Effect of anthocyanins, carotenoids, and flavonols on chlorophyll fluorescence excitation spectra in apple fruit: signature analysis, assessment, modelling, and relevance to photoprotection

Whole apple fruit (Malus domestica Borkh.) widely differing in pigment content and composition has been examined by recording its chlorophyll fluorescence excitation and diffuse reflection spectra in the visible and near UV regions. Spectral bands sensitive to the pigment concentration have been identified, and linear models for non-destructive assessment of anthocyanins, carotenoids, and flavonols via chlorophyll fluorescence measurements are put forward. The adaptation of apple fruit to high light stress involves accumulation of these protective pigments, which absorb solar radiation in broad spectral ranges extending from UV to the green and, in anthocyanin-containing cultivars, to the red regions of the spectrum. In ripening apples the protective effect in the blue region could be attributed to extrathylakoid carotenoids. A simple model, which allows the simulation of chlorophyll fluorescence excitation spectra in the visible range and a quantitative evaluation of competitive absorption by anthocyanins, carotenoids, and flavonols, is described. Evidence is presented to support the view that anthocyanins, carotenoids, and flavonols play, in fruit with low-to-moderate pigment content, the role of internal traps (insofar as they compete with chlorophylls for the absorption of incident light in specific spectral bands), affecting thereby the shape of the chlorophyll fluorescence excitation spectrum.     

Patterns of pigment changes in apple fruits during adaptation to high sunlight and sunscald development

Reflectance spectra of four apple (Malus domestica Borkh.) cultivars were studied and chlorophyll, carotenoid, anthocyanin and flavonoid content in sunlit and shaded peel was determined. In all cases sunlit peel accumulated high amounts of phenolics (flavonoid glycosides). Adaptation to strong sunlight of an apple cultivar with limited potential for anthocyanin biosynthesis (Antonovka) was accompanied by a decrease in chlorophyll and a significant increase in total carotenoid content. The increase in carotenoids also took place in sunlit sides of the Zhigulevskoye fruits, accumulating high amounts of anthocyanins, but chlorophyll content in sunlit peel was higher than that in shaded peel. Significant increases in carotenoids and anthocyanins were detected during fruit ripening when chlorophyll content fell below 1.5–1.8 nmol cm^–2. Chlorophyll in sunlit fruit surfaces of both cultivars was considerably more resistant to photobleaching than in shaded (especially of Zhigulevskoye) sides. Induced by sun irradiation, the photoadaptive responses were cultivar-dependent and expressed at different stages of fruit ripening even after storage in darkness. The development of sunscald symptoms in susceptible apple cultivars (Granny Smith and Renet Simirenko) led to a dramatic loss of chlorophylls and carotenoids, which was similar to that observed during artificial photobleaching. The results suggest that apple fruits exhibit a genetically determined strategy of adaptation of their photoprotective pigments to cope with mediated by reactive oxygen species photodynamic activity of chlorophyll under strong solar irradiation. This includes induction of synthesis and accumulation of flavonoids, anthocyanins and carotenoids that could be expressed, if necessary, at different stages of fruit development     

基于高光谱的苹果果期冠层光谱特征及其果量估测

苹果冠层光谱特征是苹果树遥感生理监测和生产管理的重要依据。对栖霞市苹果果期的冠层反射光谱进行实测,结合数码照相技术探明了苹果果期的反射光谱特性和敏感波段,并通过敏感波段与果树比指标建立回归模型,实现了对苹果冠层果量(果树比)的无损估测。结果表明:苹果树果期冠层光谱曲线总体表现为对蓝光和红光的吸收及对绿光的反射,在近红外750—1300nm之间表现为强烈的反射,且在1650nm和2200nm附近呈现两个反射峰。选择435、670、730、940、1140nm和1480nm等6个波段为苹果果期的敏感波段。利用这些波段分别构建了差值、比值及归一化植被指数,筛选了最佳光谱参数,进而构建了果量(果树比)估测模型,经筛选验证确定苹果果量的最佳估测模型为:y=0.0086[NDVI(940,730)]2-1.0934NDVI(940,730)+0.3209。模型为苹果果期果量的精确估测提供了比较快捷的方法途径。     

