Thermal and chlorophyll-fluorescence imaging distinguish plant-pathogen interactions at an early stage

Different biotic stresses yield specific symptoms, owing to their distinct influence on a plant's physiological status. To monitor early changes in a plant's physiological status upon pathogen attack, chlorophyll fluorescence imaging (Chl-FI) and thermography, which respectively visualize photosynthetic efficiency and transpiration, were carried out in parallel for two fundamentally different plant-pathogen interactions. These non-destructive imaging techniques were able to visualize infections at an early stage, before damage appeared. Under growth-room conditions, a robotized set-up captured time series of visual, thermal and chlorophyll fluorescence images from infected regions on attached leaves. As a first symptom of the plant-virus interaction between resistant tobacco and tobacco mosaic virus (TMV), thermal imaging detected a local rise in temperature while Chl-FI monitored a co-localized increase in fluorescence intensity. Chl-FI also revealed pre-symptomatic high-intensity spots for the plant-fungus system sugar beet-Cercospora beticola. Concomitantly, spots of lower temperature were monitored with thermography, in marked contrast with our observations on TMV-infection in tobacco. Knowledge of disease signatures for different plant-pathogen interactions could allow early identification of emerging biotic stresses in crops, facilitating the containment of disease outbreaks. Presymptomatic monitoring clearly opens perspectives for quantitative screening for disease resistance, either on excised leaf pieces or attached leaves.     

Uptake of diuron and concomitant loss of photosynthetic activity in leaves as visualized by imaging the red chlorophyll fluorescence

The principles of the chlorophyll (Chl) fluorescence induction kinetics (known as Kautsky effect) and their change by the photosystem II herbicide diuron are presented together with the Chl fluorescence emission spectra of a normal and diuron-inhibited leaf. By imaging the Chl fluorescence emission of green leaves the successive uptake of diuron and the concomitant loss of photosynthetic quantum conversion from the leaf base to the leaf tip are documented.     

A chlorophyll a fluorescence-based Lemna minor bioassay to monitor microbial degradation of nanomolar to micromolar concentrations of linuron

A plant-microbial bioassay, based on the aquatic macrophyte Lemna minor L. (duckweed), was used to monitor biodegradation of nano- and micromolar concentrations of the phenylurea herbicide linuron. After 7 days of exposure to linuron, log-logistic-based dose-response analysis revealed significant growth inhibition on the total frond area of L. minor when linuron concentrations > or = 80 nM were added to the bioassay. A plant-protective effect was obtained for all concentrations > 80 nM by inoculation with either a bacterial consortium or Variovorax paradoxus WDL1, which is probably the main actor in this consortium. The outcome of the plant-microbe-toxicant interaction was also assessed using pulse amplitude-modulated chlorophyll a fluorescence and chlorophyll a fluorescence imaging. Linuron toxicity to L. minor became apparent as a significant decrease in the effective quantum yield (Delta F/Fm') within 90 min after exposure of the plants to linuron concentrations > or = 160 nM. Inoculation of the bioassay with the linuron-degrading bacteria neutralized the effect on the effective quantum yield at concentrations > or = 160 nM, indicating microbial degradation of these concentrations. The chlorophyll a fluorescence-based Lemna bioassay described here offers a sensitive, fast and cost-effective approach to study the potential of biodegrading microorganisms to break down minute concentrations of photosynthesis-inhibiting xenobiotics.     

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

A new fluorescence imaging system for monitoring the uptake of the PSII-herbicide diuron (OCMU) was tested in tobacco leaves. UV-laser-induced (Λ_exc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λ_em = 440 nm), green fluorescence F520 (Λ_em = 520 nm), red chlorophyll fluorescence F690 (Λ_em = 690 nm) and for far-red chlorophyll fluorescence F740 (Λ_em = 740 nm). Diuron-treated leaf parts exhibited a higher red and far-red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) significantly decreased in diuron-treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.     

