Estudio comparativo de promotores de la Chalcon Sintasa en diferentes familias de plantas.
Flavonoids play important roles in many biological processes such as pigmentation of flowers, fruits and vegetables, plant-pathogen interactions, fertility and protection against UV light (Brouillard & Cheminat, 1988;
Gronquist et al., 2001; Bovy et al., 2007). Anthocyanins belong to the flavonoid class of molecules and contribute with the formation of color in flowers (Buchanan et al., 2000). Color in flowers has been an important subject for study in plant biotechnology and changes in flower color have been carried out by regulation or co-suppression of the genes involved in the expression of the anthocyanins (Nakatsuka et al., 2007; Nakatsuka et al., 2008; Tanaka & Ohmiya, 2008).
Chalcone synthase is a key enzyme in the production of anthocyanins and other flavonoids. Anthocyanin biosynthesis starts with the condensation of 4-coumaroil-CoA and manolil-CoA mediated by Chs (Hanumappa et al., 2007). Chalcone synthase genes form a multigene family in many species; nevertheless, some plants as those belonging to the genera Antirrhinium and Arabidopsis have a unique copy of the gene (Harborne, 1994).
Although several studies have focused on the study of the gene and particularly on its evolution (Durbin et al., 2000), the promoter sequences have been less studied and no comparative studies across plant families has been done. The chalcone synthase gene promoter has a complex series of regulator Cis elements involved in the expression control (Meer et al., 1990). Faktor et al. (1997) described two adjacent motifs, the G-Box (CACGTG) and the H Box (CCTACC) near to a TATA Box that are essential for specific expression in flowers and roots of the chalcone synthase and suggested that the three motifs act as a unit. Additionally, Koch et al. (2001) found additional regulator Cis elements in several species of the Brassicaceae family (Koch et al., 2001).
This study started as an initiative to take advantage of the available data on Cis elements on the promoter sequence of the Chs and expand this knowledge aiming for furture biotechnological applications. Comprehending Cis elements can lead to improve the regulation of protection against UV light in plants, the generation of resistant plants and the possibility of modifiying the flower colors. All of these correspond to interesting topics for future studies in our laboratory. In this research we performed a comparative study of the chalcone synthase promoters across different plant families. We describe the presence and conservation of the regulator Cis elements mentioned previously (Faktor et al., 1997).
Materials and Methods
A set of twenty-two sequences of chalcone synthase promoters were retrieved from GenBank, details of species and accession numbers are given in table 1. Sequences were aligned in the program Geneious v.4.7.5 (Biomatters Ltd., Auckland) using MUSCLE (8 iterations and the rest of parameters by default) (Edgar, 2004). Then, the alignment was constrained using the Cis elements (G-Box, H-Box and TATA Box) as guides. A different set; consisting of five chalcone synthase promoters from Visum sativum (Ch1, Ch2, Ch3, Ch5, Ch7) was also aligned in the Geneious software using MUSCLE.
A genealogy was built with the first alignment, using a bayesian approach with the software BEAST (Drummond & Rambaut, 2007). The program was run with default parameters and 10,000,000 replicates. The first 1000 iterations were discarded as burn-in. The consensus tree was visualized in FigTree (http://tree.bio. ed.ac.uk/software/figtree/) (Fig. 1).
Finding a chalcone synthase promoter in tomato
We retrieved all sequenced BAC clones of tomato (Solanum lycopersicum) from the SOL Genomics Network (http://sgn.cornell edu/) database, and performed a tBlastx search using the mRNA of Chs-A from Petunia hybrida as a query. Then, we retrieved a fragment of 2Kb upstream from the best hit and searched for any CIS motif.
