Molecular mechanisms involved in biosynthesis and regulation of carotenoids in plants P. Shilpa, K.V. Ravishankar, K.S. Shivashankara1*, A.T. Sadashiva2 and N. Sunil Kumar 3 Division of Biotechnology 1Division of Plant Physiology and Biochemistry 2Division of Vegetable Crops ICAR- Indian Institute of Horticultural Research, Bengaluru 560 089, India *Corresponding author E-mail: shiva@iihr.res.in ABSTRACT Carotenoids are coloured compounds beneficial to plants and humans. Some of the major health benefits carotenoids provide include Vitamin A precursors and, antioxidants besides being involved in several physiological functions. Even though several carotenoids are synthesised by plants, only a few like beta/ alpha carotenes and cryptoxanthin serve as Vitamin A precursors. The rest are useful as antioxidants. To draw maximum benefits from carotenoids, we need to incorporate these in crop improvement programmes for enhancing available Vitamin A precursor carotenoids. Therefore, it is essential to study biosynthesis of carotenoids, their genetics and their control. In this review, we focus on factors r egulating c ar ote noid biosynthesis, m etabolism and s tor age in plastids. Transcriptional and genetic control of carotenoid production in plants is discussed in the review using several mutants too. Further, environmental regulation of carotenoid biosynthesis is also highlighted. Carotenoid-rich fruits and vegetables have greater economic value owing to their health-promoting effects. Besides,carotenoids have several industrial applications. Therefore, knowledge of regulation mechanism in carotenoid production in plants can help develop crop varieties or technologies, thus generating carotene-rich fruits and vegetables. Key words: Carotenoid biosynthesis, regulation, plastid, fruit, transcription factor INTRODUCTION Carotenoids are naturally-occurring, lipophilic, C40 isoprenoid compounds of red, yellow and orange coloured pigments. They are usually found in all photosynthetic organisms (bacteria, algae and plants) as well as in some non-photosynthetic bacteria and fungi. Colour is an important factor that makes flowers, fruits and vegetables economically valuable. This, in turn, is directly related to accumulation of carotenoids. Orange colour from â-carotene, and red colour in tomatoes and watermelon from lycopene are some examples . Apart from the appe aling colour of carotenoids in fruits such as tomatoes, water-melon and papaya, and in vegetables such as carrot, red-bell- peppers and green leafy vegetables such as spinach, broccoli and lettuce, these are also nutritionally important to humans. Consumption of carotenoid-rich fruits and vegetables has several health benefits. These are precursors for Vitamin A synthesis - deficiency of which leads to age-related macular degeneration. Carotenoids also act as free-radical scavengers owing to their antioxidant property, and help in prevention of several degenerative diseases, cardiovascular diseases and cancer (Fraser and Bramley, 2004; Fiedor and Burda, 2014). In plants, carotenoids have diverse functions: they serve as components of the light- harvesting apparatus during photosynthesis, they attract pollinators and seed-dispersal agents (Pandurangaiah et al, 2016). In view of the importance of carotenoids in plants and humans, focus is now on carotenoid research, particularly, in horticulture crops. There are several review articles on carotenogenesis and its regulation in plants (Fraser and Bramley, 2004; Walter and Strack, 2011; Giuliano, 2014). A major focus of carotenoid research is to find compositional variation J. Hortl. Sci. Vol. 11(2): 91-103, 2017 FOCUS 92 in carotenoids and regulation thereof at different levels in various plant species. The present review purports to be an overview of the recent progress in our understanding of regulation of carotenoid biosynthesis in plants. Carotenoid biosynthesis pathway a Intermediate compounds and enzymes involved in the pathway Synthesis of carotenoids needs some precursors belonging to the family of isoprenoids. Carotenoids are derived from two isoprene isomers, isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP) (Nisar et al, 2015). Formation of isoprenoids or isopentenyl diphosphate (IPP) occurs via two pathways, the cytosolic mevalonic acid pathway (MVA) and the plastidic methyl-erythritol 4- phosphate (MEP) pathway (Rodriguez-Concepcion and Boronat, 2002; Eisenreich et al, 2004). In the MEP pathway, pyruvate and glyceraldehyde (mainly