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Plant Physiol, August 2001, Vol. 126, pp. 1493-1506
Characterization of Rice Anthranilate Synthase
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Anthranilate synthase (AS) is a key enzyme in the synthesis of
tryptophan (Trp), indole-3-acetic acid, and indole alkaloids. Two
genes, OASA1 and OASA2, encoding AS
-subunits were isolated from a monocotyledonous plant, rice
(Oryza sativa cv Nipponbare), and were characterized. A
phylogenetic tree of AS -subunits from various species revealed a
close evolutionary relationship among OASA1 and Arabidopsis ASA2,
Ruta graveolens AS2, and tobacco ASA2, whereas OASA2,
Arabidopsis ASA1, and R. graveolens AS1 were more
distantly related. OASA1 is expressed in all tissues tested, but the amount of its mRNA was greater in panicles than in
leaves and roots. The abundance of OASA2 transcripts is
similar among tissues and greater than that of OASA1
transcripts; furthermore, OASA2 expression was induced
by a chitin heptamer, a potent elicitor, suggesting that OASA2
participates in secondary metabolism. Expression of wild-type
OASA1 or OASA2 transgenes did not affect
the Trp content of rice calli or plants. However, transformed calli and plants expressing a mutated OASA1 gene,
OASA1(D323N), that encodes a protein in which
aspartate-323 is replaced with asparagine manifested up to 180- and
35-fold increases, respectively, in Trp accumulation. These transgenic
calli and plants were resistant to 300 µM 5-methyl-Trp, and AS activity of the calli showed a markedly reduced sensitivity to
Trp. These results show that OASA1 is important in the regulation of
free Trp concentration, and that mutation of OASA1 to
render the encoded protein insensitive to feedback inhibition results in accumulation of Trp at high levels. The OASA1(D323N)
transgene may prove useful for the generation of crops with an
increased Trp content.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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In higher plants, the Trp
biosynthetic pathway provides the essential amino acid Trp, as well as
produces various secondary metabolites, including the hormone
indole-3-acetic acid and plant defense compounds (Radwanski and Last,
1995
). However, the mechanisms by which these two functions are
regulated remain unknown.
Anthranilate is a common precursor of all these compounds and
anthranilate synthase (AS) catalyzes the synthesis of anthranilate from
chorismate and Gln. Purified plant AS proteins are heteromers that
consist of two subunits termed and 
(Poulsen et al., 1993; Bohlmann et al., 1995; Romero and Roberts, 1996). Although the synthesis of anthranilate from chorismate and Gln requires - and
-subunits of AS, the -subunit is able to catalyze the synthesis of anthranilate from chorismate with ammonia as an amino donor in the
presence of high concentrations of ammonium (Niyogi and Fink, 1992;
Niyogi et al., 1993; Bohlmann et al., 1995). Specific amino acids in
the -subunit of bacterial AS are important for feedback inhibition
of the enzyme by Trp (Matsui et al., 1987; Caligiuri and Bauerle,
1991).
Complementary DNAs encoding two nonallelic AS -subunit genes have
been isolated from Arabidopsis (ASA1 and ASA2)
and Ruta graveolens (AS1 and
AS2; Niyogi and Fink, 1992; Bohlmann et al., 1995).
Tobacco appears to possess more than two different AS -subunit
genes, but only one cDNA, termed ASA2, has been cloned (Song
et al., 1998). The enzymes encoded by R. graveolens AS1 (Bohlmann et al., 1996) and tobacco ASA2 (Song et al., 1998)
are Trp insensitive, with the former being implicated in alkaloid biosynthesis (Bohlmann et al., 1995, 1996). In a similar manner, the
enzyme encoded by one of the two AS genes of Pseudomonas
aeruginosa is feedback insensitive and is implicated in the
production of pynocyanin, the characteristic phenazine pigment of this
bacterium (Essar et al., 1990).
