Download LAP5 and LAP6 encode anther-specific proteins with similarity to

Plant Physiology Preview. Published on May 4, 2010, as DOI:10.1104/pp.110.157446
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Running head: CHS-like proteins in pollen exine development
Anna Dobritsa
Department of Molecular Genetics and Cell Biology
University of Chicago
929 East 57th St., W519P
Chicago, IL 60637, USA
Phone: 773-702-9559
Fax: 773-702-6648
e-mail: [email protected]
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Copyright 2010 by the American Society of Plant Biologists
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LAP5 and LAP6 encode anther-specific proteins with similarity to chalcone synthase
essential for pollen exine development in Arabidopsis thaliana
Anna A. Dobritsa*, Zhentian Lei, Shuh-ichi Nishikawa2, Ewa Urbanczyk-Wochniak3, David V.
Huhman, Daphne Preuss4, Lloyd W. Sumner
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637,
USA (A.A.D., S.N., D.P.); and Plant Biology Division, The Samuel Roberts Noble Foundation,
Ardmore, OK 73401, USA (Z. L., E.U.-W., D.V.H., L.W.S.)
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1
This project was supported in part through the National Science Foundation 2010 project grant
#MCB-0520283 (A.A.D, E.U., L.W.S, and D.P.) and through the Samuel Roberts Noble
Foundation (Z.L., E.U., D.V.H., and L.W.S.).
2
Present address: Department of Chemistry, Graduate School of Science, Nagoya University,
Chikusaku, Nagoya 464-8602, Japan
3
Present address: Monsanto Co., St. Louis, MO 63167, USA
4
Present address: Chromatin Inc., Chicago, IL 60616, USA
*
Corresponding author, e-mail [email protected]
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ABSTRACT
Pollen grains of land plants have evolved remarkably strong outer walls referred to as
exine that protect pollen and interact with female stigma cells. Exine is composed of
sporopollenin, and while the composition and synthesis of this biopolymer are not well
understood, both fatty acids and phenolics are likely components. Here we describe mutations in
the Arabidopsis thaliana LAP5 and LAP6 that affect exine development. Mutation of either gene
results in abnormal exine patterning, whereas pollen of double mutants lacked exine deposition
and subsequently collapsed, causing male sterility. LAP5 and LAP6 encode anther-specific
proteins with homology to chalcone synthase (CHS), a key flavonoid biosynthesis enzyme. lap5
and lap6 mutations reduced the accumulation of flavonoid precursors and flavonoids in
developing anthers, suggesting a role in the synthesis of phenolic constituents of sporopollenin.
Our in vitro functional analysis of LAP5 and LAP6 using 4-coumaroyl-CoA yielded bisnoryangonin (a commonly reported derailment product of CHS), while similar in vitro analyses
using fatty acyl-CoA as the substrate yielded medium-chain alkyl pyrones. Thus, in vitro assays
indicate that LAP5 and LAP6 are multi-functional enzymes and may play a role in both the
synthesis of pollen fatty acids and phenolics found in exine. Finally, the genetic interaction
between LAP5 and an anther gene involved in fatty acid hydroxylation (CYP703A2)
demonstrated that they act synergistically in exine production.
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Pollen grains of land plants are surrounded by complex cell walls that are divided into three
layers: (i) an outer exine, itself a multilayered structure, primarily made of sporopollenin, (ii) an
inner intine, made primarily of cellulose, and (iii) a lipid- and protein-rich pollen coat in the
crevices of exine. The exine is morphologically diverse, provides protection against
environmental stresses and bacterial and fungal attacks, and plays a role in species-specific
adhesion (Zinkl et al., 1999; Edlund et al., 2004).
Several studies indicate that sporopollenin is a complex polymer composed of fatty acids
and phenolic compounds (Guilford et al., 1988; Ahlers et al., 1999; Wiermann et al., 2001).
Sporopollenin is remarkably strong and chemically resistant, making it difficult to determine its
precise composition by direct chemical analysis. Ozonolysis has yielded simple straight- and
branched-chain monocarboxylic acids, typical of fatty acid breakdown (Shaw, 1971), as well as
phenolic acids, such as p-hydroxybenzoic, m-hydroxybenzoic, and protocatechuic acids.
Additional evidence for phenolic compounds came from degradation experiments or studies
showing incorporation of radiolabelled phenylalanine and p-coumaric acid into sporopollenin
(Shulze Osthoff and Wiermann, 1987; Rittscher and Wiermann, 1988; Gubatz et al., 1993), while
immunolocalization studies with anti-p-coumaric acid antibodies demonstrated the occurrence of
phenols in exines of different plant species (Niester-Nyveld et al., 1997).
While a growing number of genes have been identified that are important for exine
development, still relatively little is known about the genetic network that governs the formation
of this structure and the pathways that lead to its biosynthesis are far from being understood. In
recent years, the importance of fatty acid-derived components in sporopollenin composition has
been revealed through the identification of several Arabidopsis genes (MS2 (Aarts et al., 1997),
CYP703A2 (Morant et al., 2007), CYP704B1 (Dobritsa et al., 2009) and ACOS5 (de Azevedo
Souza et al., 2009)), which are important for exine production and involved in fatty acid
metabolism. Less is known concerning the role of phenolics in sporopollenin biosynthesis, and
the key synthetic and regulatory genes specifically associated with this aspect of sporopollenin
biosynthesis are absent from the literature.
Phenolic compounds are a large class of secondary metabolites that play a variety of
biological roles (Hahlbrock and Scheel, 1989). Most plant phenolics are products of
phenylpropanoid metabolism, including lignins, coumarins, stilbenes, and flavonoids. A wellcharacterized biosynthetic pathway leads to the biosynthesis of flavonoids (Supplemental Fig.
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S1). Chalcone synthase (CHS) catalyses the first committed step in this pathway using 4coumaroyl-CoA provided by 4-coumaroyl:CoA ligase (4CL) as a substrate. Flavonoids are
important for pollen germination and plant fertility in several plant species (Coe et al., 1981; van
der Meer et al., 1992; Fischer et al., 1997; Napoli et al., 1999), while a null mutation in the
Arabidopsis CHS gene, TT4, results in plants with normal fertility and an absence of flavonoids
in the mature stamens (Burbulis et al., 1996; Ylstra et al., 1996). This suggests that flavonoids
are either not required for Arabidopsis male fertility or that TT4-independent flavonoid synthesis
occurs in anthers, perhaps transiently and at an earlier developmental stage, through a
mechanism that has not been detected in previous experiments.
Recently, an anther-specific gene ACOS5 was described that is essential for exine
production and sporopollenin biosynthesis (de Azevedo Souza et al., 2009). ACOS5 is related to
a phenylpropanoid enzyme 4-coumaroyl:CoA ligase (4CL), but encodes a novel medium- to
long-chain fatty acyl-CoA synthetase. In this study we describe the identification and
characterization of two highly conserved anther-specific genes that are involved in pollen exine
development, likely participate in sporopollenin biosynthesis, and similar to ACOS5, are related
to, yet distinct from, an enzyme of the phenylpropanoid pathway. Our results provide further
insight into the mechanism that leads to the formation of sporopollenin.
RESULTS
lap5 and lap6 Mutants Have Defective Exine Patterning
We recovered less adhesive pollen mutants lap5 and lap6 in a screen for genes that play a
role in Arabidopsis pollen-stigma adhesion (Nishikawa et al., 2005). Both mutants had
morphological defects in exine structure that were apparent under light or scanning electron
microscopy (SEM) (Fig. 1). The mutant anthers and pollen appeared glossy under a dissection
stereomicroscope (Fig. 1, A-C). The pollen adhered tightly to the anthers and was not easily
released. Under SEM, lap5 and lap6 pollen grains exhibited abnormal exine patterning (Fig. 1,
D-I): lap5 pollen lacked the characteristic reticulate structure (Fig. 1H), and lap6 pollen grains
had a more extensively covered surface with broader muri and smaller lacunae (Fig. 1I). Pollen
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grains of both mutants collapsed more easily under vacuum than the wild type, although the
thickness of the exine layer was unaltered (data not shown).
Defects in some Arabidopsis exine mutants (ms2 (Aarts et al., 1997), flp1 (Ariizumi et
al., 2003), and cyp704b1 (Dobritsa et al., 2009)) render pollen sensitive to acetolysis, an
oxidative process often used by palynologists to prepare exine samples (Erdtman, 1960). We
incubated lap5 and lap6 pollen in a mixture of acetic anhydrate and sulfuric acid but did not
observe sensitivity. Interestingly, lap6, but not lap5 exine consistently showed reduced staining,
suggesting that the two mutants are different in the quality or amounts of the exine constituents
that react with the acetolysis mixture (Fig. 1, J-L).
Mapping LAP5 and LAP6
Both lap5 and lap6 mutations segregated as single-locus, homozygous recessive
mutations with phenotypic ratios (wild-type:mutant) of 1008:326 (lap5) and 740:219 (lap6),
consistent with 3:1 segregation (chi squared, P>0.5 and 0.5>P>0.1, respectively). Using bulked
segregant analysis (Michelmore et al., 1991), we mapped LAP5 to chromosome 4 and LAP6 to
chromosome 1 (see Materials and Methods). Additional PCR-based markers were used to refine
the locations to within 59 kb for LAP5 and 140 kb for LAP6. One gene in each interval,
At4g34850 and At1g02050, respectively, contained mutations within open reading frames that
co-segregated with the lap5 and lap6 mutant phenotypes. Both candidate genes encoded
predicted chalcone synthase (CHS) family proteins. The mutations in the genes resulted in
missense codons, causing a G227E conversion in lap5, and an I132T conversion in lap6. In
addition to the lap6-1 allele that we isolated, Arabidopsis stock centers contained two lines with
T-DNA or transposon insertions in At1g02050 (SALK_134643, lap6-2 and N172991, lap6-3)
(Fig. 2). We confirmed the positions of these insertions in the first exon (lap6-2) and in the
second exon close to the end of the ORF (lap6-3). Both insertion alleles had pollen and anther
phenotypes identical to those of lap6-1 (data not shown).
To confirm the identity of the LAP5 and LAP6 genes, complementation constructs were
created containing the entire At4g34850 or At1g02050 genes, including the native promoters,
and transformed, respectively, into lap5-1 and lap6-1. Wild-type anther and pollen phenotypes
were restored in 38/40 BASTA-resistant T1 plants for lap5. Of the remaining 2 plants, one had a
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lap5 phenotype, and the other lacked pollen and was sterile. For lap6, 35/40 BASTA-resistant T1
plants had restored the wild-type phenotype. The remaining 5 plants had the lap6 phenotypes.
We confirmed the presence of the lap5-1 and lap6-1 mutations in the rescued lines using CAPS
markers. These results indicate that wild-type copies of At4g34850 and At1g02050 can
complement the lap5-1 and lap6-1 mutants, respectively, and that the constructs used contained
sufficient cis-regulatory elements to drive expression in a spatially and temporally relevant
manner.
LAP5 and LAP6 Encode Chalcone Synthase Superfamily Proteins
The predicted LAP5 and LAP6 proteins are similar to enzymes in the chalcone synthase
superfamily (Fig. 3). Members of this superfamily are plant-specific type III polyketide
synthases (PKS) and catalyze condensing reactions that generate the backbone required for the
synthesis of a large number of phenylpropanoid compounds, such as flavonoids and stilbenes
(Schröder, 1997; Austin and Noel, 2003). BLAST results revealed that, in addition to these two
genes and a well-characterized chalcone synthase TT4 (Shirley et al., 1995), Arabidopsis
contains a third CHS-related gene, At4g00040.
CHSs (EC 2.3.1.74) from different plants often share 80-90% sequence identity
(Niesbach-Klösgen et al., 1987). In contrast, LAP5, LAP6 and At4g00040 share only 40%
sequence identity with the Arabidopsis CHS TT4 or with chalcone synthases from other
organisms.
However, they share 64% (LAP5-LAP6), 76% (At4g00040-LAP5) and 62%
(At4g00040-LAP6) identity with each other and are members of a male-organ-specific CHS-like
protein clade, which includes proteins from dicots, monocots, and gymnosperms (BA42 from B.
napus (Shen and Hsu, 1992), YY2 from O. sativa (Hihara et al., 1996), SlCHS from S. latifolia
(Ageez et al., 2005), CHSLK from N. sylvestris (Atanassov et al., 1998) (Fig. 3). The
evolutionarily distant gymnosperm Pinus radiata also has a CHS-like protein PrChS1 expressed
in male cones (Walden et al., 1999) with 68%, 68%, and 64% sequence identity with LAP5,
LAP6, and At4g00040, respectively. An ortholog of these male-organ-specific proteins exists in
the genome of the moss Physcomitrella patens (NCBI XP_001781520), in addition to orthologs
of a canonical CHS.
