Biosensing Methods to Assess Environmental Stress Encountered by Sugar Beet (Beta vulgaris L.)

C. Hermans1,2, L. Chaerle3, R.M. Rodriguez2, D. Van Der Straeten3, R.J. Strasser2 and J.P. Delhaye4

1 Laboratoire de Physiologie et de Génétique Moléculaire des Plantes, Université Libre de Bruxelles (Belgium)

2 Bioenergetics Laboratory, University of Geneva (Switzerland)

3 Department of Plant Genetics, University of Ghent (Belgium)

4 Laboratoire d’Agrotechnologies Végétales, Université Libre de Bruxelles (Belgium)

Biosensing Methods to Assess Environmental Stress Encountered by Sugar Beet (Beta vulgaris L.)

Light absorbed by photosynthetic pigments is used to drive photochemical reactions and consequently to insure plant growth. Moreover, the plant capacity to use light and to carry out photochemistry is limited and depends on several factors, including environmental stress leading the plant to suboptimality. The aim of this poster is to present some non-invasive methods which are probing the stress state by optical measurement in vivo. The pigments can be directly characterised by the reflected light which determines the colour of the leaf sample. Leaf reflectance allows to measure the sample without extracting the pigments. Light energy absorbed by the leaf is converted into chemical energy by photosynthesis or dissipated as heat or chlorophyll fluorescence. Therefore the easily measurable fluorescence signal and the expressions derived from, are strictly correlated to the photosynthetic events. More generally, the fluorescence behaviour of any plant changes continuously following its adaptation to a perpetually changing environment. The OJIP transient is a biophysical signal that is extremely rich in terms of information and reflects the time course of photosynthesis. As a complementary information, fluorescence imaging offers a spatial visualisation of the pattern of dissipation processes of the light absorbed by the leaf. In addition, thermography imaging makes it possible to visualise the differences in the surface temperature pattern by detecting infrared radiation.

Methodes de Bio-detection pour l’Estimation des Stress Environnementaux Encourus par la Betterave Sucrière

La lumière absorbée par les pigments photosynthétiques alimente les réactions photochimiques et assure en conséquence la croissance de la plante. De plus, la capacité des plantes à utiliser la lumière et à procéder à la photochimie est limitée et dépend de facteurs, dont les stress environnementaux qui mènent la plante à un état de sub-optimalité. Au travers de ce poster, quelques méthodes non destructives sont dressées afin de détecter tout état de stress encouru par la plante à partir de caractéristiques optiques mesurées in vivo. La caractérisation des pigments peut être établie directement à partir de la lumière réfléchie qui détermine la couleur de l’échantillon foliaire. La réflectance foliaire permet de mesurer l’échantillon sans avoir recours à l’extraction des pigments. L’énergie lumineuse absorbée par la feuille est convertie en énergie chimique par la photosynthèse ou dissipée sous forme de chaleur ou de fluorescence. En conséquence, le signal de fluorescence qui est facilement mesurable et les expressions qui sont dérivées de celui-ci, sont intimement corrélés aux événements photosynthétiques. De manière plus générale, l’émission de fluorescence d’une plante change constamment en réponse au changement perpétuel de l’environnement. La cinétique transitoire OJIP est un signal biophysique extrêmement riche, délivrant des informations sur l’évolution temporelle de l’activité photosynthétique. De manière complémentaire, l’imagerie de fluorescence contribue à la visualisation spatiale des processus de dissipation de la lumière absorbée par la feuille. D’autre part, l’imagerie en thermographie caractérise la répartition des zones de température par détection des ondes infrarouges.

Biotest – Methoden zur Erkennung von Umweltstress bei Zuckerrübe (Beta vulgaris L.)

Licht, das von photosynthetischen Pigmenten absorbiert wird, dient der Pflanze als Energiequelle für photochemische Reaktionen und ist letztendlich für das Wachstum verantwortlich. Die Kapazität der Pflanze, Lichtenergie zu verbrauchen und photochemische Reaktionen auszuführen, hängt von verschiedenen Faktoren ab, insbesondere auch von umweltbedingtem Stress, welcher die Pflanzen in einen suboptimalen Zustand bringt. Zweck dieses Posters ist es, einige nicht-invasive Methoden zur Erkennung von Stresszuständen mittels optischer in vivo Messungen vorzustellen. Die Pigmente können durch die Reflexion von Licht direkt charakterisiert werden, durch welches die Farbe der Probe bestimmt ist. Durch Messung der Reflexion können demnach die Pigmente analysiert werden, ohne dass sie extrahiert werden müssen. Jedes Blatt absorbiert Lichtenergie, welche durch die Photosynthese in photochemische Energie umgewandelt oder als Wärmestrahlung oder Chlorophyllfluoreszenz abgegeben wird. Somit sind die mit einfachen Mitteln messbaren Fluoreszenzsignale und die davon abgeleiteten Ausdrücke streng mit den photosynthetischen Vorgängen korreliert. Allgemein ausgedrückt verändert sich das Fluoreszenzverhalten jeder Pflanze dauernd und passt sich automatisch den sich ständig ändernden Umweltbedingungen an. Der OJIP-Transient ist ein biophysikalisches Signal, welches einen hohen Informationsgehalt aufweist und den Zeitverlauf der Photosynthese widerspiegelt. Fluoreszenzbilder als ergänzende Information erlauben eine Visualisierung der Intensität der Dissipationsvorgänge des vom Blatt absorbierten Lichts im Raum. Zusätzlich dazu machen thermographische Bilder Unterschiede in der Oberflächentemperatur durch Infrarotstrahlung sichtbar.

