Multimodal non-invasive probing of stress-induced carotenogenesis in the cells of microalga Bracteacoccus aggregatus.

Microalgae are the richest source of natural carotenoids-accessory photosynthetic pigments used as natural antioxidants, safe colorants, and nutraceuticals. Microalga Bracteacoccus aggregatus IPPAS C-2045 responds to stresses, including high light, with carotenogenesis-gross accumulation of secondary carotenoids (the carotenoids structurally and energetically uncoupled from photosynthesis). Precise mechanisms of cytoplasmic transport and subcellular distribution of the secondary carotenoids under stress are still unknown. Using multimodal imaging combining micro-Raman imaging (MRI), fluorescent lifetime (τ) imaging (FLIM), and transmission electron microscopy (TEM), we monitored ultrastructural and biochemical rearrangements of B. aggregatus cells during the stress-induced carotenogenesis. MRI revealed a decline in the diversity of molecular surrounding of the carotenoids in the cells compatible with the relocation of the bulk of the carotenoids in the cell from functionally and structurally heterogeneous photosynthetic apparatus to the more homogenous lipid matrix of the oleosomes. Two-photon FLIM highlighted the pigment transformation in the cell during the stress-induced carotenogenesis. The structures co-localized with the carotenoids with shorter τ (mainly chloroplast) shrunk, whereas the structures harboring secondary carotenoids with longer τ (mainly oleosomes) expanded. These changes were in line with the ultrastructural data (TEM). Fluorescence of B. aggregatus carotenoids, either in situ or in acetone extracts, possessed a surprisingly long lifetime. We hypothesize that the extension of τ of the carotenoids is due to their aggregation and/or association with lipids and proteins. The propagation of the carotenoids with prolonged τ is considered to be a manifestation of the secondary carotenogenesis suitable for its non-invasive monitoring with multimodal imaging.

Towards the synthesis of spectral imaging and machine learning-based approaches for non-invasive phenotyping of plants.

High-throughput phenotyping is now central to the progress of plant sciences, accelerated breeding, and precision farming. The power of phenotyping comes from the automated, rapid, non-invasive collection of large datasets describing plant objects. In this context, the goal of extracting relevant information from different kinds of images is of paramount importance. We review both the spectral and machine learning-based approaches to imaging of plants for the purpose of their phenotyping. The advantages and drawbacks of both approaches will be discussed with a focus on the monitoring of plants. We argue that an emerging approach combining the strengths of the spectral and the machine learning-based approaches will remain a promising direction in plant phenotyping in the nearest future.

Interactive Effects of Ceftriaxone and Chitosan Immobilization on the Production of Arachidonic Acid by and the Microbiome of the Chlorophyte Lobosphaera sp. IPPAS C-2047.

Pharmaceuticals including antibiotics are among the hazardous micropollutants (HMP) of the environment. Incomplete degradation of the HMP leads to their persistence in water bodies causing a plethora of deleterious effects. Conventional wastewater treatment cannot remove HMP completely and a promising alternative comprises biotechnologies based on microalgae. The use of immobilized microalgae in environmental biotechnology is advantageous since immobilized cultures allow the recycling of the microalgal cells, support higher cell densities, and boost tolerance of microalgae to stresses including HMP. Here, we report on a comparative study of HMP (exemplified by the antibiotic ceftriaxone, CTA) removal by suspended and chitosan-immobilized cells of Lobosphaera sp. IPPAS C-2047 in flasks and in a column bioreactor. The removal of CTA added in the concentration of 20 mg/L was as high as 65% (in the flasks) or 85% (in the bioreactor). The adsorption on the carrier and abiotic oxidation were the main processes contributing 65-70% to the total CTA removal, while both suspended and immobilized cells took up 25-30% of CTA. Neither the immobilization nor CTA affected the accumulation of arachidonic acid (ARA) by Lobosphaera sp. during bioreactor tests but the subsequent nitrogen deprivation increased ARA accumulation 2.5 and 1.7 times in the suspended and chitosan-immobilized microalgae, respectively. The study of the Lobosphaera sp. microbiome revealed that the immobilization of chitosan rather than the CTA exposure was the main factor displacing the taxonomic composition of the microbiome. The possibility and limitations of the use of chitosan-immobilized Lobosphaera sp. IPPAS C-2047 for HMP removal coupled with the production of valuable long-chain polyunsaturated fatty acids is discussed.