Stability of chloroplastic triazine resistance in rutabaga backcross generations

Triazine resistance originally observed in a weed biotype of birdsrape (Brassica campestris L.) has been transferred through cytoplasmic substitution into rutabaga (Brassica napus ssp. Rapifera [Metzg.] Minsk.) by conventional backcrossing. Photosynthetic function and resistance to triazines were examined in six backcross generations of rutabaga as well as in the original parents. Chloroplast thylakoid membranes were isolated and their sensitivity to atrazine, metribuzin, and diuron assayed by measuring the inhibition of photoreduction of 1,6-dichlorophenol indophenol as well as the alteration of in vitro chlorophyll fluorescence rise characteristics. Both assay methods indicated that triazine resistance persisted in all rutabaga backcross generations, and that it involved triazine binding sites in chloroplasts. There was little resistance to diuron. In vivo chlorophyll fluorescence was also monitored, in the absence of herbicides, as an indicator of the electron transfer properties of the chloroplast photosystem II complex. The results indicated that electron transport from Q_A to Q_B was slower (as indicated by a larger intermediate level fluorescence during the transient rise) in the triazine resistant parents as well as in all the rutabaga backcross generations.     

Ammonium Nutrition Enhances Chlorophyll and Glaucousness in Kohlrabi

Kohlrabi (Brassica oleracea var.gongylodes) plants were grownin the greenhouse under autumn conditions and fertilized eitherwith pellets containing nitrogen as 40% ammonium sulphate and60% urea or with nutrient solution containing nitrogen predominantlyas nitrate. Plants given nitrogen as ammonium ions developedglaucous leaves compared to those supplied with nitrate whichformed glossy leaves. Ammonium-induced glaucousness was theresult of a two-fold increase in the amount of epicuticularwax and a markedly altered fine structure. Leaves from ammoniumfertilized kohlrabi plants also showed a 21% increase in chlorophyllcontent together with a reduction in the chlorophyll a:b ratioand decreased ground state fluorescence compared to plants suppliedwith nitrate. Photosynthesis and stomatal transpiration wereunaffected by the form of supplied nitrogen. Brassica oleracea ; chlorophyll; chlorophyll fluorescence; epicuticular wax; glaucousness; photosynthesis; transpiration     

Simultaneous measurement of stomatal conductance, non-photochemical quenching, and photochemical yield of photosystem II in intact leaves by thermal and chlorophyll fluorescence imaging

A new imaging system capable of simultaneously measuring stomatal conductance and fluorescence parameters, non-photochemical quenching (NPQ) and photochemical yield of photosystem II (Phi(PSII)), in intact leaves under aerobic conditions by both thermal imaging and chlorophyll fluorescence imaging was developed. Changes in distributions of stomatal conductance and fluorescence parameters across Phaseolus vulgaris L. leaves induced by abscisic acid treatment were analyzed. A decrease in stomatal conductance expanded in all directions from the treatment site, then mainly spread along the lateral vein toward the leaf edge, depending on the ABA concentration gradient and the transpiration stream. The relationships between stomatal conductance and fluorescence parameters depended on the actinic light intensity, i.e. NPQ was greater and Phi(PSII) was lower at high light intensity. The fluorescence parameters did not change, regardless of stomatal closure levels at a photosynthetically active photon flux (PPF) of 270 micro mol m(-2) s(-1); however, they drastically changed at PPF values of 350 and 700 micro mol m(-2) s(-1), when the total stomatal conductance decreased to less than 80 and 200 mmol m(-2) s(-1), respectively. This study has, for the first time, quantitatively analyzed relationships between spatiotemporal variations in stomatal conductance and fluorescence parameters in intact leaves under aerobic conditions.     

