**3. Results**

Analyzed leaf photosynthetic rate was significantly lower in intact soybean plants grown for 3 days with continuous light than in control plants grown under daily light/dark periods of 10/14h (Fig. 1).

Leaf stomatal conductance was also significantly lower in continuous light-treated plants than in control plants (Fig. 2). Leaf intercellular CO2 concentration did not differ significantly between control and continuous light-treated plants (Fig. 2).

When activation ratio (percentage of initial activity to total activity) of Rubisco in leaf extract was calculated from analyzed initial and total activities of Rubisco in leaf extract, the ratio was significantly lower in continuous light-treated plants than in control plants (Fig. 3). The ratios in control and continuous light-treated plants were 74.2% and 56.6%, respectively.

using a portable photosynthetic analyzer (Cylus-1; Koito Industries Ltd.). After measurements, leaf disks (1.79 cm2) were cut off from fourth trifoliate leaves, immediately frozen in liquid nitrogen and stored at -80 oC until used for the other analyses described

The activity of Rubisco in leaf extract was determined at 25 oC as described previously (Kasai, 2008). For the initial activity, 20 µl of a leaf extract obtained by homogenizing a leaf disk with ice-cold buffer (100 mM HEPES-KOH, pH 7.8, 2 ml) was added to a cuvette containing 1.98 ml of assay medium [100 mM Bicine-KOH (pH 8.2), 20 mM MgCl2, 20 mM NaHCO3, 5 mM creatine phosphate, 1 mM ATP, 0.2 mM NADH, 20 units creatine kinase, 20 units 3-phosphoglycerate kinase and 20 units glyceraldehyde-3-phosphate dehydrogenase], immediately followed by the addition of RuBP (final concentration 0.6 mM) and mixed well. For total activity, RuBP was added 5 min later after 20 µl of the leaf disk extract was immediately combined with the assay medium. The change in absorbance at 340 nm was

The amount of protein-bound RuBP in leaf extract was determined as described previously (Kasai, 2008). A leaf extract (800 µl) obtained by homogenizing a leaf disk with an ice-cold buffer (100 mM HEPES-KOH, pH 7.8, 1 ml) was centrifuged (100 g, 1 min) after loading onto a column containing Sephadex G-50 (bed volume before centrifugation, 4 ml) that had been equilibrated with the same buffer. The eluent (500 µl) from the column lacking free RuBP was centrifuged (10,000 g, 10 min) after mixing with an acidic solution (5.5 M HClO4, 50 µl) to precipitate protein in the eluent. The resulting supernatant was centrifuged (10,000 g, 10 min) after neutralizing to pH 5.6 with K2CO3, and RuBP in the supernatant was determined in the assay medium for determining Rubisco activity using purified spinach

Leaf Rubisco content was determined as described by Makino et al. (1986). Leaf total protein was extracted as described by Makino et al. (1986) and quantified by the method of Bradford (1976). Leaf chlorophyll content was determined according to the method of Mackinney (1941). Leaf sucrose and starch contents were determined as described by Sawada et al. (1999). Leaf water content was analyzed by measuring fresh weight and dry weight of leaf

Analyzed leaf photosynthetic rate was significantly lower in intact soybean plants grown for 3 days with continuous light than in control plants grown under daily light/dark periods of

Leaf stomatal conductance was also significantly lower in continuous light-treated plants than in control plants (Fig. 2). Leaf intercellular CO2 concentration did not differ

When activation ratio (percentage of initial activity to total activity) of Rubisco in leaf extract was calculated from analyzed initial and total activities of Rubisco in leaf extract, the ratio was significantly lower in continuous light-treated plants than in control plants (Fig. 3). The ratios in control and continuous light-treated plants were 74.2% and 56.6%,

significantly between control and continuous light-treated plants (Fig. 2).

monitored using a spectrophotometer (Model U-2000; Hitachi Co., Tokyo, Japan).

below.

**2.3 Other analyses** 

Rubisco (0.5 units).

**3. Results** 

10/14h (Fig. 1).

respectively.

disks. Leaf disks were dried for 2 days at 75 oC.

Fig. 1. Leaf photosynthetic rate in soybean plants on day 3 after continuous exposure to light. Control plants were grown under daily light/dark periods of 10/14h for 3 days. Vertical bars indicate S.D. (n=4). \**P*<0.01 (*t*-test) when compared with control plants.

Fig. 2. Leaf stomatal conductance and leaf intercellular CO2 concentration in soybean plants on day 3 after continuous exposure to light. Control plants were grown as described in Fig. 1. Open bar, leaf stomatal conductance; closed bar, leaf intercellular CO2 concentration. Vertical bars indicate S.D. (n=4). \**P*<0.01 when compared with control plants. The intercellular CO2 concentration did not differ significantly (*P*>0.05) between control and continuous light-treated plants.

0

continuous light-treated plants.

