CHARACTERIZATION OF THE GENE AT2G45570

Cristbal Gallardo Alba

Methods and experimental technics in genetics, UMA

CHARACTERISTICS OF THE GENE SEQUENCE

The gene is located in the chromosome 2, between the positions 18779793 and 18781958, in reverse orientation, and has a length of 2166 bp. The sequence contains two intronic regions, whose localization is specified in the figure 1, in addition to the position of the 5`UTR and 3UTR regions. Bioinformatic analysis of the gene, combined with the comparisons with other related genes which share the same expression pattern (At3g28740, AT2G34500, At4g37370 and At2g24180) reveal some cis-acting regulatory sequences potentially important in the control of expression, such a Inr element (from 18781954 to 18781961), a GATA binding motif (probably from 18789214 to 18789219), a W-box (binding site for the WRKY transcription factors) probably between the positions 18789315-18789320 and one or more recognitions sites for MYB factors, probably located between the positions -95 and -129 upstream of our gene, although not very well defined. The importance of the GATA motif is supported by the experimental evidences that show that in the gnc mutant (affected in the transcription factor which recognizes the GATA motif) the expression of our gene is repressed. As to its relative position with respect to other genes, it is noteworthy that is part of a cluster that include the four members of the CYP76C2 family, all of them are oriented in antisense (figure 1): At2g45560: It codifies the CYP76C1 protein; it is downstream of our gene, with a length of 2459 bp, and functionally has been described as geraniol/nerol 10-hydroxylase.

At2g45580: It codifies the CYP76C3 protein; it is upstream of our gene, with a length of 2023 bp. Functionally, it appears associated with many metabolic pathways, like the ascorbate and aldarate metabolism, coumarine and phenylpropanoid biosynthesis and fluorene degradation. At2g45590: It codifies the CYP76C4 protein; it is upstream of or gene, with a length of 2114 bp; functionally it is associated with At2g45580.

TRANSCRIPTIONAL CHARACTERISTICS
It has been identified a single primary transcript, reconstructed from 37 cDNA clones, whose pre-mRNA has a length of 2,17 kbp, and that, after the posttranscriptional processing, results in a mature mRNA with a length of 1718 bp.

Expression pattern
The analysis of the microarray data obtained from CYPEDIA (Cytochrome P450 Expression Database using Arabidopsis) shows a complex pattern, taking place the induction at diverse developmental stages and in response to different stimulus. Thus, if we analyze the tissue expression pattern (Figure 2), it is possible to appreciate that the maximum level is achieved in sepals (concretely in the stage 15), increasing the gene expression 26 times respect to basal level. Furthermore, it also appears increased the levels of other two floral whorls, petals and stamen, both in the 15 stage. It should be pointed that is in the stage 16 when the petals and sepals begin to wither.

Figure 1: Scheme which details the disposition of the gene AT2G45570 in the chromosome

Other tissues and organs where is detected high transcription levels are the siliques in different developmental stages (mainly in mature siliques), and seeds, (mainly between the stages 8, 10 and mature seeds). In addition, it is also detected a strong correlation between the expression of our gene and the mechanical damage. On the other hand, if we analyze the expression pattern in different conditions of abiotic stress (figure 3), we can see that in both roots and leafs the strongest induction appear associated with saline and osmotic stress situations, where the maximum magnitudes of change are 13 and 557 times respectively. Also high levels have been detected in conditions that produce oxidative stress (paraquat, ozone, norfluazone and UV-B irradiation, reaching levels of 17,1 times in the treatment with ozone), cold stress (reaching maximum variations of 17 times) and metallic stress, existing experimental evidences which prove that our gene is induced by copper sulphate, cesium and lead nitrate. With regard to the induction by biotic factors, the analysis carried out on Col-0 lines highlight three microorganisms as responsible for triggering the expression of our gene: Botrytis cinerea (maximum induction of 32 times at 48 hours), Phytophtora infestans (whose effect over germination produces an increase of 3,2 times at 6 h) and Pseudomona syringae, whereof have been detected many strains that induce the expression, highlighting P. syringae DC3000 (compatible interaction) y P. syringae avrRpm1 (incompatible interaction), having detected an increase of 64 times in the expression levels.

