Biol Res41: 289-301, 2008

ARTICLES

Daucus carota as a novel model to evaluate the effect of light on carotenogenic gene expression

CLAUDIA STANGE, PAULINA FUENTES, MICHAEL HANDFORD and LORENA PIZARRO

Laboratorio de Biología Molecular Vegetal, Facultad de Ciencias, Universidad de Chile, Chile

Dirección para correspondencia

ABSTRACT

Carotenoids are synthesized in prokaryotic and eukaryotic organisms. In plants and algae, these lipophilic molecules possess antioxidant properties acting as reactive oxygen species scavengers and exert functional roles in hormone synthesis, photosynthesis, photomorphogenesis and in photoprotection. During the past decade almost all carotenogenic genes have been identified as a result of molecular, genetic and biochemical approaches utilizing Arabidopsis thaliana as the model system. Studies carried out in leaves and fruits of A. thaliana and tomato determined that light regulates carotenoid biosynthesis preferentially through the modulation of carotenogenic gene transcription. In this work we showed for the first time that light induces accumulation of psy 1, pds and zds2 transcripts in leaves of Daucus carota (carrot), a novel plant model. In addition, modified roots of carrots exposed to light accumulate zdsl, whereas the pds gene is highly repressed, suggesting that some carotenogenic genes, which are expressed in roots, are regulated by light. Additionally, light negatively regulates the development of the modified carrot root in a reversible manner. Therefore, this suggests that light affects normal growth and carotenogenic gene expression in the modified root of carrot plants. The molecular insight gained into the light-regulated expression of carotenoid genes in this and other model systems will facilitate our understanding of the regulation of carotenoid biosynthesis to improve the prospects for the metabolic engineering of carotenoid production in plants.

Key terms: carotenoid biosynthesis, carrot, gene expression, light regulation.

INTRODUCTION

Carotenoids are lipid-soluble molecules of 40 carbons that are synthesized in a wide variety of photosynthetic and non photosynthetic organisms including plants, algae, some fungi and bacteria but not animals. In plants and algae, carotenoids are synthesized in the plastids, such as chloroplasts and chromoplasts. In chloroplasts, these pigments are localized and accumulate in the thylakoid membranes (Cunningham and Gantt, 1998), acting as accessory pigments in the Light Harvesting Complex (LHC), during photosynthesis (Britton, 1995). Carotenoids are also synthesized and accumulated in lipid bodies or in crystalline structures inside chromoplasts, plastids that accumulate pigments in flowers, fruits and reserve or modifies roots (Vishnevetsky et al., 1999). In flowers and fruits, the presence of these pigmented molecules serves to attract pollinators and seed dispersal agents by the intense yellow, orange and red colors that they provide to these organs (Grotewold, 2006).

In addition, carotenoids are precursors for the biosynthesis of the plant hormone abscisic acid (Crozier et al., 2000), protect plant cells from photo-oxidative damage by quenching singlet oxygen produced from chlorophyll triplet in the reaction center of photosystem II (Takano et al., 2005; Telfer, 2005) and exert a photoprotective role during excessive light incidence through thermal dissipation by means of the xanthophyll cycle, protecting the plant from photo-oxidative damage.

Carotenoids are not synthesized by animáis. Therefore, they must be ingested in the diet for the subsequent synthesis of related molecules such as vitamin A, retinal and retinoic acid, which play essential roles in nutrition, visión and cellular difierentiation, respectively (Krinsky et al., 1994). Furthermore, these molecules 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 associated with several pathologies, including aging (Esterbauer et al., 1992), carcinogenesis (Breimer, 1990) and degenerative processes in humans, among others, can be resisted by ingestión of carotenoids (Snodderly, 1995; Mayne, 1996, Rao and Rao, 2007). Birds, fish and crustaceans utilize carotenoids for pigmentation and nutritional purposes. For example, the cetocarotenoid astaxanthin is responsible for the orange color of salmón meat and lobster shells (reviewed in Grotewold, 2006). Carotenoids also have agronomic and commercial importance in several ornamental plants, in the cosmetic and food industries (Klaui and Bauernfeind, 1981) and are employed as poultry and fish feed additives (reviewed in Bjerkeng, 2000).

Until the sixties, research in this área had been centered on the biosynthetic route of carotenoids (Cunningham and Gantt, 1998). Subsequently, almost all genes termed carotenogenic genes that codify for enzymes involved in the metabolism of carotenoids in diverse plant species, algae, fungi and bacteria have been identified and characterized (Hirschberg et al., 1997; Cunningham and Gantt 1998; Cunningham, 2002; Naik et al., 2003; Lodato et al., 2004).

In plants, carotenogenic genes are encoded in the nuclear genome and the synthesized proteins are targeted as preproteins to the plastids, where they are post-translationally processed. Carotenoid biosynthesis in chloroplasts begins with the synthesis of the isoprenoid isopentenyl pyrophosphate (IPP; Fig. 1), through the non-mevalonate route (Schwender et al., 1996; Lichtenthaler et al., 1997), by condensing D-glyceraldehyde 3-phosphate with pyruvate, forming 1-deoxy-D-xylulose-5-phosphate (DOXP) (Shanker et al., 2003; Rohmer, 1999), a reaction catalyzed by DOXP-synthase (DXS). In subsequent steps and catalyzed by DOXP reductoisomerase (DXR), hydroxymethylbutenyl diphosphate (HBMPP) synthase (HDS) and HBMPP reducíase (HDR), DOXP is transformed into IPP (Lichtenthaler, 1999). IPP molecules synthesized in the plastids are then isomerized to the allylic isomer, dimethylallyl pyrophosphate (DMAPP) by means of IPP isomerase (IPI). Subsequently, DMAPP condenses with three molecules of IPP to genérate a molecule of 20 carbons named geranylgeranyl pyrophosphate (GGPP), in a process involving GGPP synthase (GGPPS). The formation of the symmetrical 40-carbon phytoene from two molecules of GGPP is catalyzed by phytoene synthase (PSY) in a two-step reaction. Phytoene biosynthesis is the first reaction specifically related to the carotenoid biosynthesis pathway. The biosynthesis of carotenoids continúes with the desaturation of the colorless phytoene to produce the pink-colored trans-lycopene (Fig.1). These reactions are catalyzed by phytoene desaturase (PDS), forming ζ -carotene, ζ -carotene desaturase (ZDS), which synthesizes pro-lycopene (7, 9, 9', 7'-tetra-cis-lycopene) and carotene isomerase (CRTISO), which transforms pro-lycopene into lycopene (all-trans-lycopene) in plants (Isaacson et al., 2002; Park et al., 2002). In leaves, the activity of CRTISO is substituted by light that photoisomerizes ζ -carotene, neurosporene, and prolycopene (Isaacson et al., 2002). Subsequently, lycopene is transformed into different bicyclic molecules. It has been observed that in plants and algae two enzymes particípate in the cyclization of lycopene (Cunningham et al., 1996; Cunningham et al., 2007), lycopene-β-cyclase (LCYB), which converts lycopene into γ-carotene and subsequently to β-carotene and lycopene-ε-cyclase (LCYE) that cyclizes one end of the lycopene molecule with an ε-ring (δ-carotene), whereas the other ring is formed by LCYB, thus producing α-carotene (Cunningham et al., 1996). The β-carotene synthesized is utilized as substrate for the enzyme β-carotene hydroxylase (CβHx) to produce zeaxanthin, while the hydroxylation of oc-carotene by the ε-carotene hydroxylase (CεHx) and CβHx results in the formation of lutein. Finally, abscisic acid is synthesized in the cytoplasm via a series of reactions subsequent to the epoxidation of zeaxanthin by zeaxanthin epoxidase (ZEP) (Cunningham and Gantt, 1998; Cunningham, 2002; Naik et al, 2003).

The regulation of carotenoid biosynthesis has been studied in photosynthetic organs (leaves) and in non-photosynthetic organs (fruits, flowers, tubers and seeds) of traditional plant models, such as Arabidopsis thaliana, Nicotiana tabacum (tobáceo) and Solanum lycopersicon (tomato) (Rómer and Fraser, 2005; Howitt and Pogson, 2006). Almost all of these studies show that carotenogenic genes are expressed in photosynthetic organs exposed to different light qualities, during the transition of etioplasts to chloroplasts (de-etiolation) (Rómer and Fraser, 2005; Bramley, 2002). During these processes, carotenogenic gene expression is mostly regulated at the transcriptional level mediated by photoreceptors, such as the family of phytochromes (PHYA-PHYE), cryptochromes (CRY) and phototropins (Simkin et al., 2003; Woitsch and Rómer, 2003; Briggs and Olney, 2001; Scheppens et al., 2004, Franklin et al., 2005; Briggs et al., 2007).