obtained by glycolysis) are used as substrates initially for the formation of deoxy-D-xylulose 5-phosphate (DXP) which is catalyzed by DXP synthase (DXS). Next, MEP is formed from reduction of DXP by the enzyme DXP reductoisomerase (DXR). The roles of DXS and DXR in the pathway are very important, as,these enzymes have been shown to affect c arotenoid accumulation in plants (presumably through their control of IPP and DMAPP flux). In tomato, DXS gene expression exhibited developmental and organ-specific regulation and a strong correlation with carotenoid synthesis during fruit development (Lois et al., 2000). IPP is the fundamental C5 unit from which carotenoids are synthesized. DMAPP is sequentially formed from IPP, catatlyzed by IPP Isomerase. Further, by addition two IPP units, DMAPP is converted to Farnesyl diphosphate (FPP) and, later, FPP is further condensed to Geranyl geranyldi-phosphate (GGPP) - the first precursor to carotenoid biosynthesis. Formation of Phytoene from two molecules of GGPP is the first rate-limiting step in the carotenoid biosynthesis pathway (Fig.1). Biosynthesis of phytoene Shilpa et al J. Hortl. Sci. Vol. 11(2): 91-103, 2017 Fig. 1. Schematic representation of carotenoid biosynthesis pathway (Source: Current Opinion in Plant Biology, 2001, 4:210–218) 93 from GGPP is a two-step reaction catalyzed by the enzyme, phytoene synthase (PSY). PSY is encoded by multi-gene families in most plant species, except Arabidopsis where PSY is encoded by a single gene. Three PSY genes are reported in cereal crops viz., maize, rice and wheat. Tomato contains two genes, PSY1 and PSY2. The former encode s the fruit- ripening-specific isoform, whilst PSY2 predominates in green tissues, including mature green fruit, and has no role in carotenogenesis in a ripening fruit (Fraser et al, 1999). PSY gene(s) has/have also been used to increase carotenoid content in other crops such as rice, carrot and tomato. By contrast, the bacterial crtI gene encodes a single desaturase, that converts phytoene into all-trans lycopene (Fraser et al,1992). Various members of PSY family of genes are expressed differentially in various organs of a plant and are regulated by environmental factors (Li et al, 2008a,b). PSY3 in rice was recently found to control carotenoid biosynthesis in the root in response to abiotic stress (Welsch et al, 2008). A mutation in PSY1gene causes a yellow-flesh phenotype (the r, r mutant) and complete absence of carotenoids in the ripe fruit, an effect that can be mimicked with antisense PSY1 transformation in tomato (Bird et al, 1991; Bramley et al, 1992). Further, Phytoene is converted into lycopene by a multi- step process involving desaturation of phytoene by two structurally and functionally similar membrane-bound enzymes, phytoene desaturase (PDS) and æ-Zeta carotene desaturase (ZDS) in plants. Quinones act as electron acceptors for PDS and ZDS desaturation reactions, as demonstrated in daffodil and Arabidopsis (Nisar et al, 2015). PDS and ZDS are two FAD- containing enzymes that require at least a plastoquinone (Mayer et al., 1992; Norris et al, 1995) and a plastid terminal oxidase (Carol and Kuntz, 2001) as electron acceptors. Next, two cis-trans isomerases, Z-ISO (Li et al, 2007) and CRTISO (Isaacson et al, 2002) are required to convert poly-cis-configured phytoene into the all-trans form lycopene. Recently, two distantly related CRTISO-like single-copy genes (CrtISO-L1 and CrtISO-L2) have been discovered in tomato, Arabidopsis and grape. These enzymes are presumed to initiate a competing metabolic pathway, metabolizing carotenes upstream of all-trans-lycopene (Fantini et al, 2013). Isomerization of cis bonds to all-trans lycopene thus appears to be another regulatory step in carotenoid biosynthesis. Cyclization of lycopene is a crucial branching point in carotenoid biosynthesis. In plants, all-trans lycopene is a substrate for the enzyme cyclase, which further derives a diverse group of carotenes [differentiated based on their different cyclic end-groups, either addition of a beta ring (â-ring) and/or an epsilon ring (å-type ring)]. These two competing steps of lycopene cyclization determine the proportion of lycopene channelled to the two branches of the carotenoid pathway, viz., â- and á-carotenes. There are two types of lycopene cyclases: lycopene å cyclase (LCY-E), which convert lycopene into á-carotene; and, lycopene â cyclase (LCY-B & CYC-B), which convert lycopene into â-carotene (Pecker et al, 1996; Ronen et al, 