Figure 2: Expression pattern in different tissues

Other tissues and cellular states in which elevated expression levels are detected are senescing leaves and cellular suspensions, protoplasts, and leafs in dedifferentiation; dedifferentiation can be induced during normal development and as response to various stimuli, such as pathogen infection and wounding, and also characterizes the transition of differentiated leaf cells into protoplasts (plant cells devoid of cell walls), a transition accompanied by widespread chromatin decondensation. Transcriptome profiling of dedifferentiating tissues revealed striking similarities with senescing cells; both display a large increase in the expression of genes of specific transcription factor (TF) families, including ANAC, WRKY, bZIP, and C2H2. Further analysis have shown that leaves induced to senesce by exposure to dark display characteristic features of dedifferentiating cells, including chromatin decondensation, disruption of the nucleolus, and condensation of rRNA genes (Damri et al.,2009)

Figure 3: Graphic which detail the expression pattern in response to different conditions

Figure 4: Scheme that details the various components identified in our protein, which highlights the transmembrane domain at the amino terminus, and the binding region of heme group in the globular domain.

Moreover, have been identified specific elicitors which determine increase in expression, concretely hrpZ, GSTNPP1 and flg22. It should be pointed that the transcript accumulation happens on the earlier stages in the case of compatible interactions.

(because there are only evidences at transcriptional level) and, although there is little consensus about its location, recent data point that it is located in the plasmatic membrane (Heng-Hsuan Chu , 2010).

Physical and chemical characteristics

Mutant Aba1, fresh seeds Ag12, flower ARR22o, seedling Coi1, senescing leaf Gun1-gun5, whole plant Ku80, whole plant, bleomycin Leafy-GR, seedling, cyc NahG, senescing leaf Pho1, mature leaf Pho3, leaf Sfr2-1, whole rosette, 4C Ufo1, flower Cat2 Magnitude of the change 2,5 1,2 5,7 1,2 1,9 9,8 4 1,4 11,3 2,5 4,9 1,2 17,2

The protein has a molecular weight of 572562 daltons, an isoelectric point 69569 and its grand average of hydrophobicity (GRAVY) score is -0,12. The cytochromes p450 enzymes can bind carbon monoxide instead of oxygen, resulting in the displacement of the maximum absorbance of the hemo group at 450 nm, phenomenon that gave its name to such proteins as Pigment absorbing at 450 nm (P450). This property is characteristic of the p450 enzymes, whose activity is strongly inhibited by carbon monoxide association, that avoids the association with oxygen, but this inhibition can be reverted when is exposed to 450nm wavelength light.

Figure 5: Identified mutants that affect our gene expression.

Functional characteristics
Finally, other conditions where have been also detected significant increase in transcriptions levels are ABA treatment during germination (113 times), potassium deficiency (7 times), infections with RNA virus, and different mutants (Figure 5), emphasizing cat2 (172 times), pho1 (113 times), ku80 (98 times) and ARR220. Likewise, the mutant cng it is also quite interesting, on which the transcription is repressed. This enzyme has only two domains , a transmembrane domain which is extended between the positions 6 to 24, and the called P450 domain or haem domain, between the positions 39 to 540 (figure 3). This domain is involved in the activation and heterolitic cleavage of the oxygen molecule, transferring an atom to the substrate, and reducing the another one to form water. To be functional, this proteins needs to be associated to electron transfer proteins, the cytochrome P450 reductases, a family of multidomain proteins which hold three cofactor binding domains (FMN, FAD and NADPH) and a linker domain situated between the FMN and FAD/NADPH domains. However, it havent been identified the electron donators for the proteins which arent placed in the RE on plants.

TRANSLATIONAL CHARACTERISTICS
This gene codifies for the protein CYP76C2 (Cytochrome P450, Family 76, Subfamily C, Polypeptide 2), which is also known as YSL6 and F17K2.10; it is a single-pass transmembrane hemoprotein, with a predicted length of 512 aminoacids

PROTEIN FUNCTION
The references about the function of this protein are scarce, being limited to studies about expression levels in different conditions, which allow inferring its role in stress response situations, as shown in the increase of transcription associated with the hypersensitive response induced by pathogens, senescence, mechanical damage or thermal shock. In order to try to discover in which metabolic pathway is involved our gene, initially two different strategies were addressed: firstly, coexpression pattern analyses were carried out, and secondly, the mutants that showed to change the expression patters of our gene at transcriptional level were deeply analyzed. Both studies led to propose an original function for our gene, which dont appear in scientific literature: the synthesis of flavonoids, probably of anthocyanins. Once identified the possible function, it were contrasted with its potential role in different circumstances. Subsequently, analysis of orthologous genes were carried out, which provided new evidences about this proposed function and that will be detailed later.

