Discovery, biosynthesis and stress-related accumulation of dolabradiene-derived defenses in maize

62 Pages·2017·4.7 MB·English

Plant Physiology Preview. Published on February 23, 2018, as DOI:10.1104/pp.17.01351

1 Short title: Biosynthesis of maize diterpenoids
2
3 *To whom correspondence may be addressed: Philipp Zerbe; Department of Plant Biology,
4 University of California-Davis, One Shields Avenue, Davis, CA 95616, United States; phone: +1
5 530-754-9652; e-mail: [email protected]
6
7 Research Area: Biochemistry and Metabolism
8
9 Discovery, biosynthesis and stress-related accumulation of dolabradiene-derived defenses
10 in maize
11
12 Sibongile Mafu1*#, Yezhang Ding2*, Katherine M. Murphy1, Omar Yaacoobi1, J. Bennett
13 Addison3, Qiang Wang4, Zhouxin Shen2, Steven P. Briggs2, Jörg Bohlmann5, Gabriel Castro-
14 Falcon6, Chambers C. Hughes6, Mariam Betsiashvili2, Alisa Huffaker2, Eric A. Schmelz2, Philipp
15 Zerbe1*
16
17 1Department of Plant Biology, University of California Davis, One Shields Avenue, Davis, CA,
18 USA.
19 2Section of Cell and Developmental Biology, University of California San Diego, 9500 Gilman
20 Dr., La Jolla, CA, USA.
21 3Department of Chemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA,
22 USA.
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23 4College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China; Institute of
24 Ecological Agriculture, Sichuan Agricultural University, Chengdu 611130, China.
25 5Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
26 6Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography,
27 University of California San Diego, La Jolla, CA, USA.
28
29 One sentence summary
30 This study describes a class of stress-related diterpenoids, termed dolabralexins, in maize, and
31 identifies diterpene synthases and a cytochrome P450 involved in their biosynthesis.
32
33 List of Author’s contributions
34 P.Z., E.A.S., A.H. and J.B. conceived the original research, designed the experiments and
35 oversaw data analysis; S.M., Y.D., and K.M.M. performed most experiments; O.Y., M.B. and
36 E.A.S. assisted with diterpenoid metabolite purifications, B.A. G.A.C., and C.C.H. assisted with
37 NMR analyses of 3β,15,16-trihydroxydolabrene; Z.S. and S.P.B. assisted with high mass
38 resolution MS/MS experiments; Q.W. contributed to enzyme analysis; P.Z. wrote the article with
39 contributions of all the authors.
40
41 Funding sources
42 P. Z. gratefully acknowledges support by startup funds through the University of California
43 Davis and the Hellman Foundation, J.B. acknowledges financial support from the Natural
44 Sciences and Engineering Research Council (NSERC) of Canada, E.A.S and A.H. acknowledge
45 primary support from the University of California San Diego startup funds and partial support for
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46 this work through a NSF-IOS Competitive Award (#1139329). P.Z., E.A.S and A.H. collectively
47 acknowledge support the DOE Joint Genome Institute Community Science Program (grant #WIP
48 2568).

49
50 #Present address: University of Massachusetts-Amherst, Department of Biochemistry and
51 Molecular Biology, Life Science Laboratories, N431, 240 Thatcher Way, Amherst, MA, USA.
52 *Co-first author: contributed equally to the manuscript
53 Corresponding author email: [email protected]
54
55 Abstract
56 Terpenoids are a major component of maize (Zea mays) chemical defenses that mediate
57 responses to herbivores, pathogens and other environmental challenges. Here, we describe the
58 biosynthesis and elicited production of a class of maize diterpenoids, named dolabralexins.
