Effect of the Cnr mutation on carotenoid formation during tomato fruit ripening

Phytochemistry 58 (2001) 75–79 www.elsevier.com/locate/phytochem

Effect of the Cnr mutation on carotenoid formation during tomato fruit ripening P.D. Frasera, P. Bramleya, G.B. Seymourb,* a

School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 OEX, UK b Horticulture research International, Wellesbourne, Warwick CV35 9EF, UK Received 13 December 2000; received in revised form 27 March 2001

Abstract The characteristic pigmentation of ripe tomato fruit is due to the deposition of carotenoid pigments. In tomato, numerous colour mutants exist. The Cnr tomato mutant has a colourless, non-ripening phenotype. In this work, carotenoid formation in the Cnr mutant has been studied at the biochemical level. The carotenoid composition of Ailsa Craig (AC) and Cnr leaves was qualitatively and quantitatively similar. However, Cnr fruits had low levels of total carotenoids and lacked detectable levels of phytoene and lycopene. The presence of normal tocopherols and ubiquinone-9 levels in the ripe Cnr fruits suggested that other biosynthetically related isoprenoids were unaffected by the alterations to carotenoid biosynthesis. In vitro assays confirmed the virtual absence of phytoene synthesis in the ripe Cnr fruit. Extracts from ripe fruit of the Cnr mutant also revealed a reduced ability to synthesise the carotenoid precursor geranylgeranyl diphosphate (GGPP). These results suggest that besides affecting the first committed step in carotenoid biosynthesis (phytoene synthase) the Cnr mutation also affects the formation of the isoprenoid precursor (GGPP). # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Lycopersicon esculentum; Solanaceae; Tomato ripening; Pleiotropic mutant; Pigments

1. Introduction Carotenoid pigments are synthesised by all higher plants, algae, and some bacteria and fungi (Britton et al., 1995). Although essential for human health, carotenoids can not be synthesised de novo in the body and must be acquired from the diet (Parker, 1996). Tomato fruit and its products (e.g. ketchup, juices, soups and sauces) are the principal source of carotenoids in the western diet. Lycopene, the predominant carotenoid found in tomato, is a powerful antioxidant and may prevent the onset of serious disease states such as prostate cancer (Giovannucci et al., 1995). In addition, bcarotene is a provitamin A carotenoid and its deficiency can cause xerophthalmia, blindness, and premature death (Mayne, 1996). It is not surprising therefore, that the regulation of carotenoid biosynthesis in higher plants and especially crop plants, has become an area of considerable interest. * Corresponding author. Tel.: +44-1789-470382; fax: +44-1789470552. E-mail address: [email protected] (G.B. Seymour).

In higher plants, carotenoid biosynthesis and sequestration occurs in the plastid. In photosynthetic tissues, this occurs in the chloroplasts, while in non-photosynthetic plant tissues such as fruits and flowers carotenoid accumulation occurs in chromoplasts. Carotenoid formation utilizes the ubiquitous isoprenoid precursor geranylgeranyl pyrophosphate (GGPP). Two molecules of GGPP are condensed to form phytoene and this reaction is catalysed by phytoene synthase (PSY). Phytoene is then converted to lycopene in a series of dehydrogenation reactions, which introduce four double bonds into the phytoene molecule. This conversion is performed by the sequential action of phytoene and z-carotene desaturase enzymes respectively. The biosynthesis of b-carotene and a-carotene and xanthophylls then proceeds following the scheme shown in Fig. 1. Genes encoding the majority of the enzymes in the carotenoid biosynthetic pathway have been cloned. The ripening enhanced cDNA pTOM5 was identified as Psy-1 in transgenic experiments (Bramley et al., 1992). The same sequence restores lycopene accumulation in transgenic yellow-flesh mutants identifying the mutation as a lesion in a Psy-1 gene (Fray and Grierson, 1993). In both transgenic

0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00175-3

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has other pleiotropic effects including altered fruit softening (Thompson et al., 1999). Like hp, the Cnr mutation maps to chromosome 2 in a location devoid of other known fruit ripening genes. The purpose of this study was to characterise carotenoid biosynthesis in developing and ripening Cnr fruits at the biochemical level, in order to provide a greater insight into the mechanism of action of the Cnr mutation.

