Antheraxanthin

Integrative Metabolomic and Transcriptomic Analyses Reveal the Mechanism of Petal Blotch Formation in Rosa persica.[Pubmed:38612838]

Int J Mol Sci. 2024 Apr 4;25(7):4030.

Petal blotch is a specific flower color pattern commonly found in angiosperm families. In particular, Rosa persica is characterized by dark red blotches at the base of yellow petals. Modern rose cultivars with blotches inherited the blotch trait from R. persica. Therefore, understanding the mechanism for blotch formation is crucial for breeding rose cultivars with various color patterns. In this study, the metabolites and genes responsible for the blotch formation in R. persica were identified for the first time through metabolomic and transcriptomic analyses using LC-MS/MS and RNA-seq. A total of 157 flavonoids were identified, with 7 anthocyanins as the major flavonoids, namely, cyanidin 3-O-(6''-O-malonyl) glucoside 5-O-glucoside, cyanidin-3-O-glucoside, cyanidin 3-O-galactoside, cyanidin O-rutinoside-O-malonylglucoside, pelargonidin 3-O-glucoside, pelargonidin 3,5-O-diglucoside, and peonidin O-rutinoside-O-malonylglucoside, contributing to pigmentation and color darkening in the blotch parts of R. persica, whereas carotenoids predominantly influenced the color formation of non-blotch parts. Zeaxanthin and Antheraxanthin mainly contributed to the yellow color formation of petals at the semi-open and full bloom stages. The expression levels of two 4-coumarate: CoA ligase genes (Rbe014123 and Rbe028518), the dihydroflavonol 4-reductase gene (Rbe013916), the anthocyanidin synthase gene (Rbe016466), and UDP-flavonoid glucosyltransferase gene (Rbe026328) indicated that they might be the key structural genes affecting the formation and color of petal blotch. Correlation analysis combined with weighted gene co-expression network analysis (WGCNA) further characterized 10 transcription factors (TFs). These TFs might participate in the regulation of anthocyanin accumulation in the blotch parts of petals by modulating one or more structural genes. Our results elucidate the compounds and molecular mechanisms underlying petal blotch formation in R. persica and provide valuable candidate genes for the future genetic improvement of rose cultivars with novel flower color patterns.

Valorization of Nannochloropsis oceanica for integrated co-production of violaxanthin cycle carotenoids.[Pubmed:38493940]

Bioresour Technol. 2024 May;399:130597.

The development of integrated co-production of multiple high-purity carotenoids from microalgal cells holds considerable significance for the valorization of microalgae. In this study, the economical microalga Nannochloropsis oceanica was identified as an accumulator of violaxanthin cycle carotenoids, including violaxanthin, Antheraxanthin, and zeaxanthin. Notably, a novel and competent approach for the integrated co-production of violaxanthin cycle carotenoids was explored, encompassing four steps: microalgal cultivation, solvent extraction, octadecylsilyl open-column chromatography, and ethanol precipitation. Under optimal co-production conditions, the purities of the obtained violaxanthin, Antheraxanthin, and zeaxanthin all exceeded 92%, with total recovery rates of approximately 51%, 40%, and 60%, respectively. Utilizing nuclear magnetic resonance techniques, the purified violaxanthin, Antheraxanthin, and zeaxanthin were identified as all-trans-violaxanthin, all-trans-Antheraxanthin, and all-trans-zeaxanthin, respectively. This method held significance for the multiproduct biorefinery of the microalga N. oceanica and carried potential future implications for the violaxanthin cycle carotenoids.

Integrated Metabolomic and Transcriptomic Profiles Provide Insights into the Mechanisms of Anthocyanin and Carotenoid Biosynthesis in Petals of Medicago sativa ssp. sativa and Medicago sativa ssp. falcata.[Pubmed:38475545]

Plants (Basel). 2024 Feb 29;13(5):700.