1999). In fruits, carotenogenesis is carried out by two types of lycopene â cyclases,viz, chloroplast lycopene â cyclase (LCY-B), and chromoplast lycopene â cyclase (CYC-B). In higher plants, a chloroplast-specific lyc ope ne â-c yc lase enzyme (LCY-B) mediates c onve rsion of lyc ope ne into â -c a rote ne in photosynthetic tissue (Ronen et al, 2000). Lycopene to â-carotene conversion in chromoplast is mediated by a paralog of lyc ope ne â- c yclase ca lle d the chromoplast-specific lycopene beta cyclase gene (CYC-B). In tomato, LCY-B is expressed in leaves, flowers and fruits until the breaker-stage, whereas, CYC-B is expressed exclusively in flowers and in chromoplasts of fruits at breaker-and ripe-stages of fruit ripening. CYC-B retains the same catalytic function as LCY-B, but has only 55% amino acid sequence identity (Mohan et al,2016), but CCS (Capsanthin Capsorubin Synthase) of pepper has a high homology to CYC-B of tomato (Dalal et al, 2010) (Fig. 1). Xanthophylls, viz., lutein, zeaxanthin and neoxanthin, are produced by hydroxylation of á-carotene and â- carotene. á-carotene is converted in lutein by two hydroxylation reactions catalyzed by â ring carotene hydroxylases and å ring carotene hydroxylases. On the other hand, â-carotene is converted into zeaxanthin by â ring carotene hydroxylases. Then, Zeaxanthin epoxidase (ZEP) hydroxylates the â rings of zeaxanthin in two consecutive steps to yield antheraxanthin and, then, violaxanthin. Violaxanthin is converted to neoxanthin by neoxanthin synthase, which is the final step in the carotenoid biosynthesis pathway (Fig. 1). Briefly, the carotenoid pathway starts with isoprenoid units, built together to form a series of carotenoids such Biosynthesis and regulation of caratenoids in plants J. Hortl. Sci. Vol. 11(2): 91-103, 2017 94 a s phytoe ne, lyc ope ne, ca rotene a nd, f ina lly, xanthophylls, by the activity of various enzymes. Tran sge nic st udie s in p lants for alte ring carotenoid content Although conve ntiona l br ee ding a pproa che s successfully increased carotenoid content in plants, gene transfer or genetic engineering methods help faster and easier introduction of carotenogenic genes into plants, in a less laborious way. There has been significant progress in development of transgenic crop varieties that produce higher levels of carotenoids and, more recently, there have been a number of key achievements in areas of branch-point modulation (shifting the flux towards particular molecules,and away from the othe rs ), de novo ca rote noge ne sis (introduction of the entire carotenogenic pathway into plant tissues that lack carotenoids) and pathway extension (Farre et al, 2011). Plant carotenoids have been successfully engineered with either plant or bacterial genes, or, combinations of genes from the two sources. Because they display some particular fe ature s , ba c ter ial ge ne s have be e n use d for e ngine er ing both e arly (phytoe ne synthes is, desaturation and isomerization) and late (lycopene cyclization, ketocarotenoid biosynthesis) biosynthetic steps (Rosati et al, 2010). Rice, one of the non- sola naceous c rops, was choosen for carotenoid engineering. One of the major biotechnological brea kthroughs has bee n molec ular breeding of ‘GOLDEN RICE’ in both japonica a nd indica backgrounds, whose grain accumulates â-carotene (pro-vitamin A). Beyer et al (2002) introduced (in a single, combined transformation effort) the cDNA coding for phytoene synthase and lycopene cyclase- both from Narcissus pseudonarcissus and both under the control of the endosperm-specific glutelin promoter- together with a bacterial phytoene desaturase (crtI, from Erwinia uredovora, under constitutive 35S promoter control). This combination covers all the requirements for â-carotene synthesis an, as hoped, yellow â-carotene-bearing rice endosperm was obtained. Transgenic maize plants containing CrtL and CrtI genes expressed under the control of specific promoters showed increased levels of carotenoids, especially â-carotene (34-fold), contributing to the first transgenic maize developed to combat Vitamin A deficiency (Aluru et al, 2008). Jayaraj et al (2008) engineered the keto-carotenoid biosynthetic pathway in carrot tissues by introducing a â-carotene ketolase gene, isolated from the alga, Haematococcus pluvialis. Gene constructs were made with three promoters (double CaMV35S, Arabidopsis-ubiquitin, and RolD from Agrobac te rium rhizoge ne s ). Endoge nous expression of carrot â-carotene