O. B. Chivkunova

Optical properties and nondestructive estimation of anthocyanin content in plant leaves.

Abstract Absorption and reflectance spectra of maple (Acer platanoides), cotoneaster (Cotoneaster alaunica), dogwood (Cornus alba) and pelargonium (Pelargonium zonale) leaves with a wide range of pigment content and composition were studied in visible and near-infrared spectra in order to reveal specific anthocyanin (Anth) spectral features in leaves. Comparing absorption spectra of Anth-containing and Anth-free leaves with the same chlorophyll (Chl) content, absorption spectra of Anth in leaves were derived. The main spectral feature of Anth absorption in vivo was a peak around 550 nm; the peak magnitude was closely related to Anth content. A quantitative nondestructive technique was developed to subtract Chl contribution to reflectance in this spectral region and retrieve Anth content from reflectance over a wide range of pigment content and composition. Anth reflectance index in the form ARI = (R550)−1 − (R700)−1, where (R550)−1 and (R700)−1 are inverse reflectances at 550 and 700 nm, respectively, allowed an accurate estimation of Anth accumulation, even in minute amounts, in intact senescing and stressed leaves.

Assessing Carotenoid Content in Plant Leaves with Reflectance Spectroscopy

Abstract Spectral reflectance of maple, chestnut and beech leaves in a wide range of pigment content and composition was investigated to devise a nondestructive technique for total carotenoid (Car) content estimation in higher plant leaves. Reciprocal reflectance in the range 510 to 550 nm was found to be closely related to the total pigment content in leaves. The sensitivity of reciprocal reflectance to Car content was maximal in a spectral range around 510 nm; however, chlorophylls (Chl) also affect reflectance in this spectral range. To remove the Chl effect on the reciprocal reflectance at 510 nm, a reciprocal reflectance at either 550 or 700 nm was used, which was linearly proportional to the Chl content. Indices for nondestructive estimation of Car content in leaves were devised and validated. Reflectances in three spectral bands, 510 ± 5 nm, either 550 ± 15 nm or 700 ± 7.5 nm and the near infrared range above 750 nm are sufficient to estimate total Car content in plant leaves nondestructively with a root mean square error of less than 1.75 nmol/cm2.

Light-stress-induced pigment changes and evidence for anthocyanin photoprotection in apples.

Fruit of two apple (Malus domestica Borkh.) cultivars, differing in their ability to produce anthocyanin pigments when exposed to sunlight, have been studied using reflectance spectroscopy. Comparison of the spectra shows that apple anthocyanins in vivo possess a symmetric absorption band at 500-600 nm with a maximum near 550 nm. Anthocyanins considerably increase light absorption by apples. In on-tree-ripening Zhigulevskoe apples, accumulating high amounts of anthocyanin pigments, chlorophyll contents in sunlit and shaded sides of the fruits are found to be similar. In contrast, frequently considerably lower chlorophyll content is estimated in sunlit compared with shaded sides of Antonovka apples exhibiting low potential for anthocyanin formation. Sunlight also brings about an increase of carotenoid content over that of chlorophylls and accumulation of substances responsible for light absorption in the range 350-400 nm. The rates of high-light-induced chlorophyll bleaching in red zones of fruit containing anthocyanins are considerably lower than those in green zones and decrease with an increase in the pigment content. Anthocyanins show more stability to irradiation than chlorophylls. A protective function of anthocyanins against both light-induced stress in, and damage to, apples is suggested. It is proposed that anthocyanins function as an effective internal light trap filling the chlorophyll absorption gap in the green-orange part of the visible spectrum.