AtS40-3 is induced during senescence and is also regulated in response to dark treatment, ABA, salicylic acid and pathogen attack. The s40-3a mutant with a TDNA insertion in the promoter region of the gene was observed to have staygreen phenotype (FischerKilbienski et al., 2010). On the other hand, only other three genes appear on three arrays, of which one of them, At2g37770, it is also implicated in the phenylpropanoid synthesis; concretely, this gene codify for AKR4C9, a protein that shows high homology with chalcone reductase, showing a correlation coefficient of 0'8412, occupying the first position in the coexpression table (figure 7).

Coexpression pattern analysis

This analysis was done using the databases of three different platforms: the University of Leeds (Arabidopsis Coexpression Data Mining Tools) which offer two versions / platforms comprising oligonucleotide probe sets, the Affymetrix ATH1 and AtGenome arrays respectively recognizing around 22000 and 8000 Arabidopsis genes, Atteed-II and Cressexpress. All contain data from an extremely wide range of samples, covering biotic and abiotic treatments, different developmental stages and mutant analysis at different conditions. In order to do the analysis, the genes were sorted according their correlation coefficient (r), and the first forty genes of each source were selected. The initial set included a total of 130 different genes, which was processed, clustering the common genes, and selecting the genes whose correlation coefficient were above 07 (figure 7). Thus, the final set included 43 genes, of which only three of them, AT1G09500, AT4G37990 and AT4G18980 appear represented on the four expression arrays, showing very high correlation coefficients, concretely values of 0839726, 0827954 and 0,782948 respectively (figure 7). AT1G09500 codify for the protein F14J9.16 , while that AT4G37990 codify for the protein ELI3-2, both of them belonging to the cinnamyl alcohol dehydrogenase family, involved at the phenylpropanoid synthesis (Figure 6). AT4G18980, meanwhile, seems to be involved in senescence regulation; it codifies the protein AtS40-3, which belongs to a group of genes sharing the conserved DUF548.

Figure 6: Scatter diagrams; A shows the correlation between our gene and AT1G09500, and B with AT4G37990, the second and the third of the correlation table, respectively.

Figure 7: Table that includes those genes that show significant correlation values, based on data from databases Arabidopsis.Leeds, which provide two sets, 22k (A) and 8k (B),Cressexpress (C) and ATTED-II (D). The marked genes are those potentially associated with the synthesis of anthocyanins.

In addition to those described above, the correlation analysis show other genes potentially involved in phenylpropanoid biosynthesis: - AT3G55970: It codifies for the protein JRG21, a 2OG-Fe (II) oxidorreductase, which shows homology with a leuanthocianin dioxigenase involved in anthocyanin biosynthesis. - AT1G68620: It codifies a carboxylesterase of unknown function, although exist experimental evidences about the implication of some of these enzymes in priming the plant before the oxidation and deposition of phenolics (Keplan & Keen, 1980). - AT2G47190: This gene codifies for the AtMYB2, which controls the ABA induction of salt and dehydration responsive genes, although its activity effects is unknown. Several R2R3-MYBs are involved in the regulation of flavonoid biosynthesis, and bioinformatics analysis performed with different tools (JASPE, Althamap and PLACE) predict MYB binding sequences in our gene. - AT2G18980: It encodes the nuclear-targeted protein AtS40-3, that modulates senescence associated gene expression; It is a transcription factor belonging to the WRKY family .There are evidences that prove that rapid and massive induction of WRKY TF genes are correlated with the induction of genes involved in the central phenylpropanoid pathway and the downstream steps in the biosynthesis of the flavonoids and similar compounds. (Naoumkina et al.,2008). - AT2G39030 This gene codifies for GNAT, a hydroxycinnamoyl- CoA: tyramine N-hydroxycinnamoyltransferase. Likewise exists other three genes which show strong correlation, and that codify for enzymes with glycosyltransferase activity whose substrates are unknown, and that could be involved in the phenylpropanoid biosynthesis, acting as UDP-Glc: phenylpropanoid glucosyltransferases (UGTs). UGTs catalyze the transfer of glucose from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. Furthermore, the free acids rarely accumulate to high levels inside plant cells in free form; instead, they are usually conjugated to sugars (e.g., salicylate-glucose conjugates), cell wall carbohydrates (e.g., ferulate esters), or organic acids (e.g., sinapate esters, chlorogenic acid).(Schnitzler et al., 1992). Many phenylpropanoids have been shown to accumulate in the vacuole, usually as glycosides or other conjugates.