hydroxylases was up-regulated in transgenic leaves and roots, and up to 70% of the total carotenoids were converted to novel keto carotenoids, with accumulation of up to 2,400ìg/g root dry-weight. As for Solanaceous crops, tomato is the best-investigated species within Solanaceae family due to its importance as a food crop, and nutritional value of its fruits accumulating lycopene. Fruit-specific expression of a ba cterial PSY gene (CrtB from Erwinia) produced fruits with higher phytoene, â- carotene and total carotenoid levels, but with not increased lycopene content (Fraser et al, 2007). Large increases in fruit â-carotene and total carotenoids were achieved by manipulating the expression of lycopene â-cyclase genes: overexpressing Arabidopsis/ tomato LCY-b genes under the control of chromoplast-specific promoters resulted in higher â-carotene level, up to 7- fold (Rosati et al, 2000) and 32-fold (D’Ambrosio et al, 2004), respectively. In order to increase lycopene content in the fruits, antisense approach was used for silencing the LCY-b gene (Rosati et al, 2000). In a study on potato, a bacterial phytoene synthase gene under the control of patatin promoter increased â- carotene and total carotenoids (Ducreux et al, 2005). Diretto et al (2007) investigated modulation of carotenogenesis in potato leaves and tubers using bacterial phytoene desaturase, carotenoid isomerase and lycopene â cyclase. They observed an increase in metabolite as well as transcript levels in the transgenic plants. There was a 20-fold increase in expression of these genes simultaneously. The tubers showed enhanced â-carotene content and appeared deep yellow (golden) in colour. Peppers or hot chillis have peculiar caroptenoids that are different from those in other fruits. Capsanthin, capsorubin and capsanthin 5,6-epoxide are the red carotenoids exclusively accumulating in fruits of Capsicum spp. (Deli et al, 2001). The gene encoding Capsanthin-capsorubin synthase (Ccs) is involved in synthesis of these pigments. Pepper varieties can be classified according to fruit color: the two main isochromic families include red varieties-synthesizing capsanthin and capsorubin pigments; and, yellow ones accumulating a carotenoid Shilpa et al J. Hortl. Sci. Vol. 11(2): 91-103, 2017 95 pool c omprising a nthera xanthin, viola xa nthin (precursors of capsanthin and capsorubin) as well as ze a xanthin, á -cr yptoxa nthin, á -ca rote ne a nd cucurbitaxanthin A (Guil-Guerrero et al, 2006). From a molecular and biochemical viewpoint, red-fruited varieties display high Ccs and high carotenoid gene- transcript levels, besides a high red-to-yellow (R/Y) isochromic pigment fraction as also high capsanthin- to-zeaxanthin (Caps/Zeax) ratios. Regulation of carotenoid biosynthesis Transcriptional regulation Carotenoid accumulation is determined mainly by transcription regulation of the genes involved in the carotenoid pathway. Transcriptional regulation of carotenoids is extensively studied in the model plant tomato during fruit ripening. In this crop, lycopene is accumulated via upregulation of the genes DXS, PSY, PDS, CRTISO (which are the upstream genes in the pathway) and downregulation of the downstream genes, LCY-B, CYC-B and LCY-E. PSY is one of the rate-limiting enzymes and is the most targeted enzyme involved in carotenogenesis (as, it provides the initial substrate phytoene, levels of which determine the level of carotenoids synthesized). PSY1, present in very low abundance in leaves, is moderate in petals, and extremely high in fruits at the pink and mature red- stages; PSY2 is reported to be expressed in all the tissues, with the highest level in yellow petals; and PSY3 is found in the tomato genome and is predicted to regulate root carotenogenesis induced by abiotic stress, ABA and light. A tomato mutant related to lycopene biosynthesis is available, viz., yellow flesh (locus r), a loss-of-function mutant of the SlPSY1 gene, where there is lack of carotenoids; overexpression of the same restores carotenoid biosynthesis (Fray and Grierson, 1993). Gady et al (2012) identified a PSY1 knockout mutant through Targeting Induced Local Lesions IN Genomes (TILLING). The mutant fruit is yellow in colour due to the absence of carotenoids proving, that, PSY is the candidate enzyme involved in the initial step of carotenoid biosynthesis. Light-induced synthesis of carotenoids is characterized by an increase in the expression of PSY and, also, increase in enzyme activity. PSY transcript levels