**3.6 Activities of antioxidant enzymes**

248 Artificial Photosynthesis

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.

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

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

content; B: MDA content

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 in the early morning and evenfall.

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 control.

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

Fig. 9. Effect of ALA on diurnal variations of antioxidant enzymes activities of pear leaf. A: SOD; B: APX; C: CAT

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

Characteristics and Antioxidant Activity in Pear (*Pyrus Pyrifolia* Nakai) 251

Table 1. Correlations between environmental factors, gas exchange, chlorophyll fast

fluorescence, antioxidant activity and Rubisco initial activity in pear leaves

### **3.7 Analysis of correlation between environmental factors, photosynthetic parameters and antioxidant activities**

The results of Pearson correlation analysis by SPSS 13.0 showed that there were a lot of high correlations between environmental factors, gas exchange, chlorophyll fast fluorescence, antioxidant activity and Rubisco initial activity in pear leaves, significant at either the 0.05 level or the 0.01 level (Table 1).

Firstly, the light intensity (*PFD*) was positively correlated with air temperature, *Pn, Pn/Ci*, *Wk* and *φD0*, but negatively correlated with *Ci*, *φP0* and CAT activity, which means that high light intensity led to increase of temperature, net photosynthetic rate and instantaneous carboxylation efficiency, and meanwhile, OEC was possibly inactivated and the absorbed energy dissipation through heat was increased. Additionally, the intercellular CO2 concentration was prone to decrease, and the maximal photochemical efficiency and CAT activity were also inhibited under high light stress.

Secondly, air temperature was positively correlated with *Wk*, *M0*, and *φD0*, but negatively with *Ci, PIABS*, *φP0*, APX and CAT activity. This may mean that high temperature led to inactivate both donor and acceptor sides of PSII reaction center in pear leaves. Therefore, photosynthetic performance index on absorption basis and the maximal photochemical efficiency decreased under high temperature. Additionally, the activities of two H2O2 eliminating enzymes APX and CAT decreased as temperature increased.

Among the gas exchange characteristics, *Pn* was closely related with *Pn/Ci* and *Gs*, but negatively with *Ci*, which means that under the experimental condition, *Ci* was beyond a limited factor for photosynthesis. *Pn* was also positively with *PET*, SOD and Rubisco initial activity. Therefore, the electron transport activity of PSII reaction center, O2·¯ scavenging enzyme SOD activity and the enzyme for CO2 fixation were key for net photosynthetic rate.

Among the fluorescence indexes, *PIABS* was positively correlated with *PET*,Ψ*<sup>0</sup>*, *φP0* and *φE0*. Meanwhile, *PIABS* was also highly correlated with the activities of SOD, APX and CAT. Additionally, it was correlated with the relative expression of *Rubisco small unit* coded gene (*P*=0.005).

The initial activity of Rubisco was significantly correlated with *Pn*, *Pn/Ci*, *PET*, *Ψ0*, *φE0*, expression of the coding gene, and SOD activity, which means that O2 · ¯ scavenging enzyme activity and photochemical electron transfer activity were closely related with the CO2 fixation enzyme activity. Furthermore, the expression of gene coding *Rubisco small unit* was highly correlated with *PIABS*, *PET*, Ψ*<sup>0</sup>*, *φE0*, H2O2 content and three antioxidant enzyme activity.

H2O2, a famous reaction oxygen species, was correlated with *PET*,Ψ*<sup>0</sup>*, *φE0*, the activities of SOD, APX and CAT, the Rubisco initial activity and the relative expression of *Rubisco small unit* coded gene, which might imply the ROS was a active factor for photosynthesis of pear leaves under the experimental condition. MDA, a lipid peroxidation product, was the only parameter, negatively correlated with *PET*,Ψ*<sup>0</sup>*, *φE0*, and SOD activity, which might be an adverse factor for photosynthesis.

