Functional characterization of the rice kaurene synthase-like gene family

PHYTOCHEMISTRY

Phytochemistry 68 (2007) 312–326

www.elsevier.com/locate/phytochem

Functional characterization of the rice kaurene synthase-like

gene family

a

b,1

Meimei Xu , P. Ross Wilderman , Dana Morrone , Jianjun Xu

,

Arnab Roy ^b,2, Marcia Margis-Pinheiro ^c,3, Narayana M. Upadhyaya ,

c

b

a,

*

Robert M. Coates , Reuben J. Peters

a

Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011, USA

b

Department of Chemistry, University of Illinois, Urbana, IL 61801, USA

Plant Industry, Commonwealth Scientiﬁc and Industrial Research Organization (CSIRO), Canberra, ACT 2601, Australia

c

Received 6 August 2006; received in revised form 25 September 2006

Available online 1 December 2006

Abstract

The rice (Oryza sativa) genome contains a family of kaurene synthase-like genes (OsKSL) presumably involved in diterpenoid bio-

synthesis. While a number of OsKSL enzymes have been functionally characterized, several have not been previously investigated,

and the gene family has not been broadly analyzed. Here we report cloning of several OsKSL genes and functional characterization

of the encoded enzymes. In particular, we have veriﬁed the expected production of ent-kaur-16-ene by the gibberellin phytohormone

biosynthesis associated OsKS1 and demonstrated that OsKSL3 is a pseudo-gene, while OsKSL5 and OsKSL6 produce ent-(iso)kaur-

15-ene. Similar to previous reports, we found that our sub-species variant of OsKSL7 produces ent-cassa-12,15-diene, OsKSL10 pro-

duces ent-(sandaraco)pimar-8(14),15-diene, and OsKSL8 largely syn-stemar-13-ene, although we also identiﬁed syn-stemod-12-ene as

an alternative product formed in ꢀ20% of the reactions catalyzed by OsKSL8. Along with our previous reports identifying OsKSL4

as a syn-pimara-7,15-diene synthase and OsKSL11 as a syn-stemod-13(17)-ene synthase, this essentially completes biochemical charac-

terization of the OsKSL gene family, enabling broader analyses. For example, because several OsKSL enzymes are involved in phyto-

alexin biosynthesis and their gene transcription is inducible, promoter analysis was used to identify a pair of speciﬁcally conserved motifs

that may be involved in transcriptional up-regulation during the rice plant defense response. Also examined is the continuing process of

gene evolution in the OsKSL gene family, which is particularly interesting in the context of very recently reported data indicating that a

japonica sub-species variant of OsKSL5 produces ent-pimara-8(14),15-diene, rather than the ent-(iso)kaur-15-ene produced by the indica

sub-species variant analyzed here.

Keywords: Orzya sativa; Poaceae; Terpene synthase; ent-kaurene synthase; ent-isokaurene synthase; ent-sandaracopimaradiene synthase; ent-cassadiene

synthase; syn-stemarene synthase; syn-stemodene synthase; syn-pimaradiene synthase; Labdane-related diterpenoids; Phytoalexin; Natural products

biosynthesis; Plant defense; Gene family evolution

Abbreviations: AtKS, Arabidopsis thaliana kaurene synthase; CPP, copalyl diphosphate; CPS, copalyl diphosphate synthase; cv, cultivar; GGPP,

(E,E,E)-geranygeranyl diphosphate; GST, glutathione-S-transferase; KO, kaurene oxidase; KOL, kaurene oxidase-like; KS, kaurene synthase; KSL,

kaurene synthase-like; ORF, open reading frame; OsCPS, rice (Oryza sativa) copalyl diphosphate synthase; OsKS(L), rice (Oryza sativa) kaurene

synthase(-like); ssp, sub-species.

*

Corresponding author. Tel.: +1 515 294 8580; fax: +1 515 294 0453.

E-mail address: rjpeters@iastate.edu (R.J. Peters).

Present address: Neurogen Corp., 35 NE Industrial Rd., Branford, CT 06405, USA.

Present address: Albany Molecular Research, Hyderabad, India.

Present address: Laboratorio de Genetica Molecular Vegetal, Depto de Genetica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

1

2

3

doi:10.1016/j.phytochem.2006.10.016

M. Xu et al. / Phytochemistry 68 (2007) 312–326

313

1. Introduction

chi et al., 2002). The identiﬁed rice labdane-related diterp-

enoid natural products fall into ﬁve structurally related

groups (Fig. 1), with the gibberellins being elaborated from

ent-kaurene, oryzalexins A–F from ent-sandaracopimarad-

iene, phytocassanes A–E from ent-cassadiene, oryzalexin

S from syn-stemodene, and momilactones A and B from

syn-pimaradiene (Mohan et al., 1996; Wickham and West,

1992; Yajima et al., 2004).

Rice is an important food crop, and has become a model

plant for the cereal plant family with the recent availability

of draft genome sequences (Goﬀ et al., 2002; Yu et al.,

2002), as well as large numbers of deﬁned full-length

cDNAs (Kikuchi et al., 2003). This extensive sequence

information has enabled functional genomics based

approaches towards elucidating the biosynthetic machinery

underlying rice metabolism. One speciﬁc area of interest is

the production of natural products with antimicrobial

activity, which are termed phytoalexins if their biosynthesis

is induced by microbial infection and phytoanticipins if

their biosynthesis is constitutive (VanEtten et al., 1994),

as the production of these small organic compounds is

associated with resistance to microbial diseases such as that

caused by the agronomically devastating rice blast patho-

gen Magneporthe grisea.

Extensive phytochemical investigation has demon-

strated that rice produces a number of phytoalexins in

response to M. grisea infection (Peters, 2006). Intriguingly,

the rice phytoalexins, with the exception of the ﬂavonoid

sakuranetin, all fall into the large family of labdane-related

diterpenoid natural products characterized by minimally

containing a labdane type bicyclic core structure. Thus,

along with the ubiquitous gibberellin phytohormone, rice

produces more than 10 other labdane-related diterpenoids

as phytoalexins. These are momilactones A & B (Cart-

wright et al., 1977, 1981), oryzalexins A–F (Akatsuka

et al., 1985; Kato et al., 1993, 1994; Sekido et al., 1986),

oryzalexin S (Kodama et al., 1992), and phytocassanes

A–E (Koga et al., 1997; Koga et al., 1995). In addition,

momilactone B is constitutively secreted from rice roots

and acts as an allelochemical in suppressing germination

in nearby seeds (Kato-Noguchi and Ino, 2003; Kato-Nogu-

Labdane-related diterpenoids share an unusual bioge-

netic origin, as their biosynthesis is uniquely initiated by

a consecutive pair of terpene synthase catalyzed reactions.

In the ﬁrst reaction, the characteristic bicyclic core struc-

ture is formed by class II labdane-related diterpene cyc-

lases, which catalyze protonation-initiated cyclization of

the universal diterpenoid precursor (E,E,E)-geranylgeranyl

diphosphate (GGPP, 1) to produce a speciﬁc stereoisomer

of labdadienyl/copalyl diphosphate (e.g. 2 and 3) or rear-

ranged derivative structure such as clerodanyl diphosphate

(MacMillan and Beale, 1999). The resulting cyclized

diphosphate compounds can then be further cyclized

and/or rearranged by more typical class I terpene syn-

thases, which initiate catalysis via ionization of the allylic

pyrophosphate group (Davis and Croteau, 2000). Notably,

class I labdane-related diterpene synthases exhibit stereo-

speciﬁcity, e.g. all of the identiﬁed copalyl diphosphate

(CPP) speciﬁc enzymes only react with single stereoisomers

of CPP (Cho et al., 2004; Nemoto et al., 2004; Otomo et al.,

2004a; Peters et al., 2000; Wilderman et al., 2004).

Prototypical plant terpene synthases are similar in size

and consist of two structurally deﬁned domains that have

been simply termed the N- and C-terminal domains

because these are associated with the corresponding region

of their polypeptide sequence (Starks et al., 1997; Whitting-

ton et al., 2002). While both class II and class I labdane-

related diterpene synthases are phylogenetically related to

Fig. 1. Known labdane-related diterpene cyclization reactions in rice. The corresponding cyclases are indicated, along with their products and, where

known, the derived natural products (dashed arrows indicate multiple biosynthetic steps).

314

M. Xu et al. / Phytochemistry 68 (2007) 312–326

other plant terpene synthases (Bohlmann et al., 1998; Mar-

tin et al., 2004), these also invariably contain additional

N-terminal sequence (ꢀ210–240 amino acids) that has been

termed the ‘insertional’ element. However, this sequence

element was almost certainly present in the ancestral ter-

pene synthase (Bohlmann et al., 1998; Martin et al.,

2004; Trapp and Croteau, 2001), and is particularly well

conserved, along with the central region that corresponds

to the prototypical N-terminal domain, in class II diterpene

cyclases (Xu et al., 2004). Class I terpene synthases contain

a DDXXD motif in their C-terminal domain that is

involved in ligation of the divalent metal ion co-factors

required for their diphosphate ionization-initiated reaction

mechanism (Davis and Croteau, 2000). By contrast, class II

diterpene cyclases contain a DXDD motif in their central

domain that is required for their protonation-initiated

cyclization reactions (Peters and Croteau, 2002a; Peters

et al., 2001). Indeed, the C-terminal domain has been

termed the class I domain, with the prototypical N-termi-

nal or central domain termed the class II domain (Chris-

tianson, 2006; Wendt and Schulz, 1998). Given the

equally conserved nature of the ‘insertional’ element and

central domain in class II diterpene cyclases, we suggest

the use of class IIa and class IIb domains for these regions,

respectively. Hence, the domain structure of plant labdane-

related diterpene synthases consists of the class IIa, class

IIb, and class I domains, although these all seem to be

structurally interdependent and can not be divided into

separate polypeptides that exhibit the associated activity

(Peters et al., 2003).

ciﬁcally OsCPS1-4, with OsCPS1 and OsCPS2 producing

ent-CPP (2) for gibberellin and phytoalexin biosynthesis,

respectively, OsCPS3 being a pseduo-gene, and OsCPS4

producing syn-CPP (3) (Otomo et al., 2004b; Prisic et al.,

2004; Sakamoto et al., 2004; Xu et al., 2004). The class I lab-

dane-related diterpene synthases have been termed kaurene

synthases (KS), speciﬁcally OsKS1–10 (Margis-Pinheiro

et al., 2005; Otomo et al., 2004a; Sakamoto et al., 2004).

