Multicolor fluorescence imaging for early detection of the hypersensitive reaction to tobacco mosaic virus.

The physiological status of plants can nowadays be promptly monitored with non-invasive methods. This opens the possibility to continuously follow-up plant performance and permits to detect stress-induced deviations presymptomatically. Upon stress, plants may synthesize specific compounds, depending on the causal agent. Such compounds may alter the absorption of the light impinging on plant leaves, hence the spectrum of reflected, re-emitted, and transmitted light changes. UV-excited fluorescence imaging specifically allows visualization of the accumulation of phenolic compounds, e.g. those associated with the hypersensitive response to pathogens. By using imaging at regular intervals (time-lapse series) of tobacco mosaic virus (TMV) infection in resistant tobacco we aimed at the description and quantification of the kinetics of blue-green fluorescence compared to the visual development of the disease. Presymptomatic responses to TMV infection were observed with a multicolor fluorescence and reflectance imaging setup. The onset of increases in blue-green and chlorophyll fluorescence were comparable in timing, although further symptom development was strikingly different. Compounds known to accumulate during the hypersensitive response and displaying blue-green fluorescence revealed different dynamics of fluorescence evolution in time. The multichannel imaging system permitted to discern the key components salicylic acid and scopoletin. In contrast, for the compatible interaction between TMV and non-resistant tobacco, no presymptomatic responses were detected on inoculated leaves. This work proves the potential of multispectral imaging to unveil stress-associated signatures, and the power of blue-green fluorescence imaging to monitor accumulation of secondary compounds.

Multi-sensor plant imaging: Towards the development of a stress-catalogue.

Agricultural production is limited by a wide range of abiotic (e.g. drought, waterlogging) and biotic (pests, diseases and weeds) stresses. The impact of these stresses can be minimized by appropriate management actions such as irrigation or chemical pesticide application. However, further optimization requires the ability to diagnose and quantify the different stresses at an early stage. Particularly valuable information of plant stress responses is provided by plant imaging, i.e. non-contact sensing with spatial resolving power: (i) thermal imaging, detecting changes in transpiration rate and (ii) fluorescence imaging monitoring alterations in photosynthesis and other physiological processes. These can be supplemented by conventional video imagery for study of growth. An efficient early warning system would need to discriminate between different stressors. Given the wide range of sensors, and the association of specific plant physiological responses with changes at particular wavelengths, this goal seems within reach. This is based on the organization of the individual sensor results in a matrix that identifies specific signatures for multiple stress types. In this report, we first review the diagnostic effectiveness of different individual imaging techniques and then extend this to the multi-sensor stress-identification approach.

Reduced gibberellin response affects ethylene biosynthesis and responsiveness in the Arabidopsis gai eto2-1 double mutant.

Ethylene and gibberellins (GAs) control similar developmental processes in plants. The role of ethylene is at least in part to regulate the accumulation of DELLA proteins, key regulators of plant growth, which suppress the GA response. To expand our knowledge of ethylene-GA crosstalk and to reveal how the modulation of the ethylene and GA pathways affects global plant growth, the gibberellin-insensitive (gai), ethylene-overproducing 2-1 (eto2-1) double mutant, which has decreased GA signalling (resulting from gai) and increased ethylene biosynthesis (resulting from eto2-1), was characterized. Both single mutations resulted in reduced elongation growth. The double mutant showed synergistic responses in root and shoot growth, in induction of floral transition, and in inflorescence length, showing that crosstalk between the two pathways occurs in different plant organs throughout development. Furthermore, the altered ethylene-GA interactions affected root-shoot communication, as evidenced by an enhanced shoot:root ratio in the double mutant. When compared with both single mutants and the wild type, double mutants had enhanced content of active GA(4) at both the seedling and the rosette stages, and, unlike the gai mutant, they were sensitive to GA treatment. Finally, it was shown that synergistic responses in the double mutant were not caused by elevated ethylene biosynthesis but that, in the light, enhanced sensitivity to ethylene may, at least in part, be responsible for the observed phenotype.

Monitoring and screening plant populations with combined thermal and chlorophyll fluorescence imaging.

Thermal and chlorophyll fluorescence imaging are powerful tools for the study of spatial and temporal heterogeneity of leaf transpiration and photosynthetic performance. The relative advantages and disadvantages of these techniques are discussed. When combined, they can highlight pre-symptomatic responses not yet apparent in visual spectrum images and provide specific signatures for diagnosis of distinct diseases and abiotic stresses. In addition, their use for diagnosis and for selection for stomatal or photosynthetic mutants, these techniques can be applied for stress tolerance screening. For example, rapid screening for stomatal responses can be achieved by thermal imaging, while, combined with fluorescence imaging to study photosynthesis, they can potentially be used to derive leaf water use efficiency as a screening parameter. A particular advantage of imaging is that it allows continuous automated monitoring of dynamic spatial variation. Examples of applications include the study of growth and development of plant lines differing in stress resistance, yield, circadian clock-controlled responses, and the possible interactions between these parameters. In the future, such dual-imaging systems could be extended with complementary techniques such as hyperspectral and blue-green fluorescence imaging. This would result in an increased number of quantified parameters which will increase the power of stress diagnosis and the potential for screening of stress-tolerant genotypes.