Light also affeets carotenoid biosynthesis in a number of species during fruit ripening and flower development (Zhu et al., 2002, 2003; Giovanonni, 2004; Phillips et al., 2004; Kishimoto and Ohmiya, 2006). During tomato fruit ripening, expression of dxs, hdr, pds and psyl is co-ordinately upregulated, whilst at the same time the expression of lycβ and lcyε decreases (Fraser et al., 1994; Pecker et al., 1996; Roñen et al., 1999; Lois et al., 2000; Botella-Pavía et al., 2004), leading to an accumulation of lycopene in chromoplasts of ripe fruits (Pecker et al., 1996). The psy gene is upregulated during fruit development and ripening (Fraser et al., 1999, Giuliano et al., 1993) and during flower development (Zhu et al., 2002, Zhu et al., 2003). In tomato, two distantly related genes, psyl and psy2, code for phytoene synthase, and the former was found to be transcriptionally activated only in petáis and in ripening of tomato fruits (Welsch et al., 2000; Giorio et al., 2008). Psy2 is expressed in all plant organs, preferentially in tomato leaves and petáis (Giorio et al., 2008), but in green or ripe fruits it is only expressed at low levéis (Bartley and Scolnik, 1993; Giorio et al., 2008).

Carotenogenic genes are not only expressed in leaves and fruits. In potato, |3-carotene and lutein are synthesized (Nesterenko and Sink, 2003) whilst carotenoids are also present in amyloplasts of cereal seeds such as maize and wheat (Panfili et al., 2004, see Howitt and Pogson 2006 for review). Both potatoes and cereals accumulate low levéis of carotenoids in the dark (Nesterenko and Sink, 2003), in contrast to the highly pigmented modified root of carrots.

Daucus carota L. (carrot, 2n=18) is a biennal plant whose orange reserve or modified root is consumed worldwide due to its high levéis of α-carotene and β-carotene (8 mg/g dry weight, Fraser, 2004). The carotenoid composition of a typical orange colored carrot contains predominantly β-carotene (45-80%) and oc-carotene that together constitute up to 95% of total carotenoids (Simón and Wolf 1987; Baranska et al., 2006). The major physiological function of the carrot root is as a reserve of assimilates for the production of a flowering stem after appropriate stimuli (Hole 1996). Young carrot roots are palé and start to accumulate carotenoids after the first month of growth, levéis of which usually peak after three months, shortly before secondary growth is completed (Suslow et al., 1999).

The complete cDNA sequences of some carrot carotenogenic genes, such as isopentenyl pyrophosphate isomerase (ipi, DQ192183), phytoene synthase 1 and 2 (psyl, DQ192186; psy2 DQ192187), z-carotene desaturase 1 and 2 (zdsl, DO222430; zds2, D0192189). phytoene desaturase (pds, DQ222429), lycopene (3-cyclase (lcyb, DQ192190), lycopene e-cyclase (lyce, DQ192192) and capsantine capsorubine synthase (ees: DQ192191), were annotated recently at the NCBI datábase (Just et al. 2007). The kinetics of the transcript accumulation of some of these genes correlates to total carotenoid composition during the development of modified roots grown in the dark (Clotault et al., 2008). However, to date, the influence of light on carotenoid gene expression in the leaves and modified root of carrots has not been examined. In this report, we show that this environmental factor differentially affeets the accumulation of transcripts of several carotenoid genes and alters the morphology and development of modified roots grown in the presence or absence of light.

MATERIALS AND METHODS

Plant Material

Seeds of commercially-acquired carrot (Daucus carota L.) cultivar "Nantaise improved 3" were sown hydroponically and cultivated for 4, 8 and 12 weeks with a 16 h photoperiod illuminated with white fluorescent light (450 [imol m~² s¹) at 20-23°C. Leaves and modified roots of 12-week-old carrot plantlets were harvested and utilized in real time RT-PCR analysis. For light treatments of carrot roots, the upper segment was exposed to white fluorescent light (450 [imol m~² s¹ in a 16 h photoperiod) for 12 weeks, leaving the lower segment to grow in darkness. The sampled roots are depicted in Fig. 2 and 4. For dark treatment of carrot leaves, leaves of 12-week-old carrot plants were protected from light for 48 h with aluminium paper to reduce the light level to 0,06 [imol m² s¹. Leaves and modified root pieces subjected to light and dark treatment from three carrot plants were pooled, frozen in liquid nitrogen, and powdered to isolate total RNA. Each experiment was performed in triplicate.