Other genes which show strong correlation and that potentially could be associated are AT1G32940 and AT5G22860, which codify genes with high homology with serine-peptidases, and although a priori do not seem to be related with phenylpropanoid pathway, recent studies bring under the spotlight that the functional annotation based on sequence offer many unknowns, and have demonstrated the evolutionary flexibility of plant phenylpropanoid biosynthesis through the discovery that an acyltransferase involved in the biosynthesis of the major leaf hydroxycinnamate ester, sinapoyl malate, is encoded in Arabidopsis by a gene with high sequence identity to serine carboxypeptidases (Lehfeldt et al., 2000), of which there are numerous annotated yet not functionally characterized family members in the Arabidopsis genome.

Expression analysis of mutants

This hypothesis is also supported by the expression levels of our gene in the pho1 mutant, which has been recently identified to affect a protein involved in the loading of inorganic phosphate into the xylem of roots in Arabidopsis. In this mutant, which has been shown to respond to phosphate deficiency in the leaves by decreasing the amount of phosphatidylglycerol (PG), the expression levels of AT2G45570 are increased 11'3 times, which is consistent with experimental evidence indicating that accumulation of anthocyanin is one of the characteristic responses of plants to Pi starvation via a DELLA-Dependent Mechanism (Dixon et Paiva, 1995).Increases in foliar anthocyanins have been reported in phosphorus-deficient Arabidopsis (Zakhleniuk et al.,2001). Another argument that justifies this hypothesis is CAT2HP2, a transgenic A. thaliana line deficient in CAT2, a peroxisomal catalase, whose phenotype is increased sensitivity toward both ozone and photorespiratory H2O2-induced cell death , showing also significant growth retardation. Expression analysis of this lines showed an increase in gene expression levels of our gene of 172 times (Vandenabeele et al.,2004) , which is consistent with an increase in anthocyanin levels given its antioxidative properties, whose presence could protect sensitive structures such as membranes (Leng et al., 2000) or chlorophyll from degradation. It is noteworthy that also appear to induce expression of ELI3-2 (At4g37990) 23 times.

Functional implications
Now, we shall proceed to propose possible functional implications of our gene in different contexts.

- Expression in seeds and siliques: wild type seeds contain more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9), most of them have been described for the first time in Arabidopsis, and appear in most of times as glucosilconjugates. (Routaboul et al., 2006). Also in siliques have been identified accumulations of isopropanoids. However, in most cases, the functions of these compounds are unknown. - Expression in sepals and petals: although anthocyanins are the major flower pigments in higher plants, this is not its function in Arabidopsis. Weiss (2000) noted that anthocyanins might facilitate the transport of sugars in flowers since they exist almost exclusively as glycosides, in our case probably associated with wilting. Phenylpropanoid polyamine conjugates have been identified in flowers and pollen grains of Arabidopsis, having identified several genes and enzymes the biosynthetic pathway leading to flower and pollen specific phenylpropanoid conjugates in A. thaliana, but the specific function of these conjugates for flower or pollen development remains a mystery (Fellenberg et al., 2009). - Expression in stamen: Flower Flavonoid Transporter (FFT) encodes a multidrug and toxin efflux family transporter in Arabidopsis thaliana. FFT (AtDTX35) is highly transcribed in floral tissues, being localized to epidermal guard cells, including those of the anthers, stigma, siliques and nectaries. Mutant analysis demonstrates that the absence of FFT transcript affects flavonoid levels in the plant and that the altered flavonoid metabolism has wide-ranging consequences, affecting dehiscence and pollen maturation. - Expression in pathogen response: the functions of phenylpropanoid compounds in plant defence range from preformed or inducible physical and chemical barriers against infection to signal molecules involved in local and systemic signalling for defence gene induction. Defensive functions are not restricted to a particular class of phenylpropanoid compound, but are found in the simple hydroxycinnamic acids and monolignols through to the more complex flavonoids, isoflavonoids and stilbenes. - Expression in mechanical damage: many phenylpropanoid compounds are induced in response to wounding or feeding by herbivores. lncreased levels of coumestrol and coumarin are toxic to potential herbivores, causing estrogenic and anticoagulant effects, and psoralens can cause photo-induced blistering (Smith, 1982). Wound-induced chlorogenic acid, alkyl ferulate esters, and cell wall-bound phenolic esters may act directly as defense compounds or may serve as precursors for the synthesis of lignin, suberin,

