Studies on the Localization and Spectral Characteristics of the Fluorescence Emission of Differently Pigmented Wheat Leaves

Intensity, spectral characteristics and localization of the UV-laser (337 nm) induced blue-green and red fluorescence emission of green, etiolated and white primary leaves of wheat seedlings were studied in a combined fluorospectral and fluoromicroscopic investigation. The blue-green fluorescence of the green leaf was characterized by a maximum near 450 nm (blue region) and a shoulder near 530 nm (green region), whereas the red chlorophyll fluorescence exhibited maxima in the near-red (F690) and far-red (F735). The etiolated leaf with some carotenoids and traces of chlorophyll a, in turn, showed a higher intensity of the blue-green fluorescence with a shoulder in the green region and a strong red fluorescence peak near 684 to 690 nm, the far-red chlorophyll fluorescence maximum (F735) was, however, absent. The norfluorazone-treated white leaf, free of chlorophylls and carotenoids, only exhibited blue-green fluorescence of a very high intensity. In green and etiolated leaves the blue-green fluorescence primarily derived from the cell walls of the epidermis and the red fluorescence from the chlorophyll a of the mesophyll cells. In white leaves the blue-green fluorescence emanated from all cell walls of epidermis, mesophyll and leaf vein bundles. The shape and intensity of the blue-green and red fluorescence emission is determined by the reabsorption properties of chlorophylls and carotenoids in the mesophyll, thus giving rise to quite different values of the various fluorescence ratios F450/F690, F450/F530, F450/F735 and F690/F735 in green and etiolated leaves.     

Photosynthetic performance and resistance to photoinhibition of Zea mays L. leaves grown at sub-optimal temperature

The performance of the photosynthetic apparatus was examined in the third leaves of Zea mays L. seedlings grown at near-optimal (25 °C) or at suboptimal (15 °C) temperature by measuring chlorophyll (ChI) a fluorescence parameters and oxygen evolution in different temperature and light conditions. In leaf tissue grown at 25 and 15 °C, the quantum yield of PSII electron transport (ψ_PSII) and the rate of O₂ evolution decreased with decreasing temperature (from 25 to 4 °C) at a photon flux density of 125 μmol m^?2 s^?1. In leaves grown at 25 °C, the decrease of ψ_PSII correlated with a decrease of photochemical ChI fluorescence quenching (q_p), whereas in leaves crown at 15 °C q_p was largely insensitive to the temperature decrease. Compared with leaves grown at 25 °C, leaves grown at 15 °C were also able to maintain a higher fraction of oxidized to reduced Q_A (greater q_p) at high photon flux densities (up to 2000 μmol m^?2 s^?1), particularly when the measurements were performed at high temperature (25 °C). With decreasing temperature and/or increasing light intensity, leaves grown at 15 °C exhibited a substantial quenching of the dark level of fluorescence F₀ (q₀) whereas this type of quenching was virtually absent in leaves grown at 25 °C. Furthermore, leaves grown at 15 °C were able to recover faster from photo inhibition of photosynthesis after a photoinhibitory treatment (1200 μmol m^?2 s^?1 at 25, 15 or 6 °C for 8 h) than leaves grown at 25 °C. The results suggest that, in spite of having a low photosynthetic capacity, Z. mays leaves grown at sub optimal temperature possess efficient mechanisms of energy dissipation which enable them to cope better with photoinhibition than leaves grown at near-optimal temperature. It is suggested that the resistance of Z. mays leaves grown at 15 °C to photoinhibition is related to the higher content of carotenoids of the xanthophyll cycle (violaxanthin + antheraxanthin + zeaxanthin) measured in these leaves than in leaves grown at 25 °C.     

