Ontogenetic approach to the assessment of plant resistance to prolonged stress using chlorophyll fluorescence induction method

The review deals with objective reasons that limit the use of chlorophyll fluorescence induction kinetics (Chl FIK) method in plant ecology. Based on the ontogenetic approach (analysis and comparison of the dynamics of the Chl fluorescence ratio F_p/F_s and physiological characteristics of plant leaves in ontogeny) possible criteria for the estimation of general plant resistance of photosynthetic apparatus (PSA) to prolonged stress affects are proposed. One of these criteria is the presence or absence of a steady-state phase in the dynamics of Chl fluorescence ratio F_p/F_s (or R_fd) of plant leaves after they stop growing. We also propose to use the duration of the steady-state phase and variability of Chl fluorescence ratios R_fd and F_p/F_s in this period for quantitative assessment of plant PSA resistance to prolonged stress during plant leaf ontogeny. The paper is dedicated to memory of Prof. F.Ya. Sid’ko.     

The ontogenetic approach to chlorophyll fluorescence studies of plant photosynthetic apparatus under stressful conditions

Based on the analysis of reasons limiting the application of the method of chlorophyll fluorescence induction for estimating the state of the leaf photosynthetic apparatus under prolonged stress, the necessity of the ontogenetic approach consisting in a more exact determination of leaf age was substantiated. A comparison of the calendar and ontogenetic ways of determination of age of cucumber leaves under controlled conditions revealed essential distinctions in the estimation of plant leaf photosynthetic apparatus by the method of chlorophyll fluorescence induction for two variants distinguishing by the cultivation light regime ("white", 400-700 nm, and "red", 600-700 nm). It was shown that, in the case of prolonged effect of the stress factor on the plant, the unambiguity of the interpretation of chlorophyll fluorescence induction parameters in the estimation of the state of their photosynthetic apparatus depends essentially on the choice of the ontogenetic period of leaves of plants being compared and the accuracy of determination of leaf age.     

Image analysis of chlorophyll fluorescence transients for diagnosing the photosynthetic system of attached leaves

A new image instrumentation system for quantitative analysis of the rapid change in intensity of chlorophyll fluorescence during dark-light transition (CFI, chlorophyll fluorescence induction), which is a sensitive indicator of the various reactions of photosynthesis, was developed and its performance was evaluated. This system made it possible to resolve CFI at any small leaf area (about 1 square millimeter) of a whole leaf when the plant was illuminated by blue-green light at more than 50 micromoles photons per square meter per second. In order to test the usefulness of this system, we applied it to analyze the effect of SO₂ on photosynthetic apparatus in attached sunflower leaves. Dynamic CFI imaging over the whole single leaf, where there was no visible injury, indicated not only the local changes in photosynthetic activity but also the site of inhibition in photosynthetic electron transport system in chloroplasts. The new instrumentation system will be useful for the analytical diagnosis of various stress-actions on plants in situ.     

Relationships between the photochemical reflectance index (PRI) and chlorophyll fluorescence parameters and plant pigment indices at different leaf growth stages

This study aimed to evaluate the photochemical reflectance index (PRI) for assessing plant photosynthetic performance throughout the plant life cycle. The relationships between PRI, chlorophyll fluorescence parameters, and leaf pigment indices in Solanum melongena L. (aubergine; eggplant) were studied using photosynthetic induction curves both in short-term (diurnal) and long-term (seasonal) periods under different light intensities. We found good correlations between PRI/non-photochemical quenching (NPQ) and PRI/electron transport rate (ETR) in the short term at the same site of a single leaf but these relationships did not hold throughout the life of the plant. In general, changes in PRI owing to NPQ or ETR variations in the short term were <20?% of those that occurred with leaf aging. Results also showed that PRI was highly correlated to plant pigments, especially chlorophyll indices measured by spectral reflectance. Moreover, relationships of steady-state PRI/ETR and steady-state PRI/photochemical yield of photosystem II (Φ(PSII)) measured at uniform light intensity at different life stages proved that overall photosynthesis capacity and steady-state PRI were better correlated through chlorophyll content than NPQ and xanthophylls. The calibrated PRI index accommodated these pigments effects and gave better correlation with NPQ and ETR than PRI. Further studies of PRI indices based on pigments other than xanthophylls, and studies on PRI mechanisms in different species are recommended.     

