**3.4 Rubisco initial activity and RT-PCR analysis**

The diurnal variation of the Rubisco initial activity of pear leaves showed a bimodal curve, where the first maximum occurred at 8:00 am, and the second at 16:00 pm. A significant valley occurred at noontide (Fig. 7A). In most cases, ALA treatment stimulated the activity, compared with the control. From the result of RT-PCR of the coding gene (Fig.7B and C), it can be seen that expression of *Rubisco small subunit* gene in pear leaves also revealed a bimodal curve, which was similar with the change of the enzyme activity in Fig. 7A. The relative expression was significantly higher in ALA-treated leaves than that of the control, especially at 8:00, which was more than 2 times. Therefore, ALA treatment improved the expression of *Rubisco small subunit* gene at transcript level.

Effect of 5-Aminolevulinic Acid (ALA) on Leaf Diurnal Photosynthetic

content of pear leaves (Fig. 8A).

significant at *P*=0.05 level.

**3.6 Activities of antioxidant enzymes** 

in the early morning and evenfall.

control.

SOD; B: APX; C: CAT

Characteristics and Antioxidant Activity in Pear (*Pyrus Pyrifolia* Nakai) 249

stable higher level in one day than that of the control. The diurnal mean of the former was about 10% higher that of the latter, suggesting that ALA treatment could increase the H2O2

As for the MDA content of pear leaves, it revealed a wave daily variation (Fig.8B). The lowest in the diurnal variation curve was at about 10:00 am and the highest at 16:00 pm. ALA pretreatment decreased the content of MDA, and the significant differences were found in the morning and afternoon. At noon time, the MDA content in the ALA-treated leaves was also slightly higher than that of the control, however, the difference was not

The diurnal variation of SOD, APX and CAT activities in response to ALA treatment are shown in Fig. 9. In SOD, the diurnal variation was a two-peak curve, where the first big peak was recorded at 8:00 am and the second small one at 16:00 pm (Fig. 9A). Compared with the control, SOD activities in ALA-treated leaves were generally increased, especially

Different with SOD, no peak could be found in the diurnal variation of APX activities of pear leaves (Fig. 9B). Instead, it was lowest at noontide, but kept higher levels in the morning or afternoon. ALA treatment significantly stimulated the enzyme activity in all day time, and the diurnal mean in the ALA-treated was 37% higher than that of the

The diurnal variation of CAT activities in pear leaves was similar with a sine curve, which exhibited a peak at 8:00 am and valley at noontide, and then recovered to earlier levels (Fig. 9C). At any time, the activities in ALA-treated leaves were generally higher than that of the

Fig. 9. Effect of ALA on diurnal variations of antioxidant enzymes activities of pear leaf. A:

control, suggesting that ALA improved the CAT activity in pear leaves.

Fig. 7. Effect of ALA on diurnal variations of ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco) initial activity (A) and relative expression of *Rubisco small subunit gene* (B) of pear leaves. C is electrophorogram of the gene expression detected by RT-PCR.

#### **3.5 Effect of ALA treatment on the H2O2 and MDA content**

There is a difference in the H2O2 and MDA content in pear leaves between ALA treatment and control (Fig. 8). The H2O2 content in ALA treated-leaves maintained at a relatively

Fig. 8. Effect of ALA on diurnal variations of H2O2 and MDA content of pear leaves. A: H2O2 content; B: MDA content

stable higher level in one day than that of the control. The diurnal mean of the former was about 10% higher that of the latter, suggesting that ALA treatment could increase the H2O2 content of pear leaves (Fig. 8A).

As for the MDA content of pear leaves, it revealed a wave daily variation (Fig.8B). The lowest in the diurnal variation curve was at about 10:00 am and the highest at 16:00 pm. ALA pretreatment decreased the content of MDA, and the significant differences were found in the morning and afternoon. At noon time, the MDA content in the ALA-treated leaves was also slightly higher than that of the control, however, the difference was not significant at *P*=0.05 level.