Blue,Green and Red Fluorescence Signatures and Images of Tobacco Leaves*

Laser-induced fluorescence images of the leaf of an aurea mutant of Nicotiana tabacum were recorded for the blue and green fluorescence at 440 and 520 nm and the red chlorophyll fluorescence at 690 and 735 nm. The results obtained were compared with direct measurements of the fluorescence emission spectra of leaves using a conventional spectrofluorometer. The highest emission of blue (F440) and green fluorescence (F520) within the leaf was found in the leaf veins, particularly the main leaf vein. In contrast, the intercostal fields of leaves, which exhibited the highest chlorophyll content, showed only a very low blue and green fluorescence emission, which was much lower than the red and far-red chlorophyll fluorescence emission bands (F690 and F735). Correspondingly, the ratio of blue to red leaf fluorescence F440/F690 of upper and lower leaf side was much higher in the leaf veins (values 1.2 to 1.5) than in intercostal fields (values of 0.6 to 0.7). The results also demonstrated that in the intercostal fields the major part of the blue-green fluorescence was reabsorbed by chlorophylls and carotenoids. A partial reabsorption of the red fluorescence band near 690 nm by leaf chlorophyll took place, but did not affect the far-red fluorescence band near F735. As a consequence the chlorophyll fluorescence ratio F690/F735 exhibited significantly higher values in the chlorophyll-poor leaf vein regions (1.7 to 1.8) than in the chlorophyll-rich intercostal fields (0.8 to 1.3). Imaging spectroscopy of leaves was shown to be much more precise than the screening of fluorescence signatures by conventional fluorometers. It clearly demonstrated that the blue-green fluorescence and the red chlorophyll fluorescence of leaves exhibit an inverse contrast to each other. The advantage of the fluorescence imaging spectroscopy, which allows the simultaneous screening of the whole leaf surface and distinct parts of it, and its possible application in the detection of stress effects or local damage by insects and pathogens, is discussed.     

Fluorescence induction kinetics of green and etiolated leaves by recording the complete in-vivo emission spectra

Complete room temperature fluorescence emission spectra of green and etiolated leaves (Raphanus sativus L., cv. Saxa Treib, Hordeum vulgare L., cv. Villa) are continuously recorded up to 4 min after onset of excitation. In green leaves two emission bands appear, whereas in etiolated leaves only one band is observed. In both cases the emission intensity increases with time, the high-energy band of green leaves decreasing more rapidly than the low-energy band. This phenomenon can be interpreted in terms of energy transfer. During the observation time of the fluorescence induction kinetic no shift of the emission peaks is found within the accuracy of the apparatus (±2nm).     

Exogenous glycine betaine and proline play a protective role in heat-stressed barley leaves (Hordeum vulgare L.): A chlorophyll a fluorescence study

Abstract

Effects of drought and exogenous glycine betaine and proline on Photosystem II (PSII) photochemistry were studied in barley leaves under heat stress induced by exposing them to 45°C for 10 min. Polyphasic fluorescence transient (OJIP) was used to evaluate PSII photochemistry in leaves treated with either glycine betaine or proline, combined or not with heat treatment. A distinct K step in the fluorescence transient OJIP appeared in control leaves, indicating an inactivation of the oxygen evolving complex (OEC). Drought stress and exogenous glycine betaine and proline modified the shape of the OJIP curve of leaves heated at 45°C and the K step was not as pronounced. Increased thermostability of PSII may be associated with the resistance of OEC and increased energy connectivity between PSII units. The thermostability of PSII was also reflected by a lower decrease in maximum quantum yield of primary photochemistry (?_Po = F _V/F _M) and performance index (PI). Exogenous application of glycine betaine or proline can play an important role in enhancing plant stress tolerance and may help reduce effects of environmental stresses.     

Fluorescence emission spectra of Photosystem I,Photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at ?196 °C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at ?196 °C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at ?196 °C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at ?196 °C.     