The petals of Medicago sativa ssp. sativa and M. sativa ssp. falcata are purple and yellow, respectively. Free hybridization between M. sativa ssp. sativa and M. sativa ssp. falcata has created hybrids with various flower colors in nature. Moreover, the flower colors of alfalfa are closely correlated with yield, nutritional quality, stress tolerance and other agronomic characteristics. To elucidate the underlying mechanisms of flower color formation in M. sativa ssp. sativa and M. sativa ssp. falcata, we conducted an integrative analysis of the transcriptome and metabolome of alfalfa with three different petal colors (purple, yellow and cream). The metabolic profiles suggested that anthocyanins and carotenoids are the crucial pigments in purple and yellow flowers, respectively. A quantitative exploration of the anthocyanin and carotenoid components indicated that the accumulations of cyanidin, delphinidin, peonidin, malvidin, pelargonidin and petunidin derivatives are significantly higher in purple flowers than in cream flowers. In addition, the content of carotenes (phytoene, alpha-carotene and beta-carotene) and xanthophylls (alpha-cryptoxanthin, lutein, beta-cryptoxanthin, zeaxanthin, Antheraxanthin and violaxanthin derivatives) was markedly higher in yellow flowers than in cream flowers. Furthermore, we found that delphinidin-3,5-O-diglucoside and lutein were the predominant pigments accumulated in purple and yellow flowers, respectively. The transcriptomic results revealed that twenty-five upregulated structural genes (one C4H, three 4CL, twelve CHS, two CHI, one F3H, one F3'H, one F3'5'H and four DFR) are involved in the accumulation of anthocyanins in purple flowers, and nine structural genes (two PSY, one ZDS, two CRTISO, two BCH, one ZEP and one ECH) exert an effect on the carotenoid biosynthesis pathway in yellow flowers. The findings of this study reveal the underlying mechanisms of anthocyanin and carotenoid biosynthesis in alfalfa with three classic flower colors.

Tropical bloom-forming mesoalgae Cladophoropsis sp. and Laurencia sp.-responses to ammonium enrichment and a simulated heatwave.[Pubmed:38402562]

J Phycol. 2024 Apr;60(2):554-573.

Algal blooms are increasing worldwide, driven by elevated nutrient inputs. However, it is still unknown how tropical benthic algae will respond to heatwaves, which are expected to be more frequent under global warming. In the present study, a multifactorial experiment was carried out to investigate the potential synergistic effects of increased ammonium inputs (25 muM, control at 2.5 muM) and a heatwave (31 degrees C, control at 25 degrees C) on the growth and physiology (e.g., ammonium uptake, nutrient assimilation, photosynthetic performance, and pigment concentrations) of two bloom-forming algal species, Cladophoropsis sp. and Laurencia sp. Both algae positively responded to elevated ammonium concentrations with higher growth and chlorophyll a and lutein concentrations. Increased temperature was generally a less important driver, interacting with elevated ammonium by decreasing the algaes' %N content and N:P ratios. Interestingly, this stress response was not captured by the photosynthetic yield (Fv/Fm) nor by the carbon assimilation (%C), which increased for both algae at higher temperatures. The negative effects of higher temperature were, however, buffered by nutrient inputs, showing an antagonistic response in the combined treatment for the concentration of VAZ (violaxanthin, Antheraxanthin, zeaxanthin) and thalli growth. Ammonium uptake was initially higher for Cladophoropsis sp. and increased for Laurencia sp. over experimental time, showing an acclimation capacity even in a short time interval. This experiment shows that both algae benefited from increased ammonium pulses and were able to overcome the otherwise detrimental stress of increasingly emerging temperature anomalies, which provide them a strong competitive advantage and might support their further expansions in tropical marine systems.

Expression pattern of candidate genes and their correlation with various metabolites of abscisic acid biosynthetic pathway under drought stress in rice.[Pubmed:38148246]

Physiol Plant. 2023 Nov-Dec;175(6):e14102.

Drought hampers global rice production. Abscisic acid (ABA) plays versatile roles under different environmental stresses. While the link between drought and ABA is known, its effect on ABA biosynthesis genes and metabolites is unclear. This study explored the impact of drought on various metabolites, namely beta-carotene, zeaxanthin, Antheraxanthin, violaxanthin, neoxanthin, and candidate genes viz. zeaxanthin epoxidase (ZEP) and 9-cis epoxycarotenoid dioxygenase (NCED) of ABA biosynthesis pathway in rice cultivars (N22 and IR64) at anthesis 65 DAT (Days after transplanting) with different stress levels. In stressed plants, zeaxanthin significantly increased (92%), while the concentration of beta-carotene, Antheraxanthin, violaxanthin and neoxanthin decreased as drought stress progressed. The concentration of metabolites in roots was notably lower than in leaves in both genotypes. The ZEP expression was upregulated in roots (8.24-fold) under drought stress. Among five NCED isoforms, NCED3 showed significant upregulation (7.29-fold) in leaf and root tissue. NCED1 was significantly downregulated as stress progressed and was negatively correlated with ABA accumulation. NCED2, NCED4 and NCED5 showed no significant change in their expression. Drying and rolling of rice leaves was observed after imparting drought stress. The findings revealed that drought stress significantly influenced the expression of candidate genes and the concentration of metabolites of the ABA biosynthesis pathway. There was a significantly higher accumulation of ABA in N22 leaves (47%) and roots (30%) compared to IR64. The N22, a drought-tolerant genotype, exhibited significantly higher concentrations of intermediates and demonstrated increased expression of ZEP and NCED3, potentially contributing to its resilience against drought.

