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RESEARCH PAPER Analysis of elevated temperature-induced inhibition of photosystem II using chlorophyll a fluorescence induction kinetics in wheat leaves (Triticum aestivum) S. Mathur, A. Jajoo, P. Mehta & S. Bharti School of Life Science, Devi Ahilya University, Indore, MP, India INTRODUCTION Wheat is commercially one of the most important crops, especially in tropical countries. However, tropical countries also have high temperatures during the growing season. Crop yield is greatly affected by elevated temperature stress and has been directly correlated with decreased photosynthetic effi- ciency (Georgieva et al. 2000). Photosynthesis is one of the most heat-sensitive processes in plants, leading to numerous changes in structure and function of the photosynthetic apparatus. Elevated temperature-induced detrimental effects are manifested in reduced photosynthetic metabolism (Koves Pechy et al. 1998), photosynthetic capacity and photochemi- cal efficiency (Percival 2005). Within the photosynthetic apparatus, photosystem II (PSII) is the most thermolabile component of the electron transport chain (Cajanek et al. 1998). Damage to PSII is often the first manifestation of stress in a leaf. Among partial reactions of PSII, the oxygen- evolving complex (OEC) is particularly heat sensitive (Geor- gieva et al. 2000). A study of the structural and functional behaviour of PSII can allow examination of the primary effects of stress caused by elevated temperature. In recent years, measurement of chlorophyll (Chl a) fluo- rescence has become ubiquitous in plant ecophysiology stud- ies. By measuring the yield of chlorophyll fluorescence, information about changes in the efficiency of photochemis- try and heat dissipation can be gained (Maxwell & Johnson 2000). The most significant realization of the relationship between primary reactions of photosynthesis and Chl a fluo- rescence came from Kautsky & Hirsch (1931). They were the first to report that, following illumination of a dark-adapted photosynthetic sample, Chl a fluorescence emission is not constant but exhibits a fast rise to a maximum followed by a decline to a steady state over some minutes. Chlorophyll flu- orescence has been used to provide a rapid, non-destructive diagnostic method for detecting and quantifying damage to the leaf photosynthetic apparatus, particularly PSII activity in response to environmental stress. At a given moment, the shape of the fluorescence transient of any sample is deter- mined by the physiological state of the sample. In this study, we tried to exploit this characteristic of fluorescence tran- sients in order to reveal elevated temperature-induced responses in wheat plants. Analysis of the intermediate data points of the fast fluorescence rise forms the basis of what is termed the ‘OJIP curve’, whose shape is universal for all photosystems containing Chl a. Measurements of fluorescence induction kinetic have been made in many plant species, including soybean, potato and pea, where one or two parameters such as Fv ⁄Fm and Fv ⁄Fo were calculated from OJIP curves (Cajanek et al. 1998; Geor- gieva et al. 2000). Heat-induced multiple effects on PSII in wheat have also been studied (Lu & Zhang 2000), as well as the heterogeneous response of PSII to heat stress in Spirulina platensis (Zhao et al. 2008) and soybean (Li et al. 2009). However, none of these studies has provided an exclusive and extensive analysis of OJIP curves in wheat under temper- ature stress, as presented here. This study aims to investigate Chl a fluorescence induction profiles in wheat leaves under elevated temperature stress. We analysed the fluorescence transients exhibited by wheat in Keywords Chla fluorescence; elevated temperature; photosystem II; wheat. Correspondence A. Jajoo, School of Life Science, Devi Ahilya University, Indore 452017, MP, India. E-mail: anjanajajoo@hotmail.com Editor R. Leegood Received: 5 November 2009; Accepted: 2 December 2009 doi:10.1111/j.1438-8677.2009.00319.x ABSTRACT Wheat is the major crop plant in many parts of the world. Elevated temperature- induced changes in photosynthetic efficiency were studied in wheat (T. aestivum) leaves by measuring Chl a fluorescence induction kinetics. Detached leaves were subjected to elevated temperature stress of 35 �C, 40 �C or 45 �C. Parameters such as Fv/Fm, performance index (PI), and reaction centre to absorbance ratio (RC/ABS) were deduced using radial plots from fluorescence induction curves obtained with a plant efficiency analyser (PEA). To derive precise information on fluorescence induction kinetics, energy pipeline leaf models were plotted using bio- lyzer hp3 software. At 35 �C, there was no effect on photosynthetic efficiency, including the oxygen-evolving complex, and the donor side of PSII remained active. At 40 �C, activity was reduced by 14%, while at 45 �C, a K intermediate step was observed, indicating