Plant Biology ISSN 1435-8603
RESEARCH PAPER
Dissecting the mechanism of Solanum lycopersicum and Solanum chilense flower colour formation M. Gao1,2,3, H. Qu1,2, L. Gao1,2, L. Chen1,2, R. S. J. Sebastian1,2 & L. Zhao1,2,3 1 Joint Tomato Research Institute, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China 2 Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China 3 Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
Keywords Carotenoids; chromoplast; flower colour; plastoglobule; structural gene; tomato. Correspondence L. Zhao, 800 Dongchuan Road, Shanghai 200240, China. E-mail: [email protected] Editor K. Leiss Received: 5 November 2013; Accepted: 4 March 2014 doi:10.1111/plb.12186
ABSTRACT Flowers are the defining feature of angiosperms, and function as indispensable organs for sexual reproduction. Flower colour typically plays an important role in attracting pollinators, and can show considerable variation, even between closely related species. For example, domesticated tomato (S. lycopersicum) has orange/yellow flowers, while the wild relative S. chilense (accession LA2405) has bright yellow flowers. In this study, the mechanism of flower colour formation in these two species was compared by evaluating the accumulation of carotenoids, assessing the expression genes related to carotenoid biosynthetic pathways and observing chromoplast ultrastructure. In S. chilense petals, genes associated with the lutein branch of the carotenoid biosynthetic pathway, phytoene desaturase (PDS), f-carotene desaturase (ZDS), lycopene b-cyclase (LCY-B), b-ring hydroxylase (CRTR-B) and e-ring hydroxylase (CRTR-E), were highly expressed, and this was correlated with high levels of lutein accumulation. In contrast, PDS, ZDS and CYC-B from the neoxanthin biosynthetic branch were highly expressed in S. lycopersicum anthers, leading to increased b-carotene accumulation and hence an orange/yellow colour. Changes in the size, amount and electron density of plastoglobules in chromoplasts provided further evidence of carotenoid accumulation and flower colour formation. Taken together, these results reveal the biochemical basis of differences in carotenoid pigment accumulation and colour between petals and anthers in tomato.
INTRODUCTION As the sexual reproductive organ of angiosperms, flowers have evolved several remarkable traits and mating systems to promote effective reproduction (Zimmerman 1988; Li & Chetelat 2010; Craine et al. 2012; Thakur & Bhatnagar 2013). The development of insect-attracting (entomophilous) and bird-attracting (ornithophilous) flowers ended an era where successful plant pollination relied only on wind and water, a landmark event in the evolution of flowering plants. Key floral features used to attract pollinators, and which are also present in fruits to attract frugivores and promote seed dispersal, include bright colour, aroma, volatiles and accumulation of energy-rich compounds, which are present in nectar (Miller et al. 2011). Flower colour depends on the composition and content of pigments, e.g. carotenoids, anthocyanins and betalains (Kay et al. 1981; Delgado-Vargas et al. 2000; Miller et al. 2011). Carotenoids are C40 terpenoids and are widespread in plants, where they play many roles, including in photosynthetic reactions in chloroplasts. Carotenoids are also one of the most important pigments in flowers and fruits (Vishnevetsky et al. 1999; Isaacson et al. 2002; Miller et al. 2011), and help prevent photo-oxidative damage (Horton & Ruban 2005; Pascal et al. 2005; Jahns & Holzwarth 2012). Carotenoids also serve as precursors for abscisic acid (ABA) and vitamin A (Nambara & Marion-Poll 2005; DellaPenna & Pogson 2006; Barsan et al.
2010), and provide dietary components that are beneficial for human health. For example, studies have showed that lycopene, which is present at high levels in tomato fruits, has antioxidant activity and helps prevent cancers and cardiocerebrovascular disease (Marti et al. 2009; Adalid et al. 2010; Barsan et al. 2010). Carotenoids are stored at high concentrations in chromoplasts of both flowers and fruits, where they provide colours from yellow to orange and red. Chromoplasts are non-photosynthetic plastids that function as specialised organelles for synthesis and accumulation of carotenoids. They can be divided into four types: globular, tubular, membranous and crystalloid, according to shape, sub-organelle structure and types present during development in flowers and fruits (Ljubesic et al. 1991). The steps of the carotenoid biosynthesis pathway have previously been identified. The first dedicated step is the formation of geranylgeranyl diphosphate (GGPP), which involves the condensation of isopentenyl pyrophosphate and farnesyl diphosphate in the methylerythritol phosphate (MEP) pathway. Two GGPP molecules are condensed by phytoene synthase (PSY) to form phytoene, which is then converted into lycopene via four consecutive dehydrogenation reactions carried out by phytoene desaturase (PDS) and f-carotene desaturase (ZDS). Lycopene is at a key branch point in the carotenoid biosynthesis pathway, since linear lycopene can be cyclised to
Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands
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b-carotene (all trans b-carotene) or a-carotene (b-, e-carotene) by lycopene b-cyclase (LCY-B) or lycopene e-cyclase (LCY-E), respectively. In the lutein branch, a-carotene and b-carotene are then converted to lutein and zeaxanthin via consecutive hydroxylation of the e- and b-rings, respectively. Zeaxanthin is then subjected to epoxidation at the b-ring, and converted to violaxanthin via the intermediate antheraxanthin. Finally, violaxanthin is converted to neoxanthin by neoxanthin synthase (NSY) in the neoxanthin branch (Fig. S1; Ronen et al. 1999, 2000; DellaPenna & Pogson 2006; Galpaz et al. 2006; Phillips et al. 2008). In this study, we investigated the molecular basis of differences in flower colour between domesticated S. lycopersicum (cultivar P86) and its wild relative S. chilense (accession LA2405). To dissect mechanisms for formation of flower colour, we evaluated carotenoid content, expression of genes encoding key step enzymes in the carotenoid biosythesis pathway and chromoplast ultrastructure. MATERIAL AND METHODS Plant material Seeds of LA2405 (S. chilense) and P86 (S. lycopersicum) were kindly provided by the Tomato Genetics Resource Center (TGRC, http://tgrc.ucdavis.edu) and Dr. Wang Fu (Qingdao Agricultural University, China). Tomato seeds were sown between two pieces of humid filter paper in Petri dishes, and left to germinate at 25 °C in the dark. The germinated seeds were transferred to plastic pots (diameter 7.5 cm) with seedling soil, and planted in 5-l plastic pots with soil when the fourth true-leaf had fully expanded. The plants were grown in a greenhouse (25 °C day/18 °C night, 70% air humidity and natural light) in the School of Agriculture and Biology, Shanghai Jiao Tong University, China. Petals and anthers were harvested at five flower developmental stages: 5 days pre-anthesis ( 5DPA), 3 days preanthesis ( 3 DPA), 0 days post-anthesis (0 DPA), 3 days postanthesis (3DPA) and 5 days post-anthesis (5 DPA). Extraction and detection of carotenoids The extraction, separation and detection of tomato flower carotenoids were performed as described in Fraser et al. (2000). All steps were carried out on ice and shielded from strong light. Flowers of both LA2405 and P86 were harvested from 15 plants (each biological replicate included five plants) at intervals from 5 DPA to 5 DPA (days post-anthesis; as above). Anthers and petals were ground separately into a fine powder in a mortar with liquid nitrogen. Approximately 200 mg of the powder were transferred to a 15-ml conical Falcon centrifuge tube with 1.5 ml extraction buffer (60% KOH (w/v):methanol in a 1:9 ratio) and the samples were mixed using a vortexer. The mixture was incubated in a water bath at 60 °C for 30 min, before 1.5 ml Tris-HCl (50 mM, pH 7.5, containing 1 M NaCl) was added and the samples incubated at 4 °C for 10 min. After addition of 4 ml chloroform and thorough vortexing, the samples were transferred to an ice bath for 10 min for carotenoid extraction. The organic phase of the lower layer was carefully transferred into a clean centrifuge tube using a disposable syringe after centrifugation at 3000 g for 5 min, and the aqueous phase was re-extracted with 4 ml chloroform. The two 2