Light‐induced pigment degradation in leaves and ripening fruits studied in situ with reflectance spectroscopy

Pigment breakdown mediated by activated oxygen species is a consequence and a general symptom of oxidative stress and injury to plants. We have attempted to estimate the patterns of pigment bleaching and follow pigment susceptibility to irradiation as related to the process of senescence/ripening. Light‐induced pigment breakdown was studied in situ in the leaves of a shade‐requiring plant, wax flower ( Hoya carnosa R. Br.), as well as in apple ( Malus domestica Borlh. cv. Zhigulevskoe) and lemon ( Citrus limon Burm. cv. Pavlovsky) fruits, using reflectance spectroscopy. It was found that the sensitivity of plant pigments to photobleaching increases as ripening progresses in lemon fruit. Kinetic analysis showed that in all systems a rapid breakdown of the pigment occurs after a lag‐phase. The signature analysis revealed a common pattern of chlorophyll and carotenoid changes, but degradation of the individual pigments was found to be inhomogeneous. Both in lemon and apple fruits a decrease in reflectance in the band of carotenoid absorption preceded pigment photodestruction. In the fruits, the bulk of chlorophyll b and the long‐wavelength chlorophyll a forms were degraded at early stages of the process whereas the breakdown of both chlorophylls in H. carnosa leaves was more synchronous. Prolonged irradiation induced bleaching of the main chlorophyll a band with maximum at 678 nm in the difference spectra, as well as carotenoids. Some features of reflectance spectra in the bands of chlorophyll and carotenoid absorption were found to be suitable for the differentiation of photo‐induced pigment breakdown from the transformation of the pigments taking place during senescence.     

Apple flavonols during fruit adaptation to solar radiation: spectral features and technique for non-destructive assessment

Spectral properties of flavonols of three varieties (Golden Delicious, Antonovka, and Renet Simirenko) of anthocyanin-free apple fruit were investigated with reflectance spectroscopy. The results of spectral and biochemical analyses suggested that fruit reflectance in a broad spectral range 365-430 nm is strongly dependent on and, in sunlit fruit surfaces, governed by flavonols. The build up of peel flavonols (mainly rutin and other quercetin glycosides) resulted in a dramatic decrease of fruit reflectance in this range, flattening of the spectrum, and extending the region with low reflectance (4-5%) to ca. 410 nm. The spectral features observed suggest that flavonols contribute significantly to screening of excessive radiation, not only UV-A, but in the short-wave bands of chlorophyll and carotenoid absorption in the visible part of the spectrum as well. To retrieve quantitatively flavonol content from reflectance spectra, we tested the applicability of an inversion technique developed for non-destructive leaf pigment assessment. The model for flavonol content assessment was suggested in the form (R(-1)410 - R(-1)460)R800, where Rlambda is reflectance at wavelength lambda. The model was linearly related to flavonol content between 8 and 220nmol/cm2 with the coefficient of determination r2=0.92 and root mean square error of flavonol estimation of 20 nmol/ cm2 regardless of cultivar, chlorophyll, and carotenoid content.     

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.     

Non-destructive optical detection of pigment changes during leaf senescence and fruit ripening

Reflectance spectra in the visible and near infra-red range of the spectrum, acquired for maple ( Acer platanoides L.), chestnut ( Aesculus hippocastanum L.), potato ( Solanum tuberosum L.), coleus ( Coleus blumei Benth.), leaves and lemon (Citrus limon L.) and apple ( Malus domestica Borkh.) fruits were studied. An increase of reflectance between 550 and 740 nm accompanied senescence-induced degradation of chlorophyll (Chl), whereas in the range 400–500 nm it remained low, due to retention of carotenoids (Car). It was found that both leaf senescence and fruit ripening affect the difference between reflectance ( R ) near 670 and 500 nm ( R ₆₇₈− R ₅₀₀), depending on pigment composition. The plant senescing reflectance index in the form ( R ₆₇₈− R ₅₀₀)/ R ₇₅₀ was found to be sensitive to the Car/Chl ratio, and was used as a quantitative measure of leaf senescence and fruit ripening. The changes in the index were followed during leaf senescence, and natural and ethylene-induced fruit ripening. This novel index can be used for estimating the onset, the stage, relative rates and kinetics of senescence/ripening processes.     

