Sridharan Govindachary

Kinetic analyses of the OJIP chlorophyll fluorescence rise in thylakoid membranes

N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was previously used to study the kinetics of the OJIP chlorophyll fluorescence rise. The present study is an attempt to elucidate the origin of TMPD-induced delay and quenching of the I–P step of fluorescence rise. For this purpose, we analyzed the kinetics of OJIP rise in thylakoid membranes in which electron transport was modified using ascorbate, methyl viologen (MV), and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). In the absence of TMPD, the OJIP kinetics of fluorescence induction (FI) was not altered by ascorbate. However, ascorbate eliminated the I–P rise delay caused by high concentrations of TMPD. On the other hand, neither ascorbate nor DBMIB, which blocks the electron release from Photosystem II (PS II) at the cytochrome b6/f complex, could prevent the quenching of I–P rise by TMPD. In control thylakoids, MV suppressed the I–P rise of FI by about 60. This latter effect was completely removed if the electron donation to MV was blocked by DBMIB unless TMPD was present. When TMPD intercepted the linear electron flow from PS II, re-oxidation of TMPD by photosystem I (PS I) and reduction of MV fully abolished the I–P rise. The above is in agreement with the fact that TMPD can act as an electron acceptor for PS II. With MV, the active light-driven uptake of O2 during re-oxidation of TMPD by PS I contributes towards an early decline in the I–P step of the OJIP fluorescence rise.

Abolition of photosystem I cyclic electron flow in Arabidopsis thaliana following thermal-stress.

Heat tolerance of Arabidopsis thaliana (WT) and its mutants, crr2-2, lacking NADPH-dehydrogenase (Ndh-pathway), and pgr5, deficient in proton gradient regulation and/or ferredoxin-quinone-reductase (FQR-pathway), was studied from 30 to 46°C. Chlorophyll fluorescence revealed that thermal damage to photosystem II (PSII) was maximal in WT plants following short-term exposure of leaves to moderate or high temperature stress. Thermal stress impaired the photosynthetic electron flow at oxidizing and reducing sides of PSII. This was deduced from the transformation of temperature dependent OJIP to OKP patterns, changes in the relative amplitudes of K-step fluorescence rise and F(v)/F(o) ratio. The amplitude of the K-peak that corresponds to the magnitude of damage to the oxygen evolving complex (OEC) in crr2-2 mutants was about 50% of that observed in WT plants exposed to 46°C. The damage to OEC in pgr5 mutants was relatively smaller and thus their PSII complexes were more heat tolerant. P700 oxidation-reduction kinetics following heat-stress revealed that photosystem I (PSI) complexes remained oxidizable either with 10-ms multiple turn-over flashes or far-red illumination but the complementary cyclic electron flow around PSI (CEF) was abolished in both mutants. With further increase in incubation temperature, CEF was fully suppressed even in WT. Thus, P700 turn-over was not enhanced following thermal stress. Furthermore, the experimental data predicts the onset of pseudocyclic electron transport with molecular oxygen as terminal acceptor in crr2-2 and pgr5 mutants but not in wild type Arabidopsis subjected to severe thermal-stress.

Functional aspects of the photosynthetic light reactions in heat stressed Arabidopsis deficient in digalactosyl-diacylglycerol.