59 Dolabralexin biosynthesis involves the sequential activity of two diterpene synthases, the ENT-
60 COPALYL DIPHOSPHATE SYNTHASE (ZmAN2) and KAURENE SYNTHASE-LIKE 4
61 (ZmKSL4). Together, ZmAN2 and ZmKSL4 form the diterpene hydrocarbon dolabradiene. In
62 addition, we biochemically characterized a cytochrome P450 monooxygenase, ZmCYP71Z16,
63 which catalyzes the oxygenation of dolabradiene to yield the epoxides 15,16-epoxydolabrene
64 (epoxydolabrene) and 3β-hydroxy-15,16-epoxydolabrene (epoxydolabranol). Absence of
65 dolabradiene and epoxydolabranol in Zman2 mutants under elicited conditions confirmed the in
66 vivo biosynthetic requirement of ZmAN2. Combined mass spectrometry and nuclear magnetic
67 resonance experiments demonstrated that much of the epoxydolabranol is further converted into
68 3β,15,16-trihydroxydolabrene. Metabolite profiling of field-grown maize root tissues indicated
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69 that dolabralexin biosynthesis is widespread across common maize cultivars, with
70 trihydroxydolabrene as the predominant diterpenoid. Oxidative stress induced dolabralexin
71 accumulation and transcript expression of ZmAN2 and ZmKSL4 in root tissues, and metabolite
72 and transcript accumulation were up-regulated in response to elicitation with the fungal
73 pathogens Fusarium verticillioides and F. graminearum. Consistently, epoxydolabranol
74 significantly inhibited growth of both pathogens in vitro at 10 µg ml-1, while
75 trihydroxydolabrene-mediated inhibition was specific to F. verticillioides. These findings
76 suggest that dolabralexins have defense-related roles in maize stress interactions and expand the
77 known chemical space of diterpenoid defenses as genetic targets for understanding and
78 ultimately improving maize resilience.
79
80 Introduction
81 Plants produce diverse arrays of specialized metabolites that govern interactions with, and
82 adaptation to, their biotic and abiotic environments (Pichersky and Gershenzon 2002; Hartmann
83 2007; Pichersky and Lewinsohn 2011; Tholl 2015). Among these specialized metabolites,
84 diterpenoids are a large class of more than 10,000 known compounds, with diverse physiological
85 and biological functions. Plants typically harbor both a widely conserved subset of diterpenoids
86 such as the gibberellins (GAs), a class of phytohormones mediating developmental processes,
87 and a myriad of specialized compounds with diverse roles in chemical ecology and plant
88 adaptation (Keeling and Bohlmann 2006; Gershenzon and Dudareva 2007; Chaturvedi et al.
89 2012; Schmelz et al. 2014; Hedden and Sponsel 2015). Because of their agronomic importance
90 as two of the world’s major food crops, maize (Zea mays) and rice (Oryza sativa) have been
91 extensively studied for their terpenoid-based defense systems (Schmelz et al. 2014; Degenhardt,
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92 2009). In rice, a suite of momilactone, oryzalexin and phytocassane diterpenoids confer defense
93 against several major pathogens, such as rice blast (Magnaporthe oryzae) (Peters 2006; Kato-
94 Noguchi and Kobayashi 2009; Toyomasu et al. 2014). As components of rice root exudates,
95 momilactones can also exhibit allelopathic activity suppressing the development of neighboring
96 plants of other species (Kato-Noguchi and Peters 2013). These findings exemplify the diverse,
97 protective biological roles of diterpenoids in Poaceae crops. However, the potential applications
98 of these chemical defenses in agricultural improvement has been limited, in part, by knowledge
99 gaps in understanding identities, biosynthesis, regulation, and ecological roles in different crop
100 species.
