Identification of plant leaf phosphorus content at different growth stages based on hyperspectral reflectance.

BACKGROUND: Modern agriculture strives to sustainably manage fertilizer for both economic and environmental reasons. The monitoring of any nutritional (phosphorus, nitrogen, potassium) deficiency in growing plants is a challenge for precision farming technology. A study was carried out on three species of popular crops, celery (Apium graveolens L., cv. Neon), sugar beet (Beta vulgaris L., cv. Tapir) and strawberry (Fragaria × ananassa Duchesne, cv. Honeoye), fertilized with four different doses of phosphorus (P) to deliver data for non-invasive detection of P content. RESULTS: Data obtained via biochemical analysis of the chlorophyll and carotenoid contents in plant material showed that the strongest effect of P availability for plants was in the diverse total chlorophyll content in sugar beet and celery compared to that in strawberry, in which P affects a variety of carotenoid contents in leaves. The measurements performed using hyperspectral imaging, obtained in several different stages of plant development, were applied in a supervised classification experiment. A machine learning algorithm (Backpropagation Neural Network, Random Forest, Naive Bayes and Support Vector Machine) was developed to classify plants from four variants of P fertilization. The lowest prediction accuracy was obtained for the earliest measured stage of plant development. Statistical analyses showed correlations between leaf biochemical constituents, phosphorus fertilization and the mass of the leaf/roots of the plants. CONCLUSIONS: Obtained results demonstrate that hyperspectral imaging combined with artificial intelligence methods has potential for non-invasive detection of non-homogenous phosphorus fertilization on crop levels.

Recycling of Energy Dissipated as Heat Accounts for High Activity of Photosystem II.

Photosystem II (PSII) converts light into chemical energy powering almost all life on Earth. The primary photovoltaic reaction in the PSII reaction center requires energy corresponding to 680 nm, which is significantly higher than in the case of the low-energy states in the antenna complexes involved in the harvesting of excitations driving PSII. Here we show that despite seemingly insufficient energy, the low-energy excited states can power PSII because of the activity of the thermally driven up-conversion. We demonstrate the operation of this mechanism both in intact leaves and in isolated pigment-protein complex LHCII. A mechanism is proposed, according to which the effective utilization of thermal energy in the photosynthetic apparatus is possible owing to the formation of LHCII supramolecular structures, leading to the coupled energy levels corresponding to approximately 680 and 700 nm, capable of exchanging excitation energy through the spontaneous relaxation and the thermal up-conversion.

Cremophor EL Nano-Emulsion Monomerizes Chlorophyll a in Water Medium.

In this paper, the application of a non-ionic detergent Cremophor EL for monomerization of chlorophyll a in an aqueous medium is studied. The spectrophotometric properties of chlorophyll a encapsulated into the Cremophor EL nano-emulsion system were characterized by electronic absorption, steady-state and time-resolved fluorescence as well as circular dichroism spectroscopy. The results have shown that chlorophyll a dissolves more efficiently in the aqueous medium containing low-level Cremophor (5 wt%) than at an ethanolic solution even in the concentration of 10-4 M. The molecular organization of the chlorophyll a in the Cremophor EL nano-micelles was also investigated by means of Raman spectroscopy. The spectral changes in the frequency of the C=O stretching group were used to distinguish the aggregation state of chlorophyll. It was revealed that chlorophyll a exists dominantly in the monomeric form in the Cremophor EL aqueous solution. The promising aspect of the use of Cremophor EL nano-emulsion as a delivery system is to maintain stable chlorophyll monomer in an aqueous medium. It would open the potential for a new, practical application of chlorophyll a in medicine, as a dietary supplement or studies on molecular organization of chlorophyll a in the well-defined artificial system.

Light-induced formation of dimeric LHCII.

It emerges from numerous experiments that LHCII, the major photosynthetic antenna complex of plants, can appear not only in the trimeric or monomeric states but also as a dimer. We address the problem whether the dimeric form of the complex is just a simple intermediate element of the trimer-monomer transformation or if it can also be a physiologically relevant molecular organization form? Dimers of LHCII were analyzed with application of native electrophoresis, time-resolved fluorescence spectroscopy, and fluorescence correlation spectroscopy. The results reveal the appearance of two types of LHCII dimers: one formed by the dissociation of one monomer from the trimeric structure and the other formed by association of monomers into a distinctively different molecular organizational form, characterized by a high rate of chlorophyll excitation quenching. The hypothetical structure of such an energy quencher is proposed. The high light-induced LHCII dimerization is discussed as a potential element of the photoprotective response in plants.

A chloroplast "wake up" mechanism: Illumination with weak light activates the photosynthetic antenna function in dark-adapted plants.

