Computational analysis of fluorescence induction curves in intact spinach leaves treated at different pH
Introduction
Photosystem II (PS II) is an integral membrane protein complex embedded in the grana of the thylakoid membrane. PS II is unique as it is capable of oxidizing water to molecular oxygen along with the generation of reducing equivalents. The reaction centre consists of D1 and D2 proteins which contain primary electron donor P680, secondary electron donor which is redox active tyrosine residues YZ and YD, the intermediary electron acceptor pheophytin and two plastoquinone electron acceptor QA and QB along with associated non-heme iron. On the luminal side of PS II three extrinsic proteins with molecular mass of 33, 23 and 17 kDa bind and regulate the activity of oxygen evolving complex (see Ke, 2000).
The effects of pH on many physiological processes in plants are well established. Significant work has been done to explore the effects of pH on the photosynthetic process. At lower pH the lifetime of P680+ is longer entailing a greater probability of charge recombination (Lavergne and Rappaport, 1998). A low luminal pH slows down the reoxidation of plastoquinol (Hope et al., 1994). In vitro, the oxygen evolving complex loses Ca2+ at pH < 6.0, inhibiting water splitting and rendering the PS II reaction centre (Kramer et al., 2003). Recently, in isolated thylakoid membranes, we have shown pH dependent regulation of distribution of excitation energy between the PS II and PS I (Singh-Rawal et al., 2010). Acidification of the luminal pH induces qE through the protonation of PSII proteins and activation of xanthophylls synthesis by xanthophylls cycle. Together, binding of protons and xanthophylls to specific sites in the PSII antenna causes a conformational change that switches a PSII unit into a quenched state (Muller et al., 2001). Decrease in lumen pH also activates violaxanthin deepoxidase which is an enzyme of the xanthophyll cycle that catalyzes the conversion of violaxanthin to zeaxanthin via antheraxanthin (Bergantino et al., 2003). None of the above stated work investigated the effect of pH on chlorophyll a fluorescence induction kinetics in spinach leaves.
PS II is heterogeneous with respect to its antenna size and localization. The antenna size heterogeneity refers to the occurrence of distinct PSII populations with different light-harvesting chlorophyll (Chl) antenna sizes. By using kinetic analysis of fluorescence induction curve of DCMU-poisoned chloroplast, PSII was resolved into three components, i.e., PS II α, PS II β and PS II γ (Hsu and Lee, 1991) The dominant form, PSII α, is localized in the grana partition regions (Anderson and Melis, 1983) and is responsible for the majority of the water oxidation activity and plastoquinone reduction. These centers possess a Chl a core complex, an accessory Chl a–b light harvesting inner antenna (LHC II-inner), and a peripheral antenna (LHC II-peripheral) containing a combined total of about 210–250 Chl a and Chl b molecules (Morrissey et al., 1989). These have a higher absorption cross-section area due to association with the peripheral Chla/b LHCs. PSIIα are characterized by a large light harvesting antenna and possibility of excited states transfer between PSII units that is reflected in a sigmoidal fluorescence rise when measured with DCMU. On the other hand PSIIβ are mainly located in stromal region of thylakoid membranes and are characterized by about 2.5 times smaller light harvesting antenna of PSIIα and impossibility of the excited states transfer between PS IIs that is reflected in an exponential fluorescence rise when measured with DCMU. Smaller antenna size has been ascribed to the absence of peripheral LHC II in PSII. The intrinsic trapping and fluorescence property of α and β centers are considered to be similar (Melis, 1991). The antenna size of PSII γ is the smallest amongst all and is also called unpredictable centers because of their varied behavior.
Chlorophyll a fluorescence induction kinetics is a useful, highly sensitive and non-invasive parameter to study electron transfer kinetics between the various components of PS II. When dark-adapted oxygenic photosynthetic cells are illuminated, chlorophyll a fluorescence shows complex induction kinetics (FI) termed as the Kautsky Curve (Strasser et al., 2004, Zhu et al., 2005). In fluorescence induction curve, termed OJIP curve, the initial chlorophyll fluorescence at level O (Fo) reflects the minimal fluorescence yield when all the molecules of QA are in the oxidized state. The transition from phase O to J is controlled by photochemical charge separation leading to the reduction of QA to QA− while the appearance of phases I and P are limited by dark reaction. Level P (Fm) corresponds to the situation in which all molecules of QA are in the reduced state. Thus, the polyphasic rise of chlorophyll a fluorescence transient from Fo to Fm is called OJIP transient (Strasser et al., 1995). Chl a fluorescence induction curves (OJIP) enables calculation of several phenomenological and biophysical expressions for quantifying the energy fluxes and energy ratios through Photosystem II (Strasser et al., 2010, Mehta et al., 2010, Mathur et al., 2010).
The effects of different pH on Chl a fluorescence induction kinetics has been studied in spinach leaf discs. In the present study, a computational analysis of the information obtained from OJIP curves has been performed. It is evident from the results that a single measurement of fluorescence induction kinetics can be exploited to derive vast information regarding the structure and function of PS II. Such information can allow us to quickly screen various genotypes on the basis of their performance. Several parameters like Fv/Fm, ABS/RC, performance index, etc. have been derived from OJIP curves. For direct visualization and comparison, energy pipeline model and radar plot have been constructed using computer software. Effects of pH were evident on the OEC and the acceptor side of PS II. Antenna size heterogeneity of PS II is also affected by change in pH as shown by the change in the proportions of PS II α, β and γ fractions under different pH conditions.
Section snippets
Plant material: Spinach (Spinacea oleracia) leaf disc
Fresh spinach leaves were brought from the market and cut in to small discs which were transferred to the Petri plates containing 50 mM buffer solution of different pH. For pH 8.0 and 7.5 (HEPES–NaOH), pH 6.5 and 5.5 (MES–NaOH) and pH 5.0 and 4.5 (sodium acetate–acetic acid) buffers were used. Leaf discs were incubated in dark for 4 h.
Chlorophyll a fluorescence induction kinetics
Chlorophyll a fluorescence (O-J-I-P) transients were recorded at room temperature with a Plant Efficiency Analyser (PEA, Hansatech King's Lynn, Norfolk, UK). The
Analysis of OJIP transient showing pH-induced changes
Chlorophyll a fluorescence transient was measured to evaluate the effect of pH treatment on the photochemical efficiency of PS II. The OJIP transient depicts the rate of reduction kinetics of various components of PS II. When dark adapted spinach leaf is illuminated with the saturating light intensity of 3500 μmole/m2/s then it exhibits a polyphasic rise in fluorescence (OJIP). Each letter reflects distinct inflection in the induction curve as shown in (Fig. 1A). The level O represents all the
Conclusion
The work presented here demonstrates the biophysical phenomics which is based on the chlorophyll fluorescence induction kinetics measurement along with the JIP test parameters. By using computer derivations from a single measurement of chlorophyll a fluorescence induction kinetics, we have reported that acidic pH causes a significant inhibition of the donor and the acceptor side of PS II, and a change in the antenna size heterogeneity of PS II.
Acknowledgements
UGC research fellowship to TT is thankfully acknowledged. This work was financially supported, in part, by grants from the Russian Foundation for Basic Research (08-04-00241; 09-04-91219-CT; 09-04-01074) from the Molecular and Cell Biology Programs of the Russian Academy of Sciences (to DAL).
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