Scientific and technical challenges in remote sensing of plant canopy reflectance and fluorescence

State-of-the-art optical remote sensing of vegetation canopies is reviewed here to stimulate support from laboratory and field plant research. This overview of recent satellite spectral sensors and the methods used to retrieve remotely quantitative biophysical and biochemical characteristics of vegetation canopies shows that there have been substantial advances in optical remote sensing over the past few decades. Nevertheless, adaptation and transfer of currently available fluorometric methods aboard air- and space-borne platforms can help to eliminate errors and uncertainties in recent remote sensing data interpretation. With this perspective, red and blue-green fluorescence emission as measured in the laboratory and field is reviewed. Remotely sensed plant fluorescence signals have the potential to facilitate a better understanding of vegetation photosynthetic dynamics and primary production on a large scale. The review summarizes several scientific challenges that still need to be resolved to achieve operational fluorescence based remote sensing approache

Towards remote sensing of vegetation processes

The latest advances in imaging spectroscopy of vegetation enabled remote sensing (RS) of plant reflected or emitted signals associated with photosynthetic processes as the photoprotective transformation of xanthophyll pigments or the chlorophyll fluorescence (Chl-F). A potential future European Space Agency (ESA) satellite mission FLEX is expected to sense, apart from other parameters, so-called steady-state chlorophyll fluorescence (Chl-FS) signal, which may be potentially used for monitoring of photosynthesis (vegetation canopy carbon assimilation rate). Nevertheless, geometric complexity of plant canopies and signal disturbing atmospheric factors require a proper approach for scaling the information of a single leaf optical properties up to the RS image data of anisotropic vegetation canopies. Such up-scaling approach can be established only via synergic measurements of ground based and air-/space-borne optical sensors. Our initial experiment revealed that Chl-FS, being strongly driven by the air temperature, is able to accurately indicate onset and off-set of the photosynthetically active period for the evergreen plants. Next field experiment, carried out with the VNIR imaging spectroradiometer AISA Eagle (SPECIM Ltd., Finland) mounted above the montane grassland and Norway spruce (Picea abies /L./ Karst.) canopies, showed that the fluorescence signal is retrievable from passive optical imaging spectroscopy data. Further analyses revealed that some of the vegetation \u27process-related\u27 optical indices (e.g., photochemical reflectance index - PRI) are closely correlated to the parameters measured over the experimental canopies by eddy-covariance flux systems. The future objective is to continue in development the leaf-canopy Chl-F up-scaling approach by setting up local scale experiments employing the field pocket-size cost effective instruments measuring the leaf optical indices and Chl-F parameters simultaneously with canopy reflectance acquired by RS sensors from tower and aircraft platforms

Raman Spectroscopy for the characterization of algal cells

ABSTRACT Raman spectroscopy can elucidate fundamental questions about intercellular variability and what governs it. Moreover, knowing the metabolic response on single cell level this can significantly contribute to the study and use of microalgae in systems biology and biofuel technology. Raman spectroscopy is capable to measure nutrient dynamics and metabolism in vivo, in real-time, label free making it possible to monitor/evaluate population variability. Also, degree of unsaturation of the algae oil (iodine value) can be measured using Raman spectra obtained from single microalgae. The iodine value is the determination of the amount of unsaturation contained in fatty acids (in the form of double bonds). Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm −1 (cis C=C stretching mode) and 1,445 cm −1 (CH 2 scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids

Computer Reconstruction of Plant Growth and Chlorophyll Fluorescence Emission in Three Spatial Dimensions

Plant leaves grow and change their orientation as well their emission of chlorophyll fluorescence in time. All these dynamic plant properties can be semi-automatically monitored by a 3D imaging system that generates plant models by the method of coded light illumination, fluorescence imaging and computer 3D reconstruction. Here, we describe the essentials of the method, as well as the system hardware. We show that the technique can reconstruct, with a high fidelity, the leaf size, the leaf angle and the plant height. The method fails with wilted plants when leaves overlap obscuring their true area. This effect, naturally, also interferes when the method is applied to measure plant growth under water stress. The method is, however, very potent in capturing the plant dynamics under mild stress and without stress. The 3D reconstruction is also highly effective in correcting geometrical factors that distort measurements of chlorophyll fluorescence emission of naturally positioned plant leaves