collected organic phases were concentrated in Nitrogen Evaporators (MD200-2; Zhe Jiang, China), and the dried residues were dissolved in 800 ll hexane. Standards of lutein and zeaxanthin were dissolved in methanol, a-carotene and b-carotene were dissolved in methyl tert-butyl ether and lycopene was dissolved in hexane. The a-carotene standard was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the other four standards were purchased from Sigma Chemicals (St. Louis, MO, USA). Separation and detection were carried out in a Hitachi L-2000 control system (Hitachi, Tokyo, Japan). Throughout chromatography, the eluate was monitored continuously from 220 to 500 nm. A reverse-phase C30/WM-C30, 5-lm column (250 9 4.6 mm; YMC, Wilmington, NC, USA) with mobile phases consisting of methanol (A), water/methanol (v: v = 20:80) containing 0.2% ammonium acetate (B) and tertmethyl butyl ether (C) were adopted. Samples were eluted from the column with a solvent profile of 95% A, 5% B isocratically for 12 min, followed by three linear gradient steps: 80% A, 5% B, 15% C for 5 min; 50% A, 5% B, 45% C for 10 min; 25% A, 5% B, 70% C for 10 min; then a step of 25% A, 5% B, 70% C isocratically for 13 min. Another linear gradient (5 min) was used to return the gradient elution to the initial concentrations of 95% A, 5% B, and this gradient was maintained for 15 min. A flow rate of 1 ml min 1 was adopted for all experiments. Expression profiles of carotenoid biosynthetic genes Total RNA was extracted from anthers and petals of both LA 2405 and P86 (15 plants, with five plants in each biological replicate) at five flower developmental stages using the RNAprep pure Plant Kit (TIANGEN, Beijing, China). This total RNA was used as the template for cDNA synthesis using the PrimeScriptTM RT Master Mix Kit (TaKaRa, Dalian, China). The cDNA was diluted 100-fold to performed real-time quantitative RT-PCR (qRT-PCR) using gene specific primers (Table S1) and the SYBR Premix Ex TaqTM II Kit (TaKaRa) according to the manufacturer’s instructions. The programme of the qRTPCR was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 5 s and 72 °C for 20 s, finally 72 °C for 5 min and 4 °C hold. Ubiquitin (GenBank: X58253.1) was used as the reference gene for normalisation of the data. Analysis of chromoplast ultrastructure Flowers of LA2405 and P86 were harvested from nine plants (three plants for each biological replicate) at five developmental stages, and the fresh petals and anthers were separately cut into small pieces (about 1 mm2), before being immediately immersed in a pre-cooled 2.5% glutaraldehyde solution containing 4% (v:v) paraformaldehyde in a 0.1 M phosphate buffer (PB, 0.1 M Na2HPO4/0.1 M NaH2PO4 = 68.4/31.6 (v:v), pH 7.2) at 4 °C overnight. The samples were washed three times for 20 min each with PB, and were fixed in 2% osmium tetroxide in PB (w/v) for 1.5 h, and then washed three times with PB. Subsequently, the samples were gradually dehydrated in an ethanol series (50%, 70%, 90% and 100%) for 10 min per grade. The samples were kept in a mixed solution of ethanol and acetone (v:v = 1 : 1) and then 100% pure acetone for 10 min each time. Finally, the samples were transferred to the following solutions: acetone:epoxy resin 812 (1:1, 1:2 and 1:3) for 1, 2 h
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and overnight before being embedded in epon resin 812 (Hede Biotechnology, Beijing, China). The embedded specimens were sectioned using a Leica UC6-FC6 microtome with a diamond knife. The sections (50–70 nm) were stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope (Tecnai G2 Spirit Biotwin, FEI Company, Hillsboro, OR, USA). RESULTS Tomato flower colours Sepals of P86 were larger than those of LA2405, and there were visible white villi inserted on the sepal external surface of P86 but not on LA2405. The long style of LA2405 was different from that of P86 since it exerted from the anther cone, and the bright yellow petals and anthers of LA2405 were distinctly different from those of P86, which were more orange/yellow in colour (Fig. 1). Changes in carotenoids during flower development The accumulation of five types of carotenoid (lycopene, a-carotene, lutein, b-carotene and zeaxanthin) in petals and anthers of both LA2405 and P86 was measured using HPLC. The content of lycopene was very low in petals and anthers of the two tomato accessions and was below the limit of detection using this method; however, the total content of the other four carotenoids showed a ‘lower–higher–lower’ trend in the petal during the 5DPA to 5 DPA time course, reaching a peak at 0 DPA. The total carotenoid content of LA2405 petals was significantly higher than that of P86 petals. In contrast, the content of total carotenoids in tomato anthers continually increased from 0 DPA, and the amounts of the four carotenoids were higher in P86 anthers than in those of LA2405 at all developmental stages (Fig. 2, Table S2). We also found that the total content of carotenoids per gram fresh weight in petals was higher than in anthers, and the contribution of each carotenoid B
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was different between petals and anthers (Table S2). In LA2405 petals, lutein and b-carotene comprised 36% and 58%, respectively, of the carotenoids, giving a total contribution of 94% (Fig. 2A, D), while the main component of the carotenoids in P86 petals was b-carotene (90%; Fig. 2D). Although content of b-carotene in LA2405 petals was similar to that of P86 (Fig. 2D), the lutein content in LA2405 petals was significantly higher than in P86, where it was not detectable (Fig. 2A). We hypothesised that lutein accumulation might be responsible for the difference in petal colour between LA2405 and P86. b-Carotene was the most abundant carotenoid in the anthers of both species, reaching 93% and 95% of the total carotenoids in LA2405 and P86, respectively. The content of b-carotene in P86 anthers was also notably higher than in LA2405 (Fig. 2D). The zeaxanthin content in P86 anthers was significantly higher than in LA2405, but overall levels were much lower than b-carotene levels in the anthers of both species (Fig. 2C, D). The difference in anther colour between LA2405 and P86 might therefore reflect the different contribution of b-carotene and zeaxanthin in the neoxanthin branch of carotenoid biosythesis (Fig. 2C, D, Table S2, Fig. S1). Expression of carotenoid biosynthetic genes Lycopene is located at a branch point in the carotenoid biosynthetic pathway leading to lutein or neoxanthin synthesis (Fig. S1), and qRT-PCR analysis showed that the level of expression of nine carotenoid synthetic genes: b-ring hydroxylase – CRTR-B and CRTR-B2, e-ring hydroxylase – CRTR-E, chromoplast-specific lycopene b-cyclase – CYC-B, lycopene bcyclase – LCY-B, lycopene e-cyclase – LCY-E, phytoene synthase – PSY1, phytoene desaturase – PDS and f-carotene desaturase – ZDS, collectively covering both branches, was higher in LA2405 petals than those of P86 over the 5DPA to 5 DPA time course (Fig. 3). In contrast, while expression levels of PSY1, LCY-B and CRTR-B2 were also higher in LA2405 than P86 anthers, the expression levels of the other six genes were higher in P86 than in LA2405 anthers (Fig. 4). We also found
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Fig. 1. Changes in flower colour of tomato LA2405 (A–E) and P86 (F–J) during developmental stages of tomato flowers ( 5 DPA, 5 DPA). DPA, days post-anthesis.
Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands
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Fig. 2. The content and composition of carotenoids in petals and anthers of LA2405 and P86 during the time course from lutein, a-carotene, zeaxathin and b-carotene, respectively.
that PDS, CYC-B, CRTR-E and ZDS showed almost tissue-specific patterns in petals (LA2405) and anthers (P86; Figs 3, 4). Although significant differences in expression of CRTR-B during the 5DPA to 5 DPA were not found between LA2405 and P86 anthers (Fig. 4), expression in LA2405 petals was significantly higher than in P86 (Fig. 3). Ultrastructure of chloroplasts and chromoplasts The differentiation of chloroplasts to chromoplasts in petals and anthers of both LA2405 (S. chilense) and P86 (S. lycopersicum) was examined by visualising their ultrastructure using TEM. The results showed that the internal membrane system gradually disassembled in both petals and anthers, with lysis of the inter-granal thylakoids and grana in the chloroplasts and chromoplasts. While starch grains disappeared and the size and amount of plastoglobules increased, changes in the envelope system were not evident in either chloroplasts or chromoplasts (Figs 5, 6). Generally, intact internal membrane systems were maintained in the chloroplasts until 3 DPA in both petals and anthers, and then the chloroplasts differentiated into chromoplasts, accompanied by disappearance of the internal membrane system and starch gains and an increase in the number of plastoglobules (Figs 5, 6). Moreover, the number of plastoglobules in petal chromoplasts was higher than in anthers for both LA2405 and P86, and the electron density gradually increased during flower development (Figs 5, 6). 4
5 DPA to 5 DPA. A to D indicate
DISCUSSION Domesticated tomato has a range of botanical features that differ from those of its wild relatives, some of which are important for reproduction. For example, S. chilense LA2405 and S. lycopersicum P86 have different mating systems, pistil morphology and flower colour, with LA2405 being bright yellow while P86 flowers are orange/yellow. Evolution of flower colour is related to the directional selection of pollinators (Rodrıguez-Bernaldo de Quir os & Costa 2006; Miller et al. 2011), and flower colour is also one of the most attractive traits in ornamental plants (Gutterson 1995). Furthermore, several natural pigments derived from plant leaves, fruits, vegetables and flowers are used as in medicines, foods, cosmetics and other products (Delgado-Vargas et al. 2000). For example, dried stigmas of saffron (Crocus sativus) flowers provide a dye that is used for natural colour application in the costly Pashmina fibre (Raja et al. 2012). Carotenoids and anthocyanins are used as additives and colourants in foods such as butter, beverages, dairy products, syrups, flour confectionery, salad dressings, etc. (Delgado-Vargas et al. 2000). In tomato and its relatives, formation of flower colour depends on accumulation of carotenoids (Isaacson et al. 2002; Miller et al. 2011), and hence analysis of carotenoid accumulation and the expression profile of the carotenoid pathway-associated genes can provide valuable insight into the mechanisms of flower colour formation.
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Gao, Qu, Gao, Chen, Sebastian & Zhao
Development of tomato flower colour
Fig. 3. Expression profile of carotenoid biosynthetic genes in petals of LA2405 and P86 during the time course from 5 DPA to 5 DPA. CRTR-B, CRTR-B2, CRTR-E, CYCB, LCYB, LC-E, PSY1, PDS and ZDS in the figures indicate genes related to the carotenoid synthesis pathway. The 5DPA, 3DPA, 0DPA, 3DPA and 5 DPA on the horizontal axis indicate the sampled times, and values on the vertical axis indicate relative expression levels of genes related to carotenoid synthesis.
Fig. 4. Expression profile of carotenoid biosynthetic genes in anthers of LA2405 and P86 during the time course from 5 DPA to 5 DPA. CRTR-B, CRTR-B2, CRTR-E, CYC-B, LCY-B, LCY-E, PSY1, PDS and ZDS indicate genes related to the carotenoid synthesis pathway. The 5DPA, 3DPA, 0DPA, 3DPA and 5 DPA on the horizontal axis indicate the sampled times, and values on the vertical axis indicate relative expression levels of genes related to carotenoid synthesis.
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A
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Fig. 5. Ultrastructure of the chloroplasts/chromoplasts in the petals of LA2405 and P86 at different developmental stages. A to D: ultrastructure of LA 2405 petals at 5DPA, 3DPA, 0 DPA and 5 DPA, respectively. E to H: ultrastructure of the P86 petals at 5DPA, 3DPA, 0 DPA and 5 DPA, respectively. v, vacuole; cw, cell wall; g, fully-developed grana; gc, golgi complex; m, mitochondrion; p, plastoglobules; pe, plastids envelope; s, starch granules; th, fully-developed grana thylakoids. Arrows, remnant membranes. Bars=500 nm (A, C, D and E) and 200 nm(B, F, G and H)
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Fig. 6. Ultrastructure of the chloroplasts/chromoplasts in anthers of LA2405 and P86 at different developmental stages. A to D: ultrastructure of LA 2405 anthers at 5DPA, 3DPA, 0 DPA and 5 DPA, respectively. E to H: ultrastructures of the P86 anthers at 5DPA, 3DPA, 0 DPA and 5 DPA, respectively. v, vacuole; cw, cell wall; g, fully-developed grana; gc, golgi complex; m, mitochondrion; p, plastoglobules; pe, plastids envelope; s, starch granules; th, fullydeveloped grana thylakoids. Arrows, remnant membranes. Bars=500 nm(A, B, D, G and H) and 200 nm (C, E and F).
Expression of carotenoid synthetic genes and accumulation of carotenoid compounds The carotenoid pathway consists of two branches, ending with either lutein or neoxanthin, with lycopene located at the branch point (Fig. S1). Cyclisation of lycopene is therefore a critical step and one that can lead to different products depending on the balance of enzymes involved. LCYE is exclusively expressed in green tissues and is involved in the synthesis of lutein, whereas CYC-B is expressed in flowers and is involved in the synthesis of zeaxanthin (Pecker et al. 1996; Ronen et al. 2000; Kato et al. 2004; Kishimoto & Ohmiya 2006; Yamamizo et al. 2009). Up-regulation of the CYC-B gene in the previously described b-mutation results in accumulation of the orange pigment b-carotene in fruits, while up-regulation of LCY-E in the d-mutation leads to accumulation of the yellow pigment d-carotene (Ronen et al. 1999, 2000). In tomato flowers, the major pigments are xanthophylls, lutein, neoxanthin 6
and violaxanthin, which together are responsible for up to 90% of the total carotenoid content (Bramley et al. 1992; Isaacson et al. 2002). In this study, expression levels of PDS, ZDS, LCY-B, CRTRB and CRTR-E in the lutein branch were significantly higher in LA2405 petals than in P86 (Fig. 3, Table S2). This suggests increased synthesis and accumulation of a-carotene and lutein, and indeed higher accumulation in LA2405 petals was confirmed using HPLC. Furthermore, the abundance of a-carotene and lutein in LA2405 petals peaked at 0 DPA, which is consistent with the development of flower colour (Figs 1, 2A, B). Although CYC-B was expressed at higher levels in LA2405 petals than in those of P86, which would presumably favour the conversion of lycopene into carotenoids in the neoxanthin branch, no significant differences in levels of zeaxanthin and b-carotene in the petals of LA2405 and P86 was detected (Fig. 2C, D). This suggests that differences in petal colour between LA2405 and P86 might be due to a difference in lutein
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Development of tomato flower colour
Gao, Qu, Gao, Chen, Sebastian & Zhao
accumulation. On the other hand, the expression levels of PDS, ZDS and CYC-B from the neoxanthin branch were higher in P86 anthers than in LA2405, which would promote the formation of zeaxanthin and b-carotene. This result was verified as higher content of b-carotene and zeaxanthin in P86 anthers, which peaked at 3 DPA, leading to orange-coloured anthers and petals (Figs 1, 2C, D). Although PDS, ZDS, LCY-E and CRTR-E from the lutein branch were expressed at a notably higher level in P86 than LA2405 anthers, no significant differences in the abundance of a-carotene and lutein between LA2405 and P86 were detected (Figs 2A, B, 4). These results suggest that anther colour might be associated with expression of genes in the neoxanthin branch and accumulation of b-carotene and zeaxanthin. Ultrastructure of chloroplasts and chromoplasts Chromoplasts are responsible for the synthesis and accumulation of carotenoids, and some of the enzymes in the carotenoid biosynthetic pathway, PSY, PDS and ZDS, have been identified in proteomic analysis of tomato chromoplasts (Barsan et al. 2010). The structural features of the chromoplast are distinctly different from those of the proplastid, chloroplast and amyloplast (Lopez-Juez & Pyke 2005; Horner et al. 2007; Egea et al. 2010). Thus, major changes are evident when the chloroplast converts to a chromoplast, such as disappearance of internal membranes, disassembly of grana and thylakoids and the appearance of plastoglobules, which are crystalloid structures formed from disrupted thylakoid membranes (Austin et al. 2006; Vasquez-Caicedo et al. 2006). Plastoglobules consists of carotenoids, proteins and lipids that promote solubilisation of lipophilic carotenoids, a feature that