Evaluation on microalgae for the production of bio-chemicals and electricity.[Pubmed:38141667]

Chemosphere. 2024 Feb;350:141007.

Recent advancement in biophotovoltaic systems using microalgae, coupled with biorefinery approach, would improve economy-feasibility in production. The major concern is its commercial strength in terms of scalability, strain selection and extraction procedure cost. It must compete with conventional feedstocks such as fossil fuels. This project proposes to enhance the economic feasibility of microalgae-based biorefinery by evaluating their performance for bio-electricity, bio-diesel and carotenoids production in a single cycle. The first part of the study was to construct and select a Bio-bottle Voltaic (BBV) device that would allow microalgae to grow and produce bioproducts, as well as generate the maximum current output reading derived from the microalgae's photosynthesis process. The second phase consisted of a 25-day investigation into the biorefinery performance of six different microalgal species in producing bio-electricity, bio-diesel and carotenoid in a prototype BBV device. The prototype BBV device with aluminium foil and pencil lead as its anode and cathode produced the highest carotenoid and biodiesel component production from the two microalgae tested, according to the results of the first phase of the experiment. In the second portion of the study, Scenedesmus dimorphus and Chlorella vulgaris were identified as the two microalgae most capable of maintaining their growth throughout the experiment. The maximum current reading observed for C. vulgaris was 653 mV. High Performance Liquid Chromatography analysis showed four major carotenoid compounds found which were Neoxanthin, Cantaxanthin, Astaxanthin and 9-cis Antheraxanthin, and the highest carotenoid producer was C. vulgaris which recorded at 1.73 mug/mL. C. vulgaris recorded as the most alkanes producer with 22 compounds detected and Heptacosane and Heneicosane as the two major biodiesel compounds found in the extracts. Evaluation of C. vulgaris data showed that it has enormous potential for microalgal biorefinery candidates. Further ongoing research and development efforts for C. vulgaris will improve the economic viability of microalgae-based industries and reduce reliance on depleted fossil fuels.

Carotenoid Composition of Telekia speciosa.[Pubmed:38140443]

Plants (Basel). 2023 Dec 8;12(24):4116.

The carotenoid composition of the flower of Telekia speciosa was investigated for the first time by HPLC-DAD-MS. In addition to the main carotenoid lutein and its geometrical isomers, 5,6-epoxy-carotenoids, namely violaxanthin, lutein 5,6-epoxide and Antheraxanthin, were detected in larger amounts. In addition, beta-carotene 5,6-epoxide and beta-carotene 5,6,5',6'-diepoxide were found, which occurs very rarely in plants. For unambigous identification, beta-carotene 5,6-epoxide and beta-carotene 5,6,5',6'-diepoxide were prepared semisynthetically, and they were characterized by (1)H and (13)C NMR and HPLC-CD methods.

Influence of Anthocyanin Expression on the Performance of Photosynthesis in Sweet Orange, Citrus sinensis (L.) Osbeck.[Pubmed:38068602]

Plants (Basel). 2023 Nov 24;12(23):3965.