101
102 In maize, two groups of acidic terpenoids, the kauralexin class of diterpenoids and the zealexin
103 class of sesquiterpenoids (Fig. 1), are part of a complex repertoire of defense-related specialized
104 metabolites, such as phenylpropanoids, oxylipins, benzoxazinoids, and volatile terpenoids
105 (Schnee et al. 2006; Köllner et al. 2008a; Santiago and Malvar 2010; Huffaker et al. 2011;
106 Schmelz et al. 2011; Ahmad et al. 2011; Christensen et al. 2015; Richter et al. 2016; Wouters et
107 al. 2016). Inducible zealexin and kauralexin production contributes to defense, both above- and
108 below-ground, against pathogenic fungi including Fusarium, Aspergillus, and Colletotrichum
109 species (Huffaker et al. 2011; Schmelz et al. 2011; Vaughan et al. 2015). Kauralexins also
110 function as insect feeding deterrents (Schmelz et al. 2011) and accumulate in response to salt and
111 drought stress (Vaughan et al. 2015). These properties are consistent with diverse roles in stress
112 resilience that extend well beyond antimicrobial activity.
113
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114 Much of the diterpenoid chemical diversity of plants is produced through the sequential activity
115 of enzymes encoded by two functionally diverse gene families, the diterpene synthases (diTPSs)
116 and the cytochrome P450 monooxygenases (P450s). Plants employ variable combinations of
117 different diTPSs and P450s to transform a few ubiquitous diterpene precursors into thousands of
118 different diterpenoid metabolites, many of which are of limited taxonomic distribution (Peters
119 2010; Zerbe and Bohlmann 2015). Modular enzyme networks largely determine the variations of
120 diterpene profiles in closely and distantly related plant species (Peters 2010; Hamberger and Bak
121 2013; Zerbe and Bohlmann 2015). Naturally occurring modularity in diterpenoid biosynthesis
122 further enables the engineering of combinatorial diTPS and P450 expression systems in
123 microbial and plant host systems to accelerate the discovery of terpenoid pathways (Lange and
124 Ahkami 2013; Brückner and Tissier 2013; Zerbe and Bohlmann 2015; Kitaoka et al. 2015).
125
126 In monocots, diterpenoid pathway networks comprise sets of diTPSs with conserved and species-
127 specific functions in wheat (Triticum aestivum) and rice (Wu et al. 2012; Zhou et al. 2012;
128 Morrone et al. 2006; Prisic et al. 2004; Xu et al. 2004; Xu et al. 2007a; Fu et al. 2016). The
129 ensuing diTPS products often undergo further structural modifications through the activity of
130 P450 enzymes, as exemplified in rice by the P450-catalyzed formation of several bioactive
131 diterpenoid metabolites (Swaminathan et al. 2009; Wang et al. 2011; Wang et al. 2012a; Wang et
132 al. 2012b). Comparatively, less was known about the complexity of maize diterpenoid
133 biosynthesis. Characterized maize diterpenoid enzymes include the ENT-COPALYL
134 DIPHOSPHATE (CPP) SYNTHASE ZmAN1 (Bensen et al. 1995), the ENT-KAURENE
135 SYNTHASES ZmTPS1, ZmKLS3, and ZmKSL5 (Fu et al. 2016), and the ENT-KAURENE
136 OXIDASE CYP701A26 (Mao et al. 2017) (Fig. 1). All of these enzymes have either validated or
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137 predicted roles in GA metabolism. However, the frequently observed substrate promiscuity of
138 characterized enzymes (Peters 2010; Morrone et al. 2011; Zerbe and Bohlmann 2015) may also
139 enable roles in defense-related specialized metabolism. In the absence of mutant analyses, the
140 maize kauralexin and zealexin metabolic pathways have remained largely unproven, with the
141 exception of the ent-CPP synthase ZmAN2, which has been empirically demonstrated to
142 function in kauralexin formation (Harris et al. 2005; Vaughan et al. 2015). The β-macrocarpene
143 synthases ZmTPS6, ZmTPS11, and ZmCYP71Z18 have been assigned to the zealexin pathway
144 based on predictions from in vivo biochemical assays (Mao et al. 2016; Köllner et al. 2008b).