However, mutational analysis has demonstrated that only

OsKS1 is involved in gibberellin biosynthesis and, presum-

ably, produces kaurene (Margis-Pinheiro et al., 2005;

Sakamoto et al., 2004). The other characterized family

members do not produce kaurene (4) and some also have

been given alternative names (e.g. OsDTC1, OsDTC2, and

OsDTS2). Furthermore, the same OsKS nomenclature has

been assigned to diﬀerent genes by Sakamoto et al. (2004)

and Margis-Pinheiro et al. (2005). Thus, to avoid confusion

we have suggested use of OsKSL (rice kaurene synthase-

like), with the numbering scheme used by Sakamoto et al.

(2004) where appropriate, for the non-kaurene producing

class I labdane-related diterpene synthases from rice (Mor-

rone et al., 2006). Accordingly, OsKS1 retains its original

designation, but the syn-pimaradiene synthase originally

termed OsDTS2 (Wilderman et al., 2004) or OsKS4 (Otomo

et al., 2004a) should be referred to as OsKSL4, the ent-cass-

adiene synthase originally referred to as OsDTC1 (Cho

et al., 2004) is OsKSL7, the syn-stemarene synthase origi-

nally termed OsDTC2 (Nemoto et al., 2004) is OsKSL8,

and the ent-sandaracopimaradiene synthase originally

referred to as OsKS10 (Otomo et al., 2004a) is OsKSL10

(see Table 1). As indicated by this listing, a number of the

OsKSL family members were uncharacterized. Here we

report cloning and characterization of many of the OsKSL

enzymes, along with more general analysis of this gene fam-

ily, and comparison to a similar report that appeared during

the preparation of this manuscript (Kanno et al., 2006).

Based on this characteristic domain structure and the

class speciﬁc aspartate rich motifs, four class II and 11 class

I labdane-related diterpene synthases have been found in the

extensive sequence information available for rice. All four

class II genes have been characterized, demonstrated to

produce CPP, and were termed CPP synthases (CPS); spe-

Table 1

Rice kaurene synthase-like gene family

Name^a

Product

Inducible

No

Accession

Locus

Alternate

–

Reference

OsKS1

ent-Kaurene

NR^b

Os04g52230

Sakamoto et al. (2004)

Margis-Pinheiro et al. (2005)

This study

AY347876

DQ823350

DQ823351

AY616862

AB126934

DQ823352

DQ823353

DQ823354^d

AB118056

NR^b

OsKSL2

OsKSL3

OsKSL4

unknown

Æpseudo-geneæ

syn-Pimaradiene

unknown

No

Yes

Os04g52240

Os04g52210

Os04g10060

–

OsKS2^c

OsDTS2

–

This study

Wilderman et al. (2004)

Otomo et al. (2004)

This study

OsKSL5

OsKSL6

OsKSL7

OsKSL8

OsKSL9

OsKSL10

OsKSL11

a

ent-iso-Kaurene

ent-Cassadiene

syn-Stemarene^e

Æpseudo-geneæ

ent-Pimaradiene

syn-Stemodene

No

Yes

–

Os02g36220

Os02g36264

Os02g36140

Os11g28530

Os11g28500

Os12g30824

unknown

OsKS6^c

OsKS5^c

OsDTC1

OsDTC2

–

This study

Cho et al. (2004)

Nemoto et al. (2004)

Sakamoto et al. (2004)

Otomo et al. (2004)

Morrone et al. (2006)

Yes

No?

DQ823355^d

DQ100373

–

Gene numbering based on Sakamoto et al. (2004), with kaurene synthase-like (KSL) nomenclature for the nonkaurene producing family members, as

previously suggested (Morrone et al., 2006).

b

NR, not reported, Sakamoto et al. (2004) did not report any sequence information.

Partial gene sequences reported by Margis-Pinheiro et al. (2005).

Novel sequence with veriﬁed biochemical activity matching that from the original reference given on the right.

As identiﬁed in this study, OsKSL8 makes signiﬁcant amounts of stemodene in addition to stemarene.

c

d

e

M. Xu et al. / Phytochemistry 68 (2007) 312–326

315

2. Results

clone from the Rice Genome Resource Center (www.

rgrc.dna.aﬀrc.go.jp) and veriﬁed the frame-shift mutations,

partial cDNA sequences reported later (AB126935 and

AY347882) indicated OsKSL5 was a functional gene, i.e.

at least some alleles did not contain these frame-shift muta-

tions. Encouraged by this, we proceeded to clone a cDNA

covering the full ORF that also did not have the frame-shift

mutations. This OsKSL5 cDNA was 99.0% identical to

those previously reported, and the few observed diﬀerences

are presumably due to the inter-subspecies variation

between the ssp. japonica cv. Nipponbare used by others

and the ssp. indica cv. IR24 that was the source of the clone

reported here (DQ823352).

OsKSL6 was originally found among the genes pre-

dicted from the rice genome (Sakamoto et al., 2004). Two

overlapping partial cDNA sequences (AB126936 and

AY347881) that together span the entire predicted ORF

were later reported. We cloned a cDNA covering the full

ORF (DQ823353). This was 99.9% identical to the previ-

ously reported partial cDNAs, and the one observed diﬀer-

ence is presumably an allelic variant, as all sequences were

from ssp. japonica cv. Nipponbare.

OsKSL7wasoriginallyidentiﬁedasOsDTC1(AB089272),

which was cloned from cv. BL-1 rice (Cho et al., 2004). Here

we report cloning a full-length ORF for OsKSL7 from ssp.

indica cv. IR24 (DQ823354). This clone was 99.0% identical

to the BL-1 derived ORF. Again, the few observed diﬀer-

ences presumably are a result of inter-subspecies variation.

OsKSL8 was originally identiﬁed as OsDTC2 (AB118056),

which was cloned from ssp. japonica cv. Nipponbare rice

(Nemoto et al., 2004). We cloned two overlapping partial

cDNAs, also from ssp. japonica cv. Nipponbare, which

together covered the corresponding ORF. These were

spliced together to (re)create an identical copy of the full-

length ORF for OsKSL8.

OsKSL9 was originally reported to be a pseduogene

(Sakamoto et al., 2004). In particular, the genomic sequence

data indicates that the corresponding locus (Os11g28500)

only encodes a partial ORF, which was not pursued.

OsKSL10 was initially found in the rice full-length cDNA

sequencing project, although only as a single clone reported

as containing two neighboring single-base insertions that

would result in a truncated protein (AK072461). We also

obtained this clone from the Rice Genome Resource Center.

However, re-sequencing of this full-length cDNA demon-

strated that the frame-shift insertions were a result of

sequencing errors, along with two mis-called bases in the

same region. This veriﬁed sequence (DQ823355) was

99.8% identical to a partial cDNA (AB126937) reported as

OsKS10 (Otomo et al., 2004a). Both clones were from ssp.

japonica cv. Nipponbare, and the very few observed diﬀer-

ences are presumably a result of allelic variation.

2.1. Identiﬁcation of rice kaurene synthase-like genes

Because of our interest in using CPP stereospeciﬁc diter-

pene synthases as model systems for investigating substrate

and product speciﬁcity, we undertook a functional genom-

ics based approach towards identifying the enzymatic

activity of the corresponding class I labdane-related diter-

pene synthases from rice. Putative synthases were identiﬁed

in silico using the sequence of the kaurene synthase from

Arabidopsis thaliana in BLAST searches probing the exten-

sive sequence information available for rice. The OsKSL

genes were largely cloned on the basis of this information

and assigned numbers by mapping onto the genome and

comparison to the locations given by Sakamoto et al.

(2004).

OsKS1 (AY347876) was obtained after identiﬁcation of

its role in GA biosynthesis via transposon insertional

mutagenesis, as previously reported (Margis-Pinheiro

et al., 2005). This partial cDNA clone was 99.9% identical

to a recently released sequence (AB126933), and the one

observed diﬀerence presumably reﬂects allelic variation,

as both clones are from the japonica subspecies (ssp.) of

rice.

OsKSL2 was initially found as a predicted gene from

genomic sequencing (CAE05199), and we were only able

to obtain partial cDNA clones that do not cover the full

open reading frame (ORF), despite extensive eﬀorts in both

ssp. indica and japonica. We have deposited the largest such

partial cDNA from ssp. japonica (DQ823350).

OsKSL3 has previously been reported as a partial

cDNA sequence (Margis-Pinheiro et al., 2005). However,

sequencing of multiple cDNA clones from both ssp. indica

and japonica, as well as re-sequencing of the original partial

cDNA clone (AY347879), conclusively demonstrated that

this gene contains a single base insertion after nucleotide

1215 (DQ823351). The resulting frame-shift should result

in a severely truncated protein (441 instead of 764 amino

acid residues). Notably, whereas none of our other OsKSL

clones have obvious splicing errors, all three of our

OsKSL3 clones from ssp. indica cultivar (cv.) IR24 are

mis-spliced at the border between exons 10 and 11 that

joins nucleotides 1702 and 1703, suggesting degradation

of this downstream splice site in ssp. indica.

OsKSL4 is the clone (AY616862) originally reported as

OsDTS2 (Wilderman et al., 2004), which is 99.3% identical

to another cDNA (AB126934) that was separately reported

as OsKS4 (Otomo et al., 2004a). Presumably the few

observed diﬀerences are due to inter-subspecies variation,

as our OsKSL4/OsDTS2 clone is from ssp. indica cv.

IR24 and the other is from ssp. japonica cv. Nipponbare.

OsKSL5 was initially found in the rice full-length cDNA

sequencing project, although only as a single clone contain-

ing a two-base deletion that should result in a truncated pro-

tein, although there are two single-base insertions that occur

later in the sequence (AK121446). While we obtained this

OsKSL11 (DQ100373) is a full-length cDNA that has

been previously reported from both ssp. indica and japonica

(Morrone et al., 2006). Notably, corresponding sequence

still cannot be located in the currently available rice genome

(e.g. at www.gramene.org).

