Othmar Buchner

Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana

Summary.In leaf mesophyll cells of transgenic Arabidopsis thaliana plants expressing GFP in the chloroplast, stromules (stroma-filled tubules) with a length of up to 20 μm and a diameter of about 400–600 nm are observed in cells with spaces between the chloroplasts. They appear extremely dynamic, occasionally branched or polymorphic. In order to investigate the effect of temperature on chloroplasts, we have constructed a special temperature-controlled chamber for usage with a light microscope (LM-TCC). This LM-TCC enables presetting of the temperature for investigation directly at the microscope stage with an accuracy of ±0.1 °C in a temperature range of 0 °C to +60 °C. With the LM-TCC a temperature-dependent appearance of chloroplast protrusions has been found. These structures have a considerably smaller length-to-diameter ratio than typical stromules and reach a length of 3–5 μm. At 5–15 °C (low temperatures), almost no chloroplast protrusions are observed, but they appear with increasing temperatures. At 35–45 °C (high temperatures), numerous chloroplast protrusions with a beaklike appearance extend from a single chloroplast. Interaction of stromules with other organelles has also been investigated by transmission electron microscopy. At 20 °C, transverse sections of stromules are frequently observed with a diameter of about 450 nm. A close membrane-to-membrane contact of stromules with the nucleus and mitochondria has been visualised. Golgi stacks and microbodies are found in the spatial vicinity of stromules. At 5 °C, virtually no chloroplast protrusions or stromules are observed. At 35 °C, chloroplast protrusions are present as broader thylakoid-free stroma-filled areas, resulting in an irregular chloroplast appearance.

Thermotolerance of photosystem 2 of three alpine plant species under field conditions

The species specific response of photosystem 2 (PS2) efficiency and its thermotolerance to diurnal and seasonal alterations in leaf temperature, irradiance, and water relations were investigated under alpine field conditions (1 950 m) and in response to an in situ long-term heat treatment (+3 K). Three plant species were compared using the naturally occurring microstratification of alpine environments, i.e. under contrasting leaf temperatures but under similar macroclimatic conditions. Thermotolerance of PS2 showed a high variability in all three species of up to 9.6 K. Diumal changes (increases or even decreases) in PS2 thermotolerance occurred frequently with a maximum increase of +4.8 K in Loiseleuria procumbens. Increasing leaf temperatures and photosynthetic photon flux density influenced thermotolerance adjustments. Under long-term heating (+3 K) of L. procumbens canopies with infra-red lamps, the maxima of the critical (Tc) and the lethal (Tp) temperature of PS2 increased by at least 1 K. Thermotolerance of the leaf tissue (LT50) increased significantly by +0.6 K. The effects of slight water stress on thermotolerance of PS2 were species specific. High temperature thresholds for photoinhibition were significantly different between species and increased by 9 K from the species in the coldest microhabitat to the species in the warmest. Experimental heating of L. procumbens canopies by +3 K caused a significant (p>0.01) upward shift of the high temperature threshold for photoinhibition by +3 K. Each species appeared to be very well adapted to the thermal conditions of its microhabitat as under the most frequently experienced daytime leaf temperatures no photoinhibition occurred. The observed fine scale thermal adjustment of PS2 in response to increased leaf temperatures shows the potential to optimise photosynthesis under varying environmental conditions as long as the upper thermal limits are not exceeded.

A novel system for in situ determination of heat tolerance of plants: first results on alpine dwarf shrubs

BackgroundHeat stress and heat damage to plants gain globally increasing importance for crop production and plant survival in endangered habitats. Therefore the knowledge of heat tolerance of plants is of great interest. As many heat tolerance measurement procedures require detachment of plants and protocols expose samples to various heat temperatures in darkness, the ecological relevance of such results may be doubted. To overcome these constraints we designed a novel field compatible Heat Tolerance Testing System (HTTS) that opens the opportunity to induce controlled heat stress on plants in situ under full natural solar irradiation. Subsequently, heat tolerance can be evaluated by a variety of standard viability assays like the electrolyte leakage test, chlorophyll fluorescence measurements and visual assessment methods. Furthermore, recuperation can be studied under natural environmental conditions which is impossible when detached plant material is used. First results obtained on three alpine dwarf - shrubs are presented.ResultsWhen heat tolerance of Vaccinium gaultherioides Bigelow was tested with the HTTS in situ, the visual assessment of leaves showed 50% heat injury (LT50) at 48.3°C, while on detached leaves where heat exposure took place in small heat chambers this already happened at 45.8°C. Natural solar irradiation being applied during heat exposure in the HTTS had significantly protective effects: In Loiseleuria procumbens L. (Desv.), if heat exposure (in situ) took place in darkness, leaf heat tolerance was 50.6°C. In contrast, when heat exposure was conducted under full natural solar irradiation heat tolerance was increased to 53.1°C. In Rhododendron ferrugineum L. heat tolerance of leaves was 42.5°C if the exposure took place ex situ and in darkness, while it was significantly increased to 45.8°C when this happened in situ under natural solar irradiation.ConclusionsThe results obtained with the HTTS tested in the field indicate a mitigating effect of natural solar irradiation during heat exposure. Commonly used laboratory based measurement procedures expose samples in darkness and seem to underestimate leaf heat tolerance. Avoidance of detachment by the use of the HTTS allows studying heat tolerance and recuperation processes in the presence of interacting external abiotic, biotic and genetic factors under field conditions. The investigation of combined effects of heat exposure under full solar irradiation, of recuperation and repair processes but also of possible damage amplification into the results with the HTTS appears to be particularly useful as it allows determining heat tolerance of plants with a considerably high ecological significance.