145
146 In this study, we functionally characterized ZmAN2, ZmKSL4, and ZmCYP71Z16, which
147 together form a biosynthetic branch of diterpenoids, termed dolabralexins. Patterns of inducible
148 dolabralexin accumulation and gene expression in maize roots exposed to different stresses,
149 combined with antifungal bioactivity of epoxydolabranol and trihydroxydolabrene, support
150 diverse roles for dolabralexins in the maize defensive arsenal.
151
152 Results
153 The maize diterpene synthase family
154 The maize genome sequence (B73 RefGen_v3) encodes a group of four class II diTPSs
155 (ZmAN1, ZmAN2, ZmCPS3-4) and six class I diTPSs (ZmTPS1, ZmKSL1-5). Of the identified
156 class I diTPSs, three (ZmTPS1, ZmKSL3, and ZmKSL5) have been characterized as ent-kaurene
157 synthases (Fu et al. 2016), and three members (ZmKSL1, ZmKLS2, and ZmKSL4) are of
158 unknown function. Phylogenetic analysis placed ZmKSL1, ZmKLS2, and ZmKSL4 adjacent to
159 rice diTPSs of specialized metabolism (Fig. 2). Most members of the maize diTPS gene family
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160 are dispersed across the genome (Supplemental Fig. S1), which differs from the clustering of
161 genes of specialized diterpenoid pathways in rice (Schmelz et al. 2014). However, the maize ent-
162 CPP synthases, ZmAN1, which functions in GA biosynthesis (Bensen et al. 1995), and ZmAN2,
163 which has functions in specialized metabolism (Harris et al. 2005), co-localize with ZmKSL4 on
164 chromosome 1, with a distance of ~4 Mbp and ~159 Mbp, respectively.
165
166 ZmKSL4 produces an unusual diterpene scaffold
167 To test for activity of ZmKSL4 in specialized diterpenoid metabolism, we utilized the
168 combinatorial functionality of class II and class I diTPSs of different plant species, which can be
169 exploited to probe diTPS functions (Zerbe et al. 2013; Kitaoka et al. 2015; Brückner and Tissier
170 2013). An established E. coli co-expression system (Morrone et al. 2010) was used to analyze the
171 coupled activities of ZmKSL4 with (i) the ent-CPP synthase ZmAN2, (ii) the rice syn-CPP
172 synthase (OsCPS4) (Xu et al. 2004), and (iii) grand fir (Abies grandis) abietadiene synthase
173 variant D621A that produces CPP of normal (i.e. (+)) stereochemistry (Morrone et al. 2010).
174 These three different combinations were selected, since ent-, syn- and (+)-CPP represent the
175 known stereochemical variation of class II diTPS products naturally occurring in many monocots
176 (Schmelz et al. 2014; Peters 2010). When co-expressed with ZmAN2, ZmKSL4 converted ent-
177 CPP into a single product with an unusual fragmentation pattern showing major mass ions of m/z
178 204, m/z 189, and m/z 216, in addition to the mass ions m/z 257 and m/z 272 that are
179 characteristic of labdane diterpenoid structures (Fig. 2, compound 1). Preparative enzymatic
180 synthesis and purification of this product by silica chromatography and semi-preparative high
181 performance liquid chromatography (HPLC) enabled 1D and 2D nuclear magnetic resonance
182 (NMR) analysis, and identified the ZmAN2/ZmKSL4 product as dolabradiene (Fig. 2 and
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183 Supplemental Fig. S2). In addition, ZmKSL4 was active in combination with (+)-CPP to yield
184 low amounts of several other diterpenoids (compounds 2-9), of which (+)-pimara-8,15-diene 2
185 and (+)-sandaracopimaradiene 3 could be identified by comparison to authentic standards (Fig. 2
186 and Supplemental Fig. S3). Similarly, ZmKLS4 showed activity with syn-CPP, resulting in the
187 formation of syn-isopimara-7,15-diene 10, syn-pimara-7,15-diene 11 and syn-pimara-9(11),15-
188 diene 12.