Failure of Micractinium simplicissimum Phosphate Resilience upon Abrupt Re-Feeding of Its Phosphorus-Starved Cultures.

Microalgae are naturally adapted to the fluctuating availability of phosphorus (P) to opportunistically uptake large amounts of inorganic phosphate (Pi) and safely store it in the cell as polyphosphate. Hence, many microalgal species are remarkably resilient to high concentrations of external Pi. Here, we report on an exception from this pattern comprised by a failure of the high Pi-resilience in strain Micractinium simplicissimum IPPAS C-2056 normally coping with very high Pi concentrations. This phenomenon occurred after the abrupt re-supplementation of Pi to the M. simplicissimum culture pre-starved of P. This was the case even if Pi was re-supplemented in a concentration far below the level toxic to the P-sufficient culture. We hypothesize that this effect can be mediated by a rapid formation of the potentially toxic short-chain polyphosphate following the mass influx of Pi into the P-starved cell. A possible reason for this is that the preceding P starvation impairs the capacity of the cell to convert the newly absorbed Pi into a "safe" storage form of long-chain polyphosphate. We believe that the findings of this study can help to avoid sudden culture crashes, and they are also of potential significance for the development of algae-based technologies for the efficient bioremoval of P from P-rich waste streams.

Effect of Abscisic Acid on Growth, Fatty Acid Profile, and Pigment Composition of the Chlorophyte Chlorella (Chromochloris) zofingiensis and Its Co-Culture Microbiome.

Microalga Chlorella (Chromochloris) zofingiensis has been gaining increasing attention of investigators as a potential competitor to Haematococcus pluvialis for astaxanthin and other xanthophylls production. Phytohormones, including abscisic acid (ABA), at concentrations relevant to that in hydroponic wastewater, have proven themselves as strong inductors of microalgae biomass productivity and biosynthesis of valuable molecules. The main goal of this research was to evaluate the influence of phytohormone ABA on the physiology of C. zofingiensis in a non-aseptic batch experiment. Exogenous ABA stimulated C. zofingiensis cell division, biomass production, as well as chlorophyll, carotenoid, and lipid biosynthesis. The relationship between exogenous ABA concentration and the magnitude of the observed effects was non-linear, with the exception of cell growth and biomass production. Fatty acid accumulation and composition depended on the concentration of ABA tested. Exogenous ABA induced spectacular changes in the major components of the culture microbiome of C. zofingiensis. Thus, the abundance of the representatives of the genus Rhodococcus increased drastically with an increase in ABA concentration, whereas the abundance of the representatives of Reyranella and Bradyrhizobium genera declined. The possibilities of exogenous ABA applications for the enhancing of the biomass, carotenoid, and fatty acid productivity of the C. zofingiensis cultures are discussed.

Assessment of the Tolerance of a Chlorophyte Desmodesmus to CuO-NP for Evaluation of the Nanopollution Bioremediation Potential of This Microalga.

