Leaf Xanthophyll Research Articles

Generation and physiological characterization of genome-edited Nicotiana benthamiana plants containing zeaxanthin as the only leaf xanthophyll

Main conclusionSimultaneous genome editing of the two homeologousLCYeandZEPgenes ofNicotiana benthamianaresults in plants in which all xanthophylls are replaced by zeaxanthin.Plant carotenoids act both as photoreceptors and photoprotectants in photosynthesis and as precursors of apocarotenoids, which include signaling molecules such as abscisic acid (ABA). As dietary components, the xanthophylls lutein and zeaxanthin have photoprotective functions in the human macula. We developed transient and stable combinatorial genome editing methods, followed by direct LC–MS screening for zeaxanthin accumulation, for the simultaneous genome editing of the two homeologous Lycopene Epsilon Cyclase (LCYe) and the two Zeaxanthin Epoxidase (ZEP) genes present in the allopolyploid Nicotiana benthamiana genome. Editing of the four genes resulted in plants in which all leaf xanthophylls were substituted by zeaxanthin, but with different ABA levels and growth habits, depending on the severity of the ZEP1 mutation. In high-zeaxanthin lines, the abundance of the major photosystem II antenna LHCII was reduced with respect to wild-type plants and the LHCII trimeric state became unstable upon thylakoid solubilization. Consistent with the depletion in LHCII, edited plants underwent a compensatory increase in PSII/PSI ratios and a loss of the large-size PSII supercomplexes, while the level of PSI-LHCI supercomplex was unaffected. Reduced activity of the photoprotective mechanism NPQ was shown in high-zeaxanthin plants, while PSII photoinhibition was similar for all genotypes upon exposure to excess light, consistent with the antioxidant and photoprotective role of zeaxanthin in vivo.

Modulation of photosynthetic energy conversion efficiency in nature: from seconds to seasons

Modulation of the efficiency with which leaves convert absorbed light to photochemical energy [intrinsic efficiency of open photosystem II (PSII) centers, as the ratio of variable to maximal chlorophyll fluorescence] as well as leaf xanthophyll composition (interconversions of the xanthophyll cycle pigments violaxanthin and zeaxanthin) were characterized throughout single days and nights to entire seasons in plants growing naturally in contrasting light and temperature environments. All pronounced decreases of intrinsic PSII efficiency took place in the presence of zeaxanthin. The reversibility of these PSII efficiency changes varied widely, ranging from reversible-within-seconds (in a vine experiencing multiple sunflecks under a eucalypt canopy) to apparently permanently locked-in for entire seasons (throughout the whole winter in a subalpine conifer forest at 3,000m). While close association between low intrinsic PSII efficiency and zeaxanthin accumulation was ubiquitous, accompanying features (such as trans-thylakoid pH gradient, thylakoid protein composition, and phosphorylation) differed among contrasting conditions. The strongest and longest-lasting depressions in intrinsic PSII efficiency were seen in the most stress-tolerant species. Evergreens, in particular, showed the most pronounced modulation of PSII efficiency and thermal dissipation, and are therefore suggested as model species for the study of photoprotection. Implications of the responses of field-grown plants in nature for mechanistic models are discussed.

Functional integration of non-native carotenoids into chloroplasts by viral-derived expression of capsanthin-capsorubin synthase in Nicotiana benthamiana.

The biosynthesis of leaf carotenoids in Nicotiana benthamiana was altered by forced re-routing of the pathway to the synthesis of capsanthin, a non-native chromoplast-specific xanthophyll, using an RNA viral vector containing capsanthin-capsorubin synthase (Ccs) cDNA. The cDNA encoding Ccs was placed under the transcriptional control of a tobamovirus subgenomic promoter. Leaves from transfected plants expressing Ccs developed an orange phenotype and accumulated high levels of capsanthin (up to 36% of total carotenoids). This phenomenon was associated with thylakoid membrane distortion and reduction of grana stacking. In contrast to the situation prevailing in chromoplasts, capsanthin was not esterified and its increased level was balanced by a concomitant decrease of the major leaf xanthophylls, suggesting an autoregulatory control of chloroplast carotenoid composition. Capsanthin was exclusively recruited into the trimeric and monomeric light-harvesting complexes of photosystem II (PSII) and shown to significantly contribute to the light-harvesting capacity. On a chlorophyll basis, the concentrations of PSI and PSII reaction centres were not modified. This demonstration that higher plant antenna complexes can accommodate non-native carotenoids provides compelling evidence for functional remodelling of photosynthetic membranes toward a better photoreactivity by rational design of the incorporated carotenoid structures.

