Cien. Inv. Agr. 36(2):143-162. 2009
LITERATURE
REVIEW
Light-dependent regulation of carotenoid biosynthesis in plants
Lorena Pizarro, and Claudia Stange
Laboratorio de Biología Molecular Vegetal, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425,
Casilla 653 Ñuñoa, Santiago, Chile
Abstract
L. Pizarro, and C. Stange. 2009. Light-dependent regulation of carotenoid biosynthesis in plants. Cien. Inv. Agr. 36(2):143-162. Carotenoids are colored terpenes synthesized in plants, algae and some yeasts and bacteria. In plants and algae, these lipophilic molecules exert functional roles in hormone synthesis, photosynthesis, photomorphogenesis and photoprotection. Additionally, they possess antioxidant properties and act as scavengers of reactive oxygen species. During the past decade almost all of the carotenogenic genes have been identi fi ed by molecular, genetic and biochemical approaches in the Arabidopsis thaliana model system. Carotenoid biosynthesis in plants is highly regulated, although all of the processes involved have not yet been identi fi ed. In this work, we review the mechanisms involved in the light-mediated regulation of carotenoid biosynthesis and the effect of light on the levels of expression of carotenogenic genes in higher plants. It has been shown that light induces the expression of carotenogenic genes during leaf and fl ower development and during fruit ripening. During these processes, photoreceptors are activated by light and translocated to the nucleus, leading to the induction of carotenogenic gene transcription. The molecular insight gained into the light-regulated expression of carotenoid genes will facilitate our understanding of the regulation of carotenoid biosynthesis. Manipulation of light signaling is also a genetic tool for altering color and nutritional value in plants, leading to the production of novel functional foods.Key words: Carotenoid biosynthesis, gene expression, light regulation, plants.
Received 28 September 2008. Accepted 22 Decem-ber 2008.
Corresponding author: cstange@uchile.cl
Introduction
Carotenoids are lipid-soluble molecules of 40 carbons that are synthesized in a wide variety of photosynthetic and non photosynthetic or-ganisms including plants, algae, some fungi and bacteria. Over 600 carotenoid structures are known, and these are divided into non-oxygenated molecules, designated as caro-tenes, and oxygenated carotenoids, referred to as xanthophylls (Figure 1). Carotenoids act as accessory pigments and have photoprotective
functions during photosynthesis (Takano et al ., 2005, Telfer, 2005). They also protect cells from excessive light incidence through thermal dis-sipation and supply substrates for the biosynthe-sis of the plant growth regulator abscisic acid (ABA; Crozier et al ., 2000). Carotenoids also play an important role in human nutrition and health, providing provitamin A, have anti-aging and anti-cancer activities and prevent age-relat-ed macular degeneration (Krinsky et al., 1994; Mayne, 1996; Rao and Rao, 2007; Sajilata et al ., 2008). These properties have led to the develop-ment of various nutraceutical products contain-ing carotenoids.
