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.     

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.     

植物叶片花青素的光破坏防御机制研究进展

植物花青素广泛分布在植物的根、茎、叶、花和果实等器官中,是植物形态建成过程中或响应逆境而产生的一种次生代谢物质.植物叶片中的花青素具有特殊的化学结构和光谱特性,在光破坏防御机制方面发挥了重要的作用,已经成为植物光合生理生态的研究热点.本文综述了近年来植物叶片花青素与光合作用的研究进展,从叶片花青素的分布、光谱特性及其与光合色素的关系等方面说明花青素对植物光合作用的影响,重点介绍了叶片花青素通过光吸收、抗氧化剂和渗透调节等在植物光破坏防御机制方面的作用,展望了今后的主要研究方向     

冬性植物红菜薹在不同温度处理下花青素积累的分子机制

芸薹属植物红菜薹（Brassica rapa）是一种常见的蔬菜,它的花茎和叶柄表皮中均积累有花青素。为了解红菜薹中花青素合成的分子机制,进行了花青素含量的测定和花青素合成相关基因的表达分析。研究结果表明,叶柄表皮中的花青素含量显著高于叶片（去主脉）的花青素含量。同时,叶柄表皮花青素合成相关基因的表达水平高于叶柄（去表皮）和叶片（去主脉）的表达水平,这表明红菜薹中花青素的合成调控发生在转录水平。BrMYBA1仅在叶柄表皮中表达,但BrbHLH1和BrWD40在叶片和叶柄表皮中均能检测到表达。因此,BrMYBA1的转录激活可能与红菜薹的花青素合成相关。连续低温处理时,红菜薹叶柄表皮中的花青素含量逐渐增加,而该组织中花青素合成的结构基因表达水平逐渐降低。     

Antioxidant activities of red versus green leaves in Elatostema rugosum

Anthocyanin biosynthesis in leaves increases under stresses which also generate reactive oxygen species (ROS). In the present study the hypothesis that red leaves are better equipped to scavenge ROS than green leaves was tested. Antioxidants in leaf extracts from red and green morphs of Elatostema rugosum were identified, and activities quantified using enzymatic and α,α‐diphenyl‐β‐picrylhydrazyl (DPPH) assays and cyclic voltammetry. Red leaves from E. rugosum held greater amounts of superoxide dismutase, catalase, anthocyanins, and hydroxycinnamic acids, were significantly more effective at scavenging DPPH radicals, and produced higher voltammetric currents than green leaves. Anthocyanins contributed to the antioxidant pool more than all other constituent phenolics. Anthocyanin concentrations, and antioxidant activities declined with leaf age. Purified anthocyanin fractions displayed oxidative activities at both pH 7·0 and pH 5·5. Implications of the antioxidant potential of anthocyanin in its cytoplasmic and vacuolar locations are discussed.     

The anti-photooxidation of anthocyanins-rich leaves of a purple rice cultivar

In the leaf of rice (Oryza sativa L.) cultivar Yunnan purple rice,the anthocyanins with an obvious absorption peak at 530nm were distributed in the cells of upper and lower epidermis,bulliform tissue and bristle. The maximal photosynthetic oxygen evolution rate and chlorophyll content in flag leaves were 28% and 23%,respectively,more than the common green leaf rice cultivar Chijiaoru-anzhan. Higher chlorophyll content is probably one of the physiological adaptations for enhancing light harvesting capacity of the antenna in photosystems in this cyanic leaves species. Upon the photooxidation of leaf segments mediated by methyl viologen in weak light for 3 days,the distinct bleaching of anthocyanins in purple rice was associated with the reduction of scavenging ability to DPPH· free radical ability and the increase in membrane leakage rate. But almost no changes in contents of flavonoids and total phenolics were observed. Chlorophyll fluorescence parameters Fv/Fo,qP and φPSⅡ decreased with the increase in NPQ and DES of xanthophylls cycle after photooxidation treatment. Green rice leaves showed more decrease in DPPH· scavenging rate and more increase in cell membrane leakage rate but showed a trace of anthocyanins during photooxidation. It is sug-gested that anthocyanin may be a beneficial and primary antioxidant in sun cyanic rice leaves against oxidative stress induced by environmental adversity. And photooxidation could induce different changing patterns of anthocyanins between the tested purple and green rice leaves.     

