**4. Discussion**

Previous reports have demonstrated that ALA treatment can improve the net photosynthetic rate in spinach (Nishihara et al., 2003), melon (Wang et al., 2004), pakchoi (Wang et al., 2004b), radish (Hotta et al., 1997b; Wang et al., 2005b), strawberry (Liu et al., 2006) and watermelon (Sun et al., 2009a), under normal or stress conditions. The result of this work confirmed that exogenous ALA at a concentration of 0.5 mg/L could increase the net photosynthetic rate of pear leaves (Fig. 2A), which were related with the increase of *Pn/Ci* (Fig. 2B) and *Gs* (Fig.2C). This means that ALA treatment did not only promote stomatal opening, but also affect the non-stomatal factors related with photosynthesis (Farquhar and Sharkey, 1982). In other studies, exogenous ALA promotion on stomatal opening has been reported in melon (Wang et al., 2004a) or watermelon (Kang et al., 2006). A transgenic tobacco, which could over-produce endogenous ALA, also possessed higher stomatal conductivity (Zhang et al., 2010). Therefore, ALA inducing stomatal conductance might be a universal phenomena. However, the mechanism need further to be elucidated.

The shape of the OJIP transient has been found to be sensitive to stress such as excess light, temperature and drought (Appenroth et al., 2001; Thach et al., 2007). In our data (Fig. 3), the P-step in pear leaves was significantly lower at noon than that in the morning or at dusk, suggesting the environmental factors at noon impaired chlorophyll fast fluorescence yield. Wang et al. (2005a) suggested that the optimal temperature for photosynthesis of pear leaves was about 27℃. In this work, the highest temperature was 29℃ (Fig. 1), which was near to the theoretic optimal temperature, and might not be the key inhibitory factor for pear photosynthesis. Instead, the typical midday nap characteristic of pear leaves was possible to be result of the high light intensity at noontide (Fig. 2, Fig.3). ALA treatment increased the fluorescence yield, especially at noontide, suggesting that ALA could promote resistance of pear leaves against high light stress. Sun et al. (2008) also found that ALA treatment could alleviate photoinhibition of watermelon seedlings switched to high light from shaded condition. Our results here were similar with the previous observation. Because ALA treatment could also improve leaf photosynthesis of plants grown under low light condition (Wang et al., 2004; Sun and Wang, 2007), it can be deduced that ALA might stabilize photosynthetic capacity against light stresses. In fact, this effect of ALA is important because plants are not always grown under optimal light intensity condition, and low light or high light often affects plant photosynthesis. ALA improvement on photosynthesis under light stresses can increase photosynthate accumulation in many crops.

The mechanisms of ALA improvement on photosynthesis by non-stomata have been mentioned in many aspects. Firstly, ALA increased the chlorophyll content, since it is the key biosynthetic precursor of all tetrapyrrole compounds (von Wettstein et al., 1995; Jahn

