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

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 photosynthesis in pear leaves.

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: Intercellular CO2 concentration

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

Characteristics and Antioxidant Activity in Pear (*Pyrus Pyrifolia* Nakai) 245

Fig. 4. Effect of ALA on diurnal variations of performance index on absorption basis (A) and

Fig. 5 is the results of photosynthesis of pear leaves from the perspective of flux ratios of PSII photochemical reaction. There were differences in diurnal variation curve of *φPo*, *Ψo*,

Fig.5A shows that ALA treatment significantly increased the maximal photochemical efficiency of PSII (*φPo*≡*Fv/Fm*) of pear leaves compared with the control. The highest of *φPo* in diurnal variation curves was found at 8:00, and dropped to the lowest at 12:00, and then recovered in the afternoon, suggesting that the high light at noon time depressed the maximal photochemical efficiency of PSII, and ALA treatment was prone to alleviate the

In Fig. 5B, *Ψo*, a parameter of the PSII acceptor-side which means the possibility of a trapped exciton moves an electron into the electron transport chain beyond QA-, was generally higher in ALA-treated leaves than that of the control, and the diurnal means in the former

In Fig. 5C, *φEo*, another parameter of quantum yield for electron transport exhibited obvious diurnal variation in the day, which was lowest at 12:00 of noon time. ALA treatment significantly improved *φEo* of pear leaves, and the diurnal means of the former was 7%

Conversely, a single peak curve was found in the aspect of energy dissipation through heat (*φDo*), which reached the highest at 12:00, and ALA treatment depressed *φDo*, suggesting that ALA decreased non-photochemical energy dissipation in the pear leaves (Fig.5D).

performance due to electron transport probability (B) of pear leaves

**3.3.3 Flux ratios of PSII photochemical reaction** 

*φEo* and *φDo*.

photoinhibtion.

was 6.5% higher than that of the latter.

higher than that of the latter.

#### **3.3 Effects of ALA treatment on chlorophyll fast fluorescence characteristics 3.3.1 Fast induction curves of chlorophyll fluorescence**

Fig. 3 displays the fast fluorescence transients measured from 6:00 am to 18:00 pm in the control and ALA-treated pear leaves. There were many differences between OJIP curves at different time, however, the most important difference was found from the P-step, which was the highest at 6:00 am, then decreased to the lowest at 12:00 at noon, and recovered to higher levels in the afternoon. The valley of P level at noon might be a characteristic of photosynthetic midday nap, or photoinhibition under high light condition. From Fig. 3, it can be seen that the P was generally higher in ALA-treated leaves than that in the control, especially at noontide, which may suggest that ALA treatment was favorable to leaf photosynthesis against photoinhibition under high light stress.

#### **3.3.2 Performance index on absorption basis and performance index of electron transport**

The result of JIP-test showed that *PIABS*, the photosynthetic performance index on absorption basis, in the ALA treatment was generally higher than that of the control, although the trends of both diurnal variations were similar (Fig. 4A). Moreover, *PET*, the performance index of electron transport of PSII photochemical reaction was also higher in the ALA pretreatment than that of the control (Fig. 4B), suggesting that ALA treatment could improve photochemical electron transport and photosynthesis. The diurnal means of *PIABS* and *PET* were about 38% and 26% higher in the ALA treatment than that of the control, respectively.

Fig. 3 displays the fast fluorescence transients measured from 6:00 am to 18:00 pm in the control and ALA-treated pear leaves. There were many differences between OJIP curves at different time, however, the most important difference was found from the P-step, which was the highest at 6:00 am, then decreased to the lowest at 12:00 at noon, and recovered to higher levels in the afternoon. The valley of P level at noon might be a characteristic of photosynthetic midday nap, or photoinhibition under high light condition. From Fig. 3, it can be seen that the P was generally higher in ALA-treated leaves than that in the control, especially at noontide, which may suggest that ALA treatment was favorable to leaf

**3.3 Effects of ALA treatment on chlorophyll fast fluorescence characteristics** 

Fig. 3. Effect of ALA on diurnal variations of fast induction curves of chlorophyll a

**3.3.2 Performance index on absorption basis and performance index of electron** 

The result of JIP-test showed that *PIABS*, the photosynthetic performance index on absorption basis, in the ALA treatment was generally higher than that of the control, although the trends of both diurnal variations were similar (Fig. 4A). Moreover, *PET*, the performance index of electron transport of PSII photochemical reaction was also higher in the ALA pretreatment than that of the control (Fig. 4B), suggesting that ALA treatment could improve photochemical electron transport and photosynthesis. The diurnal means of *PIABS* and *PET* were about 38% and 26% higher in the ALA treatment than that of the control,

fluorescence (OJIP curve) of pear leaves. A: control, B: ALA treatment

**transport** 

respectively.

**3.3.1 Fast induction curves of chlorophyll fluorescence** 

photosynthesis against photoinhibition under high light stress.