Due to the importance of carotenoids for plant and animal health, carotenoid biosynthesis reg-
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ulation has been studied for the last 40 years, both at the pure and applied levels, and nearly all carotenogenic genes in diverse plant species, algae, fungi and bacteria have been identifi ed and characterized (Cunningham and Gantt, 1998; Cunningham et al., 2002; Howitt and Pogson, 2006). The knowledge generated from these studies has been used to improve the nu-tritional value of several plants, by engineering increased β-carotene and ketocarotenoid for-mation. (Shewmaker et al., 1999;Ye et al., 2000, 2005; Davuluri et al., 2005; Diretto et al., 2007; Aluru et al., 2008; Lamers et al., 2008). Studies in regulation of carotenoid biosynthesis in fl owers, fruits and leaves using plant models indicate that most carotenogenic genes are regu-lated at the transcriptional level. This transcrip-tional regulation has been the focus of a wide range of studies and reviews (Bramley, 2002; Cunningham, 2002; Shanker et al., 2003; Kato et al., 2004; Römer and Fraser, 2005; Howitt and Pogson 2006; Cloutault et al., 2008; Tanaka et al., 2008; Ito et al., 2009). On the other hand, light is a stimulus that activates a broad range of plant genes related to photosynthesis and photomorphogenesis. Carotenoids are required during photosynthesis in plants and algae, and, therefore, genes that direct the biosynthesis of carotenoids in these organisms are also regulat-ed by light (von Lintig et al., 1997;
Simkin et al., 2003; Woitsch and Römer, 2003; Briggs et al., 2007). However, the more recent advances in this area have not been compiled into a review of the mechanisms described to date that could modulate carotenoid gene expression in pho-toautotrophic organisms. As a result, we have prepared a timely manuscript that is aimed at a broad audience of pure and applied scientists and that synthesizes the most up-to-date litera-ture in this fast-moving fi eld of research. Improving our knowledge about physiological processes in which light is implicated will help us to understand and improve the genetic modi-fi cation processes of food crops and fruits and enhance the environmental adaptation of new transgenic plants. In this work, we review the mechanisms involved in the light-mediated reg-ulation of carotenoid biosynthesis and the effect of light on the levels of expression of caroteno-genic genes in higher plants.Biological functions of carotenoids
In plants and algae, carotenoids are synthe-sized in the plastids, such as chloroplasts and chromoplasts. In chloroplasts, carotenoids as well as other pigments, such as chlorophyll a and b,are localized and accumulate in the thy-lakoid membranes (Cunningham and Gantt, 1998), specifi cally near the reaction center of photosystem II in the light harvesting com-plexes (LHC). Carotenoids act as accessory pigments in the LHC, where they absorb light in a broader range of the blue spectrum (400-500 nm) than chlorophyll, and they transfer the absorbed energy to chlorophyll a during photosynthesi
s (Britton, 1995; Schmid 2008). In flowers and fruits, the presence of these pigmented molecules serves to attract pollina-tors and seed dispersal agents to the intense yellow, orange and red colors that they pro-vide (Grotewold, 2006). Carotenes and xan-thophylls also accumulate in lipid bodies or in crystalline structures in the chromoplasts of fl owers, fruits and reserve roots (Vishnevetsky et al., 1999). Birds, fi sh and crustaceans utilize carotenoids for pigmentation and nutrition. For example, the cetocarotenoid astaxanthin is re-sponsible for the orange color of salmon meat and lobster shells (reviewed in Grotewold, 2006). Carotenoids serve also as pigments in several ornamental plants, in the cosmetic and food industries (Klaüi and Bauernfeind, 1981) and are employed as poultry and fi sh feed ad-ditives (reviewed in Bjerkeng, 2000).
In addition, carotenoids are precursors in the biosynthesis of abscisic acid, a plant hormone involved in dormancy, maturation and differ-entiation of vegetal embryonic cells and in tol-erance to abiotic stress (Crozier et al., 2000). Carotenoids also protect plant cells from photo-oxidative damage by quenching singlet oxygen produced from the chlorophyll triplet in the reaction center of photosystem II (Takano et al., 2005; Telfer, 2005). This antioxidant char-acteristic is a result of the conjugate bonds of the polyene chain, which permit the absorption of excess energy from other molecules (Britton, 1995; Britton et al., 1998; Nelson et al., 2003). Therefore, carotenoids provide photo-oxidative protection to cells and tissue against the harm-
145 VOLUME 36 Nº2 MAY - AUGUST 2009
ful effects of singlet oxygen and lipid radicals and from the chlorophyll triplet (Stahl and Sies, 2003; Dall’Osto et al., 2006). Carotenoids also play a photoprotective role during excessive light incidence through thermal dissipation by means of the xanthophyll cycle, protecting the plant from photo-oxidative damage. This pro-cess occurs when excessive light increases the thylakoid ΔpH, activating the enzyme violax-anthin de-epoxidase (VDE), which converts violaxanthin to zeaxanthin. Zeaxanthin and protons may cause a conformational change in the LHC which favors thermal dissipation (re-viewed in Niyogi, 1999).
