Class II Cytochrome P450 reductase governs the biosynthesis of alkaloids

45 Pages·2016·1.61 MB·English

Plant Physiology Preview. Published on September 29, 2016, as DOI:10.1104/pp.16.00801
1 Running title: Class II CPR governs alkaloid biosynthesis
2 Corresponding authors :
3 Vincent Courdavault
4 Université François-Rabelais de Tours - EA2106 "Biomolécules et Biotechnologies Végétales",
5 UFR Sciences et Techniques, 37200, Tours, France
6 Tel :[phone]
7 e-mail: [email protected]
8
9 Sarah E. O’Connor
10 Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney,
11 Norwich NR4 7UH, United Kingdom
12 Tel: [phone]
13 e-mail: Sarah.O’[email protected]
14
15 Research area: Biochemistry and Metabolism
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17 One sentence summary: Class II cytochrome P450 reductase in Madagascar periwinkle
18 displays a prominent contribution towards specialized metabolism by acting as the
19 main partner of P450s dedicated to alkaloid biosynthesis.
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32 Class II Cytochrome P450 reductase governs the biosynthesis of alkaloids
33 Claire Parage1*, Emilien Foureau1*, Franziska Kellner2*, Vincent Burlat3, Samira Mahroug1, Arnaud
34 Lanoue1, Thomas Dugé de Bernonville1, Monica Arias Londono1,4, Inês Carqueijeiro1, Audrey Oudin1,
35 Sébastien Besseau1, Nicolas Papon5, Gaëlle Glévarec1, Lucia Atehortùa4, Nathalie Giglioli-Guivarc’h1,
36 Benoit St-Pierre1, Marc Clastre1,Sarah E. O’Connor2#, Vincent Courdavault1#
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38 1Université François-Rabelais de Tours, EA2106 “Biomolécules et Biotechnologies Végétales”, Tours, France.
39 2Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH,
40 United Kingdom.
41 3Université de Toulouse, UPS, UMR 5546, Laboratoire de Recherche en Sciences Végétales, BP 42617 Auzeville,
42 F-31326 Castanet-Tolosan, France.
43 4Universidad de Antioquia, Laboratorio de Biotecnología, Sede de Investigación Universitaria, Medellin,
44 Colombia.
45 5Université d'Angers, EA3142 "Groupe d'Etude des Interactions Hôte-Pathogène", Angers, France.
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47 *These authors have contributed equally to this work
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49 AUTHOR CONTRIBUTIONS
50 C.P., E.F., A.L., M.A.L, I.C., N.P., M.C. performed the biochemical characterization of CPRs;
51 E.F. studied protein subcellular localization and interaction; F.K. carried out silencing
52 experiments; V.B., S.M., B.St-P. analyzed transcript distribution by in situ hybridization;
53 TDDB, S.B. achieved bioinformatics analyses; A.O., G.G. conducted analysis of gene
54 expression; N.G.G., B.St-P., L.A., M.C. assisted in the supervision of this work; S.E.O., V.C.
55 conceived, supervised and coordinated this study and wrote the manuscript.
56 FUNDINGS
57 This research has received funding from “Région Centre” of France (ABISAL Project). C.P. and
58 I.C. were supported by a postdoctoral fellowship from “Région Centre”. E. F. was financed by
59 a fellowship from the Ministère de l'Education Nationale, de l’Enseignement Supérieur et de
60 la Recherche (France). We acknowledge support from the ERC (311363) and a BBSRC
61 Institute Strategic Programme grant (MET; BB/J004561/1) to S.E.O’C. F.K. was supported in
62 part by a PhD studentship from the University of East Anglia.