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M. Xu et al. / Phytochemistry 68 (2007) 312–326

2.2. Functional characterization of the rice kaurene synthase-

like gene products

nant preparations (i.e. for OsKSL8). The resulting recom-

binant fusion proteins were assayed with GGPP (1), ent-

CPP (2), or syn-CPP (3) as substrate, and their enzymatic

activity assessed by GC–MS analysis of organic extracts

of these reactions. Enzymatic products were identiﬁed by

GC–MS based comparison to authentic samples (Fig. 2).

All of the active enzymes were speciﬁc for either ent- (2)

or syn-CPP (3), and did not react with the alternative ste-

reoisomer, nor with GGPP (1).

All of the OsKS(L) proteins were expressed as fusions to

the C-terminus of gluthathione-S-transferase (GST-

OsKSL), which provided a convenient aﬃnity tag for single

step puriﬁcation. When activity was not detected in the

context of GST fusion proteins, it was necessary to turn

to a thioredoxin fusion construct to obtain active recombi-

Fig. 2. GC–MS analysis of products formed by selected OsKS(L). (a) Chromatograph of the product formed by OsKS1 from ent-CPP. (b) Mass spectrum

of OsKS1 product peak (RT = 13.01 min). (c) Mass spectrum of authentic ent-kaur-16-ene (4) (RT = 13.01 min). (d) Chromatograph of the product

formed by OsKSL5 and OsKSL6 (as indicated) from ent-CPP. (e) Mass spectrum of OsKSL5 product peak (RT = 12.80 min). (f) Mass spectrum of

OsKSL6 product peak (RT = 12.80). (g) Mass spectrum of authentic ent-(iso)kaur-15-ene (6) (RT = 12.81 min). (h) Chromatograph of the products

formed by OsKSL8 from syn-CPP. (i) Mass spectrum of the major OsKSL8 product peak (RT = 12.45 min). (j) Mass spectrum of authentic syn-stemar-

13-ene (8) (RT = 12.45). (k) Mass spectrum of the minor OsKSL product peak (RT = 12.54). (l) Mass spectrum of authentic syn-stemod-12-ene (10⁰)

(RT = 12.55).

M. Xu et al. / Phytochemistry 68 (2007) 312–326

317

As expected from its known role in gibberellin biosynthe-

sis (Margis-Pinheiro et al., 2005; Sakamoto et al., 2004),

OsKS1 speciﬁcally reacts with ent-CPP (2) to produce

kaur-16-ene (4) (Fig. 2A–C). OsKSL3 contains a frame-

shift mutation and appears to be a pseudo-gene. Consistent

with this interpretation, simply correcting the frame-shift

does not restore activity, suggesting that other deleterious

mutations have arisen. As previously demonstrated,

OsKSL4 speciﬁcally utilizes syn-CPP (3) to produce

pimara-7,15-diene (5) (Otomo et al., 2004a; Wilderman

et al., 2004). Interestingly, both OsKSL5 and OsKSL6 spe-

ciﬁcally react with ent-CPP (2) to produce (iso)kaur-15-ene

(6) (Fig. 2D–G). As previously reported for OsKSL7 (Cho

et al., 2004), our ssp. indica variant speciﬁcally utilizes ent-

CPP (2) to produce cassa-12,15-diene (7) (data not shown).

Unlike all the other OsKS(L), which each essentially pro-

duce only a single diterpene, it has been previously reported

that OsKSL8 speciﬁcally utilizes syn-CPP (3) to produce

stemar-13-ene (8) as its major product (ꢀ70% of the total),

along with signiﬁcant (ꢀ20%) production of another,

unidentiﬁed diterpene (Nemoto et al., 2004). Using our

clone we observed the same product mix and were able to

identify the unknown diterpene as stemod-12-ene (10⁰)

(Fig. 2H–L). As previously reported for OsKSL10 (Otomo

et al., 2004a), our allelic clone speciﬁcally reacts with ent-

CPP to produce (sandaraco/iso)pimara-8(14),15-diene (9)

(data not shown). Finally, as we have previously demon-

strated, OsKSL11 produces stemod-13(17)-ene (10) from

syn-CPP (3) (Morrone et al., 2006).

potential binding sites that were identiﬁed, the results from

searches for known transcription factor binding sites were

diﬃcult to interpret. To ﬁnd sites conserved speciﬁcally

within the inducible rice diterpene synthase promoters,

we searched for sequence elements, on either strand, com-

mon to all the inducible OsCPS and OsKSL genes, but not

found in either of the gibberellin biosynthesis associated

and non-inducible OsCPS1 and OsKS1, promoter regions.

From this analysis we identiﬁed four 6-mer motifs: GTT-

TAT, GAAATT, TGCAAT, and ATATGG.

In addition to the known involvement of labdane-related

diterpene synthases, evidence has been reported suggesting

that two other genes may be involved in rice diterpenoid

phytoalexin biosynthesis. Speciﬁcally, while rice contains

ﬁve genes homologous to kaurene oxidase (KO), the ﬁrst

cytochrome P450 involved in gibberellin biosynthesis (Ols-

zewski et al., 2002), only one of these (i.e. OsKO2) is actu-

ally involved in phytohormone metabolism (Sakamoto

et al., 2004). Transcription of the two most divergent para-

logs, termed KO-like (i.e. OsKOL4 and OsKOL5), has been

shown to be induced by either fungal elicitor or UV-irradi-

ation, leading to the suggestion that OsKOL4 and OsKOL5

are involved in rice diterpenoid phytoalexin biosynthesis

(Itoh et al., 2004). Notably, two of the speciﬁcally conserved

motifs identiﬁed above, GTTTAT and GAAATT, are also

found in the promoter regions of the similarly regulated

OsKOL4 and OsKOL5 genes, while not being found in that

of the GA biosynthesis speciﬁc and non-inducible OsKO2

paralog. By contrast, the TGCAAT motif was not found

in the promoter regions of the inducible OsKOL genes,

while the ATATGG motif was found in the promoter

region of the gibberellin biosynthesis associated and non-

inducible OsKO2, as well as that of OsKOL4 (although

not OsKOL5). Thus, it seems likely that the two somewhat

related GTTTAT and GAAATT motifs are regulatory ele-

ments involved in the observed similar transcription induc-

tion proﬁles of OsCPS2, OsCPS4, OsKSL4, OsKSL7,

OsKSL8, OsKSL10, OsKOL4, and OsKOL5. These motifs

also may be useful for identiﬁcation of other enzymatic

genes involved in rice phytoalexin biosynthesis, diterpenoid

or otherwise (i.e. the ﬂavonoid sakuranetin).

2.3. Analysis of inducible rice labdane-related diterpene

synthase gene promoters

A number of OsKSL enzymes are involved in phyto-

alexin biosynthesis (Fig. 1), and it has been reported that

transcription of that speciﬁc subset of the OsKSL family,

i.e. OsKSL4, OsKSL7, OsKSL8, and OsKSL10, is induced

by either fungal elicitor or UV-irradiation (Cho et al., 2004;

Nemoto et al., 2004; Otomo et al., 2004a; Sakamoto et al.,

2004; Wilderman et al., 2004). Also reported was similarly

induced transcription of the phytoalexin biosynthesis asso-

ciated ent- and syn-CPP synthases, OsCPS2 and OsCPS4,

respectively (Otomo et al., 2004b; Prisic et al., 2004;

Sakamoto et al., 2004; Xu et al., 2004). Given the observed

diﬀerences in transcriptional regulation of the various rice

diterpene synthase genes and their common evolutionary

origin, we expected that comparison of their promoter

regions might identify cis-acting regulatory elements, spe-

ciﬁcally potential binding sites for transcription factors

involved in the observed induction of OsKSL4, OsKSL7,

OsKSL8, and OsKSL10, as well as OsCPS2 and OsCPS4.

Because cis-acting elements are generally located within a

kilobase (kb) of the initial exon, our analyses focused on

the 1.0 kb of sequence upstream of the OsKS(L) and

OsCPS genes (except for OsCPS4 where only 776 bases

could be analyzed due to the presence of a gap in the gen-

ome sequence at this position). Due to the large number of

2.4. Sequence analysis of the rice kaurene synthase-like

genes/enzymes

As selected for by the in silico search strategy, all of the

OsKS(L) genes share signiﬁcant homology, 50–57% iden-

tity, to the kaurene synthase from A. thaliana (AtKS).

The encoded proteins also share signiﬁcant homology with

AtKS, exhibiting 39–47% identity. AtKS is most closely

related to OsKS1, sharing 47% identity at the amino acid

level. By contrast, the OsKSL enzymes are only 39–43%

identical to AtKS at the amino acid level. This is consistent

with conservation of kaurene synthase activity for AtKS

and OsKS1, and the release of such selective pressure for

the OsKSL genes, enabling the observed evolution of diver-

gent function.

318

M. Xu et al. / Phytochemistry 68 (2007) 312–326

Alignment of the OsKS(L) family revealed 53–93% iden-

tity at the nucleotide level and 42–89% identity at the

amino acid level (Fig. 3). Examining the resulting cDNA

sequence based phylogenetic relationships demonstrates

that the OsKSL genes are not broadly conserved by func-

tion (Fig. 4). For example, syn-CPP (3) and ent-CPP (2)

