**2. Materials and methods**

### **2.1. Plant materials**

Soybean (*Glycine max* L. Merr. cv. Tsurunoko) seeds were sown in plastic pots (13.5 cm in height, 8.5 cm in diameter) containing mixed vermiculite and sand (1:1 in volume) and grown in growth chambers (Koitotoron, HNL type; Koito Industries Ltd., Tokyo, Japan) un‐ der daily light/dark periods of 10/14 h, day/night temperatures of 25/17o C and relative hu‐ midity of 60 %. After 51 days, pods were all removed together with small floral organs from half of the plants, and the depodded plants were grown with the remaining plants (controls) for 3 days under same growth conditions. Nutrients were supplied twice a week with a 1000-fold diluted solution of Hyponex [6-10-5 type (N:P:K = 6:10:5); Hyponex Co., Osaka, Ja‐ pan], and tap water was supplied in sufficient amounts. Intensity of light, which was sup‐ plied with incandescent lamps, was 80 µmol photons m-2 s-1 (400-700 nm) on pots.

### **2.2. Leaf photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration**

Leaf photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 con‐ centration were determined on day 3 after pod removal in fully expanded middle trifoliate leaves at a light intensity of 800 µmol photons m-2 s-1, air flow rate of 200 ml min-1, air tem‐ perature of 25 o C, relative humidity of 60 % and CO2 concentration of 350 ppm using a port‐ able photosynthetic analyzer (Cylus-1; Koito Industries Ltd.). After measurements, leaf disks (1.79 cm2 ) were taken from the middle trifoliate leaves for the other analyses (see 2.3), as described previously (Kasai, 2008).

### **2.3. Rubisco activity, chlorophyll, protein, phosphate, sucrose and starch**

Initial and total activities of Rubisco in leaf extract were determined at 25 o C as described previously (Kasai, 2008). Leaf chlorophyll content was determined according to the method of Mackinney (1941). Leaf total protein content was determined by quantifying protein in‐ cluded in the leaf extract that had been prepared for determination of Rubisco activity by the method of Bradford (1976). Leaf inorganic phosphate content was determined according to the method of Saheki et al. (1985). Leaf sucrose and starch contents were determined as described by Sawada et al. (1995).

### **2.4. Dry weight**

mechanism(s) of regulation of photosynthetic matter production through photosynthetic source-sink balance. However, in contrast, there is also evidence suggesting that leaf photo‐ synthetic rate is not necessarily affected by accumulated photosynthetic carbohydrate(s) in leaf (Nebauer et al., 2011). Apart from the regulation of leaf photosynthesis through levels of photosynthetic carbohydrate(s), it is important to examine the mechanism(s) of regulation of photosynthetic dry matter production through photosynthetic source-sink balance by focus‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

production through photosynthetic source-sink balance (Kasai &Muto, 1990.; Schumacher et al., 1999; Li et al., 2005; Yang et al., 2007; Wang et al., 2011). However, the effect of photosyn‐ thetic source-sink balance on the activity(ies) of membrane H+ pump(s) has not been investi‐ gated. We show here experimental data of our recent study relating to this subject. We investigated in soybean plants how removal of pods, which decreases the ratio of sink to source organs, affects various characteristics related to photosynthetic dry matter produc‐ tion. Factors studied were leaf photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO2 concentration, initial and total activities of Rubisco, chlorophyll, total protein, inorganic phosphate, photosynthetic carbohydrates (sucrose and starch), and dry weights of source and sink organs. We also investigated the effect of pod removal on activi‐


in the world (Board & Kahlon, 2011; Ainsworth et al., 2012). On the basis of our experimen‐

can be important in the regulation of photosynthetic dry matter production through photo‐

Soybean (*Glycine max* L. Merr. cv. Tsurunoko) seeds were sown in plastic pots (13.5 cm in height, 8.5 cm in diameter) containing mixed vermiculite and sand (1:1 in volume) and grown in growth chambers (Koitotoron, HNL type; Koito Industries Ltd., Tokyo, Japan) un‐

midity of 60 %. After 51 days, pods were all removed together with small floral organs from half of the plants, and the depodded plants were grown with the remaining plants (controls) for 3 days under same growth conditions. Nutrients were supplied twice a week with a 1000-fold diluted solution of Hyponex [6-10-5 type (N:P:K = 6:10:5); Hyponex Co., Osaka, Ja‐ pan], and tap water was supplied in sufficient amounts. Intensity of light, which was sup‐

der daily light/dark periods of 10/14 h, day/night temperatures of 25/17o

plied with incandescent lamps, was 80 µmol photons m-2 s-1 (400-700 nm) on pots.

tal data and the other relevant information, we also consider how membrane H+

pump(s) can be important in the regulation of photosynthetic dry matter


pump(s) such


pump(s)

C and relative hu‐

Data from recent studies implicate that in plants, activity(ies) of membrane H+

pumps of the leaf plasma membrane (H+

ing on new enzyme(s) thought to be important.

as tonoplast H+

Relationships

300

ties of the H+

synthetic source-sink balance.

