2012 BRAZILIAN SOCIETY OF PLANT PHYSIOLOGY RESEARCH ARTICLE The chlorophyll a fluorescence as an indicator of the temperature stress in the leaves of Prunus persica Emanuela Garbin Martinazzo1, Aline Ramm1, Marcos Antonio Bacarin1* 1Universidade Federal de Pelotas, Instituto de Biologia, Departamento de Botânica, Laboratório de Metabolismo Vegetal, Pelotas, RS, Brazil. *Corresponding author: [email protected] Received: 11 July 2012; Accepted: 13 November 2012 ABSTRACT Plants growing in temperate regions are often exposed to stress conditions caused by high temperatures. Photosynthesis is one of the metabolic processes most sensitive to high temperature stress, and it is often inhibited before other cellular functions. Using peach leaf disks, we studied the transient chlorophyll a fluorescence along with the gas exchanges at temperatures of 25 (control) 30, 35, 40 and 45°C in the dark for a period of 30 minutes. Temperatures above 35°C caused significant changes in the transient fluorescence of chlorophyll a, including an increase in the initial fluorescence (F0), a decrease in maximum fluorescence values (FM) and the appearance of K and L bands. The values of the net assimilation rate decreased as the temperature increased and reached negative values at 45°C. Keywords: high temperature, photosynthesis, Prunus persica. INTRODUCTION reduction) due to greater reduction of the plastoquinone (PQ) pool by ferredoxin (Fd) at high temperatures (Tóth The chlorophyll (Chl) a fluorescence represents a et al., 2007). In contrast, photosystem II (PSII), particularly very small fraction of the energy that is dissipated from the oxygen-evolving complex (OEC), is deactivated even the photosynthetic mechanism, but it is widely used to at slightly elevated temperatures (Yamane et al., 1998), provide information about the structure and function demonstrating that this process is especially sensitive to of the electron transport chain (Strasser et al., 2004). temperature stress (Pushpalatha et al., 2008). Measuring Chl a fluorescence provides basic information about the photosynthetic mechanism of plants; it has When photosynthetic samples adapted to the dark been used to detect changes in photosystem II (PSII) are illuminated, changes occur in the intensity of the Chl a that occur under stress conditions, and it can easily be fluorescence. This is known as the Kautsky effect (Strauss performed using intact leaves or isolated chloroplasts et al., 2006). The transient curve displays a rapid increase (Yamane et al., 2000). that ends in less than one second, with a subsequent slow decline toward a steady state. It is postulated that the phase Photosynthesis is highly sensitive to stress caused of rapid increase represents the primary photosynthesis by extreme temperatures, and it is often inhibited before reactions (Strauss et al., 2006). When this transient other cellular functions are harmed (Chen and Cheng, phase is monitored with high-resolution fluorometers, 2009). Plants exposed to high temperatures exhibit it provides precise information on the energetic state of two opposite effects in the electron transport chain of the photosynthetic mechanisms (Strasser et al., 1995). photosynthesis (Tóth et al., 2007). Photosystem I (PSI) It is known that the kinetics of fluorescence transients is stimulated by heat (as measured by the rate of P700+ are polyphasic when plotted on a logarithmic time scale. Braz. J. Plant Physiol., 24(4): 237-246, 2012 238 MARTINAZZO E. G. et al. Such plots clearly indicate the intermediate J and I steps Analysis of the induction of Chl a fluorescence: between the initial step O and the maximum final level P Measurements of the Chl a fluorescence were taken (Strasser and Govindjee, 1992). immediately after each temperature treatment using a Handy-PEA portable fluorometer (Hansatech Instruments The JIP-test, proposed by Strasser and Strasser Ltd., King’s Lynn Norfolk, UK). The fluorescence transient (1995), is used to translate the original measurements of was induced by red light, with a peak at 650 nm and fluorescence transient into various phenomenological and approximately 3,000 µmol m-2 s-1, and was provided by biophysical expressions that quantify the function of PSII a set of three light-emitting diodes with a recording time (Tóth et al., 2007). The JIP-test can be applied to in vivo of 1 s. Fluorescence intensity was measured between 50 studies of the photosynthetic process because the typical µs and 1 s. fluorescence transient is sensitive to the stress caused by changes in many environmental conditions, making JIP-test, normalisation and subtractions of the it a very sensitive indicator of the biophysical processes fluorescence transient curves: The fluorescence intensities of the electron transport chain (ETC) (Tóth et al., 2007). determined at 50, 100 and 300 µs (F50µs, F100µs and F300µs, The inhibition of the ETC is followed by the functional respectively), 2 and 30 ms (F2ms = FJ and F30ms = FI) separation of the light-harvesting complexes of PSII and at FM (maximum fluorescence) were used to calculate (LHCII), blocking off the flux of electrons from QA to QB the JIP-test parameters (Strasser and Strasser 1995). (Petkova et al., 2007). The intensity measured at 50 µs was considered to be the initial fluorescence (F0). The Chl a fluorescence transient Thermal stress causes significant changes in the was analysed using the JIP-test using ‘Biolyzer’ software fluorescence transient of chlorophyll a, including an (Laboratory of Bioenergetics, University of Geneva, increase in the initial fluorescence (F0) and a decrease in Switzerland). the maximum fluorescence values (FM). An additional peak at approximately 0.3 ms can also be observed, which is For comparison of the events represented by the OK, called band K (Oukarroum et al., 2012). OI and IP phases, the transient curves were normalised as the relative variable fluorescence as: WOK = (Ft - F0)/ The objective of this study was to evaluate the effects (FK - F0), WOJ = (Ft - F0)/(FJ - F0), WOI = (Ft - F0)/(FI - F0) and of different temperatures on photosynthetic activity, as WIP = (Ft - FI)/(FM - FI) (Tsimilli-Michael and Strasser, 2008). measured by the Chl a fluorescence and gas exchange For analysis of the different kinetics, the divergences rates in peach leaf disc. (differences) between the relative variable fluorescence curves of the stress treatments and control were calculated (ΔW = Wtreatment - Wcontrol); this procedure reveals bands that are normally hidden between steps O and P on the relative MATERIAL AND METHODS variable fluorescence. Plant material and treatments: Adult peach plants Leaf gas exchange: After determining the fluorescence (Prunus persica) of the PE-36 cultivar were grown in an transient, the rates of CO2 exchange were measured using experimental field of the Federal University of Pelotas, a portable CO2 analyser (model LI-6400XT LI-COR, Inc., Capão do Leão Campus, RS, Brazil (31°48’11’’S and Lincoln, NE, USA). The measurements were performed in -1 52°24’58’’W). Mature completely leaves were collected, a chamber with a CO2 concentration of 380 µmol mol and a and leaf discs with an area of 1,000 mm2 were extracted. photon flux density of 1,200 µmol m-2 s-1. Twelve leaves were used per treatment, and they were conditioned using clips. The leaf disc and clip combination Statistical analysis: The results obtained for the was wrapped in absorbent paper that had been moistened parameters of the JIP test were subjected to an analysis with distilled water, and it was protected by an envelope of variance (ANOVA); when the F-test was significant, a made of aluminium foil. These packets were exposed to comparison of means was performed via the Tukey’s test. different temperatures (25, 30, 35, 40 and 45°C) for a period of 30 min by immersion in a water bath. The 25°C treatment was used as the control. RESULTS After immersion, the samples were carefully removed from the water bath so that the Chl a fluorescence transient The leaf discs incubated at 25, 30 and 35°C exhibited and gas exchange be measured. typical fluorescence transient curves. However, those that Braz. J. Plant Physiol., 24(4): 237-246, 2012 THE CHLOROPHYLL A FLUORESCENCE AS AN INDICATOR OF THE TEMPERATURE STRESS IN THE LEAVES OF PRUNUS PERSICA 239 were incubated at 40 and 45°C displayed imbalances with For the fluorescence intensity at step J (FJ), the greatest the characteristic OJIP steps (Figure 1). values were identified at a temperature of 25°C and the lowest values at 35 and 40°C. The fluorescence In Table 1, it can be observed that F0 and F300μs display intensity at step I (FI) and the maximum fluorescence significant increases at 40 and 45°C, with their highest (FM) displayed significant decreases beginning at 35°C, values measured in the leaf discs exposed to 45°C. with the lowest values obtained at 40 and 45°C. (Table 1) 1.0 25C The flux of absorption and trapping per reaction ) center (RC) of PSII, defined as ABS/RC and TR /RC, t 30C 0 respectively, were significantly greater in the leaf discs 0.8 35C 40C incubated at 40 and 45°C (Table 1). Electron transport flux per RC of PSII defined as ET /RC began increasing 45C 0 0.6 at a temperature of 35°C; this value was similar at that at 40°C, but it was lower than the value obtained at 45°C. It was not possible to estimate the flux Reduction 0.4 of End-electron acceptors (RE0/RC) for temperatures of 40 and 45°C due to a change in the shape of the kinetic Relative variable uorescente (V fluorescence emission curve (Figure 1). Thus, values were 0.2 calculated only for temperatures between 25 and 35°C, with significant increases in the values accompanying the increase in temperature (Table 1). 0.0 0.1 1 10 100 1000 With regard to the parameters that describe the Time (ms) yields and efficiency of the electron transport chain (ETC), the following can be observed in Table 1: (a) maximum Figure 1.