Nondestructive optical method for plant overall health evaluation

ABSTRACT

In vivo measurements of the optical properties of plant leaves, by spectrophotometric methods, could potentially provide very useful information for the assessment of the plant’s health. This paper describes a novel experimental set-up that enables continuous measurements of the optical reflection and transmission coefficients of broad-leaved plants. The optical spectral characteristics of leaves, in the broad wavelength range, are often a discussed topic in the scientific literature. Nevertheless, to the best of our knowledge, the real-time spectral analyses of the leaf haven’t been reported so far. Monitoring the evolution of the plant activity in real-time has resulted in the graph of the spectral Circadian rhythm as a function of time. The set-up was tested on Phaseolus vulgaris and the results of the measurements are presented and discussed.

KEYWORDS:

• Leaf reflectance
• leaf absorbance
• 665 nm red led
• Circadian rhythm
• plant monitoring
• plant health
• optical methods

Introduction

The general state of a plant is strongly correlated with the intensity of the photosynthetic processes in its leaves (Fourty et al. 1996). On the other hand, the quantity of the absorbed light represents a good measure for the intensity of the photosynthesis (Chaerle and Van der Straeten 2000). It is well known that the green plants absorb mostly in the blue and red regions of the visible light spectrum (Chaerle et al. 2007; Ferl et al. 2002). The absorption spectra of chlorophyll-a exhibit maxima at 430 and 664 nm. For chlorophyll-b the corresponding maxima are at 460 and 647 nm, respectively (Knapp and Carter 1998; Carter and Spiering 2002). Keeping in mind the correlation with the leaf spectral response at 660 nm (Knapp and Carter 1988; Yamada and Fujimura 1988; Liew et al. 2008), we have exclusively focused our research on the single-wavelength spectroscopy (660 nm).

One of the experimental challenges is the fact that during its growth a plant's leaf changes its spatial position. This effect can influence the readout results for reflection and transmission. To minimise these effects, multiple repeated measurements were performed simultaneously on a group of leaves. The systematic error, due to the change of the leaf’s orientation (with respect to the system’s optical axis), is thus randomised. A measurement apparatus, which can real-time track (in vivo) the optical transmission coefficient leaves of a plant under investigation, should provide very useful information about plants’ physiological condition (Taiz and Zeiger 2003).

In this paper we describe a measurement system that can track the optical transmission of leaves during the complete life circle of a plant. Moreover, measurement points can be taken and logged at the minute time-scale intervals. A great care has been taken to avoid any mechanical pressure to the leaves, so that the plants’ growth resembles natural conditions as close as possible (Tilman 1999). The provision for measuring of the reflection (Carter 1993; Gitelson et al. 2002; Grant 1987; Grant et al. 1993; Piekarczyk 2001) (at 45-degree incidence and reflection angle) is not the primary aim of this apparatus. It was included to provide some additional information at the very low additional expense. An example of the state-of-the-art measurements of the leaves’ reflection properties was described in Combes et al. 2007. However, our main objective is tracking of the leaf optical transmission.

To benchmark our apparatus and measurement methods, we performed a series of experiments on Phaseolus vulgaris. It is important to note that the P. vulgaris was chosen somewhat arbitrary to demonstrate the proof of concept method. The method should be applicable to any plant species with thin broad leaves.

Materials and methods

The experiment was set up in an indoor environment for the plant P. vulgaris. The experimental apparatus provides 20 independent ‘channels’ where each channel corresponds to a single leaf. The channels were distributed into four independent groups with each group containing five single channels. Each group was treated with different light conditions.

An illustration of a single measurement channel is given in Figure 1. For each channel, the source of light is a red LED with the spectral emission maximum at 656 nm. To disambiguate these light sources from the high-luminosity sources used for the plant growing, these LEDs will be called ‘Signal LEDs’. Each Signal LED was mounted in a temperature-controlled box and coupled to an optical fibre (Bornman and Vogelmann 1991; Vogelmann 1993; Vogelmann et al. 1988). The other end of this fibre was attached to the appropriate leaf holder made of a transparent plastic (methyl methacrylate), as shown in Figure 1. Signal LED power was provided with stabilised supplies that were controlled by the data-acquisition computer.

Figure 1. Single measurement channel.

Two additional optical fibres, one collecting transmitted and one collecting reflected light, are also incorporated in the arrangement. The light collection fibres were also attached to a leaf holder.

The optical fibres transmit the collected light to the photodiodes which are kept in a thermostated shielded box. The box protects the photodiodes from the stray light, electrical interference and from the temperature variations. In addition, in front of each photodiode a sharp band-pass interference filter (centred at 665 nm) was inserted, thus further reducing the ambient light leakage from the optical fibres. The leaf holder was designed to enable five degrees of freedom for the mechanical movement, thus providing the means to track the leaf’s position and orientation.