Plants are often submitted, in their natural environment, to various abiotic stresses such as heat stress. However, elevated temperature has a detrimental impact on overall plant growth and development. We have examined the physiological response of the dgd1-2 and dgd1-3 Arabidopsis mutants lacking 30-40% of digalactosyl-diacylglycerol (DGDG) exposed to heat constraint. These mutants, which grow similarly to wild type under normal conditions, were previously reported to be defective in basal thermotolerance as measured by cotyledon development. However their functional properties were not described. Chlorophyll fluorescence measurements and absorbance changes at 820nm were used to monitor photosystem II (PSII) and PSI activity, respectively. It was observed that both mutants have similar photosystem activities with some differences. The mutants were less able to use near saturation light energy and elicited higher rates of cyclic PSI electron flow compare to wild type. Arabidopsis leaves exposed to short-term (5min) mild (40°C) or strong (44°C) heat treatment have shown a decline in the operating effective quantum yield of PSII and in the proportion of active PSI reaction centers. However, cyclic PSI electron flow was enhanced. The establishment of the energy-dependent non-photochemical quenching of chlorophyll fluorescence was accelerated but its decline under illumination was inhibited. Furthermore, heat stress affected the process implicated in the redistribution of light excitation energy between the photosystems known as the light state transitions. All the effects of heat stress mentioned above were more intense in the mutant leaves with dgd1-3 being even more susceptible. The decreased DGDG content of the thylakoid membranes together with other lipid changes are proposed to influence the thermo-sensitivity of the light reactions of photosynthesis towards heat stress.

Inhibition of photosystems I and II activities in salt stress-exposed Fenugreek (Trigonella foenum graecum)

Fenugreek (Trigonella foenum graecum) seedlings were exposed to increasing NaCl concentrations in the growth medium to examine the effect of salt stress on the electron transport reactions of photosynthesis. Activities of both photosystem II (PSII), measured by chlorophyll fluorescence, and photosystem I (PSI), measured by P700 photooxidation, were decreased by salt stress. The inhibition proceeded in a two step manner. At the lower salt concentrations used and shorter exposition periods, electron transfer between the quinone acceptors of PSII, Q(A) and Q(B), was strongly retarded as shown by an increased amplitude of the OJ phase of the OJIP chlorophyll fluorescence induction traces and slowed chlorophyll fluorescence relaxation kinetics following a single turn-over flash. The above indicated a disturbance of the Q(B) binding site likely associated with the first step of photoinhibition. In the second step, strong photoinhibition was observed as manifested by increased F(0) values, declined F(v)/F(0) and loss of photoactive P700.

Effect of moderate and high light on photosystem II function in Arabidopsis thaliana depleted in digalactosyl-diacylglycerol.

The response of the heat-sensitive dgd1-2 and dgd1-3 Arabidopsis mutants depleted in the galactolipid DGDG to photoinhibition of chloroplasts photosystem II was studied to verify if there is a relationship between heat stress vulnerability due to depletion in DGDG and the susceptibility to photoinhibitory damage. Non-photochemical quenching (NPQ) is known to dissipate excessive absorbed light energy as heat to protect plants against photodamage. The main component of NPQ is dependent of the transthylakoid pH gradient and is modulated by zeaxanthin (Zx) synthesis. These processes together with chlorophyll fluorescence induction were used to characterize the response of the genotypes. The mutants were more sensitive to photoinhibition to a small extent but this was more severe for dgd1-3 especially at high light intensity. It was deduced that DGDG was not a main factor to influence photoinhibition but other lipid components could affect PSII sensitivity towards photoinhibition in relation to the physical properties of the thylakoid membrane. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Photosystem II reconstitution into proteoliposomes and methodologies for structure-function characterization.

This chapter discusses the photosystem II (PSII) reconstitution into proteoliposomes. In the first part of the chapter, protocols are outlined for the preparation of lipid bilayer vesicles (liposomes) constituted of individual thylakoid lipids or their mixtures, for the preparation of PSII particles, and for the incorporation of the PSII particles into the liposomes. In the second part of the chapter, methodologies are described for the structure-function characterization of the PSII-lipid complexes (proteoliposomes). This includes the sodium dodecylsulfate-polyacrylamide gel electrophoresis determination of the PSII proteins, the measurement of oxygen-evolving activity of PSII in the proteoliposomes, the study of structural changes of the PSII proteins upon their incorporation into the lipid bilayers by Fourier transform infrared (FT-IR) spectroscopy, and the characterization of the PSII activity by fluorescence induction.