Carotenoids are not synthesized by animals. Therefore, they must be ingested in the diet for the subsequent synthesis of related molecules such as provitamin A, retinal and retinoic acid, which play essential roles in nutrition, vision and cellular differentiation, respectively (Krinsky et al., 1994; Fraser and Bramley, 2004; Tapiero et al., 2004). Carotenoids have also been shown to delay the aging process due to their antioxidant properties (Mordi, 1993; Bartley and Scolnik, 1995). At the same time, oxidative damage, as-sociated with several pathologies, including aging (Esterbauer et al., 1992), carcinogenesis (Breimer, 1990) and degenerative processes in humans, among others, can be reduced by in-gestion of carotenoids (Snodderly, 1995; Mayne, 1996, Rao and Rao, 2007).
Biosynthesis of carotenoids in plants
Until the 1960’s, research in carotenoid bio-synthesis had been centered on characteriz-ing the enzymes involved in the biosynthetic route of carotenoids, due to the fundamental role of these pigments in plants, humans (Cun-ningham and Gantt, 1998) and other animals. However, during the past 25 years, almost all genes, termed carotenogenic genes, that encode enzymes involved in the metabolism of caro-tenoids in diverse vegetal species, algae, fungi and bacteria, have been identifi ed and charac-terized (Hirschberg et al., 1997; Cunningham and Gantt 1998; Cunningham et al., 2000; Cun-ningham, 2002; Naik et al., 2003; Lodato et al., 2004). In plants, carotenogenic genes are encoded in the nuclear genome and the synthesized pro-teins are targeted as preproteins to the plastids, where they are post-translationally processed. Some carotenoid enzymes aggregate in multi-enzyme complexes in the stroma (isopentenyl pyrophosphate isomerase (IPI), geranylgeranyl pyrophosphate synthase (GGPPS) and phytoene synthase (PSY); Camara, 1993), while others are associated with the thylakoid membranes of these organelles (phytoene desaturase (PDS), z-carotene desaturase (ZDS), lycopene β-cyclase (LCYB) and lycopene ε-cyclase (LCYE); Cun-ningham and Gantt, 1998). GGPPS, in particu-lar, is not distributed homogenously in the stro-ma; it is specifically concentrated in globules where carotenoids are accumulated (Cheniclet et al., 1992).
Carotenoid biosynthesis begins with the syn-thesis of the isoprenoid isopentenyl pyrophos-phate (IPP; Figure 1), through two alternative pathways. These pathways are termed the me-valonate acetate route, which takes place in the cytosol, and the non-mevalonate route, which takes place in the plastids (Schwender et al., 1996; Lichtenthaler et al., 1997; Lichtenthaler et al., 1999). In the mevalonate acetate route, IPP is synthesized from 3 molecules of acetyl CoA by the enzyme hydroxymethyl glutaryl CoA reductase (HMGR). IPP molecules pro-duced in the cytosol are used for the synthesis of sterols and ubiquinones, while IPP molecules synthesized in the plastids are precursors of carotenoids, chlorophylls, tocopherols and plas-toquinones. (Figure 1, adapted from Cunning-ham and Gantt, 1998, and Naik et al., 2003). Crosstalk exists between the cytosolic and the plastidial pathways of isoprenoid biosynthesis, such that plastidial IPP can be exported to the cytosol (Rodríguez-Concepción and Gruissem, 1999; Laule et al., 2003).
In the non-mevalonate route (plastidial route), IPP is synthesized by condensing D-glyceralde-hyde 3-phosphate with pyruvate, forming 1-de-oxy-D-xylulose-5-phosphate (DOXP) (Rohmer, 1999; Shanker et al., 2003), a reaction catalyzed by DOXP-synthase (DXS). In subsequent steps catalyzed by DOXP reductoisomerase (DXR), hydroxymethylbutenyl diphosphate (HBMPP) synthase (HDS) and HBMPP reductase (HDR),
CIENCIA E INVESTIGACIÓN AGRARIA 146
DOXP is transformed to IPP by adjustment of the carbon skeleton (Lichtenthaler, 1999).
