Leaky mutations in the zeaxanthin epoxidase in Capsicum annuum result in bright-red fruit containing a high amount of zeaxanthin.

Fruit color is one of the most important traits in peppers due to its esthetic value and nutritional benefits and is determined by carotenoid composition, resulting from diverse mutations of carotenoid biosynthetic genes. The EMS204 line, derived from an EMS mutant population, presents bright-red color, compared with the wild type Yuwolcho cultivar. HPLC analysis indicates that EMS204 fruit contains more zeaxanthin and less capsanthin and capsorubin than Yuwolcho. MutMap was used to reveal the color variation of EMS204 using an F3 population derived from a cross of EMS204 and Yuwolcho, and the locus was mapped to a 2.5-Mbp region on chromosome 2. Among the genes in the region, a missense mutation was found in ZEP (zeaxanthin epoxidase) that results in an amino acid sequence alteration (V291 → I). A color complementation experiment with Escherichia coli and ZEP in vitro assay using thylakoid membranes revealed decreased enzymatic activity of EMS204 ZEP. Analysis of endogenous plant hormones revealed a significant reduction in abscisic acid content in EMS204. Germination assays and salinity stress experiments corroborated the lower ABA levels in the seeds. Virus-induced gene silencing showed that ZEP silencing also results in bright-red fruit containing less capsanthin but more zeaxanthin than control. A germplasm survey of red color accessions revealed no similar carotenoid profiles to EMS204. However, a breeding line containing a ZEP mutation showed a very similar carotenoid profile to EMS204. Our results provide a novel breeding strategy to develop red pepper cultivars containing high zeaxanthin contents using ZEP mutations.

Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

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.

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.

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

MAIN CONCLUSION: Simultaneous genome editing of the two homeologous LCYe and ZEP genes of Nicotiana benthamiana results 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.

Metabolic engineering of Escherichia coli for high-level production of violaxanthin.

BACKGROUND: Xanthophylls are a large class of carotenoids that are found in a variety of organisms and play particularly important roles in the light-harvesting and photoprotection processes of plants and algae. Violaxanthin is an important plant-derived xanthophyll with wide potential applications in medicines, foods, and cosmetics because of its antioxidant activity and bright yellow color. To date, however, violaxanthins have not been produced using metabolically engineered microbes on a commercial scale. Metabolic engineering for microbial production of violaxanthin is hindered by inefficient synthesis pathway in the heterologous host. We systematically optimized the carotenoid chassis and improved the functional expression of key enzymes of violaxanthin biosynthesis in Escherichia coli. RESULTS: Co-overexpression of crtY (encoding lycopene ß-cyclase), crtZ (encoding ß-carotene 3-hydroxylase), and ZEP (encoding zeaxanthin epoxidase) had a notable impact on their functions, resulting in the accumulation of intermediate products, specifically lycopene and ß-carotene. A chassis strain that did not accumulate the intermediate was optimized by several approaches. A promoter library was used to optimize the expression of crtY and crtZ. The resulting strain DZ12 produced zeaxanthin without intermediates. The expression of ZEP was further systematically optimized by using DZ12 as the chassis host. By using a low copy number plasmid and a modified dithiol/disulfide system, and by co-expressing a full electron transport chain, we generated a strain producing violaxanthin at about 25.28 ± 3.94 mg/g dry cell weight with decreased byproduct accumulation. CONCLUSION: We developed an efficient metabolically engineered Escherichia coli strain capable of producing a large amount of violaxanthin. This is the first report of a metabolically engineered microbial platform that could be used for the commercial production of violaxanthin.

A mutation in Zeaxanthin epoxidase contributes to orange coloration and alters carotenoid contents in pepper fruit (Capsicum annuum).