Among the antioxidant enzymes, APX and CAT activities were negatively correlated with *PFD* and (or) temperature, although the coefficient between APX and *PFD* was -0.518, slight higher than the 0.05 level, which means the enzymes was affected by environmental factors. Nevertheless, SOD activity was not affected by *PFD* or temperature. SOD was significantly correlated with *Pn, PIABS*, *PET, φ0*, *φE0*, APX, CAT, Rubisco initial activity, and the relative expression of *Rubisco small unit* coded gene, but negatively with *M0* and MDA content,

**3.7 Analysis of correlation between environmental factors, photosynthetic parameters** 

The results of Pearson correlation analysis by SPSS 13.0 showed that there were a lot of high correlations between environmental factors, gas exchange, chlorophyll fast fluorescence, antioxidant activity and Rubisco initial activity in pear leaves, significant at either the 0.05

Firstly, the light intensity (*PFD*) was positively correlated with air temperature, *Pn, Pn/Ci*, *Wk* and *φD0*, but negatively correlated with *Ci*, *φP0* and CAT activity, which means that high light intensity led to increase of temperature, net photosynthetic rate and instantaneous carboxylation efficiency, and meanwhile, OEC was possibly inactivated and the absorbed energy dissipation through heat was increased. Additionally, the intercellular CO2 concentration was prone to decrease, and the maximal photochemical efficiency and CAT

Secondly, air temperature was positively correlated with *Wk*, *M0*, and *φD0*, but negatively with *Ci, PIABS*, *φP0*, APX and CAT activity. This may mean that high temperature led to inactivate both donor and acceptor sides of PSII reaction center in pear leaves. Therefore, photosynthetic performance index on absorption basis and the maximal photochemical efficiency decreased under high temperature. Additionally, the activities of two H2O2

Among the gas exchange characteristics, *Pn* was closely related with *Pn/Ci* and *Gs*, but negatively with *Ci*, which means that under the experimental condition, *Ci* was beyond a limited factor for photosynthesis. *Pn* was also positively with *PET*, SOD and Rubisco initial activity. Therefore, the electron transport activity of PSII reaction center, O2·¯ scavenging enzyme SOD activity and the enzyme for CO2 fixation were key for net photosynthetic rate.

Meanwhile, *PIABS* was also highly correlated with the activities of SOD, APX and CAT. Additionally, it was correlated with the relative expression of *Rubisco small unit* coded gene

The initial activity of Rubisco was significantly correlated with *Pn*, *Pn/Ci*, *PET*, *Ψ0*, *φE0*,

activity and photochemical electron transfer activity were closely related with the CO2 fixation enzyme activity. Furthermore, the expression of gene coding *Rubisco small unit* was

SOD, APX and CAT, the Rubisco initial activity and the relative expression of *Rubisco small unit* coded gene, which might imply the ROS was a active factor for photosynthesis of pear leaves under the experimental condition. MDA, a lipid peroxidation product, was the only

Ψ

Among the antioxidant enzymes, APX and CAT activities were negatively correlated with *PFD* and (or) temperature, although the coefficient between APX and *PFD* was -0.518, slight higher than the 0.05 level, which means the enzymes was affected by environmental factors. Nevertheless, SOD activity was not affected by *PFD* or temperature. SOD was significantly correlated with *Pn, PIABS*, *PET, φ0*, *φE0*, APX, CAT, Rubisco initial activity, and the relative expression of *Rubisco small unit* coded gene, but negatively with *M0* and MDA content,

Ψ

·

*<sup>0</sup>*, *φE0*, H2O2 content and three antioxidant enzyme

Ψ

*<sup>0</sup>*, *φE0*, and SOD activity, which might be an

*<sup>0</sup>*, *φP0* and *φE0*.

¯ scavenging enzyme

*<sup>0</sup>*, *φE0*, the activities of

eliminating enzymes APX and CAT decreased as temperature increased.

Among the fluorescence indexes, *PIABS* was positively correlated with *PET*,

expression of the coding gene, and SOD activity, which means that O2

H2O2, a famous reaction oxygen species, was correlated with *PET*,

Ψ

**and antioxidant activities** 

level or the 0.01 level (Table 1).

(*P*=0.005).

activity.

highly correlated with *PIABS*, *PET*,

adverse factor for photosynthesis.

parameter, negatively correlated with *PET*,

activity were also inhibited under high light stress.


Table 1. Correlations between environmental factors, gas exchange, chlorophyll fast fluorescence, antioxidant activity and Rubisco initial activity in pear leaves

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

maximal photochemical efficiency was improved.

factor for leaf photosynthesis.

