A common wheat (Triticum aestivum L.) variety, Norin 61, which is one of the main modern cultivars in Japan, was grown in an experimental field at Kobe University in 1997. To monitor the effect of the nitrogen supply, we set four experimental plots. In the first plot, named “No-basal”, a chemical fertilizer (N:P:K = 8:8:8) was not dressed before cultivation. In the second plot, named “Low-N”, the same chemical fertilizer was dressed at the density of 8 g/m² for each nutrient before cultivation, but no nitrogen fertilizer was applied at the heading date. The third and fourth plots were named “Middle-N” and “High-N”, respectively. In both plots, the same chemical fertilizer was dressed before cultivation, and nitrogen fertilizer was applied on the soil surface at the heading date. In the Middle-N plot, ammonium sulfate at 8.3 g/m² (0.88 g/m² of nitrogen) was applied. In the High-N plot, ammonium sulfate at 25 g/m² (2.65 g/m² of nitrogen) was applied.

Seeds were soaked on water on May 17, 1997 and incubated at 4˚C for 1 week to induce germination. The seeds were sowed onto the field. The heading date was May 19th. Seeds that were developing on June 11th were harvested and frozen immediately in liquid nitrogen for storage at −20˚C until use. On June 16th, we collected panicles and flag leaves and froze them in liquid nitrogen after measuring their fresh weights. Mature seeds were sampled at the beginning of July and weighed.

Hydroponically grown wheat was also prepared at a normal greenhouse at Kobe University in 1998 spring. On February 1st, wheat seeds were sterilized by 1% sodium hypochlorite and then sandwiched between two pieces of moist filter papers. After incubation at room temperature for 10 days, germinated seeds were planted in one-half-strength Hoagland solution [16] . After 2 weeks of cultivation, the wheat plants were transferred into Hoagland solution. The hydroponic solution was freshened once a week. On the flowering date (April 18th), on which 50% of flowers were in bloom, the plant growth environment was changed by modifying the final concentration of ammonium nitrate and using cheesecloth with the following four experimental plots: 1) low nitrogen under normal sunlight (Low-N-L); 2) low nitrogen under shade (Low-N-S); 3) high nitrogen under normal sunlight (High-N-L); and (4) high nitrogen under shade (High-N-S). In the Low-N-L and Low-N-S plots, the final concentration of ammonium nitrate was decreased from 80 to 12 mg/L, resulting in the total nitrogen concentration 214 mg/L. In the High-N-L and High-N-S plots, the final concentration of ammonium nitrate was increased to 240 mg/L, resulting in 294 mg/L as the total nitrogen concentration. In the Low-N-S and High-N-S plots, the plants were covered by cheesecloth to cut 50% of the sunlight. Developing seeds at 26 days after flowering were collected for the PEPCase assay.

The protein content in the mature seeds was determined by the Kjeldahl method [17] . The protein amount was calculated from the total nitrogen by using the general factor of 6.25.

Developing seeds of 10 gains were homogenized in an extraction buffer [100 mM Tris-HCl, pH 7.8, 1 mM EDTA, 1 mM, 2-mercaptoethanol, 10% (w/v) glycerol] with Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland) at 4˚C. The homogenate was centrifuged at 13,000 g for 15 min, and the supernatant was collected for the PEPCase assay.

PEPCase activity was assayed by coupling with a malate dehydrogenase reaction according to Echevarria’s method with modifications [18] . This assay was performed in 1 mL of a medium containing 50 mM Tricine- KOH, 5 mM MgSO₄, 0.15 mM NADH, 5 mM KHCO₃, 5 mM PEP (cyclohexylammonium salt), 4 mM DTT and 1.5 U pig heart malate dehydrogenase at 25˚C at pH 8.3. The decreasing rate of the absorbance at 340 nm was measured. One unit of enzyme activity is defined as the amount of PEPCase catalyzing the production of 1 μmol of oxaloacetate/min at 25˚C. The assay was carried out twice for each sample, and the average of measured values was adopted.

We set four growth conditions with different nitrogen supplies as described in the Plant Materials section: No- basal (without no basal fertilizer), Low-N (with the basal fertilizer), Middle-N (with the basal fertilizer and 0.88 g/m² nitrogen) and High-N (with the basal fertilizer and 2.65 g/m² nitrogen). To monitor the plant growth in the experimental plots, we weighed the panicles, flag leaves and mature seeds in each plot (Table 1). The fresh weights of the panicles and flag leaves per plant were greatly increased in the order No-basal < Low-N < Middle- N < High-N. The increases of these two parameters were in parallel. This result indicates the nitrogen use status. The weight in mature seeds per 100 grains also showed increases, in the same order of plots.

We determined the PEPCase activity in the developing seeds per fresh weight and per grain. The activities per

^*The panicle and flag leaf data are weight (g) per plant, and those in the mature seed are weight per 100 grains.

fresh weight (FW) in all of the plots were distributed around 1.0 U/g FW (Figure 1(a)). Among the three plots with the basal fertilizer, the activity was increased according to the nitrogen supply at the flowering stage. However, in the No-basal plot, activity was observed that was clearly higher than that of the Low-N plot and similar to that of the Middle-N plot. The activities per grain showed a different trend (Figure 1(b)), namely, the activities in the No-basal and Low-N plots were quite similar, although increased activity according to the nitrogen supply at the flowering stage was seen. These results showed that the application of nitrogen fertilization at the flowering stage was an effective strategy to induce an increase in PEPCase activity. The different patterns of PEPCase activity in the No-basal plot might indicate that basal fertilization caused a different carbon/nitro- gen balance in the grain-filling stages.

The protein content in the mature seeds was predicted from the nitrogen content determined by the Kjeldahl method. The lowest protein content was observed in the Low-N plot, and the highest protein content was observed in the High-N plot (Figure 2). The No-basal and Middle-N plots showed similar protein content values. The ammonium nitrogen applications of 8.3 and 25 g/m² at the flowering stage resulted in 1.15- and 1.42-fold increases in the seed protein content, respectively, compared to the Low-N plot.

We observed a rough relationship between the PEPCase activity and protein content in the four experimental plots (Figure 3). When we compared the activity per fresh weight, a linear correlation was apparent among the three plots with the basal fertilizer. This result was consistent with the concept that basal fertilization causes different carbon/nitrogen balances in grain-filling stages. In contrast, when we compared the activity per grain, the data of all four plots showed a linear correlation, suggesting that this type of correlation analysis might be effective when the samples analyzed were grown under various environmental factors.

The nitrogen application would also induce higher photosynthetic activity, and it could enhance the supply of carbon matter into seeds. Therefore, there is a possibility that the enhancement of carbon transport into seeds induces higher seed PEPCase activity. To test this possibility, we examined the effect of the nitrogen supply under normal sunlight and shaded (50% light) conditions. As shown in Figure 4, between the normal sunlight and shaded conditions, we observed no clear effect on PEPCase activity (no significant difference). In the shad- ed condition, the enzyme activity of the High-N-S plot was approx. 0.3-fold higher than that of the Low-N-S plot, although the difference was not significant (P = 0.101 by t-test). These results imply that the nitrogen sup- ply in the grain-filling stages has a greater effect on the PEPCase activity in developing seeds than the supply of sunlight.