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.     

Monitoring cashew seedlings during interactions with the fungus <Emphasis Type="Italic">Lasiodiplodia theobromae</Emphasis> using chlorophyll fluorescence imaging

The chlorophyll (Chl) fluorescence imaging technique was applied to cashew seedlings inoculated with the fungus Lasiodiplodia theobromae to assess any disturbances in the photosynthetic apparatus of the plants before the onset of visual symptoms. Two-month-old cashew plants were inoculated with mycelium of L. theobromae isolate Lt19 or Lt32. Dark-adapted and light-acclimated whole plants or previously labelled, single, mature leaf from each plant were evaluated weekly for Chl fluorescence parameters. From 21 to 28 days, inoculation with both isolates resulted in the significantly lower maximal photochemical quantum yield of PSII (F_v/F_m) than those for control samples, decreasing from values of 0.78 to 0.62. In contrast, the time response of the measured fluorescence transient curve from dark-acclimated plants increased in both whole plants and single mature leaves in inoculated plants compared with controls. The F_v/F_m images clearly exhibited photosynthetic perturbations 14 days after inoculation before any visual symptoms appeared. Additionally, decays in the effective quantum yield of PSII photochemistry and photochemical quenching coefficient were also observed over time. However, nonphotochemical quenching increased during the evaluation period. We conclude that F_v/F_m images are the effective way of detecting early metabolic perturbations in the photosynthetic apparatus of cashew seedlings caused by gummosis in both whole plants and single leaves and could be potentially employed in larger-scale screening systems.     

Cd2+胁迫对银芽柳PSⅡ叶绿素荧光光响应曲线的影响

以盆栽银芽柳为材料,利用MINI-IMAGING-PAM荧光成像测定系统,研究了Cd²⁺胁迫下叶片叶绿素荧光参数的变化及其光响应曲线。结果表明,初始荧光Fo与最大荧光Fm随着Cd²⁺浓度的增大而呈现先升后降的趋势,Fo与Fm在200 mg/LCd²⁺处理4周时达到最高值,400 mg/LCd²⁺处理则显著下降;PSⅡ最大光化学效率(Fv/Fm)与PSⅡ潜在光化学效率(Fv/Fo)显著受 Cd²⁺胁迫抑制,但随Cd²⁺浓度的增加呈先降后升的变化趋势。Cd²⁺胁迫下各叶绿素荧光参数的光响应结果表明,PSⅡ实际光量子效率Y(Ⅱ)、荧光淬灭系数(qP)随光化光强度的增加呈下降趋势,而同光强下高浓度Cd²⁺ 使Y(Ⅱ)与(qP) 显著降低;PSⅡ调节性能量耗散的量子产额Y(NPQ)、非光化学淬灭系数(qN)与表观电子传递速率(ETR)则随着光强增加呈上升趋势,同光强下高浓度Cd²⁺处理显著提高Y(NPQ)、qN 与ETR。Cd²⁺胁迫下,PSⅡ非调节性能量耗散的量子产额Y(NO)稳定在较低水平,同光强下Y(NO)随Cd²⁺浓度增加略有提高。说明,银芽柳通过调节PSⅡ反应中心开放程度与活性,对Cd²⁺胁迫表现出较强的耐性,高浓度Cd²⁺胁迫导致PSⅡ反应中心关闭或不可逆失活,表现出光抑制。     

Photosynthesis of soybean under the action of a photosystem II-inhibiting herbicide

Photosynthesis, the fundamental physiological process of plant responsible for the growth and yield of crops, is strongly affected by environmental stresses. Several methods have been used to study changes in the physiological parameters of plants exposed to stresses. The work aimed to study physiological parameters related to photosynthesis in leaf discs of soybean plants exposed to a photosystem II-inhibiting herbicide. Soybean leaf discs obtained from mature leaves of plants in the vegetative stage immersed in bentazon herbicide solutions at concentrations of 0, 100, 250 or 500 μM were evaluated. In experiment I, the effect of the herbicide on chlorophyll a fluorescence transient was measured using a portable fluorometer. In the second experiment, the effect of the herbicide on modulated chlorophyll a fluorescence and gas exchange were evaluated, with the latter being measured with an infrared gas analyzer. The evaluations of transient and modulated fluorescence provided additional information on the photosynthetic activity of soybean leaf discs exposed to the action of bentazon. For the fluorescence transient analysis, performance indices were the parameters most sensitive to the action of bentazon, showing a decrease of approximately 70 % at a dose of 500 μM. For the modulated fluorescence analysis, the photochemical quenching coefficient, the electron transport rate, the photochemical efficiency of photosystem II and the net assimilation rate, decreased in response to herbicide application, with values that were almost equal to zero at a dose of 500 µM, which are the parameters that showed the greatest sensitivity to bentazon in soybean.     