is important in their storage (Deruere et al. 1994; Bonora et al. 2000; Austin et al. 2006). Indeed, plastoglobules participate in both storage and formation of carotenoids in the chromoplasts and suppression of expression of the plastoglobule carotenoid-associated gene CHRC leads to a 30% reduction of carotenoids in tomato flowers, whereas over-expression of a pepper plastoglobule gene in tomato was reported to increase carotenoid content (LeitnerDagan et al. 2006; Simkin et al. 2007). In the early stages of tomato flower development, plastids of the petals and anthers in both LA2405 and P86 appeared as chloroplast amyloplasts, which were filled with starch granules, or with grana and thylakoids (Figs 5, 6). During development, the starch granules disappeared, the grana and thylakoids disassembled, accompanied by an increased number and size of plastoglobules, indicating conversion of plastids from chloroplast amyloplasts to chromoplasts (Figs 5, 6). We also determined that there were more plastoglobules in petals than in anthers and that their size increased during flower development, over 3 DPA to 5 DPA time course, in petals and anthers of both LA2405 and P86 (Figs 5, 6). Electron density REFERENCES Adalid A.M., Rosello S., Nuez F. (2010) Evaluation and selection of tomato accessions (Solanum section Lycopersicon) for content of lycopene, b-carotene and ascorbic acid. Journal of Food Composition and Analysis, 23, 613–618. Austin J.R., Frost E., Vidi P.A., Kessler F., Staehelin L.A. (2006) Plastoglobules are lipoprotein subcom-
analysis of the plastoglobules, as observed with TEM, indicated accumulation of carotenoids in the chromoplasts, and there was a correlation between weaker electronic density at 5 DPA than at 0 DPA and the discolouring of flowers that occurred after 3 DPA (Figs 1, 5). Finally, the increase in size and number of plastoglobules stained black with uranyl acetate and lead citrate is consistent with the accumulation of carotenoids and flower colour (Figs 1, 2, 5, 6). Taken together, the results from HPLC, qRT-PCR and ultrastructure analyses showed that tomato flower colour is mainly related to accumulation of carotenoids stored in the chromoplasts, and that this accumulation of carotenoids is regulated by the expression levels of carotenoid biosynthesis-associated genes, which are located at branches of lutein and neoxanthin synthesis, respectively. The differences in expression of these genes eventually result in differences in colour of petals and anthers in the two tomato species examined through differential regulation of carotenoid accumulation. ACKNOWLEDGEMENTS We thank TGRC and Dr. Wang Fu for providing tomato seeds. We appreciate help with TEM from the Instrumental Analysis Center of Shanghai Jiao Tong University. We thank Dr. Ming Xia (Cincinnati Children’s Hospital Medical Center, OH, USA) for help with revising the manuscript. We thank Prof. Rose (Cornell University) and PlantScribe (www.plantscribe.com) for editing this manuscript. The research was supported by funding from the National Natural Science Foundation of China (No. 31071810), the China National ‘863’ High-Tech Program (No. 2011AA100607) and the Key Technology Research and Development Program of Shanghai Science and Technology Committee (No. 13391901202). MFG and LXZ designed the research and wrote the manuscript. MFG made sections for electron microscopy, extracted and detected carotenoids, isolated RNA and performed real-time PCR analysis. HOQ extracted and detected carotenoids, LLC isolated RNA, GL performed real-time PCR and HPLC, and RSJS corrected the manuscript. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Sequences of specific PCR primers used in this study to examine expression profiles of tomato carotenoid biosynthetic genes. Table S2. Content of carotenoids in petals and anthers of LA2405 (S. chilense) and P86 (S. lycopersicum) in lg g 1 FW. Figure S1. Carotenoid biosynthetic pathway in tomato (Solanum lycopersicum) (redrawn from Fraser et al. 1999; Galpaz et al. 2006; Stigliani et al. 2011).
partments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes. Plant Cell, 18, 1693–1703. Barsan C., Sanchez-Bel P., Rombaldi C., Egea I., Rossignol M., Kuntz M., Zouine M., Latche A., Bouzayen M., Pech J.C. (2010) Characteristics of the tomato chromoplast proteome revealed by proteomic analysis. Journal of Experimental Botany, 61, 2413–2431.
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Bonora A., Pancaldi S., Gualandri R., Fasulo M.P. (2000) Carotenoid and ultrastructure variations in plastids of Arum italicum Miller fruit during maturation and ripening. Journal of Experimental Botany, 51, 873–884. Bramley P., Teulieres C., Blain I., Bird C., Schuch W. (1992) Biochemical characterisation of transgenic tomato plants in which carotenoid synthesis has been inhibited through expression of
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antisense RNA to pTOM5. Plant Journal, 2, 343–349. Craine J.M., Wolkovich E.M., Towne G.E., Kembe S.W. (2012) Flowering phenology as a functional trait in a tallgrass prairie. New Phytologist, 193, 673– 682. Delgado-Vargas F., Jimenez A.R., Paredes-L opez O. (2000) Natural pigments: carotenoids, anthocyanins, and betalains characteristics, biosynthesis, processing, and stability. Critical Reviews in Food Science and Nutrition, 40, 173–289. DellaPenna D., Pogson B.J. (2006) Vitamin synthesis in plants: tocopherols and carotenoids. Annual Review of Plant Biology, 57, 711–738. Deruere J., R€ omer S., d’Harlingue A., Backhaus R.A., Kuntz M., Camara B. (1994) Fibril assembly and carotenoid overaccumulation in chromoplasts: a model for supramolecular lipoprotein structures. Plant Cell, 6, 119–133. Egea I., Barsan C., Bian W., Purgatto E., Latche A., Chervin C., Bouzayen M., Pech J.C. (2010) Chromoplast differentiation: current status and perspectives. Plant Cell Physiology, 51, 1601–1611. Fraser P.D., Pinto M.E.S., Holloway D.E., Bramley P.M. (2000) Application of high-performance liquid chromatography with photodiode array detection to the metabolic profiling of plant isoprenoids. Plant Journal, 24, 551–558. Galpaz N., Ronen G., Khalfa Z., Zamir D., Hirschberg J. (2006) A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus. Plant Cell, 18, 1947–1960. Gutterson N. (1995) Anthocyanin biosynthetic genes and their application to flower color modification through sense suppression. HortScience, 30, 964– 966. Horner H.T., Healy R.A., Ren G., Fritz D., Klyne A., Seames C., Thornburg R.W. (2007) Amyloplast to chromoplast conversion in developing ornamental tobacco floral nectaries provides sugar for nectar and antioxidants for protection. American Journal of Botany, 94, 12–24. Horton P., Ruban A. (2005) Molecular design of the photosystem II light-harvesting antenna: photosynthesis and photoprotection. Journal of Experimental Botany, 56, 365–373. Isaacson T., Ronen G., Zamir D., Hirschberg J. (2002) Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of b-carotene and xanthophylls in plants. Plant Cell, 14, 333– 342. Jahns P., Holzwarth A.R. (2012) The role of the xanthophyll cycle and of lutein in photoprotection of
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photosystem II. Biochimica et Biophysica Acta-Bioenergetics, 1817, 182–193. Kato M., Ikoma Y., Matsumoto H., Sugiura M., Hyodo H., Yano M. (2004) Accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in citrus fruit. Plant Physiology, 134, 824–837. Kay Q.O.N., Daoud H.S., Stirton C.H. (1981) Pigment distribution, light reflection and cell structure in petals. Botanical Journal of the Linnean Society, 83, 57– 83. Kishimoto S., Ohmiya A. (2006) Regulation of carotenoid biosynthesis in petals and leaves of chrysanthemum (Chrysanthemum morifolium). Physiologia Plantarum, 128, 436–447. Leitner-Dagan Y., Ovadis M., Shklarman E., Elad Y., Rav David D., Vainstein A. (2006) Expression and functional analyses of the plastid lipid-associated protein CHRC suggest its role in chromoplastogenesis and stress. Plant Physiology, 142, 233–244. Li W., Chetelat R.T. (2010) A pollen factor linking inter- and intra-specific pollen rejection in tomato. Science, 330, 1827–1830. Ljubesic N., Wrischer M., Devise Z. (1991) Chromoplasts – the last stages in plastid development. International Journal of Developmental Biology, 35, 251– 258. Lopez-Juez E., Pyke K.A. (2005) Plastids unleashed: their development and their integration in plant development. International Journal of Developmental Biology, 49, 557–577. Marti M.C., Camejo D., Olmos E., Sandalio L.M., Fernandez-Garcia N., Jimenez A., Sevilla F. (2009) Characterisation and changes in the antioxidant system of chloroplasts and chromoplasts isolated from green and mature pepper fruits. Plant Biosystems, 11, 613–624. Miller R., Owens S.J., Rørslett B. (2011) Plants and color: flowers and pollination. Optics and Laser Technology, 43, 282–294. Nambara E., Marion-Poll A. (2005) Abscisic acid biosynthesis and catabolism. Annual Review of Plant Biology, 56, 65–85. Pascal A.A., Liu Z.F., Broess K., van Oort B., van Amerongen H., Wang C., Horton P., Robert B., Chang W., Ruban A. (2005) Molecular basis of photoprotection and control of photosynthetic light-harvesting. Nature, 436, 134–137. Pecker I., Gabbay R., Cunningham F., Hirschberg J. (1996) Cloning and characterization of the cDNA for lycopene b-cylcase from tomato reveals decrease in its expression during fruit ripening. Plant Molecular Biology, 30, 807–819.