and other wound-induced polyphenolic barriers (Hahlbrock and Scheel, 1989; Bernards and Lewis, 1992). The accumulation of flavonols such as kaempferol and its glycosides is induced by wounding in petunia (Mo et al., 1992; van der Meer et al., 1992; Vogt et al., 1994). - Expression in saline and osmotic stress: Anthocyanins are highly water soluble, especially as glycosides, and are usually found in vacuoles. The increased accumulation of solute in any plant cell will allow it to maintain tolerable water conditions even under high levels of external osmoticum. This has particularly important implications for plants resistant to saline habitats. Studies with cell cultures of various species find anthocyanin accumulation resulting from osmotic stress induced by mannitol (Tholakalabavi et al., 1997; Tholakalabavi et al., 1994; Do and Cormier, 1991a; Rajendran et al., 1992; Suzuki, 1995) . Presumably, the accumulation of anthocyanins decreases osmotic potential and thereby prevents the loss of turgor, acting as osmoregulators. Experiments in whole-plant systems have shown similar results. - Expression in response to oxidant agents: given the antioxidative properties of anthocyanins, their presence could protect sensitive structures such as membranes (Leng et al., 2000) or chlorophyll from degradation, and also increase the percentage of bound water within the cells. Many studies support the hypothesis that anthocyanin synthesis in root, stem and especially leaf tissues may allow the plant to develop resistance consequence of improved resistance to oxidative stress, caused by its contributions with peroxidase activity. (Erylmaz , 2006). - Expression in nutrient deficiency: : nutrient, and particularly nitrogen (N), deficits detrimentally affect photosynthetic function and efficiency and decrease the levels of Calvin Cycle enzymes, which commonly induces or enhances the accumulation of foliar anthocyanin in leaves of many plant species. Phosphorus (P) deficit, which restricts phosphate levels essential to energy metabolism (Salisbury & Ross, 1992), increased anthocyanin content in a range of monocotyledonous and dicotyledonous plants (Atkinson, 1973). Maximum levels of anthocyanin accumulation in Z. mays occurred after 5 days under potassium (K) deficiency (K deficit impairs photosynthetic enzyme function; Bhandal & Malik, 1988), 10 days under P deficiency, and 15 days under N deficiency (Lawanson et al., 1972). Such evidences are perfectly consistent with the expression levels of our gene, whose transcription is induced 7 times in potassium deficiency conditions, and, although on expression microarrays were not analysed the behaviour in response to nitrogen or phosphorus deficiency, the effect in response to phosphorus deficiency could be studied through the Pho1 mutant, in which the expression is induced 11,3 times.