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LAP5, LAP6 and At4g00040 have a predicted gene structure similar to that of CHS,
stilbene synthase (STS) and related type III PKS genes, including a conserved cysteine codon
(Cys60 in the numbering scheme of the well-characterized enzyme CHS2 from alfalfa Medicago
sativa) that is split by a short intron between the first and the second nucleotides (Fig 2 and 3). In
addition to this intron, LAP5, unlike the majority of this family’s members, has a second intron.
Four absolutely conserved residues (Cys164, Phe215, His303, Asn336) that define the catalytic
machinery in the active centers of CHS-related enzymes (Lanz et al., 1991; Ferrer et al., 1999;
Jez et al., 2000b; Austin et al., 2004) are present in LAP5, LAP6, At4g00040, as well as in malespecific CHS-like proteins from other species (Fig. 3). However, known male-specific CHS-like
proteins, as well as LAP5, LAP6, and At4g00040, have a large number of amino acid changes
specific to this clade (Fig. 3A).
The lap5-1 and lap6-1 point mutations affect highly conserved amino acids: Ile132 in
LAP6, replaced with threonine in lap6-1, is present in all 42 CHS, STS, and CHS-like proteins
examined (Fig. 3A and data not shown). Gly227 in LAP5, replaced with glutamic acid in lap5-1,
is conserved in all LAP5/LAP6-like proteins (Fig. 3A).
LAP5 and LAP6 Have Anther-specific Expression Patterns
We searched publicly available expression databases, including Genevestigator
(Zimmermann et al., 2004) (https://www.genevestigator.ethz.ch/) and MPSS (Meyers et al.,
2004) (http://mpss.udel.edu/at/) to identify LAP5 and LAP6 tissue expression patterns.
According to microarray and MPSS data, LAP5 and LAP6 were specifically expressed in young
anthers and in flower buds (stages 9 and 10), and transcripts were absent in flower buds from
stamen-lacking ap3 and ag mutants. In addition, a search of ATTED-II and BAR Expression
Angler databases that analyze microarray expression data and predicted cis-regulatory elements
to build networks of genes that are likely to be co-expressed (Obayashi et al., 2007; Toufighi et
al.,
2005)
(http://www.atted.bio.titech.ac.jp/),
(http://bbc.botany.utoronto.ca/ntools/cgi-
bin/ntools_expression_angler.cgi) suggested that LAP5 and LAP6 were expressed together and
highly co-expressed with several other genes involved in exine production including ACOS5 (de
Azevedo Souza et al., 2009), MS2 (Aarts et al., 1997), CYP703A2 (Morant et al., 2007),
CYP704B1 (Dobritsa et al., 2009), and DRL1 (Tang et al., 2008). To confirm LAP5 and LAP6
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expression patterns, we transformed wild-type plants with β-glucuronidase (GUS) under the
control of the LAP5 and LAP6 promoters. In the resulting T1 plants, GUS staining was observed
only in anthers of stage 9 and 10 buds (Fig. 4). Taken together, these data indicate that LAP5 and
LAP6 are male-organ-specific members of the chalcone synthase family and are expressed in
anthers coincident with the timing of exine formation.
lap5 lap6 Double Mutants Have Enhanced Exine Defects
A lap5-1 lap6-1 double mutant was created to explore the relationships between the LAP5 and
LAP6 genes in pollen development. While both single mutations resulted in fertile pollen with
abnormally patterned exine, the double mutant was sterile, produced brown shriveled anthers
with almost no pollen and failed to produce elongated seed pods or siliques (Fig. 5). Sections of
developing lap5-1 lap6-1 anthers showed normal pollen development until stage 9 (Fig. 6), but
lacked the exine typically found in wild type at stage 9 (green staining around the pollen grains,
Fig. 6C) (Sanders et al., 1999). Pollen in the double mutant subsequently collapses, with almost
no pollen remaining by the stage 11 (Fig. 6H).
Neither At4g00040 nor TT4 can Substitute for LAP5 or LAP6
Given the high similarity of the predicted At4g00040 protein to LAP5, LAP6, and malespecific CHS-like enzymes from several organisms, we tested whether this gene also plays a role
in pollen development. Three lines with DNA insertions in At4g00040, including one with an
insertion in the middle of the predicted ORF (Fig. 2), were available from Arabidopsis stock
centers. We confirmed the insertion positions in each line, and found that each contained anthers
and pollen indistinguishable from wild type.
Because gene expression databases indicated that At4g00040 was not male-specific, but
instead was expressed in various organs and stages of development, we next tested whether
driving expression of this gene in stage 9-10 anthers would rescue lap5 or lap6 defects. We
placed a wild-type copy of At4g00040 under the control of a LAP5 or LAP6 promoter and
introduced these constructs into lap5-1 and lap6-1 mutants. 10 BASTA-resistant T1 plants were
analyzed for each of the four resulting combinations, but none had reversed the mutant
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phenotype. Thus, despite its high similarity to male-specific CHS-like proteins, At4g00040
apparently is unable to substitute for LAP5 or LAP6 in pollen development.
In addition, we tested if expression of Arabidopsis CHS TT4 in stage 9-10 anthers would
rescue lap5 lap6 defects by expressing it under LAP5 promoter and introducing into the lap5-1/+
lap6-1/lap6-1 plants. None of the 36 BASTA-resistant T1 plants analyzed (11 lap5-1/lap5-1
lap6-1/lap6-1; 18 lap5-1/+ lap6-1/lap6-1; 7 lap6-1/lap6-1) had mutant phenotypes reversed.
Thus, canonical CHS is also unable to substitute for LAP5 and LAP6 in pollen development.
lap5-1 and lap6-1 Affect Production of Anther Chalcone, Naringenin, Other Flavonoids,
and Additional Metabolites
Because LAP5 and LAP6 resemble CHS proteins, we hypothesized they might catalyze
the production of phenolic components needed to form sporopollenin. We collected metabolic
profiles from stage 9/10 anther extracts from wild-type, lap5-1, lap6-1, and the lap5-1 lap6-1
double mutant (see Materials and Methods). Using ultra high performance liquid
chormotography and gas chromatography coupled to mass spectrometry (UPLC-MS and GCMS), a broad spectrum of primary and secondary metabolites were separated, detected and
characterized. 83 metabolites were characterized with UPLC-TofMS (Supplemental Tables SIIV), and 45 of these were annotated using available authentic standards or comparison to
previously published data. The annotated metabolites predominantly belonged to three chemical
groups: phenolics (mainly flavonoids), glucosinolates and long-chain fatty acids.
The levels of the phenolics, chalcone and naringenin, formed in the first committed step
of the flavonoid pathway, were significantly reduced in the lap mutants (11.8-fold, lap5 and 5.6fold, lap6) and were undetectable in the double mutant (Fig. 7; Supplemental Table SI). In
addition, several other flavonoids were reduced in both mutants, including dihydrokaempferol
and isorhamnetin 3-sophoroside. Kaempferol-3-O-gentiobioside-7-O-rhamnoside was not
detected in lap5, and kaempferol 3-glucoside-7-p-coumarylglucoside was not detected in lap6.
Levels
of
kaempferol-3-galactoside-7-rhamnoside,
luteolin
7-O-rhamnoside
and
6-
methoxytaxifolin were significantly reduced in lap6 and 6-methoxytaxifolin not detected in the
double mutant. Robinin was reduced in lap5 and almost absent in the double mutant samples.
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Surprisingly, increases (almost 6-fold) of quercetin 3-glucoside-7-rhamnoside were observed in
the lap6 mutant and another flavone, apigenin-6-C-glucoside, was detected only in lap6.
In addition to changes in the levels of phenolics, we also detected changes in the levels of
other compounds in mutant anther extracts. For instance, several glucosinolate compounds were
observed only in the mutant plants, as were some fatty acids, such as α-eleosteric acid and 10,16dihydroxypalmitic acid (Supplemental Tables SII-III). Given the possible role of 16hydroxypalmitic acid in the biosynthesis of sporopollenin (Dobritsa et al., 2009), accumulation
of this fatty acid in the lap6 and lap5 lap6 mutants could be a consequence of the overall changes
in sporopollenin composition in the mutant anthers, which could result in changes in fatty acid
polymerization. Significant changes in several unidentified compounds were also found in the
mutant extracts (Supplemental Table SIV).
The relative abundances of 96 molecules of known chemical structures, including amino
acids, Krebs cycle intermediates, carbohydrates, fatty acids derivatives and several other primary
metabolites were determined by GC/MS (Supplemental Table SV). Several primary metabolites
were altered in the mutant lines, the most noticeable being a general increase in the levels of
amino acids and their catabolic products. Levels of L-asparagine, L-valine and 4-aminobutiric
acid were increased in all mutant lines, with the most pronounced changes observed in the
double mutant samples. Additionally, levels of pipecolic acid, a degradation product of L-lysine
were increased 13.6-fold in lap5 mutant and 6.1-fold in the double mutant. Several changes in
the levels of carbohydrates were observed. The most evident change was a dramatic reduction of
maltose levels in all three mutant samples. Because maltose is a starch hydrolysis product, this
suggests a reduction in starch pools.
Enzymatic Activity of LAP5 and LAP6
The metabolic profiling data of the lap5 and lap6 mutants revealed decreased flavonoid
levels, suggesting that LAP5 and LAP6 may play a role in anther flavonoid biosynthesis.
Heterologous gene expression and in vitro enzymatic assays were performed to investigate the
function of LAP5 and LAP6 proteins. Enzymatic reactions were performed in the presence of 4coumaroyl-CoA and malonyl-CoA, CHS substrates. The reaction products were analyzed by
HPLC-UV/Vis-ITMS (Fig. 8). The empty-vector extract and the Arabidopsis CHS TT4 were
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13
used as the negative and the positive controls, respectively (Fig. 8, A and B). The product of
TT4/CHS (Fig. 8B) was identified as naringenin by comparing its retention time, UV and mass
spectrum with those obtained with the authentic naringenin standard (Figure 8C). Naringenin, the
expected CHS product, was not present in the LAP5 or LAP6 assays. However, two other
products, peaks 1 and 2, were found (Fig. 8, D and E), which were absent in both the positive
and negative controls. The mass spectra of the two peaks observed in the LAP5 and LAP6
product profiles were almost identical and dominated by three major ions m/z = 229, 185, 143
with slightly different abundance ratios (Supplemental Fig. S2). These two peaks were
unambiguously identified as trans- and cis-bisnoryangonin (i. e. 4-coumaroylacetic acid lactone)
based upon the observed ions at m/z 229 [M-H]–, 185 [M-H-CO2]– and 143 [retro Diels-Alder
reaction: M-H-C3H2O3]– as previously described (Jindaprasert et al., 2008) and characteristic UV
absorption maxima at 365 nm (Shiokawa et al., 2000). The MS assignments were further
confirmed by MS/MS of the molecular ion m/z 229 which gave two fragment ions: m/z 185 and
143 (Akiyama et al., 1999). The in vitro activities of LAP5 and LAP6 were also investigated at
different pH levels (pH 5.0, 8.0, and 9.0). Bisnoryangonin was found to be the only product in
both LAP5 and LAP6 assays at all pH levels. We also tested whether combining both LAP5 and
LAP6 would result in a different activity because chalcone and stilbene synthases are known to
function as homodimers (Ferrer et al., 1999; Shomura et al., 2005) and are also capable of
forming in vitro heterodimers with partial catalytic activities (Tropf et al., 1995). However,
bisnoryangonin was again the only detected product.
In vitro formation of trans- and cis-bisnoryangonin has been observed with other
recombinant CHS (Akiyama et al., 1999; Shiokawa et al., 2000). It is formed through the
condensation of one molecule of 4-coumaroyl-CoA and two molecules of malonyl-CoA
(Supplemental Fig. S3). Like coumaroyltriacetic acid lactone (CTAL, a condensation product of
one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA), bisnoryangonin has
been reported as an enzymatic ‘derailment’ product (i.e., an incomplete biosynthetic product that
detaches from the enzyme prior to completion of all the enzymatic steps) of CHS or CHS-like
proteins (Kreuzaler and Hahlbrock, 1975; Shiokawa et al., 2000). The formation of the
derailment products probably reflects the differences between the in vitro and the in vivo
environments, and was attributed to the lack of subsequent reactions in the in vitro environments
(Eckermann et al., 2003). Targeted analyses of the UPLC-QTOF-MS data did not show the
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14
presence of bisnoryangonin or its glycosides in the wild-type plants, indicating that
bisnoryangonin is unlikely the in vivo physiological product of LAP5 and LAP6.