Phytoene synthase (PSY1), capsanthin-capsorubin synthase (CCS), and pseudo-response regulator 2 (PRR2) are three major genes controlling fruit color in pepper (Capsicum spp.). However, the diversity of fruit color in pepper cannot be completely explained by these three genes. Here, we used an F2 population derived from Capsicum annuum 'SNU-mini Orange' (SO) and C. annuum 'SNU-mini Yellow' (SY), both harboring functional PSY1 and mutated CCS, and observed that yellow color was dominant over orange color. We performed genotyping-by-sequencing and mapped the genetic locus to a 6.8-Mb region on chromosome 2, which we named CaOr. We discovered a splicing mutation in the zeaxanthin epoxidase (ZEP) gene within this region leading to a premature stop codon. HPLC analysis showed that SO contained higher amounts of zeaxanthin and total carotenoids in mature fruits than SY. A color complementation assay using Escherichia coli harboring carotenoid biosynthetic genes showed that the mutant ZEP protein had reduced enzymatic activity. Transmission electron microscopy of plastids revealed that the ZEP mutation affected plastid development with more rod-shaped inner membrane structures in chromoplasts of mature SO fruits. To validate the role of ZEP in fruit color formation, we performed virus-induced gene silencing of ZEP in the yellow-fruit cultivar C. annuum 'Micropep Yellow' (MY). The silencing of ZEP caused significant changes in the ratios of zeaxanthin to its downstream products and increased total carotenoid contents. Thus, we conclude that the ZEP genotype can determine orange or yellow mature fruit color in pepper.

Zeaxanthin Epoxidase Activity Is Downregulated by Hydrogen Peroxide.

The xanthophyll zeaxanthin (Zx) serves important photoprotective functions in chloroplasts and is particularly involved in the dissipation of excess light energy as heat in the antenna of photosystem II (PSII). Zx accumulates under high-light (HL) conditions in thylakoid membranes and is reconverted to violaxanthin by Zx epoxidase (ZEP) in low light or darkness. ZEP activity is completely inhibited under long-lasting HL stress, and the ZEP protein becomes degraded along with the PSII subunit D1 during photoinhibition of PSII. This ZEP inactivation ensures that high levels of Zx are maintained under harsh HL stress. The mechanism of ZEP inactivation is unknown. Here, we investigated ZEP inactivation by reactive oxygen species (ROS) under in vitro conditions. Our results show that ZEP activity is completely inhibited by hydrogen peroxide (H2O2), whereas inhibition by singlet oxygen or superoxide seems rather unlikely. Due to the limited information about the amount of singlet oxygen and superoxide accumulating under the applied experimental conditions, however, a possible inhibition of ZEP activity by these two ROS cannot be generally excluded. Despite this limitation, our data support the hypothesis that the accumulation of ROS, in particular H2O2, might be responsible for HL-induced inactivation of ZEP under in vivo conditions.

Macular pigment-enriched oil production from genome-edited microalgae.

BACKGROUND: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. RESULTS: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g-1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g-1 DCW), and lipids (450.09 ± 25.48 mg g-1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g-1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g-1 lutein of oil and 7.69 ± 1.03 mg g-1 zeaxanthin of oil was produced. CONCLUSION: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

Gene silencing of BnaA09.ZEP and BnaC09.ZEP confers orange color in Brassica napus flowers.

Brassica napus is currently cultivated as an important ornamental crop in China. Flower color has attracted much attention in rapeseed genetics and breeding. Here, we characterize an orange-flowered mutant of B. napus that exhibits an altered carotenoid profile in its petals. As revealed by map-based cloning, the change in color from yellow to orange is attributed to the loss of BnaC09.ZEP (zeaxanthin epoxidase) and a 1695-bp deletion in BnaA09.ZEP. HPLC analysis, genetic complementation and CRISPR/Cas9 experiments demonstrated that BnaA09.ZEP and BnaC09.ZEP have similar functions, and the abolishment of both genes led to a substantial increase in lutein content and a sharp decline in violaxanthin content in petals but not leaves. BnaA09.ZEP and BnaC09.ZEP are predominantly expressed in floral tissues, whereas their homologs, BnaA07.ZEP and BnaC07.ZEP, mainly function in leaves, indicating redundancy and tissue-specific diversification of BnaZEP function. Transcriptome analysis in petals revealed differences in the expression of carotenoid and flavonoid biosynthesis-related genes between the mutant and its complementary lines. Flavonoid profiles in the petals of complementary lines were greatly altered compared to the mutant, indicating potential cross-talk between the regulatory networks underlying the carotenoid and flavonoid pathways. Additionally, our results indicate that there is functional compensation by BnaA07.ZEP and BnaC07.ZEP in the absence of BnaA09.ZEP and BnaC09.ZEP. Cloning and characterization of BnaZEPs provide insights into the molecular mechanisms underlying flower pigmentation in B. napus and would facilitate breeding of B. napus varieties with higher ornamental value.

Shedding light on the dark side of xanthophyll cycles.