苹果类胡萝卜素研究进展

类胡萝卜素是苹果果实色泽形成的一个重要影响因子,其种类和含量决定果实是否具有良好的外观和丰富的营养。本文综述了近年来有关苹果果实类胡萝卜素方面的研究进展,并对苹果类胡萝卜素的种类和含量,苹果发育和贮藏过程中类胡萝卜素含量的变化规律,生物合成途径中相关基因的表达,以及环境因子对类胡萝卜素积累的影响等方面进行了阐述。     

Relationships between carotenoid composition and growth habit in British plant species

The pigment composition of leaves from a number of different plant species collected from field sites in the region of Sheffield, UK, have been compared using high-performance liquid chromatography. Expression of pigment content per unit leaf area was dominated by variation in the total leaf chlorophyll. Neither chlorophyll per unit area nor the chlorophyll a/b ratio were found to be correlated with the habitat from which the plants originated. When the amounts of different carotenoids were expressed relative to the total carotenoid pool, it was found that whilst neither total carotene (α- +β-carotene) nor neoxanthin correlated with ability to grow in shade, the leaf content of both lutein and the total xanthophyll cycle carotenoids (zeaxanthin, anther-axanthin and violaxanthin) did, with lutein content being high in shade species and xanthophyll cycle intermediates low. There was a strong negative correlation between the relative amounts of each of these groups of carotenoids. The ratio of lutein to xanthophyll cycle carotenoids was strongly correlated to an index of shade tolerance.     

PIGMENT AND PHOTOSYNTHETIC RESPONSES OF OSCILLATORIA AGARDHII (CYANOPHYTA) TO PHOTON FLUX DENSITY AND SPECTRAL QUALITY1

The effects of photon flux density (PFD) and spectral quality on biomass, pigment content and composition, and the photosynthetic activity of Oscillatoria agardhii Gomont were investigated in steady-state populations. For alterations of PFD, chemostat populations were exposed to 50, 130 and 230 μmol photons·m^?2·s^?1 of photosynthetic active radiation (PAR). Decreases in biomass, chlorophyll a (Chl a) and c-phycocyanin (CPC) contents, and CPC: Chl a and CPC: carotenoid content was not altered. Increases in the relative abundances of myxoxanthophyll and zeaxanthin and deceases in the relative abundances of echinenone and β-carotene within the carotenoid pigments coincided with increasing PFD. Increases in Chl a-specific photosynthetic rates and maxima and decreases in biomass-specific photosynthetic rates and maxima with increasing PFD were attributed to increased light harvesting by carotenoids per unit Chl a and reduction in total pigment content, respectively. Responses to spectral quality were tested by exposing chemostat populations to a gradient of spectral transmissions at 50 μmol photons·m^?2·s^?1 PAR. Biomass differences among populations were likely attributable to the distinct absorption of the PAR spectrum by Chl a, CPC, and carotenoids. Although pigment contents were not altered by spectral quality, relative abundances of zeaxanthin and echinenone in the carotenoid pigments increased in populations exposed to high-wavelength PAR. The population adapted to green light possessed a greater photosynthetic maximum than populations adapted to other spectral qualities.     

Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution

The Gauss-peak spectra (GPS) method represents individual pigment spectra as weighted sums of Gaussian functions, and uses these to model absorbance spectra of phytoplankton pigment mixtures. We here present several improvements for this type of methodology, including adaptation to plate reader technology and efficient model fitting by open source software. We use a one-step modeling of both pigment absorption and background attenuation with non-negative least squares, following a one-time instrument-specific calibration. The fitted background is shown to be higher than a solvent blank, with features reflecting contributions from both scatter and non-pigment absorption. We assessed pigment aliasing due to absorption spectra similarity by Monte Carlo simulation, and used this information to select a robust set of identifiable pigments that are also expected to be common in natural samples. To test the method’s performance, we analyzed absorbance spectra of pigment extracts from sediment cores, 75 natural lake samples, and four phytoplankton cultures, and compared the estimated pigment concentrations with concentrations obtained using high performance liquid chromatography (HPLC). The deviance between observed and fitted spectra was generally very low, indicating that measured spectra could successfully be reconstructed as weighted sums of pigment and background components. Concentrations of total chlorophylls and total carotenoids could accurately be estimated for both sediment and lake samples, but individual pigment concentrations (especially carotenoids) proved difficult to resolve due to similarity between their absorbance spectra. In general, our modified-GPS method provides an improvement of the GPS method that is a fast, inexpensive, and high-throughput alternative for screening of pigment composition in samples of phytoplankton material.     

Metabolic and gene expression analysis of apple (Malus x domestica) carotenogenesis

Carotenoid accumulation confers distinct colouration to plant tissues, with effects on plant response to light and as well as health benefits for consumers of plant products. The carotenoid pathway is controlled by flux of metabolites, rate-limiting enzyme steps, feed-back inhibition, and the strength of sink organelles, the plastids, in the cell. In apple (Malus × domestica Borkh), fruit carotenoid concentrations are low in comparison with those in other fruit species. The apple fruit flesh, in particular, begins development with high amounts of chlorophylls and carotenoids, but in all commercial cultivars a large proportion of this is lost by fruit maturity. To understand the control of carotenoid concentrations in apple fruit, metabolic and gene expression analysis of the carotenoid pathway were measured in genotypes with varying flesh and skin colour. Considerable variation in both carotenoid concentrations and compound profile was observed between tissues and genotypes, with carotenes and xanthophylls being found only in fruit accumulating high carotenoid concentrations. The study identified potential rate-limiting steps in carotenogenesis, which suggested that the expression of ZISO, CRTISO, and LCY-ε, in particular, were significant in predicting final carotenoid accumulation in mature apple fruit.     

Hyperspectral imaging techniques for rapid identification of Arabidopsis mutants with altered leaf pigment status

The spectral reflectance signature of living organisms provides information that closely reflects their physiological status. Because of its high potential for the estimation of geomorphic biological parameters, particularly of gross photosynthesis of plants, two-dimensional spectroscopy, via the use of hyperspectral instruments, has been widely used in remote sensing applications. In genetics research, in contrast, the reflectance phenotype has rarely been the subject of quantitative analysis; its potential for illuminating the pathway leading from the gene to phenotype remains largely unexplored. In this study, we employed hyperspectral imaging techniques to identify Arabidopsis mutants with altered leaf pigment status. The techniques are comprised of two modes; the first is referred to as the 'targeted mode' and the second as the 'non-targeted mode'. The 'targeted' mode is aimed at visualizing individual concentrations and compositional parameters of leaf pigments based on reflectance indices (RIs) developed for Chls a and b, carotenoids and anthocyanins. The 'non-targeted' mode highlights differences in reflectance spectra of leaf samples relative to reference spectra from the wild-type leaves. Through the latter approach, three mutant lines with weak irregular reflectance phenotypes, that are hardly identifiable by simple observation, were isolated. Analysis of these and other mutants revealed that the RI-based targeted pigment estimation was robust at least against changes in trichome density, but was confounded by genetic defects in chloroplast photorelocation movement. Notwithstanding such a limitation, the techniques presented here provide rapid and high-sensitive means to identify genetic mechanisms that coordinate leaf pigment status with developmental stages and/or environmental stress conditions.     

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.     