LAP5 and LAP6 belong to the type III polyketide synthase (PKS) superfamily, which
produces a wide variety of secondary metabolites using different starter CoA esters, different
elongation cycles, and different cyclization mechanisms (Austin and Noel, 2003; Flores-Sanchez
and Verpoorte, 2009). Acetyl-CoA is one of the most common starters in polyketide synthesis
and leads to the formation of both aromatic and pyrone polyketides. To determine whether LAP5
and LAP6 proteins have PKS activity in the presence of acetyl-CoA, 4-coumaroyl-CoA was
replaced with acetyl-CoA in a separate experiment. However, no product was observed,
indicating that acetyl-CoA is unlikely a physiological substrate.
While these experiments were in progress, another group reported on the biochemical
characterization of the LAP5 and LAP6 proteins (Mizuuchi et al., 2008). The proteins were
shown in vitro to catalyze condensation reactions using medium- and long-chain fatty acyl-CoA
and malonyl-CoA as starter molecules, resulting in the formation of triketide and tetraketide αpyrone lipids. We repeated these experiments using a variety of acyl-CoA (chain length C4-C18)
as substrates. The empty-vector extract was used as a negative control. Similar to Mizuuchi et al.
(2008), we found that LAP5 and LAP6 are also capable of accepting fatty acyl-CoA as starters
and performing condensation reactions with malonyl-CoA to form triketide (major product) and
tetraketide (minor product) α-pyrone lipids (Fig. 9 and Supplemental Figs. S4-S6; data for one
protein are shown; the other demonstrated identical activity). However, unlike Mizuuchi et al.
(2008), who observed conversion of C4-C20 fatty acyl-CoA, only C6-C12 were converted into
the corresponding tri- or tetraketide pyrones under our experimental conditions. In addition to
LAP5 and LAP6, TT4/CHS was also assayed with fatty acyl-CoA as a substrate. We found that
it also possessed an in vitro activity comparable to that of LAP5 and LAP6 towards fatty acylCoAs, accepting C6-C10 substrates and catalyzing condensation reactions to form tri- and
tetraketide pyrones (Fig. 9 and Supplemental Figs. S5-S6). In reaction with hexanoyl-CoA
catalyzed by TT4/CHS, a minor product was detected, which was identified as tetraketide
resorcylic acid (Supplemental Fig. S5) based on its mass spectrum and its retention time relative
to triketide pyrone (Zhou et al., 2008). In contrast, tetraketides found in octanoyl-CoA (Fig. 9)
and decanoyl-CoA (Supplemental Fig. S6) experiments were tetraketide pyrones rather than
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15
tetraketide resorcylic acid, suggesting that tetraketides can undergo different cyclization in
solutions (Supplemental Fig. S4).
To determine if LAP5/LAP6 serve as multifunctional proteins that could generate crosslinked phenolics and fatty acids in exine, we have performed a multi-substrate assay for LAP5
and TT4/CHS using hexanoyl-CoA, 4-coumaroyl-CoA and malonyl-CoA at a 1:1:3 molar ratio
as substrates. 4-hydroxy-6-pentyl-2-pyrone and bisnoryangonin were obtained with LAP5 (at a
molar ratio of 32:1), and 4-hydroxy-6-pentyl-2-pyrone and naringenin were obtained with
TT4/CHS (at a molar ratio 11:1) (Supplemental Fig. S7). No novel products different from the
compounds obtained in separate experiments were detected, with the exception of
bisnoryangonin found in the reaction catalyzed by TT4/CHS.
Mutations Affecting LAP5 and a Gene from the Fatty Acid Hydroxylation Pathway Have
Synergistic Effects
A recently described gene CYP703A2 is necessary for normal exine development, and
encodes a protein that hydroxylates lauric acid, potentially providing fatty acid building blocks
required for sporopollenin biosynthesis (Morant et al., 2007). We created a double mutant
between lap5-1 and a hypomorphic lap4-1 allele of CYP703A2 (Dobritsa et al., 2009). The lap41 mutant causes plants to produce functional pollen that, like lap5 and lap6, appears glossy and
strongly adheres to anthers. When this pollen was stained with the exine-specific fluorescent dye
auramine O and examined under a laser scanning confocal microscope (LSCM), we observed a
strong reduction in the thickness of exine, which nonetheless exhibited reticulate pattern (Fig. 10,
C and G). Patches of exine were often missing on the surface of the lap4-1 pollen (Fig. 10C).
The lap4-1 lap5-1 double mutant was fertile, but produced glossy pollen that did not easily
separate from the anthers. When we examined the lap4-1 lap5-1 pollen grains under LSCM, we
found that they exhibited a more severe defect in their surface structure than either lap5-1 or
lap4-1 single mutants. The pollen surface showed no reticulate pattern and almost no auramine O
fluorescence (Fig. 10, D and H). When optical sections through the middle of the grains were
obtained to determine exine thickness, we found that in the lap4-1 lap5-1 double mutant exine
was virtually missing (Fig. 10H). Interestingly, in many of those pollen grains areas of somewhat
increased fluorescence were located longitudinally (Fig. 10D).
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16
DISCUSSION
Defects in LAP5 and LAP6 Proteins Have Broad Metabolic Consequences
This study underscores that a disruption of a single gene can have wide-ranging
consequences on the metabolic composition, revealing the intricacy of interactions among
metabolic pathways. Collected metabolomics data indicate that alterations in the expression of
LAP5 and LAP6 genes result in the differential accumulation of several flavonoids and of two
lipids (10,16-dihydropalmitic acid and α-eleosteric acid), and profoundly affect the biosynthesis
of a variety of metabolites outside the fatty acid or phenylpropanoid pathway. It has been
previously suggested (Rohde et al., 2004) that primary and secondary metabolism are interlocked
through the phenylalanine pool. Thus, it is possible that a reduced carbon flux into the
phenylpropanoid pathway affects the homeostasis of amino acid and carbohydrate metabolism.
Possibly, part of the surplus carbon resulting from the reduced phenolic flux in the lap5 and lap6
mutants is redirected towards increased amino acids. Under normal conditions, 20% of all fixed
carbon is directed into the shikimate pathway that generates aromatic amino acids (Herrmann,
1995). One of the major sinks of this pathway is the conversion of phenylalanine to cinnamic
acid via phenylalanine lyase (PAL), which is the first enzyme in the phenylpropanoid pathway.
Interestingly, the reduction in LAP5 and LAP6 activity has dramatic consequences for the
accumulation of several amino acids, not only the aromatic ones. Similarly, increases in the
abundance of multiple amino acids were previously observed in mutants with defective PAL
activity (Rohde et al., 2004). Also, depletion of tyrosine and tryptophan pools via overexpression of Trp and Tyr decarboxylases alters levels of both aromatic and non-aromatic amino
acids (Guillet et al., 2000). These results led to a proposed mechanism that controls amino acids
homeostasis across individual biosynthetic pathways (Guillet et al., 2000; Rohde et al., 2004).
The accumulation of amino acids in lap5 and lap6 mutants may have additional effects.
Βeta-alanine
and the L-valine biosynthetic intermediate, 2-oxoisovalerate, are used in the
formation of pantothenic acid (White et al., 2001), which is subsequently converted to coenzyme
A (Kupke et al., 2003). We interpret the accumulation of branched-chain amino acids (such as
valine and leucine), putrescine, GABA and beta-alanine in the lap5 and lap6 anthers as a result
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17
of altered CoA biosynthesis. A similar metabolic model was previously suggested by
(Broeckling et al., 2005) to describe experimentally observed changes of several metabolites.
CoA serves as a carrier for organic acids, including acetic acid (utilized in fatty acid
biosyhthesis, glycolysis, Krebs cycle, amino acids biosynthesis), 4-coumaric acid and malonic
acid that are used in flavonoid biosynthesis. Thus, levels of CoA might, in turn, provide feedback
control for the production of flavonoids and fatty acids.
Amino acids also provide a link between phenylpropanoids and glucosinolates, another
class of secondary metabolites that was increased in lap5 and lap6 mutants. Glucosinolates are
derived from various amino acids (Halkier and Gershenzon, 2006), and their close metabolic
connection (Knill et al., 2008) might account for the observed increases in glucosinolate levels in
our mutants.
LAP5 and LAP6 Are Paralogs Required for Exine Biosynthesis
Here, we have shown that LAP5 and LAP6 are essential for the production of pollen
exine. Both genes are specifically expressed during the period of exine synthesis in developing
anthers and belong to a male-specific clade of the chalcone synthase superfamily. Point
mutations lap5-1 and lap6-1 result in the formation of fertile pollen with aberrant exine
patterning; available insertions (likely null alleles) in lap6 show an identical phenotype. Double
mutants (lap5-1 lap6-1) in both genes abolish exine deposition, followed by pollen collapse and
male sterility. Given the sequence similarity of these genes and the results of in vitro enzymatic
assays, a possible interpretation of the double mutant phenotype is that the genes are functionally
redundant, with the single mutants reducing the levels of the same compound(s). On the other
hand, since the lap5 and lap6 mutants have different exine morphologies, responses to acetolysis
and metabolite profiles, it is conceivable that their functions are somewhat distinct.
In addition to LAP5 and LAP6, Arabidopsis has a third paralog, At4g00040, which,
despite sequence similarity with known male-specific CHS family proteins, does not exhibit
anther-specific expression nor appear to participate in pollen development. Several other plant
species also have multiple orthologs of LAP5 and LAP6. Poplar Populus trichocarpa has 3
orthologous proteins (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html), and at least two
orthologs are present in the Brassica rapa, tomato, maize, and rice genomes. Classical chalcone
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18
and stilbene synthases are known to function as homodimers (Ferrer et al., 1999; Shomura et al.,
2005). However, an in vitro study with mutated proteins that were inactive as homodimers
demonstrated that CHS and STS are capable of forming heterodimers with partial catalytic
activities (Tropf et al., 1995). It would be interesting to determine if LAP5 and LAP6 can
function as heterodimers, thus potentially expanding the repertoire of reaction substrates and
products. Although our experiments in combining LAP5 and LAP6 in CHS reaction have not
resulted in the change of activity in a heterodimer, it is probable that the substrates used were not
physiological.
LAP5 and LAP6: novel type III polyketide synthases
Canonical chalcone synthases, such as Arabidopsis TT4 or alfalfa CHS2, are the most
ubiquitous and best characterized members of the plant type III PKS family, which is often
referred to as the CHS family. They condense a CoA-ester from the phenylpropanoid pathway
(usually, 4-coumaroyl-CoA) with three molecules of malonyl-CoA to form a tetraketide
intermediate and a new aromatic ring of chalcone. Stilbene synthases, which are found in a
relatively small number of plants, share approximately 70% sequence identity with CHS and use
the same substrates (4-coumaroyl-CoA and malonyl-CoA) to perform three condensation
reactions. The final steps of STS tetraketide ring closure are mechanistically different than those
of CHS, enabling the formation of stilbenes, such as resveratrol. Other members of the type III
PKS superfamily, such as acridone synthase, benzophenone synthase, valerophenone synthase,
also perform three condensation reactions, but use different substrates, while still other type III
PKS enzymes utilize a different number of condensation reactions (Austin and Noel, 2003;
Karppinen et al., 2008).
Sequence comparisons among different members of CHS superfamily provide few clues
to their enzymatic functions, and amino acid residues that cause differences between catalytic
activities remain unknown. For instance, replacement of only 8 amino acids is sufficient to
convert alfalfa CHS to STS (Austin et al., 2004). Yet, there is no apparent STS consensus
sequence, as eight STS-derived mutations in pine that can convert CHS to STS are not conserved
among STS enzymes from different species. Of the two CHS-like proteins from Pinus strobus
that share 87.6% identity only one has CHS activity, whereas the other one is completely
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19
inactive with any starter CoA-esters from the phenylpropanoid pathway or with linear CoA
starters. Instead, it performs a single condensation reaction with a diketide derivative and
methylmalonyl-CoA (Schröder et al., 1998). Out of the five CHS-like genes in hop Humulus
lupulus only one encodes CHS activity, whereas the others prefer substrates unrelated to
flavonoid matabolism (Novak et al., 2006). Additionally, a 2-pyrone synthase (2PS) from
Gerbera hybrida shares 74% sequence identity with two CHSs from the same species, yet
exhibits a very different activity: it uses acetyl-CoA as a substrate, performs only two
condensation reactions, and folds the triketide intermediate into a pyrone ring structure
(Eckermann et al., 1998). Replacement of only three amino acids in CHS is sufficient to convert
it to 2PS (Jez et al., 2000a). To complicate matters further, members of the CHS family are
promiscuous in vitro. CHS is capable of accepting non-physiological substrates such as benzoylCoA or aliphatic acyl-CoA (Schüz et al., 1983); use of isovaleryl-CoA as a substrate for CHS
from pine P. sylvestris was sufficient to convert it to pyrone synthase that produces a pyrone
derivative (Zuurbier et al., 1998), and Gerbera hybrida 2PS is able to produce pyrone using
isovaleryl-CoA or benzoyl-CoA as substrates (Eckermann et al., 1998).