Fig. 4. Effect of ALA on diurnal variations of performance index on absorption basis (A) and performance due to electron transport probability (B) of pear leaves

### **3.3.3 Flux ratios of PSII photochemical reaction**

Fig. 5 is the results of photosynthesis of pear leaves from the perspective of flux ratios of PSII photochemical reaction. There were differences in diurnal variation curve of *φPo*, *Ψo*, *φEo* and *φDo*.

Fig.5A shows that ALA treatment significantly increased the maximal photochemical efficiency of PSII (*φPo*≡*Fv/Fm*) of pear leaves compared with the control. The highest of *φPo* in diurnal variation curves was found at 8:00, and dropped to the lowest at 12:00, and then recovered in the afternoon, suggesting that the high light at noon time depressed the maximal photochemical efficiency of PSII, and ALA treatment was prone to alleviate the photoinhibtion.

In Fig. 5B, *Ψo*, a parameter of the PSII acceptor-side which means the possibility of a trapped exciton moves an electron into the electron transport chain beyond QA-, was generally higher in ALA-treated leaves than that of the control, and the diurnal means in the former was 6.5% higher than that of the latter.

In Fig. 5C, *φEo*, another parameter of quantum yield for electron transport exhibited obvious diurnal variation in the day, which was lowest at 12:00 of noon time. ALA treatment significantly improved *φEo* of pear leaves, and the diurnal means of the former was 7% higher than that of the latter.

Conversely, a single peak curve was found in the aspect of energy dissipation through heat (*φDo*), which reached the highest at 12:00, and ALA treatment depressed *φDo*, suggesting that ALA decreased non-photochemical energy dissipation in the pear leaves (Fig.5D).

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

rate in pear leaves.

the origin of the fluorescence rise (*Mo*)

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

expression of *Rubisco small subunit* gene at transcript level.

Characteristics and Antioxidant Activity in Pear (*Pyrus Pyrifolia* Nakai) 247

*Mo*, an approximate slope at the origin of the fluorescence rise, represents the maximum rate QA reduction. From Fig.6A, *M0* of pear leaves rose gradually in the morning and kept high level in the afternoon. However, ALA treatment significantly reduced *M0*, which was about 80% of the control in the diurnal mean, suggesting that ALA could decrease QA reduction

Fig. 6. Effect of ALA on diurnal variations of donor side and acceptor side parameter of PSII reaction of pear leaves. A: Amplitude of the K step (*Wk*); B: An approximation of the slope at

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

Fig. 5. Effect of ALA on diurnal variations of flux ratios of PSII photochemical reaction of pear leaves. A: Maximum quantum yield for primary photochemistry (*φPo*), B: Probability that a trapped exciton moves an electron into the electron transport chain beyond QA- (*Ψo*), C: Quantum yield for electron transport (*φEo*), D: Quantum yield for heat dissipation (*φDo*).

#### **3.3.4 Activity of donor side and acceptor side of PSII reaction**

Amplitude of the K step (*Wk*) as a parameter of the PSII donor-side, expresses the inactivity of the oxygen evolving complex (OEC). The smaller of the *Wk*, the stronger of the OEC activity is. As in the Fig. 6A, the diurnal variation of *Wk* showed a plateau from 10:00 am to 16:00 pm in the control, which was obviously higher than that of ALA treatment. This suggests that ALA treatment alleviated OEC inactivity at high light environment.

Fig. 5. Effect of ALA on diurnal variations of flux ratios of PSII photochemical reaction of pear leaves. A: Maximum quantum yield for primary photochemistry (*φPo*), B: Probability that a trapped exciton moves an electron into the electron transport chain beyond QA-

C: Quantum yield for electron transport (*φEo*), D: Quantum yield for heat dissipation (*φDo*).

Amplitude of the K step (*Wk*) as a parameter of the PSII donor-side, expresses the inactivity of the oxygen evolving complex (OEC). The smaller of the *Wk*, the stronger of the OEC activity is. As in the Fig. 6A, the diurnal variation of *Wk* showed a plateau from 10:00 am to 16:00 pm in the control, which was obviously higher than that of ALA treatment. This

suggests that ALA treatment alleviated OEC inactivity at high light environment.

**3.3.4 Activity of donor side and acceptor side of PSII reaction** 

(*Ψo*),

*Mo*, an approximate slope at the origin of the fluorescence rise, represents the maximum rate QA reduction. From Fig.6A, *M0* of pear leaves rose gradually in the morning and kept high level in the afternoon. However, ALA treatment significantly reduced *M0*, which was about 80% of the control in the diurnal mean, suggesting that ALA could decrease QA reduction rate in pear leaves.

Fig. 6. Effect of ALA on diurnal variations of donor side and acceptor side parameter of PSII reaction of pear leaves. A: Amplitude of the K step (*Wk*); B: An approximation of the slope at the origin of the fluorescence rise (*Mo*)