Imaging chlorophyll a fluorescence reveals specific spatial distributions under different stress conditions

Chlorophyll a fluorescence has been adopted as a fast, non-invasive, and cheap method to detect stress effects in plants. The majority of these chl-fluorescence measurements have been carried out with ‘clamping’ fluorometers recording punctual chlorophyll a fluorescence at isolated parts of the leaf. However, this method is inherently limited in providing information on the homogeneity of responses to stresses at the leaf or whole plant level. Therefore the purpose of this study was to measure imaging chlorophyll a fluorescence and to compare the temporal and spatial distribution of this emission under allelochemical (2-3H-benzoxazolinone and 3,4-dihydroxybenzaldehyde), thermal and salt, and heavy metal (cadmium, copper and zinc) treatment in the model plant Arabidopsis thaliana (L.) Heynh. The results suggested different spatial distributions for each condition: the two allelochemicals showed inhibition spots at the edges of the oldest leaves and both did not affect the photosynthetic activity of young leaves; treatment with the three heavy metals revealed highly homogenous effects over the whole plant with a quite uniform decrease of maximum PSII efficiency (also in youngest leaves). On the contrary, temperature (heat and cold) and salt stress showed an initial decrease of fluorescence in the tissues around the vascular bundles that lasted between 2 and 3 h depending on the treatment. These irregularities in chlorophyll fluorescence make it difficult to correlate punctual measures (typical for clamping fluorometers) with the effect on the whole plant, ignoring effects that are evident when imaging is used. Therefore these results show that monitoring chlorophyll a fluorescence by imaging improves the measurement of stress effects on treated plants, suggesting that punctual fluorescence measurements do not always reveal the heterogeneity of the stress-related effects in treated plants.     

A Cheap Chlorophyll a Fluorescence Imaging System

A cheap chlorophyll (Chl) a fluorescence imaging system was developed for measuring leaf areas of 30×45 cm. Uniform saturating irradiances were created using CuSO₄ filtered radiation from stroboscopes. The system was tested using maize leaves treated with diuron. Comparison was made with a small-area-measuring pulse amplified modulation Chl fluorometer.     

Effects of silicon sources on its deposition, chlorophyll content, and disease and pest resistance in rice

Rice (Oryza sativa L.) was grown in pots with pyridine N-oxide (PNO), 4-morpholino pyridine N-oxide (MNO), and sodium meta silicate as the sources for silicon. Aliquots of these were added in fortnightly intervals to seedlings through anthesis stage. The plants were monitored for plant growth characteristics, chlorophyll content (SPAD values), photosystem 2 activity (variable to maximum fluorescence ratio of dark adapted leaves), and for blast and yellow stem borer resistance. Deposition of silica in the leaves was monitored by scanning electron microscopy and silicon mapping. PNO or MNO application resulted in significant silicon accumulation in leaf bundle sheath cells. Application of PNO and MNO imparted disease and pest resistance by increasing silicon uptake of rice plants.     

Thermographic analysis of leaf water and energy information of Japanese spindle and glossy privet trees in low temperature environment

Thermography has been used in many fields to perform non-invasive temperature measurements of natural objects. In this paper, thermography was used to determine the temperature of leaves, stems and branch kerfs of Japanese spindle (Euonymus japonicus Thunb.) and glossy privet (Ligustrum lucidum Ait.) in the city of Jinan in China during winter. The temperatures of the leaves, stems and branch kerfs were monitored as the temperature decreased after the sample was subjected to hand heating or after the branch was cut. Differences in the specific heats and the latent heats of the leaves, branches and stems with different water contents and transpiration capacities were confirmed. The significant temperature difference obtained after hand heating between different leaf sections with varied water contents made it easy to obtain the thermal images, which were clear and exhibited reduced systematic errors. After hand heating, a significantly higher temperature was found at the major vein system of both Japanese spindle and glossy privet. This increased temperature difference made it possible to detect the water and the thermal state of these leaves. Therefore, it was possible to detect scorched area of the leaves, the twig dieback and the sap warming phenomenon in the leaves using thermography. In addition, the leaf bending phenomenon observed in Japanese spindle leaves during the deep freezing process indicates that the leaf scorch symptoms result from water stress and a lack of sap warming.     

