Effect of Methanol on Photosynthesis and Chlorophyll Fluorescence of Flag Leaves of Winter Wheat

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Agricultural Sciences in China 2008. 714): 432-431

ScienceDirect

Auril 2008

Effect of Methanol on Photosynthesis and Chlorophyll Fluorescence of Flag Leaves of Winter Wheat ZHENG Yue-jin*,YANG Yue-qin*,LIANG Shan-shan and YI Xian-feng College of Agriculture, Henan University of Science and Technology, Luoyang 471003, P.R. China

Abstract Photosynthesis and chlorophyll a fluorescence parameters, photochemical efficiency of PS I1 (Fv/Fm), photochemical quenching of PS 11(qP), nonphotochemical quenching of PS I1 (NPQ), maximum activity of PS I1 (FvFo) as well as electron transport rate (ETR), and quantum yield of PS I1 (@PS 11) were measured on flag leaves of the winter wheat treated by methanol at different concentrations. The results revealed that photosynthesis was greatly improved by methanol, as indicated by higher photosynthetic rates and stomata1 conductance. The enhancement effect of methanol on photosynthesis was maintained for 3-4 days. Different methanol concentration treatments also increased intercellular CO, concentration and transpiration rates. No significant decline was found in FvfFm, Fv/Fo, and @PS 11, which revealed no photoinhibition during methanol application in different methanol concentrations. Methanol showing no apparent inhibitory effects indicated higher potential photosynthetic capacity of flag leaves of winter wheat. However, the increase in photosynthesis was not followed by an increase in the photosynthetic activity (FvEm), and fluorescence parameters did not indicate an improvement in intercellular CO, concentration and PS I1 photochemical efficiency compared with the control, thereby encouraging us to propose that lower leaf temperatures caused by applied methanol would reduce both dark respiration and photorespiration (most importantly), thus, increasing net CO, uptake and photosynthetic rates.

Key words: methanol, photosynthetic activity, chlorophyll fluorescence, flag leaf, winter wheat

INTRODUCTION In recent years, the technique of chlorophyll fluorescence has become ubiquitous in plant ecophysiology studies (Xu et al. 2005; Liu et al. 2006). No investigation into the photosynthetic performance of plants under field conditions seems complete without some fluorescence data. This trend has been fuelled, to a large degree, by the introduction of a number of highly user-friendly (portable) chlorophyll fluorometers. Chlorophyll a fluorescence has been used to evaluate photosynthetic performance under natural and some

stress conditions (Fv/Fm) (Waldhoff et al. 2002), photochemical quenching of PS I1 (qP), and nonphotochemical quenching of PS I1 (NPQ), as well as electron transport rate (ETR) and quantum yield of PS I1 (@PS 11). Organic solvents have been found to be effective on algal growth in the late 1980s (Stratton 1987; Stratton and Smith 1988) and the early1990s (Nonomura and Benson 1992; Ei Jay 1996). Methanol has been proved to show great positive effects on photosynthesis (Nonomuraand Benson 1992;Li and Yi 2004). However, understanding the effects of methanol on plants is still highly controversialbecause opposed conclusions were

Received 24 October, 2006 Accepted 22 August, 2007 Correspondence YI Xian-feng, E-mail: [email protected], Tel: +86-379-64282340. Fax:+86-379.64282340. * These authors contributed equally to this work.

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Effect of Methanol on Photosynthesis and Chlorophyll Fluorescence of Flag Leaves of Winter Wheat

made concerning the effects on the photosynthetic activity and the increase of biomass. A lot of investigations have shown that the production and photosynthetic activity of algae were increased at low concentration of methanol (Theodoridou et al. 2002). The same effects were also found in higher plant species when sprayed with 10-50% methanol (Nonomura and Benson 1992; Li et al. 1995; Li and Yi 2004)and when cultured in 0.2-5 mmol L-' methanol (Gout et al. 2000). However, these investigations mainly focused on influence of methanol on growth (Okumura et al. 2001) and its metabolic process (Gout et uE. 2000), few touched on photosynthetic performance and chlorophyll a fluorescence parameters. And at present, there is a lack of understanding on the dependency between the effect of methanol on carbon accumulation and the appropriate energy dissipation through the primary process of photosynthesis (Li et al. 1995), especially, for monocotyledons. In this study, we investigated the stirnulatory as well as the inhibitory effects of methanol on chlorophyll a fluorescence and photosynthesis when the higher plant species such as winter wheat was treated by methanol at different concentrations.