1

100

OsKS1

OsKSL7

OsKSL4

OsKSL10

OsKSL11

OsKSL8

OsKSL5

OsKSL6

(1) ------MMMLLLPSSS------------------------SSCCCRCPGGQFHGAPPRVMAPRRGVTRVY-IEKRLGVGGGNASSLQDMHRKELQARTRD

(1) ------MMLLGSPSSGGYGGKFAGASPAGGTTTMAPSAKQPSSRAPPPGITGGRNDLRILSPAAAAAAVGGLEMKKPEAEGIAESLQATHRKELEASIRK

(1) MEAVARSSLVLAPRRRRALGLLPAAAAPFVLDCRRRHNGGMRRPHVSFACSAELDTGRRQLPSTGTRAVMSS-CPGYVEGRMVGENTSQINMGREARIRR

(1) ----------MLPSSICSMGQIP-----------------RTSPHYYGMLPKQMSKGHPPMVTRAVGGVE-----KGEVGGNVRSLQVMHSKELQAKIRR

(1) ---------MMLLSSSYSGG-------------------QFPGVSPLGTRPKRSTTVVPLPVVTRATAGGVR--NNLEVVGNAGTLQGMDIDELRVIVRK

(1) ---------MMLLSSSYSGG-------------------QFPGVSPLGTRPKRSTTVVPRPVVTRAGGVRNN----LEVVGNAGTLQGMDIDELRVIVRK

(1) ---------MILPMSSACLG-------------------QFLRASPRGMIEQFNRAPPLRVSIRGAAGVEKS-LG--LGRNAG-SQQGMQKNQLQDKIRK

(1) ---------MMLPMSSACSG-----------------GQFPGASPHGIIPKQFSRAPRIRVSIRGAAGVEKS---LGLGRNAG-SQQGMHKNELHDKIRK

101

200

OsKS1

OsKSL7

(70) QLQTLELSTSLYDTAWVAMVPLRGSR--QHPCFPQCVEWILQNQQDDGSWGTRG-FGVAVTRDVLSSTLACVLALKRWNVGQEHIRRGLDFIGRNFSIAM

(95) QLQGVELSPSPYDTAWVAMVPLRGSS--HNPSFPQCVDWILENQWDDGSWSIDG-SISTANKDVLSSTLACVLALNKWNVGREHIRRGLSFIGRNFSIAM

OsKSL4 (100) HLENPEFLPSSYDIAWVAMVPLPGTDHLQAPCFPECVEWILQNQHSNGSWGVNE-FDSSASKDILLSTLACIIALEKWNVGSEQIRRGLHFIAKNFSIVI

OsKSL10

OsKSL11

OsKSL8

OsKSL5

OsKSL6

(69) QLQRVELSPSLYDTAWVAMVPERSSS--QAPCYPQCIEWILQNQHDDGSWGINS-SSLSVNKDILLSTLACVVALKKWNAGSYHIKRGLNFVGRNFSVAM

(71) QLQGVELSPSSYDTAWVAMVPVQGSP--QSPCFPQCVEWILQNQQEDGSWGHSAGPSGEVNKDILLSTLACVLALNTWNVGQDHIRRGLSFIGRNFSVAI

(69) QLQGVELSPSSYDTAWVAMVPVQGSR--QSPCFPQCVEWILQNQQEDGSWGHSAGPSGEVNKDILLSTLACVLALNIWNVGQDHIRRGLSFIGRNFSVAI

(69) QLREVQLSPSSYDTAWVAMVPVQGSH--QTPRFPQCIEWIMQNQHDDGSWGTNL-PGSVVNKDILLCTLACVVALKRWNTGRDHISRGLNFIGKNFWVAM

(71) QLRDVQLQPSSYDTAWVAMVPVQGSH--QTPRFPQSIEWILQNQYDDGSWGTNL-PGLVVNKDILLCTLACVVALKRWNTGRDHISRGPNFIGRNFSVAM

201

300

OsKS1 (167) DEQIAAPVGFNITFPGMLSLAMGMDLEFPVRQTDVDRLLHLREIELEREAGDHSYGRKAYMAYVTEGLG-NLLEWDEIMMFQRKNGSFFNCPSTTAATLV

OsKSL7 (192) DDQAVAPIGFGITFPAMLTLANGSGLEVPVRQNDIDSLNHLREMKIQREAGNHSRGRKAYMAYLAEGFG-NLLEWDEIMMFQRKNGSLFNCPSSTAGALA

OsKSL4 (199) DDQIAAPIGFNLTFPAMVNLAIKMGLEFPASEISIDQILHLRDMELKRLSGEESLGKEAYFAYIAEGLEESMVDWSEVMKFQGKNGSLFNSPAATAAALV

OsKSL10 (166) DVQNIAPVGFNVTFSGLITLASGMGLQLPVWQTDIDEIFHLRKIELERDSGGTISARKAFMAYVAEGFG-SLQDWDQVMAYQRKNGSLFNSPSTTAAAAI

OsKSL11 (169) DGQCAAPVGFNITFSGMLHLAIGMGLKFPVMETDIDSIFRLREVEFERDAGGTASARKAFMAYVSEGLG-REQDWDHVMAYQRKNGSLFNSPSTTAASAI

OsKSL8 (167) DGQCAAPVGFNITFSGMLRLAIGMGLKFPVMETDIDSIFRLREVEFERDAGGTASARKAFMAYVSEGLG-REQDWDHVMAYQRKNGSLFNSPSTTAASAI

OsKSL5 (166) DEQTIAPVGFNITFSGLLNLATGTGLEFPVMQTDIDGIFHMRKIELERDAYGTASSRRAFMAYVSEGLD-SLQDWDQVMAYQRKNRSIFNSPSATAATVI

OsKSL6 (168) DEQTVAPVGFNITFSGLLSLATRTGLELPVMQTDIDGIIHIRKIELERDAYGTASSRRAFMAYVSEGLG-NLQDWNQVMAYQRKNGSIFNSPSATAATII

301

400

OsKS1 (266) NHYNDKALQYLNCLVSKFGSAVPTVYPLNIYCQLSWVDALEKMGISQYFVSEIKSILDTTYVSWLERDEEIMLDITTCAMAFRLLRMNGYHVSSVELSPV

OsKSL7 (291) NYHDDKALQYLQSLVNKFDGVVPTLYPLNIYCQLSMVDALENMGISQYFASEIKSILDMTYSSWLGKDEEIMLDVTTCAMAFRLLRMNGYDVSSDELSHV

OsKSL4 (299) HRYDDKALGYLYSVVNKFGGEVPTVYPLNIFSQLSMVDTLVNIGISRHFSSDIKRILDKTYILWSQRDEEVMLDLPTCAMAFRLLRMNGYGVSSDDLSHV

OsKSL10 (265) HTFNDRTLNYLDSLTNKFGGPVPAMYPQNIYSQLCTVDALERTGISQKFAREIRDILDTTYRSWLHNEEEVMLDIPTCAMAFRLLRTHGYDITSDEMAHF

OsKSL11 (268) HSCNDRALDYLVSLTSKLGGPVPAIHPDKVYSQLCMVDTLEKMGISSDFACDIRDILDMTYSCWMQDEEEIMLDMATCAKAFRLLRMHGYDVSSEGMARF

OsKSL8 (266) HSCNDRALDYLVSLTSKLGGPVPAIYPDKVYSQLCMVDTLEKMGISSDFACDIRDILDMTYSCWMQDEEEIMLDMATCAKAFRLLRMHGYDVSSEGMARF

OsKSL5 (265) HGHNDSALCYLDSLVSKLHGPVPVMYPQNAYSQLCMVDTLEKMGISNNFSCEISDILDMIYRLWIHNEEELMLEMGTCAMAFRLLRMHGYDISSDGMAQF

OsKSL6 (267) HGHNYSGLAYLDFVTSKFGGPVPVMYPQNAYSQLCMVDTLERMGISESFACEISDILDMTYRLWMHNEEELMLDMRTCAMAFRLLRMHGYDITSDGMAQF

401

500

OsKS1 (366) AEASSFRESLQGYLNDKKSLIELYKASKVSKSENESILDSIGSWSGSLLKESVCSNGVKKAPIFEEMKYALKFPFYTTLDRLDHKRNIERFD------AK

OsKSL7 (391) AGASGFRDSLQGYLNDRKSVLEVYKTSKHSISENDLILDSIGSWSGGSLLKEMLSSNGKGTPGREEIEFALKYPFYSTLERLVHRKNIVLFD------AK

OsKSL4 (399) AEASTFHNSVEGYLDDTKSLLELYKASKVSLSENEPILEKMGCWSGSLLKEKLCSDDIRGTPILGEVEYALKFPFYATLEPLDHKWNIENFD------AR

OsKSL10 (365) SEQSSFDDSIHGYLNDTKTLLELFKTSQIRFSCEDLVLENIGTWSAKLLKQQLLSNKLS-TSAQSEVEYVLKFPLHSTLDRLEHRRNIEQFK------VE

OsKSL11 (368) AERSSFDDSIHAYLNDTKPLLELYKSSQLHFLEEDLILENISSWSAKLLKQQLSSNKIM-KSLMPEVEYALKYPLYSTVDALEHRGNIERFN------VN

OsKSL8 (366) AERSSFDDSIHAYLNDTKPLLELYKSSQVHFLEEDFILENIGSWSAKLLKQQLSFNKIS-KSLMPEVEYALKYPFYATVEVLEHKGNIERFN------VN

OsKSL5 (365) VEQSSFDDSIHGYLNDTKALLELYRSSQIRCLEDDLILQDIGSWSARVLQEKISSKMTHKSEMLG-VEYALKFPVYATLERLEQKRNIEQFKTKEQLKIE

OsKSL6 (367) VEQSSFDDSIHGYLNDTKALLELYKSSQLRCLEDDLILEEIGSWSARVLLEKISSKMIH-ISELPEVEYALKCPVYAILERLEQKRNIEQFKTKEQLKIE

501

600

OsKS1 (460) DSQMLKTEYLLPHANQDILALAVEDFSSSQSIYQDELNYLECWVKDEKLDQLPFARQKLTYCYLSAAATIFPRELSEARIAWAKNGVLTTVVDDFFDLGG

OsKSL7 (485) GSQMLKTACMPVHDSQDFLALAVDDFCISQSNYQNELNYLESWVKDNRLDQLHFARQKITYCYLSGAATTFRPEMGYARTSWARTAWLTAVIDDLFDVGG

OsKSL4 (493) AYQKIKTKNMPCHVNEDLLALAAEDFSFCQSTYQNEIQHLESWEKENKLDQLEFTRKNLINSYLSAAATISPYELSDARIACAKSIALTLVADDFFDVGS

OsKSL10 (458) GSKVLKSGYCGSHSNEEILALAVDYFHSSQSVYQQELKYFESWVKQCRLDELKFARVMPLIVHFSSAATIFAPELADARMVLSQTCMLITVYDDFFDCPE

OsKSL11 (461) GFQRPKSGYCGSGADKEILALAVDKFHYNQSVYQQELRYLESWVAEFGLDELKFARVIPLQSLLSALVPLFPAELSDARIAFSQNCMLTTMVDDFFDGGG

OsKSL8 (459) GFQRLKSGYCGSGADKEILALAVNKFHYAQSVYQQELRYLESWVAEFRLDELKFARVIPLQSLLSAVVPLFPCELSDARIAWSQNAILTAVVDDLFDGGG

OsKSL5 (464) GFKLLKSGYRGAITHDEILALAVDEFHSSQSVYQQELQDLNSWVAHTRLDELKFARLMPSITYFSAAATMFPSELSEARIAWTQNCILTTTVDDFFDGDG

OsKSL6 (466) GFKLLKSGYRGVIPNDEILALAVDEFHSSQSVYQQELQDLNSWVAHTRLDELKFARLMPSITYFSAAAVLLPSE--SARIAWTQNCILTTTVDDFFDGEG

601

700

OsKS1 (560) S-KEELENLIALVEKWDGH-QEEFYSEQVRIVFSAIYTTVNQLGAKASALQGRDVTKHLTEIWLCLMRSMMTEAEWQRTKYVP-TMEEYMANAVVSFALG

OsKSL7 (585) L-EQEQENLLALMEKWEEPGEDEYYSEDVKIVFQALYNTVNEIGAKASALQGHDVTKYLVDVWLHVVRCMKVEAEWQRSQHLP-TFEEYMESGMVSLGQG

OsKSL4 (593) S-KEEQENLISLVEKWDQYHKVEFYSENVKAVFFALYSTVNQLGAMASAVQNRDVTKYNVESWLDYLRSLATDAEWQRSKYVP-TMEEYMKNSIVTFALG

OsKSL10 (558) ISREEKENYIALIEKWDNHAEIGFCSKNVEIVFYAVYNTYKQIGEKAALKQNRSIMDQLVEDLVSSAKAMMVEADWTATKYIPATMEEYMSNAEVSGAFA

OsKSL11 (561) S-MEEMVNFVALIDEWDNHGEIGFCSNNVEIMFNAIYNTTKRNCAKAALVQNRCVMDHIAKQWQVMVRAMKTEAEWAASRHIPATMEEYMSVGEPSFALG