Light absorption by anthocyanins in juvenile, stressed, and senescing leaves

The optical properties of leaves from five species, Norway maple (Acer platanoides L.), cotoneaster (Cotoneaster alaunica Golite), hazel (Corylus avellana L.), Siberian dogwood (Cornus alba L.), and Virginia creeper (Parthenocissus quinquefolia (L.) Planch.), differing in pigment composition and at different stages of ontogenesis, were studied. Anthocyanin absorption maxima in vivo, as estimated with spectrophotometry of intact anthocyanic versus acyanic leaves and microspectrophotometry of vacuoles in the leaf cross-sections, were found between 537 nm and 542 nm, showing a red shift of 5–20 nm compared with the corresponding maxima in acidic water–methanol extracts. In non-senescent leaves, strong anthocyanin absorption was found between 500 nm and 600 nm (with a 70–80 nm apparent bandwidth). By and large, absorption by anthocyanin in leaves followed a modified form of the Lambert–Beer law, showing a linear trend up to a content of nearly 50 nmol cm−2, and permitting thereby a non-invasive determination of anthocyanin content. The apparent specific absorption coefficients of anthocyanins at 550 nm showed no substantial dependence on the species. Anthocyanin contribution to total light absorption at 550 nm was followed in maple leaves in the course of autumn senescence. Photoprotection by vacuolar anthocyanins is discussed with special regard to their distribution within a leaf; radiation screening by anthocyanins predominantly localized in the epidermal cells in A. platanoides and C. avellana leaves was also evaluated.

EFFECT OF NITROGEN STARVATION ON OPTICAL PROPERTIES, PIGMENTS, AND ARACHIDONIC ACID CONTENT OF THE UNICELLULAR GREEN ALGA PARIETOCHLORIS INCISA (TREBOUXIOPHYCEAE, CHLOROPHYTA) 1

Spectral properties of cell suspensions, individual cells, and extracts of the unicellular green alga Parietochloris incisa (Reisigl) Shin Watan. grown under low light were studied. Long‐term nitrogen (N) deprivation resulted in a decrease of chloroplast volume, appearance of numerous large cytoplasmic oil bodies, and the deposition of triacylglycerols with a high proportion of arachidonic acid. Chlorophylls a and b underwent a synchronous decline, whereas carotenoids (Car) showed a relative increase. Simultaneously, significant qualitative changes in the spectral properties of P. incisa individual cells, cell extracts, and cell suspensions were observed. To a large extent, the spectral changes observed in cell suspension could be attributed to a decrease in overall pigment content, leading to a gradual weakening of the so‐called package effect and accumulation of additional amounts of Car over chl, most probably, in oil bodies. Several optical characteristics of cell suspensions could serve as sensitive indicators of N‐deficiency in P. incisa. Furthermore, the absorption ratios, A476/A676 and A650/A676, showed close correlations with the Car‐to‐chl ratio and relative arachidonic acid (AA) content, respectively. The latter makes it possible to suggest that the increase in AA percentage in P. incisa proceeds in parallel with a decrease in cell chl content, accounting for the weakening of the package effect. N‐replenishment resulted in complete recovery of cell optical properties. The possible significance of the changes in cell ultrastructure, pigments, lipids, and optical properties is discussed with special reference to the ability of algae to adapt to and survive under conditions of long‐term nutrient deficiency.

Nondestructive estimation of anthocyanins and chlorophylls in anthocyanic leaves

The anthocyanin and chlorophyll contents in leaves provide valuable information about the physiological status of plants. Thus, there is a need for accurate, efficient, and practical methodologies to estimate these biochemical parameters of vegetation. In this study, we tested the performance and accuracy of several nondestructive, reflectance-based techniques for estimating anthocyanin and chlorophyll contents in leaves of four unrelated species, European hazel (Corylus avellana), Siberian dogwood (Cornus alba =Swida alba), Norway maple (Acer platanoides), and Virginia creeper (Parthenocissus quinquefolia), with widely variable pigment content and composition. An anthocyanin reflectance index, which uses reflectances in the green and red edge spectral bands, and a modified anthocyanin reflectance index, employing, in addition, the near-infrared (NIR) band, were able to accurately estimate leaf anthocyanin for all species taken together with no reparameterization of algorithms. Total chlorophyll content was accurately estimated by a red edge chlorophyll index that uses spectral bands in the red edge and the NIR. These approaches can be used to estimate anthocyanin and chlorophyll nondestructively and allow the development of simple handheld field instrumentation.