Phillips M.A., Le on P., Boronat A., Rodrıguez-Concepci on M. (2008) The plastidial MEP pathway: unified nomenclature and resources. Trends in Plant Science, 13, 619–623. Raja A.S.M., Pareek P.K., Shakyawar D.B., Wani S.A., Nehvi F.A., Sofi A.H. (2012) Extraction of natural dye from saffron flower waste and its application on pashmina fabric. Advances in Applied Science Research, 3, 156–161. Rodrıguez-Bernaldo de Quir os A., Costa H.S. (2006) Analysis of carotenoids in vegetables and plasma samples: a review. Journal of Food Composition and Analysis, 19, 97–111. Ronen G., Cohen M., Zamir D., Hirschberg J. (1999) Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant Delta. Plant Journal, 17, 341–351. Ronen G., Carmel-Goren L., Zamir D., Hirschberg J. (2000) An alternative pathway to b-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato. Proceedings of the National Academy of Sciences, USA, 97, 11102–11107. Simkin A.J., Gaffe J., Alcaraz J.P., Carde J.P., Bramley P.M., Fraser P.D., Kuntz M. (2007) Fibrillin influence on plastid ultrastructure and pigment content in tomato fruit. Phytochemistry, 68, 1545–1556. Thakur P., Bhatnagar A.K. (2013) Pollination constraints in flowering plants – Human actions undoing over hundred million years of co-evolution and posing an unprecedented threat to biodiversity. International Journal of Plant Reproductive Biology, 5, 29–74. Vasquez-Caicedo A.L., Heller A., Neidhart S., Carle R. (2006) Chromoplast morphology and b-carotene accumulation during postharvest ripening of mango cv. ‘Tommy Atkins’. Journal of Agricultural and Food Chemistry, 54, 5769–5776. Vishnevetsky M., Ovadis M., Vainstein A. (1999) Carotenoid sequestration in plants: the role of carotenoid associated proteins. Trends in Plant Science, 4, 232–235. Yamamizo C., Kishimoto S., Ohmiya A. (2009) Carotenoid composition and carotenogenic gene expression during Ipomoea petal development. Journal of Experimental Botany, 61, 709–719. Zimmerman M. (1988) Nectar production, flowering phenology, and strategies for pollination. In: Lovett Droust J., Lovett Droust L. (Eds), Plant Reproductive Ecology: Patterns and Strategies. Oxford University Press, New York, USA, pp 157–178.
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RESEARCH PAPER
Dissecting the mechanism of Solanum lycopersicum and Solanum chilense flower colour formation M. Gao1,2,3, H. Qu1,2, L. Gao1,2, L. Chen1,2, R. S. J. Sebastian1,2 & L. Zhao1,2,3 1 Joint Tomato Research Institute, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China 2 Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China 3 Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
Keywords Carotenoids; chromoplast; flower colour; plastoglobule; structural gene; tomato. Correspondence L. Zhao, 800 Dongchuan Road, Shanghai 200240, China. E-mail: [email protected] Editor K. Leiss Received: 5 November 2013; Accepted: 4 March 2014 doi:10.1111/plb.12186
ABSTRACT Flowers are the defining feature of angiosperms, and function as indispensable organs for sexual reproduction. Flower colour typically plays an important role in attracting pollinators, and can show considerable variation, even between closely related species. For example, domesticated tomato (S. lycopersicum) has orange/yellow flowers, while the wild relative S. chilense (accession LA2405) has bright yellow flowers. In this study, the mechanism of flower colour formation in these two species was compared by evaluating the accumulation of carotenoids, assessing the expression genes related to carotenoid biosynthetic pathways and observing chromoplast ultrastructure. In S. chilense petals, genes associated with the lutein branch of the carotenoid biosynthetic pathway, phytoene desaturase (PDS), f-carotene desaturase (ZDS), lycopene b-cyclase (LCY-B), b-ring hydroxylase (CRTR-B) and e-ring hydroxylase (CRTR-E), were highly expressed, and this was correlated with high levels of lutein accumulation. In contrast, PDS, ZDS and CYC-B from the neoxanthin biosynthetic branch were highly expressed in S. lycopersicum anthers, leading to increased b-carotene accumulation and hence an orange/yellow colour. Changes in the size, amount and electron density of plastoglobules in chromoplasts provided further evidence of carotenoid accumulation and flower colour formation. Taken together, these results reveal the biochemical basis of differences in carotenoid pigment accumulation and colour between petals and anthers in tomato.
INTRODUCTION As the sexual reproductive organ of angiosperms, flowers have evolved several remarkable traits and mating systems to promote effective reproduction (Zimmerman 1988; Li & Chetelat 2010; Craine et al. 2012; Thakur & Bhatnagar 2013). The development of insect-attracting (entomophilous) and bird-attracting (ornithophilous) flowers ended an era where successful plant pollination relied only on wind and water, a landmark event in the evolution of flowering plants. Key floral features used to attract pollinators, and which are also present in fruits to attract frugivores and promote seed dispersal, include bright colour, aroma, volatiles and accumulation of energy-rich compounds, which are present in nectar (Miller et al. 2011). Flower colour depends on the composition and content of pigments, e.g. carotenoids, anthocyanins and betalains (Kay et al. 1981; Delgado-Vargas et al. 2000; Miller et al. 2011). Carotenoids are C40 terpenoids and are widespread in plants, where they play many roles, including in photosynthetic reactions in chloroplasts. Carotenoids are also one of the most important pigments in flowers and fruits (Vishnevetsky et al. 1999; Isaacson et al. 2002; Miller et al. 2011), and help prevent photo-oxidative damage (Horton & Ruban 2005; Pascal et al. 2005; Jahns & Holzwarth 2012). Carotenoids also serve as precursors for abscisic acid (ABA) and vitamin A (Nambara & Marion-Poll 2005; DellaPenna & Pogson 2006; Barsan et al.