The firing sequence of the signal LEDs was controlled by a PC via a dedicated digital I/O card. The signals from the photodiodes were routed through the analogue multiplexer (HP 34970A) to the precision digital multimetre (DMM) (HP 34970A). The DMM was connected to the PC via an RS-232 interface. Block diagram of the described set-up is given in Figure 2.

Figure 2. Block diagram.

For a given group of channels, measurements were performed by the sequential firing of each channel. The duration of a single sequence used for the measurements was ∼90 s. The sequences were repeated every 15 min for the duration of the experiment (7 days). To control the experiment and collect data, we have developed an in-house code written in the so-called G language.

System calibration

To infer the values for the optical transmission and reflection coefficients, it is necessary to account for the individual sensitivity of each channel. The differences between the channels are due to the differences between the individual LED’s emission efficiency, attenuation of the individual optical fibres, as well as due to the minor mechanical positioning errors of individual channels.

For the transmission, a set of calibrated neutral density filters (NDFs) was used. For each individual channel, we measured transmitted signal for each filter in the set. Thus, we obtained relative sensitivities of the channels. The measured sensitivities were used as correction factors, thus placing readings of each channel on the same (relative) scale.

Similarly, for the reflection, an ‘ideal’ diffusive target was used. The actual target was prepared by painting a suitable plate with barium sulphate, which has almost ideal diffusive characteristics. Regarding the transmission, a set of correction factors was derived and the relative reflection coefficients were placed on the same scale.

It is worth noting that the described procedures enable accurate assessment of the relative values for the transmission and reflection and they only account for the differences between individual channels, placing the results on the same scale.

Plant material

For the current experiment, the plants of P. vulgaris L. were used. P. vulgaris belongs to C3 group of plants which have no photosynthetic adaptations. This group of plants tends to thrive in areas where sunlight intensity and temperatures are moderate and carbon dioxide concentrations are around 400 ppm or higher. Seeds of P. vulgaris were germinated in commercial humus. Seven days after germination, the plantlets were developed with the first pair of leaves, and these plantlets were used as starting plant materials. For each experimental run, 20 plantlets of the same age and similar size were used. Duration of the experiments was 13 days. Experiments were repeated in triplicate.

There were four different treated groups of plants. Each group was exposed to different lighting conditions:

Group I: Plants were grown in dark conditions (total darkness 24 h for 7 days)

Group II: Plants were grown under day-light regime (the plants were grown indoors in a room with relatively large window areas facing south during March 2020, latitude 44.8246°N 20.4581°E: sunrise about 6 am, sunset about 6 pm)

Group III: Plants were grown under LED light regime (plant growing LED was turned on for 24 h for 7 days).

Group IV: Plants were grown under combined LED light and day-light (combined day-light described in group II and LED light regime described in group III).

Chlorophylls and carotenoid quantification

Photosynthetic pigments were extracted from 100 mg frozen leaves in 96% (v:v) ethanol. The pigment content of the filtered solution was measured using the classical spectrophotometric method with a UV–visible spectrophotometer (Agilent 8453) at three wavelengths: 470, 648, and 664 nm (Socaciu 2007). The amount of pigments was calculated according to the equations of Lichtenthaler (Lichtenthaler 1987).

Results and discussion

Figures 3 and 4 show per cent variations of the coefficients of transmission/reflection as a function of time. The time interval covered by graphics was 7 days for all 4 groups.

Figure 3. Variation of transmittance T (%) as a function of time in days (started from midnight), group I (dark).

Figure 4. Variation of transmittance T (%) as a function of time in days (started from midnight), group II (day-light).

During the experimental runs, luminous flux of the plants’ illumination was also recorded.

The highest absorption coefficient was detected in plants grown in the dark conditions (Group I). The same group of plants also had the lowest value of light transmission coefficient (Figure 3). It is interesting to note the sudden increase in transmission in this group was on day 6. This may indicate that the plant lost the possibility of recovery and the cell death.

Group II (day-light illumination, Figure 4) exhibits clearly defined day-night Circadian (Figure 5) rhythms and the transmission coefficient is lowest at noon (Figure 5). This is the first experiment that presents detailed time dependence of the optical properties of plants under investigation. This could be explained by the mid-day depression in net photosynthesis (Roessel and Monson 1985). Figure 5 also suggests that during the periods of reduced illumination (e.g. in the afternoon), the value of transmission grows, reaching its highest value during the night. The overall (on one-week time scale) increase of the transmission coefficient (Figure 5) indicates that the leaf is getting older, suggesting that less chlorophyll is being synthesised.

Figure 5. Variation of luminous flux (left) and coefficient of transmission T (%) (right) as a function of time in days, group II (day-light).

The absorption coefficient value of plants exposed to LED light only (Group III, Figure 6) was lower than that of the plants that were grown under day-light regime (group II). The possible reason for this phenomenon might be the spectral distribution of LED illumination system, as shown in Figure 7.

Figure 6. Variation of transmittance T (%) as a function of time in days (started from midnight), group III (LED light).