Reverse transcription and quantitative RT-PCR

Total RNA was extracted from frozen powder using RNAsolv (Omega Biotec, USA). Genomic DNA traces were eliminated by a 20 min RNase-free DNase I treatment at 37 °C followed by addition of 25 mM EDTA-DEPC and incubation at 75°C for 5 min to inactivate the enzyme. For cDNA synthesis, 1 μg of DNA-free RNA was incubated at 70°C for 5 min with lmM of oligo dT, then cooled for 5 min on ice. Four μl of Impron II 5x buffer, 0.5 mM each dNTP, 20U of RNase Inhibitor, and 1μl of Impron II reverse transcriptase (Promega) were added to the RNAs, which were then incubated at 25 °C for 5 min and at 42 °C for 60 min. To inactivate reverse transcriptase, the reaction was incubated at 70 °C for 15 min. The cDNA was then ready for real time RT-PCR, which was performed with the LightCycler system (Stratagene), using SYBR Green I double strand DNA binding dye. Specific primers targeting carotenoid biosynthesis genes (psyl, psy2, pds, zdsl and zds2; Table 1) were designed from the 5' UTR of each gene on the basis of published cDNA sequences (Just et al., 2007). Specific primers were also designed to amplify ubiquitin, the housekeeping gene used as the reference gene. The absence of amplification from genomic DNA in cDNA samples was tested by comparison of PCR producís obtained from cDNA and from RNA templates to ubiquitin primers. These analyses confirmed the absence of genomic DNA in all cDNA samples. The amplification of the fragments was carried out in a total volume of 25 μl containing 140 nM of the sense and anti-sense primers, 12.5 μl Brillant SYBR® Green® QPCR Master Mix (Stratagene), 0.375 μl ROX and 2 μl of cDNA, prepared as described above. The following eyeling conditions were chosen: melting of the cDNA at 95 °C for 8 min, amplification with 40 eyeles with a denaturation step at 94 °C for 30 s, annealing at 52 °C for 40 s, and a final elongation at 72 °C for 30 s. A reamplifying step at the end of the process was also included: 94°C for 30s, 50°C for 30 s and 72°C for 30 s. Fluorescence data was collected after each extensión step. Fluorescence was analyzed using LightCycler Analysis Software. The crossing point for each reaction was determined using the Second Derivative Máximum algorithm and manual baseline adjustment. Gene expression levéis were calibrated to the average valué of organs analyzed to obtain a Calibrated ΔCt for each gene. Amplification efficiency was determined for each set of primers by amplification of the target from a PCR dilution series and according to the equation:

The valué obtained in this equation was used to obtain the ratio between the carotenogenic gene and the ubiquitin gene expression using the following equation (Pfaffl2001):

Efficiency valúes for carotenogenic genes and ubiquitin amplification during the standard curve calibration were between 0.75 and 0.96, and r2 valúes for these curves were over 90%. Ct valúes for ubiquitin varied by no more than 2 units among all samples analyzed for each real time experiment. Each qRT-PCR reaction was performed with three biological replicates and each sample was analyzed in duplicate (technical replicate). In all cases, the reaction specificities were tested with melt gradient dissociation curves and electrophoresis gels. To test for significant differences in gene expression, T-test 95% were carried out using the General LinearModels option in the statistical software package Graphpad Prism. Two tailed Student t-test, p< 0.05 (confidence interval 95%) was used.

RESULTS

Effect of light on carotenogenic gene expression in leaves of carrots

To evalúate the effect of light in photosynthetic organs, carotenogenic gene expression levéis were compared in leaves of 12-week oíd plants, grown in a diurnal light regime harvested in the light period, with leaves of the same plant subjected to darkness for 2 days. Transcnpt levéis of pds and zds2 were significantly higher in light-treated D. carota leaves compared to those maintained in darkness (Fig 2). This result is in agreement with the relative expression analysis of carotenogenic genes performed in A. thaliana (von Lintig et al., 1997; Welsch et al, 2000, Botella-Pavía et al, 2004). Psyl codifies for phytoene synthase, which is involved in the synthesis of phytoene, the branch point for carotenoid biosynthesis. D. carota harbors two genes that are proposed to codify for PSY, psyl and psy2, which share 73% identity at their cDNA sequences. We observed that psyl transcnpt levéis increased five-fold during light treatments, while psy2 was not affected by the same conditions. Pds and zds2 genes are involved in lycopene biosynthesis and transcnpt levéis of these genes were eleven-and four-fold greater in leaves exposed to light compared to dark-treated leaves, respectively. To date, two sequences (zdsl and zds2), sharing 87% identity, have been annotated in carrot as coding for z-carotene desaturase. In addition, no other plant species has more than one zds gene (Cunningham and Gantt, 1998; Naik et al., 2003). Therefore, their role and relative participation in carotenoid biosynthesis under different conditions and throughout development has yet to be analyzed. We observed that zds2 expression is activated by light treatments, while zdsl transcript accumulation is repressed by light (Fig 2)

.