Rapid and non-invasive detection of plants senescence using a delayed fluorescence technique.

Senescence is a phase of leaf ontogeny marked by declining photosynthetic activity that is paralleled by a decline in chloroplast function. The photosystem II in a plant is considered to be the primary site where delayed fluorescence (DF) is produced. We report here a simple, rapid, and non-invasive technique for detecting plants senescence based on quantitative measurements of DF. In the experimental study, various senescence symptoms induced by age or hormones were examined in the Catharanthus roseus L. G. Don plants. Detecting the DF emissions from leaves with a home-made DF biosensor enables DF parameters of C. roseus to be produced in a short time. Meanwhile, evaluations of leaves senescence were made from measurements of chlorophyll content, ion leakage, and net photosynthesis rate (Pn) based on the consumption of CO2 in the tested plants. The results of our investigation demonstrate that the changes in DF intensity of green plants can truly reflect the changes in photosynthetic capacity and chlorophyll content during age-dependent and hormone-modulated senescence. Moreover, the DF intensity negatively correlates with ion leakage in both types of senescence. With proper calibration, DF may provide an important approach for monitoring senescence process in vivo and quantitatively evaluating senescence extent. Therefore, a DF technique could be potentially useful for less time-consuming and automated screening of the interesting mutants with genetic modifications that change the plant senescence progress.     

Effect of light intensity on chlorophyll fluorescence in wheat leaves: Application of PAM-fluorometry

Application of pulse-amplitude-modulation (PAM) fluorometers for measuring slow stages of chlorophyll fluorescence induction (CFI) is considered. With an example of Triticum aestuvum L. plants grown under continuous illumination at a photon flux density of 600 μmol/(m² s) photosynthetically active radiation (PAR), the CFI curves were analyzed with leaves of various ages as a function of actinic light intensity. The fluorometer PAM-2100 was applied for measurements of CFI curves. The characteristic peaks of CFI curves in wheat leaves were most conspicuous and had the largest amplitudes at 600–800 μmol/(m² s) PAR, which corresponds to the middle range of actinic light intensities employed in PAM-2100 fluorometers. In plants exposed to favorable and stressful conditions, the developmental stages may proceed at different rates; thus, the comparison of fluorescence parameters for leaves of equal calendar age but having different physiological states may provide ambiguous data. Therefore, the feasibility of recording CFI curves of different types is quite important for rapid diagnostics of the age and state of plant leaves, as well as for adequate physiological conclusions.     

Growth and physiology of aspen supplied with different fertilizer addition rates

Variable internal plant nutrient content may confound plant response to environmental stress. Plant nutrient content may be controlled with relative addition rate techniques in solution culture. However, because raising large numbers of plants in flowing solution culture is difficult, we investigated the feasibility of raising plants in soil mix using relative fertilizer additions. Aspen (Populus tremuloides Michx.) clones (216, 259 and 271) planted in pots containing a peat, sand and vermiculite (2:1:1, v/v/v) soil mix were grown with exponentially increasing fertilizer concentrations and harvested periodically to assess growth. Addition rate treatments ranged from 0.01 to 0.05 day^?1. The lag phase of growth, in which plants adjusted to the fertilizer regime, lasted 40 days after which plants entered the experimental period characterized by constant relative growth rates equivalent to applied fertilizer addition rates. Total plant nutrient concentration was (1) unique for each addition rate, (2) linearly related to addition rate and growth rate, and (3) tended to increase at the highest, and decrease at the lowest addition rates. Regardless, the plants appeared to have attained steady-state conditions. Allocation of carbon to roots increased with lower addition rate treatments and was not dependent upon ontogeny. There were no treatment differences in growth response among aspen clones. Yet there were treatment differences in leaf chlorophyll and photosynthesis within the clones. For the 0.05 day^?1 addition rate treatment, chlorophyll, leaf N concentration and photosynthetic rate were strongly correlated with one another, were at a maximum in recently mature leaves, and rapidly declined with leaf age. The rate of decline in these leaf characteristics was slowest in clone 271, consistent with the leaf longevity stress response reported elsewhere. Plant responses from these relative fertilizer addition trials in soil mix agree closely with those run in hydroponics, indicating that steady-state nutrition can be achieved with a technically simple experimental assemblage.     