irreversible damage to the oxygen-evolving complex. This anal- ysis can be used to rapidly screen for vitality and stress tolerance characteristics of wheat growing in the field under high temperature stress. Plant Biology ISSN 1435-8603 Plant Biology 13 (2011) 1–6 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands 1 response to temperature stress to provide information about changes taking place in the structure, conformation and func- tion of the photosynthetic apparatus, especially in PSII. By using a single parameter of fluorescence induction kinetics, the analysis has provided valuable information on the sites of reversible and irreversible damage taking place in PSII, which directly affect the photosynthetic efficiency of wheat. The work is particularly significant in an era of global climate changes, where most crop yields are decreasing as a result of increasing temperatures. MATERIALS AND METHODS Plant material The Lok-1 cultivar of wheat (Triticum aestivum) was used for the experiments. Seeds were germinated then transferred to Petri plates containing Knop nutrient solution, under a pho- tosynthetically active photon flux density (PFD) of 300 lmol m)2 s)1, at 20 �C and a photoperiod of 20 h ⁄ 4 h light ⁄dark. The seeds were daily replenished with distilled water. The plantlets were grown to the two-leaf stage and then high tem- perature treatment was given to fully developed, cut (detached) leaves. The leaves were kept in darkness (40 min) before treatment, and the elevated temperature stress was given in the dark. Elevated temperature treatment The wheat leaves were immersed in a water bath (Julabo- F10) for 15 min at temperatures of 25 �C, 35 �C, 40 �C or 45 �C. Measurements were performed 5 cm from the tip and the base, i.e. in the middle portion on the abaxial surface of leaves. Thirty measurements were done at each temperature. Fluorescence induction transients Chl a fluorescence was measured at 25 �C using a plant effi- ciency analyser (PEA, Hansatech, King’s Lynn, Norfolk, UK). Excitation light of peak wavelength 650 nm was focused on the surface of the leaf to provide a homogeneous spot about 4 mm in diameter. Light intensity used was 3,000 lmol m)2 s)1 to generate maximal fluorescence (Fm) for all the treat- ments. For energy pipeline models, biolyzer hp3 software, a Chl a fluorescence analysis program (Bioenergetics Labora- tory University of Geneva, Switzerland) was used. RESULTS AND DISCUSSION Chlorophyll a fluorescence transients were measured in con- trol and heat-treated wheat leaves. The fluorescence parame- ters recorded were: 1 Minimal fluorescence (Fo), as a measure of the stability of the light harvesting complex (LHC) (Yamada et al. 1996). Residual dark reduction of the quinone (QA) via the plasto- quinone (PQ) pool was proposed to contribute to a rise in Fo (Bukhov & Carpentier 2000; Yamane et al. 2000). 2 The ratio of variable (Fv = Fm ) Fo) to maximal (Fm) fluorescence (Fv ⁄Fm, which represents the maximum quan- tum yield of PSII) is often correlated with the quantum yield of net photosynthesis (Adams et al. 1995; Li et al. 2008, 2009). 3 OJIP curves to monitor the effects of the PQ electron accepter reaction, the PQ pool size, of PSII (shown on log scale to allow visualization of the complete fluorescence tran- sient) (Kruger et al. 1997; Srivastava et al. 1998). OJIP curves were prepared using the PEA software, and the parameters Fv ⁄Fm, reaction centre to absorbance ratio (RC ⁄ABS) and performance index (PI) were deduced. Energy pipeline membrane models were plotted using biolyzer hp3 software. A typical Chl a induction curve is shown in Fig. 1. Leaves incubated at 25 �C exhibit a polyphasic rise, called the OJIP Chl a fluorescence transient; in which the O–J phase lasts �2 ms, J–I phase lasts �30 ms and I–P phase lasts �500 ms. The JIP test is named after the basic steps in the fluorescence transient when plotted on a logarithmic time scale (Force et al. 2003). The O–J phase is due to net photochemical reduction of QA–Q � A . The intermediate I step and final P step have been proposed to be due to the existence of fast and slow reducing plastoquinone (PQ) pools, as well to different redox states of the RC of PSII, which reduce the PQ pool (Govindjee 1995; Haldimann & Strasser 1999). Effects of elevated temperature stress on Chl a fluorescence induction kinetics are shown in Fig. 2. In response to ele- vated temperature stress (45 �C), an additional K step was observed at 300 ls. The alteration from an OJIP curve to an OKJIP curve appears to be a specific response to elevated temperature stress. Such alterations have not been recorded elsewhere in plants subjected to environmental stresses encountered in urban landscapes, such as elevated ozone, salt, CO2, heavy metals, light or water (Meinander et al. 1996; Laza´r & Iliek 1997; Lu & Zhang 1998; Pospisil et al. 1998). The K step was dominant at 45 �C, followed by a