Broad application of CuO nanoparticles (CuO-NP) for industrial and household purposes leads to a continuous increase in their discharge to, and, hence, ever-increasing environmental hazards for aquatic ecosystems. Microalgae-based technologies hold promise for bioremediation of diverse hazardous micropollutants (HMP), including NP, from wastewater. In this study, we tested the ability of the green microalga Desmodesmus sp. to accumulate CuO-NP or their components. We also assessed the tolerance of this microalga to the environmentally relevant concentrations of CuO-NP. Using scanning electron microscopy, we demonstrated that the average size of CuO-NP was 50-100 nm, and their purity was confirmed with elemental composition analysis. Tests of the colloidal suspensions of CuO-NP showed that the hydrodynamic diameter of CuO-NP and their aggregates was below 100 nm. Flow cytometry analysis showed that CuO-NP at a concentration of 100 µg L-1 slightly inhibited the viability of microalgae cells and led to an increase in their oxidative stress. The assessment of the condition of photosystem II showed that CuO-NP exert a multifaceted effect on the photosynthetic apparatus of Desmodesmus sp., depending on the concentration of and the exposure to the CuO-NP. Desmodesmus sp. turned to be relatively tolerant to CuO-NP. In addition, the ICP-MS method revealed increased bioaccumulation of copper by microalgae cells in the experimental groups. The outcomes of this study indicate that the Desmodesmus sp. has a significant potential for bioremoval of the copper-based nanostructured HMP from an aquatic environment.

Effects of Plasma-Activated Water on Leaf and Fruit Biochemical Composition and Scion Growth in Apple.

The application of plasma-activated water (PAW) in agriculture has gained the attention of researchers and practitioners. In particular, treatment with PAW is a promising method for increasing scion and rootstock survival as well as augmenting the mineral nutrition applicable to tree fruit crops. However, the applications of PAW are hampered by the lack of information about the effects of PAW on apple tree condition and yield. The increase in survival rate by PAW is believed to stem from the general stimulation of physiological processes in the plant tissue. To assess the actual effect of the PAW treatments, one needs to consider an important indicator of young tree quality such as their vegetative growth. We conducted field experiments to study the possibility of use of PAW for increase in primary nutrient contents in fruits and leaves in an orchard, as well as to assess the scion survival rate and vegetative growth of young grafts in a nursery. The application of PAW influenced the fruitset, yield, leaf nitrogen (N) and potassium (K), fruit phosphorus (P), calcium (Ca) ascorbic acid (AA) and titratable acidity (TA). Treatment with PAW did not significantly reduce the negative impact of the rootstock thickness on the survival rate of bench grafts and their subsequent development. At the same time, scion survival tended to increase in the case when the scions and the rootstocks were of compatible thickness. Further studies of the PAW treatment effects are needed to better understand its applicability in diverse fields of horticulture.

Non-Invasive Probing of Winter Dormancy via Time-Frequency Analysis of Induced Chlorophyll Fluorescence in Deciduous Plants as Exemplified by Apple (Malus × domestica Borkh.).

Dormancy is a physiological state that confers winter hardiness to and orchestrates phenological phase progression in temperate perennial plants. Weather fluctuations caused by climate change increasingly disturb dormancy onset and release in plants including tree crops, causing aberrant growth, flowering and fruiting. Research in this field suffers from the lack of affordable non-invasive methods for online dormancy monitoring. We propose an automatic framework for low-cost, long-term, scalable dormancy studies in deciduous plants. It is based on continuous sensing of the photosynthetic activity of shoots via pulse-amplitude-modulated chlorophyll fluorescence sensors connected remotely to a data processing system. The resulting high-resolution time series of JIP-test parameters indicative of the responsiveness of the photosynthetic apparatus to environmental stimuli were subjected to frequency-domain analysis. The proposed approach overcomes the variance coming from diurnal changes of insolation and provides hints on the depth of dormancy. Our approach was validated over three seasons in an apple (Malus × domestica Borkh.) orchard by collating the non-invasive estimations with the results of traditional methods (growing of the cuttings obtained from the trees at different phases of dormancy) and the output of chilling requirement models. We discuss the advantages of the proposed monitoring framework such as prompt detection of frost damage along with its potential limitations.

"Microalgae as converters of light energy into biofuels and high-value products": a session of the Russian Photobiology Society 9th Congress (Shepsi, Krasnodar region, Russia; September, 12-19, 2021).