**2. Materials and methods**

**2.1. Plant materials**

and H+

For determination of dry weights of source (leaves) and sink organs (stems, floral organs in‐ cluding pods, and roots), organs were separated from plants on day 3 after pod removal and dried at 70o C for a week.

#### **2.5. Plasma membrane, tonoplast and H+ pump activity**

The activities of H+ pumps of leaf plasma membrane and tonoplast were determined using plasma membrane vesicles and tonoplast vesicles prepared from leaves. Plasma membrane vesicles and tonoplast vesicles were prepared from leaves (25 g in fresh weight) of plants on day 3 after pod removal essentially as described by Nouri & Komatsu (2010) and Maeshima & Yoshida (1989), respectively. For preparation of plasma membrane vesicles, leaf-homoge‐ nizing medium consisted of 0.3 M sucrose, 50 mM Tris, 8 mM EDTA (acid form), 2 mM PMSF, 4 mM DTT and 0.2 % (w/v) BSA, and its volume was 200 ml. After homogenization, the medium was filtered through four layers of gauze and the filtrate was centrifuged at 10,000 g for 20 min. After supernatant was centrifuged at 80,000 g for 40 min, the pellets were suspended with a sucrose-containing medium [0.3 M sucrose, 5 mM KH2PO4, 5 mM KCl, 0.1 mM EDTA and 0.1 mM DTT (pH 7.8)], of which volume was 10 ml. The plasma membrane vesicles were prepared from the suspension by using aqueous two-phase parti‐ tioning method. Dilution of the upper layers that had been obtained was conducted with a sorbitol-containing medium [0.25 M sorbitol, 5 mM HEPES-BTP and 0.1 mM DTT (pH 7.0)]. The final pellets of the plasma membrane vesicles after centrifugation (80,000 g, 40 min) were suspended with another sorbitol-containing medium [1 M sorbitol, 5 mM HEPES-BTP

and 0.1 mM DTT (pH 7.0)] and stored at -80 o C until uses. For preparation of tonoplast vesi‐ cles, leaf-homogenizing medium consisted of 0.25 M sorbitol, 50 mM HEPES-KOH, 5 mM EGTA, 1 mM PMSF, 2.5 mM Na2S2O5 and 1.5% (w/v) PVP (pH 7.6), and its volume was 200 ml. After homogenization, the medium was filtered through four layers of gauze and the fil‐ trate was centrifuged at 4000 g for 10 min. After supernatant was centrifuged at 80,000 g for 60 min, the pellets were suspended with a sucrose-containing medium [0.3 M sucrose, 10 mM KH2PO4, 1 mM EGTA and 2 mM DTT (pH 7.8)], of which volume was 5 ml, and 3 ml of a sorbitol-containing medium [0.25 M sorbitol, 50 mM HEPES-KOH, 1 mM EGTA and 2 mM DTT (pH 7.3)] was put on the suspension and the solution was centrifuged (120,000 g, 40 min). The resulting middle layer was diluted with the same sorbitol-containing medium. The final pellets of the tonoplast vesicles after centrifugation (150,000 g, 20 min) were sus‐ pended with another sorbitol-containing medium [0.25 M sorbitol, 5 mM HEPES-BTP, 2 mM DTT (pH 7.5)] and stored at -80o C until uses.

The activity of H+ pump, i.e., H+ -ATPase of leaf plasma membrane was determined at 30o C as vanadate-sensitive ATP-hydrolytic activity (kasai & Sawada, 1994). The activities of H+ pumps, i.e., H+ -ATPase and H+ -PPase of leaf tonoplast were determined at 30o C as nitratesensitive ATP-hydrolytic activity and Na+ -sensitive PPi-hydrolytic activity (Kasai et al., 1993; Kasai & Sawada, 1994), respectively. Reaction medium (500 µl) for the activity of plas‐ ma membrane H+ pump consisted of 50 mM HEPES-BTP (pH 7.0), 3 mM MgSO4, 3 mM ATP, 1 mM EGTA, 50 mM KCl, ± 0.1 mM Na3VO4, 0.02% (w/v) Triton X-100 and membrane vesicles (10 µg). Reaction medium for the activity of tonoplast H+ -ATPase consisted of ±50 mM HEPES-BTP (pH 7.5), 3 mM MgSO4, 3 mM ATP, 1 mM EGTA, ±50 mM KCl, ± 50 mM KNO3, 0.02% (w/v) Triton X-100 and membrane vesicles (10 µg). Reaction medium for the activity of tonoplast H+ -PPase consisted of 50 mM HEPES-BTP (pH 7.5), 5 mM MgSO4, 0.5 mM PPi, 1 mM EGTA, 50 mM KNO3, ± 50 mM NaNO3, 0.02% (w/v) Triton X-100 and mem‐ brane vesicles (10 µg). Phosphate liberated from substrate ATP or PPi was determined ac‐ cording to the method of Saheki et al. (1985).