Figure 7. (a) Chlorophyll a and b absorption spectra, (b) relative emission spectra of LEDs. The curves are normalised at their respective peak intensities and (c) transmission spectra of interference filters.

Interestingly, the absorption coefficient was lowest in group IV (combined LED + day-light). The possible explanation could be the fact that those plants were over-exposed (Ruban 2009), i.e. the photosynthetic system was saturated with light (Figure 8).

Figure 8. Variation of transmittance T (%) as a function of time in days (started from midnight), group VI (LED light) and day-light.

Absorption coefficient linearly decreased starting from group II to group IV of plants (Figure 9). Similarly, the transmission coefficient was inversely proportional to time (Figure 9).

Figure 9. Average sum of absorbance (right) and transmittance (left) of each group (P. vulgaris) exposed to different light regimes.

Total chlorophyll content was highest in plants grown under LED and day-light regime, while these plants had highest transmittance and lowest absorbance level. The high level of transmittance in this plant group (IV) could be explained by the leaf morphology, thin leaves. The lowest total chlorophyll was measured in plants grown under dark conditions (Figure 10). The same pattern appears for total carotenoid content (Figure 11). Total chlorophyll content in the plants grown under day-light regime (group II) and under LED light regime (group III) was three and five times higher, respectively, than that of the group I plants (dark conditions).

Figure 10. Total chlorophyll content of each group (P. vulgaris) exposed to different light regimes.

Figure 11. Total carotenoid content of each group (P. vulgaris) exposed to different light regimes.

Carotenoid content was highest in plants grown under combined LED and day-light regime (Figure 11). This is consistent with the fact that these plants also had lowest absorption coefficient. Carotenoid content in these plants is six times higher compared to that of group II plants (day-light regime). High carotenoid content could implicate that carotenoids took the role of protective photosynthetic apparatus (Yasushi 1991; Young 1991; Demmig-Adams et al. 1996; Young and Frank 1996) and that they actively participate in thermal-energy dissipation (Figure 11). In the same group of plants (grown under combined LED and day-light regime), chlorophyll a/b ratio (Porras 2002) was lowest implying that those plants were exposed to stress conditions (high light intensity in this case) (Figure 12).

Figure 12. Chlorophyll a/b ratio of each group (P. vulgaris) exposed to different light regimes.

Chlorophyll a/b ratio in the plants in group II and III was the same (Figure 12). However, the total chlorophyll content was almost two times higher in group III in comparison to plants from group II (Figure 10).

Discussion

We have made original, experimental set-up that meets highest metrological standards. A novel instrument that enables quantitative assessment and monitoring of the optical absorption in the plants’ leaves (in real time), was described and its performance tested.

To our knowledge this is the first published time dependence of the optical transmission of the plant material. One can observe Circadian rhythms of the plant as well as long-term change (weeks) of the optical transmission during the leaves’ growth.

The instrument was tested by measuring optical properties of the P. vulgaris in a set of carefully selected growing conditions. A number of biological phenomena were observed and discussed. Monitoring the evolution of the plant activity in real-time has resulted in the graph of the spectral Circadian rhythm as a function of time. The proposed experimental technique appears to be a promising tool for the assessment of the plants’ overall condition. The presented paper is a mere proof of concept and we intend to apply the described technique on a wide range of plant species as well as to account for the wider range of biological phenomena.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work has been financially supported by the Ministarstvo Prosvete, Nauke i Tehnološkog Razvoja (the Ministry of Education, Science and Technological Development of the Republic of Serbia), (project nos. 172017 and 173024).

Notes on contributors

Becko V. Kasalica

Becko V. Kasalica is a full professor of Faculty of Physics, University of Belgrade. His research areas are applied physics and metrology, material science, spectroscopy and optical measurements.

Katarina M. Miletic

Katarina M. Miletic is a doctoral candidate, junior research assistant of Faculty of Physics, University of Belgrade. Her research fields are biophysics, applied physics and metrology.

Aneta D. Sabovljevic

Aneta D. Sabovljevic is an associate professor of Department of plant physiology, Faculty of Biology, University of Belgrade. Her research areas are physiology and molecular biology of bryophytes, physiology of plant stress, biotechnology and genetic engineering of plants.

Milorad M. Vujicic

Milorad M. Vujicic is an assistant professor of plant molecular biology and physiology, Faculty of Biology, University of Belgrade. Milorad does research in physiology ecology and botany.

Dejan A. Jeremic

Dejan A. Jeremic is a research associate of Innovation center of Belgrade, Department of General and Inorganic chemistry. His areas of scientific interest are applied chemistry, bioinorganic chemistry, inorganic material chemistry and chemical photobiology.

Ivan D. Belca

Ivan D. Belča is a full professor and dean of the Faculty of Physics. His research fields are solid state physics, applied physics-metrology. E-mail: [email protected].

Marija M. Petkovic-Benazzouz

Marija M. Petkovic-Benazzouz is a research associate of Faculty of Physics, University of Belgrade. Her areas of scientific interest are applied physics, material science and molecular modelling.