200

400

600

800

Leaf stomatal conductance (mmol m-2 s-1

or leaf intercellular CO2 concentration (ppm)

)

1000

1200

1400

1600

\*

Control plants Continuous light-treated plants

Fig. 2. Leaf stomatal conductance and leaf intercellular CO2 concentration in soybean plants on day 3 after continuous exposure to light. Control plants were grown as described in Fig. 1. Open bar, leaf stomatal conductance; closed bar, leaf intercellular CO2 concentration. Vertical bars indicate S.D. (n=4). \**P*<0.01 when compared with control plants. The intercellular CO2 concentration did not differ significantly (*P*>0.05) between control and

Fig. 3. Initial and total activities of Rubisco in leaf extract from soybean plants on day 3 after continuous exposure to light. Control plants were grown as described in Fig. 1. Open bar, initial activity; closed bar, total activity. Vertical bars indicate S.D. (n=4). In comparison with control plants of the activation ratio of Rubisco calculated as a percentage of the initial activity to total activity, \**P*<0.01.

In a study investigating the light activation of Rubisco using *Arabidopsis thalian*, it was demonstrated that the amount of protein-bound RuBP in leaf extract reflects the amount of uncarbamylated inactive Rubisco (Brooks & Portis, 1988). When the amount of proteinbound RuBP was analyzed, the amount was significantly more in continuous light-treated plants than in control plants (Fig. 4).

Fig. 4. Amount of protein-bound RuBP in leaf extract from soybean plants on day 3 after continuous exposure to light. Control plants were grown as described in Fig. 1. Vertical bars indicate S.D. (n=4). \**P*<0.05 when compared with control plants.

Contents of sucrose and starch, which are the major photosynthetic carbohydrates, in leaf were both significantly higher in continuous light-treated plants than in control plants (Fig. 5).

In a study investigating the light activation of Rubisco using *Arabidopsis thalian*, it was demonstrated that the amount of protein-bound RuBP in leaf extract reflects the amount of uncarbamylated inactive Rubisco (Brooks & Portis, 1988). When the amount of proteinbound RuBP was analyzed, the amount was significantly more in continuous light-treated

Fig. 4. Amount of protein-bound RuBP in leaf extract from soybean plants on day 3 after continuous exposure to light. Control plants were grown as described in Fig. 1. Vertical bars

Contents of sucrose and starch, which are the major photosynthetic carbohydrates, in leaf were both significantly higher in continuous light-treated plants than in control plants (Fig. 5).

indicate S.D. (n=4). \**P*<0.05 when compared with control plants.

\*

plants than in control plants (Fig. 4).

0

0.5

1

1.5

2

2.5

RuBP (nmol mg-1 protein)

3

3.5

4

4.5

Fig. 5. Leaf sucrose or starch content in soybean plants on day 3after continuous exposure to light. Control plants were grown as described in Fig. 1. Open bar, sucrose content; closed bar, starch content. Vertical bars indicate S.D. (n=4). \**P*<0.05 when compared with control plants.

Analyzed contents of chlorophyll, water, total protein and Rubisco protein in leaf did not differ significantly between control and continuous light-treated plants (Fig. 6 and 7).

Fig. 6. Leaf chlorophyll, total protein or Rubisco content in soybean plants on day 3 after continuous exposure to light. Control plants were grown as described in Fig. 1. Open bar, chlorophyll content; closed bar, total protein content; dotted bar, Rubisco content. Vertical bars indicate S.D. (n=4). The chlorophyll, total protein and Rubisco contents did not differ significantly (P>0.05) between control and continuous light-treated plants.

Analyzed leaf dry weight other than the weights of sucrose and starch was heavier a little in continuous light-treated plants than in control plants (Fig. 5 and 7). The mean dry weights

Analyzed contents of chlorophyll, water, total protein and Rubisco protein in leaf did not differ significantly between control and continuous light-treated plants (Fig. 6 and 7).

Control plants Continuous light-treated plants

Fig. 6. Leaf chlorophyll, total protein or Rubisco content in soybean plants on day 3 after continuous exposure to light. Control plants were grown as described in Fig. 1. Open bar, chlorophyll content; closed bar, total protein content; dotted bar, Rubisco content. Vertical bars indicate S.D. (n=4). The chlorophyll, total protein and Rubisco contents did not differ

Analyzed leaf dry weight other than the weights of sucrose and starch was heavier a little in continuous light-treated plants than in control plants (Fig. 5 and 7). The mean dry weights

significantly (P>0.05) between control and continuous light-treated plants.

0

2

4

6

Chlorophyll, protein or Rubisco content (g m-2)

8

10

12

14

after subtracting the weights of sucrose and starch in control and continuous light-treated plants were 49.0 g m-2 and 57.5 g m-2, respectively.

Fig. 7. Leaf water content and leaf dry weight in soybean plants on day 3 after continuous exposure to light. Control plants were grown as described in Fig. 1. Open bar, leaf water content; closed bar, leaf dry weight. Vertical bars indicate S.D. (n=4). \**P*<0.05 when compared with control plants. The leaf water content did not differ significantly (*P*>0.05) between control and continuous light-treated plants.