## **3. Results**

Analyzed leaf photosynthetic rate and transpiration rate were significantly lower in depod‐ ded plants than in control plants (Fig. 1). Leaf stomatal conductance was also lower in de‐ podded plants than in control plants, while leaf intercellular CO2 concentration did not differ significantly between control and depodded plants (Fig. 2). Initial and total activities of Rubisco in leaf extract did not differ significantly between control and depodded plants (Fig. 3). Contents of chlorophyll, total protein and inorganic phosphate in leaves were all significantly higher in depodded plants than in control plants (Fig. 4). Contents of sucrose and starch in leaves did not differ significantly between control and depodded plants (Fig. 5). Activity of H+ pump (H+ -ATPase) of leaf plasma membrane and activities of H+ pumps (H+ -ATPase and H+ -PPase) of leaf tonoplast were all significantly lower in depodded plants than in control plants (Fig. 6). Dry weights of leaves, stems and roots did not differ signifi‐ cantly between control and depodded plants (Fig. 7). When the ratio of sink (stems + floral organs including pods + roots) to source organs (leaves) was calculated, those in control and depodded plants were on the average 1.25 (100%) and 0.70 (56%), respectively.

**Figure 1.** Leaf photosynthetic rate and transpiration rate of soybean plants on day 3 after pod removal. Open bar, leaf photosynthetic rate; gray bar, leaf transpiration rate. Vertical bars indicate S.D. (n=3). \**P*<0.01 (*t*-test) when compared with control plants.

**Figure 2.** Leaf stomatal conductance and intercellular CO2 concentration of soybean plants on day 3 after pod remov‐ al. Open bar, leaf stomatal conductance; gray bar, leaf intercellular CO2 concentration. Vertical bars indicate S.D. (n=3). \**P*<0.01 when compared with control plants. The leaf intercellular CO2 concentration did not differ significantly (*P*>0.05) between control and depodded plants.

### **4. Discussion**

and 0.1 mM DTT (pH 7.0)] and stored at -80 o

DTT (pH 7.5)] and stored at -80o

pump, i.e., H+


sensitive ATP-hydrolytic activity and Na+

cording to the method of Saheki et al. (1985).

pump (H+

The activity of H+

ma membrane H+

activity of tonoplast H+

**3. Results**

5). Activity of H+


(H+

pumps, i.e., H+

Relationships

302

C until uses. For preparation of tonoplast vesi‐




pump consisted of 50 mM HEPES-BTP (pH 7.0), 3 mM MgSO4, 3 mM




C

C as nitrate-

pumps


cles, leaf-homogenizing medium consisted of 0.25 M sorbitol, 50 mM HEPES-KOH, 5 mM EGTA, 1 mM PMSF, 2.5 mM Na2S2O5 and 1.5% (w/v) PVP (pH 7.6), and its volume was 200 ml. After homogenization, the medium was filtered through four layers of gauze and the fil‐ trate was centrifuged at 4000 g for 10 min. After supernatant was centrifuged at 80,000 g for 60 min, the pellets were suspended with a sucrose-containing medium [0.3 M sucrose, 10 mM KH2PO4, 1 mM EGTA and 2 mM DTT (pH 7.8)], of which volume was 5 ml, and 3 ml of a sorbitol-containing medium [0.25 M sorbitol, 50 mM HEPES-KOH, 1 mM EGTA and 2 mM DTT (pH 7.3)] was put on the suspension and the solution was centrifuged (120,000 g, 40 min). The resulting middle layer was diluted with the same sorbitol-containing medium. The final pellets of the tonoplast vesicles after centrifugation (150,000 g, 20 min) were sus‐ pended with another sorbitol-containing medium [0.25 M sorbitol, 5 mM HEPES-BTP, 2 mM

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

as vanadate-sensitive ATP-hydrolytic activity (kasai & Sawada, 1994). The activities of H+

1993; Kasai & Sawada, 1994), respectively. Reaction medium (500 µl) for the activity of plas‐