An outline of the "Microalgae as converters of light energy into biofuels and high-value products" section of the 9th Congress of the Russian Photobiological Society is presented. Key talks and poster presentations are briefly introduced along with key findings made by their authors. We conclude that this section was a success with many interesting talks and a vigorous follow-up discussion indicative of the keen interest of Russian researchers in microalgae and biotechnologies based on these microorganisms.

Chlorophyll fluorescence as a valuable multitool for microalgal biotechnology.

Variable fluorescence of chlorophyll (CF) of the photosynthetic apparatus is an ample source of valuable information on physiological condition of photosynthetic organisms. Currently, the most widespread CF-based technique is represented by recording pulse-amplitude modulated (PAM) induction of CF by saturating light. The CF-based monitoring techniques are increasingly employed for characterization of performance and stress resilience of microalgae in microalgal biotechnology. Analysis of CF induction curves reveals the fate of light energy absorbed by photosynthetic apparatus, the proportions of the energy that have been utilized for photochemistry (culture growth), and heat dissipated by photoprotective mechanisms. Hence CF and its derived parameters are an accurate proxy of the metabolic activity of the photosynthetic cell and the engagement of photoprotective mechanisms. This information is a solid foundation for making decisions on the microalgal culture management during the lab-scale and industrial-scale cultivation. Applications of CF and PAM include the monitoring of stressor (high light, nutrient deprivation, extreme temperatures, etc.) effects for assessment of the culture robustness. It also serves as a non-invasive express test for gauging the effect of assorted toxicants in microalgae. This approach is becoming widespread in ecological toxicology and environmental biotechnology, particularly for bioprospecting strains capable of the destruction of dangerous pollutants such as pharmaceuticals. In the review, we discuss the advantages and drawbacks of using CF-based methods for assessment of the culture conditions. Special attention is paid to the potential caveats and applicability of different variations of CF and PAM measurements for solving problems of microalgal biotechnology.

Photosynthetic and ultrastructural responses of the chlorophyte Lobosphaera to the stress caused by a high exogenic phosphate concentration.

Biotechnology of microalgae holds promise for sustainable using of phosphorus, a finite non-renewable resource. Responses of the green microalga Lobosphaera sp. IPPAS C-2047 to elevated inorganic phosphate (Pi) concentrations were studied. Polyphosphate (PolyP) accumulation and ultrastructural rearrangements were followed in Lobosphaera using light and electron microscopy and linked to the responses of the photosynthetic apparatus probed with chlorophyll fluorescence. High tolerance of Lobosphaera to ≤ 50 g L-1 Pi was accompanied by a retention of photosynthetic activity and specific induction of non-photochemical quenching (NPQ up to 4; Fv/Fm around 0.7). Acclimation of the Lobosphaera to the high Pi was accompanied by expansion of the thylakoid lumen and accumulation of the carbon-rich compounds. The toxic effect of the extremely high (100 g L-1) Pi inhibited the growth by ca. 60%, induced a decline in photosynthetic activity and NPQ along with contraction of the lumen, destruction of the thylakoids, and depletion of starch reserves. The Lobosphaera retained viability at the Pi in the range of 25-100 g L-1 showing moderate an increase of intracellular P content (to 4.6% cell dry weight). During the initial high Pi exposure, the vacuolar PolyP biosynthesis in Lobosphaera was impaired but recovered upon acclimation. Synthesis of abundant non-vacuolar PolyP inclusions was likely a manifestation of the emergency acclimation of the cells converting the Pi excess to less metabolically active PolyP. We conclude that the remarkable Pi tolerance of Lobosphaera IPPAS C-2047 is determined by several mechanisms including rapid conversion of the exogenic Pi into metabolically safe PolyP, the acclamatory changes in the cell population structure. Possible involvement of NPQ in the high Pi resilience of the Lobosphaera is discussed.

