Plant Molecular Biology 27: 425-428, 1995. © 1995 Kluwer Academic Publishers. Printed in Belgium. 425

Short communication

Structure of a functional geranylgeranyl pyrophosphate synthase gene from Capsicum annuum

Alfredo Badillo, Johannes Steppuhn, Jean Deru6re, Bilal Camara and Marcel Kuntz* Institut de Biologie Mol~culaire des Plantes du CNRS and UniversitO Louis Pasteur, Strasbourg, 12 r u e

du GdnOral Zimmer, 67084 Strasbourg, France (*author for correspondence)

Received 15 July 1994; accepted in revised form 10 November 1994

Key words: bell pepper, carotenoid, chromoplast, fruit ripening

Abstract

A geranylgeranyl pyrophosphate synthase (GGPPS) gene from Capsicum annuum (bell pepper) was cloned. The nucleotide sequence shows that this gene, like the capsanthin/capsorubin gene but unlike the phytoene synthase gene from C. annuum, is not interrupted by an intron. Southern blot analysis of C. annuum genornic DNA suggests the presence of a single gene highly similar to the cDNA and also of additional related sequences. The present data suggest that this cloned gene is functional.

Geranylgeranyl pyrophosphate synthase (GGPPS, EC 2.5.1.29) catalyses the sequential addition of 3 molecules of isopentenyl pyrophos- phate (IPP) onto the starter molecule, dimethy- lallyl pyrophosphate (an isomer of IPP), to pro- duce the C20 molecule which is the precursor of several terpenoids (e.g. carotenoids, kaurene, diterpenes, side-chain of chlorophylls, quinones, tocopherols, etc.) [for recent reviews see 2, 15]. The purification to homogeneity of GGPPS from Capsicum annuum chromoplasts has been re- ported previously [7]. We have also cloned the corresponding cDNA and shown that this gene is expressed in all tissues in which carotenoid bio- synthesis occurs [11]. In addition, the expression of the GGPPS gene was found to be increased in

ripening fruits in parallel with the increase in carotenoid biosynthesis during chloroplast to chromoplast differentiation in fruits.

In contrast with this observation, the expres- sion of pds, the gene encoding phytoene/phyto- fluene desaturase, is not significantly stimulated during C. annuum fruit ripening [8], while the expression of psy, the gene encoding phytoene synthase, is only weakly stimulated [14]. There- fore, we anticipate that the GGPPS gene will be a good model to study the regulatory mechanisms controlling carotenoid accumulation at a tran- scriptional level during C. annuum fruit ripening. A prerequisite for such a study is to clone a func- tional GGPPS gene.

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X80267.

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Cloning and sequencing of a GGPPS gene from C. annuum

About 10 6 plaques from a genomic DNA library from C. annuum, constructed in 2Gem11 [5], was screened using an internal fragment of the GGPPS cDNA which was [32p]dCTP-labelled by random priming [11 ]. A positive 2 clone was plaque-purified and a preliminary map of restric- tion enzyme cleavage sites was determined for the insert DNA (Fig. 1). Hybridization of the radio- active probe to the cleaved insert DNA revealed that the coding region of the GGPPS gene spans over 2 Xho I-Eco RI DNA fragments of about 3 and 4 kb, respectively, and is located on a Hind III-Hind III fragment of about 4 kb (Fig. 1). The 3 kb Xho I-Eco RI fragment and the 4 kb Hind III-Hind III fragment were cloned in the plasmid vector pBluescriptKS and subclones were generated in order to determine the nucle- otide sequence of the GGPPS gene (Fig. 1). The region corresponding to the 5' portion of the cDNA and the 5'-flanking region are located on the 3 kb Xho I-Eco RI fragment, while most of the coding region and the 3'-flanking region are located on the 4 kb Hind III-Hind III fragment (Fig. 1).

The nucleotide sequence of the GGPPS gene revealed the presence of an open reading frame of 369 codons which is identical to that of the cDNA ([ 11], database accession number X80267). The

3 kbp m, 4 kbp

9

probe

Xhol Hindlll EooRI Hindlll I "'°~'T' ~,n.,,, .%. ~ J

CMT...2~p...TATM...98~...ATa

Fig. 1. Schematic representation of the cloned GGPPS gene from C. annuum. The coding region is boxed. Arrows delin- eate the fragments that were subcloned. The bar shows the DNA fragment which was random-prime-labelled (probe). A simplified structure of the region upstream of the ATG initia- tion codon is shown in the lower part of this figure. This re- gion contains potential CAAT and TATA boxes. The nucle- otide sequence of this region, of the coding region and of the 3'-flanking region is available in the databases under the accession number X80267.