MATERIALSAND METHODS Study area The study was conducted in 2006 in the arid and semiarid transition area in Luoyang City (elevation averaged at 600 m, 34" 35'N, 112"24'E), Henan Province, China. The climate of experimental site was dominated by the warm and temperate zonal continental monsoon. The annual average air temperature was 14.8"C. The average annual precipitation was at 578 mm.

Experimentaldesign Each plot in this experiment was 5 m2 and arranged in the north-south direction. Winter wheat variety, Jinfeng 3 , was selected and planted. A total of 12 plots were established and randomly distributed, spaced 1 m apart along a transect line in a 200 m2area. Three duplications were, respectively, set for the control and methanol treatment at different concentrations.

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Protocols Flag leaves of winter wheat were evenly sprayed by methanol in 0 (distilled water), 2, 5, and 8% concentrations from 1O:OO to 11:00 am in natural conditions at the milk stage in the field. Photosynthetic rate, stomata1conductance (GS),transpiration rate and intercellular CO, concentration were measured using portable photosynthesis systems (CIRA-1, PP Systems, Ltd., UK) 20 min after spraying. Photosynthesis induction light intensity was set at 1000 pmol m-2s-I. The measurement of Chl a fluorescence induction and parameters was done at field temperatureby using a pulse amplitude-modulated fluorometer (FMS-2, Hansatech Ltd., Norfolk, UK) as described by Rohacek and Bartak (1999). The minimal (dark) (Fo) and maximal (Fm) fluorescence yield was obtained with weak modulated light (0.04 pmol m-2 s-I), then with a 1 s pulse of saturating light (5 OOO p o l m-* s-'). The ratio of Fv/Fm was served as a measure of the maximum photochemical efficiency of PS II. Photochemical quenching (qP) and nonphotochemical quenching (NPQ) were calculated according to Schreiber et al. (1986). The efficiency of energy conversion in PS I1 (@PSIT) was calculated as (Fm'- Fs)/Fm' (Fs = stationary level of fluorescence emission, Fm' = maximum fluorescence during illumination) (Genty et al. 1989). The level of (1- qP) was calculated after van Kooten and Snel(l990). Before taking fluorescence measurements, the leaves were adapted to darkness for 20 min. To evaluate the electron transport condition, total electron flow rate (J,) was calculated according to Krall and Edward (1992): J,=@PS 11x PPFD x a x f, where PPFD refers to photosynthetic photon flux density, a indicates the proportion of light that leaves absorb, and f represents the proportion of light energy distributed to PS 11. The noncyclic photosynthetic electron flow in photorespiration (J,) and carbon reduction (J,) are expressed as J, = 2/3 [J, - 4 (P, +RJ and J, = 1/3 [J, + 8 (P, + Rp)], respectively (Epron et al. 1995). Rates of oxidation and carboxylation are obtained from: J, =4V, +6V, and V,=P,+Rp+0.5Vo(Di et al. 1994),where Pn symbolizes net photosynthetic rate and RPrefers to respiration rate of mitochondrion under light, i.e., photorespiration rate.

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test whether there was a long-term effect of methanol. The results proved a 3-4 day potential of methanol to enhance flag leave photosynthesis of winter wheat (Fig. l), especially, at the concentration of 5%.

RESULTS Photosynthesis As shown in Table 1, photosynthetic CO, assimilation (photosynthetic rate) of flag leaves of winter wheat treated by methanol at different concentrations (2-8%) increased significantly compared with the control, with the highest effect being that of 5% methanol (t=-2.573, df= 13, P = 0.023). No significant difference was observed between three treatments (chi-square= 0.105, df= 2, P = 0.949). Methanol application at different concentrations (2-8%) also apparently enhanced stomata1 conductance of flag leaves (chi-square = 10.271, df= 3, P = 0.016) (Table 1). Stomata conductance induced by 5% methanol was 2.42 times as that of the control. Corresponding with stomata conductance, transpiration rates increased with the treatments of methanol but did not show a significance (chi-square = 4.827, df= 3, P = 0.185). The higher intercellular CO, concentrations were also observed when administered with different methanol concentrations (chi-square = 6.967, df= 3, P = 0.07)

Chlorophyll a fluorescence parameters No significant changes were observed in PS I1 photochemical activity (chi-square = 1.985, df= 3, P = 0.576), i.e., FvFm (Table 2), indicating no occurrence of photoinhibition under administration of methanol. Simultaneously, the maximum ratio of photochemical

l9

r

+

Control

+ 0%

18 17

.-

16 15 14 I

8

13

1

12 1

(Table 1). Application of distilled water (0%methanol) had slight but no significant influence on photosynthetic process, as indicated by the net photosynthetic rate, stomata conductance' and transpiration rate' we continuously measured photosynthesis for 5 days to

I

2

1

3

5

4

Days after application of methanol at different concenrrations(d)

Fig. 1 Changes in photosynthetic rates in winter wheat flag leaves treated with different concentration methanol.