OsKSL8 (559) S-MEEMLNLVALFDKWDDHGEIGFCSSNVEIMFNAVYNTTKRIGAKAALVQKRCVIDHIAEQWQVMVRAMLTEAEWAAGKHIPATMGEYMSVAEPSFALG

OsKSL5 (564) S-KEEMENLVKLIKKWDGHGEIGFSSECVEILFYAIYNTSKQIAEKAVPLQKRNVVDHIAESWWFTVRGMLTEAEWRMDKYVPTTVEEYMSAAVDSFAVG

OsKSL6 (564) S-KEEMENLVKLIEKWDDHGEIGFSSECVEILFYAVYNTSKQIAEKAMPLQKRNAVDHIAESWWFTVRGMLTEAEWRMDKYVPTTVEEYMSAAVDSFAVG

701

800

OsKS1 (657) PIVLPTLYFVGPKLQEDVVRDHEYNELFRLMSTCGRLLNDSQGFERESLEGKL-NSVSLLVHHSGGSI-------------SIDEAKMKAQKSIDTSRRN

OsKSL7 (683) CTVMSALFLIGEKLPEGIVELEEYDELFRLMGTCGRLLNDIRGIEREESDGKMTNGVSLLVHASGGSM-------------SVDEAKTEVMKRIDASRRK

OsKSL4 (691) PTILIALYFMGQNLWEDIVKNAEYDELFRLMNTCGRLQNDIQSFERECKDGKLNSVSLLVLDSKDVMS--------------VEEAKEAINESISSCRRE

OsKSL10 (658) SFVCPPLYFLGLKLSEEDVKSHEYTQLLKLTNVIGRLQNDSQTYRKEILAGKVNSVLLRALTDSGNTSPE-----------SIEAAKEIVNRDAESSMVE

OsKSL11 (660) PIVPLSAYLLGEELPEEAVRSPEYGQLLRHASAVGRLLNDVMTYEKEVLTWTPNSVLLQALAAARGGGESP----TPPSPACAEAARGEVRRAIQASWRD

OsKSL8 (658) PIVPVSAYLLGEELPEEAVRSPEYGRLLGLASAVGRLLNDVMTYEKEMGTGKLNSVVLLQPLAAGGAASRGGGGAPAPAPASVEAARAEVRRAIQASWRD

OsKSL5 (663) PIITSAALFVGPELSEEVFRSEEYIHLMNLANTIGRLLNDMQTYEKEIKMGKVNSIMLHALSHSGGGRGSP--------EASMEEAKREMRRVLQGSRCD

OsKSL6 (663) PIITSAALFVGPELSEEVFRSEEYIHLMNLANTIGRLLNDMQTYEKEIKMGKVNSVMLHALSHSGGGRGSP--------EASMEEAKREMRRVLQGCRFE

801

875

OsKS1 (743) LLRLVLGEQ-----GAVPRPCKQLFWKMCKIVHMFYSRTDGFSSPKEMVSAVNAVVKEPLKLKVSDPYGSILSGN

OsKSL7 (770) LLSLVVSEQE----GPIPRPCKQLFWKMCKILHLFYYQTDGFSSPKEMVSAVDAVINEPLQLRLL----------

OsKSL4 (777) LLRLVVRED-----GVIPKSCKEMFWNLYKTSHVFYSQADGFSSPKEMMGAMNGVIFEPLKTRGN----------

OsKSL10 (747) MRSLVFSEG-----GPIPRPCKDRFWEMCKIVFYFYSEDDAYRTPKETMSSARAVILDPLRLIPPPSCPETLSS-

OsKSL11 (756) LHRLVFRDDDG--SSIVPRACRELFWGTAKVANVFYQEVDGYTP-KAMRGMANAVILDPLHLQE-----------

OsKSL8 (758) LHGLVFGSGGGSSSSIIPRPCREVFWHTGKVASVFYQEGDGYAR-KAMRSMANAVILEPLHLQE-----------

OsKSL5 (755) LLRLVTRDG-----GVVPPPCRKLFWFMSKVLHFVYMEKDGYFTADGMMASANAVILDPLQVTLLPSGLGTL---

OsKSL6 (755) LLGLVTRDA-----GVVPPPCRKLFWLMSKVLHFVYMEKDRYFTAEGMMASANAVILDPLQVTLPPSDSGTL---

Fig. 3. Sequence comparison of the rice kaurene synthase-like enzymatic family. Shown are all the active enzymes, with the DDXXD motif underlined.

M. Xu et al. / Phytochemistry 68 (2007) 312–326

319

Fig. 4. Phylogenetic tree illustrating the relationship of the OsKSL gene

family based on alignment of the corresponding cDNA (ORF only)

sequences. Indicated is the enzymatic stereospeciﬁcity and inducible or

non-inducible nature of the corresponding gene transcription.

speciﬁc OsKSL enzymes do not group together, as the syn-

CPP (3) speciﬁc OsKSL4 is more closely related to the ent-

CPP (2) speciﬁc OsKS1, OsKSL7, and OsKSL10 than the

similarly syn-CPP (3) speciﬁc OsKSL8 and OsKSL11.

There also is no clear phylogenetic relationship among

the inducible versus non-inducible OsKSL genes. Although

the inducible OsKSL4, OsKSL7, and OsKSL10 may share

a common ancestor, these are only distantly related to the

similarly regulated OsKSL8, being much more closely

related to the un-inducible OsKS1 instead.

Nevertheless, there are two closely related pairs of

OsKSL genes that do have some functional characteristics

in common. OsKSL5 and OsKSL6 are the two most closely

related OsKSL family members, exhibiting 93.0% identity

at the nucleotide level and 89.0% identity at the amino acid

level, and both are ent-CPP (2) speciﬁc and produce iso-

kaurene (6). Further, both are similarly regulated, as their

transcription is not inducible by UV-irradiation or fungal

elicitor (Otomo et al., 2004a; Sakamoto et al., 2004).

OsKSL8 and OsKSL11 are similarly closely related, exhib-

iting 92.0% identity at the nucleotide level and 88.6% iden-

tity at the amino acid level, and both are syn-CPP (3)

speciﬁc and produce biogenetically related stemarane (e.g.

8) and stemodane (e.g. 10 and 10⁰) type diterpenes, whose

cyclization mechanisms can be envisioned as largely over-

lapping (Fig. 5). Indeed, as demonstrated here, OsKSL8

produces signiﬁcant amounts of both skeletal types

(Fig. 2H–L). However, whereas OsKSL8 is associated with

phytoalexin biosynthesis and exhibits inducible gene tran-

scription (Nemoto et al., 2004), the physiological role of

OsKSL11 remains unclear as its transcription does not

appear to be inducible (Morrone et al., 2006).

Fig. 5. Proposed biogenetic cyclization mechanisms leading to formation

of stemarene (8) and stemodene (10) with the product outcomes catalyzed

by OsKSL8 and OsKSL11 indicated (dotted bonds indicate alternative

locations for the carbon–carbon double bond).

et al., 2004), class I labdane-related diterpene synthases

[e.g. KS(L)] are conserved across the entire mature protein

sequence (Fig. 6), i.e. excluding the poorly conserved plas-

tid-directing transit sequence found in all mono- and di-ter-

pene synthases (Davis and Croteau, 2000). The observed

conservation of the class I domain is consistent with previ-

ous suggestions that class I activity in labdane-related

diterpene synthases is carried out in an active site that is

essentially entirely located in this DDXXD containing

structural domain (Peters et al., 2003; Peters and Croteau,

2002b; Peters et al., 2001), just as found in other terpene

synthases (Christianson, 2006).

Consistent with their observed enzymatic activity, all the

OsKS(L) enzymes contain the class I activity associated

DDXXD divalent metal binding motif, and not the class

II activity associated DXDD motif. Comparison of the

OsKS(L) with KS from other plant species revealed that,

unlike the class II labdane-related diterpene synthases

(e.g. CPS) that are highly conserved across the class IIa

and class IIb regions, but not the class I domain (Xu

Fig. 6. Histograph depicting amino acid sequence similarity over all

KS(L), i.e. class I labdane-related diterpene synthases (numbering based

on the consensus sequence). Also depicted is the approximate corre-

sponding domain structure deﬁned in the text, along with approximate

location of the class I associated DDXXD motif (T indicates transit

peptide region).