2010), and provide dietary components that are beneficial for human health. For example, studies have showed that lycopene, which is present at high levels in tomato fruits, has antioxidant activity and helps prevent cancers and cardiocerebrovascular disease (Marti et al. 2009; Adalid et al. 2010; Barsan et al. 2010). Carotenoids are stored at high concentrations in chromoplasts of both flowers and fruits, where they provide colours from yellow to orange and red. Chromoplasts are non-photosynthetic plastids that function as specialised organelles for synthesis and accumulation of carotenoids. They can be divided into four types: globular, tubular, membranous and crystalloid, according to shape, sub-organelle structure and types present during development in flowers and fruits (Ljubesic et al. 1991). The steps of the carotenoid biosynthesis pathway have previously been identified. The first dedicated step is the formation of geranylgeranyl diphosphate (GGPP), which involves the condensation of isopentenyl pyrophosphate and farnesyl diphosphate in the methylerythritol phosphate (MEP) pathway. Two GGPP molecules are condensed by phytoene synthase (PSY) to form phytoene, which is then converted into lycopene via four consecutive dehydrogenation reactions carried out by phytoene desaturase (PDS) and f-carotene desaturase (ZDS). Lycopene is at a key branch point in the carotenoid biosynthesis pathway, since linear lycopene can be cyclised to
Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands
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b-carotene (all trans b-carotene) or a-carotene (b-, e-carotene) by lycopene b-cyclase (LCY-B) or lycopene e-cyclase (LCY-E), respectively. In the lutein branch, a-carotene and b-carotene are then converted to lutein and zeaxanthin via consecutive hydroxylation of the e- and b-rings, respectively. Zeaxanthin is then subjected to epoxidation at the b-ring, and converted to violaxanthin via the intermediate antheraxanthin. Finally, violaxanthin is converted to neoxanthin by neoxanthin synthase (NSY) in the neoxanthin branch (Fig. S1; Ronen et al. 1999, 2000; DellaPenna & Pogson 2006; Galpaz et al. 2006; Phillips et al. 2008). In this study, we investigated the molecular basis of differences in flower colour between domesticated S. lycopersicum (cultivar P86) and its wild relative S. chilense (accession LA2405). To dissect mechanisms for formation of flower colour, we evaluated carotenoid content, expression of genes encoding key step enzymes in the carotenoid biosythesis pathway and chromoplast ultrastructure. MATERIAL AND METHODS Plant material Seeds of LA2405 (S. chilense) and P86 (S. lycopersicum) were kindly provided by the Tomato Genetics Resource Center (TGRC, http://tgrc.ucdavis.edu) and Dr. Wang Fu (Qingdao Agricultural University, China). Tomato seeds were sown between two pieces of humid filter paper in Petri dishes, and left to germinate at 25 °C in the dark. The germinated seeds were transferred to plastic pots (diameter 7.5 cm) with seedling soil, and planted in 5-l plastic pots with soil when the fourth true-leaf had fully expanded. The plants were grown in a greenhouse (25 °C day/18 °C night, 70% air humidity and natural light) in the School of Agriculture and Biology, Shanghai Jiao Tong University, China. Petals and anthers were harvested at five flower developmental stages: 5 days pre-anthesis ( 5DPA), 3 days preanthesis ( 3 DPA), 0 days post-anthesis (0 DPA), 3 days postanthesis (3DPA) and 5 days post-anthesis (5 DPA). Extraction and detection of carotenoids The extraction, separation and detection of tomato flower carotenoids were performed as described in Fraser et al. (2000). All steps were carried out on ice and shielded from strong light. Flowers of both LA2405 and P86 were harvested from 15 plants (each biological replicate included five plants) at intervals from 5 DPA to 5 DPA (days post-anthesis; as above). Anthers and petals were ground separately into a fine powder in a mortar with liquid nitrogen. Approximately 200 mg of the powder were transferred to a 15-ml conical Falcon centrifuge tube with 1.5 ml extraction buffer (60% KOH (w/v):methanol in a 1:9 ratio) and the samples were mixed using a vortexer. The mixture was incubated in a water bath at 60 °C for 30 min, before 1.5 ml Tris-HCl (50 mM, pH 7.5, containing 1 M NaCl) was added and the samples incubated at 4 °C for 10 min. After addition of 4 ml chloroform and thorough vortexing, the samples were transferred to an ice bath for 10 min for carotenoid extraction. The organic phase of the lower layer was carefully transferred into a clean centrifuge tube using a disposable syringe after centrifugation at 3000 g for 5 min, and the aqueous phase was re-extracted with 4 ml chloroform. The two 2
collected organic phases were concentrated in Nitrogen Evaporators (MD200-2; Zhe Jiang, China), and the dried residues were dissolved in 800 ll hexane. Standards of lutein and zeaxanthin were dissolved in methanol, a-carotene and b-carotene were dissolved in methyl tert-butyl ether and lycopene was dissolved in hexane. The a-carotene standard was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the other four standards were purchased from Sigma Chemicals (St. Louis, MO, USA). Separation and detection were carried out in a Hitachi L-2000 control system (Hitachi, Tokyo, Japan). Throughout chromatography, the eluate was monitored continuously from 220 to 500 nm. A reverse-phase C30/WM-C30, 5-lm column (250 9 4.6 mm; YMC, Wilmington, NC, USA) with mobile phases consisting of methanol (A), water/methanol (v: v = 20:80) containing 0.2% ammonium acetate (B) and tertmethyl butyl ether (C) were adopted. Samples were eluted from the column with a solvent profile of 95% A, 5% B isocratically for 12 min, followed by three linear gradient steps: 80% A, 5% B, 15% C for 5 min; 50% A, 5% B, 45% C for 10 min; 25% A, 5% B, 70% C for 10 min; then a step of 25% A, 5% B, 70% C isocratically for 13 min. Another linear gradient (5 min) was used to return the gradient elution to the initial concentrations of 95% A, 5% B, and this gradient was maintained for 15 min. A flow rate of 1 ml min 1 was adopted for all experiments. Expression profiles of carotenoid biosynthetic genes Total RNA was extracted from anthers and petals of both LA 2405 and P86 (15 plants, with five plants in each biological replicate) at five flower developmental stages using the RNAprep pure Plant Kit (TIANGEN, Beijing, China). This total RNA was used as the template for cDNA synthesis using the PrimeScriptTM RT Master Mix Kit (TaKaRa, Dalian, China). The cDNA was diluted 100-fold to performed real-time quantitative RT-PCR (qRT-PCR) using gene specific primers (Table S1) and the SYBR Premix Ex TaqTM II Kit (TaKaRa) according to the manufacturer’s instructions. The programme of the qRTPCR was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 5 s and 72 °C for 20 s, finally 72 °C for 5 min and 4 °C hold. Ubiquitin (GenBank: X58253.1) was used as the reference gene for normalisation of the data. Analysis of chromoplast ultrastructure Flowers of LA2405 and P86 were harvested from nine plants (three plants for each biological replicate) at five developmental stages, and the fresh petals and anthers were separately cut into small pieces (about 1 mm2), before being immediately immersed in a pre-cooled 2.5% glutaraldehyde solution containing 4% (v:v) paraformaldehyde in a 0.1 M phosphate buffer (PB, 0.1 M Na2HPO4/0.1 M NaH2PO4 = 68.4/31.6 (v:v), pH 7.2) at 4 °C overnight. The samples were washed three times for 20 min each with PB, and were fixed in 2% osmium tetroxide in PB (w/v) for 1.5 h, and then washed three times with PB. Subsequently, the samples were gradually dehydrated in an ethanol series (50%, 70%, 90% and 100%) for 10 min per grade. The samples were kept in a mixed solution of ethanol and acetone (v:v = 1 : 1) and then 100% pure acetone for 10 min each time. Finally, the samples were transferred to the following solutions: acetone:epoxy resin 812 (1:1, 1:2 and 1:3) for 1, 2 h
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and overnight before being embedded in epon resin 812 (Hede Biotechnology, Beijing, China). The embedded specimens were sectioned using a Leica UC6-FC6 microtome with a diamond knife. The sections (50–70 nm) were stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope (Tecnai G2 Spirit Biotwin, FEI Company, Hillsboro, OR, USA). RESULTS Tomato flower colours Sepals of P86 were larger than those of LA2405, and there were visible white villi inserted on the sepal external surface of P86 but not on LA2405. The long style of LA2405 was different from that of P86 since it exerted from the anther cone, and the bright yellow petals and anthers of LA2405 were distinctly different from those of P86, which were more orange/yellow in colour (Fig. 1). Changes in carotenoids during flower development The accumulation of five types of carotenoid (lycopene, a-carotene, lutein, b-carotene and zeaxanthin) in petals and anthers of both LA2405 and P86 was measured using HPLC. The content of lycopene was very low in petals and anthers of the two tomato accessions and was below the limit of detection using this method; however, the total content of the other four carotenoids showed a ‘lower–higher–lower’ trend in the petal during the 5DPA to 5 DPA time course, reaching a peak at 0 DPA. The total carotenoid content of LA2405 petals was significantly higher than that of P86 petals. In contrast, the content of total carotenoids in tomato anthers continually increased from 0 DPA, and the amounts of the four carotenoids were higher in P86 anthers than in those of LA2405 at all developmental stages (Fig. 2, Table S2). We also found that the total content of carotenoids per gram fresh weight in petals was higher than in anthers, and the contribution of each carotenoid B