[FIGURE 1 OMITTED]
Results and Discussion
All the sequences corresponding to the Brassicaceae grouped in a monophyletic clade with high branch support (Figure 1, blue clade). All the Cis elements described by Faktor et al. (1997) were found in this family. The G-Box (a typical Cis element of the family) was detected at the beginning of these sequences. In this plant family, besides having the three Cis elements, an extra G-Box was found after the first G-Box identified by Faktor et al. This Cis element may be acting as an extra site for binding nuclear proteins for a successful expression of Chs (Harter et al., 1994).
All of the sequences from the genus Arabidopsis were grouped in a monophyletic clade. Arabis pauciflora grouped with Choclearia excelesa showing that this promoter is highly variable in the genus Arabis an insertion consisting of one adenine at the position 6 of the 3' end of the G-Box was found in Arabis pauciflora and six Arabidopsis spp. This insertion may be modifying the function of the Cis element since it has been shown that the G-box and the H-box make major contributions to the transcription of some promoters in vivo (Hartmann et al., 1998). Further studies should be done in order to know if the insertion is modifying the function of this Cis element. The species Aethionema grandiflora (Brassicaceae) and Pisum sativum (Fabaceae) presented the three Cis elements described by Faktor et al. (1997), but did not show an extra G-Box, characteristic of the Brassicaceae family. The position of A. grandiflora at the base of the Brassicaceae family may suggest that the additional G-Box might be a derived character that characterizes some genus of this family. Additionally, we found one transition in the H-Box from cytosine to thymine in Pisum sativum.
Two members of the Solanaceae family used in this study, Petunia hybrida and Nicotiana tabacum, formed a single group with a posterior probability of 0.96 (figure 1, orange group). This group showed the three Cis elements and an additional G-Box before the G-Box and HBox, as described by Faktor et al. (1997). Thus, we found four Cis elements: G-G-H-TATA Boxes. As mentioned before, the extra G-Box could act like an extra element for specific expression or like an extra site for binding nuclear proteins for a successful expression of chalcone synthase (Harter et al., 1994). This might be due to the fact that the G-Box sometimes carries out its regulatory function combined with other Cis elements (Donald & Cashmore, 1990). Additionally, both species have one transition in the H-Box from a cytosine to a thymine, a feature also found in Pisum sativum.
[FIGURE 2 OMITTED]
Four species, Ginko biloba, Gentiana triflora, Pinus radiata, and Phaseolus vulgaris, did not present an H-Box. This absence could have implications in UV light resistance; the LRU (Light Regulator Unit) that contains the G-Box and the H-Box is sufficient for UV/blue light-regulated expression of Chs (Hartmann et al., 1998). These two Cis elements are accounted as the most common in some Chs promoters (Meer et al., 1990; Hartmann et al., 1998; Koch et al., 2001). One possibility is that the regulation of the promoter is mediated by undetected Cis elements. Our results showed that the three Cis elements described by Faktor et al. (1997) are not present in all species. This may be indicative that in some plant families these three Cis elements play an important role in the expression of the chalcone synthase gene, while others may present different and currently un-described Cis elements.
Only some gene copies from the Chalcone Synthase gene in Pisum sativum presented all the Cis elements identified by Faktor et al. (1997) (figure 2). In the promoter Chs1 we found the three Cis elements with the previously mentioned transition in the H-Box. In the Chs2 the transition in the H-Box was also found but no G-Box was found. In the Chs3 and Chs4, the three Cis elements were found, but the H-box presented a transversion of one cytosine for one guanine in the first position. These changes or absences of the boxes in these promoters could change the functionality or regulation of the promoter (Hartmann et al., 1998).
Probably these Chs promoters (Chs2, Chs3, Chs5) are only expressed in the plant under stress conditions or are non-functional promoters, flavonioids (mediated in the first step of biochemical reaction by chalcone synthase) can attenuate some adverse effects like heat stress on fertilization or early seed maturation. (Coberly & Rausher, 2003) in the Chs7 promoter no Cis elements were found, in this case, the Chs7 could be a duplicate non-functional copy.