The anti-photooxidation of anthocyanins-rich leaves of a purple rice cultivar

In the leaf of rice (Oryza sativa L.) cultivar Yunnan purple rice, the anthocyanins with an obvious absorption peak at 530nm were distributed in the cells of upper and lower epidermis, bulliform tissue and bristle. The maximal photosynthetic oxygen evolution rate and chlorophyll content in flag leaves were 28% and 23%, respectively, more than the common green leaf rice cultivar Chijiaoruanzhan. Higher chlorophyll content is probably one of the physiological adaptations for enhancing light harvesting capacity of the antenna in photosystems in this cyanic leaves species. Upon the photooxidation of leaf segments mediated by methyl viologen in weak light for 3 days, the distinct bleaching of anthocyanins in purple rice was associated with the reduction of scavenging ability to DPPH · free radical ability and the increase in membrane leakage rate. But almost no changes in contents of flavonoids and total phenolics were observed. Chlorophyll fluorescence parameters Fv/Fo, qP and ϕ_PSII decreased with the increase in NPQ and DES of xanthophylls cycle after photooxidation treatment. Green rice leaves showed more decrease in DPPH · scavenging rate and more increase in cell membrane leakage rate but showed a trace of anthocyanins during photooxidation. It is suggested that anthocyanin may be a beneficial and primary antioxidant in sun cyanic rice leaves against oxidative stress induced by environmental adversity. And photooxidation could induce different changing patterns of anthocyanins between the tested purple and green rice leaves.     

Red light stimulates flowering and anthocyanin biosynthesis in American cranberry

Morphological responses of American cranberry (Vacciniummacrocarpon Ait, Ericaceae) to different light conditions (red,far-red, white light and sunlight) were examined. Root growth and development,stem elongation, leaf enlargement, de-etiolation of stem and leaf, flower budformation, and flowering of American cranberry were measured under each lightcondition and in the dark. It was found that red light promotes development ofroots and leaves, flowering, and de-etiolation of stem and leaf of Americancranberry. Stem elongation and etiolation of stem and leaf were shown infar-redlight and dark. Anthocyanin biosynthesis as phytochemical response in cranberryplants was most sensitive to red light. Estimation of anthocyanin levels indifferent parts of cranberry plant suggested that anthocyanins were presentonlyin red fruit skins, and not in peeled fruits, green fruits, green leaves, greenstems, roots and seeds.     

A field study with geometrid moths to test the coevolution hypothesis of red autumn colours in deciduous trees

Red autumn colouration of trees is the result of newly synthesized anthocyanin pigments in senescing autumn leaves. As anthocyanin accumulation is costly and the trait is not present in all species, anthocyanins must have an adaptive significance in autumn leaves. According to the coevolution hypothesis of autumn colours, red autumn leaves warn herbivorous insects – especially aphids that migrate to reproduce in trees in the autumn – that the tree will not be a suitable host for their offspring in spring due to a high level of chemical defence or lack of nutrients. The signalling allows trees to avoid herbivores and herbivores to choose better host trees. In this study the coevolution hypothesis was tested with four deciduous tree species that have red autumn leaf colouration – European aspen (Populus tremula L.) (Salicaceae), rowan (Sorbus aucuparia L.) (Rosaceae), mountain birch [Betula pubescens ssp. czerepanovii (NI Orlova) Hämet‐Ahti], and dwarf birch (Betula nana L.) (Betulaceae), and with two generalist herbivores, the autumnal moth [Epirrita autumnata (Borkhausen)] and the winter moth [Operophtera brumata (L.)] (both Lepidoptera: Geometridae). Anthocyanin concentrations of autumn leaves were determined from leaf samples and the growth performance parameters of the moth larvae on the study trees were measured in the spring. Trees with higher anthocyanin concentration in the autumn were predicted to be low‐quality food for the herbivores. Our results clearly showed that anthocyanin concentration was not correlated with the growth performance of the moths in any of the studied tree species. Thus, our study does not support the coevolution hypothesis of autumn colours.     

Functional role of anthocyanins in high-light winter leaves of the evergreen herb Galax urceolata

High-light leaves of the evergreen herb Galax urceolata exhibit a striking color change from green to red during winter months due to anthocyanin synthesis in outermost mesophyll cells. Here we investigate three possible functions of this color change. To test the hypothesis that anthocyanins function as light attenuators, maximum photosystem II efficiency (F(v)/F(m)) of red and green leaves was measured during and after exposure to wavelengths either strongly or poorly absorbed by anthocyanin. To determine whether anthocyanins elevate radical-scavenging capacity, antioxidant activity of red and green leaves was assessed using the alpha,alpha-diphenyl-beta-picrylhydrazyl assay. Nonstructural carbohydrate levels were analyzed to test the hypothesis that anthocyanins function as a carbon sink. Declines in F(v)/F(m) under white and green light were significantly greater for green than red leaves, but were comparable under red light. Anthocyanin content positively correlated with antioxidant activity. Although levels of anthocyanins did not appear to be related to nonstructural carbohydrate concentration, high levels of sugars may be necessary for their photo-induction. Results suggest that anthocyanins function as light attenuators and may also contribute to the antioxidant pool in winter leaves.     

