Biophysical studies of photosystem II-related recovery processes after a heat pulse in barley seedlings (Hordeum vulgare L.)
Introduction
Several photosynthetic processes are sensitive to heat stress. Rubisco activase, the protein that facilitates the release of sugar phosphates from Rubisco ceases to function properly at elevated temperatures, inactivating rubisco (Crafts-Brandner and Law, 2000). Heat stress also affects processes related to membrane integrity, ion conductivity, and phosphorylation activity. It has been shown that elevated temperatures accelerate the decay of the flash induced absorbance change at 518 nm (Weis, 1982; Havaux et al., 1996), which is thought to be related to ion transport across thylakoid membranes (Peters et al., 1984). Gounaris et al. (1984) demonstrated that a heat treatment induced grana destacking and the formation of cylindrical-inverted lipid micelles. With respect to the photosynthetic electron transport chain, exposure of plants to elevated temperatures has two contrasting effects. It was observed that photosystem I (PSI) (measured as the reduction rate of P700+) is stimulated by heat, which is related to a reduction of the plastoquinone pool by electrons from the stroma (Havaux, 1996; Bukhov et al., 1999). In contrast, PSII and especially the oxygen-evolving complex is very heat sensitive. Heat induces the dissociation of the manganese-stabilizing 33 kDa protein from the photosystem II (PSII) reaction center complex followed by release of the manganese atoms (Enami et al., 1994; Yamane et al., 1998). Heat-inactivation of PSII may be followed by dissociation of the light-harvesting Chl a/b protein complexes of PSII (LHCII) (Schreiber and Armond, 1978; Takeuchi and Thornber, 1994; Yamane et al., 1997). A further heat-induced effect that has been described is a shift of the redox equilibrium between QA and QB, increasing the residence time of an electron on QA relative to QB (Ducruet and Lemoine, 1985; Havaux, 1989).
For plants, the rate of recovery from heat-induced damage might be even more important than the extent of the damage itself. Gour et al. (1997) reported that in cyanobacteria, the degree of recovery depends very much on the presence of light during the repair processes. It was also shown (Heckathorn et al., 1997) that heat-related effects on PSII and particularly on the oxygen evolution are important limitations to post-stress recovery. In general, however, little is known about the processes involved in post-stress repair.
In this work, we focus our attention on the recovery processes related to the repair of heat damage to PSII in planta. To this end, full inhibition of oxygen evolution was induced using heat pulses: immersion of leaves of young barley seedlings for 40 s in a water bath of 50 °C. Pilot experiments showed that homogeneous starting material of whole plants with fully inhibited oxygen evolving complex could most readily be obtained with pulses, rather than with prolonged heat treatments at somewhat lower (40–45 °C) temperatures. This treatment, while it induces the typical effects of heat stress, virtually causes no visible symptoms. As a comparison, we also subjected some plants to a shorter, 20 s, heat pulse that caused partial inhibition of oxygen evolving activity. Several biophysical techniques were used to investigate the recovery kinetics of heat-inactivated PSII and also the role of light in these processes.
Section snippets
Plant material and growth conditions
Barley (Hordeum vulgare L. cv. Triangel) seeds were soaked in tap water for 4 h and then sown on perlite. The seedlings were grown in a growth chamber (Conviron S10) under long day conditions (16 h light and 8 h darkness) at 22 °C and 100 μmol photons m−2 s−1 photosynthetic photon flux density. Seedlings were watered with Hoagland solution and heat treatments were carried out when the barley seedlings were 7 days old.
Heat treatment and plant growth conditions following the treatment
The heat treatment of 7-day-old barley seedlings consisted of turning a pot
Results
Leaves of barley seedlings were given a heat pulse of 50 °C for 20 s or 40 s. Measurements of various parameters were carried out 30 min, 4 h, 24 h and 48 h after the heat treatment. These time points were chosen in preliminary experiments using chlorophyll-a fluorescence transients. The main consideration was to choose characteristic points defining the recovery processes occurring after the heat treatment and to carry out the measurements until considerable (for the 40 s heat pulse) or almost full
Discussion
The aim of this work was to study the recovery kinetics of PSII activity after heat stress in planta. In order to facilitate these investigations, we induced complete or partial inhibition of the oxygen evolving activity with heat pulses, immersing leaves of barley seedlings into a water bath of 50 °C for 40 s or 20 s. This kind of heat-treatment has the advantage that it induces very homogeneous effects and avoids several secondary effects, such as desiccation, metabolic changes, etc. Similar
Note added in proof
At the meeting “Photosynthesis and Post-Genomic Era” (25–28 August 2004, Trois-Rivières, Canada), prof. Y. Yamamoto (Okayama University, Japan) presented data on the cleavage of the D1-protein after a heat treatment. These not yet published biochemical data show that the initial plant responses to light and heat stresses are very similar; it provides additional support for the model we present.
Acknowledgements
S. Z. T. is grateful to Dr. Tibor Janda for his encouragement to write the manuscript and also for the critical reading. This work was supported by the Hungarian Research Foundation (T 026075 and T 34188).
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