LAP5, LAP6, and other members of their clade in the CHS superfamily share
approximately 40% sequence identity with CHS. At the amino acid level, they are much more
dissimilar to the canonical CHS than 2PS and other members of this superfamily with
demonstrated non-CHS activity. While there are limitations to primary structure analysis for
members of this family, several notable sequence differences could be important for LAP5 and
LAP6 enzymatic activity: 1) The conserved residue, Thr197, is important for determining the
volume of the coumaroyl-binding pocket in the active center of CHS and possibly for the
preference for 4-coumaroyl-CoA as a substrate (Jez et al., 2000a). In addition, the hydroxyl of
Thr197 interacts with the carbonyl of the chalcone product (Ferrer et al., 1999). LAP5, LAP6,
and other members of the male-specific CHS-like clade contain an invariant glycine at the
corresponding position, potentially affecting a substrate preference. lap5-1 mutation highlights
the importance of this residue, replacing it with the larger and negatively charged amino acid,
glutamate. 2) Lys62, an amino acid that sits at the entrance to the CoA-binding tunnel of CHS2
and forms a hydrogen bond with a phosphate of CoA (Ferrer et al., 1999), is replaced with Thr or
Ser in LAP5, LAP6 and related proteins. 3) Homodimers of CHS have two independent active
sites, each of which is composed almost entirely of residues from a single monomer. The only
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20
residue that comes from a different monomer is Met137 that protrudes into a hole on the surface
of the adjoining monomer to form a part of the cyclization pocket (Ferrer et al., 1999). This Met
residue is conserved in all CHS, STS, and most other members of the superfamily with identified
biochemical activity. Met137 is replaced by Leu or Phe in the LAP5/LAP6-related proteins.
The physiological role of LAP5 and LAP6, as that of many other members of type III
PKS superfamily, remains unclear. Given that very few amino acid changes are sufficient to
convert CHS into a protein with a different activity and the level of LAP5/LAP6 divergence
from CHS, we did not readily expect LAP5 and LAP6 to catalyze CHS-type reactions. This
assumption is consistent with the results of the in vitro protein assays and with the apparent
inability of the TT4/CHS to rescue the lap5 and lap6 pollen defects. Interestingly, despite the
fact that LAP5 and LAP6 do not exhibit in vitro CHS activity, the metabolic profiles of the lap5
and lap6 mutants revealed clear reduction in anther chalcones and flavonoids. It is presently
unknown whether this reduction is directly caused by the absence of LAP5 and/or LAP6
activities or whether this is an indirect consequence of the general changes stemming from
altered sporopollenin synthesis and exine development. Thus, a strong possibility remains that
LAP5 and LAP6 function in the production of phenylpropanoid precursors of sporopollenin.
The biochemistry results presented here and by Mizuuchi et al. (Mizuuchi et al., 2008)
demonstrated that in vitro LAP5 and LAP6 are also able to accept a variety of fatty acyl-CoA
and convert them to corresponding pyrone lipids in the condensation reactions with malonylCoA. Homology modeling of LAP5 and LAP6 predicted that, while they share the threedimensional overall fold with other plant type III PKS proteins, they have an unusually long
substrate-binding cavity (Mizuuchi et al., 2008), consistent with their ability to accept long
substrates. This feature resembles the long substrate-binding tunnel of the bacterial PKS-18 from
M. tuberculosis, whose crystal structure was resolved (Sankaranarayanan et al., 2004) and which
also produces long-chain pyrones from fatty acyl-CoA starters (Saxena et al., 2003).
Interestingly, however, we also observed similar activity towards fatty acid derivatives with the
bona fide CHS, TT4, which is known to accept other aliphatic acyl-CoA substrates (Schüz et al.,
1983). This further highlights the in vitro multi-functionality of the type III PKS superfamily
enzymes and demonstrates that conclusions on the physiological roles of these proteins from in
vitro studies should be approached with caution.
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21
If long-chain pyrones were produced in the developing anthers by LAP5 and LAP6, one
would expect to see a differential accumulation of these compounds in the wild type and the lap5
and lap6 mutants. However, the metabolite analyses did not identify long-chain pyrones in the
developing anthers. One possibility is that these compounds are among the metabolites, which
were not positively identified (Supplemental Table SIV) and some of which were significantly
reduced in mutants. Another possibility is that they undergo rapid polymerization and
incorporation into sporopollenin and, therefore, escape detection during a metabolomics analysis.
It remains to be demonstrated that fatty acyl-CoA molecules serve as in vivo substrates for LAP5
and LAP6.
Despite the overlapping in vitro biochemical activity, TT4/CHS does not rescue the lap5
and lap6 pollen phenotypes when expressed under the control of the LAP5 promoter. It is
possible that the intracellular environment may affect the in vivo activity of this enzyme or that
TT4/CHS does not get access to the same substrates as LAP5 and LAP6 because it is sequestered
in a metabolon dedicated to flavonoid metabolism (Winkel, 2004).
Phenylpropanoid Pathway and Sporopollenin Biosynthesis Pathway: Possible Parallels
While the precise structure of sporopollenin remains undetermined, chemical studies
have suggested that it is composed of fatty acid and phenolic compounds (Guilford et al., 1988;
Wiermann et al., 2001). Several genes and corresponding mutants have been described that
confirm the important role of fatty acid metabolism in sporopollenin production. CYP703A2 and
CYP704B1 are cytochromes P450 that have fatty acid hydroxylase activities and use lauric acid
and long-chain fatty acids, respectively, as preferred substrates (Morant et al., 2007; Dobritsa et
al., 2009), whereas MS2 is an anther-specific gene encoding a fatty acyl reductase (Aarts et al.,
1997; Doan et al., 2009). Mutations in each of these genes severely impair pollen wall
production. A recently described gene ACOS5 encodes a fatty acyl-CoA synthetase, which has
an in vitro preference for medium-to long-chain fatty acids (de Azevedo Souza et al., 2009).
ACOS5 protein is related to 4-coumaroyl-CoA ligase, an enzyme of the general phenylpropanoid
pathway that acts directly upstream of and provides the substrate (4-coumaroyl-CoA) for
chalcone synthase. Thus, it is very tempting to speculate that LAP5 and LAP6, which are related
to CHS and are able to utilize medium-chain fatty acyl-CoA in vitro, function immediately
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22
downstream of ACOS5. This is in agreement with mutant analysis of these three genes. An
insertion in ACOS5 results in the formation of brown shriveled anthers that lack pollen, causing
male sterility ((de Azevedo Souza et al., 2009) and A.A.D., unpublished data). Sections of
developing anthers from this mutant revealed that pollen develops normally until stage 9 when
an exine deficiency was observed, followed by subsequent collapse of the pollen grains (de
Azevedo Souza et al., 2009; Supplemental Fig. S8). This phenotype is identical to that of the
lap5 lap6 double mutant and is very distinct from the phenotype of the cyp703a2 cyp704b1 ms2
triple mutant, which has viable pollen despite its lack of the normal exine layer and a
characteristic zebra exine phenotype (Dobritsa et al., 2009). The more severe phenotypes point to
a more central role that the ACOS5/LAP5/LAP6 pathway plays in the sporopollenin biosynthesis
compared with the fatty acid-modifying CYP703A2, CYP704B1, and MS2.
Similarly to CYP703A2, CYP704B1, and MS2 (Morant et al., 2007; Dobritsa et al.,
2009), ACOS5 (de Azevedo Souza et al., 2009) and LAP5/LAP6 are highly conserved in plant
lineages, with homologs present in the genomes of angiosperms, gymnosperms and bryophytes
but absent in green algae. The ability to synthesize sporopollenin is believed to be a critical
evolutionary innovation at a time when plants colonized land, which allowed plants to protect
spores from harmful effects of UV irradiation and desiccation (Bowman et al., 2007; Morant et
al., 2007; de Azevedo Souza et al., 2009). Existence of ACOS5 and LAP5/LAP6 orthologs in the
genome of moss P. patens indicates that this possible variation of the phenylpropanoid pathway
was an ancient adaptation.
DRL1, another anther-specific protein in Arabidopsis related to a protein from the
phenylpropanoid pathway (dihydroflavonol 4-reductase (DFR, Supplemental Fig. S1), whose
function is presently unknown, has also been recently found to be important for exine
development (Tang et al., 2008). DRL1 is similarly conserved in land plants. Expression analysis
of microarray data from the wild type and several transcription factor mutants with abnormal
anther and pollen development (Wijeratne et al., 2007, Yang et al., 2007; Xing and Zachgo,
2008, Xu et al., 2010) demonstrates that DRL1 is co-expressed with ACOS5, LAP5, LAP6,
CYP703A2, CYP704B1, and MS2. This makes DRL1 another strong candidate for an enzyme
that is involved in the biochemical pathway leading to sporopollenin biosynthesis.
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23
MATERIALS AND METHODS
Plant Material
lap5-1, lap6-1, and the lap4-1 allele of CYP703A2 were isolated as described (Nishikawa et al.,
2005) from CS6242, a cer6-2 Landsberg erecta (Ler) variety, mutagenized with 1.25 mM ethyl
nitrosourea. lap6-2 (SALK_134643) and lap6-3 (N172991) were from the Arabidopsis
Biological Resource Center (ABRC) and the European Arabidopsis Stock Center (NASC),
respectively. T-DNA insertions in At4g00040 were from NASC (N180467, N162784; both in the
3’ end of the ORF) and from ABRC (SAIL_829_D04; in the middle of the ORF). A transposon
insertion in At1g62940 (N123936) was from NASC. Plants were grown in a greenhouse at 22°C
with a 16-h-light/8-h-dark photoperiod.
Genetic Mapping
Positional cloning was used to map lap5-1 and lap6-1 (backcrossed three times to CER6 Ler) in
populations generated by crossing to wild-type Columbia. Bulked segregant analysis
demonstrated linkage between lap5-1 and CIW7 and NGA1107 on the bottom of chromosome 4
and between lap6-1 and ZFPG on the top of chromosome 1 (markers are available at the
Arabidopsis Information Resource http://www.arabidopsis.org/). Fine mapping of lap5-1 in 657
F2 plants localized it to a 59 kb region between markers T4L20E2 and F11I11P1; similarly,
mapping analysis of 378 F2 plants localized lap6-1 to 140 kb between T1N6A and T7I23G1.
Candidate genes were sequenced and polymorphisms relative to wild-type Ler were found in
At4g34850 for lap5-1 and At1g02050 for lap6-1. The lap5-1 mutation removes a BamHI site. A
corresponding CAPS marker was designed (lap5-850-CF, GAGCCTGCATCAAGAACTGG and
lap5-850-CR, CGGTTCAAGATGGCTGGTC) to score the lap5-1 alleles in the segregating
populations. To score the lap6-1 alleles, a dCAPS marker was designed (lap6-2050-CF,
GCAGCTCCAACTAGGTCG,
lap6-2050-BR1,
GGTTGCATCAAGGAATGGGGAAGGCCAGTGGAAGATA, EcoRV cleaves the wild-type
allele).
Complementation Constructs
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24
LAP5 (from 631 nt upstream of the start codon to 264 nt downstream of the stop codon) was
amplified
with
Platinum
Pfx
DNA
polymerase
(Invitrogen)
(GTGTACCGGTCATCAGTGGGAAATGTCTCTCAG)
and
using
lap5-850-EF2
lap5-850-ER
(CCATGGATGAGAAGATCCAACTCCTGGAC). LAP6 (from 479 nt upstream of the start
codon to 148 nt downstream of the stop codon) was amplified using lap6-2050-ER2
(GTGTACCGGTTGAAGGATAGACCGTGCATCTGG)
and
lap6-2050-EF
(CCATGGGCACTTGCTTTAACGACACGTGGTGC). PCR products were digested with
AgeI/NcoI, and incorporated into a modified binary vector pGreenII02229 (Hellens et al., 2000;
von Besser et al., 2006). lap5-1 and lap6-1 plants were transformed by the floral dip method
(Clough and Bent, 1998) and transgenic plants were selected with BASTA.