Anthocyanins are a class of natural pigments that accumulate transiently or permanently in plant tissues, often in response to abiotic and biotic stresses. They play a photoprotective role by attenuating the irradiance incident on the photochemical apparatus and quenching oxyradicals through their powerful anti-oxidative function. The objective of the current study is to understand the impact of introducing Vitis vinifera mybA1 (VvmybA1) in 'Hamlin' sweet orange trees on various aspects, including photosynthetic performance, pigment composition, and gene expression related to photosynthesis and light harvesting. We describe the relationship between anthocyanin accumulation and photosynthetic measurements in genetically modified 'Hamlin' sweet orange trees expressing the grapevine-derived Vitis vinifera mybA1 (VvmybA1). The juvenile leaves of transgenic plants displayed an intense purple color compared to the mature leaves, and microscopic visualization showed anthocyanin accumulation primarily in the leaf epidermal cells. Under optimal growth conditions, there were no significant differences in leaf gas exchange variables, suggesting normal photosynthetic performance. The chlorophyll fluorescence maximum quantum yield of PSII was slightly reduced in VvmybA1 transgenic leaves compared to the performance of the control leaves, while the total performance index per absorbance remained unaffected. Comparison of the chlorophyll and carotenoid pigment contents revealed that chlorophyllide a and carotenoid pigments, including trans-neoxanthin, trans-violaxanthin, cis-violaxanthin, zeaxanthin, Antheraxanthin, and total xanthophylls were enhanced in VvmybA1 transgenic leaves. Although there were no significant changes in the rates of the gas exchange parameters, we recorded a high relative expression of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBP) and rubisco activase (RCA) in the mature leaves of transgenic plants, indicating activation of Rubisco. Our findings confirm an efficient photoacclimation of the photosynthetic apparatus, allowing the transgenic line to maintain a photosynthetic performance similar to that of the wild type.

Changing Despicable Me: Potential replacement of azo dye yellow tartrazine for pequi carotenoids employing ionic liquids as high-performance extractors.[Pubmed:37986530]

Food Res Int. 2023 Dec;174(Pt 1):113593.

Color is a crucial sensory attribute that guides consumer expectations. A high-performance pequi carotenoid extraction process was developed using ionic liquid-based ethanolic solutions and a factorial design strategy to search for a potential substitute for the artificial azo dye yellow tartrazine. All-trans-Antheraxanthin was identified with HPLC-PAD-MSn for the first time in pequi samples. [BMIM][BF4] was the most efficient ionic liquid, and the maximization process condition was the solid-liquid ratio R((S/L)) of 1:3, the co-solvent ratio R((IL/E)) of 1:1 ([BMIM][BF4]: ethanol), and three cycles of extraction with 300 s each and yielded 107.90 mug carotenoids/g of dry matter. The ionic liquid-ethanolic solution recyclability was accomplished by freezing and precipitating with an average recovery of 79 %. In CIELAB parameters, pequi carotenoid extracted with [BMIM][BF4] was brighter and yellower than the artificial azo dye yellow tartrazine. A color change of 11.08 and a hue* difference of 1.26 degrees were obtained. Furthermore, carotenoids extracted with [BMIM][BF4] showed antioxidant activity of 35.84 mumol of alpha-tocopherol. These findings suggest the potential of employing the pequi carotenoids to replace the artificial azo dye yellow tartrazine in foods for improved functional properties.

Integrated transcriptome and metabolome analysis unveils the mechanism of color-transition in Edgeworthia chrysantha tepals.[Pubmed:37968605]

BMC Plant Biol. 2023 Nov 16;23(1):567.

BACKGROUND: Edgeworthia chrysantha, a deciduous shrub endemic to China, is known for its high ornamental value, extensive cultivation history, and wide-ranging applications. However, theoretical research on this plant is severely lacking. While its flowering process displays striking color transitions from green (S1) to yellow (S2) and then to white (S3), the scientific exploration of this phenomenon is limited, and the underlying regulatory mechanisms are yet to be elucidated. RESULTS: Correlation analysis between phenotypic measurements and pigment content revealed that carotenoids and chlorophyll are the key pigments responsible for the color changes. Metabolomic analysis of carotenoids demonstrated that lutein and beta-carotene were present at higher levels in S1, while S2 exhibited increased diversity and quantity of carotenoids compared to other stages. Notably, Antheraxanthin, zeaxanthin, lycopene, and alpha-cryptoxanthin showed significant increases. In S3, apart from the colorless phytoene, other carotenoid metabolites were significantly reduced to extremely low levels. Transcriptomic data indicated that PSY, Z-ISO, crtZ, ZEP, PDS and ZDS are key genes involved in carotenoid biosynthesis and accumulation, while NCED plays a crucial role in carotenoid degradation. SGR was identified as a key gene contributing to the progressive decline in chlorophyll content. Additionally, three transcription factors potentially regulating carotenoid metabolism were also identified. CONCLUSIONS: This study represents the first systematic investigation, spanning from phenotypic to molecular levels, of the color-changing phenomenon in E. chrysantha. The study elucidates the crucial pigments, metabolites, genes, and transcription factors responsible for flower color changes during the flowering process, thereby providing preliminary understanding of the intrinsic regulatory mechanisms. These findings establish a theoretical foundation for the genetic improvement of flower color in E. chrysantha.