Table 1 Photosynthetic parameters of flag leaves of winter wheat treated by methanol at different concentrations Treatments Control 0% methanol 2% methanol 5% methanol 8% methanol

Transpiration rate ( k o l H.0 m - 2 sI) 5.29 f 0.41 5.03 f 0.32 5.76 i 0.32 5.92 i 0.30 6.16 i 0.29

Stomatal conductance (urn01co, m - 2 s1) 1007.63 f 215.63 984.35 f 119.28 1836.33 f 383.48 2439.63 f 289.72 2 371.63 f 650.63

Photosynthetic parameters Photosynthetic rate (urn01co. m-2 SI) 14.85 i 2.23 14.44 f 1.82 17.79 f 3.88 18.13 f 1.12 18.08 i 0.87

Intercellular CO, concentration (Darn) 282.00 f 8.25 286.76 f 9.03 288.67 i 14.30 302.38 f 2.63 300.00 f 5.00

Leaf temperature ( 0 0 ~~

~

32.4 f 1.2 31.5 f 1.0 30.6 f 1.8 27.3 f 1.1 25.0 f 0.8

Table 2 Chlorophyll a fluorescence parameters of flag leaves of winter wheat treated by methanol at different concentrations Chlorophyll fluorescence parameters FvFo

FvlFm QPS I1 qp

NPQ ETR

Treatments Control 6.18 0.72 0.86 f 0.01 0.20 f 0.02 0.35 f 0.10 1.31 iO.39 84.28 f 2.2

*

0% methanol 5.92 f 0.68 0.85 0.02 0.20 f 0.01 0.36 f 0.08 1.30 f 0.28 85.34 f 3.7

*

2% methanol 5.69 0.82 0.85 0.02 0.21 f 0.06 0.37 i0.07 1.22 i 0.48 104.16 i 5.8

* *

5% methanol 5.73 i 0.55 0.85 f 0.01 0.21 f 0.03 0.34 f 0.09 1.29 f 0.47 88.23 i 3.3

8% methanol 5.60 f 0.24 0.85 f 0.01 0.20 i 0.02 0.31 f 0.05 1.23 f0.15 84.04 f 3.2

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Effect of Methanol on Photosynthesis and Chlorophyll Fluorescence of Flag Leaves of Winter Wheat

quantum yields and concurrent nonphotochemical processes, Fv/Fo, decreased slightly along the methanol concentrations (chi-square = 2.005, df= 3, P = 0.571). OPS 11, which reflects the actual quantum yield of electron transport in PS 11, did not show significant change (chi-square= 1.819, df=3, P=0.611) (Table 2). Application of distilled water (0% methanol) showed no significant effect on Fv/Fm and QPS I1 compared with the control.

Quenching coefficients Table 2 indicated that qP increased by 2%methanol treatment but decreased by 8%, and no change was found when treated with 5 % methanol. Nonphotochemical quenching (NPQ) is thought to protect the photochemical apparatus against the destructive effects of excess light energy (Weis and Berry 1987). However, no significant decrease in NPQ was induced by methanol with different concentrations compared with the control. ETR was not improved when methanol was supplied at different concentrations (Table 2).

The ratio of noncyclic photo-respiratory electron flow to total electron flow rate, Jo/J, of winter wheat flag leaves accounted for 12.81, 6.84, 6.05, and 3.46% in the control and three treatments, respectively. However, the ratio of noncyclic electron flow for photosynthetic carbon reduction to total electron flow rate, JJJ, of winter wheat flag leaves showed no

0%

2%

5%

significant change in the control and the three treatments (40.06, 40.81, 40.91 and 41.23% respectively) (Fig. 2). Rates of oxidation of Rubisco (V,) were recorded as 2.03, 1.13, 1.00, and 0.55 pmol m-'s-' in controlled and treated leaves, whereas carboxylation rates of Rubisco (V,) were 18.03,20.37,20.55,and 20.19 pmol rn-, s-' in the corresponding leaves. The ratio of V0N, was significantly declined in the methanol treatment (Fig. 3). However, we did not witness the differences in V,, V,, and V,N,of winter wheat leaves treated by 0% methanol and the control.

