of continuous exposure to light for intact soybean plants also increased significantly both the contents of sucrose and starch, which are the major photosynthetic carbohydrates, in leaf (see Fig. 5), indicating that the light treatment could result in an excessive photosynthetic source capacity in the plants. The present study also shows that analyzed leaf chlorophyll, total protein and water contents were not affected significantly by the treatment of continuous exposure to light (see Fig. 6 and 7). Therefore, results obtained in the present study strongly suggest that the decrease in leaf photosynthetic rate in intact soybean plants caused by treatment of continuous exposure to light is unlikely to be due to simple damages such as the breakdown of cellular compartments, but is likely to be due to deactivation of Rubisco, which is associated with accumulation of photosynthetic carbohydrates (sucrose

and starch) in leaf under excessive photosynthetic source capacity.

As described in the Introduction, single-rooted soybean leaves have quite been helpful to study the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity, since the plants have simple source-sink organization and have excellent characteristics [growing only the sink organs (roots) without growing source organ (leaf)], which have not been found in other plants (Sawada et al., 1986, 2003). However, as already mentioned, as the plant leaf is constituted from only the primary leaf in intact soybean plants, there is the possibility that properties of single-rooted soybean leaves may not reflect those of the original intact soybean plants or the other intact plants. However, results obtained in the present study of the changes in leaf photosynthetic rate, initial activity and activation ratio of Rubisco in leaf extract, and contents of major photosynthetic carbohydrates (sucrose and starch) and chlorophyll in leaf caused by treatment of continuous exposure to light corresponded with results from studies that have performed similar experiments of continuous exposure to light using single-rooted soybean leaves (Sawada et al., 1986, 1990, 1992). Leaf intercellular CO2 concentration, amount of proteinbound RuBP in leaf extract and leaf Rubisco content have not been analyzed in the singlerooted soybean leaves. As already mentioned, the present study used the original intact soybean plants from which single-rooted soybean leaves can be made. Therefore, the correspondence of data from original intact soybean plants and those from single-rooted soybean leaves highlights that properties of single-rooted soybean leaves and those of original intact soybean plants are very similar, thus suggesting that properties of singlerooted soybean leaves and those of original intact soybean plants can reflect each other. As described in the Introduction, studies using single-rooted soybean leaves have implicated that there is a regulatory mechanism of leaf photosynthetic rate through deactivation of Rubisco, which is associated with accumulation of photosynthetic carbohydrates in leaf under excessive photosynthetic source capacity (Sawada et al., 1986, 1989, 1990, 1992, 1999, 2003). Data from the present study using the original intact soybean plants have also suggested the same regulatory mechanism of leaf photosynthetic rate. Therefore, the suggested regulatory mechanism of leaf photosynthetic rate may be a common mechanism in plants. With respect to the excellent characteristic of single-rooted soybean leaves that do not change the leaf dry weight other than the weights of major photosynthetic carbohydrates (sucrose and starch) (Sawada et al., 1986), a little change (increase) of leaf (fourth trifoliate leaves) dry weight other than the weights of major photosynthetic carbohydrates (sucrose and starch) was observed by treatment of continuous exposure to light in the original intact soybean plants (see Fig. 5 and 7). Although the present study conducted various analyses to examine the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity, the same series of analyses have not been conducted together in other studies that have performed the treatment of continuous exposure to light using plants.

Treatment of continuous exposure to light for plants results, in most cases, in accumulation of photosynthetic carbohydrate(s) in leaf and decrease in leaf photosynthetic rate. However, in addition to these effects of the light treatment, there are other effects of the light treatment that are different from those indicated by the present study. In tomato, egg plant, peanut and potato, treatment of continuous exposure to light has been shown to result in leaf decolorization (Bradley & Janes, 1985; Globig et al., 1997; Murage et al., 1996, 1997; Rowell et al., 1999; Wheeler & Tibbitts, 1986; Tibbitts et al., 1990). In young leaves of potato and *Arabidopsis*, the continuous light treatment has been shown to accelerate expressions of photosynthetic genes, pigments and proteins, and subsequent declines of the expressions (Cushman et al., 1995; Stessman et al., 2002). In a study using young apple, a decrease in leaf photosynthetic rate caused by treatment of continuous exposure to light was suggested to be due to stomatal limitation of CO2 diffusion rather than a reduction of Rubisco activity, although, in the study, leaf water content, which is likely to affect stomatal aperture (Brodribb & McAdam, 2011), was not analyzed (Cheng et al., 2004). Therefore, leaf photosynthetic rate may also be regulated through changes in expressions of photosynthetic genes, pigments and proteins and through a regulation of stomata under excessive photosynthetic source capacity in plants.