ATP, 1 mM EGTA, 50 mM KCl, ± 0.1 mM Na3VO4, 0.02% (w/v) Triton X-100 and membrane

mM HEPES-BTP (pH 7.5), 3 mM MgSO4, 3 mM ATP, 1 mM EGTA, ±50 mM KCl, ± 50 mM KNO3, 0.02% (w/v) Triton X-100 and membrane vesicles (10 µg). Reaction medium for the

mM PPi, 1 mM EGTA, 50 mM KNO3, ± 50 mM NaNO3, 0.02% (w/v) Triton X-100 and mem‐ brane vesicles (10 µg). Phosphate liberated from substrate ATP or PPi was determined ac‐

Analyzed leaf photosynthetic rate and transpiration rate were significantly lower in depod‐ ded plants than in control plants (Fig. 1). Leaf stomatal conductance was also lower in de‐ podded plants than in control plants, while leaf intercellular CO2 concentration did not differ significantly between control and depodded plants (Fig. 2). Initial and total activities of Rubisco in leaf extract did not differ significantly between control and depodded plants (Fig. 3). Contents of chlorophyll, total protein and inorganic phosphate in leaves were all significantly higher in depodded plants than in control plants (Fig. 4). Contents of sucrose and starch in leaves did not differ significantly between control and depodded plants (Fig.

than in control plants (Fig. 6). Dry weights of leaves, stems and roots did not differ signifi‐ cantly between control and depodded plants (Fig. 7). When the ratio of sink (stems + floral

C until uses.

vesicles (10 µg). Reaction medium for the activity of tonoplast H+

As described in the Introduction, it is important to examine the mechanism(s) of regulation of plant photosynthetic dry matter production through photosynthetic source-sink balance by focusing on a new enzyme(s). We focused on H+ pumping enzymes of leaf plasma mem‐ brane (H+ -ATPase) and tonoplast (H+ -ATPase and H+ -PPase), and investigated in soybean plants how removal of pods, which decreases the ratio of sink to source organs, affects vari‐

ous characteristics related to photosynthetic dry matter production and the activities of H+ pumps. Pod removal was shown to decrease largely leaf photosynthetic rate, transpiration rate and stomatal conductance without affecting significantly leaf intracellular CO2 concen‐ tration (Fig. 1 and 2). These results imply that pod removal decreased equally the rate of CO2 diffusion via leaf stomata and the rate of CO2 fixation in leaf photosynthetic cells. In plants, Rubisco is a major protein in leaves (Furbank et al., 1996; von Caemmerer et al., 2005), and there is evidence from studies altering the expressions of Rubisco or its activation enzyme, Rubisco activase, that changes in the activity or the amount of Rubisco in leaves significantly affect leaf photosynthetic rate (Furbank et al., 1996; von Caemmerer et al., 2005). There is also a report demonstrating that a rough and positive correlation exists be‐ tween leaf chlorophyll content and photosynthetic rate (Arp, 1991). Therefore, it is speculat‐ ed that the pod removal-induced decrease in leaf photosynthetic rate might have resulted from a decrease in the activity or the amount of Rubisco in the leaf or the content of leaf chlorophyll. However, data of Figure 3 and 4 indicate that pod removal did not significantly affect potential activity of Rubisco in the leaf and could not decrease the content of Rubisco or chlorophyll, suggesting that the pod removal-induced decrease in leaf photosynthetic rate did not result from either of these factors.

**Figure 3.** Initial and total activities of Rubisco in leaf extract from soybean plants on day 3 after pod removal. Open bar, initial activity; gray bar, total activity. Vertical bars indicate S.D. (n=3). Both initial and total activities did not differ significantly (*P*>0.05) between control and depodded plants.

Previously, in single-rooted soybean leaves that are the same species as we used in our pod removal study, it was demonstrated that a decrease in leaf inorganic phosphate content can result in a decrease in leaf Rubisco activity in vivo (Sawada et al., 1990, 1992). In vitro, inor‐ ganic phosphate has been found to promote the binding of activator CO2 to uncarbamylated inactive Rubisco (Bhagwat, 1981; McCurry et al., 1981; Anwaruzzaman et al., 1995). Data of Figure 4 indicate that pod removal did not decrease leaf inorganic phosphate content. This result indicates that the pod removal-induced decrease in leaf photosynthetic rate did not result from a decrease in leaf inorganic phosphate content.