More tests are needed to study the effect against nitrogen limitation. - Expression in freezing: low temperature has been shown to induce anthocyanin synthesis and accumulation in leaves of a number of species. Anthocyanins would serve to depress the freezing point of water in these tissues, reducing the likelihood of the internal spread of ice from nucleating events on the leaf surface. Additionally, the presence of cellular anthocyanins would decrease the solute potential and inhibit water migration into extracellular spaces Recently, McKown et al. (1996) described four Arabidopsis mutants deficient in freezing tolerance were unable to accumulate anthocyanins, suggesting some commonality between anthocyanin biosynthesis and freezing tolerance. - Expression in senescence: the accumulation of anthocyanins in temperate, senescing deciduous leaves has been intensively studied both descriptively and experimentally. It is known that anthocyanins are synthesized de novo in response to the shorter days and cooler nights of autumn. The ability of anthocyanins to bind with otherwise reactive sugars such as glucose makes these pigments logical candidates for carbohydrate transport during senescence. Indeed, the presence of foliar sugars has been shown to induce anthocyanin synthesis as discussed earlier. Anthocyanins in these tissues could also depress the osmotic potential of the leaf so as to decrease water loss and support those biochemical pathways associated with programmed senescence. - Expression in response to ABA: treatment with ABA stimulated accumulation of anthocyanin and phenolics as well as increased ethylene production. Parallel changes in anthocyanin accumulation and PAL activity apparently reflect control of anthocyanin synthesis by PAL, presumably through the supply of component cinnamic acid molecules. (Jiang et Joyce, 2003). - Expression in UV exposition: flavonoids and other phenylpropanoid have long been thought to play a role in protecting against UV irradiation, because they accumulate primarily in the epidermal and hypodermal layers of leaves and stems (the most illuminated layers) and strongly absorb light in the UV-6 wavelengths. Recent studies have demonstrated that in Arabidopsis leaves, levels of flavonoids (such as kaempferol conjugates) and sinapate esters increase in response to UV irradiation (Li et al.,1993; Lois, 1994), supporting this proposed protective role. - Expression in metallic stress: experimental efforts have linked anthocyanin accumulation to exposure to heavy metals, including cobalt, iron, manganese, vanadium, and zinc. The mechanism(s) behind heavy metal tolerance is not clearly understood. A possible phytochelating effect is described by Hale et al. (2001),

whereby anthocyanins bind to molybdenum and are sequestered in the vacuoles of peripheral tissues. An inability to form phytochelators (including anthocyanins) was found in Arabidopsis mutants with hypersensitivity to cadmium exposure (Xiang et al. 2001). Other evidence suggests that heavy metals affect plant water relations.

PHILOGENETIC ANALYSIS
Homologous genes
After searching for proteins with sequence homology, 279 proteins showed significative values (for = 0,01), all belonging to the cytochrome P450 superfamily, reflecting the enormous diversity of this group. Indeed, one of the surprising findings when Arabidopsis genome sequence was completed was the extraordinary diversity of this superfamily. The final P450 count in the complete genome is 244 genes, some of which produce different genic products by alternative splicing, and 28 pseudogenes, which makes P450s one of the largest families of enzyme proteins in plants. Among the genes that show greater degree of homology are those belonging to the CYP76 family, which include nine genes in the Arabidopsis genome, eight belong to the CYP76C subfamily (including one pseudogene) and a single one to the subfamily CYP76G. The function of these P450s is not known, except for a patent claiming geraniol 10-hydroxylase activity for CYP76C1 (Otah and Mizutani, 1998). CYP76C1 to C4 form a small cluster on chromosome 2, and appear to have arisen by gene duplication. CYP76C1 was shown to be highly expressed in flowers, and at lower levels in stems, siliques and leaves (Mizutani et al., 1998). Its expression is decreased by wounding. It gradually increases upon illumination and drops when plants are returned to the dark.

Figure 8: Alignment of genes which show strongest homology degree in Arabidopsis thaliana

Orthologous genes
P450 cytochromes have been identified in all domains of life, that is, in animals, plants, fungus, protists, archeas and even some virus, having been identified more than 11500 different CYP proteins; because this huge diversity, for the analysis were selected the first 39 proteins belonging to different genus. The sequence alignment showed three clusters clearly different; AT2G45570 is grouped with other 6 genes (red branch in figure 9A), although are also uncharacterized CYP of unknown function.

On the other hand, despite the diversity of taxa, the alignment shows 26 completely conserved aminoacids among all the sequences, highlighting a region around the active site which includes 21 of these residues (figure 9C). Additional studies about the functional implication of such residues could be very useful to assign functions to these proteins, because, given the enormous functional plasticity of CYP in the nature, some of these conserved residues could be responsible of substrate specificity on which they act. This proposal is supported by the fact that, despite the unknown function of most of these proteins, 13 of them (33%) has been identified as Flavonoid 3',5'hydroxylases, which also supports the hypothesis of the involvement of our gene in anthocyanins metabolism.