Differential Responses to UV-A Stress Recorded in Carotenogenic Microalgae Haematococcus rubicundus, Bracteacoccus aggregatus, and Deasonia sp.

UV-A is the main ultraviolet component of natural (solar) radiation. Despite it, its effect on phototrophs is studied less than UV-B. Effects of UV-A on photosynthetic apparatus of three carotenoid-producing microalgae were elucidated. Photosynthetic activity was studied using chlorophyll fluorescence analysis. Cell extracts were evaluated by absorbance spectroscopy. On the one hand, there were some common features of three strains. In all cases the changes involved PSII primary photochemistry and antennae size. All strains accumulated UV-absorbing polar compounds. On the other hand, some responses were different. Upregulation of non-photochemical quenching was observed only in B. aggregatus BM5/15, whereas in other cases its level was low. H. rubicundus BM7/13 and Deasonia sp. NAMSU 934/2 accumulated secondary carotenoids, whereas B. aggregatus BM5/15 accumulated primary ones. Microscopic features of the cultures were also different. H. rubicundus BM7/13 and Deasonia sp. NAMSU 934/2 were represented mostly by solitaire cells or small cell clusters, lacking their green color; the cells of B. aggregatus BM5/15 formed aggregates from green cells. Cell aggregation could be considered as an additional UV-protecting mechanism. Finally, the strains differed by their viability. B. aggregatus BM5/15 was most resistant to UV-A, whereas massive cell death was observed in two other cultures.

Chilling Upregulates Expression of the PsbS and LhcSR Genes in the Chloroplasts of the Green Microalga Lobosphaera incisa IPPAS C-2047.

Non-photochemical quenching (NPQ) of excited chlorophyll states is essential for protecting the photosynthetic apparatus (PSA) from the excessive light-induced damage in all groups of oxygenic photosynthetic organisms. The key component of the NPQ mechanism in green algae and some other groups of algae and mosses is the LhcSR protein of the light harvesting complex (LHC) protein superfamily. In vascular plants, LhcSR is replaced by PsbS, another member of the LHC superfamily and a subunit of photosystem II (PSII). PsbS also performs the photoprotective function in mosses. For a long time, PsbS had been believed to be nonfunctional in green algae, although the corresponding gene was discovered in the genome of these organisms. The first evidence of the PsbS accumulation in the model green alga Chlamydomonas reinhardtii in response to the increase in irradiance was obtained only six years ago. However, the observed increase in the PsbS content was short-termed (on an hour-timescale). Here, we report a significant (more than three orders of magnitude) and prolonged (four days) upregulation of PsbS expression in response to the chilling-induced high-light stress followed by a less significant (~ tenfold) increase in the PsbS expression for nine days. This is the first evidence for the long-term upregulation of the PsbS expression in green alga (Chlorophyta) in response to stress. Our data indicate that the role of PsbS in the PSA of Chlorophyta is not limited to the first-line defense against stress, as it was previously assumed, but includes full-scale participation in the photoprotection of PSA from the environmental stress factors.

The Effect of Chilling on the Photosynthetic Apparatus of Microalga Lobosphaera incisa IPPAS C-2047.