In addition to differences in feedback inhibition of the encoded
proteins, the two different AS -subunit genes appear to be subject
to distinct mechanisms of transcriptional regulation. Accumulation of
Arabidopsis ASA1 mRNA is induced by wounding and bacterial
infection, whereas ASA2 mRNA is present in small amounts and
does not respond to these stimuli. In a similar manner, the abundance
of R. graveolens AS1 transcripts increases by a factor of
approximately 100 in response to treatment with a fungal elicitor, whereas the number of AS2 transcripts is not affected and
the gene is expressed constitutively. The marked increases in the steady-state amounts of ASA1 and AS1 mRNAs
induced by wounding or pathogen-related stimuli suggest a role for
these genes in the production of defense-associated substances (Niyogi
and Fink, 1992; Bohlmann et al., 1996).
Although ASA1 is thought to participate in the synthesis of secondary
metabolites, ASA1 mutants of Arabidopsis exhibit increased concentrations of soluble Trp as a result of altered feedback regulation of AS activity (Kreps et al., 1996; Li and Last, 1996). The
tobacco ASA2 enzyme is thought to be related to R. graveolens AS1 because of its insensitivity to Trp.
Overexpression of tobacco ASA2 in the forage legume
Astragalus sinicus resulted in an increase of the
concentration of free Trp (Cho et al., 2000). These observations indicate that despite their predicted role in alkaloid synthesis, feedback-insensitive AS enzymes mediate Trp accumulation. It might, therefore, be expected that the AS enzymes not linked to alkaloid synthesis, such as those encoded by AS2 and Arabidopsis
ASA2, may possess an even greater potential for inducing Trp accumulation.
Given that cereal crops such as rice, maize, wheat, and oats exhibit a
relatively low content of Trp in seed storage proteins, an increase in
the abundance of this amino acid would improve their nutritional value.
We now describe the identification and characterization of two AS
-subunit genes (OASA1 and OASA2) from rice
(Oryza sativa cv Nipponbare), a monocotyledonous plant, and we demonstrate an important role for OASA1 in Trp synthesis.
We generated feedback-insensitive mutants of OASA1 whose expression resulted in the accumulation of free Trp at markedly increased concentrations in calli and plant leaves.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Isolation of Two AS -Subunit Genes from Rice
To isolate rice AS genes we screened a rice cDNA library with a probe based on Arabidopsis ASA1. Of 900,000 clones screened, 11 positive clones were isolated, sequenced, and categorized. The positive clones corresponded to two distinct cDNA sequences showing similarity to Arabidopsis ASA1 or ASA2. The clone with the largest insert, number 5-1, contained a complete open reading frame (ORF), which we designated OASA1 because it showed greater sequence similarity to Arabidopsis ASA1 than it did to ASA2 from this plant. The other cDNA represented in the positive clones was designated OASA2; however, the nucleotide sequence corresponding to the NH2-terminal region of the encoded protein was missing from the OASA2 clones.
To determine the nucleotide sequence of the missing 5' region of the
OASA2 ORF we partially sequenced a genomic DNA clone. Digestion of genomic DNA with EcoRI yielded fragments of
approximately 6.2 and 6.4 kb, which hybridized on Southern-blot
analysis with the Arabidopsis ASA1 probe used for cDNA
library screening. Size-fractionated EcoRI-generated DNA
fragments of approximately 6.0 to 6.5 kb were cloned into a 
ZAPII
vector; of 270,000 clones screened with the ASA1 cDNA probe,
23 positive clones were isolated and analyzed. Sequence analysis
revealed that a clone corresponding to the 6.4-kb hybridizing fragment
was derived from OASA1, whereas another clone corresponding
to the 6.2-kb hybridizing fragment appeared to be derived from
OASA2. Both clones contained the entire respective ORFs.
To clone the 5' portion of the OASA2 ORF we designed primers to amplify the region containing the putative AUG start codon by reverse transcription and PCR. The resulting cDNA fragments were cloned into the pCRII vector (Invitrogen, Carlsbad, CA) and their sequences were confirmed to be consistent with the genomic DNA sequence, with the exception of the spliced introns. This 5' region of the ORF was ligated to the cDNA fragment that lacked this region to generate the full-length OASA2 ORF.