Interaction of Photosynthetic Source-Sink Balance and Activities of Membrane H+ Pumps in Soybean http://dx.doi.org/10.5772/50544 305

ous characteristics related to photosynthetic dry matter production and the activities of H+ pumps. Pod removal was shown to decrease largely leaf photosynthetic rate, transpiration rate and stomatal conductance without affecting significantly leaf intracellular CO2 concen‐ tration (Fig. 1 and 2). These results imply that pod removal decreased equally the rate of CO2 diffusion via leaf stomata and the rate of CO2 fixation in leaf photosynthetic cells. In plants, Rubisco is a major protein in leaves (Furbank et al., 1996; von Caemmerer et al., 2005), and there is evidence from studies altering the expressions of Rubisco or its activation enzyme, Rubisco activase, that changes in the activity or the amount of Rubisco in leaves significantly affect leaf photosynthetic rate (Furbank et al., 1996; von Caemmerer et al., 2005). There is also a report demonstrating that a rough and positive correlation exists be‐ tween leaf chlorophyll content and photosynthetic rate (Arp, 1991). Therefore, it is speculat‐ ed that the pod removal-induced decrease in leaf photosynthetic rate might have resulted from a decrease in the activity or the amount of Rubisco in the leaf or the content of leaf chlorophyll. However, data of Figure 3 and 4 indicate that pod removal did not significantly affect potential activity of Rubisco in the leaf and could not decrease the content of Rubisco or chlorophyll, suggesting that the pod removal-induced decrease in leaf photosynthetic

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

**Figure 3.** Initial and total activities of Rubisco in leaf extract from soybean plants on day 3 after pod removal. Open bar, initial activity; gray bar, total activity. Vertical bars indicate S.D. (n=3). Both initial and total activities did not differ

Previously, in single-rooted soybean leaves that are the same species as we used in our pod removal study, it was demonstrated that a decrease in leaf inorganic phosphate content can result in a decrease in leaf Rubisco activity in vivo (Sawada et al., 1990, 1992). In vitro, inor‐ ganic phosphate has been found to promote the binding of activator CO2 to uncarbamylated inactive Rubisco (Bhagwat, 1981; McCurry et al., 1981; Anwaruzzaman et al., 1995). Data of Figure 4 indicate that pod removal did not decrease leaf inorganic phosphate content. This result indicates that the pod removal-induced decrease in leaf photosynthetic rate did not

rate did not result from either of these factors.

Relationships

304

significantly (*P*>0.05) between control and depodded plants.

result from a decrease in leaf inorganic phosphate content.

**Figure 4.** Leaf chlorophyll, total protein and inorganic phosphate contents in soybean plants on day 3 after pod re‐ moval. Open bar, chlorophyll; gray bar, total protein; black bar, inorganic phosphate. Vertical bars indicate S.D. (n=3). \**P*<0.01 (\*\**P*<0.05) when compared with control plants.

**Figure 5.** Leaf sucrose and starch contents in soybean plants on day 3 after pod removal. Open bar, sucrose content; gray bar, starch content. Vertical bars indicate S.D. (n=3). Both sucrose and starch contents did not differ significantly (*P*>0.05) between control and depodded plants.

There is a hypothesis of inhibition of photosynthesis through accumulation of sucrose in the leaf, although the detailed mechanism(s) is still unclear (Kasai, 2008). For example, in a study having continuous exposure to light of single-rooted soybean leaves, a significant negative correlation was shown between leaf sucrose content and photosynthetic rate (Sa‐ wada et al., 1986). It is thought that in both control and depodded plants, sucrose-in‐ duced inhibition of leaf photosynthesis was, if any, very small. Leaf sucrose content of control plants, which was higher on the average than that of depodded plants (Fig. 5), corresponded with a content that led to a very small decrease in leaf photosynthetic rate of single-rooted soybean leaves (Sawada et al., 1986). Leaf sucrose content of control plants did not seem to decrease leaf photosynthetic rate in our previous pod removal study us‐

ing soybean plants (Kasai et al., 2008). There is also a hypothesis of inhibition of photosyn‐ thesis through accumulation of starch in leaves (Kasai, 2008). In the same study, continuous exposure to light of single-rooted soybean leaves resulted in a significant negative correla‐ tion between leaf starch content and photosynthetic rate (Sawada et al., 1986). Another study using single-rooted soybean leaves demonstrated that accumulation of starch decreas‐ es the rate of CO2 diffusion (Sawada et al., 2001; Kasai et al., 1996). In our pod removal study, pod removal did not significantly affect leaf starch content (Fig. 5). Therefore, it is suggested that the pod removal-induced decrease in leaf photosynthetic rate did not re‐ sult from accumulation of sucrose or starch in leaves.