Design and construction of a new temperature-controlled chamber for light and confocal microscopy under monitored conditions: biological application for plant samples.

A new light microscope–temperature‐controlled chamber (LM–TCC) has been constructed. The special feature of the light microscope–temperature‐controlled chamber is the Peltier‐element temperature control of a specimen holder for biological samples, with a volume capacity of 1 mL. This system has marked advantages when compared to other approaches for temperature‐controlled microscopy. It works in a temperature range of −10°C to +95°C with an accuracy of ±0.1°C in the stationary phase. The light microscope–temperature‐controlled chamber allows rapid temperature shift rates. A maximum heating rate of 12.9°C min−1 and a maximum cooling rate of 6.0°C min−1 are achieved with minimized overshoots (≤1.9°C). This machinery operates at low cost and external coolants are not required. Especially with samples absorbing irradiation strongly, temperature control during microscopy is necessary to avoid overheating of samples. For example, leaf segments of Ficaria verna exposed to 4500 μmol photons m−2 s−1 in a standard microscopic preparation show a temperature increase (δT) of 18.0°C, whereas in the light microscope–temperature‐controlled chamber this is reduced to 4°C. The kinetics of microscope‐light induced δT are described and infrared thermography demonstrates the dissipation of the temperature. Chloroplasts of the cold adapted plant Ranunculus glacialis show the tendency to form stroma‐filled protrusions in relation to the exposure temperature. The relative number of chloroplasts with protrusions is reduced at 5°C when compared to 25°C. This effect is reversible. The new light microscope–temperature‐controlled chamber will be useful in a wide range of biological applications where a rapid change of temperature during microscopic observations is necessary or has to be avoided allowing a simulation of ecologically relevant temperature scenarios.

Readiness to frost harden during the dehardening period measured in situ in leaves of Rhododendron ferrugineum L. at the alpine timberline

Summary The readiness to frost harden during the dehardening period can be crucial to the frost survival of Rhododendron ferrugineum leaves. When already dehardened plants occasionally fall snow free in late winter or spring still heavy night frosts can occur in the subalpine environment. R. ferrugineum shrubs were treated in situ using a newly developed field portable freezing chamber. They were exposed to controlled night frosts of close to the lowest temperature sustained without frost damage (LT 0 ) to determine the potential frost hardening response under otherwise completely natural conditions. There was a lag period of 3 days during which no significant increase of frost resistance was observed. After three days frost resistance increased suddenly by 5.9°C within 24 hours. High daytime leaf temperatures (+19°C) combined with night frosts further retarded the rate of frost hardening. The in situ frost treatment generally yielded frost resistances (LT 10 ) approaching the highest ever measured in that season. In early spring the in situ frost hardening response was three times greater than early reports for detached twigs with a total hardiness gain between 7.8°C and 9.2°C. It is suggested that the limiting step in frost hardening in leaves of Rhododendron ferrugineum in spring is not the extent of frost resistance achieveable but rather the slow rate of frost hardening.

Dynamics of Tissue Heat Tolerance and Thermotolerance of PS II in Alpine Plants

At first sight heat may not be expected to be an environmental constraint of significant importance in alpine environments, as low atmospheric temperatures are among the well-known common features of the alpine macroclimate (see Korner 2003). Although atmospheric temperatures are low, alpine plants – due to their small, prostrate growth form – often grow very close to the soil surface and can be surrounded by bare soil, causing a decoupling from ambient air temperature. In addition, the decoupling effect is promoted by an appropriate protection from cooling winds, a favourable slope, and exposure to the usually increased solar irradiation at high altitudes.

Application of heat stress in situ demonstrates a protective role of irradiation on photosynthetic performance in alpine plants

The impact of sublethal heat on photosynthetic performance, photosynthetic pigments and free radical scavenging activity was examined in three high mountain species, Rhododendron ferrugineum, Senecio incanus and Ranunculus glacialis using controlled in situ applications of heat stress, both in darkness and under natural solar irradiation. Heat treatments applied in the dark reversibly reduced photosynthetic performance and the maximum quantum efficiency of photosystem II (Fv/Fm), which remained impeded for several days when plants were exposed to natural light conditions subsequently to the heat treatment. In contrast, plants exposed to heat stress under natural irradiation were able to tolerate and recover from heat stress more readily. The critical temperature threshold for chlorophyll fluorescence was higher under illumination (Tc′) than in the dark (Tc). Heat stress caused a significant de-epoxidation of the xanthophyll cycle pigments both in the light and in the dark conditions. Total free radical scavenging activity was highest when heat stress was applied in the dark. This study demonstrates that, in the European Alps, heat waves can temporarily have a negative impact on photosynthesis and, importantly, that results obtained from experiments performed in darkness and/or on detached plant material may not reliably predict the impact of heat stress under field conditions.

