Ralf Welsch

A Third Phytoene Synthase Is Devoted to Abiotic Stress-Induced Abscisic Acid Formation in Rice and Defines Functional Diversification of Phytoene Synthase Genes

We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5′ untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1.

Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase

BackgroundPotato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity.ResultsWe silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e), by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold). Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed.ConclusionThe data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified.

Transcription factor RAP2.2 and its interacting partner SINAT2: stable elements in the carotenogenesis of Arabidopsis leaves.

The promoter of phytoene synthase, the first specific enzyme of carotenoid biosynthesis, shows two main regulatory regions: a G-box-containing region located near the TATA box, and a TATA box distal region containing the cis-acting element ATCTA, which mediates strong basal promoter activity. This second element was also present in the promoter of phytoene desaturase, the next step of the carotenoid pathway, suggesting a common regulatory mechanism. In this work, we demonstrate that AtRAP2.2, a member of the APETALA2 (AP2)/ethylene-responsive element-binding protein transcription factor family, binds to the ATCTA element. In Arabidopsis (Arabidopsis thaliana) leaves, AtRAP2.2 transcript and protein levels were tightly controlled as indicated by unchanged transcript and protein levels in T-DNA insertion mutants in the AtRAP2.2 promoter and 5′ untranslated region and the lack of change in AtRAP2.2 protein levels in lines strongly overexpressing the AtRAP2.2 transcript. Homozygous loss-of-function mutants could not be obtained for the AtRAP2.2 5′ untranslated region T-DNA insertion line indicating a lethal phenotype. In AtRAP2.2 overexpression lines, modest changes in phytoene synthase and phytoene desaturase transcripts were only observed in root-derived calli, which consequently showed a reduction in carotenoid content. The RING finger protein SEVEN IN ABSENTIA OF ARABIDOPSIS2 (SINAT2) was identified as an AtRAP2.2 interaction partner using a two-hybrid approach. The structure of SINAT2 and related proteins of Arabidopsis show homology to the SEVEN IN ABSENTIA protein of Drosophila that is involved in proteasome-mediated regulation in a variety of developmental processes. The action of SINAT2 may explain the recalcitrance of AtRAP2.2 protein levels to change by altering AtRAP2.2 transcription.

Provitamin A Accumulation in Cassava (Manihot esculenta) Roots Driven by a Single Nucleotide Polymorphism in a Phytoene Synthase Gene

Cassava is a very important staple crop, especially in the arid tropics where it is a chief source of carbohydrates, but the provitamin A content in the storage root is insufficient to sustain a healthy life. The work presented shows that a single amino acid exchange in a conserved region of the enzyme phytoene synthase leads to substantive accumulation of β-carotene (provitamin A) in the roots. Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.

Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

Background As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism. Methology/Principal Findings In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals. Conclusions The sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

Abstract. During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents.

Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers

BackgroundBeta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold.ResultsIn this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species.ConclusionTogether with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

Structural and functional characterization of the phytoene synthase promoter from Arabidopsis thaliana

Abstract. The expression of the gene coding for the carotenogenic enzyme phytoene synthase is highly regulated. To study this, its promoter and truncated versions thereof were translationally fused to the luciferase gene as a reporter and these constructs were used to transform Arabidopsis thaliana. The full-length promoter was shown to be active in the dark, but mediated positive responses towards different light qualities (far-red, red, blue and white light). Among the herbicides tested, norflurazon and gabaculine showed no notable effects, while CPTA abolished light induction completely. Response towards different light qualities was mediated by a TATA box-proximal promoter region up to position –300, containing G-box-like elements involved in the distinction of different monochromatic light qualities applied. This is detected in electrophoretic mobility shift assays (EMSAs), which reveal differential complex formation. A TATA box distal region of the promoter was shown to be responsible for a high basal promoter activity that was not modulated by different light qualities. Using EMSAs, a novel cis-acting element ATCTA occurring in tandem between positions –854 and –841 proved to be decisive in this respect. The motif was found in several other promoter regions involved in carotenoid and tocopherol biosynthesis, as well as in the promoter regions mediating the expression of photosynthesis-related genes. The functional equivalence of the motifs was shown by successfully using the respective regions in EMSAs. We conclude that the ATCTA motif represents an element capable of mediating a coordinated regulation of these pathways at the transcriptional level.

Identification of a novel gene coding for neoxanthin synthase from Solanum tuberosum

The polymerase chain reaction analysis of potato plants, transformed with capsanthin capsorubin synthase ccs, revealed the presence of a highly related gene. The cloned cDNA showed at the protein level 89.6% identity to CCS. This suggested that the novel enzyme catalyzes a mechanistically similar reaction. Such a reaction is represented by neoxanthin synthase (NXS), forming the xanthophyll neoxanthin, a direct substrate for abscisic acid formation. The function of the novel enzyme could be proven by transient expression in plant protoplasts and high performance liquid chromatography analysis. The cloned NXS was imported in vitro into plastids, the compartment of carotenoid biosynthesis.

Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis

Significance Carotenoids are indispensable to plants and humans. Despite significant achievements in carotenoid research, we still lack the fundamental knowledge of the regulatory mechanisms underlying carotenogenesis in plants. Phytoene synthase (PSY) and ORANGE (OR) are the two key proteins for carotenoid biosynthesis and accumulation in plastids. This study shows that OR family proteins interact directly with PSY and function as the major regulators of active PSY protein abundance in mediating carotenoid biosynthesis. The findings establish posttranscriptional regulation of PSY as a novel way to control carotenoid biosynthesis in plants and provide strategies for crop nutritional quality improvement. Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in the carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control carotenoid biosynthesis and accumulation in plants. However, little is known about the mechanisms underlying their posttranscriptional regulation. Here we report that PSY and OR family proteins [Arabidopsis thaliana OR (AtOR) and AtOR-like] physically interacted with each other in plastids. We found that alteration of OR expression in Arabidopsis exerted minimal effect on PSY transcript abundance. However, overexpression of AtOR significantly increased the amount of enzymatically active PSY, whereas an ator ator-like double mutant exhibited a dramatically reduced PSY level. The results indicate that the OR proteins serve as the major posttranscriptional regulators of PSY. The ator or ator-like single mutant had little effect on PSY protein levels, which involves a compensatory mechanism and suggests partial functional redundancy. In addition, modification of PSY expression resulted in altered AtOR protein levels, corroborating a mutual regulation of PSY and OR. Carotenoid content showed a correlated change with OR-mediated PSY level, demonstrating the function of OR in controlling carotenoid biosynthesis by regulating PSY. Our findings reveal a novel mechanism by which carotenoid biosynthesis is controlled via posttranscriptional regulation of PSY in plants.