It is believed that in plants, P-type H+ pump (H+ -ATPase) exists in the plasma membrane, and V-type H+ pumps (H+ -ATPase and H+ -PPase) exist in the tonoplast (Hall & Williams, 1991; Barkla et al., 2008). In plant leaves, a decrease in H+ pump activity of the guard cell plasma membrane can induce decreases of stomatal conductance and transpiration rate by inducing a decrease in stomatal pore size (Tominaga et al., 2001). Although we did not ana‐ lyze the H+ pump activity of the guard cell plasma membrane, it was shown that pod re‐ moval largely decreased the H+ pump activity of the leaf plasma membrane (Fig. 6). Essentially, almost the same method is used for isolation of the plasma membrane from leaves and guard cells (Becker et al., 1993). Therefore, it is suggested that a large decrease in H+ pump activity of the guard cell plasma membrane could cause the pod removal-induced decreases of leaf stomatal conductance and transpiration rate. In plant cells, a decrease in H+ pump activity of the plasma membrane can induce a decrease in the electrochemical poten‐ tial difference of H+ across the plasma membrane (Hall & Williams, 1991; Barkla et al., 2008). There is increasing evidence that depolarization-activated Ca2+ channel, Ca2+-activated anion (e.g., Cl- ) channel, depolarization-activated anion channel (e.g., HCO3 - ), electrogenic Ca2+/H+ antiporter (which has a stoichiometry higher than 2H+ /Ca2+), and CO2-transportable and Ca2+-inhibitable water channel are present in the plant plasma membrane (Thuleau et al., 1994; Roberts, 2005; Frachisse et al., 1999, Kasai et al., 1990; Song et al., 2011; Chaumont et al., 2005). Therefore, it is speculated that the observed decrease in H+ pump activity of the leaf plasma membrane could cause the decrease in the rate of CO2 transport in leaf photo‐ synthetic cells by inducing a depolarization of the plasma membrane, a decrease in the pro‐ ton motive force across the plasma membrane and a rise of Ca2+ concentration inside the plasma membrane in the leaf photosynthetic cells. The suggestion drawn on the basis of da‐ ta of Figure 1 and 2 that pod removal decreased equally the rate of CO2 diffusion via leaf stomata and the rate of CO2 fixation in leaf photosynthetic cells is roughly consistent with the above-mentioned suggestion and speculation proposing the regulation of leaf stomatal conductance, transpiration rate and CO2 transport in leaf photosynthetic cells by activity of H+ pump of leaf plasma membrane. As shown in Figure 6, pod removal also greatly de‐ creased activities of the H+ pumps (H+ -ATPase and H+ -PPase) of the leaf tonoplast. There is increasing evidence that electrogenic Ca2+/H+ antiporter (which has a stoichiometry higher than 2H+ /Ca2+) is also present in the plant tonoplast (Blackford et al., 1990; Mei et al., 2007). Therefore, it is speculated that a large decrease in H+ pump activity of the leaf plasma mem‐ brane and a large decrease in activities of H+ pumps of the leaf tonoplast could cause coop‐ eratively the pod removal-induced decrease in leaf photosynthetic rate by inducing equal decreases in the rate of CO2 diffusion via leaf stomata and the rate of CO2 fixation in leaf photosynthetic cells. To verify our speculations, more evidence is needed. We emphasize, however, that until now, in similar studies other than our study, activities of the H+ pumps of plasma membrane and tonoplast have not been analyzed.

ing soybean plants (Kasai et al., 2008). There is also a hypothesis of inhibition of photosyn‐ thesis through accumulation of starch in leaves (Kasai, 2008). In the same study, continuous exposure to light of single-rooted soybean leaves resulted in a significant negative correla‐ tion between leaf starch content and photosynthetic rate (Sawada et al., 1986). Another study using single-rooted soybean leaves demonstrated that accumulation of starch decreas‐ es the rate of CO2 diffusion (Sawada et al., 2001; Kasai et al., 1996). In our pod removal study, pod removal did not significantly affect leaf starch content (Fig. 5). Therefore, it is suggested that the pod removal-induced decrease in leaf photosynthetic rate did not re‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

pump (H+

plasma membrane can induce decreases of stomatal conductance and transpiration rate by inducing a decrease in stomatal pore size (Tominaga et al., 2001). Although we did not ana‐

Essentially, almost the same method is used for isolation of the plasma membrane from leaves and guard cells (Becker et al., 1993). Therefore, it is suggested that a large decrease in