Xanthophyll cycles are broadly important in photoprotection, and the reversible de-epoxidation of xanthophylls typically occurs in excess light conditions. However, as presented in this review, compiling evidence in a wide range of photosynthetic eukaryotes shows that xanthophyll de-epoxidation also occurs under diverse abiotic stress conditions in darkness. Light-driven photochemistry usually leads to the pH changes that activate de-epoxidases (e.g. violaxanthin de-epoxidase), but in darkness alternative electron transport pathways and luminal domains enriched in monogalactosyl diacyl glycerol (which enhance de-epoxidase activity) likely enable de-epoxidation. Another 'dark side' to sustaining xanthophyll de-epoxidation is inactivation and/or degradation of epoxidases (e.g. zeaxanthin epoxidase). There are obvious benefits of such activity regarding stress tolerance, and indeed this phenomenon has only been reported in stressful conditions. However, more research is required to unravel the mechanisms and understand the physiological roles of dark-induced formation of zeaxanthin. Notably, the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in darkness is still a frequently ignored process, perhaps because it questions a previous paradigm. With that in mind, this review seeks to shed some light on the dark side of xanthophyll de-epoxidation, and point out areas for future work.

Violaxanthin: natural function and occurrence, biosynthesis, and heterologous production.

Violaxanthin is biosynthesized from zeaxanthin with zeaxanthin epoxidase (ZEP) by way of antheraxanthin only in photosynthetic eukaryotes including higher plants and involved in the xanthophyll cycle to eliminate excessive light energy. Violaxanthin and antheraxanthin have commercially been unavailable, in contrast to commercial production of other carotenoids contained in higher plants, e.g., lycopene, ß-carotene, lutein, zeaxanthin, ß-cryptoxanthin, and capsanthin. One of the reasons is considered that resource plants or other resource organisms do not exist for enabling efficient supply of the epoxy-carotenoids, which are expected to be produced through (metabolic) pathway engineering with heterologous microbial hosts such as Escherichia coli and Saccharomyces cerevisiae. In this Mini-Review, we show heterologous production of violaxanthin with the two microorganisms that have exhibited significant advances these days. We further describe natural function and occurrence, and biosynthesis involving violaxanthin, antheraxanthin, and their derivatives that include auroxanthin and mutatoxanthin. KEY POINTS: • A comprehensive review on epoxy-carotenoids violaxanthin and antheraxanthin. • Pathway engineering for the epoxy-carotenoids in heterologous microbes. • Our new findings on violaxanthin production with the budding yeast.

Pathway engineering for efficient biosynthesis of violaxanthin in Escherichia coli.

Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 µg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3).

The peculiar NPQ regulation in the stramenopile Phaeomonas sp. challenges the xanthophyll cycle dogma.

In changing light conditions, photosynthetic organisms develop different strategies to maintain a fine balance between light harvesting, photochemistry, and photoprotection. One of the most widespread photoprotective mechanisms consists in the dissipation of excess light energy in the form of heat in the photosystem II antenna, which participates to the Non Photochemical Quenching (NPQ) of chlorophyll fluorescence. It is tightly related to the reversible epoxidation of xanthophyll pigments, catalyzed by the two enzymes, the violaxanthin deepoxidase and the zeaxanthin epoxidase. In Phaeomonas sp. (Pinguiophyte, Stramenopiles), we show that the regulation of the heat dissipation process is different from that of the green lineage: the NPQ is strictly proportional to the amount of the xanthophyll pigment zeaxanthin and the xanthophyll cycle enzymes are differently regulated. The violaxanthin deepoxidase is already active in the dark, because of a low luminal pH, and the zeaxanthin epoxidase shows a maximal activity under moderate light conditions, being almost inactive in the dark and under high light. This light-dependency mirrors the one of NPQ: Phaeomonas sp. displays a large NPQ in the dark as well as under high light, which recovers under moderate light. Our results pinpoint zeaxanthin epoxidase activity as the prime regulator of NPQ in Phaeomonas sp. and therefore challenge the deepoxidase-regulated xanthophyll cycle dogma.

A functional zeaxanthin epoxidase from red algae shedding light on the evolution of light-harvesting carotenoids and the xanthophyll cycle in photosynthetic eukaryotes.

