**Regulation of Leaf Photosynthesis Through Photosynthetic Source-Sink Balance in Soybean Plants**

Minobu Kasai

*Department of Biology, Faculty of Agriculture and Life Science, Hirosaki University Japan* 

#### **1. Introduction**

442 Soybean Physiology and Biochemistry

Zhao, L.; Zhang, F.; Guo, J.; Yang, Y.; Li, B. & Zhang, L. (2004). Nitric oxide functions as a

*communis Trin*.). *Plant Physiology,* Vol.134, pp.849–857. ISSN 0032-0889. Zilli, C.; Santa-Cruz, D.; Yannarelli, G.; Noriega, G.; Tomaro, M. & Balestrasse, K. (2009).

PMCID: PMC2809017.

signal in salt resistance in the calluses from two ecotypes of reed (*Phragmites* 

Heme Oxygenase Contributes to Alleviate Salinity Damage in *Glycine max* L. Leaves, *International Journal of Cell Biology.* Published online (September 2009),

> Plant photosynthesis is the basis for matter production needed for all living organisms. In the future, plant photosynthesis would be more important, since environmental problems such as climatic warming due to increasing environmental CO2 concentration and problems of food and energy shortages due to increasing populations may be severer (von Caemmerer & Evans, 2010; Raines, 2011). Increasing plant leaf photosynthesis and thereby increasing plant matter production would be expected as a realistic way to resolve the problems. There is, however, a well-known hypothesis that in plants leaf photosynthesis can be down regulated through accumulated photosynthetic carbohydrates in leaf under excessive photosynthetic source capacity, which also means sink limitation, although the detailed mechanism is not clear (see Kasai, 2008). Actually, for example, there is evidence for the excessive photosynthetic source capacity causing down regulation of photosynthesis in crop plants under field conditions (Okita et al., 2001; Smidansky et al., 2002, 2007). Therefore, for the better improvement of leaf photosynthesis in plants, it is important to elucidate the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity and thereby clarify way of the improvement of leaf photosynthesis.

> To elucidate the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity, experimental construction of the excessive photosynthetic source capacity is important. Excising sink organs such as pods, fruits or flowers from plant materials is a way to construct excessive photosynthetic source capacity, and it has often been conducted to study the regulatory mechanism of photosynthetic source-sink balance in plants (see Kasai, 2008). However, the way excising sink organs results not directly but indirectly in excessive photosynthetic source capacity by diminishing sink capacity, and can give some damages to plant materials. 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 significantly (Raines, 2003, 2006)**.** Therefore, the use of the transgenic plants with improved higher leaf photosynthetic rate may be useful to study the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity, since the higher photosynthetic rate is likely to result in excessive photosynthetic source capacity. However, it seems difficult to analyze the down regulation of

photosynthesis that may hide in the improved photosynthetic rate. Actually, down regulation of photosynthesis that is associated with excessive photosynthetic source capacity has not been analyzed in the transgenic plants with improved higher photosynthetic rate. Exposure to high CO2 or continuous exposure to light of plant materials is thought as the other way to construct excessive photosynthetic source capacity. It is well known that leaf photosynthetic rate, especially, in C3 plants does not reach the saturation at the present atmospheric CO2 concentration and thus the rate increases initially under high CO2 conditions (Ward et al., 1999). Therefore, in C3 plants, exposure to high CO2 is expected to result in excessive photosynthetic source capacity. However, the way of exposure to high CO2 may be not suitable to analyze the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity, because of the same reason described for the transgenic plants with improved photosynthetic rate and well-known action of high CO2 to decrease stomatal aperture (Bredmose & Nielsen, 2009). In contrast, continuous exposure to light of plant materials, which prolongs photosynthetic period, can result in excessive photosynthetic source capacity without affecting directly the sink organs, leaf photosynthetic rate and stomatal aperture and giving direct damage to the plant materials. Soybean plants, although it is single-rooted soybean leaves, have largely contributed to study the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity through the experimental system using continuous exposure to light. Singlerooted soybean leaves are source-sink model plants with a simple organization of a leaf, a short petiole and roots developed from the petiole in individuals and were developed by Sawada et al. (1986) using the primary leaves of intact soybean plants (*Glycine max* L. Merr. cv. Tsurunoko). Studies using single-rooted soybean leaves have shown that treating the plants with continuous light results in accumulation of photosynthetic carbohydrates (sucrose and starch) in the leaf and decrease in the leaf photosynthetic rate, which correlates with the increase in leaf carbohydrate (sucrose or starch) content (Sawada et al., 1986, 1989, 1990, 1992). Also, it has been shown in the single-rooted soybean leaves that deactivation of Rubisco, a CO2-fixing enzyme is caused by the treatment of continuous exposure to light (Sawada et al., 1990, 1992). As continuous exposure to light of single-rooted soybean leaves also increased the leaf phosphorylated intermediates' contents (Sawada et al., 1989), and there have been findings that in vitro, inorganic phosphate promotes activation of Rubisco by enhancing the affinity of uncarbamylated inactive Rubisco to CO2 (Bhagwat, 1981; McCurry et al., 1981; Anwaruzzaman et al., 1995), the studies using single-rooted soybean leaves have suggested 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, and that the deactivation of Rubisco may be caused by limitation of inorganic phosphate (Sawada et al., 1990, 1992). Data from a study using single-rooted soybean leaves demonstrate that the plants do not change the leaf area and leaf dry weight other than the weights of major photosynthetic carbohydrates (sucrose and starch) and grow only the roots during experimental period, irrespective of whether light conditions are normal (daily light/dark periods of 10/14 h) or continuous without darkness (Sawada et al., 1986). Although the source-sink model plants with simple source-sink organization have been developed from various plant species, only the single-rooted soybean leaves have been demonstrated to show almost no growth in the source organ (Sawada et al., 2003). No growth of the source organ and the simple organization of source and sink in the single-rooted soybean leaves are