干旱和复水对草莓叶片叶绿素荧光特性的影响

采用日本丰香草莓(Fragaria×ananassa Duch.cv.Toyonoka)品种进行实验,研究干旱和复水对其叶片叶绿素荧光特性的影响。结果表明,随着干旱胁迫程度的加剧,草莓叶片的最大荧光(Fm)、PSⅡ原初光能转化效率(Fv/Fm)、PSⅡ实际光化学效率(Yield)、光化学猝灭系数(qP)都随干旱胁迫的加剧而下降。干旱胁迫14d后,不同处理组草莓叶片的叶绿素荧光参数存在着显著的差异(P0.05)。复水后,低度胁迫和中度胁迫处理组能较快地恢复到正常水平,但重度胁迫组与对照组存在着显著的差异(P0.05)。     

Effects of nitrogen on the activity of antioxidant enzymes and gene expression in leaves of Populus plants subjected to cadmium stress

The research aimed to verify the important physiological effect of nitrogen (N) on plants exposed to cadmium (Cd). The poplar plants were grown in a Hoagland nutrient solution and treated with extra N, Cd, and N + Cd. After treatment, plant growth and chlorophyll content were recorded. The oxidative stress, the activity of antioxidant enzymes, and the expression of related genes were also examined. The results indicated the plants treated with sole Cd presented obvious toxicity symptoms, i.e. growth inhibition, reactive oxygen species accumulation, and chlorophyll content decrement. However, when N was added to the plants under Cd stress, plant growth was enhanced, chlorophyll synthesis was promoted, and the oxidative stress was alleviated. Further, the expression of antioxidant enzymes genes was upregulated by N. The results indicated that N partially reversed the toxic effect of Cd on poplar plants, which can provide new methodology to enhance the phytoremediation technology for heavy metal pollution soil.     

Managing heavy metal toxicity stress in plants: Biological and biotechnological tools

The maintenance of ion homeostasis in plant cells is a fundamental physiological requirement for sustainable plant growth, development and production. Plants exposed to high concentrations of heavy metals must respond in order to avoid the deleterious effects of heavy metal toxicity at the structural, physiological and molecular levels. Plant strategies for coping with heavy metal toxicity are genotype-specific and, at least to some extent, modulated by environmental conditions. There is considerable interest in the mechanisms underpinning plant metal tolerance, a complex process that enables plants to survive metal ion stress and adapt to maintain growth and development without exhibiting symptoms of toxicity. This review briefly summarizes some recent cell biological, molecular and proteomic findings concerning the responses of plant roots to heavy metal ions in the rhizosphere, metal ion-induced reactions at the cell wall-plasma membrane interface, and various aspects of heavy metal ion uptake and transport in plants via membrane transporters. The molecular and genetic approaches that are discussed are analyzed in the context of their potential practical applications in biotechnological approaches for engineering increased heavy metal tolerance in crops and other useful plants.     

In situ detection of heavy metal substituted chlorophylls in water plants

The in vivo substitution of magnesium, the central atom of chlorophyll, by heavy metals (mercury, copper, cadmium, nickel, zinc, lead) leads to a breakdown in photosynthesis and is an important damage mechanism in heavy metal-stressed plants. In this study, a number of methods are presented for the efficient in situ detection of this substitution (i.e. in whole plants or in chloroplasts). While macroscopic observations point to the formation of heavy metal chlorophylls at higher concentrations, fluorescence microscopy enables the detection of this reaction at very low substitution rates. Therefore, the course of the reaction can be followed by continuously measuring the fluorescence of whole plants. Furthermore absorbance spectroscopy of whole cells or isolated chloroplasts also enables the in situ detection of heavy metal chlorophylls. These methods provide practicable approaches in detecting the formation of these compounds in situ, avoiding artefacts that might occur using extraction methods based on polar solvents. In addition to the new methods for in situ detection, an extreme heterogeneity in the reaction of cells in the same tissue upon heavy metal stress was observed: while some cells are already disintegrating, others still show normal fluorescence and photosynthetic activity. Measurements of fluorescence kinetics gave a further hint that in high light intensity a substitution of Mg by heavy metals might take place specifically in PS II reaction centres.     