Correlations between the temperature dependence of chlorophyll fluorescence and the fluidity of thylakoid membranes

To monitor changes in membrane fluidity in Arabidopsis leaves and thylakoid membranes, we investigated the temperature dependence of a chlorophyll fluorescence parameter, minimum fluorescence (Fo), and calculated the threshold temperature [T(Fo)] at which the rise of the fluorescence level Fo was considered to be started. For the modification of membrane fluidity we took three different approaches: (1) an examination of wild‐type leaves initially cultured at room temperature (22°C), then exposed to either a lower (4°C) or higher (35°C) temperature for 5 days; (2) measurements of the shift in T(Fo) by two mutants deficient in fatty acid desaturase genes – fad7 and fad7fad8 and (3) an evaluation of the performance of wild‐type plants when leaves were infiltrated with chemicals that modify fluidity. When wild‐type plants were grown at 22°C, the T(Fo) was 48.3 ± 0.3°C. Plants that were then transferred to a chamber set at 4 or 35°C showed a shift in their T(Fo) to 42.7 ± 0.9°C or 48.9 ± 0.1°C, respectively. Under low‐temperature acclimation, the decline in this putative transition temperature was significantly less in fad7 and fad7fad8 mutants compared with the wild‐type. In both leaf and thylakoid samples, values for T(Fo) were reduced in samples treated with benzyl alcohol, a membrane fluidizer, whereas T(Fo) rose in samples treated with dimethylsulfoxide, a membrane rigidifier. These results indicate that the heat‐induced rise of chlorophyll fluorescence is strongly correlated with the fluidity of thylakoid membranes.     

Assessment of the frost sensitivity of Trifolium species by chlorophyll fluorescence analysis

The potential of the chlorophyll fluorescence technique in screening for frost sensitivity in a range of Trifolium species from different geographical origins was assessed by measuring the decrease in variable chlorophyll fluorescence (F_var) of leaves after freezing at - 5°C for 60 min. The method was rapid and the results obtained agreed well with a visual assessment of freezing injury carried out after leaves were returned to optimal growth conditions for 72 h. Trifolium alexandrinum (Berseem clover) cv. Tabor originating from Israel was shown to be the most frost sensitive species studied and Trifolium subterraneum (subterranean clover) cv. Mt. Barker, from temperate regions of Australia, the most frost resistant. On extended periods of freezing, frost damage increased and this was associated with a further reduction in variable chlorophyll fluorescence and in quenching capacity of the thylakoid membranes. These results thus indicate that substantial thylakoid membrane dysfunction is induced at freezing temperatures. Furthermore, it was found that frost hardening of the frost sensitive species T. alexandrinum for 21 days at 5°C reduced the extent of damage sustained by the thylakoid membranes as shown by higher fluorescence quenching capacity, smaller reduction in variable fluorescence (F_var) and higher initial fluorescence (F_o) when leaves of hardened plants were frozen at -5°C and -7°C.     

The arrangement of chloroplasts in cells influences the reabsorption of chlorophyll fluorescence emission. The effect of desiccation on the chlorophyll fluorescence spectra of Rhizomnium punctatum leaves

Chlorophyll fluorescence spectra measured with leaves are distorted by the effect of fluorescence reabsorption. A heterogeneous theoretical model simulating the effect of chloroplast arrangement in a cell on the distortion of chlorophyll fluorescence spectra due to reabsorption was formulated. Desiccation of leaves of the moss Rhizomnium punctatum was carried out as a simple model experiment. The parameters entering the model (maximal number of chloroplasts forming columns in a cell, chloroplast size and chlorophyll concentration in a chloroplast) were estimated by means of light microscopy and spectrophotometry. During the desiccation, a grouping of chloroplasts was observed by light microscopy and the chlorophyll fluorescence emission and excitation spectra of the leaves were measured at room temperature and at 77 K. The leaves were infiltrated with DCMU. The ratio F685/F735 of the main emission bands decreased by about 50% at room temperature and by about 30% at 77 K upon decreasing the leaf water content. No significant changes were found in the ratio E475/E436 of the bands of the leaf fluorescence excitation spectra at 77 K for both 685- and 735-nm emission wavelengths. The excitation spectra and mechanical dilution experiments indicated that no functional changes appeared upon desiccation at the level of energy transfer. Theoretical simulations were in a good agreement with the experimental dependencies. We were able to conclude that the grouping of chloroplasts in cells may enhance the effect of chlorophyll reabsorption and thereby cause a significant decrease of the F685/F735 ratio in the chlorophyll fluorescence spectrum.     