have been reported to increase in response to light of the wavelength far-red (Welsch et al, 2000). The light-induced increase in PSY expression ha s been shown to be mediate d by phytochrome (PHYs) photoreceptors. Light-induced activation makes the cytoplasmic localized PHYs to translocate to the nucleus where they interact with, and me dia te , de gra da tion of the Phytochrome Interacting transcription Factors (PIFs); these factors bind to G-boxes in the promoters of light-induced genes, and negatively regulate their expression (Leivar et al, 2009). Transgenic lines over-expressing PSY1 gene significantly increased carotenoid content in the tomato fruit (Fraser et al, 2007). In tomato fruits, the MADS box transcription factor, RIN (Ripening Inhibitor), has been confirmed to regulate carotenoid accumulation by interacting with the SlPSY1 promoter (Martel et al, 2011). According to Luo et al (2013), SlSGR1 (STAY GREEN PROTEIN) regulates carotenoid accumulation through directly interacting with SlPSY1 during tomato ripening and, therefore, repression of SlSGR1 significantly increases lycopene and â- carotene accumulation. Issacson et al (2002) e luc idated the molecular mechanism of carotenoid isomerization by studying tangerine fruits in tomato. Fruit of tangerine are orange, and accumulate prolycopene (cis-lycopene) instead of all-trans-lycopene, which is normally synthesized in the wild type. Map-based cloning of the tangerine locus of tomato identified CRTISO, which encodes an authentic carotenoid isomerase that func tions dur ing c ar ote noid de sa tura tion. In Arabidopsis, a gene designa ted Pdh e ncodes a polypeptide 75% identical to the CRTISO from tomato (86% identical in the predicted mature-polypeptide region). Evidence that Arabidopsis CRTISO ortholog is involved in carotenoid biosynthesis is described by Park et al (2002). It is interesting to note that CRTISO activity can be partially substituted by exposure to light in green tissues via photoisomerization (Isaacson et al, 2002; Park et al, 2002). Cyclization of lycopene is a key branching-point in the carotenoid pathway. Lycopene cyclases are also important determinants of carotenoid content in different plants. The two cyclases, viz., â cyclases and å cyclases are associated with differences in lycopene, â-carotene and, further, to xanthophylls by the former, and ä-carotene to lutein by the latter. Tissue- specific isoforms are involved in fine-tuning carotenoid composition in a few plants. In Arabidopsis and rice, only single-copy of these cyclases is expressed, whereas in plants like tomato, water melon and citrus, there are two types of cyclases: chloroplast-specific Biosynthesis and regulation of caratenoids in plants J. Hortl. Sci. Vol. 11(2): 91-103, 2017 96 and chromopla st-specific (Tadmor et al, 2005; Alque´zar et al, 2009; Devitt et al, 2010). Chloroplast- specific cyclases are expressed in leaves and in the green tissues of fruit. In contrast, chromoplast-specific cyclases are expressed in flowers and chromoplasts of the fruit. The chloroplast to chromoplast transition is a remarkable event during the fruit-ripening process, as, chlorophyll content decreases and carotenoid content increases. Downregulation of â cyclase leads to a n ac c umula tion of lyc ope ne, where as , its upregulation leads to synthesis of â-carotene from lycopene. Expression of chromoplast-specific â cyclase has been found to correlate with accumulation of â-carotene and/or the downstream xanthophylls in tomato and citrus (Ronen et al, 2000; Alque´ zar et al, 2009). Increased level of â-carotene is due to the fruit-specific chromoplast lycopene â cyclase (CYC-B). This phenotype was named Beta (B gene). Beta is a partially dominant, single-locus mutation that imparts an orange colour to the fully-ripened fruit owing to accumulation of â-carotene at the expense of lycopene (Ronen et al, 2000). In wild tomatoes, the B gene is expressed at low leve ls, wherea s in the Beta mutant, its transcription dramatically increases. Null mutations in the B gene are responsible for the phenotype of og (old gold), indicating that og is an allele of B. Two recessive allelic mutations, oldgold (og) and old-gold- crimson (ogc), have the same phenotype of deep red fruits, rich in lycopene but lacking â-carotene (Ronen et al, 2000). Lycopene å cyclase (LCY-E), in wild tomatoes is downregulated at the breaker-stage of ripening. In contrast, it increases approximately 30- fold during fruit ripening in Delta plants. This