63 Corresponding authors : [email protected] & Sarah.O’[email protected]
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65 Abstract
66 Expansion of the biosynthesis of plant specialized metabolites notably results from the
67 massive recruitment of cytochrome P450s that catalyze multiple types of conversion of
68 biosynthetic intermediates. For catalysis, P450s require a two-electron transfer catalyzed by
69 shared cytochrome P450 oxido-reductases (CPR), making these auxiliary proteins an
70 essential component of specialized metabolism. CPR isoforms usually group into two distinct
71 classes with different proposed roles, namely involvement in primary and basal specialized
72 metabolisms for class I and inducible specialized metabolism for class II. By studying the role
73 of CPR in the biosynthesis of monoterpene indole alkaloids (MIA), we provide compelling
74 evidence of an operational specialization of CPR isoforms in Catharanthus roseus. Global
75 analyses of gene expression correlation combined with transcript localization in specific leaf
76 tissues and gene silencing experiments of both classes of CPR all points to the strict
77 requirement of class II CPR for MIA biosynthesis with a minimal or null role of class I. Direct
78 assays of interaction and reduction of P450s in vitro however showed that both classes of
79 CPR performed equally well. Such high specialization of class II CPR in planta highlights the
80 evolutionary strategy that ensures an efficient reduction of P450s in specialized metabolism.
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82 Key-words : Catharanthus, cytochrome P450 reductase, specialized metabolism, alkaloids
83 Abbreviations: BiFC, bimolecular fluorescence complementation; C4H, cinnamate 4-
84 hydroxylase; C. roseus, Catharanthus roseus; CPR, NADPH-cytochrome P450 reductase; CFP,
85 cyan fluorescent protein; DFR, diflavin reductase; DIC, differential interphase contrast; FAD,
86 Flavin adenine dinucleotide; FMN, flavin mononucleotide; G8H, geraniol 8-hydroxylase; IPAP,
87 internal phloem associated parenchyma; IO, iridoid oxidase; PCC, Pearson correlation
88 coefficient; SLS, secologanin synthase; T3O, 16-methoxytabersonine 3-oxygenase; T16H,
89 tabersonine 16-hydroxylase; T19H, tabersonine 19-hydroxylase; YFP, yellow fluorescent
90 protein; VIGS, virus-induced gene silencing; 7DLH, 7-deoxyloganic acid 7-hydroxylase.
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94 INTRODUCTION
95 With more than 200,000 distinct molecules, the specialized metabolism of plants constitutes
96 one of the main sources of bioactive natural compounds, with this large number reflecting
97 the capacity of these sessile organisms to adapt to and interact with the environment. This
98 singular complexity of compounds results from an evolutionary process that involves a
99 dramatic diversification of plant metabolic pathways and the genes encoding the associated
100 metabolic enzymes. From this perspective, cytochromes P450 (P450s) are a prototypical
101 example of ubiquitous enzymes encoded by a gene superfamily that can carry out multiple
102 types of reactions ranging from hydroxylation, epoxidation, oxygenation, dealkylation,
103 decarboxylation, C-C cleavage or ring opening (Bak et al., 2011; Guengerich and Munro,
104 2013). P450s catalyze a considerable array of chemically challenging reactions in the
105 biosynthesis of plant specialized metabolites including phenylpropanoids, terpenoids,
106 cyanogenic glycosides and alkaloids (Mizutani and Ohta, 2010; Mizutani and Sato, 2011), in
107 addition to various roles in plant primary metabolism, such as the biosynthesis of hormones
108 (Bak et al., 2011; Takei et al., 2004). Most characterized P450s perform single oxidation
109 reactions but growing evidence now also points to the existence of multi-step oxidations
110 catalyzed by a single P450 (Guengerich et al., 2011). However, in all P450 catalyzed
111 reactions, the catalytic cycle always requires two one-electron transfer steps from NADPH
112 into the prosthetic heme. This transfer occurs through the flavin adenine dinucleotide (FAD)
113 and flavin mononucleotide (FMN) domains of NADPH-cytochrome P450 reductases (CPR),
114 though in some cases, the second electron transfer can also arise from cytochrome b5
115 (Munro et al., 2013). The absolute requirement of CPR thus makes this flavoprotein a
116 cornerstone in P450 activities and, consequently, in specialized and primary metabolic
117 pathways. As such, the physical interactions between P450s and CPR that guide electron
118 shuffling directly influence P450 activities and are facilitated by membrane anchoring of
119 both types of proteins, which are typically localized to endoplasmic reticulum (Hasemann et
120 al., 1995; Ro et al., 2002; Denisov et al., 2007). Elucidation of the three-dimensional
121 structure of the rat CPR suggested that P450s/CPR interactions occur through both ionic
122 interactions involving the FMN domain of CPR and hydrophobic interactions between the
123 membrane domains of CPR and P450s (Wang et al., 1997). The low CPR/P450 ratio,
124 measured at approximately 1:15 in liver, also implies a potential competition between P450s
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125 for these accessory proteins in plants and as a consequence, some kind of logistic control
126 must be employed to ensure the coordinated operation of all P450s belonging to similar
127 biosynthetic pathways (Shephard et al., 1983).