Effect of light on modified root development and carotenogenic gene expression

D. carota has a large reserve root that develops in darkness and where high levéis of α and β-carotene accumulate (Fraser and Bramley, 2004). We studied the effect of light on the development of the modified root of carrot. As shown in figure 3, a 4-week-old carrot plant can be segmented into three sections: leaves (L), roots (R) and a root that will become the modified root of the plant (Fig 3A, Rd). When the upper part of the future modified root was exposed to light (Rl), and the lower part (Rd) kept in darkness for 8 weeks, only the latter developed into a normal, expanded orange carrot (Fig 3B). However, when the Rl segment was later protected from light (R1-d) for a further 4 weeks, this segment developed as a normal modified root (Fig 3C R1-d and Rd), becoming indistinguishable from the portion of the root grown in continuous darkness (not shown). Therefore, the D. carota modified root develops only in darkness (Rd) and light treatments (R1) inhibit normal development in a reversible manner (R1-d).

In order to determine the effect of light on carotenogenic gene expression during modified root development, the relative abundance of some carotenogenic genes in roots exposed to light (Rl) and in modified roots grown in normal development conditions (Rd) from a 12-week-old carrot plant was measured by real time RT-PCR (Fig .4).

Psy1, psy2, pds, zds1 and zds2 genes were expressed in both conditions; however, only pds and zds2 expressions were induced in dark grown roots (Rd) relative to the segment of root that was exposed to light (R1, Fig 4A and B). On the other hand, the relative abundance of zdsl was higher in roots exposed to light (R1) than in the 12-week-old normal modified root (Rd, Fig 4B), while psyl, psy2 and zds2 transcript levéis did not change significantly between the two conditions. These results suggest that the expression of pds and zdsl is regulated by light.

DISCUSSION

In this study, morphological analysis and qRT-PCR were used to determine the effect of light on root development and carotenogenic gene expression in Daucus carota L, a novel plant model. This plant produces and accumulates carotenoids in leaves that are exposed to light and in the modified root, grown in darkness. No other plant model synthesizes such amounts of carotenoids in an organ that is not exposed to light. All analyzed carotenogenic genes were expressed in leaves and in modified roots of carrot, although expression levéis differed between these organs, suggesting that different regulatory mechanisms leading to carotenoid accumulation are involved in leaves and roots. To our knowledge, these results demónstrate for the first time that the expression profiles of selected carotenogenic genes are differentially influenced in carrots after dark or light treatments. Clotault et al., (2008), analyzed the transcript accumulation of psyl, psy2, pds, lcyb, zdsl, zds2, lcye and zep during modified root development in four carrot varieties. It was show that the expression was correlated to the accumulation of carotenoid pigments. However one of them, Blanche, a white variety, does not accumulate carotenoids, but expresses almost all genes during root development (Clotault et al., 2008). Therefore, the expression of carotenogenic genes does not always lead to a functional carotenogenic enzyme, by the presence of non-functional alleles.

We determined that light induces expression of pds and zds2 in leaves and represses the accumulation of zdsl transcripts, whilst psyl and psy2 were not affected significantly by light or dark treatments. This suggests that carrot pdsl, zdsl and zds2 genes respond to light and may have light responsive elements (LRE) in their promoters, as was shown for the psy gene promoter of A. thaliana, which has LREs, specifically G-box and G box-like elements that respond to light (Welsch et al., 2003; 2007). The results obtained in carrot leaves are in agreement with the relative expression analysis of carotenogenic genes performed in A. thaliana and tomato, in which psy and hdr transcripts increased during the transfer of leaves to light after being in darkness (von Lintig et al, 1997; Welsch et al, 2000, Botella-Pavía et al., 2004). However in D. carota leaves, psy2 expression was not affected by light or dark treatments and the induction of psyl by light was not statistically significant in relation to the dark treatment (Fig 2). We are currently isolating and characterizing the promoter of pds and zdsl genes to determine whether LREs are present in the promoter sequences.