is due to a single, dominant gene, Del, in the tomato mutant Delta, which changes fruit colour to orange from accumulation of ä-carotene at the expense of lycopene (Ronen et al, 1999). LCY-E plays a key role in determining â-carotene/á-carotene ratio (Harjes et al, 2008). Lutein and zeaxanthin are produced next, by å- carotene hydroxylase and â-carotene hydroxylase. Further epoxidation of zeaxanthin by zeaxanthin epoxidase (ZEP) produces violaxanthin. This reaction is reversed by violaxanthin de-epoxidase (VDE) to give rise to the xanthophyll cycle in plants to adapt to high light-stress. Violaxanthin is converted into neoxanthin by neoxanthin synthase (NSY) (Shan Lu and Li, 2008). A study shows that a novel gene-product of ABA4 is needed for neoxanthin synthesis (North et al, 2007). Environmental regulation Light plays an important role too in carotenoid biosynthesis. Light intensity, duration, direction, spectral quality are all key factors for modulating plant carotenoids during fruit development. Photomorpho- genic mutants have been reported in a number of species, including Arabidopsis, sorghum, brassica, tobacco, tomato and pea (Levin et al, 2006). The tomato high pigment 1 (hp1) and high pigment 2 (hp2) are regulated by light, which controls plastid development in these mutants. Light-signalling proteins are responsible for hp1 and hp2 phenotypes in tomato. Two re gula tory ge ne s, UV-DA MA GED DNA- BI NDING P ROTE IN1 (DDB1) and DE- ETIOLATED1 (DET1), c ontrol light-signalling pathways. Mutations in these genes are responsible for high-pigment mutants (hp1 and hp2) in tomato (Shan Lu and Li, 2008). Carotenoid accumulations in these mutants is linked to early plastid-biogenesis, number and size to provide a large compartment for carotenoid biosynthesis and deposition (Liu et al, 2004). The red-ripe fruits of these mutants are characterized by an intense red colour mainly from increased levels of carotenoids, primarily lycopene. These hp mutants have proved to be excellent tools in the study of complex inte rac tions betwee n light a nd plant development. Some of these have also been harnessed in breeding programs. Similarly, like hp1 and hp2, high pigment 3 (hp3) mutant in tomato is caused by a mutation in the gene of ZEP, which confers an enhanced level of carotenoid accumulation by increasing the size of plastid compartment in the cells, to enable greater biosynthesis and higher storage capacity (Galpaz et al, 2008). Apart from these mutants, light-regulated carotenoid synthesis is observed through LREs (Light Responsive Elements) and G box elements present in the promoter region of the genes involved in the pathway. The most common type of LREs present in genes activated by light are ATCTA element, and, G1 (CACGAG) and G2 motifs (CTCGAG) (von Lintig et al, 1997). Therefore, light acts as an inducer of photo- morphogenesis, and carotenoid biosynthesis through photoreceptors and activation of known transcription factors (Pizarro and Stange, 2009). PSY activity is regulated by red and far-red light as a consequence of phytochrome activation, leading to increase in PSY activity only under red light conditions (Schofield and Paliyath, 2005). Shilpa et al J. Hortl. Sci. Vol. 11(2): 91-103, 2017 97 Temperature has a significant influence on growth and development of tomato fruits. It has been reported that high temperature (35°C) can specifically inhibit accumulation of lycopene by stimulating conversion of lycopene into carotene (Hamauzu et al, 1998). Biosynthesis of lycopene is affected by environmental conditions. If the temperature of the fruits exceeds 30°C, synthesis of lycopene is inhibited in tomato (Brandt et al, 2006). Another study by Dumas et al (2003) showed that temperatures below 12°C strongly inhibited lycopene biosynthesis, while, temperatures above 32°C stopped this process altogether in tomato. Carotenoid accumulation in plants is regulated by hormones such as ethylene, jasmonates (JA), ABA,etc. Ethylene plays a central role in fruit-ripening. Effect of ethylene in regulating carotenoid accumulation during fruit de ve lopme nt in toma to ha s bee n increasingly reviewed. Ethylene production strongly correlates with rapid accumulation of -carotene and lycopene, and expression of SlPSY1 and SlPDS. This shows that the process is dependent on ethylene (Marty et al, 2005). Many transcription factors have been shown to affect carotenoid accumulation in the fruit of tomato through regulation of ethylene biosynthesis and