**2.4 Determination of Rubisco initial activity and its coding gene expression**

Rubisco activity was determined spectrophotometrically by monitoring NADH oxidation at A340 (Lilley et al., 1974; Xia et al., 2009). Leaf samples were homogenized in a chilled mortar within the ice-cold extraction buffer solution, 40mM Tris–HCl (pH 7.6), which contained 10mM MgCl2, 0.25mM EDTA, 5mM glutathione, 2% β-mercaptoethanol and 1.5% PVP (W/V). The homogenate was centrifuged at 4℃ for 15 min at 10,000g. The resulting supernatant was used for assay of the enzyme. The reaction mixture contained 100 mM Tris–HCl buffer solution, which contained 12 mM MgCl2 and 0.4 mM EDTA (pH 7.8), 0.2 M NaHCO3, 5 mM NADH, 50 mM ATP, 50 mM creatine phosphate, 1 U of glyceraldehyde 3 phosphodehydrogenase and 1 U of 3-phosphoglycerate kinase. The activity was estimated after the addition of enzyme extract and 0.2 mM ribulose-1, 5- bisphosphate (RuBP). Enzyme activity was expressed as mmol CO2 fixed per min g FW.

To measure the gene expression, leaf RNA was extracted according to Louime et al. (2008). Firstly, a washing buffer, which contained 100 mM Tris-boric acid (pH 7.4), 0.35 mol/L sorbitol and 10% (w/v) PEG-6000, was added to remove the secondary material of samples in the free state. Then an extraction buffer, containing 0.25 M Tris-Boric acid (pH 7.4), 0.05 M EDTA, 2.5 M NaCl, 2% CTAB, 3% PVP, and 5% β-mercaptoethanol was used. The yield and

The experiment was started in the late of May 2010 in the Horticultural Experimental Station, Nanjing Agricultural University, Jiangsu Province China. Ten years old pear trees (*Pyrus pyrifolia* Nakai. 'Akemizu') were used, which were grew in the brown yellow soil with space of 4×5m. ALA solution in concentration of 0.5 mg·L-1 was sprayed to the leaves, with the distilled water as the control. The experimental trees were arranged in complete random design, with ten tree repeats for ALA treatment or control, respectively. The measurements of the gas exchange and chlorophyll fast fluorescence parameters were conducted one week after ALA spray at an interval of 2 hours from morning to dusk. And meanwhile, the leaf

The measurements of diurnal gas exchange parameters were carried out according to the method described by Wang et al. (2004) with a portable photosynthesis system CIRAS-2 (PP Systems, UK). At each time point, from 6:00 am to 18:00 pm, the net photosynthetic rate (*Pn*), intercellular CO2 concentration (*Ci*) and stomata conductance (*Gs*) were measured simultaneously under the photon flux densities (PFD) from a built-in light source equal to the natural light intensities with the ambient temperatures. Each measurement was

Chlorophyll fast fluorescence transient was measured by a Plant Efficiency Analyzer (Hansatech, UK), according to methods of Strasser et al. (1995) and Sun et al. (2009a). All the leaves were immediately exposed to a saturating light pulse (3000 mmol·m-2·s-1 PFD) for 1 s after dark adapted for 20min. Each transient obtained from the dark-adapted samples was analyzed according to the JIP-test (Srivastava et al., 1997; Li et al., 2005; Sun et al., 2009a).

Rubisco activity was determined spectrophotometrically by monitoring NADH oxidation at A340 (Lilley et al., 1974; Xia et al., 2009). Leaf samples were homogenized in a chilled mortar within the ice-cold extraction buffer solution, 40mM Tris–HCl (pH 7.6), which contained 10mM MgCl2, 0.25mM EDTA, 5mM glutathione, 2% β-mercaptoethanol and 1.5% PVP (W/V). The homogenate was centrifuged at 4℃ for 15 min at 10,000g. The resulting supernatant was used for assay of the enzyme. The reaction mixture contained 100 mM Tris–HCl buffer solution, which contained 12 mM MgCl2 and 0.4 mM EDTA (pH 7.8), 0.2 M NaHCO3, 5 mM NADH, 50 mM ATP, 50 mM creatine phosphate, 1 U of glyceraldehyde 3 phosphodehydrogenase and 1 U of 3-phosphoglycerate kinase. The activity was estimated after the addition of enzyme extract and 0.2 mM ribulose-1, 5- bisphosphate (RuBP).

To measure the gene expression, leaf RNA was extracted according to Louime et al. (2008). Firstly, a washing buffer, which contained 100 mM Tris-boric acid (pH 7.4), 0.35 mol/L sorbitol and 10% (w/v) PEG-6000, was added to remove the secondary material of samples in the free state. Then an extraction buffer, containing 0.25 M Tris-Boric acid (pH 7.4), 0.05 M EDTA, 2.5 M NaCl, 2% CTAB, 3% PVP, and 5% β-mercaptoethanol was used. The yield and

conducted at least 10 times, and the means were used to compare the ALA's effect.

**2.4 Determination of Rubisco initial activity and its coding gene expression** 

Enzyme activity was expressed as mmol CO2 fixed per min g FW.

samples were taken and stored in liquid nitrogen for subsequent analysis.

**2. Materials and methods 2.1 Plant growth and treatment** 

**2.2 Gas exchange parameters** 

**2.3 Chlorophyll a fast fluorescence and JIP test** 

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

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 electrophoresis on a 1% agarose gel containing ethidium bromide.