Mannose metabolism in corn and its impact on leaf metabolites, photosynthetic gas exchange, and chlorophyll fluorescence

When intact corn leaves were provided millimolar concentrations of d-mannose through the transpiration stream photosynthesis was inhibited; 5.7 millimolar resulted in a 50% inhibition of the carbon exchange rate. This inhibition was partially reversible by the addition of orthophosphate to the feeding solution. Mannose metabolism by corn leaves was limited in that it did not act as a resource for sucrose or starch synthesis. Mannose 6-phosphate accumulated in the leaf tissues and was slowly metabolized by a pathway involving mannose 1-phosphate. Correlated with the mannose-6-phosphate accumulation were decreases in ATP, orthophosphate, sucrose, and phosphoenolpyruvate and increases in starch and maltose. When provided in the transpiration stream mannose had access to both mesophyll and bundle sheath cells. Mannose feeding led to oscillations in steady state chlorophyll fluorescence emission (680 nanometers) and an elimination of the Kautsky effect during fluorescence induction. Pyridoxal 5-phosphate and 2,4-dinitrophenol were found to be inhibitors of CO₂ exchange when provided in the transpiration stream of intact corn leaves. However, Pyridoxal 5-phosphate induced a quenching of steady state fluorescence while 2,4-dinitrophenol led to an increase in fluorescence emission.     

2种补血草属植物幼苗对NaCl胁迫的生理响应

以盐生植物大叶补血草和黄花补血草为实验材料,研究了NaCl胁迫对其幼苗叶绿素、可溶性蛋白、丙二醛含量等的影响,探讨这些生理指标的变化与补血草耐盐性的关系。结果表明,150mmol/LNaCl胁迫均使大叶补血草和黄花补血草幼苗叶片的叶绿素a(Chla)、叶绿素b(Chlb)和叶绿素总量降低,而过氧化氢(H2O2)和丙二醛(MDA)含量增加,且这种胁迫效应随处理时间的延长而增强,在相同胁迫时间段,大叶补血草各指标的减少或增加幅度明显大于黄花补血草;NaCl胁迫使大叶补血草叶片可溶性蛋白含量降低,却使黄花补血草可溶性蛋白含量增加。表明,NaCl胁迫对大叶补血草的伤害更大,黄花补血草的耐盐性可能强于大叶补血草。     

Salt tolerance in crop plants monitored by chlorophyll fluorescence in vivo

The potential of measurements of chlorophyll fluorescence in vivo to detect cellular responses to salinity and degrees of salt stress in leaves was investigated for three crop plants. Sugar beet (Beta vulgaris L.) (salt tolerant), sunflower (Helianthus annuus L.) (moderately salt tolerant), and bean (Phaseolus Vulgaris L. cv Canadian Wonder) (salt intolerant) were grown in pots and watered with mineral nutrient solution containing 100 millimolar NaCl. The fast rise in variable chlorophyll fluorescence yield that is correlated with photoreduction of photosystem II acceptors increased in leaves of sugar beet plants treated with salt suggesting stimulation of photosystem II activity relative to photosystem I. In sunflower, this fast rise was depressed by approximately 25% and the subsequent slow rate of quenching of the chlorophyll fluorescence was stimulated. These differences were more marked in the older mature leaves indicating an increasing gradient of salt response down the plant. The salt effect in vivo was reversible since chloroplasts isolated from mature leaves of salt-treated and control sunflower plants gave similar photosystem II activities. Unlike in sugar beet and sunflower, leaves of salt-treated bean progressively lost chlorophyll. The rate of slow quenching of chlorophyll fluorescence decreased indicating development of a partial block after photosystem II and possible initial stimulation of photosystem II activity. With further loss of chlorophyll photosystem II activity declined. It was concluded that measurements of chlorophyll fluorescence in vivo can provide a rapid means of detecting salt stress in leaves, including instances where photosynthesis is reduced in the absence of visible symptoms. The possible application to screening for salt tolerance is discussed.     