Photosynthesis dynamics and regulation sensed in the frequency domain

Foundations of photosynthesis research have been established mainly by studying the response of plants to changing light, typically to sudden exposure to a constant light intensity after dark acclimation or light flashes. This approach remains valid and powerful, but can be limited by requiring dark acclimation before time-domain measurements and often assumes that rate constants determining the photosynthetic response do not change between dark and light acclimation. We show that these limits can be overcome by measuring plant responses to sinusoidally modulated light of varying frequency. By its nature, such frequency-domain characterization is performed in light-acclimated plants with no need for prior dark acclimation. Amplitudes, phase shifts, and upper harmonic modulation extracted from the data for a wide range of frequencies can target different kinetic domains and regulatory feedbacks. The occurrence of upper harmonic modulation reflects non-linear phenomena, including photosynthetic regulation. To support these claims, we measured chlorophyll fluorescence emission of the green alga Chlorella sorokiniana in light that was sinusoidally modulated in the frequency range 1000 – 0.001 Hz. Based on these experimental data and numerical as well as analytical mathematical models, we propose that frequency-domain measurements can become a versatile tool in plant sensing

Metabolic rhythms of unicellular, nitrogen fixing cyanobacteria and possible interplay with modeled KaiABC circadian oscillator

Circadian clocks of living organisms provide evolutionary advantage and make living in dynamic environment more efficient. The clocks affect human lives and health as well as control the simplest organisms such as prokaryotic cyanobacteria. The cyanobacteria represent an excellent model that is amendable to a multitude of genetic, biochemical, and biophysical methods. The cyanobacterium, Cyanothece sp. ATCC 51142 relies on the circadian clock to permit, in the same cell, anoxygenic nitrogen fixation at night and oxygenic photosynthesis during solar day. We measured real-time, in-situ photosynthetic and respiratory activities as well as the culture growth under light forcing conditions and also under constant light, i.e. free-running mode. Interestingly, the experiments show a strong 24h-period dynamic pattern that is replaced by apparent 12h-period in free running mode. The 24h-pattern does not change significantly when changing the light/dark ratio from 16hL/8hD to 12hL/12hD and 8hL/16hD. Furthermore, we tried to elucidate connection between these metabolic rhythms and known structure of KaiABC circadian oscillator by means of mathematical modeling. One of simulation results show a strong correlation between the presumed catabolic event indicated by significant peak in respiratory activity, and simulated dynamics of KaiB4 complex in modeled circadian pacemaker. A causal relationship between these 2 events is suggested because KaiB4 facilitates dephosphorylation of KaiC6 hexamer, which is known to signal upcoming dark period

Complex metabolic oscillations in plants forced by harmonic irradiance.

Plants exposed to harmonically modulated irradiance, approximately 1 + cos(omegat), exhibit a complex periodic pattern of chlorophyll fluorescence emission that can be deconvoluted into a steady-state component, a component that is modulated with the frequency of the irradiance (omega), and into at least two upper harmonic components (2omega and 3omega). A model is proposed that accounts for the upper harmonics in fluorescence emission by nonlinear negative feedback regulation of photosynthesis. In contrast to simpler linear models, the model predicts that the steady-state fluorescence component will depend on the frequency of light modulation, and that amplitudes of all fluorescence components will exhibit resonance peak(s) when the irradiance frequency is tuned to an internal frequency of a regulatory component. The experiments confirmed that the upper harmonic components appear and exhibit distinct resonant peaks. The frequency of autonomous oscillations observed earlier upon an abrupt increase in CO(2) concentration corresponds to the sharpest of the resonant peaks of the forced oscillations. We propose that the underlying principles are general for a wide spectrum of negative-feedback regulatory mechanisms. The analysis by forced harmonic oscillations will enable us to examine internal dynamics of regulatory processes that have not been accessible to noninvasive fluorescence monitoring to date

