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Optical methods for phenotyping the response to abiotic and biotic stress.

Karel Klem, Global Change Research Centre AS CR, v.v.i.

A number of methods was tested to compare new high-throughput optical methods potentially useful for phenotyping the response / tolerance / resistance to abiotic and biotic stress. The main methods tested include

  • spectral reflectance,
  • thermal imaging,
  • chlorophyll fluorescence imaging,
  • UV and blue excitation spectra of chlorophyll fluorescence,
  • chlorophyll fluorescence emission spectra

The measurement parameters resulting from these methods are usually correlated to physiological parameters reflecting the sensitivity / tolerance / resistance to a given abiotic or biotic stress. This is primarily a measurement of CO2 assimilation rate (A), stomatal conductance (Gs), water use efficiency (WUE), leaf relative water content (RWC), leaf water potential (y), leaf area (LA), leaf mass area (LMA), nitrogen content (N), primary and secondary metabolite analysis (metabolomics LC / MS and GC / MS) etc. Simultaneously with the use of commonly utilized parameters also new parameters are developed, providing higher response, less variability and greater linear range. A typical example is particularly the detailed analysis of hyperspectral reflectance curves to develop new spectral indices (e.g. new index NRERI providing greater reliability of estimation the response to nitrogen deficiency (75)).

Here we focus on a number of parameters which can be used to describe and quantify different abiotic and biotic stress factors described below. A matrix with measurement parameters and the environmental stress they can be used for is shown in Table 1:

a)      Drought stress

High potential for phenotyping of drought stress is provided by the thermal imaging, due to the correlation heat transfer to the transpiration. Passive and active approaches can be applied (10, 11, 97) In the case of thermal imaging there is, however, very important the process of calibration and standardization of the measurement conditions.

The evaluation of response to drought stress were also achieved using chlorophyll fluorescence imaging particularly for the parameters of non-photochemical quenching (NPQ) and actual quantum yield of PSII photochemistry (fPSII)(5,6).

The use of hyperspectral reflectance in drought stressed Arabidopsis thaliana show highly variable results in comparison with the literature, especially if the effect of drought stress is slow and is more reflected in the leaf area change and leaf thickness (LMA). It is always useful to calibrate the method and reflectance parameters to the type of drought induction and even better to evaluate drought response as a combination of reflectance and growth parameters (e.g. leaf area) (1, 2, 20, 21, 37). The most appropriate reflectance parameters to evaluate the response to drought appears the indices based on SWIR region (1400-3000 nm) (18), where the strong absorption bands of water are located (e.g. NDWI) or NIR bands around 970 nm (e.g. WI) (19).

b)      Heat stress

There are only little information available in the literature concerning the response of optical methods to heat stress .The most promising methods are based on fast chlorophyll fluorescence kinetics (OJIP) (40, 41) and IR thermal imaging (12). In addition to these also other chlorophyll fluorescence parameters (eg. fPSII) (39, 42, 43) and spectral reflectance indices utilizing the red-ege and NIR reflectance (R740 / R800) (37) can be potentially used to estimate response to heat stress.

c)       Low temperature stress

The evaluation of the response to low temperatures can be performed using parameters of chlorophyll fluorescence and particularly when the entire kinetics is analysed in combinatorial imaging (7, 8, 9). Promising are also methods based on IR thermal imaging (14), UV screening of chlorophyll fluorescence (27) and spectral reflectance in red-edge region (80, 81).

d)      Salinity

The plant response to higher salt concentration in the rhizosphere is from physiological point of view very similar to drought response. For this reason, to evaluate the tolerance to salinity, similar optical methods can be used. These are mainly the IR thermal imaging (13), where the response is based on changes of stomatal conductance, hereinafter chlorophyll fluorescence, especially parameters NPQ and fPSII (44, 45, 46, 47), and finally indices based on spectral reflectance, particularly WI, PRI and parameters based on reflectance in the red-edge region (82, 83, 84, 85).

e)      Nitrogen deficiency

The potentially most reliable method for evaluating the response to nitrogen deficiency and nitrogen use efficiency is the spectral reflectance preferably as imaging method (3, 4). Although a number of vegetation indices sensitive to nitrogen deficiency can be used, it is preferable to use rather indices based on reflectance in red-ege or green bands and also the ratio of the reflectance in the main absorption bands of chlorophyll (red and blue) (NRERI, REIP, SRPI) (75, 79). These indices are not so affected by total biomass, achieve higher linear range and are not saturated at higher nitrogen concentrations. As a very good auxiliary indicator of response to nitrogen deficiency may serve the methods based on UV screening of chlorophyll fluorescence (29, 30, 31). Here the knowledge, that nitrogen deficiency leads to increased accumulation of epidermal phenolic compounds (flavonoids) at the expense of chlorophyll is exploited. Additionally a number of fluorescence parameters can be used (48, 49, 50, 51, 52, 53).

f)       Phosphorus deficiency

Probably none of the optical methods cannot provide a reliable estimation of response to phosphorus deficiency. Nevertheless, it is evident that as good indicator may serve indices utilizing spectral absorption bands of anthocyanins (400-450 nm) (88, 98) and also the indices based on reflectance in the red-edge band (86, 87).

g)      UV radiation

One of the most important protective mechanisms of plants against high UV radiation is the accumulation epidermal flavonoids which act not only as UV screening compounds but also as antioxidants. Therefore, the most promising optical method for plant phenotyping tolerance to UV is measurement of UV screening or UV excitation spectra of chlorophyll fluorescence (32, 33, 34). This approach can also be used as imaging method. From the parameters of chlorophyll fluorescence the best results were achieved with maximum quantum yield of PSII photochemistry (Fv/Fm) (60, 62, 64). The utilization of spectral reflectance is also associated with the accumulation of flavonoids, wherein the spectral indices used are based on the absorption properties of flavonoids and anthocyanins (absorption band around 400 to 450 nm) (34, 90). Presumably, also the use of UV reflectance (200-400 nm) or blue-green fluorescence have considerable potential in phenotyping of tolerance to UV, but these methods require further validation.

h)      Biotic stress

Even if we turn our attention within biotic stress only on the diseases, it is still very variable group of stress factors with very different physiological effects that require the use of different optical methods and parameters for diagnostics of plant response. We can observe, for example, different effects of plant viruses, obligate and facultative fungal diseases on plant physiology. Generally, the recent results indicate perspective use of blue-green fluorescence and IR thermal imaging (15, 16, 17, 35, 71, 72). From the spectral reflectance parameters appear to be promising indices based on absorption bands of carotenoids and anthocyanins (400-550 nm) and also complex computational methods utilizing a larger number of reflectance wavelengths such as neural network (91, 92, 93, 94, 95).