Soil Temperature Determines the Reaction of Olive Cultivars to Verticillium dahliae Pathotypes

Background

Development of Verticillium wilt in olive, caused by the soil-borne fungus Verticillium dahliae, can be influenced by biotic and environmental factors. In this study we modeled i) the combined effects of biotic factors (i.e., pathotype virulence and cultivar susceptibility) and abiotic factors (i.e., soil temperature) on disease development and ii) the relationship between disease severity and several remote sensing parameters and plant stress indicators.

Methodology

Plants of Arbequina and Picual olive cultivars inoculated with isolates of defoliating and non-defoliating V. dahliae pathotypes were grown in soil tanks with a range of soil temperatures from 16 to 32°C. Disease progression was correlated with plant stress parameters (i.e., leaf temperature, steady-state chlorophyll fluorescence, photochemical reflectance index, chlorophyll content, and ethylene production) and plant growth-related parameters (i.e., canopy length and dry weight).

Findings

Disease development in plants infected with the defoliating pathotype was faster and more severe in Picual. Models estimated that infection with the defoliating pathotype was promoted by soil temperatures in a range of 16 to 24°C in cv. Picual and of 20 to 24°C in cv. Arbequina. In the non-defoliating pathotype, soil temperatures ranging from 16 to 20°C were estimated to be most favorable for infection. The relationship between stress-related parameters and disease severity determined by multinomial logistic regression and classification trees was able to detect the effects of V. dahliae infection and colonization on water flow that eventually cause water stress.

Conclusions

Chlorophyll content, steady-state chlorophyll fluorescence, and leaf temperature were the best indicators for Verticillium wilt detection at early stages of disease development, while ethylene production and photochemical reflectance index were indicators for disease detection at advanced stages. These results provide a better understanding of the differential geographic distribution of V. dahliae pathotypes and to assess the potential effect of climate change on Verticillium wilt development.     

A retrieval algorithm to evaluate the Photosystem I and Photosystem II spectral contributions to leaf chlorophyll fluorescence at physiological temperatures

A new computational procedure to resolve the contribution of Photosystem I (PSI) and Photosystem II (PSII) to the leaf chlorophyll fluorescence emission spectra at room temperature has been developed. It is based on the Principal Component Analysis (PCA) of the leaf fluorescence emission spectra measured during the OI photochemical phase of fluorescence induction kinetics. During this phase, we can assume that only two spectral components are present, one of which is constant (PSI) and the other variable in intensity (PSII). Application of the PCA method to the measured fluorescence emission spectra of Ficus benjamina L. evidences that the temporal variation in the spectra can be ascribed to a single spectral component (the first principal component extracted by PCA), which can be considered to be a good approximation of the PSII fluorescence emission spectrum. The PSI fluorescence emission spectrum was deduced by difference between measured spectra and the first principal component. A single-band spectrum for the PSI fluorescence emission, peaked at about 735?nm, and a 2-band spectrum with maxima at 685 and 740?nm for the PSII were obtained. A linear combination of only these two spectral shapes produced a good fit for any measured emission spectrum of the leaf under investigation and can be used to obtain the fluorescence emission contributions of photosystems under different conditions. With the use of our approach, the dynamics of energy distribution between the two photosystems, such as state transition, can be monitored in vivo, directly at physiological temperatures. Separation of the PSI and PSII emission components can improve the understanding of the fluorescence signal changes induced by environmental factors or stress conditions on plants.     