硫对烟草叶片光合特性和叶绿素荧光参数的影响

通过液培试验,测定了不同硫营养水平(0～32 mmol/L)下烟草叶片的叶绿素含量、气体交换参数和叶绿素荧光参数.结果表明,随着硫浓度的升高,烟草叶片的Chl.a、Chl.b、总Chl的含量和Chl.a/b逐渐增加;类胡萝卜素呈先下降后升高的趋势;烟草叶片的净光合速率(Pn)、蒸腾速率(Tr)、气孔导度(Gs)、胞间CO2浓度(Ci)下降显著,气孔限制值(Ls)明显升高,这表明高硫对烟草叶片的气体交换参数影响较大.随着硫浓度的升高,烟草叶片光合有效量子产量(EQY)、光合电子传递速率(ETR)、光化学淬灭(qP)都表现为先升高后降低,一般是4 mmol/L和8 mmol/L处理相对较高,浓度超过8 mmol/L以后,以上参数显著下降.非光化学淬灭(NPQ) 0,16 mmol/L和32 mmol/L处理表现相对较高,2、4、8 mmol/L处理的相对较低,2～8 mmol/L浓度的处理最适宜烟草的生长,高硫或低硫都会导致烟草EQY、ETR、qP降低和NPQ的升高.     

Short-term responses of Photosystem I to heat stress

When 23°C-grown potato leaves (Solanum tuberosum L.) were exposed for 15 min to elevated temperatures in weak light, a dramatic and preferential inactivation of Photosystem (PS) II was observed at temperatures higher than about 38°C. In vivo photoacoustic measurements indicated that, concomitantly with the loss of PS II activity, heat stress induced a marked gas-uptake activity both in far-red light (>715 nm) exciting only PS I and in broadband light (350–600 nm) exciting PS I and PS II. In view of its suppression by nitrogen gas and oxygen and its stimulation by high carbon-dioxide concentrations, the bulk of the photoacoustically measured gas uptake by heat-stressed leaves was ascribed to rapid carbon-dioxide solubilization in response to light-modulated stroma alkalization coupled to PS I-driven electron transport. Heat-induced gas uptake was observed to be insensitive to the PS II inhibitor diuron, sensitive to the plastocyanin inhibitor HgCl₂ and saturated at a rather high photon flux density of around 1200 E m^–2 s^–1. Upon transition from far-red light to darkness, the oxidized reaction center P700⁺ of PS I was re-reduced very slowly in control leaves (with a half time t_1/2 higher than 500 ms), as measured by leaf absorbance changes at around 820 nm. Heat stress caused a spectacular acceleration of the postillumination P700⁺ reduction, with t_1/2 falling to a value lower than 50 ms (after leaf exposure to 48°C). The decreased t_1/2 was sensitive to HgCl₂ and insensitive to diuron, methyl viologen (an electron acceptor of PS I competing with the endogenous acceptor ferredoxin) and anaerobiosis. This acceleration of the P700⁺ reduction was very rapidly induced by heat treatment (within less than 5 min) and persisted even after prolonged irradiation of the leaves with far-red light. After heat stress, the plastoquinone pool exhibited reduction in darkness as indicated by the increase in the apparent Fo level of chlorophyll fluorescence which could be quenched by far-red light. Application (for 1 min) of far-red light to heat-pretreated leaves also induced a reversible quenching of the maximal fluorescence level Fm, suggesting formation of a pH gradient in far-red light. Taken together, the presented data indicate that PS I responded to the heat-induced loss of PS II photochemical activity by catalyzing an electron flow from stromal reductants. Heat-stress-induced PS I electron transport independent of PS II seems to constitute a protective mechanism since block of this electron pathway in anaerobiosis was observed to result in a dramatic photoinactivation of PS I.Abbreviations PFD photon flux density - PS Photosystem - Apt and Aox amplitude of the photothermal and photobaric components of the photoacoustic signal, respectively - P700 reaction center pigment of PS I - Fo and Fm initial and maximal levels of chlorophyll fluorescence, respectively - Fv=Fm Fo-variable chlorophyll fluorescence - Q_A primary (stable) electron acceptor of PS II - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Cyt cytochrome     

Delayed fluorescence imaging of photosynthesis inhibitor and heavy metal induced stress in potato