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

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

Previous reports have demonstrated that ALA treatment can improve the net photosynthetic rate in spinach (Nishihara et al., 2003), melon (Wang et al., 2004), pakchoi (Wang et al., 2004b), radish (Hotta et al., 1997b; Wang et al., 2005b), strawberry (Liu et al., 2006) and watermelon (Sun et al., 2009a), under normal or stress conditions. The result of this work confirmed that exogenous ALA at a concentration of 0.5 mg/L could increase the net photosynthetic rate of pear leaves (Fig. 2A), which were related with the increase of *Pn/Ci* (Fig. 2B) and *Gs* (Fig.2C). This means that ALA treatment did not only promote stomatal opening, but also affect the non-stomatal factors related with photosynthesis (Farquhar and Sharkey, 1982). In other studies, exogenous ALA promotion on stomatal opening has been reported in melon (Wang et al., 2004a) or watermelon (Kang et al., 2006). A transgenic tobacco, which could over-produce endogenous ALA, also possessed higher stomatal conductivity (Zhang et al., 2010). Therefore, ALA inducing stomatal conductance might be a universal phenomena. However, the mechanism need further to be elucidated. The shape of the OJIP transient has been found to be sensitive to stress such as excess light, temperature and drought (Appenroth et al., 2001; Thach et al., 2007). In our data (Fig. 3), the P-step in pear leaves was significantly lower at noon than that in the morning or at dusk, suggesting the environmental factors at noon impaired chlorophyll fast fluorescence yield. Wang et al. (2005a) suggested that the optimal temperature for photosynthesis of pear leaves was about 27℃. In this work, the highest temperature was 29℃ (Fig. 1), which was near to the theoretic optimal temperature, and might not be the key inhibitory factor for pear photosynthesis. Instead, the typical midday nap characteristic of pear leaves was possible to be result of the high light intensity at noontide (Fig. 2, Fig.3). ALA treatment increased the fluorescence yield, especially at noontide, suggesting that ALA could promote resistance of pear leaves against high light stress. Sun et al. (2008) also found that ALA treatment could alleviate photoinhibition of watermelon seedlings switched to high light from shaded condition. Our results here were similar with the previous observation. Because ALA treatment could also improve leaf photosynthesis of plants grown under low light condition (Wang et al., 2004; Sun and Wang, 2007), it can be deduced that ALA might stabilize photosynthetic capacity against light stresses. In fact, this effect of ALA is important because plants are not always grown under optimal light intensity condition, and low light or high light often affects plant photosynthesis. ALA improvement on photosynthesis under light

transfer and photosynthetic dark reaction.

not adverse in the experimental condition.

stresses can increase photosynthate accumulation in many crops.

The mechanisms of ALA improvement on photosynthesis by non-stomata have been mentioned in many aspects. Firstly, ALA increased the chlorophyll content, since it is the key biosynthetic precursor of all tetrapyrrole compounds (von Wettstein et al., 1995; Jahn

**4. Discussion** 

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 factor for leaf photosynthesis.

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 maximal photochemical efficiency was improved.

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

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 ALA regulation on gene transcription needs to be elucidated further.

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

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

Science+Business Media. 2009, 29-42

synthase. Photosynthetica, 2008, 46: 3-9

Michx.). Euro J Sci Res, 2008, 22:232-240

Occident Sin, 2006, 26:57-62

388-395

1997, 1320: 95-106

47(3):347-354

occident Sin, 2008, 28: 1384-1390

Characteristics and Antioxidant Activity in Pear (*Pyrus Pyrifolia* Nakai) 255

Hotta Y, Tanaka T, Takaoka H, Takeuchi Y, Konnai M. New physiological effects of 5-

Hotta Y, Tanaka T, Takaoka H, Takeuchi Y, Konnai M. Promotive effects of 5-aminolevulinic acid on the yield of several crops. Plant Growth Regul, 1997a, 22:109-114 Jahn D, Heinz DW. Biosynthesis of 5-aminolevulinic acid. In: Tetrapyrroles: Birth, Lift and

Jung S, Back K, Yang K, Kuk YI, Chon SU. Defence response produced during

Kang L, Cheng Y, Wang LJ. Effects of 5-aminoluveulinic acid (ALA) on the photosyntehsis

Li PM, Gao HY, Strasser RJ. Application of the fast chlorophyll fluorescence induction

Lilley RM, Walker DA. An improved spectrophotometric assay for ribulose-bisphosphate

Liu WQ, Kang L, Wang LJ. Effect of 5-aminolevulinic acid (ALA) on photosynthesis and its

Liu YM, Ai XZ, Yu XC. Effects of ALA on photosynthesis of cucumber seedlings under suboptimal temperature and light intensity. Acta Hort Sin, 2010, 37: 65-71 Louime C, Vasanthaiah HKN, Jittayasothorn Y, Jiang L, Basha SM, Thipyapong P, Boonkerd

Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in

Neill S, Desikan R, Hancock J. Hydrogen peroxide signaling. Curr Opin Plant Biol, 2002, 5:

Nishihara E, Kondo K, Parvez MM, Takahashi K, Watanabe K, Tanaka K. Role of 5-

Srivastava A, Guissé B, Greppin H, Strasser RJ. Regulation of antenna structure and electron

Strasser RJ, Srivastava A, Govindjee. Polyphasic chlorophyll-alpha fluorescence transient in

Sun YP, Wang LJ. Effects of 5-aminolevulinic acid (ALA) on chlorophyll fluorecence dynamics of watermelon seedlings under shade condition. Acta Hort Sin, 2007, 34: 901-908 Sun YP, Wei ZY, Zhang ZP, Wang LJ. Protection of 5-aminolevulinic acid (ALA) on high

Sun YP, Zhang ZP, Wang LJ. Promotion of 5-aminolevulinic acid treatment on leaf

spinach chloroplasts. Photochem Photobiol, 1981, 37: 679-690

spinach (*Spinacia oleracea*). Plant Physiol, 2003, 160: 1085- 1091

plants and cyanobacteria. Photochem Photobiol, 1995, 61: 32-42

and plant growth. Biosci Biotech Biochem, 1997b, 61:2025-2028

and winter. Acta Bot Boreal-Occident Sin, 2006, 26: 2297-2301

carboxylase. Biochim Biophys Acta, 1974, 358: 226-229

aminolevulinic acid in plants: the increase of photosynthesis, chlorophyll content,

Death, ed by Warren M, Smith AG. Landes Bioscience and Springer

photodynamic damage in transgenic rice overexpressing 5-aminolevulinic acid

and anti-oxidative enzyme activity of leaves of greenhouse watermelon in summer

dynamics analysis in photosynthesis study. J Plant Physiol Mol Biol, 2005, 31: 559-566

relationship with antioxidant enzymes of strawberry leaves. Acta Bot Boreal

N. A simple and efficient protocol for high quality RNA extraction and cloning of chalcone synthase partial cds from muscadine grape cultivars (*Vitis Rotundifolia*

aminolevulinic acid (ALA) on active oxygen-scavenging system in NaCl treated

transport in PSII of *Pisum sativum* under elevated temperature probed by the fast polyphasic chlorophyll a fluorescence transient: OKJIP. Biochim Biophys Acta,

light photoinhibition of watermelon grown under shade condition. Acta Bot Boreal-

photosynthesis is related with increase of antioxidant enzyme activity in watermelon seedlings grown under shade condition. Photosynthetica, 2009a,

activity in transgenic rice than in the wild type. The same was true in transgenic tobacco and *Arabidopsis* (Wang et al., unpublished). Thus, enhancement of SOD activity was accompanied with increase of endogenous or exogenous ALA levels. However, the mechanism of SOD activity induced by ALA has no been known.

That ALA induced the increase of enzymes eliminating H2O2, such as APX, CAT and POD, has been suggested (Nishihara et al., 2003; Liu et al., 2006). Since ALA is the essential biosynthetic precursor of tetrapyrrole compounds including heme, and the latter is a necessary component for the activity of all three enzymes (Tsiftsoglou et al., 2006), it is reasonable to deduce that ALA induces heme accumulation, which is beneficial for H2O2 eliminating enzyme activity. In this work, it was also observed that ALA induced increase of activities of APX and CAT in pear leaves (Fig. 9). However, ALA treatment also increased the content of H2O2 in pear leaves, which might be at a safe level, because MDA content with ALA treatment was significantly lower than the control (Fig. 8). In Table 1, H2O2 level was correlated with *PET, Ψ0*, *φE0*, the activities of SOD, APX, CAT and Rubisco initial activity, as well as transcript of gene coding *Rubisco small unit*. This means that H2O2 might be an active signal molecule rather than an adverse ROS, involved in regulation of antioxidant enzyme activity and physiological or molecular processes. H2O2 has been suggested as a cellular signal, and has wide-ranging effects in many biological processes (Finkel and Holbrook, 2000). It can also regulate gene expression in plants (Neill et al., 2002; Apel and Hirt, 2004). However, whether ALA promotion on plant photosynthesis is dependent on H2O2 signal need further study.