signalling. In fruit tissues, MADS box transcription factor, ripening inhibitor (RIN), has been confirmed to regulate carotenoid accumulation by interacting with SlPSY1 promoter (Martel et al, 2011) (Fig. 2). RIN interacts directly with the promoters of ethylene biosynthesis genes, ACC SYNTHASE2 (ACS2) and ACS4, also the ethylene perception gene, ETHYLENE RECEPTOR 3 (ETR3/NR) (Fig. 2). The corresponding mutant of RIN, rin, fails to synthesize climacteric ethylene and a ccumulates lycopene in the fruit (Vrebalov et al, 2002). TAGL1 and FRUITFULL 1 and 2 (which belong to MADS box transcription factor) Biosynthesis and regulation of caratenoids in plants J. Hortl. Sci. Vol. 11(2): 91-103, 2017 Fig. 2. Schematic representation of role of ethylene in carotenoid accumulation are positive regulators of ethylene biosynthesis (Vrebalov et al, 2009). ETHYLENE RESPONSE FACTOR 6 (SlERF6), a tomato Ethylene Response Factor, is also found to be a negative carotenoid modulator. Reduced expression of SlERF6 by RNAi enhanc ed both ethyle ne re lease and ca rotenoid accumulation during fruit ripening in tomato (Lee et al, 2012). The role of ethylene in carotenoid formation is further demonstrated by a tomato ethylene-insensitive mutant, Never ripe (Nr), which exhibits a ripening- defective fruit phenotype, and fails to accumulate lycope ne (Lanahan et al, 1994). J A is a lso of importance in positively controlling carotenoid accumulation. In tomato, lycopene content is greatly reduced in the fruits of JA-deficient mutants, and, is increased in transgenic lines having enhanced JA levels. Exogenous MeJA treatment of an ethylene-insensitive tomato mutant (Nr) can dramatically enhance lycopene ac cumulation in the fruit. ABA has also be en documented to control carotenoid biosynthesis by regulating plastid development. A study of ABA- deficient tomato mutants, namely, hp3, flacca (flc), and sitiens (sit), shows an inverse correlation between ABA levels and plastid number. In these mutant fruits, both plastid number and lycopene levels are enhanced (Galpaz et al, 2008) indicating that ABA, a carotenoid 98 derivative, may be implicated in build-up of storage capacity in plastids (Liu et al, 2015). Deficiency of ABA in hp-3 mutant led to enlargement in plastid compartment size, probably by increasing plastid division. By doing so, it enabled greater pigment biosynthesis, and 30% more storage capacity for carotenoids in the mature fruit (Galpaz et al, 2008). S om e s tu di e s a re re por t e d on th e ro le o f transcription factors in regulation of carotenoid biosynthesis. Lee et al (2012) characterized one c a ndi da te for im pa c t o n tr a ns -lyc ope n e a nd -carotene accumulation, viz., SlERF6, revealing that it indeed influenced carotenoid biosynthesis and other ripening phenotypes. Reduced expression of SlERF6 by RNAi enhanced both carotenoid and ethylene levels during fruit ripening, demonstrating an important role for SlERF6 in ripening through integrating the ethylene and carotenoid synthesis pa thwa ys . A numbe r of tr ans c ription fa ct ors im pa c ti ng r ipe nin g a nd , t hu s, c a r ot e no id accumulation, have been identified including RIN- MADS (Vrebalov et al, 2002), CNR SQUAMOSA promoter binding protein (Manning et al, 2006), TAGL1 MADS box (Vrebalov et al, 2009), LeHB- 1 HBzip (Lin et al, 2008) and SlAP2a, an AP2 gene (Karlova et al, 2011). More specific carotenoid regulators have been identified in non-fruit tissues. In Ar abidops is, modula ting mRNA l eve ls of ethylene response factor (ERF) RAP2.2 (which is capable of binding the PSY promoter), resulted in small carotenoid alterations in root calli (Welsch et al, 2007). AtRAP2.2 belongs to AP2/EREBP family of transcription fa ctors and binds to upstream regulatory elements of the genes PSY and PDS. AtRAP2.2 recognizes cis-acting element ATCTA, which mediates a strong basal promoter activity (Welsch et al , 2007). A putative tomato NAC transcription fa ctor named SlNAC4 ac ts a s a positive regulator of fruit carotenoid synthesis in tomato. SlNAC4 plays an important role in response to a bio tic st re s s , but, tr a ns ge nic re pre s s ion de monstrates tha t SlNA C4 also participates in normal fruit-ripening as a positive regulator by modula ting the c limac te ric ripening hormone , ethylene, and carotenoid pigmentation (Zhu et al, 2013) (Table 1) . Carotenoid catabolism A metabolic equilibrium between biosynthesis and catabolism of carotenoids is essential for maintaining the content and