photosynthesis that may hide in the improved photosynthetic rate. Actually, down regulation of photosynthesis that is associated with excessive photosynthetic source capacity has not been analyzed in the transgenic plants with improved higher photosynthetic rate. Exposure to high CO2 or continuous exposure to light of plant materials is thought as the other way to construct excessive photosynthetic source capacity. It is well known that leaf photosynthetic rate, especially, in C3 plants does not reach the saturation at the present atmospheric CO2 concentration and thus the rate increases initially under high CO2 conditions (Ward et al., 1999). Therefore, in C3 plants, exposure to high CO2 is expected to result in excessive photosynthetic source capacity. However, the way of exposure to high CO2 may be not suitable to analyze the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity, because of the same reason described for the transgenic plants with improved photosynthetic rate and well-known action of high CO2 to decrease stomatal aperture (Bredmose & Nielsen, 2009). In contrast, continuous exposure to light of plant materials, which prolongs photosynthetic period, can result in excessive photosynthetic source capacity without affecting directly the sink organs, leaf photosynthetic rate and stomatal aperture and giving direct damage to the plant materials. Soybean plants, although it is single-rooted soybean leaves, have largely contributed to study the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity through the experimental system using continuous exposure to light. Singlerooted soybean leaves are source-sink model plants with a simple organization of a leaf, a short petiole and roots developed from the petiole in individuals and were developed by Sawada et al. (1986) using the primary leaves of intact soybean plants (*Glycine max* L. Merr. cv. Tsurunoko). Studies using single-rooted soybean leaves have shown that treating the plants with continuous light results in accumulation of photosynthetic carbohydrates (sucrose and starch) in the leaf and decrease in the leaf photosynthetic rate, which correlates with the increase in leaf carbohydrate (sucrose or starch) content (Sawada et al., 1986, 1989, 1990, 1992). Also, it has been shown in the single-rooted soybean leaves that deactivation of Rubisco, a CO2-fixing enzyme is caused by the treatment of continuous exposure to light (Sawada et al., 1990, 1992). As continuous exposure to light of single-rooted soybean leaves also increased the leaf phosphorylated intermediates' contents (Sawada et al., 1989), and there have been findings that in vitro, inorganic phosphate promotes activation of Rubisco by enhancing the affinity of uncarbamylated inactive Rubisco to CO2 (Bhagwat, 1981; McCurry et al., 1981; Anwaruzzaman et al., 1995), the studies using single-rooted soybean leaves have suggested 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, and that the deactivation of Rubisco may be caused by limitation of inorganic phosphate (Sawada et al., 1990, 1992). Data from a study using single-rooted soybean leaves demonstrate that the plants do not change the leaf area and leaf dry weight other than the weights of major photosynthetic carbohydrates (sucrose and starch) and grow only the roots during experimental period, irrespective of whether light conditions are normal (daily light/dark periods of 10/14 h) or continuous without darkness (Sawada et al., 1986). Although the source-sink model plants with simple source-sink organization have been developed from various plant species, only the single-rooted soybean leaves have been demonstrated to show almost no growth in the source organ (Sawada et al., 2003). No growth of the source organ and the simple organization of source and sink in the single-rooted soybean leaves are attractive characteristics to analyze comprehensively the regulatory mechanism of photosynthetic source-sink balance in plants, including the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity. Actually, as mentioned above, various analyses have been conducted in the single-rooted soybean leaves, especially in studies for elucidating the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity. Therefore, the single-rooted soybean leaves are important plant materials to elucidate further the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity. However, the plants are made artificially, and do not exist in nature, and in addition, as already mentioned, the plant leaf originates from only the primary leaf in intact soybean plants (Sawada et al., 1986). Therefore, 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. Thus, it is important to examine the regulatory mechanism for leaf photosynthesis under excessive photosynthetic source capacity using the original, intact soybean plants.

The present study used the original intact soybean plants, and it was analyzed how continuous exposure to light affects the leaf photosynthetic rate and related characteristics, such as leaf stomatal conductance and intercellular CO2 concentration, contents of water, chlorophyll, major photosynthetic carbohydrates (sucrose and starch), total protein and Rubisco protein in leaf, and activity and activation ratio (ratio of initial to total activity) of Rubisco and amount of protein-bound ribulose-1,5-bisphosphate (RuBP) in leaf extract, which were analyzed to evaluate the amount of uncarbamylated inactive Rubisco (Brooks & Portis, 1988). The same series of analyses have not been conducted together in studies that have performed the experiment of continuous exposure to light using plants.