Application of Reflectance Spectroscopy for Analysis of Higher Plant Pigments

Nondestructive techniques developed by the authors for assessment of chlorophylls, carotenoids, and anthocyanins in higher plant leaves and fruits are presented. The spectral features of leaf reflectance in the visible and near infrared regions are briefly considered. For pigment analysis only reflectance values at several specific wavelengths are required. The chlorophyll (Chl) content over a wide range of its changes can be assessed during leaf ontogeny using reflectance near 700 nm and, in the absence of anthocyanins, at 550 nm. The approaches used for elimination of Chl interference in the analysis of carotenoids (reflectance at 520 nm) and anthocyanins (at 550 nm) are described. The suitability of reflectance spectroscopy for estimates of carotenoid/chlorophyll ratios during leaf senescence and fruit ripening is demonstrated. The algorithms developed for pigment analysis are presented, and the conditions of their applicability are considered. Further perspectives for the application of reflectance spectroscopy including remote sensing for estimation of plant pigment content and physiological states are discussed.

Does a leaf absorb radiation in the near infrared (780–900 nm) region? A new approach to quantifying optical reflection, absorption and transmission of leaves

The following question is addressed here: do healthy leaves absorb, as the spectra published over the last 50 years indicate, some 5–20% of incident radiation in the 780–900 nm region? The answer is found to be negative, and previous findings result from incomplete collection of the transmitted light by the detection system (even when the leaf is placed next to, but outside, the entrance port of an integrating sphere). A simple remedy for this inherent flaw in the experimental arrangement is applied successfully to leaves (of 10 unrelated species) differing in thickness, age and pigment content. The study has shown that, from an optical standpoint, a leaf tissue is a highly scattering material, and the infinite reflectance of a leaf is exceedingly sensitive to trace amounts of absorbing components. It is shown that water contributes, in a thick leaf (Kalanchoe blossfeldiana), an easily detectable signal even in the 780–900 nm region. The practical benefits resulting from improved measurements of leaf spectra are pointed out.

A Spectrophotometric Analysis of Pigments in Apples

Methods of pigment extraction using traditional polar organic solvents (acetone or methanol) were compared to those employing a chloroform–methanol mixture. We found that, for spectrophotometric pigment analysis in the apple peel, the cuticular lipids must be preliminarily extracted from the samples with chloroform and MgO must be added during homogenization to prevent pigment degradation. The traditional extraction did not result in the complete extraction of intact pigments, and such extracts contained a considerable amount of light-absorbing impurities. The application of chloroform–methanol extraction allowed us to markedly reduce the content of such impurities and to increase the accuracy and sensitivity of the measurement of the content of chlorophylls and carotenoids. In addition, this extraction method proved useful for the analysis of phenolic substances (anthocyanins and flavonoids) in the water-methanol fraction of the extracts.

Pigment composition, optical properties, and resistance to photodamage of the microalga Haematococcus pluvialis cultivated under high light

The relationships between changes in cell suspension absorbance, pigment composition, and resistance to photodamage were investigated in the microalga Haematococcus pluvialis Flotow em. Wille (Chlorophyta) IPPAS H-239 cultivated under high level of photosynthetically active radiation (PAR, 50 W/m2). When the green flagellated cells of H. pluvialis lacking astaxanthin (Ast) and possessing low (<0.5) carotenoid/chlorophyll ratio were irradiated by intense light (2500 W/m2 PAR), rapid and synchronous photobleaching of 70–80% of chlorophyll (Chl) and carotenoids (Car) was observed. By contrast, the rate of pigment photobleaching in cells with Car/Chl > 1, which retained high Chl content (> 0.6 fmol/cell) and accumulated significant amounts of Ast, was two times lower than in the green cells. Red aplanospores, with Car/Chl > 10, containing high amounts of Ast and low amounts of Chl (> 0.8 and < 0.1 fmol/cell, respectively) were resistant to photodestruction. The extent of cell resistance to photobleaching correlated closely with an increase in contribution of Car to light absorption by H. pluvialis cell suspensions. The build up of Ast during acclimation to high light was accompanied by a gradual increase in the optical density ratio OD480/OD678, whereas synchronous (OD480/OD678 ≈ const; r2 > 0.99) and profound (>20%) bleaching of Car and Chl absorption bands was characteristic of photodamage. The spectral features of photoacclimation and photodamage revealed in this work can be used for nondestructive diagnostics of photodamage in H. pluvialis cultures and for on-line assessment of cell resistance to photooxidative death. The results are discussed with respect to the nondestructive monitoring of laboratory and production cultures of H. pluvialis and their protection from photooxidative death.