320

M. Xu et al. / Phytochemistry 68 (2007) 312–326

Finally, it seems worth mentioning that the observed

and OsKSL3 are group together in a tandem array, and

OsKSL8 is found next to the partial pseudo-gene OsKSL9

(Sakamoto et al., 2004). Further, a similar partial gene

sequence for another class I labdane-related diterpene syn-

thase pseudo-gene is located near OsKSL10 (rice gene locus

Os12g30800). Hence, OsKSL4 appears to be the only

OsKSL not located near another such gene in the rice gen-

ome. While OsKSL11 still can not be found in the currently

available rice genome, there is a ‘gap’ in the genome

sequence near OsCPS4 and OsKSL4, and it is tempting

to speculate that OsKSL11 resides in this ‘gap’, which

would be consistent with clustering of the OsKSL genes,

as well as functional clustering by enzymatic stereospeciﬁc-

ity (i.e. the shared syn-CPP (3) speciﬁc nature of OsCPS4,

OsKSL4, and OsKSL11). In any case, it has been noted

that these gene clusters all contain retrotransposon-like ele-

ments, which has been suggested to underlie the observed

extensive duplication of CPS and KSL genes in rice

(Sakamoto et al., 2004), and presumably also enabled their

assembly into the functional biosynthetic modules noted

above.

Intriguingly, it has been recently reported that barley

(Hordeum vulgare) and wheat (Triticum aestivum) similarly

contain multiple copies of CPS- and KS-like genes (Spiel-

meyer et al., 2004). This was based on chromosomal map-

ping using barley gene probes, which mapped to regions of

the barley and wheat genomes that are syngenic with the

rice labdane-related diterpene synthase gene clusters, indi-

cating that these gene clusters were assembled prior to

the evolutionary divergence between barley, wheat, and

rice. Furthermore, a number of the barley genes mapped

onto the rice genome most strongly to rice genes now

known to be involved in phytoalexin biosynthesis (e.g.

OsCPS4 and OsKSL4), additionally suggesting that lab-

dane-related diterpenoid secondary metabolism is wide-

spread in the grass/cereal family (Poaceae). Consistent

with this hypothesis, maize (Zea mays) has been shown

to produce labdane-related diterpenes in response to fungal

infection (Mellon and West, 1979), and phylogenetic anal-

ysis of the CPS gene family within the Poaceae has been

used to suggest early CPS gene duplication and evolution

of labdane-related diterpenoid biosynthesis in cereal crop

plants (Harris et al., 2005; Prisic et al., 2004).

diﬀerence between allelic copies of the various OsKSL

genes is limited to P99.8% identity, while the diﬀerence

between inter-subspecies orthologs is 99.0–99.3% identity.

The corresponding 0.7–1.0% inter-subspecies variation in

the OsKSL gene family approximates the 0.5% rate of

occurrence for single nucleotide polymorphisms/diﬀerences

observed between the non-repetitive regions of the genomic

sequences of ssp. indica and japonica (Yu et al., 2002).

3. Discussion

The speciﬁcity of class I labdane-related diterpene syn-

thases for particular stereoisomers of CPP provides a

potential model system for investigating the underlying ste-

ric constraints in the active sites of these and, by extension,

other terpene synthases. Such studies would be greatly

assisted by the identiﬁcation of closely related yet function-

ally distinct class I labdane-related diterpene synthases.

Notably, terpene synthases are generally conserved by tax-

onomic origin rather than enzymatic activity (Bohlmann

et al., 1998; Martin et al., 2004). Thus, in order to provide

a model system for investigation of class I labdane-related

diterpene synthases, we have been engaged in a functional

genomics based eﬀort to elucidate the individual biochem-

ical functions of each member of the rice kaurene synthase-

like (OsKSL) gene family. While the biochemical function

of some of the corresponding enzymes have been previ-

ously reported, a number remained uncharacterized. Here

we report the completion of functional characterization

of essentially all the individual family members (Table 1),

which further enabled analysis of this gene family more

generally.

One notable ﬁnding from our earlier studies was the

functional pairing of the gene encoding the syn-CPP syn-

thase OsCPS4 near that for the syn-pimaradiene synthase

(OsKSL4) in the rice genome (Wilderman et al., 2004).

We also noted at that time that the genes encoding phyto-

alexin associated ent-CPP synthase OsCPS2 and the ent-

cassadiene synthase OsKSL7 were similarly clustered

together, along with OsKSL5 and OsKSL6, and hypothe-

sized that the enzymes encoded by these latter two gene

also would prove to be speciﬁc for ent-CPP (2), which

has now been veriﬁed (Fig. 2D–G). Thus, the two gene

clusters containing both class I and II labdane-related

diterpene synthase are grouped by biochemical function

and not co-regulation. In particular, each cluster represents

a functional biosynthetic module encoding enzymes that

act consecutively to produce and then use a speciﬁc stereo-

isomer of CPP (2, 3), but the genes are not necessarily reg-

ulated in the same manner (i.e. while transcription of

OsCPS2 and OsKSL7 is inducible, that of OsKSL5 and

OsKSL6 in the same gene cluster is not).

Given the number of genes examined here it is perhaps

not surprising that evidence can be found for the on-going

process of evolution in the rice KS(L) gene family. For

example, OsKSL3 appears to be a pseudo-gene undergoing

gene ‘decay’. The corresponding cDNA clearly contains a

frame-shifting single base insertion that severely truncates

the encoded polypeptide. Furthermore, this sequence

appears to have accumulated a number of other deleterious

mutations, as simply restoring the ‘correct’ reading frame

does not result in the production of an active class I lab-

dane-related diterpene synthase, and the observed down-

stream mis-splicing of the corresponding cDNA

sequences from ssp. indica is a further indication of gene

‘decay’, at least in this sub-species.

In addition to these two class I and II labdane-related

diterpene synthase containing gene clusters, the other

mapped OsKSL genes cluster together. OsKS1, OsKSL2,

M. Xu et al. / Phytochemistry 68 (2007) 312–326

321

The physiological role(s) of the isokaurene (6) produced

by OsKSL5 and OsKSL6, as well as the stemodene (10)

produced by OsKSL11, is not clear. Nevertheless, it seems

likely that these diterpene hydrocarbons will be elaborated

to more complex diterpenoid natural products that may

have signiﬁcant biological function(s). Given the important

role in plant defense that the other non-gibberellin lab-

dane-related diterpenoids play in rice and the non-induc-

ible nature of the associated cyclases, one plausible role

for isokaurene- and stemodene-derived diterpenoids would

be to act as phytoanticipins and/or allelochemicals. An

interesting alternative is that one or more such labdane-

related diterpenoid could act as distinct (i.e. non-gibberel-

lin) signaling molecule(s), analogous to the recently

reported role of labda-11,13-diene-8a,15-diol in tobacco

(Seo et al., 2003).

As a ﬁnal note, while this manuscript was in preparation

a similar study was published (Kanno et al., 2006), which

reported identical ﬁndings regarding OsKSL3 (i.e. this is

a pseudo-gene containing a frame-shifting single base inser-

tion) and OsKSL6 (i.e. this encodes an ent-(iso)kaur-15-ene

synthase). However, Kanno et al. (2006) do not report gene

promoter analysis, nor do they discuss KS(L) gene evolu-

tion. There also is one signiﬁcant diﬀerence. Whereas our

ssp. indica variant of OsKSL5 (OsKSL5i) encodes an ent-

isokaurene synthase (Fig. 2D–G), their ssp. japonica

variant of OsKSL5 (OsKSL5j; AB126935) encodes an

ent-pimara-8(14),15-diene synthase, which represents

deprotonation of the probable pimarenyl⁺intermediate in

cyclization of ent-CPP (2) to kaurane type diterpenes

(Fig. 7). This diﬀerence in product outcome represents an

intriguing example of sub-species speciﬁc diﬀerence in

natural products metabolism, although the corresponding

physiological signiﬁcance is not clear as there are no cur-

rently known rice metabolites derived from either diter-

pene. Regardless, such emerging functional divergence in

product outcome, at least in ssp. japonica, between the clo-

sely related and, therefore, presumably recently duplicated

OsKSL5 and OsKSL6 is consistent with the hypothesis that

there is selective pressure to diﬀerentiate similarly regulated

genes, and serves as a further example of the continuing

process of gene evolution in the KS(L) gene family.

OsKSL5i and OsKSL5j share 98.1% identity at the

amino acid level, and of the ﬁfteen diﬀerences between their

sequences, only three appear in or near the active site in

modeled structures, and only two of these are conserved

between OsKSL5i and OsKSL6, which both produce iso-

kaurene. Because it has been demonstrated that very small

numbers of substitutions in the active site of class I terpene

synthases are suﬃcient to alter product proﬁle (Greenha-

gen et al., 2006; Kollner et al., 2004; Yoshikuni et al.,

2006), it seems likely that these two or three changes in

active site residues between OsKSL5i and OsKSL5j (i.e.

Val661, Ile664, and Ile718 in OsKSL5i, which are Leu,

Thr, and Val, respectively, in OsKSL5j) may be responsible

for the observed diﬀerence in product outcome (i.e. abortive

production of pimara-8(14),15-diene (11) from ent-CPP (2)

by OsKSL5j, rather than the more complex cyclization to

isokaurene (6) catalyzed by OsKSL5i), which will be used

to guide future structure-function investigations.

4. Conclusions

In summary, we report here cloning and/or biochemical

characterization of seven members of the rice kaurene syn-

thase-like gene family (Table 1). While some of these previ-

ously had been analyzed either in vitro or by in planta

mutagenesis, we veriﬁed here the expected production of

ent-kaurene (4) by the gibberellin biosynthesis associated

OsKS1, and have clariﬁed the product composition result-

ing from OsKSL8 catalyzed cyclization (Fig. 2). In addi-

tion, novel data are reported demonstrating that OsKSL3

is a pseudo-gene, while OsKSL5i and OsKSL6 encode

active enzymes that produce isokaurene (6) from ent-CPP

(2). With essentially all of the rice kaurene synthase-like

genes characterized, it also was possible to carry out anal-

ysis of this gene family more broadly, revealing a pair of

potential defense-related inducible promoter motifs, and

enabling more detailed examination of the labdane-related

diterpene synthase gene clusters found in the rice genome.