GUS, At4g00040, and TT4 Expression Constructs
LAP5
and
LAP6
promoters
GGATCCCATCAGTGGGAAATGTCTCTCAG
were
amplified
and
(lap5-850-EF3,
lap5-850-FR,
ACCGGTTTGTATCTGTTTGTTGAAGACTC;
lap6-2050-DF2,
ACCGGTTTCTTTTCAAGCGCAGAAGG and lap6-2050-ER2), digested with BamHI/AgeI,
and incorporated into a modified vector pGreenII02229, creating LAP5pr-pGR111 or LAP6prpGR111 constructs. The β-glucuronidase gene (GUS) was amplified with GUS-AF
(CACCATGGATGTTACGTCCTGTAGAAACCC)
and
GUS-CR
(CCACTAGTTCATTGTTTGCCTCCCTGCTGC), digested with NcoI/SpeI, and cloned into
NcoI/SpeI-cut LAP5pr-pGR111 or LAP6pr-pGR111. TT4 was amplified with TT4-AF
(TCTCCCATGGATGGTGATGGCTGGTGCTTC)
and
TT4-CR
(AGGGACTAGTTTAGAGAGGAACGCTGTGC), and similarly cloned in LAP5pr-pGR111.
At4g00040 was amplified with 040-AF (ACCGGTATGTTGGTGTCCGCAAGGGTAGAG) and
040-AR2
(CCATGGGTCAATAGCATTGAGTTGTAAATAAGTCTC),
digested
AgeI/NcoI, and cloned into AgeI/NcoI-digested LAP5pr-pGR111 or LAP6pr-pGR111.
Phylogenetic Analysis
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with
25
CHS family proteins analyzed and their accession numbers from the Entrez Protein database are:
Brassica napus CHS A2 (AAC31912), Zea mays CHS C2 (P24825) and CHS Whp (P24824),
Bromheadia finlaysonia CHS (O23729), Hypericum androsaemum CHS (Q9FUB7), Ipomoea
purpurea CHS-D (AAK39111), Lycopersicon esculentum CHS (P23419), Pinus sylvestris CHS
(P30079), Medicago sativa CHS2 (P30074), Daucus carota CHS9 (Q9SB26), Allium cepa CHS
(AAO63020), Petunia x hybrida CHSA (P08894) and CHSJ (P22928),
Ipomaea nil CHS
(O22046), Gerbera hybrida 2-PS (P48391), Ruta graveolens ACS (Q9FSC0), Rheum palmatum
ALS (AAS87170), Pinus radiata PrCHS1(AAB80804), Silene latifolia SlCHS (BAE80096),
Vitis vinifera STS (CAA54221), Nicotiana sylvestris CHSLK (CAA74847), Oryza sativa YY2
(BAA23618) and CHSS6 (BAC21541), Physcomitrella patens CHS-like (XP_001781520).
Additional
sequences
were
obtained
from
The
Institute
for
Genomic
Research
(http://www.tigr.org) Zea mays CHSS1 and CHSS2, Brassica rapa CHSS1 and CHSS2, and
Lycopersicon esculentum CHS1 and CHS2. Predicted proteins were aligned using ClustalW
(MegAlign, DNAStar, Lasergene, Madison, WI, USA). A phylogenetic tree was created using
the neighbor-joining method; bootstrap values were obtained (trials=1000, seed=111). Polyketide
synthase PKS18 from Mycobacterium tuberculosis (A70958) was used as an outgroup.
Microscopy
Dissection stereomicroscopy (50X-90X magnification) was used to assess anther/pollen
phenotypes. Anther images were captured using Zeiss SteReo Lumar V12 (80X magnification)
fitted with a Zeiss AxioCam MRc5 digital camera.
SEM was performed as described
(Nishikawa et al., 2005). For developmental analysis, inflorescences were fixed in 4%
paraformaldehyde, 0.5% glutaraldehyde, 0.05 M phosphate buffer, pH 7.2 for 2 h (RT), washed
in phosphate buffer (4 times, 15 min), dehydrated in ethanol (15%, 30%, 45%, 60%, 70%),
ethanol:n-butanol (50%:35%, 40%:55%:, 25%:75%, v/v), and 100% n-butanol, infiltrated with
paraffin, and cut into 5 μm sections. Slides were processed in two changes of xylene, gradually
rehydrated in an ethanol series followed by water, and stained with 0.1% toluidine blue.
Auramine O staining, GUS staining and acetolysis were performed as described (Dobritsa et al.,
2009).
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26
Metabolomics
Anthers from stage 9 and 10 flower buds (staged according to (Smyth et al., 1990)) were
collected on dry ice; three replicates composed of 60 anthers each were analyzed per genotype.
Samples were lyophilized, homogenized and extracted in 50 μl of 80% methanol with the
internal standard 2 µg umbelliferone for 2 h. Half the sample was used for ultra performance
liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer (UPLCQTOFMS), using an ACQUITY UPLC system (Waters Corp.) equipped with a binary solvent
delivery system and an autosampler. The mobile phases were 0.1% acetic acid in water (eluent
A) and 100% acetonitrile (eluent B). UPLC separations used a Waters 2.1 x 100 mm, BEH C18
column with 1.7 μm particles, a linear gradient of 95%:5% to 30%:70% eluents A:B, a flow rate
of 0.56 ml/min, column temperature of 60°C, and sample temperature of 4°C (autosampler).
Each wash cycle used 0.8 ml of 100% methanol and 2 ml of 0.1% acetic acid in water. A blank
(80% methanol) was injected between every five samples. Mass spectrometry was performed
using a Waters QTOFMS Premier operating in the negative electrospray ionization mode, with
the nebulization gas at 850 l/h (350°C), the cone gas at 50 l/h (120°C). QTOFMS data were
collected between 50 and 2000 m/z and were acquired using an independent reference lock-mass
ion via the LockSpray interface, with the LockSpray frequency set at 10 s in the centroid mode.
Raffinose (m/z 503.1612) was used as the reference compound and delivered at a concentration
of 50 fmol/mL and flow rate of 0.2 mL/h.
GC/MS was performed on the remaining portion of the anther extracts (three replicates,
~25 μl each). 500 µl of 80% MeOH containing an internal standard of 2.5 μg ribitol was added
and samples were incubated for 1 h (50°C), equilibrated to room temperature, and incubated for
1 h at 50°C following the addition of 500 μl of chloroform containing 1 μg of docosanol (an
internal standard). 400 μl of H20 was added and phases were separated by centrifugation at 2900
g (30 min, 4°C). Equal amounts of each phase were collected, transferred to 2 ml vials, and dried
at room temperature under nitrogen (organic phase) or in air (aqueous phase). Polar extracts were
methoximated in pyridine with 20 μl of 1.5% methoxyamine–HCl, briefly sonicated, suspended
at 50°C, and incubated with 20 μl commercial derivatization solution containing MSTFA+1%
TMCS (1 h, 50°C). Non-polar extracts were suspended in 0.5 ml chloroform, hydrolyzed with
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
27
0.5 ml 1.25M HCl in MeOH (4 h, 50°C), and then evaporated under nitrogen. Polar and nonpolar extracts were analyzed with an Agilent 6890 GC coupled to a 5973 MSD. Samples were
injected at a 1:1 split ratio, and the inlet and transfer lines were maintained at 280°C. Separation
was achieved with a temperature program of 80°C for 2 min, ramped at 5°C /min to 315°C and
held for 12 min on a 60 m DB-5MS column (J&W Scientific, 0.25 mm ID, 0.25 lm film
thickness) and a constant flow of 1 ml/min helium. Mass spectra were collected from 50 to 800
m/z. For each compound, a Student’s t-test was performed to assess statistical differences.
Protein Expression and Purification
LAP5, LAP6 and TT4 ORFs were PCR amplified from cDNA that was prepared from
RNA isolated from stage 9-10 buds (Dobritsa et al., 2009). The following primers were used:
lap5-start1
(CACCATGGGTAGCATCGACGCAGCAGTGTTGGGT)
(TCAGACATCAAGGTTTCGAGC),
lap5-stop1
lap6-start1
(CACCATGTCTAATTCTCGTATGAATGGTGTTGAG)
and
(TTAGGAAGAGGTGAGGCTGCGGATG),
(CACCATGGTGATGGCTGGTGCTTCTTC)
and
lap6-stop1
TT4-start1
and
TT4-stop1
(TTAGAGAGGAACGCTGTGCAAGAC). Start codons (ATG) are underlined in the start
primers; stop codons are underlined in the stop primers. Bold corresponds to the mutagenized
nucleotides that introduce silent mutations for improved expression in E. coli). The PCR
products were cloned into pET100-D/Topo vector from Invitrogen (Carlsbad, CA, USA), and
resulting constructs were transformed into E. coli BL21 Star (DE3). 500 mL cultures of
transformed E. coli were grown at 37°C in LB media containing 50 μg/mL kanamycin until
OD600=0.8. After induction with 0.3 mM isopropyl 1-thio-β-D-galactopyranoside, the cultures
were grown for 16 h at 16°C. Cells were pelleted, harvested and resuspended in extraction buffer
(50 mM NaH2PO4, pH 8.0, 500 mM NaCl, 10% glycerol, 10 mM β-mercaptoethanol, 10 mM
imidazole) supplemented with protease inhibitors. After brief sonication, cells were
homogenized in an EmulsiFlex-C3 high-pressure homogenizer (Avestin, Ottawa, Canada). The
homogenate was clarified at 30,000 and 50,000 x g for 20 min each, and incubated with Talon
Metal Affinity Resin (Clontech) for 1 h at 4°C and then loaded onto a disposable column. After a
50-mL extraction buffer wash, proteins were eluted with Talon elution buffer (extraction buffer
supplemented with 250 mM imidazole) and 1 mL fractions were collected. Protein purity was
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
28
analyzed on 12.5% SDS-PAGE. Protein-rich elution fractions (1-6) were pooled (fractions 1-6
were similarly pooled for vector-only control; fractions 1-4 from vector-only control contained
proteins detectable by Coumassie blue staining), dialyzed in 2 L storage buffer (20 mM HEPES,
1 mM EDTA, 200 mM KCl, 0.01% NaN3, 10% (v/v) glycerol, pH 7.4) overnight, concentrated
using Centriprep filters (Millipore) to approximately 1 mg/mL, and stored on ice. Similarly
processed extracts from vector-only transformed E. coli were used as negative controls.
Synthesis of 4-coumaroyl-CoA
4-coumaroyl-CoA was synthesized as previously described (Stoeckigt and Zenk, 1975). The Nhydroxysuccinimide ester of 4-coumaric acid was first synthesized from 4-coumaric acid (C9008, Sigma-Aldrich) and N-hydroxysuccinimide (130672, Sigma-Aldrich) in the presence of
N,N’-dicyclohexylcarbodiimide
(36650,
Sigma-Aldrich).
After
purification,
the
N-
hydroxysuccinimide ester of 4-coumaric acid was then converted into 4-coumaroyl-CoA through
thioester exchange between the N-hydroxysuccinimide ester and coenzyme A. The resulting 4coumaroyl CoA was purified on a Diethylaminoethyl (DEAE)-Sephacel (GE Healthcare) column
(1.6 x 20 cm) and lyophilized.
Protein Activity Assays
The activities of LAP5 and LAP6 were determined by monitoring the enzymatic reaction
products with HPLC-UV/Vis-ITMS (ion trap mass spectrometry).
The TT4 gene product,
TT4/CHS, was used as a positive control and the empty-vector extract was used as a negative
control. The enzymatic reactions were performed in a 100-µL reaction buffer (100 mM of
potassium phosphate buffer, pH 7.0) containing 15 µM of 4-coumaroyl-CoA, 45 µM of malonylCoA (M4263, Sigma-Aldrich), and 50 µg of the purified proteins. In the separate assays, 4coumaroyl-CoA was replaced with acetyl-CoA (A2181, Sigma-Aldrich) with other conditions
remaining unchanged. Reactions were allowed to continue for 4 hours at 37°C and quenched
with the addition of 20 µl of formic acid and extracted with 200 µL of ethyl acetate twice. The
mixtures were vortexed and briefly centrifuged to assist in phase partitioning. The ethyl acetate
phases were collected, combined and evaporated to dryness under nitrogen stream, redissolved in
methanol (35 µL), and subjected to HPLC-UV/Vis-ITMS analyses as previously described
(Farag et al., 2007). Briefly, 30 µL aliquots of the reaction products were injected onto an
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
29
Agilent 1100 series II HPLC system (Hewlett-Packard, Palo Alto, CA) which was coupled to a
Bruker Esquire ion-trap mass spectrometer. Separations were achieved using a reverse-phase
C18 column (5 µm, 4.6 mm I.D. x 250 mm, J.T.Baker, Phillipsburg, NJ) and a linear gradient of
5–70% B (v/v) over 60 min at a flow rate of 0.8 mL/min. The mobile phases consisted of solvent
A (0.1% acetic acid (v/v) in water) and solvent B (acetonitrile). UV spectra were obtained by
scanning from 200 to 600 nm and the mass spectra were acquired in negative electrospray
ionization mode with a mass scan range of 50-2200 m/z using an electrospray ionization voltage
of 4.0 kV. Tandem mass spectrometry (MS/MS) was performed with manual selection of
precursor ion and the fragmentation energy set at 1.5. To determine the activities of LAP5, LAP6
and TT4/CHS towards fatty acyl-CoA, 4-coumaroyl-CoA was replaced with 8 n-alkyl fatty acylCoA (butyryl-, hexanoyl-, octanoyl-, decanoyl-, lauroyl-, myristoyl-, palmitoyl-, stearoyl-CoA
(Sigma-Aldrich), with the reaction conditions, product processing and analyses remaining
unchanged.