2. Results and discussion 2.1. Carotenoid and isoprenoid analysis of Ailsa Craig (AC) and Cnr tissues

Fig. 1. Summary of the biosynthetic pathway for carotenoids. Numbers indicate enzyme responsible for the conversion. 1. Isopentenyl diphosphate isomerase. 2. Geranylgeranyl diphosphate synthase. 3. Phytoene synthase. 4. Phytoene desaturase. 5. z-Carotene desaturase. 6. b-Cyclase. 7. b- and "-Cyclase. 8. b-Hydroxylase. 9. b- and "-Hydroxylase.

plants containing Psy-1 in an antisense orientation and in the yellow flesh mutant there are virtually no carotenoids in the ripe fruits (Fray and Grierson, 1993), although carotenoid levels in leaf and green fruit are essentially unaffected. This is due to the presence of another phytoene synthase, Psy-2, which is principally responsible for phytoene formation in chloroplast containing tissues (Fraser et al., 1999). The regulation of carotenoid biosynthesis in higher plants is poorly understood. Tomato ripening mutants such as high-pigment (hp) (Yen et al., 1997) and colourless non-ripening (Cnr) (Thompson et al., 1999) are useful tools for exploring the regulation of this process. In hp, the level of carotenoids is twice that in normal fruits, but with a quantitatively very similar pattern (Bramley, 1997). This difference almost certainly reflects an increase in plastid number (Yen et al., 1997). The hp gene maps to chromosome 2 and is not allelic with any known carotenoid biosynthetic genes. In the Cnr mutant, carotenoid biosynthesis is all but abolished resulting in mature fruit with a white pericarp. This mutation also

HPLC-PDA (photodiode array) analysis of carotenoids extracted from AC and Cnr chloroplast-containing tissues (leaf and developing fruit) show a very similar pigment profile (Table 1). The predominant pigment was the xanthophyll lutein (55–80%) also present in appreciable amounts were violaxanthin and b-carotene. Although not drastically reduced, less total carotenoid was found in the leaf and mature green fruit stage of the Cnr mutant (70 and 57% less respectively). As fruit ripening proceeded the characteristic accumulation of lycopene was not observed in the Cnr mutant, the intermediate phytoene also appeared to be absent. The total carotenoid content of AC fruit was 100-fold higher than in the mutant (Table 1), confirming previous data (Thompson et al., 1999). The profile of carotenoids in the ripe Cnr fruit resembles that found in green fruit or leaf tissue (Fig. 2, Table 1). However, the mature green and ripe Cnr fruits appear to lack violaxanthin, which would presumably result in an inhibition of ABA biosynthesis. Determination of other related isoprenoids e.g. tocopherols and ubiquinone-9 revealed no differences between Cnr and AC fruits (Table 2). The apparent reduction in leaf carotenoid levels in Cnr may reflect an affect of the mutation in tissues other than fruits. The data for the leaf samples was, however, quite variable due probably to effects of position of leaf with respect to others in the canopy and leaf carotenoid levels requires further investigation. Comparison of the carotenoids present in AC and the Cnr mutant indicates that Cnr lacks the characteristic accumulation of ripening (or chromoplast) related carotenoids (e.g. lycopene and phytoene). Previous studies (Thompson et al., 1999) have shown that Psy-1 is not expressed in the Cnr mutant fruit. Characterisation of AC and Cnr carotenoids supports the absence of Psy-1 expression and show that Psy-2 can not compensate for its absence in the formation of carotenoid in ripe fruit, as postulated previously (Fraser et al., 1999). Both Psy1 down-regulated mutants (Bramley et al., 1992; Fray and Grierson, 1993) and the Cnr have a characteristic

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Table 1 Carotenoid content of Ailsa Craig and Cnr leaves and fruit. The tissue samples were prepared from three fruit or leaves. Fruits were cut in half, the seeds removed and freeze dried from which a ground powder was produced. Determinations were carried out in triplicate Carotenoid (mg/g DW) Phytoene (%)

Phytofluene (%)

Lycopene (%)

b-Carotene (%)