This particularly included examination of related gene clus-

ters, and associated diterpene metabolism, in other species

from the grass/cereal family, suggesting the widespread

occurrence, and continuing evolution, of defensive lab-

dane-related diterpenoid secondary metabolism through-

out this important crop plant family. Notably, such

widespread retention of this type of defensive metabolism

indicates that labdane-related diterpenes are particularly

good scaﬀolds for assembling antibiotic compounds and

further suggests that these natural products will prove to

Fig. 7. Proposed biogenetic cyclization mechanism catalyzed by OsKSL5i

to produce iso-kaurene (6), along with the abortive production of pimara-

8(14),15-diene (11) catalyzed by OsKSL5j. The further cyclization

catalyzed by OsKSL5i is indicated by the arrow marked ‘i’, the

deprotonation catalyzed by OsKSL5j is indicated by the arrow marked ‘j’.

322

M. Xu et al. / Phytochemistry 68 (2007) 312–326

be important components of the defense response in many,

if not all, cereal crop plants.

resin as previously outlined (Mohan et al., 1996). The mod-

iﬁed procedures for conversion of ent-copalol to the

diphosphate is presented below.

5. Experimental

5.4.1. Ent-copalyl chloride

The preparation of ent-copalyl chloride followed the

method of Collington and Meyers (Meyers and Collington,

1971). A solution of ent-copalol (0.050 g, 0.171 mmol) and

2,4,6-collidine (0.208 g, 1.71 mmol) under N₂was stirred

and cooled at 0 ꢁC as a suspension of LiCl (0.073 g,

1.712 mmol) partially dissolved in dry DMF (3.0 mL)

was added. CH₃SO₂Cl (0.059 g, 0.514 mmol) was added

dropwise. After 1.5 h at 0 ꢁC, the pale yellow reaction mix-

ture was poured into ice-water (10 mL), and the product

was extracted with cold ether (4 · 20 mL). The combined

organic phases were washed with saturated aqueous

Cu(NO₃)₂(2 · 10 mL), 5% NaHCO₃(2 · 10 mL) and brine

(2 · 10 mL), dried (MgSO₄), and evaporated to yield a col-

orless oil (0.050 g; 95% yield), which was used without fur-

5.1. General chemicals

Unless otherwise noted, all chemicals were purchased

from Fisher Scientiﬁc (Loughborough, Leicestershire,

UK) and molecular biology reagents from Invitrogen

(Carlsbad, CA, USA). Prior to use CH₃CN and DMF were

˚

distilled from P₂O₅and stored over 3 A molecular sieves.

5.2. Instrumentation

All NMR spectroscopy was carried out in the School of

Chemical Sciences NMR facility at the University of Illi-

1

nois. H NMR spectra in CDCl₃and CD₃OD were refer-

1

enced internally with CHCl₃(7.26 ppm) or with CD₃OH

(4.78 ppm). ³¹P NMR spectra was carried out in CD₃OD

with 85% H₃PO₄as external reference (0.00 ppm). Gas

chromatography–mass spectrometry (GC–MS) analyses

were performed using an HP1-MS column on an Agilent

(Palo Alto, CA, USA) 6890N GC instrument with a

5973N mass selective detector located in the W.M. Keck

Metabolomics Research Laboratory at Iowa State Univer-

sity, as previously described (Xu et al., 2004). Brieﬂy, 5 lL

samples were injected at 40 ꢁC in splitless mode, the oven

temperature held for 3 min, then raised at 20 ꢁC/min to

300 ꢁC, and held there for 3 min. MS data were collected

from 50 to 500 m/z during the temperature ramp.

ther puriﬁcation. H NMR (400 MHz, CDCl₃)d 5.40 (tm,

J = 8.1 Hz, 1H, @CHCH₂), 4.81 (br s, 1H, @CH₂), 4.48

(br s, 1H, @CH₂), 4.09 (d, J = 7.9 Hz, 2H, CH₂OPP),

2.37 (ddd, J = 12.7, 4.0, 2.4 Hz, 1H), 2.16 (tt, J = 9.9,

4.3 Hz, 1H), 1.95 (td, J = 13.0, 4.9 Hz, 1H), 1.80–1.88 (m,

1H), 1.73–1.75 (m, 1H), 1.71 (s, 3H, CH₃C@), 1.53–1.57

(m, 2H), 1.44–1.52 (m, 1H), 1.38–1.40 (m, 1H), 1.35–1.37

(m, 1H), 1.27–1.33 (m, 1H), 1.24–1.26 (m, 1H), 1.15–1.20

(m, 1H), 1.06 (dd, J = 12.5, 2.6 Hz, 1H), 0.95–1.02 (m,

1H), 0.85 (s, 3H), 0.78 (s, 3H), 0.66 (s, 3H) ppm; ¹³C

NMR (100 MHz, CD₃OD) d 148.3, 143.5, 124.3, 106.1,

55.8, 55.3, 41.9, 41.1, 38.8, 38.0, 33.4, 30.1, 24.2, 21.7,

21.5, 19.9, 16.0, 14.3 ppm.

5.3. Diterpene substrates and standards

5.4.2. Ent-CPP

The conversion of ent-copalyl chloride to ent-CPP (2)

was modeled after two methods already in the literature

(Mohan et al., 1996; Woodside et al., 1993) using previ-

ously described ion exchange and puriﬁcation methods

(Zhao, 2005). In a dry vial equipped with a stirring bar

were placed ent-copalyl chloride (0.050 g, 0.162 mmol)

GGPP (1) was purchased from Sigma–Aldrich. The

acquisition of reference samples of sandaracopimaradiene,

ent-kaurene (4), syn-pimaradiene (5), and syn-stemarene

(8), along with the preparation of syn-CPP (3), have been

previously described (Mohan et al., 1996). A reference sam-

ple of ent-isokaurene (6) was prepared by separation of a 1:1

mixture of endo- and exo-cyclic isomers (6 and 4, respec-

tively) obtained by I₂equilibration of ent-kaurene (4) (Bell

et al., 1966) using AgNO₃-silica gel chromatography (Wil-

liams and Mander, 2001). The exo- and endo-isomers of

syn-stemodene (10 and 10⁰, respectively) have also been pre-

viously described (White and Somers, 1994), and later sep-

arated and characterized (Morrone et al., 2006). A reference

sample of ent-cassadiene (7) was kindly provided by Drs.

Arata Yajima and Goro Yabuta (Yajima et al., 2004).

˚

and dry 4 A molecular sieves (0.70 g). Dry CH₃CN

(2.0 mL) and HOPP (NBu₄)₃(0.296 g, 0.324 mmol) were

added quickly. The vial was sealed with a septum and

purged with N₂, and the suspension was stirred at room

temperature for 12 h. The solids were removed by ﬁltra-

tion and washed with CH₃CN (70 mL). The CH₃CN ﬁl-

trate was washed with pentane (3 · 10 mL), and CH₃CN

was removed with a rotary evaporator. The remaining tet-

rabutylammonium salt was dissolved in 18 mL of 0.2%

(w/v) NH₄HCO₃and 2% (v/v) isopropyl alcohol in H₂O

(solvent A) and applied to a Bio-Rad AG50W-X8 ion

exchange column (d 1.2 cm, h 25 cm, volume 30 mL) that

had previously been freshly washed with saturated

NH₄OH and deionized water until pH 7.0, and the col-

umn was eluted with solvent A (42 mL). The total eluate

(ꢀ60 mL) was lyophilized three times for 3 h each. The

crude white powder was suspended in dry MeOH

5.4. Synthesis of ent-CPP

While the preparation of ent-CPP (2) has been previ-

ously described (Mohan et al., 1996), it was prepared for

this study using a modiﬁed procedure. For this process

ent-copalol (0.73 g) was obtained from Brazilian copal

M. Xu et al. / Phytochemistry 68 (2007) 312–326

323

Table 2

OsKSL cloning primers

(8 mL), vortexed, and centrifuged, then the supernatant

removed and concentrated to 0.5–0.6 mL in vacuo. This

extraction/vortexing/centrifugation/concentration proce-

dure was repeated three times. ³¹P NMR analyses of the

four supernatant fractions were conducted to determine

the ratio of product diphosphate (2d, d_Pꢁ8 to ꢁ9 ppm)

to PPi (s, d_P+ 3 ppm). Supernatant fractions 1 and 2 were

combined and concentrated to give ent-CPP (2) NH₄salt

as a white powder, which was not further puriﬁed (yield:

17.6 mg, purity 82%), in 19% overall yield for the two

steps from ent-copalol. ³¹P NMR (376 MHz, CD₃OD) d

ꢁ8.25 (d, J = 16.6 Hz, 1P), ꢁ8.85 (d, J = 19.3 Hz, 1P)

OsKS1-F

OsKS1-R

CACCATGAGGGACCAGCTCCAGACATTGGA

TCAATTGCCCGACAAAATAGAGCCATATGGAT

OsKSL2frag- CAGCTCGGCTCCTCGCCGGAGA

F

OsKSL2frag- ACATACCGGCTCTGCCTCCACTC

R

OsKSL3-F

OsKSL3-R

OsKSL5-F

OsKSL5-R

OsKSL6-F

OsKSL6-R

OsKSL7-F

OsKSL7-R

OsKSL8-

3⁰RACE-

F

OsKSL8-

3⁰frag-F

OsKSL8-

3⁰frag-R

OsKSL10-F

OsKSL10-R

CACCATGTTTCAGTTAGAATTAGTGAACGTCGTC

TCACGAAGCAGGAATGATATATATGGGTTC

CACCATGATACTTCCTATGAGTTCAGCATGCTT

TCACAGCGTTCCCAAACCAGATGGAAG

CACCATGATGCTTCCTATGAGTTCAGCATGC

TCACAGTGTTCCCGAATCAGATGGAG

CACCATGATGCTGCTAGGTTCCCCT

ppm; ¹H NMR (400 MHz, CD₃OD)

d 5.37 (tm,

CTACAATAATCTGAGTTGAAG

CACCATGATGCTGCTGAGTTCCTC

J = 8.0 Hz, 1H, @CHCH₂), 4.83 (s, 1H, @CH₂), 4.52 (s,

1H, @CH₂), 3.35 (br s, 2H, CH₂OPP), 2.39 (ddd,

J = 12.5, 4.0, 2.4 Hz, 1H), 2.11–2.18 (m, 1H), 1.95–2.01

(m, 1H), 1.82–1.87 (m, 1H), 1.73–1.80(m, 1H), 1.69 (s, 3

H, CH₃C@), 1.57–1.64 (m, 2H), 1.46–1.51 (m, 1H),

1.40–1.44 (m, 1H), 1.36–1.40 (m, 1H), 1.33 (dd,

J = 13.0, 4.3 Hz, 1H), 1.26–1.30 (m, 1H), 1.17–1.25 (m,

1H), 1.12 (dd, J = 12.7, 2.6 Hz, 1H), 1.00–1.07 (m, 1H)

ppm.