Adaptive plasticity in response to light and nutrient availability in the clonal plant Duchesnea indica

克隆植物蛇莓对光照强度和养分条件的适应性可塑性 表型可塑性可帮助植物缓冲环境压力并使其表型与当地环境相匹配,但目前仅少数性状的可塑性被广泛认为是适应性的。为充分理解可塑性的适应性意义,仍需进一步研究更多的植物功能性状及其环境因子。本研究将匍匐茎克隆植物蛇莓(Duchesnea indica)的21个基因型种植于不同的光照和养分条件下,并利用选择梯度分析检测了形态和生理可塑性对光照强度和养分有效性变化的适应性值。在遮荫条件下,蛇莓适合度(果实数、分株数和生物量)降低,节间缩短变细,成熟叶叶绿素含量降低,但叶柄长度、比叶面积、老叶叶绿素含量均增加。在低养分条件下,植株叶柄缩短,叶面积缩小变厚,叶绿素含量降低,但果实数量和根冠比增加。选择梯度分析表明,叶柄长度和老叶叶绿素含量对光照变化的可塑性是适应性的,老叶和成熟叶叶绿素含量对养分变化的可塑性也是适应性的。因此,不同性状的可塑性适应值取决于特定的生态背景。该研究的发现有助于理解克隆植物表型可塑性响应环境变化的适应性意义。     

Physiological effects of the induction of resistance by compost or Trichoderma asperellum strain T34 against Botrytis cinerea in tomato

Certain types of compost used as growth media can induce resistance to foliar pathogens in above-ground parts of a plant. The induction of resistance can sometimes be associated with growth impairment and yield reduction. The objective of this study was to establish whether plants grown in olive marc compost had enhanced resistance against Botrytis cinerea at the cost of growth or physiological performance.Tomato plants grown in mature olive marc compost had approximately 60% less disease severity than plants grown in perlite. As a reference, plants grown in perlite enriched with the known inducer of resistance Trichoderma asperellum strain T34 (T34) had 35% less disease severity than plants grown in perlite. The salicylic acid (SA) pathway/abscisic acid (ABA) is involved in compost induced systemic resistance. Instead, perlite enriched with T34 is not linked to SA pathway/ABA. Physiological measures of water status, root/shoot ratio, stable isotopes of C and chlorophyll fluorescence showed that the plants grown in compost were close to a stress situation. However, growth measured as biomass and plant height of plants grown in compost was higher than in plants grown in perlite suggesting that plants in compost were not grown in a stress situation, but in a eustress. Tomato plants grown in perlite enriched with T34 had better growth, measured as total leaf area, biomass, height and nutrient uptake, than plants grown in perlite. Physiological measures showed that plants grown either in perlite or perlite enriched with T34 did not show any abiotic stress situation.     

NaCl-induced Senescence in Leaves of Rice (Oryza sativa L.) Cultivars Differing in Salinity Resistance

The influence of NaCl on senescence-related parameters (proteinand chlorophyll concentrations, membrane permeability and chlorophyllfluorescence) was investigated in young and old leaves of fiverice cultivars differing in salt resistance. NaCl hastened thenaturally-occurring senescence of rice leaves which normallyappears during leaf ontogeny: it decreased chlorophyll and proteinconcentrations and increased membrane permeability and malondialdehydesynthesis. Such an acceleration of deteriorative processes affectedall leaves in salt-sensitive cultivars while it was more markedin oldest than in youngest leaves of salt-resistant genotypes.NaCl-induced senescence also involved specific modifications,such as an increase in basal non-variable chlorophyll fluorescence(F ₀) recorded in all cultivars or a transient increase in solubleprotein concentration recorded in salt-resistant genotypes only.Alteration of membrane permeability appeared as one of the firstsymptoms of senescence in rice leaves and allowed discriminationamong cultivars after only 7 d of stress. In contrast, F _v/F _mratio (variable fluorescence/maximal fluorescence) was thesame for all cultivars during the first 18 d of stress and thuscould not be used for identifying salt-resistant rice exposedto normal light conditions. Relationships between parametersinvolved in leaf senescence are discussed in relation to salinityresistance of rice cultivars. Chlorophyll concentration; chlorophyll fluorescence; electrolyte leakage; magnesium; malondialdehyde; membrane permeability; NaCl; Oryza sativa L.; protein; rice; salinity resistance; senescence; UV absorbing substances     

