**1. Introduction**

238 Artificial Photosynthesis

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Photosynthesis is the basis of fruit growth and development. The higher photosynthetic efficiency of tree leaves, the more photosynthate is accumulated, which is beneficial to tree growth, root development, flower bud initialization, and the ultimate guarantee of quality and yield of fruits.

5-Aminolevulinic acid (ALA) is a key precursor of all porphyrin compounds, such as chlorophyll (Chl), heme, and phytochrome (von Wettstein et al., 1995). Exogenous application of ALA at low concentrations was found to promote growth and yield of several crops and vegetables (Hotta et al., 1997a). It also improved chlorophyll content and gas exchange capacity of melon seedlings under low light and chilling conditions (Wang et al., 2004), increased CO2 fixation in the light, and suppressed the release of CO2 in darkness (Hotta et al., 1997a), and promoted salt tolerance of cotton plants by manipulating the Na+ uptake (Watanabe et al., 2000). ALA-based fertilizer also enhanced the photosynthetic rate, chlorophyll content, and stomatal conductance in spinach and date palm seedlings under salinity (Nishihara et al., 2003; Youssef and Awad, 2008). However when the plant was treated with exogenous ALA at high concentrations (such as ≥1000 mg/L), it was assumed that the induced chlorophyll intermediate accumulation acted as a photosensitizer for the formation of 1O2, triggering photodynamic damage in ALA-treated plants (Chakrabory et al., 1992). Thus, ALA could be used as a natural bioherbicide.

ALA has been suggested to be a new natural and environmental friendly regulator, which can be widely used in agriculture (Wang et al., 2003). However, whether it can be used in woody trees such as pear has not been reported, and the mechanisms of ALA regulation on plant growth have not yet been elucidated. In the work, we found ALA promotion on pear photosynthesis might be related with the increase of antioxidant enzyme activities, and well as H2O2, which might act as signaling molecules involved in the regulation process.

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

electrophoresis on a 1% agarose gel containing ethidium bromide.

recorded within 3 min after the start of the reaction at 1 min intervals.

**2.5 Determination of antioxidant enzymes** 

reduction of NBT (Beauchamp et al., 1971).

et al., 2008).

3 min.

Characteristics and Antioxidant Activity in Pear (*Pyrus Pyrifolia* Nakai) 241

quality of total RNA were measured by absorbance at 230, 260, and 280 nm (A260/A230 and A260/A280 ratios) using UV-spectrophotometer and by running samples on a 1.5% nondenaturing agarose gel electrophoresis. Message RNAs in total RNA solution were reversed transcribed to their complementary cDNA (I strand) by oligo-dT primer using MLV reverse

To amplify the cDNA produced from RNA by the RT reaction, PCR was performed according to the protocol. As a template, the RT product was used. According to published pear *Actin* (GenBank: GU830958.1) and *Rubisco small subunit* sequences (GenBank: D00572.1), two pairs of oligonucleotide primers were designed for expression analysis. Gene-specific primers for *Actin* (forward: 5'-CAATGTGCCTGCCATGTATG-3'; reverse: 5'- CCAGCAGCTTCCATTCCAAT-3') and for *Rubisco small subunit* (forward: 5'- CTTGGAATTTGAGTTGGAGAC-3'; reverse: 5'-GTAA GCGATGAAACTGATGC-3') were used in RT-PCR. Each pair of primers cycling parameters were *Actin*: 29 cycles, Tm 51℃ and *Rubisco small subunit*: 32 cycles, Tm 59℃. PCR products were analyzed following

One hundred milligrams of leaves were homogenized in 2 ml of 50 mM phosphate buffer (pH 7.8) which contained 0.4 % polyvinyl pyrrolidone (PVP), an inhibitor of phenolic compounds in a pre-chilled mortar and pestle on ice. The homogenate was centrifuged at 10 000×g for 20 min at 4 °C and the supernatant was collected as crude enzyme extraction (Tan

Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured by monitoring the inhibition of nitro blue tetrazolium (NBT) reduction at 560 nm. The reaction mixture (3 ml) contained 195 mM methionine, 1.125 mM NBT, 3 μM EDTA, and 100 μl of enzyme extract in 50 mM PBS (pH 7.8). After addition of 20 μM riboflavin, the cuvettes were exposed to a 15- W circular "white light" tube for photoreaction 10 min. Then the reaction mixture was measured as absorbance of 1 cm cuvette at 560 nm. One unit of SOD activity was defined as the amount of enzyme per fresh mass sample causing 50 % inhibition of the photochemical

Ascorbate peroxidase (APX, EC 1.11.1.11) activity was assayed by measuring the oxidation of ascorbate at 290 nm according to Nakano and Asada (1981). Total 3 ml of reaction solution contained 50 mM PBS (pH 7.0), 1 mM H2O2, and 1mM ascorbate. The reaction was started by adding 100 μl of enzyme extraction. Changes of absorbance at 290 nm were then

Catalase (CAT, EC 1.11.1.6) activity was determined according to the method of Zavaleta-Mancera et al. (2007). The total reaction mixture (3 mL) contained 50 mM PBS pH 7.0 and 100 μL of enzyme extract. The reaction was initiated by the addition of 10 mM H2O2. The decomposition was followed directly by the decrease in absorbance at 240 nm every 20 s for

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

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

transcriptase Kit (Takara Bio) according to the manufacturer's recommendations.