128 In contrast to yeasts and mammals that harbor only a single CPR, vascular plants have
129 evolved 2 or 3 CPR isoforms, as reported for instance in Jerusalem artichoke (Benveniste et
130 al., 1991), poplar (Ro et al., 2002), parsley (Koopmann and Hahlbrock, 1997), cotton (Yang et
131 al., 2010) and winter cherry (Rana et al., 2013).The Arabidopsis genome contains two genes
132 encoding functionally active CPR genes (Arabidopsis Thaliana P450 Reductase), named ATR1
133 and ATR2, and a third more distant gene (ATR3) whose expression product was not able to
134 reduce P450s in vitro (Urban et al., 1997; Mizutani and Ohta, 1998; Varadarajan et al., 2010).
135 By contrast, poplar and Nothapodytes foetida possess three genes encoding genuine CPRs
136 that are similar to ATR1 and ATR2 and an additional predicted ATR3-like isoform (Ro et al.,
137 2002; Huang et al., 2012). Homologs of ATR1 and ATR2 in flowering plants are highly
138 conserved (65%-80%) and are clustered into two distinct phylogenic groups on the basis of
139 the N-terminal sequences. The first cluster (ATR1 homologs, class I) contains sequences from
140 eudicotyledons while the second (class II) are found in both monocotyledons and
141 eudicotyledons (Ro et al., 2002). Homologs of ATR3 fall in a distinct third cluster
142 (Varadarajan et al., 2010). While CPRs from both class I and class II can reduce P450 with an
143 apparent similar efficiency, their expression profiles are different (Urban et al., 1997;
144 Mizutani and Ohta, 1998; Ro et al., 2002). CPR1s belonging to class I, such as ATR1, are
145 constitutively expressed, while CPR2s from class II (ATR2 for example) are inducible by
146 environmental stimuli such as wounding, pathogen infection or light exposure (Koopmann
147 and Hahlbrock, 1997; Mizutani and Ohta, 1998; Ro et al., 2002; Schwarz et al., 2009; Yang et
148 al., 2010, Rana et al., 2013). These observations suggest that each CPR isoform has dedicated
149 physiological roles, ensuring that plants can meet the reductive demand of P450-mediated
150 reactions. It is now believed that constitutively expressed CPRs (CPR1s- class I) ensure basal
151 P450 activities in primary metabolism or in constitutive synthesis of specialized metabolism,
152 while inducible CPRs (CPR2s- class II) serve in adaptation mechanisms or in defense reactions
153 including the elicited biosynthesis of specialized metabolites. This hypothesis has been
154 partially confirmed by establishing a correlation between ATR2 activity and lignin
155 biosynthesis in Arabidopsis as well as between CPR1 expression and basal pungent alkaloid
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156 synthesis in Capsicum spp. (Mazourek et al., 2009; Sundin et al., 2014). In contrast, CPR-like
157 (ATR3) homologs are poorly characterized but appear to be essential for embryo
158 development (Varadarajan et al., 2010). However, more investigations are required to firmly
159 establish the functional specificity of all CPRs in plants.