Ca2+-inhibitable water channel are present in the plant plasma membrane (Thuleau et al., 1994; Roberts, 2005; Frachisse et al., 1999, Kasai et al., 1990; Song et al., 2011; Chaumont et al., 2005). Therefore, it is speculated that the observed decrease in H+ pump activity of the leaf plasma membrane could cause the decrease in the rate of CO2 transport in leaf photo‐ synthetic cells by inducing a depolarization of the plasma membrane, a decrease in the pro‐ ton motive force across the plasma membrane and a rise of Ca2+ concentration inside the plasma membrane in the leaf photosynthetic cells. The suggestion drawn on the basis of da‐ ta of Figure 1 and 2 that pod removal decreased equally the rate of CO2 diffusion via leaf stomata and the rate of CO2 fixation in leaf photosynthetic cells is roughly consistent with the above-mentioned suggestion and speculation proposing the regulation of leaf stomatal conductance, transpiration rate and CO2 transport in leaf photosynthetic cells by activity of

pump of leaf plasma membrane. As shown in Figure 6, pod removal also greatly de‐

/Ca2+) is also present in the plant tonoplast (Blackford et al., 1990; Mei et al., 2007).


Therefore, it is speculated that a large decrease in H+ pump activity of the leaf plasma mem‐

eratively the pod removal-induced decrease in leaf photosynthetic rate by inducing equal

) channel, depolarization-activated anion channel (e.g., HCO3 -

 pump activity of the guard cell plasma membrane could cause the pod removal-induced decreases of leaf stomatal conductance and transpiration rate. In plant cells, a decrease in H+ pump activity of the plasma membrane can induce a decrease in the electrochemical poten‐ tial difference of H+ across the plasma membrane (Hall & Williams, 1991; Barkla et al., 2008). There is increasing evidence that depolarization-activated Ca2+ channel, Ca2+-activated anion

pump activity of the guard cell plasma membrane, it was shown that pod re‐


pump activity of the guard cell

), electrogenic Ca2+/H+

/Ca2+), and CO2-transportable and


antiporter (which has a stoichiometry higher

pumps of the leaf tonoplast could cause coop‐


pump activity of the leaf plasma membrane (Fig. 6).

sult from accumulation of sucrose or starch in leaves.

1991; Barkla et al., 2008). In plant leaves, a decrease in H+

antiporter (which has a stoichiometry higher than 2H+

pumps (H+


It is believed that in plants, P-type H+

moval largely decreased the H+

pumps (H+

and V-type H+

Relationships

306

lyze the H+

H+

(e.g., Cl-

H+

than 2H+

creased activities of the H+

increasing evidence that electrogenic Ca2+/H+

brane and a large decrease in activities of H+

With respect to the mechanism(s) of why pod removal decreased the H+ pump activity of the leaf plasma membrane and activities of the H+ pumps of the leaf tonoplast, it is inferred that plant hormones abscisic acid and cytokinin could be involved in the mechanism(s). In general, cytokinin is known to have positive effect in synthesizing chlorophyll and protein, and its content in plant cells is known to decrease under deficiency of mineral nutrients such as P and N. In contrast, abscisic acid antagonizes the effects of cytokinin, and its content in plant cells increases under conditions of mineral nutrient deficiencies (Pozsar et al., 1967; Kusnetsov et al., 1998; Salama & Wareing, 1979; Mizrahi & Richmond, 1972; Battal et al., 2003). In our pod removal study, it was shown that pod removal, which decreased the ratio of sink to source organs (Fig. 7), increased significantly the contents of chlorophyll, total protein and inorganic phosphate in the leaf (Fig. 4), implicating that pod removal might have increased cytokinin content relative to abscisic acid content in the leaf by influencing the partitioning of mineral nutrients such as P and N within the plant. In barley, it was dem‐ onstrated that abscisic acid has stimulatory effects on activities of tonoplast H+ pumps, whereas cytokinin has opposite effects antagonizing the effects of abscisic acid (Kasai et al., 1993; Fukuda & Tanaka, 2006). In *Phaseolus vulgaris*, excessive levels of cytokinin were dem‐ onstrated to decrease leaf stomatal conductance, transpiration rate and photosynthetic rate (Pospisilova, 2003).

**Figure 6.** Activities of H+ pumps of leaf plasma membrane and tonoplast from soybean plants on day 3 after pod re‐ moval. Open bar, plasma membrane H+-ATPase; gray bar, tonoplast H+-ATPase; black bar, tonoplast H+-PPase. Vertical bars indicate S.D. (n=3). \**P*<0.01 (\*\**P*<0.05) when compared with control plants.