Effects of different processing conditions on the carotenoid's composition, phenolic contents, and antioxidant activities of Brassica campestris leaves.[Pubmed:37908713]

Heliyon. 2023 Oct 22;9(11):e21191.

Leafy vegetables are enriched with health-promoting compounds such as carotenoids and polyphenols. Different processing treatments have been shown to affect the amounts of these compounds. In this study, mustard (Brassica campestris) leaves were subjected to various processing treatments, boiling, frying, freezing, sonication, microwaving, and blanching. Carotenoid contents were determined using HPLC-DAD while the total phenolic, flavonoids, anthocyanin, and antioxidant activities were determined using established spectroscopic protocols. It has been found that different processing treatments concentrated the lutein, flavoxanthin, and beta-carotene contents of mustard leaves, while frying has been found to have deleterious effects on these compounds. During boiling the concentration of violaxanthin, Antheraxanthin, flavoxanthin, and lutein was significantly increased to 87.4, 29.9, 20.4, and 340.8 mug/g respectively versus control. The total anthocyanin and phenolic contents of mustard leaves were better preserved during frying having values of 6.2 mg/L and 1281.2 mg/100g, respectively, whereas the total flavonoid contents (TFC) in the control sample was 111.8 mg/100g. Among the studied treatments the highest TFC was reported in the blanched samples (108.7 mg/100g), followed by sonication (107.1 mg/100g). During microwave and sonication, the antioxidant potential of the treated samples had significantly increased while in other treatments, it was reduced.

Differences in photoprotective strategy during winter in Eastern white pine and white spruce.[Pubmed:37861656]

Tree Physiol. 2024 Feb 6;44(1):tpad131.

Conifers growing in temperate forests utilize sustained forms of thermal dissipation during winter to protect the photosynthetic apparatus from damage, which can be monitored via pronounced reductions in photochemical efficiency (Fv/Fm) during winter. Eastern white pine (Pinus strobus L.) and white spruce (Picea glauca (Moench) Voss) are known to recover from winter stress at different rates, with pine recovering more slowly than spruce, suggesting different mechanisms for sustained dissipation in these species. Our objectives were to monitor pine and spruce throughout spring recovery in order to provide insights into key mechanisms for sustained dissipation in both species. We measured chlorophyll fluorescence, pigments, and abundance and phosphorylation status of key photosynthetic proteins. We found that both species rely on two forms of sustained dissipation involving retention of high amounts of Antheraxanthin (A) + zeaxanthin (Z), one that is very slowly reversible and temperature independent and one that is more dynamic and occurs only on subzero days. Differences in protein abundance suggest that spruce, but not pine, likely upregulates cyclic or alternative pathways of electron transport involving the cytochrome b6f complex and photosystem I (PSI). Both species show an increased sustained phosphorylation of the D1 protein on subzero days, and spruce additionally shows dramatic increases in the sustained phosphorylation of light-harvesting complex II (LHCII) and other PSII core proteins on subzero days only, suggesting that a mechanism of sustained dissipation that is temperature dependent requires sustained phosphorylation of photosynthetic proteins in spruce, possibly allowing for direct energy transfer from PSII to PSI as a mechanism of photoprotection. The data suggest differences in strategy among conifers in mechanisms of sustained thermal dissipation in response to winter stress. Additionally, the flexible induction of sustained A + Z and phosphorylation of photosynthetic proteins in response to subzero temperatures during spring recovery seem to be important in providing photoprotection during transitional periods with high temperature fluctuation.

Multiomics Analysis Reveals the Chemical and Genetic Bases of Pigmented Potato Tuber.[Pubmed:37856829]

J Agric Food Chem. 2023 Nov 1;71(43):16402-16416.