The epoxy-xanthophylls antheraxanthin and violaxanthin are key precursors of light-harvesting carotenoids and participate in the photoprotective xanthophyll cycle. Thus, the invention of zeaxanthin epoxidase (ZEP) catalyzing their formation from zeaxanthin has been a fundamental step in the evolution of photosynthetic eukaryotes. ZEP genes have only been found in Viridiplantae and chromalveolate algae with secondary plastids of red algal ancestry, suggesting that ZEP evolved in the Viridiplantae and spread to chromalveolates by lateral gene transfer. By searching publicly available sequence data from 11 red algae covering all currently recognized red algal classes we identified ZEP candidates in three species. Phylogenetic analyses showed that the red algal ZEP is most closely related to ZEP proteins from photosynthetic chromalveolates possessing secondary plastids of red algal origin. Its enzymatic activity was assessed by high performance liquid chromatography (HPLC) analyses of red algal pigment extracts and by cloning and functional expression of the ZEP gene from Madagascaria erythrocladioides in leaves of the ZEP-deficient aba2 mutant of Nicotiana plumbaginifolia. Unlike other ZEP enzymes examined so far, the red algal ZEP introduces only a single epoxy group into zeaxanthin, yielding antheraxanthin instead of violaxanthin. The results indicate that ZEP evolved before the split of Rhodophyta and Viridiplantae and that chromalveolates acquired ZEP from the red algal endosymbiont and not by lateral gene transfer. Moreover, the red algal ZEP enables engineering of transgenic plants incorporating antheraxanthin instead of violaxanthin in their photosynthetic machinery.

M-type thioredoxins are involved in the xanthophyll cycle and proton motive force to alter NPQ under low-light conditions in Arabidopsis.

KEY MESSAGE: M-type thioredoxins are required to regulate zeaxanthin epoxidase activity and to maintain the steady-state level of the proton motive force, thereby influencing NPQ properties under low-light conditions in Arabidopsis. Non-photochemical quenching (NPQ) helps protect photosynthetic organisms from photooxidative damage via the non-radiative dissipation of energy as heat. Energy-dependent quenching (qE) is a major constituent of NPQ. However, the mechanism underlying the regulation of qE is not well understood. In this study, we demonstrate that the m-type thioredoxins TRX-m1, TRX-m2, and TRX-m4 (TRX-ms) interact with the xanthophyll cycle enzyme zeaxanthin epoxidase (ZE) and are required for maintaining the redox-dependent stabilization of ZE by regulating its intermolecular disulfide bridges. Reduced ZE activity and accumulated zeaxanthin levels were observed under TRX-ms deficiency. Furthermore, concurrent deficiency of TRX-ms resulted in a significant increase in proton motive force (pmf) and acidification of the thylakoid lumen under low irradiance, perhaps due to the significantly reduced ATP synthase activity under TRX-ms deficiency. The increased pmf, combined with acidification of the thylakoid lumen and the accumulation of zeaxanthin, ultimately contribute to the elevated stable qE in VIGS-TRX-m2m4/m1 plants under low-light conditions. Taken together, these results indicate that TRX-ms are involved in regulating NPQ-dependent photoprotection in Arabidopsis.

Loss of Function in Zeaxanthin Epoxidase of Dunaliella tertiolecta Caused by a Single Amino Acid Mutation within the Substrate-Binding Site.

The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found that the zea1 mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and zea1 were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in zea1 was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of zea1, we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function.

[Cloning and expression analysis on full length CDS of zeaxanthin epoxidase gene in Pseudostellaria heterophylla].

Zeaxanthin epoxidase plays an important role in indirect pathway of plant abscisic acid biosynthesis. According to the data of Pseudostellaria heterophylla transcriptome, zeaxanthin epoxidase gene was isolated and named as PhZEP. The results of bioinformatics analysis showed that the coding sequence of PhZEP was 1 263 bp long and encoded 420 amino acids. The putative protein molecular weight was 47.34 kDa and its theoretical isoelectric point was 6.64. The characteristic structure domains were predicted, including binding site of lipoprotein and flavoprotein monooxyenase. A signal peptide was discovered at the N-terminal of amino acids. The Real-time PCR revealed that PhZEP had a higher expression level in leaves than other tissues of P.heterophylla. Highly expressed PhZEP was also observed at 10 d and 40 d tuberous root after flowering. PhZEP presented a different expression after treatment with ABA, fluridone and ABA +fluridone compared to the control. The expression of PhZEP in tuberous root after ABA treatment was close to that in control group, while PhZEP showed significant up-regulation in the fluridone treatment group. In this study, the PhZEP gene from P. heterophylla was cloned and this result has important significance for its functional identification. This research provides a basis for the further analysis on functional mechanism of ABA during development of P. heterophylla.