The Arabidosis Genome Initiative locus numbers for the genes analyzed in this article are as
follows: LAP5 (At4g34850), LAP6 (At1g02050), At4g00040, CYP703A2 (At1g01280), TT4
(At5g13930).
Supplemental material
The following materials are available in the online version of this article.
Supplemental Figure S1. Biosynthesis of plant phenolic compounds.
Supplemental Figure S2. Mass spectra of the two products from the LAP6 enzymatic assay.
Supplemental Figure S3. In vitro formation of trans- and cis-bisnoryangonin.
Supplemental Figure S4. Condensation of fatty acyl-CoA with malonyl-CoA leads to the
formation of triketide pyrones. tetraketide pyrones and tetraketide resorcylic acid.
Supplemental Figure S5. HPLC-ITMS UV analyses of the products of the enzymatic reactions
with hexanoyl-CoA and malonyl-CoA as substrates.
Supplemental Figure S6. HPLC-ITMS UV analyses of the products of the enzymatic reactions
with decanoyl-CoA and malonyl-CoA as substrates.
Supplemental Figure S7. HPLC chromatograms of the enzymatic assays with multiple
substrates (coumaroyl-CoA, hexanoyl-CoA and malonyl-CoA).
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
30
Supplemental Figure S8. Defects of pollen and anther development in acos5 mutant strongly
resemble those in lap5-1 lap6-1 mutant.
Supplemental Table SI.
Supplemental Table SII.
Supplemental Table SIII.
Supplemental Table SIV.
Supplemental Table SV.
ACKNOWLEDGEMENTS
We are grateful to J.J. Lado and A. Shicoff for technical help, to B. Scott for advice and help
with protein purification, and to E. Bray, M. Jones-Rhoades, R. Swanson and A. Fiebig for
critical reading of the manuscript.
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
31
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Zhou H, Zhan J, Watanabe K, Xie X, Tang Y (2008) A polyketide microlactone synthase
from the filamentous fungus Gibberella zeae. Proc. Natl. Acad. Sci. 105: 6251-6254
Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR.
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Zuurbier KW, Leser J, Berger T, Hofte AJ, Schroder G, Verpoorte R, Schroder J (1998) 4Hydroxy-2-pyrone formation by chalcone and stilbene synthase with nonphysiological
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
39
FIGURE LEGENDS
Figure 1. lap5 and lap6 mutants have defects in anther and pollen exine morphology. (A-C)
Compared to the wild-type anthers (A), anthers of lap5-1 (B) and lap6-1 (C) mutants appear
glossy and do not easily shed pollen (arrowhead in A). (D-I) SEM of pollen grains and exine.
Wild-type grains (D,G) have a regular reticulate exine pattern, while lap5-1 (E,H) and lap6-1
(F,I) mutations cause pollen to collapse more easily and disrupt exine, changing the pattern or
resulting in a more extensively covered surface. (J-L) Similar to wild type (J), lap5-1 (K) and
lap6-1 (L) pollen does not demonstrate sensitivity to acetolysis. The lap6-1 grains, however,
exhibit decreased reactivity to acetolysis-dependent staining. Scale bars: 100 μm (A-C), 10 μm
(D-F and J-L), 5 μm (G-I).
Figure 2. The lap5 and lap6 defects map to At4g34850 and At1g02050, respectively. LAP5,
LAP6, and At4g00040 have gene structures similar to CHS/STS family members, including the
conserved position of an intron separating the first and second nucleotides in a cysteine codon
(TGC). Exons are shown as black rectangles. Positions of the lap5-1 and lap6-1 point mutations
and the lap6-2, lap6-3, and At1g00040 insertions are indicated with black triangles.
Figure 3. LAP5, LAP6 and At1g00040 belong to a male-specific clade of the chalcone/stilbene
synthase family. (A) Amino acid alignment of protein sequences from LAP5, LAP6, At1g00040,
CHS, STS and CHS/STS-like proteins from several plant species (two-thirds of the protein
length is shown, the remainder shows a similar trend). Residues identical to LAP5 are shaded
blue; those identical to the A. thaliana CHS TT4 are shaded yellow; those that match the
majority consensus from 17 sequences are orange. Amino acid numbers correspond to CHS2
from M. sativa. Proteins with demonstrated male-specific expression patterns are marked with
asterisks; the lap5-1 and lap6-1 lesions are indicated, and two of the four amino acids critical for
active center formation (C164 and F215) are boxed. M137, important for dimer formation, is
conserved in the CHS-like enzymes (boxed) but replaced with L/F in enzymes more similar to
LAP5. (B) A phylogenetic tree demonstrates a clear segregation of the LAP5/LAP6-like proteins
in a clade separate from the other members of the CHS superfamily. Red bar, chalcone
synthases; blue bar, sequences similar to LAP5; green bar, four CHS-like proteins with
demonstrated non-CHS activity (2-pyrone synthase (2PS), aloesone synthase (ALS), acridone
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
40
synthase (ACS), stilbene syntase (STS)). Numerical values indicate bootstrap support for each
node. Polyketide synthase PKS18 from M. tuberculosis was used as an outgroup.
Figure 4. LAP5 and LAP6 are expressed specifically in developing anthers. LAP5pr::GUS (AD) and LAP6pr::GUS (E-H) promoter fusion constructs are expressed in anthers of stage 9 and
10 buds (B,F), but not at earlier stages (A,E), or later stages (stage 11 (C,G), mature anthers
(D,H)). Scale bar, 100 μm. All images are to the same magnification.
Figure 5. A lap5-1 lap6-1 double mutant is sterile and lacks pollen. (A) Left to right: siliques
from wild-type, lap5-1, lap6-1, and a lap5-1 lap6-1 double mutant. (B) Mature anther from lap51 lap6-1 demonstrates complete absence of pollen grains (compare with Fig. 1A for a wild-type
anther).
Figure 6. Pollen produced by a lap5-1 lap6-1 double mutant lacks exine and degenerates. Anther
development in the wild-type Arabidopsis (A-D) and a lap5-1 lap6-1 double mutant (E-H).
Anther locules at stages 6 (A,E), 7 (B,F), 9 (C,G), and 11 (D,H) are shown. Exine is deposited
around developing pollen grains at stage 9 in the wild type (green halo around the pollen grains,
C), but not in lap5 lap6 (G). Insets in (C,G) are higher magnification views of boxed areas. Little
pollen is visible in lap5 lap6 by stage 11 (H). Scale bars: 20 μm (A-H); 10 μm, insets.
Figure 7. Levels of chalcone and naringenin are affected in the lap5 and lap6 mutants. Example
of UPLC-QTOFMS chromatograms obtained from lap5-1, lap6-1, the lap5-1 lap6-1 double
mutant and wild-type (WT) anther extracts.
Figure 8. HPLC-UV-MS chromatograms of the enzymatic assays with 4-coumaroyl-CoA and
malonyl-CoA as substrates. (A) Empty-vector control; (B) TT4/CHS. Inserts are UV and MS
spectra of the enzymatic reaction product (naringenin); (C) Naringenin standard. The inserts are
the UV and MS spectra; (D) LAP5. The inserts are the close-up view of peaks 1 and 2, and their
UV spectra; (E) LAP6. The inserts are the close-up view of peaks 1 and 2, and their UV spectra.
Figure 9. HPLC-ITMS UV analyses of the products of the enzymatic reactions with octanoylCoA and malonyl-CoA as substrates. (A-C) UV chromatograms (287 nm) of the products of
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
41
enzymatic reactions in the presence of (A) empty-vector control; (B) LAP5; (C) TT4/CHS; (D)
Mass spectrum of the major enzymatic product, 4-hydroxy-6-heptyl-2-pyrone, of the reaction
catalyzed by LAP5; (E) Mass spectrum of the major enzymatic product, 4-hydroxy-6-heptyl-2pyrone, of the reaction catalyzed by TT4.
Figure 10. Exine from the lap5-1 and cyp703a2 (lap4-1) double mutant has defects stronger than
either single mutant. (A-D) Confocal images of the exine surface from wild-type (A,E), lap5-1
(B,F), lap4-1 (C,G), and a lap4-1 lap5-1 double mutant (D,H) pollen grains stained with
auramine O. Arrow in (A) indicates an aperture. Arrow in (D) indicates a longitudinal area of
increased fluorescence. (E-F) Optical sections through the middle of pollen grains allow
visualization of exine thickness. Scale bar, 10 μm. All images are to the same magnification.
Supplemental Figure S1. Biosynthesis of plant phenolic compounds. Shown are reactions
catalyzed by phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4coumaroyl ligase (4CL), chalcone synthase (CHS), stilbene synthase (STS), chalcone isomerase
(CHI), flavonoid 3-hydroxylase (F3H), flavanone 3’-hydroxylase (F3’H), flavonol synthase (FS),
and dihydroflavonol reductase (DFR).
Supplemental Figure S2. Mass spectra of the two products observed in the LAP6 enzymatic
assay. Upper panel (A) is the spectrum of product 1 and the lower panel (B) is product 2.
Supplemental Figure S3. In vitro formation of trans- and cis-bisnoryangonin.
Supplemental Figure S4. Condensation of fatty acyl-CoA with malonyl-CoA leads to the
formation of triketide pyrones. tetraketide pyrones and tetraketide resorcylic acid.
Supplemental Figure S5. HPLC-ITMS UV analyses of the products of the enzymatic reactions
with hexanoyl-CoA and malonyl-CoA as substrates. (A-C) UV chromatograms (287 nm) of the
products of enzymatic reactions in the presence of (A) empty-vector control; (B) LAP6; (C)
TT4/CHS; (D) Mass spectrum of the major enzymatic product, 4-hydroxy-6-pentyl-2-pyrone, of
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
42
the reaction catalyzed by LAP6; (E) Mass spectrum of the major enzymatic product, 4-hydroxy6-pentyl-2-pyrone, of the reaction catalyzed by TT4.
Supplemental Figure S6. HPLC-ITMS UV analyses of the products of the enzymatic reactions
with decanoyl-CoA and malonyl-CoA as substrates. (A-C) UV chromatograms (287 nm) of the
products of enzymatic reactions in the presence of (A) empty-vector control; (B) LAP6; (C)
TT4/CHS; (D) Mass spectrum of the major enzymatic product, 4-hydroxy-6-nonyl-2-pyrone, of
the reaction catalyzed by LAP6; (E) Mass spectrum of the major enzymatic product, 4-hydroxy6-nonyl-2-pyrone, of the reaction catalyzed by TT4.
Supplemental Figure S7. HPLC chromatograms of the enzymatic assays with multiple
substrates (coumaroyl-CoA, hexanoyl-CoA and malonyl-CoA). (A) Empty-vector control. (B)
LAP5. Triketide pyrone from the condensation of hexanoyl-CoA and malonyl-CoA was the
major product. (C) TT4/CHS. Naringenin, bisnoryangonin, triketide pyrone, and tetraketide
resorcylic acid were identified. Triketide pyrone from the condensation of hexanoyl-CoA and
malonyl-CoA was the major product.
Supplemental Figure S8. Defects of pollen and anther development in the acos5 mutant
strongly resemble those in the lap5-1 lap6-1 double mutant (compare with Fig. 6). Mutation in
ACOS5 impairs exine deposition and leads to pollen degeneration and male sterility. Anther
development in wild-type (A-D) and acos5 mutant (E-H). Anther locules at stages 6 (A,E), 7
(B,F), 9 (C,G), and 11 (D,H) are shown. Exine (visible as green halo in C and D) is not deposited
around developing pollen grains in the acos5 mutant (G). Pollen undergoes degeneration in the
mutant, with almost none visible by stage 11 (H). Scale bar, 40 μm. All images are to the same
magnification.