Lutein (%)

Violaxanthin (%)

AC Leaf IMG MG B B+7

0 0 0 14
1.10 (8.6) 238
12 (5.6)

0 0 0 0 17
1.4 (0.4)

0 0 0 31.0
1.0 (19) 3746
326 (87)

514
15 (30) 20
7 (32) 11
0.7 (31) 84
1 (52) 200
9 (4.7)

953
6 (55) 40.4
15 (65) 23.2
1.4 (67) 32.9
1.3 (20) 20.4
2.4 (0.5)

260
6.52 (15) 1.1
0.46 (1.7) 0.7
0.1 (2.0) 0.82
0.03 (0.8) 3.9
0.04 (0.09)

Cnr Leaf IMG MG B B+7

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

312
97 (31) 29.1
8.4 (37) 4.1
0.04 (20) 12.7
1.0 (26) 14.0
2.7 (37)

536
166 (54) 45.3
0.7 (58) 16
0.26 (80) 36.9
3.0 (74) 23.7
4.5 (63)

153
49 (15) 3.7
0.2 (4.7) 0 0 0

Total 1727
27 62.0
22 35.0
2.2 161.8
4.4 4225
351 1000.9
312 78.1
9.3 20.1
0.3 49.6
4 37.7
7.2

Table 2 Levels of selected isoprenoids in red ripe (breaker+7) from Ailsa Craig and Cnr pericarp. The tissue samples were prepared from three fruit or leaves. Fruits were cut in half, the seeds removed and freeze dried from which a ground powder was produced. Determinations were carried out in triplicate Isoprenoid mg/g DW

AC Breaker+7 Cnr Breaker+7

Tocopherols

Ubiquinone- 9

6.0
1.0 7.3
2.0

79
7.4 75
17

ripening. Unlike both the carotenoids and tocophorols, which are synthesised in the plastid from isopentenyl pyrophosphate (IPP) formed by the ‘Rohmer’ pathway, ubiquinone is a non-plastidic isoprenoid derived from IPP resulting from the mevalonate pathway. In the Cnr mutant ubiquinone content was unaffected. 2.2. In vitro analysis of phytoene synthesis from IPP

Fig. 2. HPLC trace of major carotenoids from (A) AC (B+7) and (B) Cnr (B+7). Peaks correspond to (1) lutein, (2) b-carotene, (20 ) cis-bcarotene, (3) cis-lycopene and (4) lycopene. Traces were recorded at 450 nm.

yellow skin colour. This is due to the presence of the flavonoid naringen chalcone and not the presence of carotenoids (Bolwell, Fraser and Bramley, unpublished data). Structurally, tocopherols possess a prenyl moiety, which is derived from GGPP. In Cnr tocopherols were not affected, suggesting that a pool of GGPP is available and that the Cnr mutation does not result in an absolute block in the formation of GGPP during fruit

In order to confirm phytoene synthase as the biochemical block in Cnr, in vitro analysis was performed. Stromal extracts were prepared from both the AC and Cnr. The ability of these extracts to incorporate the precursor isopentenyl pyrophosphate (IPP) was determined. Data were expressed on a protein and fresh weight (FW) basis to eliminate possible discrepancies that may result from alterations in textural properties of the Cnr mutant. Comparisons clearly show (Table 3) that phytoene formation from [14C]-IPP is significantly reduced (27-fold) in the Cnr mutant. Although not reduced as dramatically (5-fold), the levels of GGPP are also affected in Cnr. In contrast the total acid hydrolysable prenyl phosphates formed from IPP are not affected in the Cnr mutant. These data confirm that phytoene formation in the Cnr fruit is virtually abolished. Furthermore, besides

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Table 3 In vitro analysis of phytoene formation in red ripe fruit of Ailsa Craig and Cnr IPP isomerase activity