Southern-blot analysis of EcoRI-digested rice genomic DNA
with the Arabidopsis ASA1 cDNA probe yielded no signals
other than the 6.2- and 6.4-kb fragments under low-stringency
hybridization conditions. We, therefore, concluded that the rice genome
possesses only two homologous AS -subunit genes, OASA1
and OASA2, which encode proteins of 577 and 606 amino acids, respectively.
The amino acid sequences deduced from the OASA1 and
OASA2 cDNAs were aligned with those of AS -subunits of
dicotyledonous plants and microorganisms (Fig.
1). The
NH2-terminal regions of OASA1 and OASA2 exhibited
almost no sequence similarity to each other. Likewise, the
NH2-terminal sequences of other plant homologs are highly variable and are predicted to contain plastid-localization signals (Zhao and Last, 1995). These regions of OASA1 and OASA2 are
relatively rich in specific amino acid residues, including Ser, Thr,
Lys, and Arg, and are relatively deficient in acidic amino acids. These
characteristics thus suggest that the NH2-termini of OASA1 and OASA2 also act as signal peptides for plastid localization (Gavel and von Heijne, 1990). The mature 521- and 523-residue OASA1 and
OASA2 proteins (assuming cleavage to be determined by an Asn residue at
positions 62 and 89, respectively) show extensive sequence homology to
each other (72% identity) and to other AS -subunits (71%-74%);
the sequence identity shared with Saccharomyces cerevisiae
TRP2 and Escherichia coli TrpE was only 23% and 33%, respectively. There was no significant difference in sequence identity
between AS -subunits of dicotyledons and those of the monocotyledon
rice. However, a phylogenetic tree based on deduced amino acid
sequences revealed a close evolutionary relationship among Arabidopsis
ASA2, R. graveolens AS2, tobacco
ASA2, and rice OASA1 (Fig.
2). In contrast, Arabidopsis
ASA1, R. graveolens AS1, and rice
OASA2 exhibited a level of evolutionary diversity similar to
that of their microbial homologs.
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The OASA2 cDNA was expressed by transformation of the
E. coli mutant strain B666 (
trpE), which is
deficient in the endogenous AS -subunit. The Trp requirement of B666
cells was reduced by transformation with the plasmid pTASA2F, which
contains the full-length OASA2 ORF. Complementation was
apparent under the restricted conditions, with a requirement for 50 µM
isopropyl--D-thiogalactopyranoside, 100 mM NH4Cl, and 1% (w/v)
casamino acids (data not shown).
Analysis of Gene Expression
We examined the expression of OASA1 and OASA2 in rice tissue with the use of RNA gel-blot analysis and digoxigenin-labeled antisense riboprobes prepared from the corresponding cDNAs; hybridization was performed under high-stringency conditions to prevent cross-hybridization of OASA1 and OASA2 mRNAs with the two probes. The sizes of the OASA1 and OASA2 transcripts, estimated from the sizes of ribosomal RNAs, were 2.1 and 2.4 kb, respectively. The two genes were shown to be expressed differentially in adult plants (Fig. 3A). OASA1 mRNA was detected in immature panicles, but was ambiguous in leaves and root, when total RNA was used for analysis (Fig. 3). We confirmed the expression of OASA1 in leaves and roots using enriched poly(A)+ RNA samples (data not shown). The result of Figure 3 using total RNA presented different expression of OASA1 in tissues. In contrast, the amount of OASA2 mRNA was similar in all tissues analyzed and was greater than that of OASA1 mRNA. Both genes were expressed at a relatively high level in cultured cells.
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The expression of Arabidopsis ASA1 and R. graveolens
AS1 is induced by an elicitor or by pathogen invasion (Niyogi
and Fink, 1992; Bohlmann et al., 1995). To test the inducibility of the two rice AS -subunit genes, we added
N-acetylchitohepalose (1 µg mL
1),
a potent elicitor (Yamada et al., 1993), to suspension cultures of rice
calli. The elicitor induced a time-dependent increase in the abundance
of OASA2 mRNA, but had no effect on that of OASA1 mRNA (Fig. 3B); the maximal effect of chitin heptamer was apparent after approximately 2 h.