Photosynthetic organisms have developed a set of mechanisms aimed at preventing photo-oxidative reactions in the photosynthetic apparatus (PSA) initiated by excessively absorbed light energy. Along with high irradiance, other stressors, e.g., chilling temperatures, can lead to the absorption of the excess of light energy and hence to photo-oxidative stress. Here, we studied induction of photoprotective mechanisms in response to chilling (0°C) at a low irradiance (50 µmol PAR photons m-2·s-1) in the cells of microalga Lobosphaera incisa IPPAS C-2047. After 4 days of incubation at a low temperature, L. incisa IPPAS C-2047 cells showed a notable decrease in the photochemical activity of photosystem II (PSII) and in the efficiency of photosynthetic electron transport, as well as a significant increase in the thermal dissipation of the absorbed light energy in the light-harvesting antenna. In contrast, most conventional markers of PSA acclimation to excess light energy [total chlorophyll and carotenoid content; violaxanthin cycle pigment content and de-epoxidation state; photosynthetic antenna, PSII, and photosystem I (PSI) ratio] remained virtually unchanged. The content of major unsaturated fatty acids also remained almost unaffected, except for arachidonic acid (increased by 40%) recently assumed to activate violaxanthin de-epoxidase by adjusting its lipid microenvironment. Significant changes (4-7-fold increase) were observed in the expression of the gene encoding protective protein LhcSR. Pre-conditioning at 5°C prior to the acclimation to 0°C augmented the PSA photochemical activity. Our data show that the mid-term (4-d) acclimation of L. incisa IPPAS C-2047 to a chilling temperature at a low irradiance triggers the PSA response resembling, in part, the response to high light but relying mostly on the LhcSR protein-dependent quenching of excitation in the photosynthetic antenna.

Essential Role of Potassium in Apple and Its Implications for Management of Orchard Fertilization.

K (K) is of paramount importance for apple (Malus × domestica Borkh.), not only for tree growth and development but also for the size and quality of fruit yield. The apple plant's demand for K varies, along with the progression of phenological phases, during the growing season. The K demand peaks during ripening of fruits featuring relatively high concentration of K comparable to that of the leaves. The mainstream method of apple tree K fertilization is through application of the fertilizer to the soils to improve K uptake by the roots. The bioavailability of K depends on assorted various factors, including pH, interaction with other nutrients in soil solution, temperature, and humidity. An important role in making the K from soil available for uptake by plants is played by plant growth-promoting microorganisms (PGPM), and the specific role of the PGPM is discussed. Advantages of fertigation (the combination of irrigation and fertilization) as an approach include allowing to balance application rate of K fertilizer against its variable demand by plants during the growing season. Excess K in the soil leads to competitive inhibition of calcium uptake by plants. The K-dependent deficiency of Ca leads to its predominant channeling to the leaves and hence to its decline in fruits. Consequently, the apple fruits affected by the K/Ca imbalance frequently develop physiological disorders in storage. This emphasizes the importance of the balanced K application, especially during the last months of the growing season, depending on the crop load and the actual K demand. The potential use of modern approaches to automated crop load estimation through machine vision for adjustment of K fertilization is underlined.

Simulating the Interplay between the Uptake of Inorganic Phosphate and the Cell Phosphate Metabolism under Phosphorus Feast and Famine Conditions in Chlorella vulgaris.

Using a mathematical simulation approach, we studied the dynamics of the green microalga Chlorella vulgaris phosphate metabolism response to shortage and subsequent replenishing of inorganic phosphate in the medium. A three-pool interaction model was used to describe the phosphate uptake from the medium, its incorporation into the cell organic compounds, its storage in the form of polyphosphates, and culture growth. The model comprises a system of ordinary differential equations. The distribution of phosphorous between cell pools was examined for three different stages of the experiment: growth in phosphate-rich medium, incubation in phosphate-free medium, and phosphate addition to the phosphorus-starving culture. Mathematical modeling offers two possible scenarios for the appearance of the peak of polyphosphates (PolyP). The first scenario explains the accumulation of PolyP by activation of the processes of its synthesis, and the decline in PolyP is due to its redistribution between dividing cells during growth. The second scenario includes a hysteretic mechanism for the regulation of PolyP hydrolysis, depending on the intracellular content of inorganic phosphate. The new model of the dynamics of P pools in the cell allows one to better understand the phenomena taking place during P starvation and re-feeding of the P-starved microalgal cultures with inorganic phosphate such as transient PolyP accumulation. Biotechnological implications of the observed dynamics of the polyphosphate pool of the microalgal cell are considered. An approach enhancing the microalgae-based wastewater treatment method based on these scenarios is proposed.