Xanthophyll-cycle dynamics and rapid induction of anthocyanin synthesis in Eucalyptus nitens seedlings transferred to photoinhibitory conditions

The effects of rapid induction and subsequent relaxation of cold-induced photo-inhibition on pigment chemistry of Eucalyptus nitens (Deane and Maiden) Maiden seedling leaves was assessed. The seedlings were subjected to four treatments in a nursery, fertilised or non-fertilised and shaded or non-shaded, before induction of photoinhibition in a growth chamber. Within 2 days, growth chamber conditions decreased photochemical efficiency (Fv/Fm) and increased xanthophyll-cycle conversion ratio. This was associated with decreased levels of total chlorophyll and increased levels of xanthophyll-cycle pigments. After 8 days, Fv/Fm of all treatments rose. Anthocyanin Levels gradually increased until day eight, except in non-shaded, non-fertilised seedlings, which had high levels before the induction of photoinhibition. Visible spectroscopy indicated increased absorption between 500 and 590nm, indicative of anthocyanin absorption. Electron transport rate after xanthophyll-cycle relaxation was half that measured before induction of photoinhibition. The findings indicate that anthocyanins may provide a photoprotective role in E. nitens seedlings under conditions of photoinhibition.     

Anthocyanin contribution to chlorophyll meter readings and its correction

Leaf chlorophyll content is an important physiological parameter which can serve as an indicator of nutritional status, plant stress or senescence. Signals proportional to the chlorophyll content can be measured non-destructively with instruments detecting leaf transmittance (e.g., SPAD-502) or reflectance (e.g., showing normalized differential vegetation index, NDVI) in red and near infrared spectral regions. The measurements are based on the assumption that only chlorophylls absorb in the examined red regions. However, there is a question whether accumulation of other pigments (e.g., anthocyanins) could in some cases affect the chlorophyll meter readings. To answer this question, we cultivated tomato plants (Solanum lycopersicum L.) for a long time under low light conditions and then exposed them for several weeks (4 h a day) to high sunlight containing the UV-A spectral region. The senescent leaves of these plants evolved a high relative content of anthocyanins and visually revealed a distinct blue color. The SPAD and NDVI data were collected and the spectra of diffusive transmittance and reflectance of the leaves were measured using an integration sphere. The content of anthocyanins and chlorophylls was measured analytically. Our results show that SPAD and NDVI measurement can be significantly affected by the accumulated anthocyanins in the leaves with relatively high anthocyanin content. To describe theoretically this effect of anthocyanins, concepts of a specific absorbance and a leaf spectral polarity were developed. Corrective procedures of the chlorophyll meter readings for the anthocyanin contribution are suggested both for the transmittance and reflectance mode.     

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.     

Attenuation of incident light in Galax urceolata (Diapensiaceae): concerted influence of adaxial and abaxial anthocyanic layers on photoprotection

Although anthocyanin coloration in lower (abaxial) leaf cells has been documented for numerous species, the functional significance of this character has not been comprehensively investigated according to habitat or leaf orientation. Here, we demonstrate that abaxial anthocyanin may function as a photoprotectant, similarly to its purported role in upper (adaxial) cells, in leaves vulnerable to high irradiance incident on abaxial surfaces. Spectral scans were derived for Galax urceolata leaves with the following phenotypes: abaxial or adaxial anthocyanin only, abaxial and adaxial anthocyanin, and no anthocyanin. To determine whether anthocyanins conferred protection from photoinhibition, maximum photosystem II efficiencies of red (anthocyanic) and green (acyanic) surfaces were compared during and after exposure to photoinhibitory conditions. Leaves were either positioned with their adaxial surfaces facing the light source or inverted to expose abaxial surfaces. Spectral scans showed increased absorptance of 500-600 nm wavelengths by red surfaces (consistent with the absorbance spectrum of anthocyanin), regardless of whether that surface was abaxial or adaxial. Leaves with anthocyanin in either illuminated surface were also photoinhibited less than leaves lacking anthocyanin in that surface. These results suggest that anthocyanic layers reduce absorbed sunlight in the mesophyll not only for adaxial surfaces, but also for the abaxial. Adaxial/abaxial anthocyanin plasticity may therefore be adaptive in high-light environments or during light-sensitive developmental stages where leaf orientation and/or substrate albedo are variable.     