Generation of Transgenic Rice
Two types of mutated OASA1 cDNAs were constructed by
PCR-mediated site-directed mutagenesis (Fig.
4). One mutated cDNA,
OASA1(D323N), was designed to encode a protein in which
Asp-323 was replaced by Asn. This Asp residue is well conserved among
plant AS -subunits, with the only exception of OASA2, and the same
mutation in ASA1 was previously shown to render Arabidopsis
resistant to 5-methyl-Trp (5MT; Kreps et al., 1996; Li and Last, 1996).
The second mutated OASA1 cDNA, designated
OASA1(M340T), was designed to encode a protein in which
Met-340 was replaced by Thr; the corresponding mutation has been
identified in a mutant of Salmonella typhimurium (Caligiuri
and Bauerle, 1991). Each mutated cDNA as well as wild-type OASA1 and OASA2 cDNAs were introduced
individually into the T-vector using the maize Ubi1 promoter
for high-level expression in rice (Fig. 4). Transformed calli and
regenerated plants were established by transfection with
Agrobacterium tumefaciens EHA101 harboring each of the four
cDNA constructs.
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Many independent transformants and transgenic plants were generated. Expression of the introduced cDNAs was examined by RNA gel-blot analysis (Fig. 5). Almost all calli and regenerated plants analyzed exhibited a high level of expression of the transgenes; one exception was number D5 in which the abundance of OASA1(D323N) mRNA was low, although hygromycin resistance was maintained. Because of high-level expression of the transgenes, film-exposing time of Figure 5 was shorter than that of Figure 3. In Figure 5, this time was not enough for capturing endogenous OASA1 and OASA2 signals.
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5MT Resistance of Transformed Calli and Progeny Seedlings
The various types of transformed calli were tested for their
sensitivity to 100 and 300 µM 5MT (Fig.
6). Those expressing the
OASA1(D323N) transgene exhibited marked resistance to 5MT. Calli that had been maintained on hygromycin-containing medium during
subculture exhibited growth on medium containing 300 µM 5MT that was 30% to 70% of that apparent
on control medium without 5MT or hygromycin (N6 medium [Chu et
al., 1975] supplemented with 2,4-dichlorophenoxyacetic acid [2,4-D]
and casamino acid [2N6]; data not shown). Calli that had been
maintained on medium without hygromycin showed the same amount of
growth on medium containing 300 µM 5MT as they
did on 2N6 medium (Fig. 6B). In contrast, untransformed calli (Fig. 6A)
as well as calli expressing OASA1 (Fig. 6C) or OASA1(M340T) (Fig. 6D) transgenes did not grow in the
presence of 100 or 300 µM 5MT. Of 61 transformed callus lines expressing the OASA2 transgene
examined, 31 lines grew on medium containing 100 µM 5MT (Fig. 6, E and F; Table
I); however, none of the calli was able
to grow in the presence of 300 µM 5MT. Thus,
only approximately 50% of the calli transformed with the
OASA2 transgene exhibited resistance to 5MT, and the level
of resistance shown was moderate. Given that RNA gel-blot analysis
showed that all OASA2-transformed calli examined expressed
the transgene at a similar level (Fig. 5B), the difference in
resistance did not reflect a difference in the level of transgene
expression.
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The inheritance of 5MT resistance in OASA1(D323N) transgenic rice was confirmed by a germination test. Growth of resistant seedlings in transgenic line number D1-1-5 was thus vigorous on medium containing 300 µM 5MT (Fig. 7). There was no difference in growth of seedlings on medium without 5MT between parental variety Nipponbare and line number D1-1-5. The progeny seeds of all tested transgenic plants showed segregation of the 5MT sensitivity trait. Calli derived from resistant progeny seeds also exhibited resistance to 5MT (data not shown). These observations indicated that only the OASA1(D323N) transgene conferred 5MT resistance to transformed calli and progeny seedlings.