Chloroplast protrusions in leaves of Ranunculus glacialis L. respond significantly to different ambient conditions, but are not related to temperature stress

Abstract The occurrence of chloroplast protrusions (CPs) in leaves of R anunculus glacialis  L. in response to different environmental conditions was assessed. CPs occur highly dynamically. They do not contain thylakoids and their physiological function is still largely unknown. Controlled in situ sampling showed that CP formation follows a pronounced diurnal rhythm. Between 2 and 27 °C the relative proportion of chloroplasts with CPs (rCP) showed a significant positive correlation to leaf temperature (TL; 0.793, P < 0.01), while irradiation intensity had a minor effect on rCP. In situ shading and controlled laboratory experiments confirmed the significant influence of TL. Under moderate irradiation intensity, an increase of TL up to 25 °C significantly promoted CP formation, while a further increase to 37 °C led to a decrease. Furthermore, rCP values were lower in darkness and under high irradiation intensity. Gas treatment at 2000 ppm CO2/2% O2 led to a significant decrease of rCP, suggesting a possible involvement of photorespiration in CP formation. Our findings demonstrate that in R . glacialis, CPs are neither a rare phenomenon nor a result of heat or light stress; on the contrary, they seem to be most abundant under moderate temperature and non‐stress irradiation conditions.

Winter frost resistance of Pinus cembra measured in situ at the alpine timberline as affected by temperature conditions.

Winter frost resistance (WFR), midwinter frost hardening and frost dehardening potential of Pinus cembra L. were determined in situ by means of a novel low-temperature freezing system at the alpine timberline ecotone (1950 m a.s.l., Mt Patscherkofel, Innsbruck, Austria). In situ liquid nitrogen (LN₂)-quenching experiments should check whether maximum WFR of P. cembra belonging to the frost hardiest conifer group, being classified in US Department of Agriculture climatic zone 1, suffices to survive dipping into LN₂ (-196 °C). Viability was assessed in a field re-growth test. Maximum in situ WFR (LT₅₀) of leaves was <- 75 °C and that of buds was less (-70.3 °C), matching the lowest water contents. In midwinter, in situ freezing exotherms of leaves, buds and the xylem were often not detectable. Ice formed in the xylem at a mean of -2.8 °C and in leaves at -3.3 °C. In situ WFR of P. cembra was higher than that obtained on detached twigs, as reported earlier. In situ LN₂-quenching experiments were lethal in all cases even when twigs of P. cembra were exposed to an in situ frost hardening treatment (12 days at -20 °C followed by 3 days at -50 °C) to induce maximum WFR. Temperature treatments applied in the field significantly affected the actual WFR. In January a frost hardening treatment (21 days at -20 °C) led to a significant increase of WFR (buds: -62 °C to <- 70 °C; leaves: -59.6 °C to -65.2 °C), showing that P. cembra was not at its specific maximum WFR. In contrast, simulated warm spells in late winter led to premature frost dehardening (buds: -32.6 °C to -10.2 °C; leaves: -32.7 to -16.4 °C) followed by significantly earlier bud swelling and burst in late winter. Strikingly, both temperature treatments, either increased air temperature (+10.1 °C) or increased soil temperature (+6.5 °C), were similarly effective. This high readiness to frost harden and deharden in winter in the field must be considered to be of great significance for future winter survival of P. cembra. Determination of WFR in field re-growth tests appears to be a valuable tool for critically judging estimates of WFR obtained on detached twigs in an ecological context.

A low-temperature freezing system to study the effects of temperatures to 70 C on trees in situ

The ability to determine winter frost resistance of woody plants is limited for two reasons: (1) assessment of frost damage in midwinter is extremely difficult because results obtained by the currently available viability assays deviate greatly and (2) equipment that allows plants to be frozen at controlled freezing and thawing rates to below the midwinter frost resistance of most Northern Hemisphere woody plants is unavailable. To overcome these limitations, we developed a novel low-temperature freezing system (LTFS) that makes it possible to conduct in situ freezing experiments in midwinter with full control of cooling and thawing rates down to -70 degrees C. Frost resistance can be determined unequivocally by the regrowth test. The LTFS was tested on various, mostly subalpine, woody plants. Results obtained demonstrate the importance of conducting frost tests in situ. In needles of Picea abies (L.) Karst., frost injuries were not visible immediately after the frost test but took several weeks to develop fully. The low-freezing temperatures attained and the small control oscillations (typically +/-0.1 K) of the LTFS during cooling permitted in situ detection of low-temperature freezing exotherms in xylem of Quercus robur L. and in buds of P. abies and Rhododendron ferrugineum L., all of which showed supercooling. With the LTFS, the effects of low temperatures on plants can be specified directly by in situ assessment and regrowth tests.