TCTGCTGTTGTCCCCTTGTTCCCCTGC

TTACTCTTGCAGGTGCAGTGGCTCCAGA

CACCATGGTAAGAAAACAGTTGCAGAGAG

TCATGAGGACAACGTTTCTGG

Italicized CACC indicates introduced sequence required for directional

topoisomerization mediated cloning.

5.5. Plant material

For OsKS1 we used the partial cDNA (AY347876)

previously reported as OsKS1A (Margis-Pinheiro et al.,

2005). For OsKSL2, despite repeated attempts with both

RT-PCR and RACE reactions from both ssp. japonica

cv. Nipponbare and ssp. indica cv. IR24, it was only pos-

sible to generate fragmentary sequence information. The

largest such partial cDNA (1545 nucleotides), from ssp.

japonica cv. Nipponbare, was deposited (DQ823350).

For OsKSL3 three independent full-length cDNAs were

obtained from both ssp. japonica cv. Nipponbare and

ssp. indica cv. IR24 rice. All six clones contained an

‘extra’ adenosine base after nucleotide 1215 of the origi-

nally predicted sequence. This was further conﬁrmed by

re-sequencing of the originally reported partial cDNA

clone (AY347879), which also proved to contain this

frame-shift insertion. The three clones from ssp. indica

cv. IR24 were additionally mis-spliced between exons 10

and 11 (joining nucleotides 1702 and 1703), with one

being un-spliced (i.e. containing the corresponding intron)

and the other two missing nucleotides 1703–1709 (i.e. as a

result of being spliced at the wrong nucleotide). Outside

of these splicing diﬀerences the six full-length clones were

approximately 99% identical, with almost all of the diﬀer-

ences arising from inter-subspecies variation between the

ssp. indica and ssp. japonica clones. A representative

cDNA clone from ssp. japonica cv. Nipponbare has been

deposited (DQ823351). The frame-shift insertion in this

particular cDNA was also ‘removed’ by PCR-based muta-

genesis to generate a ‘corrected’ OsKSL3. Cloning of

OsKSL4 from ssp. indica cv. IR24 has been previously

reported (Wilderman et al., 2004). For OsKSL5 two inde-

pendent clones were obtained from ssp. indica cv. IR24

Rice plants (Orzya sativa L. ssp. indica cv. IR24), and

seedlings (ssp. japonica cv. Nipponbare) were those previ-

ously described (Xu et al., 2004). Brieﬂy, leaves were

detached from 4-week-old greenhouse grown plants and

UV-irradiated for 25 min from 15 cm with 254 nm wave-

length light. These were then incubated for 24 h in dark

humid conditions at 30 ꢁC. Seedlings were germinated

from surface sterilized seeds on ﬁlter paper on top of

1.2% agar plates (eight seeds per plate) incubated in the

dark at 30 ꢁC for one week. The seedlings were then

sprayed with approximately 2 mL 0.1% Tween 20 contain-

ing 0.5 mM methyl jasmonate per plate and incubated

under the same conditions for 24 h. Total RNA was

extracted using the Concert Plant RNA Reagent, and

mRNA puriﬁed using Dynabeads Oligo(dT)₂₅(Dynal Bio-

tech, Oslo, Norway).

5.6. Cloning

The OsKSL genes were largely obtained via RT-PCR

using the primer pairs shown in Table 2. All of the genes

were initially cloned into pCR-BluntII-TOPO and com-

pletely sequenced. The corresponding ORFs were then

transferred to the Gateway vector system via PCR ampliﬁ-

cation and directional topoisomerization based insertion

into pENTR/SD/D-TOPO and veriﬁed by complete

sequencing. The resulting clones were subsequently trans-

ferred via directional recombination to the T7-based N-ter-

minal GST fusion expression vector pDEST15 and, for

OsKSL8, the T7-based N-terminal thioredoxin fusion

expression vector pTH8 (Hammarstro¨m et al., 2002).

324

M. Xu et al. / Phytochemistry 68 (2007) 312–326

and fully sequenced, demonstrating complete identity, to

verify the deposited sequence (DQ823352). For OsKSL6

two independent clones were obtained from ssp. japonica

cv. Nipponbare and fully sequenced, demonstrating com-

plete identity, to verify the deposited sequence

(DQ823353). For OsKSL7 two independent clones were

obtained from ssp. indica cv. IR24 and fully sequenced,

demonstrating complete identity, to verify the deposited

sequence (DQ823354). For OsKSL8 we were not able to

clone a full-length cDNA, instead consistently ﬁnding

the closely related OsKSL11 in both ssp. indica and japon-

ica (Morrone et al., 2006). It was possible to clone partial

cDNA fragments separately corresponding to the ﬁrst

1715 and last ꢀ900 nucleotides of the reported ORF of

OsKSL8 (AB118056; 2463 nucleotides total). However,

sequencing of the 3⁰end fragment revealed a 21 nucleo-

tide deletion in the last exon (i.e. nucleotides 2171–2192

were missing), which is extremely GC-rich and contains

quite repetitive sequence elements in this region. Attempts

to clone a 3⁰end fragment with the correct sequence were

unsuccessful, and ultimately the missing nucleotides were

inserted via PCR extension, and the full-length ORF sub-

sequently re-created using the 5⁰end and corrected 3⁰end

5.8. Functional characterization

Assays were generally carried out with GGPP (5 lg) as

substrate in coupled reactions with either the syn-CPP syn-

thase OsCPS4 or ent-CPP synthase ZmCPS2/An2 (Harris

et al., 2005), both expressed and puriﬁed as GST-fusion pro-

teins. For every gene product individual enzyme assays also

were run with 5 lg GGPP (1), ent-CPP (2), or syn-CPP (3)

directly. Reactions were run in 0.5 mL assay buﬀer (50 mM

Hepes, pH 7.2, 10 mM MgCl₂, 10% glycerol, and 5 mM

DTT) using 25 lL recombinant CPS and/or 25 lL recombi-

nant OsKS(L) for 3–16 h at room temperature. These assays

were extracted three times with an equal volume of hexanes,

which were pooled, dried under nitrogen, and re-dissolved in

100 lL of hexanes prior to GC–MS analysis.

5.9. Sequence analysis

BLAST searches were carried out on-line at both Gen-

Bank (www.ncbi.nih.gov) and TIGR (www.tigr.org). The

cloned cDNAs were mapped back onto the rice genome

by web-based alignment at Gramene (www.gramene.org),

and the corresponding up-stream sequences also obtained

here. Notably, 776 bases upstream of the OsCPS4 gene

there seems to be a gap in the genome sequence, as repre-

sented by a stretch of several hundred unidentiﬁed bases

(i.e. ‘N’s). Promoter analysis was done on-line at PlantCare

(bioinformatics.psb.ugent.be/webtools/plantcare/html) and

TAIR (www.arabidopsis.org/tools/bulk/motiﬃnder/index.

jsp). All other sequence analysis was carried out with the

VectorNTI software package (Invitrogen) using standard

parameters.

fragments in

a mega-primed PCR approach. For

OsKSL10 a full-length cDNA was obtained from the Rice

Genome Resource Center (ssp. japonica cv. Nipponbare),

re-sequenced, and the corrected sequence deposited

(DQ823355). Cloning of OsKSL11 from both ssp. indica

and japonica has been previously reported (Morrone

et al., 2006).

5.7. Recombinant expression

Heterologous expression was performed using the Over-

Express C41 strain of E. coli (Avidis, France), as previously

described (Xu et al., 2004). Brieﬂy, 50-mL NZY cultures

inoculated from 5 to 7 individual transformants/colonies

were grown at 37 ꢁC to mid-log phase (A₆₀₀ꢀ 0.6), then

transferred to 16 ꢁC for 1–2 h prior to induction with

1 mM IPTG for overnight (14–18 h) expression. Cells were

harvested by centrifugation, resuspended in 1 mL cold lysis

buﬀer (50 mM Bis–Tris, pH 6.8, 1 mM DTT), lysed by mild

sonication (15 s, continuous output, half-maximum

power), and clariﬁed by centrifugation (40,0000g, 20

min). Recombinant GST-tagged proteins were puriﬁed

using GST-agarose beads (Sigma–Alrich, St. Louis, MO,

USA) in batch mode. The thioredoxin-OsKSL8 fusion

protein was only partially puriﬁed using ceramic hydroxya-

petite type II (BioRad, Hercules, CA, USA) in batch mode.

The batch puriﬁcation was carried out at 4 ꢁC during which

clariﬁed extract was incubated 5–10 min with ꢀ0.3 mL

beads, washed 5 times with 1 mL lysis buﬀer, then eluted

in 0.5 mL (either 10 mM glutathione in lysis buﬀer for

GST-OsKSL bound to glutathione beads, or 0.2 M sodium

phosphate, pH 6.8, for thioredoxin-OsKSL8 bound to

ceramic hydroxyapetite), the resulting eluate was ﬁltered

(0.2 lm) and immediately used in enzymatic assays.

Acknowledgements

We thank Drs. Arata Yajima and Goro Yabuta (Tokyo

University of Agriculture) for graciously providing an

authentic sample of synthetic ent-cassa-12,15-diene, and

Dr. Gustavo MacIntosh (Iowa State University) for assis-

tance with promoter analysis. This study was generously

supported by grants from the USDA-CSREES (2005-

35318-15477) to R.J.P., and from the NIH (GM13956) to

R.M.C., along with fellowship support from Iowa State Uni-

versity (to D.M.), while the work of M.M.-P. and N.M.U. in

the CSIRO Plant Industry’s ‘‘Rice Functional Genomics’’

group (http://www.pi.csiro.au/fgrttpub/home.htm) was

supported by GrainGene (Australia), Rural Industries Re-

search and Development Corporation (RIRDC), Australia,

and the NSW Agricultural Genomics Centre.

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Article Doi

Functional characterization of the rice kaurene synthase-like gene family

DOI: 10.1016/j.phytochem.2006.10.016

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Full text of DOI:10.1016/j.phytochem.2006.10.016

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