Figure 9: A) Phylogenetic tree of the 39 proteins with more homology degree. B) Details of the branch which includes our gene. C) Details of the alignment regions which show higher conservation degree. The asterisk indicate that these proteins havent been 9 characterized.

TOOLS FOR ANALYSIS OF GENE

We have four tools for the analysis of our gene: cDNA clones, mutant lines, silenced lines by iRNA and the employ of the Oxygen Biosensor System for enzymatic analysis.

Determination of catalytic activity and kinetic parameters

The vast majority of the P450-catalysed reactions imply consumption of one molecule of NADPH (or of electrons from a donor source) and of one molecule of atmospheric oxygen:

cDNA clones
In the Tair database there are eight cDNA clones of our gene, of which three, U16830, GSLTLS3ZF04 and RAFL08-19-C07 are quite interesting: - U16830 Clone: It is between the positions 18779872 to 18781922; it has a length of 2051 bp and includes the translatable region of the transcript, and a 3'UTR fragment. It was cloned into a PENTR/SD-DTOPO vector. - GSLTLS3ZF04: It is between the positions 18779793 and 18781929; it has a length of 2137 bp and includes the whole transcript except an initial 5 UTR fragment. - RAFL08-19-C07: It is between the positions 18779815 18780174; it has a length of 360 bp and includes the third exon and the 3UTR, both incomplete. However, the only currently available is U16830.

Alexandre Olrys group proposed in 2007 a new method based on the detection of oxygen consumed during P450-catalysed reactions, broadly applicable to any P450 enzyme and a wide array of substrates. This method is a microplate-based assay that uses the Oxygen Biosensor System developed by BD Biosciences .Its first application is the functional characterization of the orphan plant cytochromes P450 revealed by genome analyses. Others include fast determination of catalytic parameters, screening for and design of P450 inhibitors, and screening of potential alternative substrates for industrial applications including biotechnology, biocatalysis and drug design. The monitoring of oxygen consumption in a microplate assay offers a powerful approach for the detection of cytochrome P450-catalysed reactions in membrane fractions of recombinant organisms.

Mutant lines
There are five SALK lines especially interesting for the study of our gene: SALK_011865C, which has been generated by T-DNA insertion in the position 18782032, upstream of the 5`UTR; this mutant would let us study the promoter sequence of the gene. Other interesting lines are SK22500, SK2250 (both resulted of the insertion of T-DNA in the second exon), SALK_107835.34.05.x and SALK_107836.16.70.x, (both generated by T-DNA insertion next to 5`end and middle position of the third exon, respectively). However, dont exist SALK lines that affect to the first exon, although could be possible to employ the mutant GT18181.Ds3.07.29.2005.jy52.342, which was generated by insertion in this region of a transponible element.

BIBLIOGRAPHY
Laurence Godiarda, Laurent Sauviaca, Nathalie Dalbin, Laurence Liaubet, Didier Callard, Pierre Czernic, Yves Marco (1998) CYP76C2, an Arabidopsis thaliana cytochrome P450 gene expressed during hypersensitive and developmental cell death.FEBS Ludmila Rizhsky, Hongjian Liang, Joel Shuman, Vladimir Shulaev, Sholpan Davletova, and Ron Mittler (2004) When Defense Pathways Collide. The Response of Arabidopsis to a Combination of Drought and Heat Stress. Plant Physiol. Yong Wang, Cecile Ribot, Enea Rezzonico2, and Yves Poirier (2004) Structure and Expression Profile of the Arabidopsis PHO1 Gene Family Indicates a Broad Role in Inorganic Phosphate Homeostasis. Plant Physiol. Richard A. Dixon' and Nancy L. Paiva (1995) Stresslnduced Phenylpropanoid Metabolism. The Plant Cell Richard A.Dixon, Lahocine Achnine, Parvathi Kota, Chang-Jun Liu, M.S. Srinivasa Reddy and Liangjiang Wang (2002) The phenylpropanoid pathway and plant defence-a genomics perspective. Molecular Plant Pathology.

Lines with gene silencing

Other tool that could let us study this gene is the line CATMA2a43970, which has AT2G45570 silenced by iRNA.

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