160 For more than forty years, specialized metabolism has been investigated using the
161 Madagascar periwinkle, Catharanthus roseus (C. roseus), which serves as an excellent case
162 study for study of plant specialized metabolism. This plant most notably synthesizes alkaloids
163 from the monoterpene indole alkaloid (MIA) family that includes valuable compounds such
164 as the antineoplastic vinblastine and vincristine. These MIAs result from a long and complex
165 biosynthetic pathway whose characterization has made great progress over the last five
166 years due to the development of large sets of transcriptomic data and a draft genome
167 sequence (Góngora-Castillo et al., 2012; Van Moerkercke et al., 2013 ; Xiao et al., 2013; Dugé
168 de Bernonville et al., 2015a; Kellner et al., 2015a). Within the 30-50 enzymatic steps
169 predicted to form the network of reactions of the MIA biosynthetic pathway, no less than
170 eleven P450s have been successively identified to date including (Figure 1): geraniol 8-
171 hydroxylase (G8H, CYP76B6; Collu et al., 2001), iridoid oxidase (IO, CYP76A26; Salim et al.,
172 2014: Miettinen et al., 2014), 7-deoxyloganic acid 7-hydroxylase (7DLH, CYP72A224; Salim et
173 al., 2013; Miettinen et al., 2014), four isoforms of secologanin synthase (SLS1-4, CYP72A1;
174 Irmler et al., 2000; Dugé de Bernonville et al., 2015b; Brown et al., 2015), two isoforms of
175 tabersonine 16-hydroxylase (T16H1-2, CYP71D12-CYP71D351; Schröder et al., 1999;
176 Guirimand et al., 2011; Besseau et al., 2013), tabersonine 19-hydroxylase (T19H, CYP71BJ1;
177 Giddings et al., 2011 ) and 16-methoxytabersonine 3-oxygenase (T3O, CYP71D1; Kellner et
178 al., 2015b; Qu et al., 2015a). While some of these enzymes catalyze a single oxygenation
179 reaction including hydroxylation (7DLH, T16H1, T16H2, T19H) or epoxidation (T3O), unusual
180 reactions have been also reported such as the ring opening of loganin to yield secologanin
181 (SLS) or the three step oxidation of nepetalactol to form 7-deoxyloganetic acid (IO). In
182 addition to this diversity of reactions, a complex spatiotemporal organization of these P450-
183 mediated enzymatic steps has been also reported in periwinkle leaves with the first MIA
184 biosynthetic conversions (G8H to 7DLH) occurring in internal phloem associated parenchyma
185 (IPAP) and the remaining reactions until T3O and the next two enzymatic steps in epidermis
186 (Figure 1; Courdavault et al., 2014; Salim et al., 2014; Miettinen et al., 2014; Qu et al.,
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187 2015a). As a matter of fact, this multicellular organization of the MIA biosynthetic pathway
188 constitutes the first layer of the physiological processes regulating MIA formation.
189 Interestingly, the periwinkle CPR was one of the first plant CPRs to be purified and cloned
190 (Madyastha and Coscia, 1979; Meijer et al., 1993). Due to the presence of a hydrophobic
191 residue region at its N-terminal end, this protein groups with class II CPRs in agreement with
192 its transcriptional regulation in response to fungal elicitor preparation and to jasmonate
193 (Meijer et al., 1993; Collu et al., 2001; Ro et al., 2002). Although only one CPR had been
194 cloned in C. roseus at the start of this study, occurrence of multiple isoforms was expected
195 and is now supported by transcriptomic and genomic data (Canto-Canché and Loyolas-
196 Vargas, 2001). However, no formal relationship had been established between the
197 periwinkle CPRs and the biosynthesis of MIA. This prompted us to take advantage of the
198 richness of the C. roseus MIA metabolism to accurately explore the role of each class of CPRs
199 in specialized metabolism. By combining biochemical characterization, protein interaction
200 analyses, mapping of cellular gene expression profiles and gene silencing approaches, our
201 data provide new evidence for the predominant role of class II CPR in MIA/specialized
202 metabolism.
