**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, 12.5 cm in diameter) containing almost equal volumes of vermiculite and sand that had been mixed, and were grown in growth chambers (Koitotoron, HNL type; Koito Industries Ltd., Tokyo, Japan) under daily light/dark periods of 10/14 h, day/night temperatures of 24/17oC and relative humidity of 60 %. After 8 weeks, plants were divided into two groups, and one group was grown for 3 days with continuous light, and another group was grown for 3 days under daily light/dark periods of 10/14 h as controls. Nutrients were supplied once a week with a 1000-fold diluted solution of Hyponex [6-10-5 type (N:P:K = 6:10:5); Hyponex Co., Osaka, Japan], and tap water was supplied in sufficient amounts. Light was supplied with incandescent lamps at an intensity of 480 µmol photons m-2 s-1 (400-700 nm) at the middle height of plants grown for 8 weeks.

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

Leaf photosynthetic rate, stomatal conductance and intercellular CO2 concentration were determined in fully expanded fourth trifoliate leaves at a light intensity of 1000 µmol photons m-2 s-1, air flow rate of 200 ml min-1, air temperature of 25 oC, relative humidity of 60 % and CO2 concentration of 350 ppm on day 3 after treating plants with continuous light 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 below.

#### **2.3 Other analyses**

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

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 Rubisco (0.5 units).

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