:

0 Control

0.00

0%

2%

5%

8%

Methanol concentrations

Fig, 3 Changes in the ratios of oxidation to carboxylation (V,/V,) in winter wheat flag leaves treated with different concentration methanol.

Photosynthetic electron distribution

Control

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8%

Methanol concentrations

Fig. 2 Changes in the ratios of J,/J, and JJJ, in winter wheat flag leaves treated with different concentration methanol.

DISCUSSION The results were consistent with our previous study on leaf-used lettuce (Li and Yi 2004). Treatments by methanol at different concentrationspromoted stomatal behavior, thereby enhancing the transpiration rate and stomatal conductance (Table 1 ) . Increase in photosynthetic rate induced by methanol was similar with those by David et al. (2003), who showed that the rate of oxygen evolution and photosynthetic rate of Lemna gibba increased under lower methanol concentrations. Slight increases of intercellular CO, concentration were observed when administered with different concentrations of methanol, which may be due to an increase in the availability of CO, via the cellular transformation of methanol (Nonomura and Benson 1992), and consequently a decrease in nonphotochemical energy dissipation was induced, which resulted in efficient nicotinamide adenine

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dinucleotide phosphate (reduced) and adenosine triphosphate synthesis, therefore benefiting photosyntheticaccumulation (Table 2). The combination of methanol as carbon source has been investigated by Cossin (1964) and Gout et al. (2000). It was postulated that methanol might be incorporated into carbon metabolism as a single carbon compound (Cossin 1964; Nonomura and Benson 1992; David et al. 2003), like formaldehyde and serine as primary products (Gout et al. 2000). But whether there is methanol oxidase or not in higher plants still remain controversial, despite its existence in methanol-used alga. As seen from Tables 1 and 2, the increase in the photosynthetic rate induced by methanol was not followed by an increase in the photosynthetic activity. Actually, the PS I1 activity supported by the water splitting system and the oxygen evolution were not significantly increased (Table 2). Moreover, there was no improvement in the PS I1 photochemical efficiency (FvLFm) compared with the control. When compared with the control, no change was seen for fluorescence parameters concerning the PS I1 activity, such as the PS I1 quantum yield (FvLFm), the basal quantum yield of nonphotochemical processes (Fo/Fm), the maximum ratio of photochemical quantum yields (FvLFo), and the photochemical quenching value (qP) under methanoltreated conditions (Table 2). Although NPQ plays an important role in excessive solar energy dissipation, no change was detected after administration of methanol with different concentrations. However, it is still unknown why the increase in photosynthesis was not linearly correlated with the photosynthetic activity as indicated by Fv/Fm and @PS 11. Therefore, the improvement in the photosynthesis treated with a higher concentrationof methanol appeared to be related to other mechanisms. As reported in Table 1, most remarkable differences were found in leaf stomatal conductance, accompanied by only modest changes in transpiration. The only way by which this could occur, physically, is that the leaf temperatures were lower in the methanol treated leaves than in the control. As we know, lower leaf temperatures would reduce both dark respiration and photorespiration (most importantly). The inconsistency between transpiration rate and stomatal conductance encouraged us to propose that lower leaf temperatures caused by applied methanol would reduce

both dark respiration and (most importantly) photorespiration, thus, increasing net CO, uptake and photosynthetic rate. The effect is even maintained for several days as revealed by a 3-4 day potential of methanol to enhance photosynthesis in flag leaves of winter wheat. This hypothesis was not followed by David et al. (2003), who proposed that energy distribution and decline in nonphotochemical quenching were related to the increasing net CO, uptake of Lemna gibba under lower methanol concentrations. For flag leaves of winter wheat treated by methanol at different concentrations, the ratio Fv/Fo, as the maximal quantum yield of photochemical and concurrent non-photochemical processes of PS I1 (Rohacek and Bartak 1999), was decreased. Therefore, toxicity of methanol with higher concentrations to some degrees is shown, here, on flag leaves of winter wheat. The real effect of methanol shown as an inhibition or a stimulation of photosynthesis has to be further investigated at the molecular level because it appeared, here, that the effect of methanol on flag leaves of winter wheat is a complex of interactions between energy storage via photosynthesis and nonphotochemical energy-dissipative processes. The mechanism of methanol by which a decrease in leaf temperature and consequent depression in the photorespiration rates needs further investigation.