pro- nounced dip and later by a slight increase to a highly sup- pressed P step. The appearance of a K step may be caused by inhibition of the OEC (Guisse´ et al. 1995a,b; Laza´r et al. 1997b; Srivastava et al. 1997; Laza´r & Pospisil 1999), inhibi- tion of electron transport from pheophytin to QA (Guisse´ et al. 1995b) or may reflect changes in the structure of the LHC of PSII (Srivastava et al. 1997). The K step arises when elec- tron flow to the acceptor side exceeds electron flow from the Fig. 1. A typical Chla polyphasic fluorescence rise O-J-I-P in an untreated wheat leaf. Elevated temperature-induced changes in PSII of wheat Mathur, Jajoo, Mehta & Bharti 2 Plant Biology 13 (2011) 1–6 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands donor side, leading to oxidation of the RC. Thus, injury to the OEC due to heat stress, for example, induces the K step, by inhibiting efficient electron donation to the RC (Strasser 1997; Strasser et al. 2004). From the induction curves in Fig. 2, parameters like Fv ⁄Fm and PI were deduced (Tables 1 and 2). The expression Fv ⁄Fm is an excellent measure of the quan- tum yield of primary photochemistry of PSII. It has been suggested that elevated temperature stress decreases the quan- tum efficiency of PSII photochemistry through: (i) a decrease in the rate of primary charge separation; (ii) a reduction in the stabilisation of charge separation; (iii) an increase in the recombination rate constant of the radical pair of the RC; and (iv) the disconnection of some minor antenna from PSII (Briantais et al. 1996). As the temperature increased, a dra- matic decline in Fv ⁄Fm was observed, together with an increase in Fo. This decrease shows that the photochemical yield of PSII has been affected. However, since the Fv ⁄Fm ratio alone cannot accurately reveal alterations in PSII behav- iour, we also studied other parameters. An increase in Fo has been attributed to physical separation of the PSII RC from associated pigment antennae, resulting in blocked energy transfer of the PSII traps, although a part of this phenome- non could possibly reflect accumulation of the reduced form of Q�A . Fo can be used as an indicator for irreversible damage in PSII, associated with LHCII dissociation and blocking of electron transference on the reductant side of PSII (Costa et al. 2002). The value of Fm declined dramatically with the rise in temperature, indicating that elevated temperature caused damage to the donor side of PSII. After elevated temperature stress, a significant decrease in the intensity of P was observed (Fig. 2). The decrease in P is related to heat inacti- vation of the OEC. Thus, the decrease in the value of P again indicates that the inactive donor side has very limited capac- ity for donating electrons, and that the donor side of PSII, especially the OEC, is damaged. Quantitative analysis of the fluorescence transients, the so- called JIP test, can be used to explain the stepwise flow of energy through PSII at the RC level, as well as the level of the PSII cross-section. The most popular parameter of the JIP test is the performance index (PI). The photosynthetic PI is an indicator of sample vitality. It is the combined measure- ment of the RC ⁄ABS, maximal energy flux that reaches the PSII RC and electron transport at the onset of illumination. PI can be calculated as: PIABS ¼ RC=ABS � UPO=ð1� UPOÞ �W0=ð1�W0Þ where FPo, is the exciton trapped per photon absorbed, and Wo, is the probability that an electron can move further than Q�A (Strasser et al. 2000, 2004). The PI combines three independent functional steps of photosynthesis, the density of RCs in the chlorophyll bed, excitation energy trapping and conversion of excitation energy to electron transport, into a single multi-parametric expression (Strasser et al. 1999; Tsimilli-Michael et al. 2000). This is a function of Wo, FPo, and RC ⁄ABS (Srivastava et al. 1999). The value RC ⁄ABS represents the antenna size of chlo- rophyll molecules. Efficiency with which a trapped exciton can move an electron into the electron transport chain fur- ther than Q�A is Wo. A decrease in RC ⁄ABS means a decrease in the size of the chlorophyll antenna serving each RC. The PI decreased with the increase in temperature. Com- pared to the control, 40 �C reduced PI to 51% (49% dam- age) and 45 �C left only 1.5% (99% damage), which indicates that the leaves were in an absolute stress condition (Table 2) and also that their vitality had been damaged due to the rise Table 1. Effect of elevated temperature on Chla fluorescence parameters as derived from the fluorescence induction curves shown in Fig. 2. treatment Fo Fm Fv Fv ⁄ Fm 25 �C 233 ± 10.29 (100) 1353 ± 62.29 (100) 1120 ± 52.44 (100) 0.828 ± 0.01 (100) 35 �C 253 ± 10.19 (108) 1348 ± 44.32 (99) 1095 ± 36.44 (97) 0.812 ± 0.01 (98) 40 �C 283 ± 9.11 (121) 995 ± 34.77 (73) 712 ± 34.91 (63) 0.716 ± 0.01 (86) 45 �C 490 ± 18.20 (210) 737 ± 29.46 (54) 247 ± 12.32 (22) 0.335 ± 0.01 (40) values at 35 �C are insignificant as only less than 5% damage was observed at this temperature, but above 35 �C the changes are significant. Table 2. Effect of elevated temperature treatment on performance index (PI) and its components as derived from the fluorescence induction curves in Fig. 2. treatment FPo ⁄ (1 ) FPo) RC ⁄ABS PI 25 �C 4.806 ± 0.07 (100) 0.711 ± 0.043 (100) 2.014 ± 0.20 (100) 35 �C 4.327 ± 0.12 (90) 0.680 ± 0.05 (95) 1.718 ± 0.21 (85) 40 �C 2.514 ± 0.15 (52) 0.571 ± 0.08 (80) 1.033 ± 0.28 (51) 45 �C 0.503 ± 0.01 (10) 0.101 ± 0.01 (14) 0.031 ± 0.01 (1.5) changes in the values of various parameters at all temperatures are signifi- cant. Fig. 2. Changes in Chla fluorescence induction curves in wheat leaves treated at elevated temperatures. 25 �C (•), 35 �C (�), 40 �C (h) and 45 �C (D). Mathur, Jajoo, Mehta & Bharti Elevated temperature-induced changes in PSII of wheat Plant Biology 13 (2011) 1–6 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands 3 in temperature, leading to a decrease in FPo with elevated temperature. The FPo (Fv ⁄Fm = TRO ⁄ABS) is a parameter that expresses maximum efficiency of PSII, which is con- trolled by the primary photochemistry of PSII (charge separa- tion, recombination and stabilisation), non-radiative loss of excited states in light-harvesting antennae and excited states quenched by oxidised PQ molecules from the PQ pool. The low value of FPo in leaves resulted from inactivity of RC, which might favour higher energy dissipation as heat and flu- orescence, as deduced from the high values for FDo (Laza´r 2003; Gonc¸alves et al. 2007). The value of FPo ⁄ (1 ) FPo) (=Fv ⁄Fo) (Strasser et al. 2000) decreased with an increase in temperature; at 40 �C it was only 50%, while it decreased by more than 90% at 45 �C, indicating that structural alterations on the donor side of PSII had occurred. The relative values of selected expressions can be plotted using a spider plot. This is a multiparametric description of structure and function of each photosynthetic sample, pre- sented using an octagonal line. This type of plot provides a direct visualisation of the behaviour of a sample and thus facilitates comparison of plant materials, as well as effects of different stresses (Fig. 3). In our experiments, the most affected parameters that could be easily viewed through a spi- der plot were Fv ⁄Fm, Fo and PI. The energy pipeline leaf model as an effect of elevated tem- perature was deduced using biolyzer hp3 software (Fig. 4). This model gives information about the efficiency of flow of energy from antennae to the electron transport chain compo- nents through the RC of PSII. The area of the arrows for each of the parameters, ABS ⁄CSo, TRo ⁄CSo, ETo ⁄CSo and DIo ⁄CSo, indicate the efficiency of light absorption, trapping, electron transport and dissipation per cross-section of PSII, respectively, in control and elevated temperature-treated sam- ples. The following effects were observed in wheat leaves [phenomenologically per excited cross-section (CS) area]: ABS ⁄CSo, ETo ⁄CSo decreased. ABS ⁄CSo describes the num- ber of photons absorbed by antenna molecules of active and inactive PSII RCs over the excited cross-section of the tested sample and is represented by the dark-adapted Fo. The ABS ⁄CSo can be substituted as an approximation by the flu- orescence intensity, Fo. A decrease in ABS ⁄CSo at high Fig. 3. A spider plot presentation of selected parameters quantifying the behaviour of PSII of wheat leaves exposed for 15 min to 35 �C, 40 �C or 45 �C relative to that of wheat leaves at 25 �C. Fig. 4. Energy pipeline leaf model of wheat leaves at elevated temperatures. Elevated temperature-induced changes in PSII of wheat Mathur, Jajoo, Mehta & Bharti 4 Plant Biology 13 (2011) 1–6 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands temperature indicates a decrease in the energy absorbed per excited cross-section. ETo ⁄CSo represents electron transport in a PSII cross-section and indicates the rate of reoxidation of reduced QA via electron transport over a cross-section of active RCs (Force et al. 2003). A decrease in this ratio indi- cates inactivation of RC complexes and the OEC and also suggests that the donor side of PSII has been affected. DIo ⁄CSo represents the total dissipation measured over the cross-section of the sample that contains active and inactive RCs. A decrease in the density of active RCs (indicated as open circles) and an increase in the density of inactive RCs (indicated as filled circles) was observed in response to ele- vated temperature. Dissipation occurs as heat, fluorescence and energy transfer to other systems. An increase in energy dissipation at high temperature indicates that energy available for photochemistry is reduced under stress conditions (Strasser 1987; Strasser et al. 1996, 2000; Kruger et al. 1997; Force et al. 2003). Recovery The extent of recovery after damage induced by