During darkness, biosynthesis of carotenoids in leaves is stopped due principally to the very low level of expression of carotenogenic genes. In C. annum, psy, pds, zds and lcyb, genes are down regulated under these conditions (Simkin et al., 2003) and in A. thaliana the psy and hdr genes are active in darkness only at basal levéis (Welsch et al, 2003, Botella-Pavía et al., 2004). In carrot, psyl, pds and zds2 genes are also expressed at basal levéis in leaves treated for 2 days in the dark.

On the other hand, the accumulation of zdsl mRNA was repressed in the presence of light. Carotenoids are synthesized during light exposure, but it was shown that when light intensity increases from 150 to 280 mmol m² s¹ the rate of photo oxidation is higher than the rate of synthesis and carotenoids are destroyed, reaching a basal level (Simkin et al, 2003). The level of expression of some carotenogenic genes are reduced following prolonged illumination at modérate light intensities (Woitsch and Rómer, 2003), as was shown for pds transcript accumulation in tomato seedlings and was referred to as inhibition by final product (lycopene) (Corona et al., 1996; Giuliano et al., 1993). This phenomenon could also explain the behavior of carrot zds1.

In roots, pds and zds1 were affected significantly by light treatment, whereas zds2 was not, indicating that pds and zdsl genes might have LREs in the promoters. This observation is in agreement with the results obtained in carrot leaves. In A. thaliana leaves, it has been shown that light, through PHYA, plays a role in the transcriptional induction of psy in A. thaliana (von Lintig et al., 1997), by promoting the binding of the transcription factor HY5 to LREs located in the promoter. It is possible that during modified root development other transcription factors are involved, which are activated by dark conditions and repressed when roots are exposed to light. In this way, pds gene accumulation in modified carrot root could be explained, whereas the expression of zdsl could be associated with a basal level of expression in the modified root through the absence of a specific transcription factor, which is only present in light conditions or could be also associated with repression by final product. Through these results, we can conclude that pds and zds2 genes are important during carotenoid biosynthesis in leaves and in roots, associated directly with normal developmental conditions of each organ.

In contrast to leaves, in fruits, flowers, seeds, root tubers, and reserve roots, carotenoids accumulate in chromoplasts. In these plastids, carotenoids are stored in plastoglobuli, where they are more photo-stable than in chloroplasts (Merzlyak and Solovchenko, 2002). Therefore, photo-oxidation does not affect carotenoid content in these organs, even when they are exposed to light. Our results indicate that light has a negative effect on modified carrot root development, possibly because some transcriptional or growth factors are inhibited, although more extensive studies are needed to investigate this phenomenon. Roots exposed to light did not develop normally (Fig.3) and did not synthesize carotenoids compared to roots developed in darkness (data not shown). Therefore, microarray studies could be a helpful tool for the global comparison of genes that are induced or repressed in roots grown in darkness or exposed to light conditions. To date, many specific factors that are implicated in the transcriptional activation of genes regulated by light, such as carotenogenic genes, have been identified by means of microarray analyses. During initial exposure of A. thaliana seedlings to light, many transcription factors (HY5, CCA1, LHY, APRR9, APRR5, HYH, SPA1, PKS1) are early over-expressed (Quail, 2007; Tepperman et al., 2001). However, it has yet to be established whether these transcription factors play a role in the induction of carotenogenic genes.

Conventional studies focused on a specific gene or step in the carotenoid pathway combined with new technologies permitting an analysis of the entire pathway will be needed to understand the role of light on carotenoid biosynthesis in diverse organisms. Transcriptome analysis will provide insights into regulatory branch points of the pathway, whilst proteomic studies could help to associate the protein/ enzyme component profiles with the carotenoid content in plants. Without doubt, aspects associated with the effect of light on carotenoid biosynthesis regulation will be avenues warranting more intensive research efforts. Research to alter the light-mediated signal transduction machinery would also be an effective approach for modulating chlorophyll and fruit carotenoid content in plants.

ACKNOWLEDGMENTS

Supported by grant DI 12 05/06-2 from the Universidad de Chile.

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Corresponding Author: cstange@uchile.cl Las Palmeras 3425, Ñuñoa, Santiago, Casilla 653, Phone: 56 2 978 7361 Fax: 56 2 2712983.

Received: July 13, 2008. In Revised Form: September 15, 2008. Accepted: October 21, 2008