Acknowledgements The study was supported by the Student Research Training Program of Henan University of Science and Technology (2007142) and Program for Science and Technology Innovation Talents in Universities of Henan Province, China (2008 HASTIT003).

References Cossin R. 1964. The utilization of carbon-I compounds by plants. The metabolism of methanol-14Cand its role in amino acid biosynthesis. Canadian Journal of Biochemistry, 44, 1739-1802. David D, Claire D, Phillippe J, Guy V, Radovan P. 2003. Effects of methanol on photosynthetic processes and growth of Lemna gibba. Photochemistry and Photobiology, 78, 420424. Di M, Iannell M A, Loreto. 1994. Relationship between photosynthesis and photorespiration in field-grown wheat

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Effect of Methanol on Photosynthesis and Chlorophyll Fluorescence of Flag Leaves of Winter Wheat

leaves. Photosynthetica, 3 0 , 4 5 51. Ei Jay A. 1996. Effects of organic solvents and solvent-atrazine interations on two algae, Chloralla vulgaris and Alenastrum capricomutum. Archive of Environmental Contamination and Toxicology, 31, 84-90. Epron D, Godard D, Comic G, Genty B. 1995. Limitation of net CO, assimilation rate by internal resistance to CO, transfer in the leaves of two tree species (Fagus sylvatica L. and Castanea sativa Mill). Plant Cell and Environment, 18,4351. Genty B, Briantais J M, Baker N R. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta, 99,87-92. Gout E, Aubert S , Bligny R, Rebeille F, Nonomura A R, Benson A A, Douce R. 2000. Metabolism of methanol on plant cells. Carbon- 13 nuclear magnetic resonance studies. Plant Physiology, 123,287-296. van Kooten 0, Snel J F H. 1990. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynthetic Research, 25, 147-150. Krall J P, Edward G E. 1992. Relationshipbetween photosystem I1 activity and CO, fixation in leaves. Physiology Plant, 86, 180-187. Li Z R, Yi, X F. 2004. Effect of methanol with different concentration on photosynthesis and yield of leaf-used lettuce. Journal of Inner Mongolia Normal University, 33, 71-73. (in Chinese) Li Y, Gupta G, Joshi J M, Siyumbano A K. 1995. Effect of methanol on soybean photosynthesis and chlorophyll. Journal of Plant Nutrition, 18, 1875-1880. Liu W H, Gao D S, Shu H R. 2006. Effects of different photon flux density on the characteristics of photosynthesis and chlorophyll fluorescence of peach trees in protected culture. Scientia Agricultura Sinica, 39,2069-2075. (in Chinese)

437

Nonomura A R, Benson A A . 1992. The path of carbon in photosynthesis: improved crop yields with methanol. Proceedings of the National Academy of Sciences of the USA, 89, 9794-9798. Okumura Y, Koyama J, Takaku H, Satoh H. 2001. Influence of organic solvents on the growth of marine microalgae. Archive of Environmental Contamination and Toxicology, 41, 123128. Rohacek K, Bartak M. 1999. Technique of modulated chlorophyll fluorescence: basic concepts, useful parameters, and some applications. Photosysthetica, 37,339-363. Schreiber U, Schliwa U, Bilger W. 1986.Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluometer. Photosynthetic Research, 10,5 1-62. Stratton G W. 1987. Toxic effects of organic solvents on the growth of blue-green algae. Bulletin of Environmental Contamination and Toxicology, 38, 1012-1019. Stratton G W, Smith T M. 1988. Interaction of organic solvents with the green alga Chlorella pyrenoidosa. Bulletin of Environmental Contamination and Toxicology, 40,736-742. Theodoridou A, Dornemann D, Kotzabasis K. 2002. Lightdependent induction of strongly increased mocroalgal growth by methanol. Biochimica et Biophysica Acta, 1573,189-198. Waldhoff D, Furch B, Junk W J. 2002. Fluorescence parameters, chlorophyll concentration, and anatomical features as indicators for flood adaptation of an abundant tree species in central Ammonia: Symmeria paniculata. Environmental and Experimental Botany, 48,225-235. Weis E, Berry J A. 1987. Quantum efficiency of photosystem I1 in relation to energy-dependent quenching of chlorophyll fluorescence.Biochimica et Biophysica Acta, 894,198-208. Xu K, Guo Y P, Zhang S L. 2004. Effect of light quality on photosynthesis and chlorophyll fluorescence in strawberry leaves. Agricultural Sciences in China, 3,369-375. (Edited by ZHANG Yi-min)

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