Anthocyanins and carotenoids determine the diversity of potato tuber flesh pigmentation; here, the underlying chemical and genetic bases were elucidated by multiomics analyses. A total of 31 anthocyanins and 30 carotenoids were quantified in five differently pigmented tubers. Cyanidin and pelargonidin derivatives determined the redness, while malvidin, petunidin, and delphinidin derivatives contributed to purpleness. Violaxanthin derivatives determined the light-yellow color, while zeaxanthin and Antheraxanthin derivatives further enhanced the deep-yellow deposition. Integrated transcriptome and proteome analyses identified that F3'5'H highly enhanced anthocyanin biosynthesis in purple flesh and was responsible for metabolic divergence between red and purple samples. BCH2 significantly enhanced carotenoid biosynthesis in yellow samples and along with ZEP, NCED1, and CCD1 genes determined metabolic divergence between light and deep-yellow samples. The weighted correlation network analysis constructed a regulatory network revealing the central role of AN1 in regulating anthocyanin biosynthesis, and 10 new transcription factors related to anthocyanin and carotenoid metabolism regulation were identified. Our findings provide targeted genes controlling tuber pigmentation, which will be meaningful for the genetic manipulation of tuber quality improvement.

A Comprehensive Genome-Wide Association Study of Carotenoid and Capsaicinoid Contents in Capsicum chinense Germplasm.[Pubmed:37762188]

Int J Mol Sci. 2023 Sep 9;24(18):13885.

Pepper is a highly important vegetable globally, both economically and nutritionally. However, to efficiently select and identify genetic resources for pepper breeding programs, it is crucial to understand the association between important traits and genetic factors. In this study, we investigated the genetic basis of carotenoid and capsaicinoid content in 160 Capsicum chinense germplasms. The study observed significant variability in carotenoid and capsaicinoid content among the germplasms. Correlation analysis revealed a strong positive correlation between violaxanthin and Antheraxanthin. In contrast, capsaicin and dihydrocapsaicin displayed negative correlations with individual carotenoids but exhibited a strong positive correlation between the two compounds (r = 0.90 ***). Genotyping-by-sequencing (GBS) was performed on 160 genotypes of pepper germplasm, which identified 47,810 high-quality SNPs. A comprehensive genome-wide association analysis was performed using these SNPs to identify SNPs associated with carotenoids and capsaicinoids, revealing 193 SNPs that exhibited significant associations. Specifically, 4 SNPs were associated with violaxanthin, 2 with Antheraxanthin, 86 with capsorubin, 5 with capsanthin, 63 with zeaxanthin, 3 with beta-cryptoxanthin, and 2 with alpha-carotene. With further studies, the significantly associated SNPs identified in this study have the potential to be utilized for selecting pepper accessions with high carotenoid and capsaicinoid contents. Additionally, the genes associated with these significant SNPs will be used to understand their roles and involvement in the biosynthesis pathway of carotenoids and capsaicinoids. Understanding the function of these genes can provide insights into the molecular mechanisms underlying the production of these bioactive compounds in pepper. The findings of this study hold valuable implications for selecting pepper varieties with desirable traits and developing breeding programs aimed at enhancing the nutritional and medicinal properties of pepper.

The Amount of Zeaxanthin Epoxidase But Not the Amount of Violaxanthin De-Epoxidase Is a Critical Determinant of Zeaxanthin Accumulation in Arabidopsis thaliana and Nicotiana tabacum.[Pubmed:37556318]

Plant Cell Physiol. 2023 Oct 16;64(10):1220-1230.

The generation of violaxanthin (Vx) de-epoxidase (VDE), photosystem II subunit S (PsbS) and zeaxanthin (Zx) epoxidase (ZEP) (VPZ) lines, which simultaneously overexpress VDE, PsbS and ZEP, has been successfully used to accelerate the kinetics of the induction and relaxation of non-photochemical quenching (NPQ). Here, we studied the impact of the overexpression of VDE and ZEP on the conversion of the xanthophyll cycle pigments in VPZ lines of Arabidopsis thaliana and Nicotiana tabacum. The protein amount of both VDE and ZEP was determined to be increased to about 3- to 5-fold levels of wild-type (WT) plants for both species. Compared to WT plants, the conversion of Vx to Zx, and hence VDE activity, was only marginally accelerated in VPZ lines, whereas the conversion of Zx to Vx, and thus ZEP activity, was strongly increased in VPZ lines. This indicates that the amount of ZEP but not the amount of VDE is a critical determinant of the equilibrium of the de-epoxidation state of xanthophyll cycle pigments under saturating light conditions. Comparing the two steps of epoxidation, particularly the second step (Antheraxanthin to Vx) was found to be accelerated in VPZ lines, implying that the intermediate Ax is released into the membrane during epoxidation by ZEP.