IPP molecules synthesized in the plastids are then isomerized to the allylic isomer dimethy-lallyl pyrophosphate (DMAPP) by means of IPP isomerase (IPI). Escherichia coli transformed with IPI cDNA of plant, algal and yeast origins showed an increase in the production of carote-noids (Kajiwara et al., 1997; Sun et al., 1998), suggesting that the IPI enzyme could partici-pate in a limiting step in the biosynthesis of car-otenoids, since its overexpression is suffi cient to increase the amount of carotenoids. Subsequently, DMAPP condenses with three molecules of IPP to generate a molecule of 20 carbons named geranylgeranyl pyrophosphate (GGPP), in a process involving GGPP synthase (GGPPS, in bacteria). In eukaryotes, the soluble DXS, IPI and GGPPS enzymes are located in the stroma of the chloroplast where they cata-lyze the formation of IPP, DMAPP and GGPP. These molecules are required for the synthe-sis of carotenoids and other isoprenoids such as gibberellins, tocopherols and chlorophylls (Cunningham, 2002).
The formation of the symmetrical 40-carbon phytoene from two molecules of GGPP is catalyzed by phytoene synthase (PSY, CRTB in bacteria) in a two-step reaction (Figure 1). Phytoene biosynthesis is
the fi rst reaction spe-cifi cally related to the carotenoid biosynthesis pathway. The amino acid sequence of PSY from Arabidopsis thaliana, tomato (Solanum lyco-persicum), algae and cyanobacteria resembles those of bacterial (CRTB) origin and shares a conserved prenyl-transferase domain (Cun-ningham and Gantt, 1998).
The biosynthesis of carotenoids continues with the desaturation of the colorless phytoene to produce the pink-colored trans-lycopene. These reactions are catalyzed by phytoene desatu-rase (PDS) that catalyzes the biosynthesis of ζ-carotene, ζ-carotene desaturase (z-CDS) which synthesizes pro-lycopene (7, 9, 9’, 7’-tetra-cis-lycopene) and carotene isomerase (CRTISO) that transforms the pro-lycopene into lycopene (all-trans-lycopene) in plants (Isaacson et al., 2002; Park et al., 2002; Isaacson et al., 2004). In
reactive materials studiesleaves, the activity of CRTISO is substituted by photoisomerization of ζ-carotene, neurosporene and prolycopene in the chloroplasts (Isaacson et al., 2002). Therefore, it has been hypothesized that the function of CRTISO in plants is to en-able carotenoid biosynthesis in the dark, or in chromoplasts of nonphotosynthetic tissues such as fruits and roots (Isaacson et al. 2002, 2004). In almost all fungi and nonphotosynthetic bac-teria, the enzymatic reactions that convert phy-toene into lycopene are performed by a mul-tifunctional enzyme encoded by the crtI gene (Misawa and Shimada, 1998).
Figure 1. Schematic representation of carotenoid synthesis in prokaryotic and eukaryotic organisms. The isopentenyl pyrophosphate (IPP) molecule is synthesized via two pathways. The mevalonate route involves the synthesis of IPP via hydroxymethyl glutaryl CoA reductase (HMGR), whereas in the non-mevalonate route, IPP is synthesized by DOXP synthase (DXS), DOXP reductoisomerase (DXR), hydroxymethylbutenyl diphosphate (HBMPP) synthase (HDS) and HBMPP reductase (HDR). The other enzymes that participate in the biosynthesis of carotenoids and abscisic acid are: isopentenyl pyrophosphate synthase (IPI), geranylgeranyl pyrophosphate synthase (GGPPS), phytoene synthase (PSY), phytoene desaturase (PDS), z-carotene desaturase (z-CDS), carotene isomerase (CRTISO), lycopene β cyclase (LCYB), lycopene ε cyclase (LCYE), β-carotene hydroxylase (CβHx), ε-carotene hydroxylase (CεHx) and zeaxanthin epoxidase (ZEP). The
parentheses.