Tissue-specific accumulation and regulation of zeaxanthin epoxidase in Arabidopsis reflect the multiple functions of the enzyme in plastids.

The enzyme zeaxanthin epoxidase (ZEP) catalyzes the conversion of zeaxanthin to violaxanthin, a key reaction for ABA biosynthesis and the xanthophyll cycle. Both processes are important for acclimation to environmental stress conditions, in particular drought (ABA biosynthesis) and light (xanthophyll cycle) stress. Hence, both ZEP functions may require differential regulation to optimize plant fitness. The key to understanding the function of ZEP in both stress responses might lie in its spatial and temporal distribution in plant tissues. Therefore, we analyzed the distribution of ZEP in plant tissues and plastids under drought and light stress by use of a ZEP-specific antibody. In addition, we determined the pigment composition of the plant tissues and chloroplast membrane subcompartments in response to these stresses. The ZEP protein was detected in all plant tissues (except flowers) concomitant with xanthophylls. The highest levels of ZEP were present in leaf chloroplasts and root plastids. Within chloroplasts, ZEP was localized predominantly in the thylakoid membrane and stroma, while only a small fraction was bound by the envelope membrane. Light stress affected neither the accumulation nor the relative distribution of ZEP in chloroplasts, while drought stress led to an increase of ZEP in roots and to a degradation of ZEP in leaves. However, drought stress-induced increases in ABA were similar in both tissues. These data support a tissue- and stress-specific accumulation of the ZEP protein in accordance with its different functions in ABA biosynthesis and the xanthophyll cycle.

Epoxycarotenoid-mediated synthesis of abscisic acid in Physcomitrella patens implicating conserved mechanisms for acclimation to hyperosmosis in embryophytes.

Plants acclimate to environmental stress signals such as cold, drought and hypersalinity, and provoke internal protective mechanisms. Abscisic acid (ABA), a carotenoid-derived phytohormone, which increases in response to the stress signals above, has been suggested to play a key role in the acclimation process in angiosperms, but the role of ABA in basal land plants such as mosses, including its biosynthetic pathways, has not been clarified. Targeted gene disruption of PpABA1, encoding zeaxanthin epoxidase in the moss Physcomitrella patens was conducted to determine the role of endogenous ABA in acclimation processes in mosses. The generated ppaba1 plants were found to accumulate only a small amount of endogenous ABA. The ppaba1 plants showed reduced osmotic acclimation capacity in correlation with reduced dehydration tolerance and accumulation of late embryogenesis abundant proteins. By contrast, cold-induced freezing tolerance was less affected in ppaba1, indicating that endogenous ABA does not play a major role in the regulation of cold acclimation in the moss. Our results suggest that the mechanisms for osmotic acclimation mediated by carotenoid-derived synthesis of ABA are conserved in embryophytes and that acquisition of the mechanisms played a crucial role in terrestrial adaptation and colonization by land plant ancestors.

Cloning and Functional Analysis of a Zeaxanthin Epoxidase Gene in Ulva prolifera.

The xanthophyll cycle is a photoprotective mechanism in plants and algae, which protects the photosynthetic system from excess light damage under abiotic stress. Zeaxanthin is considered to play a pivotal role in this process. In this study, the relative content of xanthophylls was determined using HPLC-MS/MS in Ulva prolifera exposed to different salinities. The results showed that high-salt stress significantly increased the relative content of xanthophylls and led to the accumulation of zeaxanthin. It was speculated that the accumulated zeaxanthin may contribute to the response of U. prolifera to high-salt stress. Zeaxanthin epoxidase (ZEP) is a key enzyme in the xanthophyll cycle and is also involved in the synthesis of abscisic acid and carotenoids. In order to explore the biological function of ZEP, a ZEP gene was cloned and identified from U. prolifera. The CDS of UpZEP is 1122 bp and encodes 373 amino acids. Phylogenetic analysis showed that UpZEP clusters within a clade of green algae. The results of qRT-PCR showed that high-salt stress induced the expression of UpZEP. In addition, heterologous overexpression of the UpZEP gene in yeast and Chlamydomonas reinhardtii improved the salt tolerance of transgenic organisms. In conclusion, the UpZEP gene may be involved in the response of U. prolifera to high-salt stress and can improve the high-salt tolerance of transgenic organisms.