An insight into spectral composition of light available for photosynthesis via remotely assessed absorption coefficient at leaf and canopy levels.

Non-invasive comparative analysis of the spectral composition of energy absorbed by crop species at leaf and plant levels was carried out using the absorption coefficient retrieved from leaf and plant reflectance as an informative metric. In leaves of three species with contrasting leaf structures and photosynthetic pathways (maize, soybean, and rice), the blue, green, and red fractions of leaf absorption coefficients were 48, 20, and 32%, respectively. The fraction of green light in the total budget of light absorbed at the plant level was higher than at the leaf level approaching the size of the red fraction (24% green vs. 25.5% red) and surpassing it inside the canopy. The plant absorption coefficient in the far-red region (700-750 nm) was significant reaching 7-10% of the absorption coefficient in green or red regions. The spectral composition of the absorbed light in the three species was virtually the same. Fractions of light in absorbed PAR remained almost invariant during growing season over a wide range of plant chlorophyll content. Fractions of absorption coefficient in the green, red, and far-red were in accord with published results of quantum yield for CO2 fixation on an absorbed light basis. The role of green and far-red light in photosynthesis was demonstrated in simple experiments in natural conditions. The results show the potential for using leaf and plant absorption coefficients retrieved from reflectance to quantify photosynthesis in each spectral range.

The Dynamics of the Bacterial Community of the Photobioreactor-Cultivated Green Microalga Haematococcus lacustris during Stress-Induced Astaxanthin Accumulation.

Haematococcus lacustris is a natural source of a valuable ketocarotenoid astaxanthin. Under autotrophic growth conditions, it exists in the form of a community with bacteria. The close coexistence of these microorganisms raises two questions: how broad their diversity is and how they interact with the microalga. Despite the importance these issues, little is known about microorganisms existing in Haematococcus cultures. For the first time, we characterize the dynamic of the H. lacustris microbiome of the microbiome of Haematococcus (a changeover of the bacterial associated species as function of the time) cultivated autotrophically in a photobioreactor based on 16S rRNA metabarcoding data. We found that Proteobacteria and Bacteroidetes are predominant phyla in the community. The Caulobacter bacterium became abundant during astaxanthin accumulation. These data were supported by microscopy. We discuss possible roles and interactions of the community members. These findings are of potential significance for biotechnology. They provide an insight into possible bacterial contamination in algal biomass and reveal the presence of bacteria essential for the algal growth.

Linking Tissue Damage to Hyperspectral Reflectance for Non-Invasive Monitoring of Apple Fruit in Orchards.

Reflected light carries ample information about the biochemical composition, tissue architecture, and physiological condition of plants. Recent technical progress has paved the way for affordable imaging hyperspectrometers (IH) providing spatially resolved spectral information on plants on different levels, from individual plant organs to communities. The extraction of sensible information from hyperspectral images is difficult due to inherent complexity of plant tissue and canopy optics, especially when recorded under ambient sunlight. We report on the changes in hyperspectral reflectance accompanying the accumulation of anthocyanins in healthy apple (cultivars Ligol, Gala, Golden Delicious) fruits as well as in fruits affected by pigment breakdown during sunscald development and phytopathogen attacks. The measurements made outdoors with a snapshot IH were compared with traditional "point-type" reflectance measured with a spectrophotometer under controlled illumination conditions. The spectra captured by the IH were suitable for processing using the approaches previously developed for "point-type" apple fruit and leaf reflectance spectra. The validity of this approach was tested by constructing a novel index mBRI (modified browning reflectance index) for detection of tissue damages on the background of the anthocyanin absorption. The index was suggested in the form of mBRI = (R640-1 + R800-1) - R678-1. Difficulties of the interpretation of fruit hyperspectral reflectance images recorded in situ are discussed with possible implications for plant physiology and precision horticulture practices.