Multispectral fluorescence and reflectance imaging at the leaf level and its possible applications.

Images taken at different spectral bands are increasingly used for characterizing plants and their health status. In contrast to conventional point measurements, imaging detects the distribution and quantity of signals and thus improves the interpretation of fluorescence and reflectance signatures. In multispectral fluorescence and reflectance set-ups, images are separately acquired for the fluorescence in the blue, green, red, and far red, as well as for the reflectance in the green and in the near infrared regions. In addition, 'reference' colour images are taken with an RGB (red, green, blue) camera. Examples of imaging for the detection of photosynthetic activity, UV screening caused by UV-absorbing substances, fruit quality, leaf tissue structure, and disease symptoms are introduced. Subsequently, the different instrumentations used for multispectral fluorescence and reflectance imaging of leaves and fruits are discussed. Various types of irradiation and excitation light sources, detectors, and components for image acquisition and image processing are outlined. The acquired images (or image sequences) can be analysed either directly for each spectral range (wherein they were captured) or after calculating ratios of the different spectral bands. This analysis can be carried out for different regions of interest selected manually or (semi)-automatically. Fluorescence and reflectance imaging in different spectral bands represents a promising tool for non-destructive plant monitoring and a 'road' to a broad range of identification tasks.

Robotized thermal and chlorophyll fluorescence imaging of pepper mild mottle virus infection in Nicotiana benthamiana.

After infecting a susceptible host, pathogens spread throughout the plant. Depending on pathogen type and strain, the severity of symptoms varies greatly. In the case of pepper mild mottle virus (PMMoV) infection in Nicotiana benthamiana, newly developing leaves display visual symptoms (symptomatic leaves). In this study, two PMMoV strains were used, the Spanish strain (PMMoV-S) being more virulent than the Italian strain (PMMoV-I). Plants infected with PMMoV-I could recover from the virus-induced symptoms. Leaves that were fully developed at the start of PMMoV infection remained symptomless. In these asymptomatic leaves, an increase in temperature, initiating from the tissue adjacent to the main veins, was observed 7 d before the Chl fluorescence pattern changed. Virus immunolocalization on tissue prints matched well with the concomitant pattern of Chl fluorescence increase. The temperature increase, associated with the veins, was shown to be related to stomatal closure. Upon PMMoV-I infection, the appearance of thermal and Chl fluorescence symptoms as well as virus accumulation were delayed by 3 d compared with PMMoV-S-induced symptoms. The temperature increase of whole symptomatic leaves was also correlated with a decrease in stomatal aperture. In contrast to the persistent increase in symptomatic leaf temperature observed during PMMoV-S infection, the temperature of symptomatic leaves of PMMoV-I-infected plants decreased gradually during recovery. We propose that the earliest temperature increase is caused by a systemic plant response to the virus infection, involving the control of water loss. In conclusion, thermography has potential as an early reporter of an ongoing compatible infection process.

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.

The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin.

Ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) can stimulate hypocotyl elongation in light-grown Arabidopsis seedlings. A mutant, designated ACC-related long hypocotyl 1 (alh1), that displayed a long hypocotyl in the light in the absence of the hormone was characterized. Etiolated alh1 seedlings overproduced ethylene and had an exaggerated apical hook and a thicker hypocotyl, although no difference in hypocotyl length was observed when compared with wild type. Alh1 plants were less sensitive to ethylene, as reflected by reduction of ACC-mediated inhibition of hypocotyl growth in the dark and delay in flowering and leaf senescence. Alh1 also had an altered response to auxin, whereas auxin levels in whole alh1 seedlings remained unaffected. In contrast to wild type, alh1 seedlings showed a limited hypocotyl elongation when treated with indole-3-acetic acid. Alh1 roots had a faster response to gravity. Furthermore, the hypocotyl elongation of alh1 and of ACC-treated wild type was reverted by auxin transport inhibitors. In addition, auxin up-regulated genes were ectopically expressed in hypocotyls upon ACC treatment, suggesting that the ethylene response is mediated by auxins. Together, these data indicate that alh1 is altered in the cross talk between ethylene and auxins, probably at the level of auxin transport.