A
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was different between petals and anthers (Table S2). In LA2405 petals, lutein and b-carotene comprised 36% and 58%, respectively, of the carotenoids, giving a total contribution of 94% (Fig. 2A, D), while the main component of the carotenoids in P86 petals was b-carotene (90%; Fig. 2D). Although content of b-carotene in LA2405 petals was similar to that of P86 (Fig. 2D), the lutein content in LA2405 petals was significantly higher than in P86, where it was not detectable (Fig. 2A). We hypothesised that lutein accumulation might be responsible for the difference in petal colour between LA2405 and P86. b-Carotene was the most abundant carotenoid in the anthers of both species, reaching 93% and 95% of the total carotenoids in LA2405 and P86, respectively. The content of b-carotene in P86 anthers was also notably higher than in LA2405 (Fig. 2D). The zeaxanthin content in P86 anthers was significantly higher than in LA2405, but overall levels were much lower than b-carotene levels in the anthers of both species (Fig. 2C, D). The difference in anther colour between LA2405 and P86 might therefore reflect the different contribution of b-carotene and zeaxanthin in the neoxanthin branch of carotenoid biosythesis (Fig. 2C, D, Table S2, Fig. S1). Expression of carotenoid biosynthetic genes Lycopene is located at a branch point in the carotenoid biosynthetic pathway leading to lutein or neoxanthin synthesis (Fig. S1), and qRT-PCR analysis showed that the level of expression of nine carotenoid synthetic genes: b-ring hydroxylase – CRTR-B and CRTR-B2, e-ring hydroxylase – CRTR-E, chromoplast-specific lycopene b-cyclase – CYC-B, lycopene bcyclase – LCY-B, lycopene e-cyclase – LCY-E, phytoene synthase – PSY1, phytoene desaturase – PDS and f-carotene desaturase – ZDS, collectively covering both branches, was higher in LA2405 petals than those of P86 over the 5DPA to 5 DPA time course (Fig. 3). In contrast, while expression levels of PSY1, LCY-B and CRTR-B2 were also higher in LA2405 than P86 anthers, the expression levels of the other six genes were higher in P86 than in LA2405 anthers (Fig. 4). We also found
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Fig. 1. Changes in flower colour of tomato LA2405 (A–E) and P86 (F–J) during developmental stages of tomato flowers ( 5 DPA, 5 DPA). DPA, days post-anthesis.
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3 DPA, 0 DPA, 3 DPA and
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A
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Fig. 2. The content and composition of carotenoids in petals and anthers of LA2405 and P86 during the time course from lutein, a-carotene, zeaxathin and b-carotene, respectively.
that PDS, CYC-B, CRTR-E and ZDS showed almost tissue-specific patterns in petals (LA2405) and anthers (P86; Figs 3, 4). Although significant differences in expression of CRTR-B during the 5DPA to 5 DPA were not found between LA2405 and P86 anthers (Fig. 4), expression in LA2405 petals was significantly higher than in P86 (Fig. 3). Ultrastructure of chloroplasts and chromoplasts The differentiation of chloroplasts to chromoplasts in petals and anthers of both LA2405 (S. chilense) and P86 (S. lycopersicum) was examined by visualising their ultrastructure using TEM. The results showed that the internal membrane system gradually disassembled in both petals and anthers, with lysis of the inter-granal thylakoids and grana in the chloroplasts and chromoplasts. While starch grains disappeared and the size and amount of plastoglobules increased, changes in the envelope system were not evident in either chloroplasts or chromoplasts (Figs 5, 6). Generally, intact internal membrane systems were maintained in the chloroplasts until 3 DPA in both petals and anthers, and then the chloroplasts differentiated into chromoplasts, accompanied by disappearance of the internal membrane system and starch gains and an increase in the number of plastoglobules (Figs 5, 6). Moreover, the number of plastoglobules in petal chromoplasts was higher than in anthers for both LA2405 and P86, and the electron density gradually increased during flower development (Figs 5, 6). 4
5 DPA to 5 DPA. A to D indicate
DISCUSSION Domesticated tomato has a range of botanical features that differ from those of its wild relatives, some of which are important for reproduction. For example, S. chilense LA2405 and S. lycopersicum P86 have different mating systems, pistil morphology and flower colour, with LA2405 being bright yellow while P86 flowers are orange/yellow. Evolution of flower colour is related to the directional selection of pollinators (Rodrıguez-Bernaldo de Quir os & Costa 2006; Miller et al. 2011), and flower colour is also one of the most attractive traits in ornamental plants (Gutterson 1995). Furthermore, several natural pigments derived from plant leaves, fruits, vegetables and flowers are used as in medicines, foods, cosmetics and other products (Delgado-Vargas et al. 2000). For example, dried stigmas of saffron (Crocus sativus) flowers provide a dye that is used for natural colour application in the costly Pashmina fibre (Raja et al. 2012). Carotenoids and anthocyanins are used as additives and colourants in foods such as butter, beverages, dairy products, syrups, flour confectionery, salad dressings, etc. (Delgado-Vargas et al. 2000). In tomato and its relatives, formation of flower colour depends on accumulation of carotenoids (Isaacson et al. 2002; Miller et al. 2011), and hence analysis of carotenoid accumulation and the expression profile of the carotenoid pathway-associated genes can provide valuable insight into the mechanisms of flower colour formation.
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Development of tomato flower colour
Fig. 3. Expression profile of carotenoid biosynthetic genes in petals of LA2405 and P86 during the time course from 5 DPA to 5 DPA. CRTR-B, CRTR-B2, CRTR-E, CYCB, LCYB, LC-E, PSY1, PDS and ZDS in the figures indicate genes related to the carotenoid synthesis pathway. The 5DPA, 3DPA, 0DPA, 3DPA and 5 DPA on the horizontal axis indicate the sampled times, and values on the vertical axis indicate relative expression levels of genes related to carotenoid synthesis.
Fig. 4. Expression profile of carotenoid biosynthetic genes in anthers of LA2405 and P86 during the time course from 5 DPA to 5 DPA. CRTR-B, CRTR-B2, CRTR-E, CYC-B, LCY-B, LCY-E, PSY1, PDS and ZDS indicate genes related to the carotenoid synthesis pathway. The 5DPA, 3DPA, 0DPA, 3DPA and 5 DPA on the horizontal axis indicate the sampled times, and values on the vertical axis indicate relative expression levels of genes related to carotenoid synthesis.
Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands
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A
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Fig. 5. Ultrastructure of the chloroplasts/chromoplasts in the petals of LA2405 and P86 at different developmental stages. A to D: ultrastructure of LA 2405 petals at 5DPA, 3DPA, 0 DPA and 5 DPA, respectively. E to H: ultrastructure of the P86 petals at 5DPA, 3DPA, 0 DPA and 5 DPA, respectively. v, vacuole; cw, cell wall; g, fully-developed grana; gc, golgi complex; m, mitochondrion; p, plastoglobules; pe, plastids envelope; s, starch granules; th, fully-developed grana thylakoids. Arrows, remnant membranes. Bars=500 nm (A, C, D and E) and 200 nm(B, F, G and H)
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H
Fig. 6. Ultrastructure of the chloroplasts/chromoplasts in anthers of LA2405 and P86 at different developmental stages. A to D: ultrastructure of LA 2405 anthers at 5DPA, 3DPA, 0 DPA and 5 DPA, respectively. E to H: ultrastructures of the P86 anthers at 5DPA, 3DPA, 0 DPA and 5 DPA, respectively. v, vacuole; cw, cell wall; g, fully-developed grana; gc, golgi complex; m, mitochondrion; p, plastoglobules; pe, plastids envelope; s, starch granules; th, fullydeveloped grana thylakoids. Arrows, remnant membranes. Bars=500 nm(A, B, D, G and H) and 200 nm (C, E and F).