Photosynthetic responses of Oryza sativa L. seedlings to cadmium stress: physiological,biochemical and ultrastructural analyses

In the present study, photosynthetic responses induced by cadmium stress in chlorophyll biosynthesis, photochemical activities, the stability of thylakoid membranes chlorophyll-protein complexes and the chloroplast ultrastructure of the cereal crop Oryza sativa L. were characterized. Cadmium inhibited the biosynthesis of chlorophyll by interfering with activity of δ-aminolevulinic acid dehydratase in the rice seedlings. For the photochemical activities analyses, the extent of the decrease in photosystem II activity was much greater than that in the PS I activity. The variations in the chlorophyll a fluorescence parameters also indicated that cadmium toxicity drastically affected the photochemistry of PS II. Biochemical analyses by BN-PAGE and protein immunoblot showed that cadmium toxicity considerably affected the stability of PS II-core, cytb ₆ /f, RuBisCO, PSI + LHCI and LHCII (Trimeric). We observed the rate of the thylakoid membranes protein degradation, was mainly at the level of RbcL, PsaA, Lhca1 and D1. In addition, the damages to chloroplast structure and thylakoid stacking analyzed by transmission electron microscopy were indicative of general disarray in the photosynthetic functions exerted by cadmium toxicity. These results are valuable for understanding the biological consequences of heavy metals contamination particularly in soils devoted to organic agriculture.     

He–Ne laser illumination ameliorates photochemical impairment in ultraviolet-B stressed-wheat seedlings via detoxifying ROS cytotoxicity

The protective effect and physiochemical mechanism of He-Ne laser illumination on photochemical impairment were evaluated by investigating chlorophyll fluorescence characteristics, photochemical activities of two photosystems, reactive oxygen species (ROS) levels and antioxidant enzyme activities in UV-B stressed-wheat (Triticum aestivum L.) seedlings. The results showed that enhanced UV-B stress significantly inhibited plant growth, reduced photosynthetic pigment content and antioxidant enzyme activities, while increased intracellular ROS levels. Meanwhile, UV-B stress also altered chlorophyll fluorescence characteristics and photochemical activities of seedlings. However, He-Ne laser illumination markedly improved photochemical activities and photosynthetic efficiency of two photosystems through detoxifying excessive ROS productions. Illumination with white fluorescent lamps (W), red light (R), or red light, then far-red light (R + FR) had not alleviated the inhibitory effect of UV-B stress on plant growth, suggesting that He-Ne laser illumination might be responsible for UV-B-stressed seedlings due to its regulation for intracellular ROS levels and plant oxidant/antioxidant balance. Furthermore, the laser alone also showed a positive impact on photochemical activities of photosystem I and photosystem II in plants.     

Investigation of deleterious effects of chromium phytotoxicity and photosynthesis in wheat plant

Increasing human and industrial activities lead to heavy metal pollution. Heavy metal chromium (Cr) is considered to be a serious environmental contaminant for the biota. Phytotoxic effects of Cr were studied in wheat plants. Growth parameters were largely inhibited as a result of disturbances in the plant cell metabolism in response to Cr toxicity. Chromium toxicity led to decline in a number of active reaction centres of PSII, rate of electron transport, and change in PSII heterogeneity. Chromium did not cause any change in heterogeneity of the reducing side. A significant change in antenna size heterogeneity of PSII occurred in response to Cr toxicity. Chromium seems to have extensive effects on the light harvesting complex of PSII.     