Modelling phosphorus uptake in microalgae

Phosphorus (P) is an essential non-renewable nutrient that frequently limits plant growth. It is the foundation of modern agriculture and, to a large extent, demand for P is met from phosphate rock deposits which are limited and becoming increasingly scarce. Adding an extra stroke to this already desolate picture is the fact that a high percentage of P, through agricultural runoff and waste, makes its way into rivers and oceans leading to eutrophication and collapse of ecosystems. Therefore, there is a critical need to practise P recovery from waste and establish a circular economy applicable to P resources. The potential of microalgae to uptake large quantities of P and use of this P enriched algal biomass as biofertiliser has been regarded as a promising way to redirect P from wastewater to the field. This also makes the study of molecular mechanisms underlying P uptake and storage in microalgae of great interest. In the present paper, we review phosphate models, which express the growth rate as a function of intra-and extracellular phosphorus content for better understanding of phosphate uptake and dynamics of phosphate pools

New Insights into Photosynthetic Oscillations Revealed by Two-dimensional Microscopic Measurements of Chlorophyll Fluorescence Kinetics in Intact Leaves and Isolated Protoplasts

Chlorophyll fluorescence kinetic microscopy was used to analyze photosynthetic oscillations in individual cells of leaves and in isolated leaf cell protoplasts. Four Brassicaceae species were used: Arabidopsis halleri (L.) O Kane & Al-Shehbaz, Thlaspi fendleri (Nels.) Hitchc, Thlaspi caerulescens J.&C. Presl and Thlaspi ochroleucum Boiss et Helder. With the latter two, the measurements were extended also to isolated protoplasts. The oscillations were induced under the microscope by exposing dark-adapted samples to actinic irradiance. Detailed analysis of the induced transients revealed that they consist of several processes oscillating with different frequencies and not only one component as reported earlier. Furthermore, it was found that most of these processes are controlled inside each individual cell. This was shown by differences in oscillations in neighboring cells and protoplasts that share a uniform intercellular environment. The frequency of the dominant oscillation frequency depended neither on irradiance nor on CO2 concentration and is, therefore, not controlled by the photosynthetic rate. Characteristic differences in the frequency spectrum and damping of oscillations have been found among the plant species examined

Raman microscopy shows that nitrogen-rich cellular inclusions in microalgae are microcrystalline guanine

Microalgal cells possess a vast diversity of subcellular structures and cytoplasmic inclusions differing in theirmorphology, functionality, and composition, some of them giving rise to distinct Raman spectral signaturesallowing their identification, localization, and visualization in situ. Here,we showthat certain Raman features observedin Raman spectra of microalgae can be unambiguously attributed to guanine microcrystals because theyare clearly distinct from Raman fingerprints of closely related purine species. Using confocal Raman microscopy,we have localized crystalline guanine as a part of cellular inclusions in the chlorophyte Desmodesmus quadricaudaand in the eustigmatophyte Trachydiscus minutus. Wepropose that this finding also explains the chemical natureof similar nitrogen-rich crystalline structures recently documented in a number of other chlorophyte species byenergy-dispersive X-ray spectroscopy. To the best of our knowledge, this is the first Raman microscopy-based directevidence of the presence of guanine microcrystalline inclusions within microalgal cells. We tentatively proposethat the crystalline guanine serves as a very compact, long-termdepot of nitrogen in microalgae. Simplicityof specimen preparation requiring no fixation, labeling, or staining of the cells predetermines Raman microscopyas a method of choice for more advanced studies of the physiological role of guanine particles, as well as othercrystalline inclusions in situ within intact cells