Formation from IPP GGPP

dpm/g FW AC Cnr

799
129 1186
190

dpm/mg Protein 6

6.010
1.0 3.0106
0.5

dpm/g FW 68.0
22 18
4.0

phytoene synthesis, it would appear that the formation of GGPP is also reduced in the mutant. The reduction in GGPP does not, however, affect tocopherols suggesting that another pool of GGPP separate from that utilized for carotenoid biosynthesis is available. To date only one GGPP synthase has been isolated from tomato (Ro¨mer et al., 1993) but several GGPP synthases have been identified in Arabidopsis (Scolnik and Bartley, 1996). It is, therefore, feasible that alternative GGPP synthase(s) exist in tomato one of which is responsible for the supply of GGPP into the carotenoid pathway. The Cnr mutation is the result of a lesion in a single gene which has pleiotropic effects on ripening including abnormal softening and colour development . The Cnr gene is likely to be involved in the regulation of normal ripening in tomato (Thompson et al., 1999). Data presented in this article show that carotenoid formation is an important component of the co-ordinated fruit ripening processes upon which the Cnr gene product exerts its effects. Typically PSY-1 is regarded as the most influential step for carotenoid formation in tomato, but the biochemical data obtained from the Cnr mutant suggest that an accessible pool of GGPP is a prerequisite for carotenoid accumulation. Future isolation of the Cnr gene could provide an important tool in the engineering of quality traits into tomato fruit.

Phytoene dpm/mg Protein 5

3.310
0.7 6.7104
1.3

dpm/g FW

dpm/mg Protein

4.1
0.6 0.47
0.1

3.0104
0.4 1.1103
0.2

International, Amersham, Bucks, UK. Biochemical reagents were supplied by Sigma, Poole, UK, unless otherwise stated. 3.2. Extraction, separation, and identification of carotenoids and isoprenoids Procedures for the solvent extraction, HPLC separation, identification (PDA) and quantification are provided in Fraser et al. (2000a). 3.3. Preparation of subcellular extracts Pericarp tissue typically from three to four tomatoes (65–100 g per tomato) were homogenised in a Waring blender at 4 C. The homogenisation buffer contained 0.4 M sucrose, 1 mM EDTA, and 1 mM DTT, buffered with 50 mM Tris–HCl (pH 8.0). Ice-cold buffer was added until it covered the pericarp tissue (about 1:3/1:4 fruit vol: buffer). The mixture was left for 20 min before homogenising for two 3 s bursts. The suspension was filtered through cotton wool sandwiched by two layers of muslin. The filtrate (homogenate) was subjected to further subcellular fractionation as described in Fraser et al. (2000b). 3.4. In vitro assays

3. Experimental 3.1. Plant material, radiolabelled substrates and reagents Tomato (Lycoperiscon esculentum Mill. cv Ailsa Craig) fruit and a near isogenic line of Cnr, produced by back-crossing five times to Ailsa Craig, followed by selfing to generate a homozygous mutant line.mutant line, were grown in a heated greenhouse using standard cultural practices with regular additions of N,P,K fertilizer and supplementary lighting when required. Fruits were harvested at the following days post anthesis (DPA): immature green (IMG, c. 20 days DPA), mature-green (MG, 35 DPA), breaker (B, 49 DPA) and breaker+7 (B+7, 56 DPA). The radiolabelled [1-14C]IPP (2.2 GBq/mmol) was purchased from Amersham

[14C]-Isopentenyl diphosphate (19 kBq) was used to ascertain incorporation into prenyl diphosphates as well as phytoene. Incubations were buffered with 0.4M Tris– HCl (pH 8.0), containing 1 mM DTT, 4 mM MgCl2, 6 mM MnCl2, 3 mM ATP, 0.1%(w/v) Tween 60, 1 mM KF and substrate. The total volume of the buffer mixture was 150 ml, to which 150 ml of the enzyme preparation was added. Incubations were shaken for 3–4 h at 28 C unless stated otherwise. Radiolabelled products were separated and identified as described in Fraser et al. (2000b). 3.5. Other determinations Protein contents were estimated as detailed previously (Lowry et al., 1951). Prior to determination, proteins were precipitated (Wessel and Flugge, 1984). Determination of

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radioactivity by liquid scintillation counting was carried out according Bramley et al., 1973). Acknowledgements This work was funded in part by the UK Biotechnology and Biological Sciences Research Council (GBS), an EU Programme PL 962077 (P.B.) and The Royal Society G503 (P.D.F.).

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