CHROMOPLASTS OF TOMATO FRUITS. I. ULTRASTRUCTURE OF LOW-PIGMENT AND HIGH-BETA MUTANTS. CAROTENE ANALYSES

Three pigment lines of the tomato cultivar ‘Pearson’ with isogenic backgrounds were studied to determine the relationship between certain carotenoids and the development of chromoplasts during fruit ripening. The lines were normal red (r⁺/r⁺), in which about 90% of the carotenoids in the ripe fruit is lycopene; high-beta (B/B) mutant, in which beta-carotene is the major pigment and the mature fruit color is deep orange ; and low-pigment (r/r) mutant, in which carotenoids are drastically reduced and the mature fruit is pale yellow-orange. This paper reports pigment analyses for the three lines and the ultrastructural changes in plastids of the two mutant lines. Very young, pale green fruits contain proplastids with limited lamellar structure. As the fruits reach the mature green stage, the plastids in all three lines develop into typical chloroplasts. Differences in pigment content and in ultrastructure among the lines are not apparent until ripening commences. In the low-pigment mutant carotenoids are reduced as ripening progresses and no carotenoid crystalloids are formed. As chlorophyll decreases the fruits become pale yellow. The grana become disorganized and the thylakoids appear to separate at the partitions and tend to be arrayed in lines, some still with their ends overlapping. Globules increase slightly in number. In the high-beta mutant the grana break down during ripening and globules increase greatly in size and number. Beta-carotene, presumed to be largely in the globules, crystallizes into elongated or druse type forms which may distort the globules. The crystals may affect the shape of the chromoplasts; long crystals may extend the length of the plastid to over 15 μ. Thylakoid plexes with a regular lattice structure sometimes occur in the chromoplasts of the high-beta mutant. Granules resembling aggregations of phytoferritin particles occur in the chromoplasts of both of these mutants.     

Properties of pupil mechanisms in owl-fly Ascalaphus macaronius (Neuroptera)

Regulation of light flux by pupil mechanisms in the UV-sensitive superposition eye of owl-fly Ascalaphus macaronius (Neuroptera) was studied with a fast reflection microspectrophotometric technique. The spectral sensitivity of pupil reaction, which was calculated on the basis of changes of transient amplitude reflection, was almost identical with the one of Deilephila eye. This indicates that in spite of different life styles and spectral sensitivities of photoreceptors, pupil closing is triggered by the same photosensitive structure in both eyes. By measuring the spectra of reflected light from the Ascalaphus eye between 400 and 700 nm after different dark periods following light stimulation, it was established that the restoration of reflection was much faster in the red than in the blue spectral range. Based on this, we propose that two different pupil mechanisms with different spectral absorption characteristics are involved in light-flux regulation. Fast-reacting pupil is probably represented by screening pigment migration in the secondary pigment cells and a slow blue-absorbing system by the activity in primary pigment cells. The importance of two different pupils for the photoregeneration of visual pigment is discussed. Accepted: 1 October 1998     

Functional Organization of Chlorophyll a and Carotenoids in the Alga, Nannochloropsis salina

Chlorophyll-protein complexes were isolated from a yellow-green alga, Nannochloropsis salina after mild detergent treatment and gel electrophoresis. Three different complexes were obtained which correspond to the three major kinds of chlorophyll-proteins isolated from spinach chloroplasts by the same procedure and previously identified as reaction center complexes for photosystems I and II and a light-harvesting complex. The analogy between the algal complexes and those from spinach was drawn from their absorption and fluorescence spectra and relative pigment content. The identities and amounts of the major carotenoids associated with each isolated complex were determined by HPLC. Although the reaction center complexes accounted for only 14% of the total chlorophyll, they were highly enriched in β-carotene, whereas the light-harvesting complex contained a high proportion of xanthophylls (mainly violaxanthin and vaucheriaxanthin-ester). Fluorescence excitation spectra of the algal membranes showed that one or both of the major xanthophylls may act as antenna pigment for photosynthesis.     

Reflectance Indices Indicative of Changes in Water and Pigment Contents of Peanut and Wheat Leaves

Measurements of reflectance in visible and near-infrared spectral regions were made on detached leaves of two crop species of different leaf morphology, structure, and water content (peanut and wheat) throughout progressive desiccation. Relative water content (RWC) was well correlated with water index (WI) but even better with the ratio of WI and normalized difference vegetation index. RWC was also significantly correlated with structural independent pigment index indicative of carotenoids/chlorophyll ratio. New indication is thus provided to assess leaf water content and apply simple and fast radiometric techniques for plant water stress management. This revised version was published online in June 2006 with corrections to the Cover Date.