Changes of the chlorophyll fluorescence induction kinetics of C3 and CAM plants during day/night cycles

The induction kinetics of the 680 nm chlorophyll fluorescence were measured on attached leaves of Kalanchoë daigremontiana R. Hamet et Perr. (CAM plant), Sedum telephium L. and Sedum spectabile Bor. (C₃ plant in spring, CAM plant in summer) and Raphanus sativus L. (C₃ plant) at three different times during a 12/12h day/night cycle. During the fluorescence transient the fluorescence intensity at the O, P and T-level (f_O, f_max, f_st,) was different for the plant species tested; this may be due to their different leaf structure, pigment composition and organization of their photosystems. The kinetics of the fluorescence induction depended on the time of preillumination or dark adaptation during the light/dark cycle but not on the type of primary CO₂ fixation mechanism (C₃ and CAM). For dark adapted leaves measured either at the end of the dark phase or after dark adaptation of plants taken from the light phase a higher P-level fluorescence, a higher variable fluorescence (P-O) and a larger complementary area were found than for leaves of plants taken directly from the light phase. This indicates the presence of largely oxidized photosystem 2 acceptor pools during darkness. During the light phase the fluorescence decline after the P-level was faster than during the dark phase; from this we conclude that the light adaptation of the photosynthetic apparatus (state 1→ state 2 transition, Δ pH) during the induction period proceeded faster in plants taken from the light phase than in plants taken from the dark phase.     

Applications of chlorophyll fluorescence can improve crop production strategies: an examination of future possibilities

Chlorophyll fluorescence has been routinely used for many years to monitor the photosynthetic performance of plants non-invasively. The relationships between chlorophyll fluorescence parameters and leaf photosynthetic performance are reviewed in the context of applications of fluorescence measurements to screening programmes which seek to identify improved plant performance. The potential role of chlorophyll fluorescence imaging in increasing both the sensitivity and throughput of plant screening programmes is examined. Finally, consideration is given to possible specific applications of chlorophyll fluorescence for screening of plants for tolerance to environmental stresses and for improvements in glasshouse production and post-harvest handling of crops.     

Radiation Use Efficiency,Chlorophyll Fluorescence,and Reflectance Indices Associated with Ontogenic Changes in Water-Limited Chenopodium quinoa Leaves

Net photosynthetic rate, radiation use efficiency, chlorophyll (Chl) fluorescence, photochemical reflectance index (PRI), and leaf water potential were measured during a 25-d period of progressive water deficit in quinoa plants grown in a glasshouse in order to examine effects of water stress and ontogeny. All physiological parameters except F_v/F_m were sensitive to water stress. Ontogenic variations did not exist in F_v/F_m and leaf water potential, and were moderate to high in the other parameters. The complete recovery of photosynthetic parameters after re-irrigation was related with the stability in F_v/F_m. PRI showed significant correlation with predawn leaf water potential, F_m, and midday F_v/F_m. Thus PRI and Chl fluorescence may help in assessing physiological changes in quinoa plants across different developmental stages and water status.     

干旱胁迫下黄条金刚竹的光合和叶绿素荧光特性

通过盆栽试验,研究了自然干旱胁迫处理下,黄条金刚竹植株形态、土壤含水量(SWC)、叶片含水量(LWC)、叶片水势(LWP)、光合作用参数和叶绿素荧光参数的变化,及与环境因素的关系.结果表明:自然干旱胁迫后第17天,黄条金刚竹出现干旱伤害症状,叶片失水下垂、内卷,第43天地上部分失水死亡;复水后10 d,叶片叶绿素荧光参数与第43天无显著差异.在干旱胁迫0～21 d,SWC显著下降,而在17 d以后,LWP急剧下降,29 d以后LWC显著降低.随着干旱胁迫程度的加剧,叶片蒸腾速率(T_r)、气孔导度(g_s)持续下降,而光合速率(P_n)波动较大,瞬时水分利用效率（PWUE）则先升高后降低;17 d后,P_n由气孔限制为主转为非气孔限制为主,实际光合效率(Φ_PSⅡ)开始下降;25 d后,黄条金刚竹的光合机构被破坏,最大光化学效率(F_v/F_m)和PSⅡ潜在热耗散能力(q_N)分别由0.64、0.79下降到-0.11、0.33.T_r、g_s、PWUE与LWP密切相关,大气湿度对P_n、T_r、g_s的影响显著,气孔是光合参数变化的主导因素.本试验条件下,黄条金刚竹适宜在土壤相对含水率≥12%的土壤中生长,且干旱胁迫持续时间不超过25 d.     