An analysis of the chlorophyll-fluorescence transients from pea leaves generated by changes in atmospheric concentrations of CO2 and O2

Removal of CO₂ from pea leaves, which were either grown in a glasshouse at 15°C or in a controlled environment at 23°C, produced an initial increase in chlorophyll fluorescence emission followed by a slow decrease to steady state. From estimations of the redox state of Q, using a nondestructive in vivo technique, the contributions of photochemical and nonphotochemical quenching processes to these fluorescence transients were determined. The fluorescence changes observed on removal of CO₂ from the two types of pea leaves were mainly attributable to changes in nonphotochemical quenching although markedly different changes in photochemical quenching were also observed. When leaves grown at 23°C were depleted of CO₂, Q immediately became more reduced, whereas in leaves grown at 15°C Q, unexpectedly, became more oxidised. On return of CO₂ to the leaves these phenomena were reversed, i.e., in leaves grown at 23°C Q became more oxidised and in the 15°C grown leaves Q became more reduced. Increased electron transport to O₂ may account for the oxidation of Q on depletion of CO₂ from 15°C grown leaves. The generation of fluorescence transients on removal and return of CO₂ to the leaf required the presence of oxygen. The fast fluorescence kinetics observed on exposure of the leaf at steady state to a second saturating irradiation suggest that O₂ may accept electrons directly from Photosystem II at a site between Q and B.     

Imaging of the Blue,Green, and Red Fluorescence Emission of Plants: An Overview

An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing.     

Multicolour Fluorescence Imaging of Sugar Beet Leaves with Different Nitrogen Status by Flash Lamp UV-Excitation

Fluorescence images of leaves of sugar beet plants (Beta vulgaris L. cv. Patricia) grown on an experimental field with different fertilisation doses of nitrogen [0, 3, 6, 9, 12, 15 g(N) m^–2] were taken, applying a new multicolour flash-lamp fluorescence imaging system (FL-FIS). Fluorescence was excited by the UV-range (280–400 nm, _max = 340 nm) of a pulsed Xenon lamp. The images were acquired successively in the four fluorescence bands of leaves near 440, 520, 690, and 740 nm (F₄₄₀, F₅₂₀, F₆₉₀, F₇₄₀) by means of a CCD-camera. Parallel measurements were performed to characterise the physiological state of the leaves (nitrogen content, invert-sugars, chlorophylls and carotenoids as well as chlorophyll fluorescence induction kinetics and beet yield). The fluorescence images indicated a differential local patchiness across the leaf blade for the four fluorescence bands. The blue (F₄₄₀) and green fluorescence (F₅₂₀) were high in the leaf veins, whereas the red (F₆₉₀) and far-red (F₇₄₀) chlorophyll (Chl) fluorescences were more pronounced in the intercostal leaf areas. Sugar beet plants with high N supply could be distinguished from beet plants with low N supply by lower values of F₄₄₀/F₆₉₀ and F₄₄₀/F₇₄₀. Both the blue-green fluorescence and the Chl fluorescence rose at a higher N application. This increase was more pronounced for the Chl fluorescence than for the blue-green one. The results demonstrate that fluorescence ratio imaging of leaves can be applied for a non-destructive monitoring of differences in nitrogen supply. The FL-FIS is a valuable diagnostic tool for screening site-specific differences in N-availability which is required for precision farming.     