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Subsequently, lycopene is transformed into dif-ferent bicyclic molecules. It has been observed in plants and algae, that two enzymes partici-pate in the cyclization of lycopene (Cunningham et al. 1996, 2007), whereas in bacteria a single protein carries out this function; CRTL in photo-synthetic bacteria and CRTY in non-photosyn-thetic bacteria. In plants, one of these cyclases is lycopene-β-cyclase (LCYB), which converts lycopene into γ-carotene and subsequently to β-carotene. The other enzyme is lycopene-ε-cyclase (LCYE), which cyclizes one end of the lycopene molecule with an ε-ring (δ-caroteno); the other ring is formed by LCYB, thus produc-ing α-carotene (Cunningham et al., 1996). The β-carotene synthesized is utilized as a substrate for the enzyme β-carotene hydroxylase (CβHx, CRTZ) to produce zeaxanthin, while the hy-droxylation of α-carotene by the ε-carotene hydroxylase (CεHx) and CβHx results in the formation of lutein. Using A. thaliana T-DNA knockout mutants, it has been shown that CβHx also possesses ε-hydroxylation activity and that it could act as a CεHx enzyme (reviewed in Tian and Della Penna, 2004). Finally, abscisic acid is synthesized in the cytoplasm via a se-ries of reactions subsequent to the epoxidation of zeaxanthin by zeaxanthin epoxidase (ZEP) (Figure1, Cunningham and Gantt, 1998; Cun-ningham, 2002; Naik et al., 2003).
Carotenoid gene activation mediated by photoreceptors in plants
The regulation of carotenoid biosynthesis has been studied in photosynthetic organs (leaves) and in non-photosynthetic organs (fruits, fl ow-ers, tubers and seeds) (Römer and Fraser, 2005; Howitt and Pogson, 2006). Almost all of these studies determined that light plays a consider-able role in the induction of carotenogenic gene expression during the transition of etioplasts to chloroplasts (de-etiolation) and during fruit and fl ower development (Bramley, 2002; Römer and Fraser, 2005). During these processes, carote-nogenic gene expression is mostly regulated at the transcriptional level and photoreceptors are involved in this process (Simkin et al., 2003; Woitsch and Römer, 2003). Plant photorecep-tors include the family of phytochromes (PHYA-PHYE) that absorb in the red and far red range as well as cryptochromes (Cry) and phototropins that absorb in the blue and UV-A range (Briggs and Olney, 2001; Franklin et al., 2005; Briggs et al., 2007). Phytochromes (PHY) are the most characterized type of photoreceptor and their photosensitivity is due to their reversible conver-sion between two isoforms: the Pr isoform that absorbs light at 660 nm (red light), resulting in its transformation to the Pfr isoform that absorbs light radiation at 730 nm (far red). Once Pr is ac-tivated, it is translocated to the nucleus as a Pfr homodimer or heterodimer (Huq et al., 2003; Matsushita et al., 2003; Sharrock and Clack, 2004; Franklin et al., 2005; Figure 2) where it ac-cumulates in subnuclear b
odies, called speckles (Nagy and Schafer, 2002; Nagatani, 2004). PHY acts as irradiance sensor through its active Pfr form, contributing to the regulation of growth and development in plants (Franklin et al., 2007), and a stable balance between these two isoforms regulates the light-mediated activation of signal transduction in plants.