3D lidar imaging for detecting and understanding plant responses and canopy structure

Understanding and diagnosing plant responses to stress will benefit greatly from three-dimensional (3D) measurement and analysis of plant properties because plant responses are strongly related to their 3D structures. Light detection and ranging (lidar) has recently emerged as a powerful tool for direct 3D measurement of plant structure. Here the use of 3D lidar imaging to estimate plant properties such as canopy height, canopy structure, carbon stock, and species is demonstrated, and plant growth and shape responses are assessed by reviewing the development of lidar systems and their applications from the leaf level to canopy remote sensing. In addition, the recent creation of accurate 3D lidar images combined with natural colour, chlorophyll fluorescence, photochemical reflectance index, and leaf temperature images is demonstrated, thereby providing information on responses of pigments, photosynthesis, transpiration, stomatal opening, and shape to environmental stresses; these data can be integrated with 3D images of the plants using computer graphics techniques. Future lidar applications that provide more accurate dynamic estimation of various plant properties should improve our understanding of plant responses to stress and of interactions between plants and their environment. Moreover, combining 3D lidar with other passive and active imaging techniques will potentially improve the accuracy of airborne and satellite remote sensing, and make it possible to analyse 3D information on ecophysiological responses and levels of various substances in agricultural and ecological applications and in observations of the global biosphere.     

不同水氮梯度下典型挺水植物叶绿素荧光的响应特性

叶绿素荧光测量分析可以揭示植物叶片光化学效率的变化,已越来越多地应用于植物生态监测。以再生水为主要补给水源的北京门城湖湿地公园为研究区,选取典型湿地挺水植物芦苇(Phragmites australis)、香蒲(Typha angustifolia)和茭白(Zizania latifolia)为研究对象,通过野外测量叶片尺度的叶绿素荧光参数和室内测定对应样点的水体总氮含量指标,研究了再生水补给条件下,不同水氮梯度植物叶绿素荧光的响应特性。结果表明,3种典型挺水植物的初始荧光(Fo)与最大荧光(Fm)随着水体总氮含量的增加呈现上升的趋势;PSII的量子效率(F_v/F_m)与实际量子效率(ΦPSII)受水氮含量的影响先升高,达到15–20 mg·L~(–1)区间时,则与之持平;光化学淬灭(qP)参数则呈现先升高后降低的变化趋势,而非光化学淬灭(NPQ)参数的变化没有明显的规律。当水氮含量为15–20 mg·L~(–1)时,光化学反应减弱,光合作用出现抑制。不同类型植物的荧光参数也有所不同,处于生长期(6月)植物的光合作用显著强于生长成熟期(9月)。     

Insights into the Roles of Melatonin in Alleviating Heavy Metal Toxicity in Crop Plants

Alleviating heavy metal pollution in farmland soil, and heavy metal toxicity in plants is the focus of global agricultural environmental research. Melatonin is a kind of indoleamine compound that wide exists in organisms; it is currently known as an endogenous free radical scavenger with the strongest antioxidant effect. As a new plant growth regulator and signaling molecule, melatonin plays an important role in plant resistance to biotic or abiotic stress. Recent studies indicate that melatonin can effectively alleviate heavy metal toxicity in crop plants, which provides a new strategy to minimize heavy metal pollution in crop plants. This study summarizes the research progress on the role of melatonin in alleviating heavy metal toxicity in crop plants and the related physiological and ecological mechanisms such as reducing the concentration of heavy metals in the rhizosphere, fixing and regionally isolating of heavy metals, maintaining the mineral element balance, enhancing the antioxidant defense system and interacting with hormonal signaling. Furthermore, future prospects for the mechanism of melatonin in regulating heavy metal toxicity, the pathway regulating synthesis and catabolism, and the interaction mechanism of melatonin signaling and other phytohormones are presented in this paper, with the goal of providing a theoretical basis for controlling heavy metal ion accumulation in crop plants grown in contaminated soil.     

Natural and commercial Salix clones differ in their ecophysiological response to Zn stress

This study was carried out to determine the effect of different zinc concentrations on the ecophysiological response of Salix clones: four commercial clones (“1962”, “1968”, “Drago”, and “Levante”) selected for short rotation coppice, and one natural clone, “Sacco”, obtained from a contaminated area. Gas exchanges, chlorophyll a fluorescence (JIP-test), relative chlorophyll content, and biometric parameters were measured in plants grown for fifteen days in soil containing Zn concentrations of 0, 300, 750, and 1,500 mg(ZnCl₂) kg^?1. Ecophysiological response to metal stress differed in dependence on the Zn concentration and clone. At the low Zn concentration (300 mg kg^?1), the absence of any significant reductions in parameters investigated indicated an efficient plant homeostasis to maintain the metal content within phytotoxic limits. Stomatal limitation, observed at 750 and 1,500 mg kg^?1, which was found in all clones after three days of the treatment, might be caused by indirect effects of metal on guard cells. Among commercial clones, “Drago” was more sensitive to Zn stress, showing inhibition of growth, while “1962” clone showed a downregulation of PSII photochemistry following the slowdown in the Calvin-Benson cycle. On the contrary, the natural Salix clone (“Sacco”) performed better compared to the other clones due to activation of a photosynthetic compensatory mechanism.     