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
lap5-1
WT
lap6-1
A
B
C
D
E
F
G
H
I
J
K
L
Figure 1. lap5 and lap6 mutants have defects in anther and pollen exine morphology. (AC) Compared to the wild-type anthers (A), anthers of lap5-1 (B) and lap6-1 (C) mutants
appear glossy and do not easily shed pollen (arrowhead in A). (D-I) SEM of pollen grains
and exine. Wild-type grains (D,G) h ave a regular reticulate exine pattern, while lap5-1
(E,H) and lap6-1 (F,I) mutations cause pollen to collapse more easily and disrupt exine,
changing the pattern or resulting in a more extensively covered surface. (J-L) Similar to
wild type (J), lap5-1 (K) and lap6-1 (L) pollen does not demonstrate sensitivity to
acetolysis. The lap6-1 grains, however, exhibit decreased reactivity to acetolysisdependent staining. Scale bars: 100 m (A-C), 10 m (D-F and J-L), 5 m (G-I).
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2010 American Society of Plant Biologists. All rights reserved.
T
lap5-1
GC
LAP5
lap6-2
T GC
lap6-1
lap6-3
LAP6
T GC
At4g00040
Figure 2. The lap5 and lap6 defects map to At4g34850 and At1g02050, respectively.
LAP5, LAP6, and At4g00040 have gene structures similar to CHS/STS family members,
including the conserved position of an intron separating the first and second nucleotides
in a cystein codon (TGC). Exons are shown as black rectangles. Positions of the lap5-1
and lap6-1 point mutations and the lap6-2, lap6-3, and At1g00040 insertions are
indicated with black triangles.
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
A
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S
N
N
K
N
L
L
L
L
L
L
L
L
L
L
L
L
L
L
M
V
M
M
M
I
M
F
I
F
M
M
I
I
V
I
A
T
A
A
R
A
T
A
A
A
A
K
A
A
C
C
C
C
C
C
C
C
C
C
T
C
C
C
C
G
I
T
T
T
T
M
T
I
T
V
Q
M
Q
V
A
E
A
A
A
A
A
E
E
E
A
E
E
A
E
A
E
E
E
E
E
A
E
E
E
E
E
E
E
E
Y
Y
Y
Y
Y
F
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
G
G
G
G
G
G
G
G
G
G
G
G
G
G
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
G
S
S
T
T
Q
S
G
T
L
L
T
Q
S
A
A
S
A
A
A
A
A
A
A
A
A
A
A
A
A
S
P
P
P
P
A
P
S
P
P
P
P
P
A
P
P
P
P
P
P
P
P
P
S
P
P
P
P
P
P
90
T
T
T
T
T
T
T
T
T
T
T
T
T
T
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
V
I
I
I
L
V
I
V
I
V
M
L
M
V
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
T
K
K
K
T
K
K
K
N
K
N
T
K
R
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
N
Q
Q
Q
Q
P
Q
Q
Q
Q
Q
Q
P
Q
Q
T
A
A
A
A
A
A
A
A
A
A
A
A
A
A
I
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
L
L
L
L
L
L
L
L
L
L
L
L
L
L
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
D
E
E
E
D
D
E
D
E
D
D
D
D
E
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
E
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
M
I
V
V
I
I
I
I
I
I
I
I
I
M
I
C
A
A
A
C
C
A
C
A
C
S
C
S
S
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
I
100
N D A
N E A
N D A
N P A
N A A
N D A
N E A
N D A
N E A
N A A
N A A
N A A
N K A
N V A
V V E
V V E
V V E
V V E
V T E
V T E
V V E
V V E
V V E
V N E
V V E
V V E
V V E
V V E
V V E
T A E
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
I
V
V
V
V
V
T
V
V
V
L
T
V
T
V
T
T
I
T
T
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
E
E
Q
E
E
E
E
E
E
E
Q
D
Q
D
K
R
K
K
K
K
K
K
K
K
K
K
K
K
R
K
M
M
M
M
L
M
M
M
M
M
M
L
M
M
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
A
A
A
A
G
A
A
A
A
A
A
G
A
A
G
G
G
G
G
A
G
G
G
G
G
G
G
G
G
G
V
L
Y
K
A
I
L
V
K
I
T
A
T
V
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
E
E
E
Q
T
D
E
E
E
E
E
A
E
D
E
E
E
A
A
E
E
E
E
E
S
E
E
E
E
E
110
A S R
A S L
A S L
A S Q
A A R
A S K
A S L
A S R
A S L
A S Q
A S L
A A R
A S L
A C R
A A V
A A V
A A V
A A Q
A A Q
A A V
A A V
A A Q
A A Q
A A L
A A Q
A A Q
A A Q
A A Q
A A A
A A L
A
G
V
A
A
A
G
S
A
S
S
A
A
D
K
K
K
K
E
R
K
S
K
R
A
K
K
K
K
K
C
C
C
C
A
C
C
C
C
C
C
A
C
C
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
I
I
I
I
L
I
I
I
I
I
V
L
V
L
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
L
K
K
K
K
G
S
K
K
K
K
R
H
R
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
N
E
E
E
E
D
E
N
E
K
S
E
S
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
R
R
R
R
G
R
G
R
Q
Q
Q
Q
Q
H
Q
Q
Q
R
R
Q
Q
Q
Q
Q
V
V
V
I
L
A
L
V
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
E
S
D
S
S
A
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
D
D
E
D
S
D
E
E
K
K
K
R
R
R
K
K
K
K
H
K
K
K
K
K
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
T
T
S
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
L
I
V
L
L
L
L
V
L
V
L
L
L
L
V
V
L
L
L
L
L
V
L
V
V
V
V
V
I
V
V
V
I
V
V
V
I
V
I
V
I
I
V
V
V
I
V
Y
Y
Y
Y
Y
Y
Y
Y
F
V
F
F
F
F
F
F
F
F
F
F
F
F
F
F
S
S
S
S
S
S
S
S
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
S
S
S
S
S
S
S
S
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
E
E
E
E
E
E
E
E
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
S
R
S
R
H
S
K
S
R
N
S
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
Q
R
E
P
N
N
N
P
P
S
P
S
P
P
P
P
S
P
P
P
P
P
P
P
P
P
P
P
S
P
P
P
D
D
E
D
N
E
D
E
D
E
D
N
D
D
S
Y
S
S
V
S
S
S
S
S
S
S
S
S
S
S
T
V
V
I
T
T
V
T
V
T
V
T
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
H
N
Q
G
V
N
N
H
A
K
R
V
R
S
K
K
K
N
N
N
K
K
K
K
N
K
K
K
K
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
V
V
V
V
T
V
V
V
V
V
V
T
V
V
L
Y
L
L
L
F
L
L
L
F
F
L
L
F
V
V
L
M
M
M
S
M
M
L
M
M
M
S
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
160
L Y F
L Y F
L Y F
L Y F
L L F
L Y F
L Y F
L Y F
L Y F
L Y F
L A F
L L F
L A F
L Y F
M Y Q
M Y Q
M Y Q
M Y Q
M Y Q
L Y Q
M Y Q
M Y Q
M Y Q
M Y Q
M Y Q
M Y Q
M Y Q
M Y Q
M Y Q
L Y H
V
L
L
L
L
S
L
V
L
A
T
L
T
L
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
V
V
L
V
A
V
V
V
V
V
V
A
V
V
G
G
S
G
G
G
G
G
G
G
G
G
G
G
G
G
170
A G L
T G L
S G L
T G L
A A L
A G F
T G L
A G L
T G L
A G L
A G L
A A L
A G L
T G L
T V L
T V L
T V L
T V L
T V L
T V L
T V L
T V L
T V I
T V L
T V L
T V L
T V L
T V I
T V L
T V L
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
V
V
V
V
T
V
V
V
V
V
V
T
V
V
I
L
L
V
V
L
L
V
L
L
M
L
L
L
V
T
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
D
D
D
D
D
D
D
D
D
D
G
D
G
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
I
I
I
I
I
I
I
I
I
I
L
I
L
L
L
L
L
L
V
L
L
L
L
L
L
L
L
L
L
L
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
180
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E S C
E N N
E S C
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
E N N
P
P
P
P
P
P
P
P
P
Q
P
P
P
P
R
K
R
R
R
A
K
K
K
K
R
K
K
K
R
A
G
G
G
G
G
G
G
G
G
E
G
G
G
G
G
G
G
G
G
G
G
G
G
N
G
G
G
G
G
G
S
S
S
S
S
S
S
S
S
A
A
S
A
S
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
R
R
R
R
R
R
R
R
R
E
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
V
V
V
V
V
V
V
V
V
X
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
M
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
V
L
L
L
L
L
L
V
L
L
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
190
A T S
T T S
T T S
T T S
V A A
A T S
T T S
A T S
T T S
A T S
A T S
T A A
A T S
A T S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
V C S
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
200
K P P
R P P
R P P
R P P
R P P
K P P
R P P
K P P
R P P
K P P
R P P
R P P
R P P
R P P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
R G P
S
N
K
N
S
N
N
S
N
S
S
S
S
N
S
S
S
S
S
S
T
N
S
N
S
N
N
S
S
S
V
K
K
K
P
P
K
K
N
G
P
Y
P
P
D
D
D
E
E
E
D
E
D
D
D
D
D
D
D
E
D
A
A
A
D
D
A
D
A
D
D
D
D
E
T
T
T
S
S
S
T
T
T
T
T
T
T
A
T
D
R
R
R
R
R
R
R
R
R
R
R
R
R
R
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
A
P
P
P
P
P
P
P
P
P
P
P
P
P
P
L
L
L
L
V
L
L
L
L
L
L
L
L
L
L
L
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
210
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
L V G
M V G
L V G
M V G
L V G
L V G
L V G
M V G
L V G
V
A
A
A
A
V
A
V
A
V
V
A
V
A
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
C164
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
S
Y
Y
Y
S
S
Y
S
Y
S
S
S
S
Y
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Y
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
30
L
V
V
L
V
L
V
L
L
L
L
V
L
L
H
C
H
C
C
A
C
C
C
C
V
C
C
C
A
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
I
V
I
I
I
I
I
I
I
I
I
I
I
I
I
I
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
I
I
V
I
V
I
I
I
I
I
I
V
I
I
A
A
A
A
A
A
A
V
A
V
A
A
A
A
A
V
I
L
L
L
L
I
L
I
L
I
V
L
V
L
V
V
V
V
V
V
V
V
V
I
V
V
V
V
V
V
G
G
G
G
G
G
G
G
G
G
G
G
G
G
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
40
Y
F
Y
Y
Y
Y
F
Y
Y
Y
F
Y
F
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
F
L
L
I
L
F
L
F
M
F
M
L
M
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
K
R
R
R
Q
R
R
K
R
K
K
Q
R
R
R
K
R
R
R
R
R
R
R
R
R
R
R
R
K
R
T
D
E
D
D
N
D
T
D
N
N
E
N
N
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
V
T
T
I
T
T
T
T
T
T
T
T
S
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
K
K
K
N
I
N
K
N
N
N
N
S
N
N
N
N
N
K
K
K
N
K
N
N
K
N
N
N
N
K
C
C
C
C
C
C
C
C
C
C
C
C
C
C
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
D
D
D
Q
D
D
D
D
K
D
D
D
D
Q
E
E
E
E
D
E
E
D
E
E
E
E
E
E
E
E
D
D
D
D
D
D
D
D
D
D
D
D
D
D
H
H
H
H
H
H
H
H
H
D
H
H
H
H
H
H
M
K
M
L
L
L
K
M
M
K
M
K
K
L
M
M
50
P
A
L
L
P
P
P
P
L
P
P
P
P
P
T
T
T
T
T
T
A
T
T
P
T
T
T
V
T
T
E
F
S
A
A
E
F
E
R
E
E
D
E
V
D
E
D
D
D
E
E
D
E
E
D
D
E
E
E
A
L
I
I
I
T
L
I
L
I
L
L
T
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
K
K
K
K
R
K
K
K
K
K
K
R
K
R
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
Q
E
D
E
A
Q
E
Q
E
Q
E
A
E
Q
E
E
E
E
E
E
E
Q
E
E
E
E
E
E
E
K
K
K
K
K
K
K
K
K
K
R
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
L
L
L
L
L
L
L
L
L
L
L
L
L
L
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
T
E
Q
E
E
T
E
T
E
T
T
E
T
E
K
Q
K
K
K
K
Q
Q
K
R
K
K
Q
K
R
N
R
H
H
R
R
R
H
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
L
L
L
L
L
L
L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
I
60
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
K
K
K
K
K
K
K
K
K
K
K
T
K
K
D
D
D
D
D
D
D
D
D
E
E
E
D
E
D
D
T
T
S
T
T
T
T
T
T
T
T
T
T
T
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
T
T
T
T
T
T
T
T
T
T
T
T
T
T
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
M
T
M
M
M
Q
M
M
M
M
M
M
M
A
M
V
V
V
V
V
V
V
V
V
V
V
V
V
V