Finding a Chs promoter in tomato
In the sequence of 2Kb of the BAC C12HBa0183M06.1.v1 corresponding to the best tBlastx hit (87% of identity), no Cis elements were found. This sequence may correspond to a non-functional promoter, as in the case of Chs7 of Pisum sativum.
There is an apparent evolution of Cis elements that grouped by families as it was seen for the Brassicaceae and Solanaceae family. This must be confirmed with a larger set of sequences from related species and other botanical families. The three Cis elements described by Faktor et al. (1997) may be present in promoters of some species or families with different modifications or duplications but were not present in all of the promoters under study. Thus indicating that there may be other Cis elements involved in the expression. Additionally, in Pisum sativum, a species with several copies of the Chs gene, the regulation of the expressed promoters of the Chs are carried out by the recognized Cis elements (G-Box, H-Box and Tata-Box) and promoters without this Cis elements may be expressed under stress conditions.
Recibido: agosto 21 de 2009 Aprobado: noviembre 23 de 2009
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Buitrago, F. (1), Sierra, R. (2), Grajales, A. (3), Rodriguez-R, L.M. (4), Jimenez, P. (5), Bernal, A. (6), Restrepo, S. (7)
(1) Francisco Buitrago, B. Sc. Cra 1 No. 18A-10 Bloque J205 Laboratorio de Micologia y Fitopatologia, Universidad de los Andes, Bogota, Colombia. email@example.com
(2) Roberto Sierra, M.Sc. Cra 1 No. 18A-10 Bloque J205 Laboratorio de Micologia y Fitopatologia, Universidad de los Andes, Bogota, Colombia. firstname.lastname@example.org
(3) Alejandro Grajales, M.Sc. Cra 1 No. 18A-10 Bloque J205 Laboratorio de Micologia y Fitopatologia, Universidad de los Andes, Bogota, Colombia. email@example.com
(4) Luis Miguel Rodriguez-R, B. Sc. Cra 1 No. 18A-10 Bloque J205 Laboratorio de Micologia y Fitopatologia, Universidad de los Andes, Bogota, Colombia. firstname.lastname@example.org
(5) Pedro Jimenez, Ph.D. Cra. 11 No. 101-80 Laboratorio de Fitopatologia, Universidad Militar Nueva Granada, Bogota, Colombia. email@example.com
(6) Adriana Bernal, Ph.D. Cra 1 No. 18A-10 Bloque J205 Laboratorio de Micologia y Fitopatologia, Universidad de los Andes, Bogota, Colombia. firstname.lastname@example.org
(7) Silvia Restrepo, Ph.D. Cra 1 No. 18A-10 Bloque J205 Laboratorio de Micologia y Fitopatologia, Universidad de los Andes, Bogota, Colombia. email@example.com
Table 1. Summary table of the sequences and taxa in this study. GenBank Taxa Accesion No. Length (bp) Gene (1) Aethionema grandiflora AF249000 424 Chs Arabidopsis griffitihana AF248989 1184 Chs Arabidopsis halleri AF248986 575 Chs Arabidopsis lyrata AF248987 605 Chs Arabidopsis thaliana AF248988 581 Chs Arabis alpina AF248995 632 Chs Arabis jacquinii AF248994 616 Chs Arabis pauciflora AF248988 485 Chs Arabis turrita AF248996 547 Chs Barbarea vulgaris AF249991 483 Chs Cardamine amara AF248993 476 Chs Cochlearia excelsa AF248999 468 Chs Gentiana triflora AB005484 1162 Chs Ginkgo biloba EF091691 871 Chs Lepidium campestre AF248990 513 Chs Matthiola incana AF248997 479 Chs Nicotiana tabacum FJ655994 529 Chs Petunia hybrida EF199747 550 ChsA Phaseolus vulgaris AY268022 1453 Chs8 Pinus radiata AF337656 1055 Chs1 Pisum sativum AF060238.1 314 Chs1 Rorippa amphibia AF248992 469 Chs (1) Chs: chalcone synthase