Other ways, which indirectly construct excessive photosynthetic source capacity as described in the Introduction, have also been shown to result in accumulation of photosynthetic carbohydrate(s) in leaf and decrease in leaf photosynthetic rate. With respect to the cause(s) of why leaf photosynthetic rate declines under the excessive photosynthetic source capacity, for example, data from photosynthetic carbohydrate-feeding or high CO2 treatment experiments suggest that decreased expressions of photosynthetic genes, including genes for chlororphyll-related protein and Rubisco protein can be causes (Paul & Foyer, 2001; Martin et al., 2002; Paul & Pellny, 2003). However, there is also evidence from high CO2 treatment experiments using various C3 plants that decreased Rubisco activity in leaf rather than changes in leaf Rubisco content is likely to be a main cause (Sage et al., 1989). Data from experiments conducting excisions of sink organs (pods or flower buds and flowers) or petiole girdling suggest that a decrease of stomatal conductance or Rubisco activity or Rubisco content in leaf, or both decreases of Rubisco activity and Rubisco content in leaf can be responsible for the decrease in leaf photosynthetic rate under excessive photosynthetic source capacity (Mondal et al., 1978; Setter & Brun, 1980; Setter et al., 1980; Wittenbach, 1982, 1983; Xu et al., 1994; Crafts-Brandner & Egli, 1987; Cheng et al., 2008). As described in the Introduction, excising sink organs or high CO2 treatment can have side effect(s) other than inducing excessive photosynthetic source capacity. In the present study using intact soybean plants in which excessive photosynthetic source capacity was constructed by treatment of continuous exposure to light, visible damages such as leaf decolorization and wilt were not observed. Treatment of continuous exposure to light did not affect significantly leaf chlorophyll, total protein and water contents analyzed. However, as mentioned above, totally, the effects of indirectly constructed excessive photosynthetic source capacity on leaf carbohydrate status, photosynthetic rate, stomatal conductance, Rubisco activity and photosynthetic gene expressions including Rubisco gene expression are similar to those of excessive photosynthetic source capacity that is constructed by treatment of continuous exposure to light.

Regarding the detailed mechanism(s) of why leaf photosynthetic rate declines under excessive photosynthetic source capacity, recent studies using transgenic plants show that hexokinase could be involved in carbohydrate-mediated repression of photosynthetic gene expression (Jang et al., 1997; Dai et al., 1999; Moore et al., 2003). Other recent study shows that protein kinases (KIN10 and KIN11) may be involved in governing the entirety of carbohydrate metabolism, growth and development in response to carbohydrates in plants (Baena-Gonzalez et al., 2007). Data from a study investigating the effect of chilling stress on leaf photosynthetic rate suggest that H2O2, a reactive oxygen species can induce deactivation of Rubisco (Zhou et al., 2006). As described in the Introduction, inorganic phosphate has been found to promote activation of Rubisco by enhancing the affinity of uncarbamylated inactive Rubisco to CO2 (Bhagwat, 1981; McCurry et al., 1981; Anwaruzzaman et al., 1995). Data from a more recent study suggest that pH within the chloroplasts can be an important factor affecting leaf photosynthetic rate, since the study has demonstrated that pH can affect distribution of Rubisco activase within the chloroplasts by affecting binding of the enzyme to the thylakoid membranes (Chen et al., 2010). Distribution of Rubisco activase within the chloroplasts can affect activation state of Rubisco, since Rubisco activase plays a role in promoting the activation of Rubisco by dissociating RuBP from uncarbamylated inactive Rubisco (Crafts-Brandner & Salvucci, 2000), which tightly binds RuBP (Jordan & Chollet, 1983). Since ATP is needed for the catalytic action of Rubisco activase (Crafts-Brandner & Salvucci, 2000) and it is well known that ATP is needed for regeneration of RuBP, a substrate for Rubisco in Calvin cycle (see Kasai, 2008), it is evident that ATP is also an important factor affecting leaf photosynthetic rate. However, the precise mechanism of how hexokinase and protein kinases exercise regulation of photosynthetic carbohydrate metabolism including the carbohydrate-mediated repression of photosynthetic gene expression is not yet clear. In addition, effects of excessive photosynthetic source capacity on the levels of H2O2, inorganic phosphate, pH and ATP within the chloroplasts in which central photosynthesis is performed have not been analyzed in intact plants at real times under light. A main reason seems to be the lack of appropriate methods. Therefore, further researches including those following the development of new methods are important to elucidate further the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity.

Recent studies using transgenic plants have shown that overexpression of Calvin cycle enzymes (sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase) or leaf plasma membrane CO2 transport protein increases the leaf photosynthetic rate and the biomass production (Raines, 2003, 2006). Increasing plant leaf photosynthesis and thereby increasing plant matter (biomass) production seems to be an effective way to resolve the serious problems such as climatic warming and food and energy shortages. However, data obtained in the present study and those from other studies strongly suggest that excessive photosynthetic source capacity decreases the efficiency of leaf photosynthetic matter production. This means that under excessive photosynthetic source capacity, efficiency of plant matter (biomass) production decreases. There is also evidence for the excessive photosynthetic source capacity causing down regulation of photosynthesis in plants under field conditions (Okita et al., 2001; Smidansky et al., 2002, 2007). Therefore, it is strongly suggested that for the efficient improvement of plant matter (biomass) production, wellbalanced improvement of source and sink would be essential. Further studies are desired for deeper and more comprehensive understanding of the regulatory mechanism of photosynthetic source-sink balance including the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity. Soybean plants (*Glycine max* L. Merr. cv. Tsurunoko) used in the present study from which single-rooted soybean leaves can be made are one of the important experimental materials.