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
K
K
K
K
R
K
K
K
K
K
R
R
K
K
R
K
R
R
R
K
K
T
N
N
E
K
K
K
K
K
T
T
T
T
T
T
T
T
T
T
T
T
T
T
K
R
K
K
K
K
K
K
K
T
K
K
K
K
K
K
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
H
Y
H
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
70
V
T
T
T
T
V
T
V
T
V
V
T
V
V
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
I
**
LAP5 At4g34850
LAP6 At1g02050
At4g00040
CHSLK-N.sylvestris
YY2-O.sativa
SlChs-S.latifolia
ChSS1-B.rapa
ChSS2-B.rapa
ChS1-L.esculentum
ChS2-L.esculentum
ChSS1-Z.mays
ChSS2-Z.mays
ChSS6-O.sativa
PrChS1-P.radiata
ChS TT4-A.thaliana
ChS2-M.sativa
ChS A2-B.napus
ChS C2-Z.mays
ChS Whp-Z.mays
ChS-B.finlaysoniana
ChS-H. androsaemum
ChS-I.purpurea
ChS-L.esculentum
ChS9-D.carota
ChSA-A.cepa
ChSA-P.hybrida
ChSJ-P.hybrida
ChSE-I.nil
ChS-P.sylvestris
StS-V.vinifera
* **
*
120
130
140
150
M137
lap6-1:T
R S I S D I T H V V Y V S S S E A R L P G G D L Y L A K G L G L LAP5 At4g34850
R P V E D I T H I V Y V S S S E I R L P G G D L Y L S A K L G L LAP6 At1g02050*
*
R A V E D I T H L V Y V S S S E F R L P G G D L Y L S A Q L G L At4g00040
R S A E E I T H I V Y V S S S E I R L P G G D L Y L A T E L G L CHSLK-N.sylvestris
R P A A D I T H L V Y I S S S E L R L P G G D L F L A T R L G L YY2-O.sativa
* **
R P I S D I T H L V Y V S S S E A R L P G G D L Y L A K G L G L SlChs-S.latifolia
lap5-1:E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
G
G
E
G
T
G
G
G
G
G
G
S
G
G
Y
F
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
F
T
S
S
P
A
P
A
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
V
V
V
V
V
I
V
V
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
I
A
I
A
A
L
A
I
V
V
V
V
V
I
V
V
V
V
G
V
V
V
V
V
R
R
R
R
R
R
R
R
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
E
L
L
L
L
F
L
F
L
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
F215
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
G
G
G
G
G
G
G
G
G
C
G
G
G
G
S
G
S
G
G
G
G
G
G
G
G
G
G
G
G
G
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
R
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
A
A
A
A
A
A
A
A
A
A
C
A
A
A
A
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
L
L
L
L
L
L
L
L
Y
Y
Y
Y
Y
Y
Y
Y
Y
F
Y
Y
Y
Y
Y
Y
Y
F
H
Y
H
H
H
Y
Q
Q
Q
Q
Q
Q
Q
Q
Q
R
Q
Q
Q
Q
Q
K
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
S
A
S
A
A
A
A
A
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
A
A
K
T
K
R
A
R
S
K
K
K
K
K
R
K
K
K
K
K
K
K
K
K
N
K
G
E
G
A
R
A
R
L
L
L
A
A
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
ChSS1-B.rapa
ChSS2-B.rapa
ChS1-L.esculentum
ChS2-L.esculentum
ChSS1-Z.mays
ChSS2-Z.mays
ChSS6-O.sativa
PrChS1-P.radiata
ChS TT4-A.thaliana
ChS2-M.sativa
ChS A2-B.napus
ChS C2-Z.mays
ChS Whp-Z.mays
ChS-B.finlaysoniana
ChS-H. androsaemum
ChS-I.purpurea
ChS-L.esculentum
ChS9-D.carota
ChSA-A.cepa
ChSA-P.hybrida
ChSJ-P.hybrida
ChSE-I.nil
ChS-P.sylvestris
StS-V.vinifera
220
A G A
A A A
A A A
A A A
A S A
A G A
A A A
A G A
A A A
A G A
A G A
A S A
A G A
A A A
A A A
A A A
A A A
A A A
A A A
A A A
A A A
A A A
A A A
A G A
A A A
A G A
A A A
A A A
A A A
S A A
M
V
L
V
A
M
V
M
V
M
A
V
A
M
L
L
L
V
G
I
I
I
M
V
L
I
I
L
L
V
I
I
I
I
I
I
I
I
I
N
V
I
V
V
I
I
I
V
R
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
V
L
V
V
V
V
G
V
I
V
I
V
V
I
I
I
V
V
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
S
A
A
T
A
S
A
S
T
S
T
A
A
T
S
S
S
A
A
S
S
S
S
S
S
S
S
S
A
S
D
D
D
E
G
D
D
D
E
N
D
G
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
D
R
T
I
I
N
L
D
I
I
A
M
T
R
D
D
-
P
E
E
M
A
P
P
I
R
A
P
T
V
T
D
D
D
I
T
L
V
D
I
L
D
V
D
LAP5 At4g34850
LAP6 At1g02050
At4g00040
CHSLK-N.sylvestris
YY2-O.sativa
SlChs-S.latifolia
ChSS1-B.rapa
ChSS2-B.rapa
ChS1-L.esculentum
ChS2-L.esculentum
ChSS1-Z.mays
ChSS2-Z.mays
ChSS6-O.sativa
PrChS1-P.radiata
ChS TT4-A.thaliana
ChS2-M.sativa
ChS A2-B.napus
ChS C2-Z.mays
ChS Whp-Z.mays
ChS-B.finlaysoniana
ChS-H. androsaemum
ChS-I.purpurea
ChS-L.esculentum
ChS9-D.carota
ChSA-A.cepa
ChSA-P.hybrida
ChSJ-P.hybrida
ChSE-I.nil
ChS-P.sylvestris
StS-V.vinifera
B
60.6
37.7
41.9
77.1
*
**
* **
*
66.5
49.0
NA
39.2
68.9
ChS-L.esculentum
ChSJ-P.hybrida
ChSE-I.nil
ChSA-P.hybrida
ChS-I.purpurea
ChS-H. androsaemum
ChS2-M.sativa
ChS-P.sylvestris
ChS TT4-A.thaliana
ChS A2-B.napus
ChSA-A.cepa
ChS C2-Z.mays
ChS Whp-Z.mays
ChS-B.finlaysoniana
2PS-G.hybrida
ALS-R. palmatum
ACS-R. graveolens
StS-V.vinifera
LAP5 At4g34850
ChSS2-B.rapa
SlChs-S.latifolia
ChS2-L.esculentum
ChSS1-Z.mays
ChSS6-O.sativa
PrChS1-P.radiata
LAP6 At1g02050
ChSS1-B.rapa
At4g00040
CHSLK-N.sylvestris
ChS1-L.esculentum
YY2-O.sativa
ChSS2-Z.mays
ChS-like-P.patens
PKS18 M.tuberculosis
100.0
46.3
59.0
100.0
70.1
57.9
79.5
54.7
97.3
67.0
67.8
62.1
99.9
31.0
100.0
94.5
100.0
59.1
97.7
100.0
100.0
34.6
68.6
114.3
100
80
60
Nucleotide Substitutions (x100)
Bootstrap Trials = 1000, seed = 111
40
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20
0
Figure 3. LAP5, LAP6 and At1g00040 belong to a male-specific clade of the
chalcone/stilbene synthase family. (A) Amino acid alignment of protein sequences from
LAP5, LAP6, At1g00040, CHS, S TS and CHS/STS-like proteins from several plant
species (two-thirds of the protein length is shown, the remainder shows a similar trend).
Residues identical to LAP5 are shaded blue; those identical to the A. thaliana CHS TT4
are shaded yellow; those that match the majority consensus from 17 sequences are
orange. Amino acid numbers correspond to CHS2 from M. sativa. Proteins with
demonstrated male-specific expression patterns are marked with asterisks; the lap5-1 and
lap6-1 lesions are indicated, and two of the four amino acids critical for active center
formation (C164 and F215) are b oxed. M137, important for dimer formation, is
conserved in the CHS-like enzymes (boxed) but replaced with L/F in enzymes more
similar to LAP5. (B) A phylogenetic tree demonstrates a clear segregation of the
LAP5/LAP6-like proteins in a clade separate from the other members of the CHS
superfamily. Red bar, chalcone synthases; blue bar, sequences similar to LAP5; green
bar, four CHS-like proteins with demonstrated non-CHS activity (2-pyrone synthase
(2PS), aloesone synthase (ALS), acridone synthase (ACS), stilbene syntase (STS)).
Numerical values indicate bootstrap support for each node. Polyketide synthase PKS18
from M. tuberculosis was used as an outgroup.
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LAP6pr::GUS
A
E
B
F
C
G
D
H
mature
stage 11
stage 9-10
young
LAP5pr::GUS
Figure 4. LAP5 and LAP6 are expressed specifically in developing anthers.
LAP5pr::GUS (A-D) and LAP6pr::GUS (E-H) promoter fusion constructs are expressed
in anthers of stage 9 and 10 b uds (B,F), but not at earlier stages (A,E), or later stages
(stage 11 (C,G), mature anthers (D,H)). Scale bar, 200 m. All images are to the same
magnification.
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A
B
Figure 5. A lap5-1 lap6-1 double mutant is sterile and lacks pollen. (A) Left to right:
siliques from wild-type, lap5-1, lap6-1, and a lap5-1 lap6-1 double mutant. (B) Mature
anther from lap5-1 lap6-1 demonstrates complete absence of pollen grains (compare with
Fig. 1A for a wild-type anther).
A
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WT
lap5-1 lap6-1
E
B
F
C
G
D
H
stage 11
stage 9
stage 7
stage 6
A
Figure 6. Pollen produced by a lap5-1 lap6-1 double mutant lacks exine and degenerates.
Anther development in the wild-type Arabidopsis (A-D) and a lap5-1 lap6-1 double
mutant (E-H). Anther locules at stages 6 (A,E), 7 (B,F), 9 (C,G), and 11 (D,H) are shown.
Exine is deposited around developing pollen grains at stage 9 in the wild type (green halo
around the pollen grains, C), but not in lap5 lap6 (G).
Insets in (C,G) are higher
magnification views of boxed areas. Little pollen is visible in lap5 lap6 by stage 11 (H).
Scale bars: 20 m (A-H); 10 m, insets.
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naringenin
chalcone
WT
lap5-1
lap6-1
lap5-1 lap6-1
Figure 7. Levels of chalcone and naringenin are affected in the lap5 and lap6 mutants.
Example of UPLC-QtofMS chromatograms obtained from lap5-1, lap6-1, the lap5-1
lap6-1 double mutant and wild-type (WT) anther extracts.
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A
D
mAU 287 nm
mAU 287 nm
UV
UV
nm
nm
12
12
min
min
B
E
UV
MS
mAU 287 nm
mAU 287 nm
UV
nm
UV
nm
nm
1 2
12
min
min
C
mAU 287 nm
UV
MS
nm
min
Figure 8. HPLC-UV-MS chromatograms of the enzymatic assays with 4-coumaroyl-CoA and
malonyl-CoA as substrates. (A) Empty-vector control; (B) TT4/CHS. Inserts are UV and MS
spectra of the enzymatic reaction product (naringenin); (C) Naringenin standard. The inserts are
the UV and MS spectra; (D) LAP5. The inserts are the close-up view of peaks 1 and 2, and their
UV spectra; (E) LAP6. The inserts are the close-up view of peaks 1 and 2, and their UV spectra.
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Empty-vector control
LAP5
TT4/CHS
Figure 9. HPLC-ITMS UV analyses of the products of the enzymatic reactions with octanoylCoA and malonyl-CoA as su bstrates. (A-C) UV chromatograms (287 nm) of the products of
enzymatic reactions in the presence of (A) empty-vector control; (B) LAP5; (C) TT4/CHS; (D)
Mass spectrum of the major enzymatic product, 4-hydroxy-6-heptyl-2-pyrone, of the reaction
catalyzed by LAP5; (E) Mass spectrum of the major enzymatic product, 4-hydroxy-6-heptyl-2pyrone, of the reaction catalyzed by TT4.
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WT
lap4-1
lap5-1
lap4-1 lap5-1
A
B
C
D
E
F
G
H
Figure 10. Exine from the lap5-1 and cyp703a2 (lap4-1) double mutant has defects
stronger than either single mutant. (A-D) Confocal images of the exine surface from
wild-type (A,E), lap5-1 (B,F), lap4-1 (C,G), and a lap4-1 lap5-1 double mutant (D,H)
pollen grains stained with auramine O. Arrow in (A) indicates an aperture. Arrow in (D)
indicates a longitudinal area of increased fluorescence. (E-F) Optical sections through the
middle of pollen grains allow visualization of exine thickness. Scale bar, 10 m. All
images are to the same magnification.
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