Early chemical-induced stress in Solanum tuberosum leaves was visualized using delayed fluorescence (DF) imaging. The ability to detect spatially heterogeneous responses of plant leaves exposed to several toxicants using delayed fluorescence was compared to prompt fluorescence (PF) imaging and the standard maximum fluorescence yield of PSII measurements (Fv/Fm). The toxicants used in the study were two photosynthesis inhibitors (herbicides), 100 μM methyl viologen (MV) and 140 μM diuron (DCMU), and two heavy metals, 100 μM cadmium and 100 μM copper. The exposure times were 5 and 72 h. Significant photosynthesis-inhibitor effects were already visualized after 5 h. In addition, a significant reduction in the DF/PF index was measured in DCMU- and MV-treated leaves after 5 h. In contrast, only DCMU-treated leaves exhibited a significant decrease in Fv/Fm after 5 h. All treatments resulted in a significant decrease in the DF/PF parameter after 72 h of exposure, when only MV and Cd treatment resulted in visible symptoms. Our study highlights the power of delayed fluorescence imaging. Abundant quantifiable spatial information was obtained with the instrumental setup. Delayed fluorescence imaging has been confirmed as a very responsive and useful technique for detecting stress induced by photosynthesis inhibitors or heavy metals.     

毛白杨幼苗叶片生长及其光合参数和硼转运蛋白基因对高硼胁迫的响应

该研究采用毛白杨(Populus tomentosa)为试验材料,分析了温室条件下沙培幼苗对短期高硼胁迫(1、5、10 mmol/L硼酸)下的叶片生长、光合参数和硼转运蛋白的响应特征。结果显示:(1)与对照(0.05 mmol/L硼酸)相比,1 mmol/L硼酸处理导致毛白杨幼苗叶片叶绿素荧光参数上调,活性氧含量上升,树苗基部叶片出现少量黑色坏死斑;5 mmol/L硼酸胁迫下,叶片净光合速率、气孔导度和蒸腾速率下调,胞间二氧化碳浓度上升,叶绿素荧光参数和过氧化氢含量进一步上调,超氧阴离子含量较1 mmol/L硼酸胁迫时下调但仍然高于对照,除顶部叶片之外的其他叶片上出现大量坏死斑;10 mmol/L硼酸胁迫下,气体交换参数、叶绿素荧光参数和活性氧含量与5 mmol/L硼酸胁迫时相似,所有叶片均在平行于次级叶脉的方向出现呈带状分布的坏死斑。(2)毛白杨幼苗根和茎硼含量在硼胁迫条件下与对照相比变化幅度较小,而叶片硼含量在5 mmol/L和10 mmol/L硼酸胁迫下比对照显著上升,此时硼转移系数和生物富集系数均维持较高的水平。(3)硼转运蛋白(BOR)基因家族成员中PtoBOR4和PtoBOR8在根中的表达水平随着外界硼浓度的增加呈先上升后下降的趋势;在茎中,PtoBOR3基因下调表达,PtoBOR5上调表达;在叶片中,PtoBOR4表达先上升后下降,而PtoBOR7和PtoBOR8上调表达。研究表明,毛白杨幼苗叶片叶绿素荧光参数、活性氧、气体交换参数及硼转运蛋白基因家族表达对高硼胁迫较为敏感,硼胁迫症状在较短的时间内在叶片上以坏死斑的形式出现,可能与其较强的控制根系硼浓度的能力和向地上部分迅速运输硼的能力有关。     

Effects of polyethylene-glycol-induced osmotic stress on transpiration and photosynthesis in pinto bean leaf discs

A new leaf disc chamber allows measurements of chlorophyll fluorescence and CO₂ and H₂O vapor exchanges during infusion of solution into the cut edge of the disc. Polyethylene glycol (molecular weight, 6000) was used to apply a mild external osmotic stress to the leaf disc within this chamber. This stress rapidly caused a temporary increase in transpiration. This increase was reversed (5-6 minutes later) and after 20 to 25 min, the stomates nearly completely closed. Internal CO₂ (calculated) and leaf temperature followed the transpiration measurements. However, chlorophyll fluorescence (small rise) followed internal CO₂ (small rise). This complete sequence of events resembles those caused by exposure of leaves to certain air pollutants which have been seen to cause such a transient increase followed by a decrease in stomatal closure.     

Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.