189
190 ZmAN2, ZmKSL4, and ZmCYP71Z16 function together to form 15,16-epoxydolabrene
191 To gain insight into the structural elaboration of the predominant product of ZmAN2/ZmKSL4,
192 namely dolabradiene, we tested its further modification by maize P450s. To this end, we
193 performed a BLAST search of the B73 RefGen_v3 genome against monocot P450s of the
194 CYP71, CYP76, and CYP99 families known to function in terpenoid metabolism. This search
195 identified a member of the CYP71 family, ZmCYP71Z16, which showed high protein sequence
196 similarity (89%) to the recently reported maize ZmCYP71Z18 involved in zealexin biosynthesis
197 (Mao et al. 2016). Phylogenetic analysis placed ZmCYP71Z16 adjacent to ZmCYP71Z18 within
198 a clade that also contained the rice P450s CYP71Z1, 6 and 7, of which CYP71Z6 and CYP71Z7
199 catalyze reactions in oryzalide and phytocassane biosynthesis (Wu et al. 2011) (Fig. 3).
200
201 To biochemically characterize ZmCYP71Z16, a codon-optimized and N-terminally modified
202 coding sequence was synthesized and co-expressed in E. coli with ZmAN2 and ZmKSL4, as
203 well as the maize cytochrome P450 reductase ZmCPR2. Co-expression of the two diTPSs and
204 ZmCPR2 without ZmCYP71Z16 served as a negative control. When all four enzymes were co-
205 expressed, the presence of ZmCYP71Z16 resulted in the conversion of dolabradiene into two
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206 new products with fragmentation patterns featuring m/z 288 (compound 13) and m/z 304
207 (compound 14) mass ions that are characteristic of diterpenoid scaffolds containing one and two
208 oxygen atoms, respectively (Fig. 3). High resolution liquid chromatography-mass spectrometry
209 (LC-MS) analysis identified the exact mass of compound 14 as m/z 304.2478, consistent with a
210 molecular formula of C H O . Subsequent HPLC-purification of compounds 13 and 14
20 32 2
211 followed by NMR analysis verified the structures as 15,16-epoxydolabrene (epoxydolabrene)
212 and 3β-hydroxy-15,16-epoxydolabrene (epoxydolabranol), respectively (Fig. 3 and Supplemental
213 Fig. S4). The biochemical characterization of ZmCYP71Z16 adds a previously unknown
214 function to the diverse activities of members of the plant CYP71 family, facilitating the regio-
215 specific oxygenation of dolabradiene at C-3 and C-16, presumably in a sequential reaction
216 process. The formation of the mono-epoxide 13, in the absence of detectable formation of a
217 mono-hydroxyl intermediate, is consistent with a P450 reaction sequence of epoxidation at C-16
218 prior to hydroxylation at C-3. To further investigate the catalytic activity of ZmCYP71Z16, we
219 performed E. coli co-expression assays with ZmCPR2 and fed 10 µM purified epoxydolabrene
220 and epoxydolabranol to the culture 5 h post induction. Expectedly, no new products were
221 identified when feeding epoxydolabranol to the culture. In contrast, epoxydolabrene was
222 converted to the hydroxylated derivative (Supplemental Fig. S5), supporting our proposed
223 reaction sequence from epoxydolabrene to epoxydolabranol.
224
225 The phylogenetic relatedness of ZmCYP71Z16 and the previously reported zealexin-forming
226 ZmCYP71Z18 (Mao et al. 2016) indicated that ZmCYP71Z16 may be active on other substrates
227 beyond a role in epoxydolabranol biosynthesis. To test this hypothesis, we conducted E. coli co-
228 expression assays of ZmCYP71Z16 with ZmCPR2 and ZmTPS11, which form the zealexin
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