Black leaf-clips of a commercial fluorometer increased leaf temperature during dark adaptation under high solar radiation

The use of black leaf-clips for dark adaptation under high solar radiation conditions is reported to underestimate the maximum quantum yield of PSII photochemistry (F_v/F_m) measured by the continuous-excitation fluorometer Pocket PEA. The decrease in F_v/F_m was due to a rise in minimum fluorescence emission (F_o), probably resulting from increased leaf temperature (T_l). In field-grown tomato and pepper, fluorescence parameters and T_l in the region covered by the black leaf clip were measured in clipped leaves exposed to solar radiation during dark adaptation (clipped-only leaves) and in clipped leaves protected from solar radiation by aluminium foil (shrouded clipped leaves). Results confirmed significant F_v/F_m underestimates in clipped-only leaves primarily due to increased F_o. In one tomato experiment, T_l increased from 30 to 44.5°C in clipped-only leaves, with a negligible rise in shrouded clipped leaves. In two respective pepper experiments, T_l in clipped-only leaves increased from 27 to 36.2°C and 33 to 40.9°C. Based on the results of this study, a clip-effect parameter (P_CE) on fluorescence emission is proposed as the difference for F_v/F_m (or ?F_o/F_m) between shrouded clipped leaves and clipped-only leaves, which resulted to be 0.706 for tomato, and 0.241 and 0.358 for the two pepper experiments.     

Carnation Italian ringspot virus

Carnation Italian ringspot virus (CIRV) was obtained only twice in tests on several thousand carnations in Britain during 15 yr. The two isolates, from cultivars ‘Dusty Sim’ imported from Italy and ‘Orchid Beauty’ from the U.S.A., were indistinguishable serologically and in host reactions. CIRV was cultured in Nicotiana clevelandii and assayed in Chenopodium amaranti-color; it was readily transmitted by leaf-rubbing inoculation to 62 of 104 plant species tested. Virus-free carnations were infected only by injecting purified preparations into the stem, and developed chlorotic spots and oval rings in the younger leaves. CIRV was eliminated from Nicotiana clevelandii plants grown for 8 weeks at 36°C. CIRV presents no threat to carnation growing in Britain. In N. clevelandii sap, CIRV was infective at a dilution of 1/50000 to 1/100000, after heating 10 min at 85 °C (but not 90 °C), and after 16 weeks at 16 °C or 23 weeks at 2 °C. After freeze-drying, the virus survived at least 7 yr storage under vacuum at room temperature. CIRV was still infective and antigenic after treatment for 30 min at 18 °C with ultraviolet radiation (750 μW/cm²), ultrasound, 2% formaldehyde or 0.2% tri-sodium ortho-phosphate (TSP). Infectivity was not wholly abolished in 30 min by 2% TSP. The virus was readily purified by overnight maceration of N. clevelandii leaves extracted in phosphate buffer + butanol, followed by differential centri-fugation. Purified preparations contained abundant isometric particles c. 29 nm diameter, and like other serotypes of the tomato bushy stunt-pelargonium leaf curl group, gave three or four specific bands in density-gradient centri-fugation. The bands corresponded to four Schlieren peaks in analytical centrifugation. Virus from the lower bands was usually less invasive in N. clevelandii than from the upper bands, although the material in the different bands contained similar amounts of nucleic acid. Only one antigenic component was found by Immunoelectrophoresis; different serotypes of the TBSV-PLCV group differed widely in immunoelectrophoretic behaviour. The present cryptogram of CIRV is */*:*/*:S/S:S/*.     

Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature

This investigation determined whether thylakoid proteins would be degraded more rapidly or not in senescing wheat (Triticum aestivum L. em. Thell.) leaves concurrently exposed to high temperatures. Excised leaves were pulse-labelled with [³⁵S]-methionine for a 12 h period, and then incubated at 22,32 or 42°C for 0, 1, 2, or 3 d, before extracting a thylakoid enriched membrane sample. After electrophoretic separation, two prominent [³⁵S]-labelled protein bands were chosen for further analyses. Band A contained the D-1 thylakoid protein and band B contained thylakoid proteins of the light harvesting complex (LHCII) associated with photosystem II (PSII). Total protein, [³⁵S]-labelled protein, band A protein, and band B protein within the thylakoid enriched membrane samples were measured. Unlabelled thylakoid enriched membrane samples, extracted from leaves given similar treatments, were used to measure uncoupled whole-chain and photosystem II (PSII) electron transport and chlorophyll fluorescence. Accentuated decline in whole-chain and PSII electron transport, increasing F_o values, and decreasing F_max values were a result of high temperature injury in leaves treated at 42°C. None of the thylakoid enriched membrane protein fractions were degraded more rapidly in high-temperature treated leaves. Degradation of the total [³⁵S]-labelled membrane proteins and band B was inhibited by the 42°C treatment. The results indicate that high temperature stress may disrupt some aspects of normal senescence.     

Photochemical reactions of chlorophyll in dehydrated Photosystem II: two chlorophyll forms (680 and 700 nm)

Lichens and phototolerant poikilohydric mosses differ from spinach leaves, fern fronds or photosensitive mosses in that they show strongly decreased F_o chlorophyll fluorescence after drying. This desiccation-induced fluorescence loss is rapidly reversible under rehydration. Fluorescence emission from Photosystem II at 685 nm was decreased more strongly by dehydration than 720 nm emission. Reaction centers of Photosystem II lose activity on dehydration and regain it on hydration. Heating of desiccated lichens increased F_o chlorophyll fluorescence. The activation energy for the reversible part of the temperature-dependent fluorescence increase was 0.045 eV, which corresponds to the energy difference between the 680 and 697 nm absorption bands. In desiccated chlorolichens such as Parmelia sulcata, heating induces the appearance of positive variable fluorescence related to the reversible reduction of Q_A due to overcoming the energy barrier. This is interpreted to provide information on the mechanism of photoprotection: energy is dissipated by changing Chl680 or P680 into a chlorophyll form, which absorbs at 700 nm and emits light at 720 nm (Chl-720 or P680(700)) with a low quantum yield. Dissipation of light energy in this trap is activated by desiccation.     

Absorbance and fluorescence properties of protochlorophyllide in etiolated bean leaves

Primary leaves of 7-to-9 day-old etiolated bean seedlings contain a species of protochlorophyllide which is not transformed to chlorophyllide by light; this pigment species exhibits an absorption peak at 631nm $\text{in}\text{vivo}$ at ?196° and a fluorescence emission peak at 639nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of etiolated leaves converts the phototransformable protochlorophyllide holochrome to a pigment species with $\text{in}\text{vivo}$ absorption and fluorescence peaks identical to those of endogenous nontransformable protochlorophyllide. Administration of δ-amino-levulinic acid to etiolated leaves causes the synthesis of non-transformable protochlorophyllide with an absorption peak also at 631nm $\text{in}\text{vivo}$ at ?196° but with a fluorescence emission peak at 643nm $\text{in}\text{vivo}$ at room temperature. Heat-treatment of such leaves does not affect the position of these bands. The results indicate that protochlorophyllide which is derived from exogenous δ-amino-levulinic acid is in a physically different state from other forms of protochlorophyllide in the leaf.     