The efficient and fluent operation of photosynthesis in plants relies on activity of pigment-protein complexes called antenna, absorbing light and transferring excitations toward the reaction centers. Here we show, based on the results of the fluorescence lifetime imaging analyses of single chloroplasts, that pigment-protein complexes, in dark-adapted plants, are not able to act effectively as photosynthetic antennas, due to pronounced, adverse excitation quenching. It appeared that the antenna function could be activated by a short (on a minute timescale) illumination with light of relatively low intensity, substantially below the photosynthesis saturation threshold. The low-light-induced activation of the antenna function was attributed to phosphorylation of the major accessory light-harvesting complex LHCII, based on the fact that such a mechanism was not observed in the stn7 Arabidopsis thaliana mutant, with impaired LHCII phosphorylation. It is proposed that the protein phosphorylation-controlled change in the LHCII clustering ability provides mechanistic background for this regulatory process.

Two wavelength-dependent mechanisms of sensitisation of light-induced quenching in the isolated light-harvesting complex II.

The efficiency of visible light in inducing fluorescence quenching in the isolated light-harvesting complex II (LHCII) of higher plants is investigated by action spectroscopy in the visible portion of photosynthetic active radiation. The efficiency spectrum displays a relatively homogenous quenching yield across the most intense electronic transitions of the chlorophyll a and carotenoid pigments, indicating that quenching proceeds from the equilibrated state of the complex. Larger yields are observed in the 510-640-nm window, where weak transitions of LHCII-bound chromophores occur. This observation is interpreted in terms of an additional quenching sensitisation process mediated by these electronic transitions.

Light-Driven Reconfiguration of a Xanthophyll Violaxanthin in the Photosynthetic Pigment-Protein Complex LHCII: A Resonance Raman Study.

Resonance Raman analysis of the photosynthetic complex LHCII, immobilized in a polyacrylamide gel, reveals that one of the protein-bound xanthophylls, assigned as violaxanthin, undergoes light-induced molecular reconfiguration. The phototransformation is selectively observed in a trimeric structure of the complex and is associated with a pronounced twisting and a trans-cis molecular configuration change of the polyene chain of the carotenoid. Among several spectral effects accompanying the reconfiguration there are ones indicating a carotenoid triplet state. Possible physiological importance of the light-induced violaxanthin reconfiguration as a mechanism associated with making the pigment available for enzymatic deepoxidation in the xanthophyll cycle is discussed.

The xanthophyll cycle pigments, violaxanthin and zeaxanthin, modulate molecular organization of the photosynthetic antenna complex LHCII.

The effect of violaxanthin and zeaxanthin, two main carotenoids of the xanthophyll cycle, on molecular organization of LHCII, the principal photosynthetic antenna complex of plants, was studied in a model system based on lipid-protein membranes, by means of analysis of 77 K chlorophyll a fluorescence and "native" electrophoresis. Violaxanthin was found to promote trimeric organization of LHCII, contrary to zeaxanthin which was found to destabilize trimeric structures. Moreover, violaxanthin was found to induce decomposition of oligomeric LHCII structures formed in the lipid phase and characterized by the fluorescence emission band at 715 nm. Both pigments promoted formation of two-component supramolecular structures of LHCII and xanthophylls. The violaxanthin-stabilized structures were composed mostly of LHCII trimers while, the zeaxanthin-stabilized supramolecular structures of LHCII showed more complex organization which depended periodically on the xanthophyll content. The effect of the xanthophyll cycle pigments on molecular organization of LHCII was analyzed based on the results of molecular modeling and discussed in terms of a physiological meaning of this mechanism. Supramolecular structures of LHCII stabilized by violaxanthin, prevent uncontrolled oligomerization of LHCII, potentially leading to excitation quenching, therefore can be considered as structures protecting the photosynthetic apparatus against energy loses at low light intensities.

Photothermal microscopy: imaging of energy dissipation from photosynthetic complexes.

An idea of a photothermal imaging microscopy (PTIM) is proposed, along with its realization based on a dependence of fluorescence anisotropy of dye molecules on heat emission in their nearest vicinity. Erythrosine B was selected as a fluorophore convenient to report thermal deactivation of the excited pigment-protein complex isolated from the photosynthetic apparatus of plants (LHCII), owing to the relatively large spectral gap between the fluorescence emission bands of chlorophyll a and a probe. Comparison of the simultaneously recorded images based on fluorescence lifetime of LHCII and fluorescence anisotropy of erythrosine shows a high rate of thermal energy dissipation from the aggregated forms of the complex and, possibly, thermal energy transmission along the protein supramolecular structures. Relatively high resolution of this novel microscopic technique, comparable to the fluorescence lifetime microscopy, enables its application in a nanoscale imaging and in nanothermography.

Is It Beneficial for the Major Photosynthetic Antenna Complex of Plants To Form Trimers?

The process of primary electric charge separation in photosynthesis takes place in the reaction centers, but photosynthesis can operate efficiently and fluently due to the activity of several pigment-protein complexes called antenna, which absorb light quanta and transfer electronic excitations toward the reaction centers. LHCII is the major photosynthetic pigment-protein antenna complex of plants and appears in the trimeric form. Several recent reports point to trimeric organization of LHCII as a key factor responsible for the chloroplast architecture via stabilization of granal organization of the thylakoid membranes. In the present work, we address the question of whether such an organization could also directly influence the antenna properties of this pigment-protein complex. Chlorophyll fluorescence analysis reveals that excitation energy transfer in LHCII is substantially more efficient in trimers and dissipative energy losses are higher in monomers. It could be concluded that trimers are exceptionally well suited to perform the antenna function. Possibility of fine regulation of the photosynthetic antenna function via the LHCII trimer-monomer transition is also discussed, based on the fluorescence lifetime analysis in a single chloroplast.

Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes.

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.

The negative feedback molecular mechanism which regulates excitation level in the plant photosynthetic complex LHCII: towards identification of the energy dissipative state.

Overexcitation of the photosynthetic apparatus is potentially dangerous because it can cause oxidative damage. Photoprotection realized via the feedback de-excitation in the pigment-protein light-harvesting complex LHCII, embedded in the chloroplast lipid environment, was studied with use of the steady-state and time-resolved fluorescence spectroscopy techniques. Illumination of LHCII results in the pronounced singlet excitation quenching, demonstrated by decreased quantum yield of the chlorophyll a fluorescence and shortening of the fluorescence lifetimes. Analysis of the 77K chlorophyll a fluorescence emission spectra reveals that the light-driven excitation quenching in LHCII is associated with the intensity increase of the spectral band in the region of 700nm, relative to the principal band at 680nm. The average chlorophyll a fluorescence lifetime at 700nm changes drastically upon temperature decrease: from 1.04ns at 300K to 3.63ns at 77K. The results of the experiments lead us to conclude that: (i) the 700nm band is associated with the inter-trimer interactions which result in the formation of the chlorophyll low-energy states acting as energy traps and non-radiative dissipation centers; (ii) the Arrhenius analysis, supported by the results of the FTIR measurements, suggests that the photo-reaction can be associated with breaking of hydrogen bonds. Possible involvement of photo-isomerization of neoxanthin, reported previously (Biochim. Biophys. Acta 1807 (2011) 1237-1243) in generation of the low-energy traps in LHCII is discussed.

Light-induced isomerization of the LHCII-bound xanthophyll neoxanthin: possible implications for photoprotection in plants.

Light-harvesting pigment-protein complex of Photosystem II (LHCII) is the largest photosynthetic antenna complex of plants and the most abundant membrane protein in the biosphere. Plant fitness and productivity depend directly on a balance between excitations in the photosynthetic apparatus, generated by captured light quanta, and the rate of photochemical processes. Excess excitation energy leads to oxidative damage of the photosynthetic apparatus and entire organism and therefore the balance between the excitation density and photosynthesis requires precise and efficient regulation, operating also at the level of antenna complexes. We show that illumination of the isolated LHCII leads to isomerization of the protein-bound neoxanthin from conformation 9'-cis to 9',13- and 9',13'-dicis forms. At the same time light-driven excitation quenching is observed, manifested by a decrease in chlorophyll a fluorescence intensity and shortened fluorescence lifetimes. Both processes, the neoxanthin isomerization and the chlorophyll excitation quenching, are reversible in dim light. The results of the 77K florescence measurements of LHCII show that illumination is associated with appearance of the low-energy states, which can serve as energy traps in the pigment-protein complex subjected to excess excitation. Possible sequence of the molecular events is proposed, leading to a protective excess excitation energy quenching: neoxanthin photo-isomerization→formation of LHCII supramolecular structures which potentiate creation of energy traps→excitation quenching.

Investigation of the molecular mechanism of the blue-light-specific excitation energy quenching in the plant antenna complex LHCII.

Excitation of the major photosynthetic antenna complex of plants, LHCII, with blue light (470nm) provides an advantage to plants, as it gives rise to chlorophyll a fluorescence lifetimes shorter than with excitation with red light (635nm). This difference is particularly pronounced in fluorescence emission wavelengths longer than 715nm. Illumination of LHCII preparation with blue light additionally induces fluorescence quenching, which develops on a minute timescale. This effect is much less efficient when induced by red light, despite the equalized energy absorbed in both the spectral regions. Simultaneous analysis of the fluorescence and photoacoustic signals in LHCII demonstrated that the light-driven fluorescence quenching is not associated with an increase in heat emission. Instead, a reversible light-induced conformational transformation of the protein takes place, as demonstrated by the FTIR technique. These findings are discussed in terms of the blue-light-specific excitation energy quenching in LHCII, which may have photoprotective applications.

Blue-light-controlled photoprotection in plants at the level of the photosynthetic antenna complex LHCII.

Plants have developed several adaptive regulatory mechanisms, operating at all the organization levels, to optimize utilization of light energy and to protect themselves against over-excitation-related damage. We report activity of a previously unknown possible regulatory mechanism that operates at the molecular level of the major photosynthetic pigment-protein complexes of plants, LHCII. This mechanism is driven exclusively by blue light, operates in the trimeric but not in the monomeric complex, and results in singlet excitation quenching leading to thermal energy dissipation. The conclusions are based on single molecule fluorescence lifetime analysis, direct measurements of thermal energy dissipation by photo-thermal spectroscopy, and on fluorescence spectroscopy. Possible molecular mechanisms involved in the blue-light-induced photoprotective effect are discussed, including xanthophyll photo-isomerization and the thermo-optic effect.