酸雨胁迫对秃瓣杜英幼苗叶片叶绿素荧光特性和生长的影响

采用盆栽试验和模拟酸雨喷淋的试验方法,研究了重度酸雨(pH 2.5)、中度酸雨(pH 4.0)和对照(pH 5.6)处理下,不同季节秃瓣杜英幼苗叶片的叶绿素荧光特性及其生长差异.结果表明：不同季节相同处理下,秃瓣杜英幼苗叶片的相对叶绿素含量(SPAD)、PSⅡ的最大光化学效率(F_v/F_m)、PSⅡ实际光化学量子产量(Φ_PSⅡ)、株高和地径均为10月＞7月＞4月＞1月;同一季节不同处理中,SPAD、F_v/F_m、Φ_PSⅡ、株高和地径为重度酸雨＞中度酸雨＞对照;不同pH酸雨梯度处理和季节的交互作用对秃瓣杜英的SPAD、F_v/F_m、株高和地径的影响显著,而对Φ_PSⅡ、光化学淬灭系数和非光化学淬灭系数的影响不显著.     

Photosynthetic response of three climber plant species to osmotic stress induced by polyethylene glycol (PEG) 6000

We examined the photosynthesis response to osmotic stress in three climber plant species, Pharbitis nil (Linn.) Choisy, Lonicera japonica Thunb, and Parthenocissus tricuspidata (Sieb.et Zucc.) Planch. All climber plants were exposed to osmotic stress induced by polyethylene glycol (PEG) 6000 at 4 levels (slight, moderate, severe osmotic and the control) for 30?days. Photosynthesis response was determined by measuring leaf photosynthesis, chlorophyll fluorescence, carbonic anhydrase activity and stable carbon isotope ratios. P. nil maintained high photosynthetic activity under long-term moderate osmotic stress due to both stable photosystem II photochemical efficiency and high carbonic anhydrase activity. L. japonica maintained high photosynthetic activity under long-term moderate stress due to high carbonic anhydrase activity rather than photosystem II photochemical efficiency. P. tricuspidata tolerated only short-term moderate osmotic stress and long-term slight osmotic stress because its response was mainly stomatal limitation, with the lowest photosynthetic activity and hardly any carbonic anhydrase activity. Carbonic anhydrase activity was inversely correlated with stable carbon isotope ratios. The regulation by carbonic anhydrase was probably the reason for P. nil and L. japonica to tolerate long-term moderate osmotic stress. The selection on the species should consider the differential adaptation mechanism to osmotic stress during the development of drought-resistant plants.     

Two-photon excitation chlorophyll fluorescence lifetime imaging: a rapid and noninvasive method for in vivo assessment of cadmium toxicity in a marine diatom Thalassiosira weissflogii

We demonstrate that a two-photon excitation fluorescence lifetime imaging technology can rapidly and noninvasively assess the cadmium (Cd)-induced toxic effects in a marine diatom Thalassiosira weissflogii. The chlorophyll, an intrinsic fluorophore, was used as a contrast agent for imaging of cellular structures and for assessment of cell toxicity. The assessment is based on an imaging-guided statistical analysis of chlorophyll fluorescence decay. This novel label-free imaging method is physically based and free of tedious preparation and preprocessing of algal samples. We first studied the chlorophyll fluorescence quenching induced by the infrared two-photon excitation laser and found that the quenching effects on the assessment of Cd toxicity could be well controlled and calibrated. In the toxicity study, chlorophyll fluorescence lifetime images were collected from the diatom samples after exposure to different concentrations of Cd. The alteration of chloroplast structure at higher Cd concentration was clearly identified. The decay of chlorophyll fluorescence extracted from recorded pixels of high signal-to-noise ratio in the fluorescence lifetime image was analyzed. The increase of average chlorophyll fluorescence lifetime following Cd treatment was observed, indicating the Cd inhibition effect on the electron transport chain in photosynthesis system. The findings of this study show that the temporal characteristics of chlorophyll fluorescence can potentially be utilized as a biomarker for indicating Cd toxicity noninvasively in algal cells.