Photosynthetic induction in C4 leaves

Simultaneous measurements of CO₂ uptake, transpiration rate, and chlorophyll a fluorescence in leaf strips of C₄ plants during the induction phase of photosynthesis are described. The timecourse of CO₂ fixation is biphasic with the initial phase occurring within the first 1 to 5 min and the secondary phase consisting of a slow rise to the steady-state rate of photosynthesis. Transpiration rate follows the CO₂-fixation timecourse closely but the intercellular CO₂ concentration never falls below saturation for C₄ plants. Chlorophyll a fluorescence quenching occurs exclusively during the initial fast phase of the CO₂-fixation timecourse. The effect of duration of dark pretreatment of leaves on these parameters and the effects of light intensity and CO₂ concentration are examined. These results are discussed with respect to the C₄ cycle and photochemical and non-photochemical chlorophyll fluorescence quenching.Abbreviations IRGA infra-red gas analyser - NADP-ME, NAD-ME and PEP-CK the three groups of C₄ plants utilising the enzymes NADP-malic enzyme, NAD-malic enzyme and phosphoenolpyruvate carboxykinase, respectively, for C₄-acid decarboxylation - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglyceric acid     

Increase of the chlorophyll fluorescence ratio F690/F735 during the autumnal chlorophyll breakdown

Summary The chlorophyll content and the fluorescence induction kinetics at two wavelengths (690 nm and 735 nm) have been measured in leaves of nine common broadleaf tree species during the autumnal chlorophyll breakdown. The ratio of the chlorophyll fluorescence maxima F690/F735 was determined at fluorescence maximum (fm) and at steady-state conditions (fs) by the laser-induced fluorescence emission using the two-wavelength fluorometer. The ratio F690/F735 increases with the leaf discolouring during the autumnal chlorophyll breakdown. The relationship between the chlorophyll content and the ratio F690/F735 can be expressed by a power function (curvilinear relationship) which is valid for all the species examined. In most cases the ratio F690/F735 measured in the upper leaf side is lower than that in the lower leaf side, but the trend is the same along the decreasing chlorophyll content. The ratio F690/F735 is always higher at maximum fluorescence than at steady-state fluorescence in the upper as well as lower leaf side and these values are well fitted in a linear correlation. This study confirms the usefulness of the ratio F690/F735 as a suitable non-destructive indicator of the in-vivo chlorophyll content, especially at medium and low chlorophyll content.     

不同时间的UV-B辐射对拟南芥幼苗生长的影响

以哥伦比亚野生型拟南芥(Arabidopsis thaliana)为实验材料, 用辐射功率为16.67 μW·cm^-2但不同时间(0.5、1、1.5、2、2.5和3小时)的UV-B辐射对拟南芥幼苗进行处理, 观察叶片形态, 并测定其根长、叶绿素含量、可溶性蛋白含量、丙二醛(MDA)浓度、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性及叶绿素荧光参数。结果显示, 短时间UV-B辐射可促进拟南芥根的伸长, 叶绿素和可溶性蛋白含量升高; 长时间UV-B辐射则抑制拟南芥根的生长, 使叶绿素含量、可溶性蛋白含量、叶绿素荧光参数F_v/F_m及qP逐渐降低, MDA浓度、SOD活性、CAT活性和qN值升高, 并随着时间的延长逐渐降低或升高。当辐射功率为16.67 μW·cm^-2时, 其最佳辐射时间为1.5小时。UV-B辐射作为一种环境胁迫, 其胁迫程度都是在一定的范围内, 当胁迫达到极限时, 植株都会对UV-B辐射产生一定的适应效应而使损伤降低。     

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.