Introduction

Further advances in precision agriculture will require plant metabolism spectroscopy to sustain crop production. Actually, photosynthesis restricts to various environmental stresses that affect biomass production. For that reason, it becomes crucial to assess presymptomaticaly any stress state. Biosensing methods at plant scale increases the understanding of crop physiological needs. Therefore, it has become prevalent in agricultural project management to apply optical techniques, such as leaf reflectance, chlorophyll fluorometry and thermography to probe plant behaviour in vivo. The potential of these techniques to monitor beet cropping is considered.

1.- Leaf reflectance

Information can be gained from visible and near-infrared reflectance about the spectral range in which the pigments molecule capture light quanta and about leaf architecture (Fig 1). As the pigments contribute to light harvesting and to photochemical energy transformation, they provide a way to diagnose the physiological status of the plant. Actually, pigments can be directly characterised base of the reflectance spectrum and furthermore by derived parameters from (PeNuellas, J. & Filella, I., 1998).

Fig 1: Reflectance spectrum of a healthy beet (black trace) and Mg deficient beet (grey trace) gained with the reflectometer GER 1500 (GER Corporation) - mean of 20 spectra.

The Normalized Difference Vegetation Index NDVI (plotted in the insert) is a mathematical expression defined as (NIR-R) / (NIR+R) that characterize the green biomass. NIR: reflectance in the near-infrared range, R: reflectance in the red range.

2.- Chlorophyll fluorOMETRY

Quantitative and qualitative evaluation of the photosynthetic activity can be gathered from the chlorophyll fluorescence emission because fluorescence changes are associated with changes in photochemical flux. Two instrumentations exist to induce fluorescence: continuous and modulated light excitation measurements. Continuous excitation fluorometers (PEA, Hansatech Instrument Ltd.) resolve early induction events of the OJIP transient (Strasser , R.J, et al., 2000) with an integration time of 10 µs. Pulse modulated fluorometers (FMSII, Hansatech Instrument Ltd.) can provide information on physiology of plants under ambient light.

3.- CHLOROPHYLL FLUORESCENCE IMAGING

The two-dimensional image analysis of the fluorescence signals is used to evaluate the physiological status of beet plants suffering different mineral deficiencies. The chlorophyll fluorescence emission from the leaves in stressed plants shows different patterns compared to the spatial distribution patterns registered from the control plants (Fig 2), in direct correlation to an altered photosynthetic functionality. Quantitative evaluation of the photosynthetic activity can be done by simple pixel value extraction and by arithmetic manipulation for calculation of fluorescence yield images. The calculated yield images are found to be consistent with those obtained by an in situ fluorometer (PEA) by applying the JIP-test procedure, in agreement with early reported data (Rolfe, S.A. & Scholes, J.D., 1995). However, plant stress that causes localized damage may not be easily detected with small field of view point measurements. Imaging of a whole leaf provides better means to study the effects of non-systemic stresses.

Fig 2: Laboratory measurements of chlorophyll fluorescence kinetics (Kaustky effect) on dark-adapted leaf samples of beet have been performed with the Fluorcam M690 (PSI instruments, Czech Republic), by illuminating the leaf targets with continuous actinic light provided with orange (635 nm) light emitting diodes panels that generate a uniform irradiance field.

4.- Thermography

Leaf or canopy temperature depends highly on the rate of transpiration and can therefore be used as an indicator of stomatal opening. Detectors sensitive in the 8-14 micrometer wavelength band convert infrared radiation emitted by a body to a temperature reading. Scanning systems based on these sensors convert patterns of radiation to visual pseudocolor images representing temperature levels (Fig 3). Infrared thermography has been developed as a means for irrigation scheduling (Jones, H.G., 1999). Recently, disease-like cell death in tobacco and Arabidopsis were visualised presymptomatically (Chaerle, L. et al., 2001). Local alterations in stomatal aperture result in changes in transpiration, affecting the distribution of temperature at the leaf surface. In the here depicted experiment, a beet plant was subjected to drought stress, resulting in a progressive increase in leaf temperature, starting before visible wilting (Fig 3 and Fig 4).

Fig 4 (above): Evolution of leaf surface temperature in function of the drought treatment. The circle defines the measurement area of Fig 5.

Fig 5 (against) : The variation in leaf temperature is apparent for the 2 days before the onset of drought-stress. These data show that thermal imaging provides an early warning for emerging drought stress.

References

1.  Chaerle, L., De Boever, F., Van Montagu, M. and Van Der Straeten, D.: Thermographic visualisation of cell death in tobacco and Arabidopsis. Plant Cell Environ, 24: 15-26, 2001.

2.  Jones, H.G.: Use of infrared thermometry for estimation of stomatal conductance as a possible aid to irrigation scheduling. Agricultural and Forest Meteorology, 95: 139-149, 1999.

3.  PENUELLAS, J. and FILELLA, I.: Visible and near-infrared reflectance techniques for diagnosing plant physiological status. Trends in Plant Science, Vol 3: 4, 151-155, 1998.

4.  Rolfe, S.A., Scholes, J.D.: Quantitative imaging of chlorophyll fluorescence, New Phytologist, 131, 69-79, 1995.

5.  Strasser, R.J., Srivastava, A., Tsimilli-Michael, M.: The fluorescence transient as a tool to characterize and screen photosynthetic samples, in Probing Photosynthesis: mechanisms, regulation and adaptation, M.Yunus, U. Pathre, P. Mohanty (eds.), Taylor & Francis, London. Ch.25, 445-483. 2000.

Proceedings of the 64th IIRB Congress, 26-27 June 2001, Bruges (B)