5' and 3' untranslated regions are also identical in the genomic clone and the cDNA. Additional downstream and upstream sequences were also determined. A putative TATA box and CAAT box are present in the latter region (Fig. 1).

Intron distribution in carotenogenic genes from higher plants

Our data show that the cloned GGPPS gene is not interupted by an intron. We have previously reported that the capsanthin-capsorubin (CCS) gene from C. annuum is uninterrupted while the fibrillin gene is interrupted by 2 introns of 805 and 94bp, respectively [5]. It is striking that the GGPPS gene is also uninterrupted since the available data suggest that this gene is co- expressed with the CCS gene during fruit ripen- ing in C. annuum [ 3 ]. In contrast, the fibrillin gene appears to be induced earlier during fruit ripen- ing and shows a different pattern of expression as judged by RNA gel blot analysis [6].

It is not known whether the presence ofintrons influences the expression of carotenoid biosyn- thetic genes. However, it should be noted that multiple introns occur in some of these genes. This is the case for the tomato [13] and C. an- nuum [our unpublished data] psy genes and for the tomato pds [1, 12]. In addition, unspliced transcripts have been detected for the tomato pds gene [1, 12] and for the C. annuum fibrillin gene [ 5 ]. In the latter case, no link between spicing rate and carotenoid biosynthesis could be observed [51.

Characterization of the cloned GGPPS gene

Hybridization of a labelled internal fragment (see Fig. 1) of the GGPPS cDNA to C. annuum genomic DNA blots were performed. Under standard hybridization conditions (6x SSPE, 65 ° C), these experiments suggested the presence of only one gene (Fig. 2a). However, since in non- stringent conditions additional bands were visible (Fig. 2b), the C. annuum genome must contain

F~g. 2. Autoradiographs of a C. annuum genomic DNA blot hybridized with the GGPPS cDNA under standard condi- tions (a) or under non-stringent conditions (b). The hybrid- ization probe was labelled by random priming of the subfrag- merit shown in Fig. 1. Eco RV does not cleave in the coding region. Hind III cleaves near the 5' end of the coding region. Eco RI cleaves in the central part of the coding region, but asymmetrically the fragment that was radiolabelled (which results in different hybridization intensities for the 2 Eco RI fragments). Only partial Hind III digestion of genomic DNA could be obtained. * Indicate bands not observed under stan- dard conditions (even after long exposure of the autoradio- graph). Standard hybridization and wash conditions were 65 °C, 6 x SSPE, 0.1~o SDS, 1 x Denhardt's solution. To minimize background under non-stringent conditions, the pro- tocole described by Church and Gilbert [4] was used at 60 ° C.

sequences weakly homologous to the G G P P S cDNA. These sequences could either correspond to a second G G P P S gene or to a pseudogene. The presence of a second G G P P S gene has been postulated in Arabidopsis [ 16]. On the other hand, since the presence of a pseudogene for the CCS gene has been observed and cloned from C. an-

nuurn (F. Bouvier etal . , unpublished data) it seems possible that pseudogenes also exist for GGPPS. In addition, the case ofpsy , analysis of C. annuum genomic DNA blots was ambiguous because multiple weak bands were observed (data not shown). Taken together, these observations suggest that the large size of the nuclear genome of C. annuum could be explained in part by the presence of numerous non-functional sequences which have been duplicated from functional genes. The fact that the sequence of the G G P P S coding region is 100 ~o identical to the sequence

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of the cDNA strongly suggests that we have cloned a functional gene.

This is also confirmed by our observation that the 5'-flanking region of the G G P P S gene is able to drive expression of a reporter gene in transient expression assays. In these assays the GUS gene was used as a reporter gene [9] and a biolistic method [ 10] was used to introduce recombinant DNA into C. annuum cells. When a construct consisting of about 2.5 kb of 5'-flanking region of the G G P P S gene (including the CAAT box, the TATA box and the whole 5'-untranslated leader) was used, GUS activity was detected in both leaf and fruit cells (data not shown). As a control, most of this 2.5 kb region (including the CAAT box and the TATA box) was deleted and only the 5'-untranslated leader was kept in the construct. In this case only background GUS activity was observed.

The data reported here open the way for further work in order to delineate the regions of the G G P P S gene which are involved in the control of gene expression.

Acknowledgements

We wish to thank A. Klein for excellent techni- cal assistance and Prof. J. H. Weil for his inter- est in this work. This work was supported by a grant from the EEC (Biotech, PTP 'Plant Mo- lecular Genetics for an Environmentally Compat- ible Agriculture').

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