composition of ca rotenoids in pho tos yn the ti c t is s ue s (Be i se l e t al , 20 10) . Carotenoid degradation is brought about by a group of e nz ym e s kno wn a s Caro te no id Cle av ag e Dioxygenases (CCDs). Thus, catalytic activity of c a rot e no id c l e a v ag e d io x y g e na s e s (CCD s ) , wh ic h le a ds t o e n z yma tic tu r nov e r of C 4 0 ca rote noids into apocarotenoids, is c ritica l in regulating carotenoid accumulation. In Arabidopsis, the CCD gene family consists of nine members and can be divided into two groups: four CCDs (CCD1, 4, 7 a n d 8 ) a nd f ive 9- c is - e po x y c ar ote n oid dioxygenases (NCE D 2, 3, 5, 6 and 9). T he se enzymes cleave different carotenoids and some exhibit unique substrate specificity (Auldridge et al, 2006). Diffe rent CCDs and NCEDs rec ogniz e diffe re nt c arote noid substra te s a nd c le a ve a t different sites, producing various apocarotenoids (Walter and Strack, 2011). In vitro expression of CCD1 in E. coli from a number of horticultural crops such as tomato fruit saffron flower, and melon fruit indicates that CCD1 cleaves -carotene and other carotenoids into a range of volatiles. CCD1 transcription is associated with carotenoid levels in some horticultural crops. Although a negatively correlated change between CCD1 or CCD4 and carotenoid content has been observed in various fruits and vegetables, regulation of CCD expression is n ot w e l l-u nd e r s too d ( Yua n e t al , 20 15 ) . Investigation on the activity of SlCCD1B and its homolog, SlCCD1A, provides direct evidence for involvement of CCD in carotenoid degradation. In vitro assays showed that SlCCD1A and SlCCD1B target double bonds in all trans configured and cis- Shilpa et al J. Hortl. Sci. Vol. 11(2): 91-103, 2017 Table 1. List of some transcription factors and type of regulation involved in carotenoid synthesis Tra nsc rip ti on Family Type of factor reg ula tio n S l E R F 6 M ADS RIN Positive regulation TA G L 1 M ADS Box Positive regulation L eH B - 1 H B Zip Positive regulation S l A P 2 a A PE TA L A 2/E R F Negative regulation R A P 2 . 2 E R F Positive regulation S l N A C 4 NA C Positive regulation 99 Biosynthesis and regulation of caratenoids in plants J. Hortl. Sci. Vol. 11(2): 91-103, 2017 configured carotenoids, ac yclic carotene s and apocarotenoids, contributing to the production of a vast majority of tomato isoprenoid volatiles (Ilg et al, 2014). Fruit specific RNAi-mediated suppression of SlNCED1 produces deep-red fruits with reduced SlNCED1 transcription and ABA biosynthe sis, besides increased accumulation of lycopene and b- carotene (Sun et al, 2012). Besides catabolism and turnover of carotenoids, the ability of a cell to store and sequester carotenoids plays a significant role in determining the level of carotenoid accumulation in a given cell. Nearly all of carotenoid biosynthesis enzymes are located in the plastid, but all their genes are encoded by the nuclear genome (Cazzonelli et al, 2009). Carotenoids in plants are synthesized de novo in nearly all types of plastids, but accumulate in large quantities in chloroplasts and chromoplasts (Howitt and Pogson, 2006). Carotenoids in the chloroplast are integrated with chloroplast binding proteins and, in chromoplast, carotenoids are associated with polar lipids and carotenoid associated proteins, to form carotenoid-lipoprotein sequestering substructures to effectively sequester and retain a large quantity of carotenoids (Vishnevetsky et al, 1999). Chromoplast development and differentiation plays a crucial role in carotenoid biosynthesis, as, chromoplasts provide a site not only for active carotenoid biosynthesis, but also for carotenoid storage. Size and structure of the plastids is also important for accumulation of carotenoids. Thus, carotenoid accumulation is a net result of biosynthesis, turnover and, finally, stable storage of end- products (Shan Lu and Li, 2008). CONCLUSION An understanding of carotenoid biosynthesis and regulatory mechanism has been of interest from several years, as, carotenoid content is one of the most important quality traits in fruits and vegetables with industrial, health and nutritional attributes. The high value of naturally-occurring carotenoids (which are economically important) has triggered special interest in uncovering the several levels/ modes of carotenoid regulation. Several attempts have been made to improve plant carotenoid content and composition. 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