Influence of solar radiation and leaf angle on leaf xanthophyll concentrations in mangroves.

Mangroves have similar xanthophyll cycle components/chlorophyll ratios [i.e. (V+A+Z)/chl] to other plant species. (V+A+Z)/chl ratios were sensitive to the light environment in which leaves grew, decreasing as light levels decreased over a vertical transect through a forest canopy. The (V+A+Z)/chl ratio also varied among species. However, in sun leaves over all species, the (V+A+Z)/chl ratios correlate with the proportion of leaf area displayed on a horizontal plane, which is determined by leaf angle. Thus, leaf angle and the xanthophyll cycle may both be important in providing protection from high light levels in mangrove species. A canopy survey assessed whether (V+A+Z)/chl ratios could be correlated with species dominance of exposed positions in forest canopies.Rhizophora mangroves, with near-vertical leaf angles, andBruguiera parviflora, with small, horizontal, xanthophyllrich leaves, dominated the canopy, whileB. gymnorrhiza, a species with large, horizontally arranged leaves, was less abundant at the top of the canopy. Thus, two different strategies for adapting to high solar radiation levels may exist in these species. The first strategy is avoidance through near vertical leaf angles, and the second is a large capacity to dissipate energy through zeaxanthin. The (V+A+Z)/chl ratio was also negatively correlated with the epoxidation state of the xanthophyll cycle pool (the proportion present as violaxanthin and half that present as antheraxanthin) at midday. This suggested that the requirement for dissipation of excess light (represented by the midday epoxidation state) may influence the (V+A+Z)/chl ratio.

Leaf Xanthophyll content and composition in sun and shade determined by HPLC

As a part of our investigations to test the hypothesis that zeaxanthin formed by reversible de-epoxidation of violaxanthin serves to dissipate any excessive and potentially harmful excitation energy we determined the influence of light climate on the size of the xanthophyll cycle pool (violaxanthin + antheraxanthin + zeaxanthin) in leaves of a number of species of higher plants. The maximum amount of zeaxanthin that can be formed by de-epoxidation of violaxanthin and antheraxanthin is determined by the pool size of the xanthophyll cycle. To quantitate the individual leaf carotenoids a rapid, sensitive and accurate HPLC method was developed using a non-endcapped Zorbax ODS column, giving baseline separation of lutein and zeaxanthin as well as of other carotenoids and Chl a and b.The size of the xanthophyll cycle pool, both on a basis of light-intercepting leaf area and of light-harvesting chlorophyll, was ca. four times greater in sun-grown leaves of a group of ten sun tolerant species than in shade-grown leaves in a group of nine shade tolerant species. In contrast there were no marked or consistent differences between the two groups in the content of the other major leaf xanthophylls, lutein and neoxanthin. Also, in each of four species examined the xanthophyll pool size increased with an increase in the amount of light available during leaf development whereas there was little change in the content of the other xanthophylls. However, the α-carotene/β-carotene ratio decreased and little or no α-carotene was detected in sun-grown leaves. Among shade-grown leaves the α-carotene/β-carotene ratio was considerably higher in species deemed to be umbrophilic than in species deemed to be heliophilic.The percentage of the xanthophyll cycle pool present as violaxanthin (di-epoxy-zeaxanthin) at solar noon was 96-100% for shade-grown plants and 4-53% for sun-grown plants with zeaxanthin accounting for most of the balance. The percentage of zeaxanthin in leaves exposed to midday solar radiation was higher in those with low than in those with high photosynthetic capacity.The results are consistent with the hypothesis that the xanthophyll cycle is involved in the regulation of energy dissipation in the pigment bed, thereby preventing a buildup of excessive excitation energy at the reaction centers.

Xanthophylls and Abscisic Acid Biosynthesis in Water-Stressed Bean Leaves

Experiments were designed to obtain evidence about the possible role of xanthophylls as abscisic acid (ABA) precursors in water-stressed leaves of Phaseolus vularis L. Leaves were exposed to (14)CO(2) and the specific activities of several major leaf xanthophylls and stress-induced ABA were determined after a chase in (12)CO(2) for varying periods of time. The ABA specific radioactivities were about 30 to 70% of that of lutein and violaxanthin regardless of the chase period. The specific activity of neoxanthin, however, was only about 15% of that of ABA. The effects of fluridone on xanthophyll and ABA levels and the extent of labeling of both from (14)CO(2) were determined. Fluridone did not inhibit the accumulation of ABA when leaves were stressed once, although subsequent stresses in the presence of fluridone did lead to a reduced ABA accumulation. The incorporation of (14)C from (14)CO(2) into ABA and the xanthophylls was inhibited by fluridone and to about the same extent. The incorporation of (18)O into ABA from violaxanthin which had been labeled in situ by means of the violaxanthin cycle was measured. The results indicated that a portion of the ABA accumulated during stress was formed from violaxanthin which had been labeled with (18)O. The results of these experiments are consistent with a preformed xanthophyll(s) as the major ABA precursor in water-stressed bean leaves.