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205 RESULTS
206 Identification of C. roseus CPR homologs
207 In addition to a contig corresponding to the CPR cDNA (X69791), which was
208 previously cloned by Meijer et al. (1993), interrogation of C. roseus transcriptomic resources
209 led to the identification of two additional contigs homologous to CPR (Supplemental Table
210 1). Proteins deduced from “contig CPR candidate 1” and “contig CPR candidate 2” displayed
211 67% and 25% of identity with the originally identified CPR, respectively (Supplemental
212 Figure 1). According to previously published classifications of CPRs (Ro et al., 2002; Jensen
213 and Moller, 2010; Varadarajan et al., 2010), phylogenetic analyses demonstrated that each
214 deduced protein clusters in distinct phylogenetic subgroups (Supplemental Figure 2). While
215 the original CPR is categorized as a class II CPR, “contig CPR candidate 1” falls into the class I
216 CPR, which is hypothesized to be mostly dedicated to basal metabolism. “Contig CPR
217 candidate 2” clusters in class III CPR whose prominent member in Arabidopsis corresponds
218 to ATR3. Based on this result and on the Arabidopsis nomenclature, “contig CPR candidate
219 1”, the original CPR and “contig CPR candidate 2” were renamed CPR1, CPR2 and
220 CPR3/diflavin reductase (DFR), respectively. C. roseus genome analysis also revealed that the
221 three corresponding genes were present at one copy per haploid genome and spanned over
222 18 exons for CPR1 (CRO_T001672) and CPR2 (CRO_T031702) and and 12 exons for CRP3
223 (CRO_T033752) (Supplemental Figure 3). Genomic organization of CPR1 and CPR2 is similar
224 regarding intron positions and intron/exon sizes except for the first intron of CPR1 that is
225 roughly 7-fold longer than the first intron of CPR2. Such similarity may reflect the gene
226 duplication event that led to both CPR appearances. Therefore, these results suggest that C.
227 roseus contains two CPRs, CPR1 (KJ701028) and CPR2 (X69791) potentially associated to
228 basal and inducible/specialized metabolism, respectively, and one more distant copy
229 CPR3/DFR (KM111538), a likely ortholog of ATR3, the function of which remains unclear.
230 Sequence analysis and subcellular localization of C. roseus CPRs
231 Analysis of the deduced protein sequences of C. roseus CPRs revealed that both CPR1
232 and CPR2 are characterized by (i) the presence of conserved FMN-, FAD- and NADPH binding
233 domains that have been implicated in electron transfer, and (ii) identical residues predicted
234 to be involved in interactions with P450s (Jensen and Moller, 2010) (Supplemental Figure 4,
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235 Supplemental Figure 5). Both proteins also bear a predicted membrane spanning domain at
236 their N-terminal end (Supplemental Figure 6) that has been shown to mediate endoplasmic
237 reticulum (ER) anchoring in poplar (Ro et al., 2002). To investigate whether this sequence
238 does in fact anchor the protein to the ER membrane, the subcellular localization of C. roseus
239 CPR1 and CPR2 were expressed as a C-terminal yellow fluorescent protein (YFP) fusion
240 proteins (CPR1-YFP, CPR2-YFP) to avoid interference with the membrane spanning domain.
241 In C. roseus transiently transformed cells, the fusion proteins exhibited a network-shaped
242 fluorescent signal that perfectly merged with the signal of the “ER”-cyan fluorescent protein
243 (CFP) marker (Figure 2A-H), suggesting that both CPR1 and CPR2 are anchored to the ER, in
244 agreement with the classical localization pattern of P450s. When mutants of CPR1 and CPR2
245 that lacked this predicted transmembrane domain were expressed as YFP labeled fusions,
246 the previously observed localization pattern was disrupted, while the fusion of these
247 membrane domains to YFP enabled ER anchoring. These experiments clearly demonstrate
248 that this predicted spanning membrane domain is necessary and sufficient to ensure ER
249 localization/anchoring (Supplemental Figure 7). In addition, in agreement with the assigned
250 CPR classification, we noted that CPR1 and CPR2 differ in the length of the protein sequence
251 preceding the membrane spanning domain; CPR1 only has a short stretch of residues while
252 CPR2 exhibits an extended amino acid sequence, enriched in serine residues, which was
253 initially, but wrongly, considered as a plastid targeting sequence (Supplemental Figure 1; Ro
254 et al., 2002). CPR3/DFR also displays conserved FMN-, FAD- and NADPH binding domains but
255 shows substantial differences in the P450 interacting region compared to CPR1 and CPR2
256 (Supplemental Figure 5). Moreover, CPR3/DFR lacks the N-terminal membrane spanning
257 domain, explaining the nucleocytosolic localization observed in C. roseus cells transformed
258 with CPR3-YFP fusion protein (Figure 2I-L). This nucleocytosolic localization has been
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