Characteristics and Antioxidant Activity in Pear (*Pyrus Pyrifolia* Nakai) 253

and Heinz, 2009), which has been suggested to contribute to increase of photosynthesis (Tanaka et al., 1993). However, in the most cases, the chlorophyll content was not a limiting

ALA has been suggested to increase the activity of OEC at the donor side of PSII reaction center under stress condition. Sun et al. (2009b) found that *Wk*, which represented the inhibition of OEC activity, was lower in ALA-treated leaves than that in the control of watermelon seedlings under chilling stress. Zhang et al. (2010) observed that *Wk* in the transgenic tobacco with capacity to over- produce endogenous ALA was also lower than that of the wild type. In the work, *Wk* in ALA-treated pear leaves was always lower than that of the control (Fig. 6A). Thus, ALA promotion in OEC activity might be a general effect. ALA might improve the photochemical efficiency of PSII reaction center. Whether the darkadapted or light-adapted maximal photochemical efficiency, it has been reported that ALA had significant effect (Sun et al., 2009a; Wang et al., 2010). Recently, the similar effect was confirmed in the transgenic tobacco (Zhang et al., 2010). In this work, we observed that *φP0* of pear leaves treated by ALA was higher than that of the control (Fig. 5A), suggesting the

ALA might also improve the activity of acceptor side of PSII reaction center. Two important fluorescence parameters *Mo* and *ψ0*, where the *M0* represents the proximate rate of QA completely being reduced, and *ψ0* was the probability of a trapped exciton moves an electron into the electron transport chain beyond QA- , often responded to ALA treatment. In most situations, ALA decreased *M0* but stimulated *ψ0*, which was beneficial to electron transfer through QA- electron acceptor of PSII reaction center (Strasser et al., 1995; Li et al., 2005). The results in the work approved the previous observations that QA was retardant to be completely reduced (Fig. 6B) and the electron was easily transferred to the downstream

Liu et al. (2010) suggested that ALA treatment alleviated the decrease of Rubisco activity of cucumber under suboptimal temperature and light intensity stress. In this work, we found the diurnal variation of Rubisco initial activity in pear leaves (Fig. 7A), which was improved by ALA treatment and highly correlated with *Pn* (r=0.835, *P*<10-5). It was the first time to observe that ALA could up-regulate transcription of gene coding *Rubisco small unit* in pear leaves (Fig. 7). The level of transcript in ALA-treated leaves at 8:00 am was more than 2 times as high as that of the control, which means that ALA treatment did not only affect light reaction of photosynthesis, but also dark reaction, even the expression of the key enzyme. The effect of ALA has been not mentioned before. However, the mechanism of

In aspect of antioxidant enzymes, it has been suggested that ALA treatment stimulated SOD activity around PSI reaction center, which can scavenge ROS aroused from photosynthetic electron transport in electron transfer chain to improve photochemical electron transfer rate (Sun et al., 2009a, b). Diethyldithiocarbamate (DDC), an inhibitor of Cu-Zn-SOD, could eliminate ALA' effect on photochemical efficiency (Liu et al., 2006; Sun et al., 2009a), which suggested the important role of SOD on ALA promotion. In this work, ALA treatment also induced SOD activity in pear leaves (Fig. 9A), which was positively correlated with many photosynthesis and chlorophyll fluorescence parameters but negatively with *M0* and the MDA content (Table 1), suggesting that it might play an important role on the acceptor side activity of PSII reaction center and preventing lipid peroxidation of photosynthetic apparatus during daytime. On the other hand, Jung et al. (2008) found higher levels of SOD

electron acceptors beyond QA- in the chain after ALA treatment (Fig. 5B).

ALA regulation on gene transcription needs to be elucidated further.

suggesting that SOD was not only important in prevention of lipid peroxidation, but also in prevention PSII reaction center close, and therefore promotion of photochemical electron transfer and photosynthetic dark reaction.

Additionally, the correlations of APX and CAT activities were similar with the SOD in the most parameters. This means that three antioxidant enzymes synergized in eliminating reaction oxygen species to prevent peroxidation of lipid in plant cells. However, the correlations of MDA with APX and CAT were not significant (*P*>0.05), implying the role of APX and CAT activity was not enough to impact lipid peroxidation, i.e, the H2O2 level was not adverse in the experimental condition.