Influence of heat shock on chlorophyll fluorescence of white oak (<Emphasis Type="Italic">Quercus pubescens</Emphasis> Willd.) leaves

Chlorophyll fluorescence parameters of Quercus pubescens Willd. as response to heat shock (HS) by immersing leaves for 5 and 15 min in water of temperatures between 38 and 59 °C were examined. Fluorescence was measured after different periods of recovery (15, 30, 90, 210, and 1 440 min at 24/26 °C night/day temperature and 100 % humidity). The effective quantum yield of photosystem 2 (Y) in control and HS-treated leaves was always measured after previous 15 min irradiation. Under a 5 min HS, Y did not change after using temperatures below 44 °C, was rapidly restored after HS of moderate temperatures (44–48 °C), and progressively decreased and recovered eventually to the initial value after HS of high temperatures (48–52 °C). Y did not recover after HS with temperatures higher than 52 °C. Increase in the duration of HS from 5 to 15 min lead to change of the initial Y at each HS temperature, but the recovery processes were similar to those characteristic after 5 min incubation. The processes of recovery may depend mainly on the specificity of injuries caused by different heat shock temperatures. Thus Q. pubescens is able to preserve and recover the functional potential of its photosynthetic apparatus in response to HS up to 52 °C.     

Linking isoprene with plant thermotolerance, antioxidants and monoterpene emissions

The purpose of the present study was to test the possible plant thermotolerance role of isoprene and to study its relationship with non-enzymatic antioxidants and terpene emissions. The gas exchange, chlorophyll fluorescence, extent of photo- and oxidative stress, leaf damage, mechanisms of photo- and antioxidant protection, and terpene emission were measured in leaves of Quercus ilex seedlings exposed to a ramp of temperatures of 5 °C steps from 25 to 50 °C growing with and without isoprene (10 µL L⁻¹) fumigation. The results showed that isoprene actually conferred thermotolerance (shifted the decrease of net photosynthetic rates from 35 to 45 °C, increased F_v/F_m at 50 °C from 0.38 to 0.65, and decreased the leaf area damaged from 27 to 15%), that it precluded or delayed the enhancement of the antioxidant non-enzymatic defence conferred by α-tocopherol, ascorbic acid or β-carotene consumption in response to increasing temperatures, and that it decreased by approximately 70% the emissions of monoterpenes at the highest temperatures. This suggests that there are inducible mechanisms triggered by the initial stages of thermal damage that up-regulate these antioxidant compounds at high temperatures and that these mechanisms are somehow suppressed in the presence of exogenous isoprene, which seems to already exert an antioxidant-like behaviour.     

Light and temperature dependence of the emission of cyclic and acyclic monoterpenes from holm oak (Quercus ilex L.) leaves

In a laboratory study, we investigated the monoterpene emissions from Quercus ilex, an evergreen sclerophyllous Mediterranean oak species whose emissions are light dependent. We examined the light and temperature responses of individual monoterpenes emitted from leaves under various conditions, the effect of heat stress on emissions, and the emission-onset during leaf development. Emission rate increased 10-fold during leaf growth, with slight changes in the composition. At 30 °C and saturating light, the monoterpene emission rate from mature leaves averaged 4·1 nmol m^–2 s^–1, of which α-pinene, sabinene and β-pinene accounted for 85%. The light dependence of emission was similar for all monoterpenes: it resembled the light saturation curve of CO₂ assimilation, although monoterpene emission continued in the dark. Temperature dependence differed among emitted compounds: most of them exhibited an exponential increase up to 35 °C, a maximum at 42 °C, and a slight decline at higher temperatures. However, the two acyclic isomers cis-β-ocimene and trans-β-ocimene were hardly detected below 35 °C, but their emission rates increased above this temperature as the emission rates of other compounds fell, so that total emission of monoterpenes exponentially increased from 5 to 45 °C. The ratio between ocimene isomers and other compounds increased with both absolute temperature and time of heat exposure. The light dependence of emission was insensitive to the temperature at which it was measured, and vice versa the temperature dependence was insensitive to the light regime. The results demonstrated that none of the models currently applied to simulate isoprene or monoterpene emissions correctly predicts the short-term effects of light and temperature on Q. ilex emissions. The percentage of fixed carbon lost immediately as monoterpenes ranged between 0·1 and 6·0% depending on temperature, but rose up to 20% when leaves were continuously exposed to temperatures between 40 and 45 °C.