Data from recent studies using transgenic plants and those from physiological studies impli‐ cate that in plants, activity(ies) of membrane H+ pump(s) can be important in the regulation of photosynthetic dry matter production through photosynthetic source-sink balance. For

example, in a study using *Arabidopsis* plants, overexpression of tonoplast H+ -PPase was shown to result in an increase in whole plant growth, in particular, growth of sink organ roots (Li et al., 2005). In another study using *Arabidopsis* plants, it was shown that overex‐ pression of tonoplast H+ -PPase resulted in a 1.9-fold increase in root dry weight and a 1.5 fold increase in shoot dry weight (Yang et al., 2007). There is also a report that transgenic manipulation increasing tonoplast H+ -ATPase resulted in an increase in root length (Wang et al., 2011). In a study using rye plants, increases of root/shoot ratio and activities of tono‐ plast H+ pumps (H+ -PPase and H+ -ATPase) were shown to occur under growth conditions inducing deficiency of mineral nutrients (Kasai et al., 1998). Whereas conditions inducing deficiency of mineral nutrients can increase abscisic acid level in plant cells, it can also de‐ crease cytokinin level (Salama & Wareing, 1979; Mizrahi & Richmond, 1972; Battal et al., 2003). In a recent study using *Arabidopsis* plants, transgenic manipulation decreasing cytoki‐ nin level was shown to enhance root/shoot ratio (Werner et al., 2003). In plant cells, levels of abscisic acid and cytokinin can also be affected by levels of photosynthetic carbohydrates, which can interact with the levels of mineral nutrients (Rolland et al., 2002). Interestingly, a more recent study using *Arabidopsis* plants showed that overexpression of tonoplast H+ - PPase resulted in an increase in cell abscisic acid level and a decrease in cell cytokinin (bio‐ logically active cytokinin) level (Gonzalez et al., 2010). In the other study using *Arabidopsis* plants, it was shown that a mutant losing about 60% of tonoplast H+ -ATPase activity had a morphology resembling cytokinin-treated plants (Schumacher et al., 1999). As already men‐ tioned, abscisic acid had stimulatory effects on activities of tonoplast H+ pumps, whereas cy‐ tokinin had opposite effects antagonizing the effects of abscisic acid in barley (Kasai et al., 1993; Fukuda & Tanaka, 2006). In our pod removal study using soybean plants, decreasing the ratio of sink to source organs by conducting pod removal was shown to result in a large decrease in activities of H+ pumps of the leaf plasma membrane and tonoplast. Although characteristics such as leaf photosynthetic rate related to photosynthetic dry matter produc‐ tion were not analyzed in the above studies other than our study, information from these studies and the other information of our data from pod removal study implicate that in plants, changes in activity(ies) of membrane H+ pump(s) can actually play key roles in the regulation of photosynthetic matter production through photosynthetic source-sink balance. It is suggested from experimental evidence that abscisic acid and cytokinin can be involved at least in the regulation of activities of tonoplast H+ pumps. Although no direct evidence exists, data from more recent studies let us suppose that now well-known 2C-type protein phosphatase and salt-overly-sensitive 2 protein kinase may be involved (in part) in the regu‐ lation of activities of tonoplast H+ pumps by abscisic acid and cytokinin (Batelli et al., 2007; Huertas et al., 2012). In plants, abscisic acid, cytokinin and membrane H+ pump(s) are specu‐ lated to be symbolic biomolecules that are able to induce or respond to a variety of internal and external environmental changes that cause the regulation of photosynthetic dry matter production through photosynthetic source-sink balance. Many experimental data suggest that interaction actually exists between environmental change and cell abscisic acid and cy‐ tokinin levels and environmental change and, for example, activities of tonoplast H+ pumps and environmental change and photosynthetic matter production and environmental change and photosynthetic source-sink balance (Board & Kahlon, 2011; Peleg & Blumwald, 2011; Kasai, 1999; Rolland et al., 2002). On the basis of a lot of evidence, it seems evident that photosynthetic source-sink balance is an essential factor regulating photosynthetic dry mat‐ ter production (Kasai, 2008, 2011; Rolland et al., 2002). As described in the Introduction, plant photosynthetic dry matter production is essential for all living organisms and is also essential for creating sound environments. Therefore, further studies are important for eluci‐ dation of the detailed mechanism(s) of how membrane H+ pump(s) are involved in the regu‐ lation of photosynthetic dry matter production through photosynthetic source-sink balance. We emphasize that for improvement of plant productivity and for creating sound environ‐ ments, well-balanced improvement of source and sink would be essential.

**Figure 7.** Dry weights of leaves, stems, floral organs including pods, and roots in soybean plants on day 3 after pod removal. Open bar, leaves; gray bar, stems; black bar, floral organs including pods; dotted bar, roots. Vertical bars indi‐ cate S.D. (n=3). Dry weights of leaves, stems and roots did not differ significantly (*P*>0.05) between control and depod‐ ded plants.