Variation in the xanthophyll and carotene content of lucerne, clovers and grasses

AbstractThe xanthophyll and carotene contents of eleven lucerne clones were found to be higher during September and November than during June and July. Both carotenoids reached a maximum at or before first flowering and then declined as blossoming progressed. Lutein decreased in November while violaxanthin increased. Six grasses and four legumes were analysed for the principal leaf xanthophylls, lutein, violaxanthin and neoxanthin, as well as total xanthophyll and carotene.

Biosynthetic origin of oxygen in the leaf xanthophylls

Isotope enrichment was studied in the xanthophylls from Chlorella vulgaris grown in the dark and in the presence of either molecular oxygen or water enriched with O 18. All three leaf xanthophylls—lutein, violaxanthin, and neoxanthin—incorporate oxygen from the atmosphere. Oxygen from water was incorporated by neoxanthin but not detectably by lutein. The pattern of incorporation suggests that O 2 is utilized for the hydroxyl groups and H 2O for the epoxides.

XANTHOPHYLLS AND THE HILL REACTION*

SummaryThe incorporation of oxygen from water enriched in H218O into the leaf xanthophylls during the Hill reaction was investigated. No enrichment was detectable in violaxanthin and neoxanthin. A low incorporation into lutein, which was not dependent on the Hill reaction, was observed.The results indicate that the epoxy pigments do not function as an oxygen transport system in photosynthesis under the conditions of these experiments.

Extracted herbage leaf protein for poultry feeding I. Introduction and feeding trial with laying hens

1. The extraction of leaf protein from autumn-grown perennial ryegrass is described.2. The composition, chemical analysis and rate of feeding of two mashes compared in the experiment are given. The same basal cereal mixture was used in each ration, with fish meal added to one and leaf protein and minerals to the other.3. Two groups of three birds were kept on each ration. Nine-month-old Brown Leghorn × Light Sussex pullets, in full lay, were used.4. The birds were given a 6 weeks' preliminary period on a uniform ration to accustom them to the experimental conditions and to ascertain their potential egg production.5. In a 10-week feeding trial no difference occurred in the number of eggs or the total weight of eggs laid by the birds fed on either ration.6. A small loss of body weight occurred in the birds in both treatments.7. Egg-quality determinations indicated that the specific gravity of the eggs was not affected by the differential feeding. Yolk height was greater in eggs from birds fed on the ration containing fish meal. This ration also resulted in a higher number of eggs with rough shells and containing a higher content of foreign bodies.8. The eggs from both treatments had low ‘Yolk-colour indices’, showing that the leaf xanthophylls were not present in quantities in the leaf-protein extract.

Improved Methods for the Purification of the Common Carotenoids, and the Quantitative Measurement of Their Absorption Spectra

1. Simple improved methods for the purification of three of the common carotenoids have been developed. The methods of Tswett and Escher have been combined for the purification of the carotene isomers. The methods are described in detail. 2. A critical discussion of chromatographic analysis has been made. The necessary precautions are listed. 3. The quantitative absorption spectra of alpha and beta carotene, leaf xanthophyll, and lycopene were measured with an error of 2.0 per cent or less between 3950 and 5500 A. units. These spectra were measured by a spectro-photoelectric method which is accurate and precise. 4. Spectroscopic data show that certain tests of purity which have been considered accurate are only approximate. It is concluded that a critical examination of the absorption spectrum is the most reliable. 5. The absorption spectra of the carotenoids are employed as the basis for quantitative spectral analytical methods which have been applied to binary mixtures. 6. The preparative procedure incl...

A Rapid and Accurate Quantitative Method for the Determination of the Common Carotenoids; Analyses of Beta-Carotene and Leaf Xanthophyll in Thirteen Plant Tissues1

A Rapid and Accurate Quantitative Method for the Determination of the Common Carotenoids; Analyses of Beta-Carotene and Leaf Xanthophyll in Thirteen Plant Tissues¹