**2.6 Determination of hydrogen peroxide (H2O2) and malondialdehyde (MDA)**

Hydrogen peroxide content was estimated through the formation of a titanium-hydro peroxide complex (Agarwal et al., 2005). One hundred milligram of leaf sample was ground with liquid nitrogen and the fine powdered material was mixed with 2 ml cooled acetone. Then the mixture was centrifuged at 10,000×g for 10 min and the supernatant was collected

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

photosynthesis in pear leaves.

Intercellular CO2 concentration

**3.2 Effect of ALA on gas exchange characteristics in pear leaves** 

Characteristics and Antioxidant Activity in Pear (*Pyrus Pyrifolia* Nakai) 243

was about 19℃,which linearly increased to the maximum temperature about 29℃ at noon (12:00), then decreased to 25.5℃ at dusk (6:00 pm). In the aspect of light intensity, the PFD was 65.6 μmol·m-2·s-1 in the morning, and the maximum was 1612 μmol·m-2·s-1 at noon.

The measurement of diurnal variations of leaf gas exchange characteristics showed that the net photosynthetic rate (*Pn*) possessed a twin-peaks curve (Fig. 2A) and ALA treatment significantly increased *Pn* of pear leaves compared with the control, especially at noon time. *Pn*/*Ci*, representing the instantaneous carboxylation efficiency, exhibited a single peak curve in diurnal variation (Fig. 2B), where ALA treatment generally promoted *Pn*/*Ci* of pear leaves, especially at noontide. Changes in stomatal conductance was similar with *Pn*, and ALA treatment promoted stomtal open in most of day time (Fig. 2C). However, there was no difference in the intercellular CO2 concentrations of pear leaves between control and treatment (Fig. 2D), suggesting that 150 μmol/mol CO2 in the experiment did not limit

Fig. 2. Effect of ALA on diurnal variations of gas exchange parameters of pear leaves. A: Net photosynthetic rate; B: Instantaneous carboxylation efficiency; C: Stomatal conductance; D:

as hydrogen peroxide extraction. One milliliter of the extract was added by 0.1 ml 5% titanium sulfate and 0.2 ml ammonium solution to precipitate the titanium-hydro peroxide complex. Reaction mixture was centrifuged at 5,000×g for 10 min in the centrifuge and the precipitate was washed 5 times by cooled acetone. Precipitate was dissolved in 10 ml 2 M H2SO4 and then recentrifuged. Supernatant was read at 415 nm against reagent blank in UVspectrophotometer. The hydrogen peroxide content was calculated by comparing with a standard curve drawn with known hydrogen peroxide concentrations.

The MDA content was measured following the method of Heath et al. (1968) with modification and expressed as nmol per g of fresh weight. Five hundred milligram of frozen powder was added to about 5 ml of 5% trichloroacetic acid (TCA) and centrifuged at 10,000×g for 5 min. Two milliliter aliquot of supernatant was added to 2 ml of 0.67% 2 thiobarbituric acid (TBA). The mixture was incubated in boiling water for 30 min and then quickly cooled in an ice bath. After centrifugation at 10,000×g for 10 min, A532, A600 and A450 of the supernatant were recorded. MDA content was estimated by the formula C (μmol/L) =6.45(A532-A600)-0.56A450.

#### **2.7 Statistical analysis**

All data were subjected to ANOVA test and the means were compared by the Duncan's test. Comparisons with p<0.05 were considered significant difference. Pearson correlation analysis between parameters was performed to test for relationships between variables by SPSS. 13 software.

### **3. Results**

#### **3.1 Diurnal variation of air temperature and light intensity in the orchard**

On the testing day, the weather was fine when the temperature and photon flux density (*PFD*) exhibited a single peak curve (Fig.1). In the morning (6:00 am), the air temperature

Fig. 1. Diurnal variations of air temperature and light intensity in pear orchard

as hydrogen peroxide extraction. One milliliter of the extract was added by 0.1 ml 5% titanium sulfate and 0.2 ml ammonium solution to precipitate the titanium-hydro peroxide complex. Reaction mixture was centrifuged at 5,000×g for 10 min in the centrifuge and the precipitate was washed 5 times by cooled acetone. Precipitate was dissolved in 10 ml 2 M H2SO4 and then recentrifuged. Supernatant was read at 415 nm against reagent blank in UVspectrophotometer. The hydrogen peroxide content was calculated by comparing with a

The MDA content was measured following the method of Heath et al. (1968) with modification and expressed as nmol per g of fresh weight. Five hundred milligram of frozen powder was added to about 5 ml of 5% trichloroacetic acid (TCA) and centrifuged at 10,000×g for 5 min. Two milliliter aliquot of supernatant was added to 2 ml of 0.67% 2 thiobarbituric acid (TBA). The mixture was incubated in boiling water for 30 min and then quickly cooled in an ice bath. After centrifugation at 10,000×g for 10 min, A532, A600 and A450 of the supernatant were recorded. MDA content was estimated by the formula C (μmol/L)

All data were subjected to ANOVA test and the means were compared by the Duncan's test. Comparisons with p<0.05 were considered significant difference. Pearson correlation analysis between parameters was performed to test for relationships between variables by

On the testing day, the weather was fine when the temperature and photon flux density (*PFD*) exhibited a single peak curve (Fig.1). In the morning (6:00 am), the air temperature

**3.1 Diurnal variation of air temperature and light intensity in the orchard** 

Fig. 1. Diurnal variations of air temperature and light intensity in pear orchard

standard curve drawn with known hydrogen peroxide concentrations.

=6.45(A532-A600)-0.56A450.

**2.7 Statistical analysis** 

SPSS. 13 software.

**3. Results** 

was about 19℃,which linearly increased to the maximum temperature about 29℃ at noon (12:00), then decreased to 25.5℃ at dusk (6:00 pm). In the aspect of light intensity, the PFD was 65.6 μmol·m-2·s-1 in the morning, and the maximum was 1612 μmol·m-2·s-1 at noon.