The signal transduction machinery activated by PHY requires transcription factors such as PIF, HY5 and LAF1, which act in the de-etiolation process (Chattopadhyay et al., 1998; Ballesteros et al., 2001; Tepperman et al., 2001; Al-Sady et al., 2006). Recently, studies on phytochrome-regulated gene expression by means of mi-croarray analyses showed that many other tran-scription factors are also early over-expressed through PHYA and PHYB (Quail, 2007).
It has been shown that PHYA, but not PHYB, plays a role in the transcriptional induction of phytoene synthase (psy) in Arabidopsis (von Lintig et al., 1997), by promoting the binding of HY5 to white, blue, red and far red light respon-sive elements (LREs) located in its promoter (Figure 2). LREs are described to be suffi cient to confer responsiveness to light in promoters that are not induced normally by light (Roa-Rodríguez, 2003). The most common type of LREs that are present in genes activated by light are the ATCTA element, and the G1 (CACGAG) and G2 motifs (CTCGAG) (von Lintig et al., 1997). PHYA, PHYB and CRY1, can also ac-tivate the Z-box (ATCTATTCGTATACGTGT-CAC), anothe
r LRE present in light-inducible promoters (Yadav et al., 2002).
CIENCIA E INVESTIGACIÓN AGRARIA
148The involvement of the b-zip transcription factor HY5 in tomato carotenogenesis was also proven with Lehy5 transgenic tomatoes that carry an antisense sequence or RNAi of the PHYA tran-scription factor-activated hy5 gene. The trans-genic Lehy5 antisense plants contained 24–31% less leaf chlorophyll compared with non-trans-genic plants (Liu et al ., 2004), while immature fruit from Lehy5 RNAi plants exhibited an even greater reduction in chlorophyll and carotenoid accumulation.
The cryptochrome CRY, another type of pho-toreceptor, is also involved in carotenoid light-mediated gene activation. CRY1 localizes to the nucleus when plants are grown in light; however, during darkness it is exported into the cytoplasm (Guo et al., 1999; Yang et al ., 2000; Schepens et al ., 2004). CRY2, which belongs to the same family as CRY1, is localized to the nucleus of plant cells during both light and dark periods (Guo et al ., 1999). Overexpression of Cry2 in tomato causes repression of lycopene cyclase genes, resulting in an overproduction of fl avonoids and lycopene in fruits (Giliberto et al ., 2005). In vivo assays showed that blue and UV-A light trigger the phosphorylation of CRY1
and CRY2 (Shalitin et al . 2002, 2003, Figure 2) and that PHYA phosphorylates cryptochrome in vitro (Ahmad et al ., 1998). Therefore, phyto-chrome and cryptochrome signal transduction events are coordinated (Casal, 2000).
It has been reported that zeaxanthin acts as a chromophore of CRY1 and CRY2, leading to stomatal opening when guard cells are exposed to light (Winslow and Briggs, 1999). The blue/green light absorbed by these photoreceptors induces a conformational change in the zeax-anthin molecule, resulting in the formation of a physiologically active isomer leading to the opening and closing of stomata (Talbott et al., 2002). Interestingly however, it has been dem-onstrated that this carotenoid can itself act as a photoreceptor. However, to date there are no other reports of zeaxanthin functioning as a photoreceptor.
Cryptochrome and PHY bind and inactivate COP1 through direct protein-protein contact (Wang et al ., 2001; Seo et al ., 2004, Figure 2). COP1 is a ring fi nger ubiquitin ligase protein as-sociated with the signalosome complex involved
in protein degradation processes via the 26S
Figure 2. Model of the light-mediated activation of carotenogenic gene transcription in plants. A. On perception of light, the photoreceptors phytochrome (Pr inactive and Pfr active) and cryptochromes (CRY inactive and CRY-P active) are activated and translocated to the nucleus where they release the transcription factors (TF) from the signalosome (S). DET1, COP1 and DDB1 are some of the compone
nts of the signalosome. B. Direct phytochrome-mediated up-regulation of transcription factor expression. In both instances, free TF binds to light responsive elements (LRE) located in promoters, activating the transcription of carotenogenic genes.
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