Expression of carotenoid synthetic genes and accumulation of carotenoid compounds The carotenoid pathway consists of two branches, ending with either lutein or neoxanthin, with lycopene located at the branch point (Fig. S1). Cyclisation of lycopene is therefore a critical step and one that can lead to different products depending on the balance of enzymes involved. LCYE is exclusively expressed in green tissues and is involved in the synthesis of lutein, whereas CYC-B is expressed in flowers and is involved in the synthesis of zeaxanthin (Pecker et al. 1996; Ronen et al. 2000; Kato et al. 2004; Kishimoto & Ohmiya 2006; Yamamizo et al. 2009). Up-regulation of the CYC-B gene in the previously described b-mutation results in accumulation of the orange pigment b-carotene in fruits, while up-regulation of LCY-E in the d-mutation leads to accumulation of the yellow pigment d-carotene (Ronen et al. 1999, 2000). In tomato flowers, the major pigments are xanthophylls, lutein, neoxanthin 6
and violaxanthin, which together are responsible for up to 90% of the total carotenoid content (Bramley et al. 1992; Isaacson et al. 2002). In this study, expression levels of PDS, ZDS, LCY-B, CRTRB and CRTR-E in the lutein branch were significantly higher in LA2405 petals than in P86 (Fig. 3, Table S2). This suggests increased synthesis and accumulation of a-carotene and lutein, and indeed higher accumulation in LA2405 petals was confirmed using HPLC. Furthermore, the abundance of a-carotene and lutein in LA2405 petals peaked at 0 DPA, which is consistent with the development of flower colour (Figs 1, 2A, B). Although CYC-B was expressed at higher levels in LA2405 petals than in those of P86, which would presumably favour the conversion of lycopene into carotenoids in the neoxanthin branch, no significant differences in levels of zeaxanthin and b-carotene in the petals of LA2405 and P86 was detected (Fig. 2C, D). This suggests that differences in petal colour between LA2405 and P86 might be due to a difference in lutein
Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands
Development of tomato flower colour
Gao, Qu, Gao, Chen, Sebastian & Zhao
accumulation. On the other hand, the expression levels of PDS, ZDS and CYC-B from the neoxanthin branch were higher in P86 anthers than in LA2405, which would promote the formation of zeaxanthin and b-carotene. This result was verified as higher content of b-carotene and zeaxanthin in P86 anthers, which peaked at 3 DPA, leading to orange-coloured anthers and petals (Figs 1, 2C, D). Although PDS, ZDS, LCY-E and CRTR-E from the lutein branch were expressed at a notably higher level in P86 than LA2405 anthers, no significant differences in the abundance of a-carotene and lutein between LA2405 and P86 were detected (Figs 2A, B, 4). These results suggest that anther colour might be associated with expression of genes in the neoxanthin branch and accumulation of b-carotene and zeaxanthin. Ultrastructure of chloroplasts and chromoplasts Chromoplasts are responsible for the synthesis and accumulation of carotenoids, and some of the enzymes in the carotenoid biosynthetic pathway, PSY, PDS and ZDS, have been identified in proteomic analysis of tomato chromoplasts (Barsan et al. 2010). The structural features of the chromoplast are distinctly different from those of the proplastid, chloroplast and amyloplast (Lopez-Juez & Pyke 2005; Horner et al. 2007; Egea et al. 2010). Thus, major changes are evident when the chloroplast converts to a chromoplast, such as disappearance of internal membranes, disassembly of grana and thylakoids and the appearance of plastoglobules, which are crystalloid structures formed from disrupted thylakoid membranes (Austin et al. 2006; Vasquez-Caicedo et al. 2006). Plastoglobules consists of carotenoids, proteins and lipids that promote solubilisation of lipophilic carotenoids, a feature that is important in their storage (Deruere et al. 1994; Bonora et al. 2000; Austin et al. 2006). Indeed, plastoglobules participate in both storage and formation of carotenoids in the chromoplasts and suppression of expression of the plastoglobule carotenoid-associated gene CHRC leads to a 30% reduction of carotenoids in tomato flowers, whereas over-expression of a pepper plastoglobule gene in tomato was reported to increase carotenoid content (LeitnerDagan et al. 2006; Simkin et al. 2007). In the early stages of tomato flower development, plastids of the petals and anthers in both LA2405 and P86 appeared as chloroplast amyloplasts, which were filled with starch granules, or with grana and thylakoids (Figs 5, 6). During development, the starch granules disappeared, the grana and thylakoids disassembled, accompanied by an increased number and size of plastoglobules, indicating conversion of plastids from chloroplast amyloplasts to chromoplasts (Figs 5, 6). We also determined that there were more plastoglobules in petals than in anthers and that their size increased during flower development, over 3 DPA to 5 DPA time course, in petals and anthers of both LA2405 and P86 (Figs 5, 6). Electron density REFERENCES Adalid A.M., Rosello S., Nuez F. (2010) Evaluation and selection of tomato accessions (Solanum section Lycopersicon) for content of lycopene, b-carotene and ascorbic acid. Journal of Food Composition and Analysis, 23, 613–618. Austin J.R., Frost E., Vidi P.A., Kessler F., Staehelin L.A. (2006) Plastoglobules are lipoprotein subcom-
analysis of the plastoglobules, as observed with TEM, indicated accumulation of carotenoids in the chromoplasts, and there was a correlation between weaker electronic density at 5 DPA than at 0 DPA and the discolouring of flowers that occurred after 3 DPA (Figs 1, 5). Finally, the increase in size and number of plastoglobules stained black with uranyl acetate and lead citrate is consistent with the accumulation of carotenoids and flower colour (Figs 1, 2, 5, 6). Taken together, the results from HPLC, qRT-PCR and ultrastructure analyses showed that tomato flower colour is mainly related to accumulation of carotenoids stored in the chromoplasts, and that this accumulation of carotenoids is regulated by the expression levels of carotenoid biosynthesis-associated genes, which are located at branches of lutein and neoxanthin synthesis, respectively. The differences in expression of these genes eventually result in differences in colour of petals and anthers in the two tomato species examined through differential regulation of carotenoid accumulation. ACKNOWLEDGEMENTS We thank TGRC and Dr. Wang Fu for providing tomato seeds. We appreciate help with TEM from the Instrumental Analysis Center of Shanghai Jiao Tong University. We thank Dr. Ming Xia (Cincinnati Children’s Hospital Medical Center, OH, USA) for help with revising the manuscript. We thank Prof. Rose (Cornell University) and PlantScribe (www.plantscribe.com) for editing this manuscript. The research was supported by funding from the National Natural Science Foundation of China (No. 31071810), the China National ‘863’ High-Tech Program (No. 2011AA100607) and the Key Technology Research and Development Program of Shanghai Science and Technology Committee (No. 13391901202). MFG and LXZ designed the research and wrote the manuscript. MFG made sections for electron microscopy, extracted and detected carotenoids, isolated RNA and performed real-time PCR analysis. HOQ extracted and detected carotenoids, LLC isolated RNA, GL performed real-time PCR and HPLC, and RSJS corrected the manuscript. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Sequences of specific PCR primers used in this study to examine expression profiles of tomato carotenoid biosynthetic genes. Table S2. Content of carotenoids in petals and anthers of LA2405 (S. chilense) and P86 (S. lycopersicum) in lg g 1 FW. Figure S1. Carotenoid biosynthetic pathway in tomato (Solanum lycopersicum) (redrawn from Fraser et al. 1999; Galpaz et al. 2006; Stigliani et al. 2011).
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