Association of red coloration with senescence of sugar maple leaves in autumn

We evaluated the association of red coloration with senescence in sugar maple (Acer saccharum Marsh.) leaves by assessing differences in leaf retention strength and the progression of the abscission layer through the vascular bundle of green, yellow, and red leaves of 14 mature open-grown trees in October 2002. Computer image analysis confirmed visual categorization of leaves as predominantly green, yellow or red, and chemical quantification of leaf pigment concentrations verified that leaf color reflected underlying differences in leaf biochemistry. Significantly lower chlorophyll concentrations within red and yellow leaves indicated that senescence was more advanced in leaves from these color categories relative to green leaves. Among leaf types, only red leaves contained high concentrations of anthocyanins. There were significant differences in leaf retention capacity among color categories, with the petioles of green leaves being the most firmly attached to twigs, followed by red and then yellow leaves. Microscopic analysis indicated that yellow leaves had the most advanced extension of the abscission layer through the vasculature, with green and red leaves having significantly less abscission layer progression than yellow. A more limited progression of the abscission layer through vascular bundles may be evidence of delayed leaf senescence that could extend resorption of mobile leaf constituents. Together, results from this study suggest an association between leaf anthocyanin content and functional delays in senescence.     

Osservazioni Sulla Germinazione Asimbiotica e Simbiotica di Alcune Orchidee

Abstract

Anthocyanins are secondary metabolites, which play important roles in the physiology of plants. In tomato (Solanum lycopersicum L.), anthocyanins are normally synthesized only in vegetative tissues. M375 is a mutant unable to produce anthocyanins in leaves and stems. In this study, we investigated the anthocyanin biosynthetic pathway in M375 and in its genetic background, Alice, in order to find out where the anthocyanin biosynthesis is blocked, along the pathway, in the mutant. Anthocyanins accumulation was enhanced by sucrose only in the wild type, even though the expression of several genes involved in anthocyanin biosynthesis was normal in both the genotypes. Genes coding for the final steps along the anthocyanin biosynthetic pathway were, however, less expressed in the M375 when compared to the wild type.     

Anthocyanin inheritance and instability in purple basil (Ocimum basilicum L.)

The instability of the purple pigments (anthocyanins) in purple basil varieties (Ocimum basilicum L.) limits their use as ornamental plants and as a potential anthocyanin source. Several self-pollinated generations of all purple plants were unsuccessful in stabilizing anthocyanin expression. In this study we investigated the inheritance and stability patterns of leaf traits using the Purple Ruffles variety. The results from the complete diallele crosses indicated anthocyanin expression in vegetative tissue is controlled by two dominant genes and ruffled leaf texture is controlled by a single recessive gene. Genes controlling leaf margin and leaf base structures were tightly linked to leaf texture. Essential oil production and oil constituents in leaves did not change as a result of the reversion in color. Color stability in cuttings was affected by the environment and the location where cuttings were taken. An accumulation of secondary metabolites (apigenin, genistein, and kaempferol) in green-reverted sectors on purple leaves was detected using reverse-phase high-performance liquid chromatography (HPLC) analysis; this suggested a potential block in the anthocyanin pathway. We hypothesize the reversion mutation is occurring in an anthocyanin regulatory gene.     

The ecophysiology of foliar anthocyanin

The accumulation of foliar anthocyanins can be consistently attributed to a small range of contexts. Foliar anthocyanin accumulates in young, expanding foliage, in autumnal foliage of deciduous species, in response to nutrient deficiency or ultraviolet (UV) radiation exposure, and in association with damage or defense against browsing herbivores or pathogenic fungal infection. A common thread through these causative factors is low photosynthetic capacity of foliage with accumulated anthocyanin relative to leaves at different ontogenetic stages or unaffected by the environmental factor in question. The ecophysiological function of anthocyanin has been hypothesized as: 1) a compatible solute contributing to osmotic adjustment to drought and frost stress; 2) an antioxidant; 3) a UV protectant; and 4) protection from visible light. Review of the internal leaf distribution of anthocyanin, of experimental evidence using seedlings, and of studies that directly investigated light absorption by anthocyanin and its development relative to recognized processes of photoprotection support the hypothesis that anthocyanins provide protection from visible light.     

Nuclear magnetic resonance study of spin relaxation and magnetic field gradients in maple leaves.

1H Nuclear magnetic resonance techniques were used to measure the distributions of spin-spin relaxation times, T2, and of magnetic field gradients in both the chloroplast and nonchloroplast water compartments of maple leaves (Acer platanoides). Results showed that encounters between water molecules and membranes inside chloroplasts provide an inefficient relaxation mechanism; i.e., chloroplast membranes interact weakly with water molecules. Gradient measurements indirectly measured the sizes of chloroplasts by showing that water in the chloroplasts is confined to small compartments a few microns in diameter. A comparison between measured gradients and gradients calculated for a model leaf indicated that chloroplasts are somewhat more likely to occupy positions along cell walls adjacent to air spaces, but also they may be found in the interiors of cells.