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AS Activity of Transformants
The 5MT resistance of transgenic lines expressing OASA1(D323N) suggested that the D323N mutation affected feedback regulation of OASA1. To characterize the mutant enzyme we performed in vitro AS activity assays with calli expressing OASA1(D323N), OASA1, or OASA2 transgenes. Extracts were prepared from calli growing on 2N6 plates and were subjected to gel filtration chromatography to remove endogenous Trp, chorismic acid, Gln, and other low Mr molecules. The AS activity of untransformed calli and of transformed calli with a high level of expression of the OASA1 transgene was inhibited by 71% in the presence of 50 µM Trp, compared with the activity apparent in the absence of this amino acid (Fig. 8A). In contrast, the AS activity of calli expressing the OASA1(D323N) transgene was inhibited by only 26% in the presence of 50 µM Trp and by 47% in the presence of 100 µM Trp. The latter calli thus exhibited substantial Trp-insensitive AS activity. The AS-specific activity of these calli was, however, similar to that of untransformed cells or OASA1-expressing lines.
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Transformants expressing OASA2 showed two patterns of AS activity (Fig. 8B). One line, number 25-2, exhibited a level of AS activity substantially less than that of untransformed calli, whereas others (nos. 14-1, 46-2, and 49-1) showed markedly increased levels of AS activity in the presence of low concentrations of Trp. The AS activity of these latter transgenic lines was, however, inhibited by Trp in an apparently normal manner; activity in the presence of 50 µM Trp was only approximately 5% of that in its absence.
Trp Content of Transformants
The feedback-insensitive AS activity as well as 5MT
resistance of calli expressing the OASA1(D323N) transgene
might be expected to result in Trp accumulation. We, therefore, next
measured the soluble amino acids of calli after culture on 2N6 medium
for 2 weeks. The soluble Trp content of transformed calli expressing OASA1(D323N) was increased by a factor of up to 180 compared
with that of control calli (Fig. 9; Table
II). The abundance of the other 19 amino
acids or of anthranilate was not affected in the transformed
calli. A high Trp content was also detected in the leaves of progeny
seedlings from transgenic rice expressing OASA1(D323N) (Table III). Compared with untransformed
plants, the soluble Trp content of the transgenic rice was increased by
a factor of approximately 25 to 35, whereas transgenic plants
expressing OASA1 (W) did not increase the level of Trp. All
progeny seedlings analyzed were selected on medium containing 40 mg
L1 hygromycin and were grown under the same
condition. These results revealed that supplement of hygromycin in
medium did not cause increased level of soluble Trp in transgenic
plants expressing OASA1(D323N).
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The Trp content of 10 callus lines expressing the OASA2 transgene was not increased, even though three of these lines (nos. 24-2, 46-1, and 49-1) showed resistance to 100 µM 5MT (Table I). Thus, the moderate resistance to 5MT induced by expression of the OASA2 transgene did not result from altered feedback regulation of AS.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Two Rice Genes Encoding AS -Subunits
We have isolated two rice genes, OASA1 and
OASA2, that encode AS -subunits. The predicted proteins
contain putative plastid transit peptides at their
NH2-termini. Although we have not demonstrated that the two proteins are localized in plastids, fusion proteins comprising the respective putative transit peptides linked to green
fluorescent protein exhibited a plastid localization in transformed
calli (Y. Tosawa and K. Wakasa, unpublished data). The mature
rice AS -subunits shared marked (72%) sequence identity; however,
they occupied distinct positions in a phylogenetic tree. Whereas rice
OASA1, Arabidopsis and tobacco ASA2, and R. graveolens AS2 appeared to constitute a monophyletic group, rice OASA2, Arabidopsis ASA1, and R. graveolens AS1 were more
distantly related.
Expression and Predicted Function of OASA1 and OASA2
The abundance of OASA1 transcripts was greater in
immature panicles than in leaves and roots. This distribution differs
from that of Arabidopsis ASA2 mRNA, which is not detectable
in flowers (Niyogi and Fink, 1992). The amount of
OASA2 mRNA was similar in all tissues analyzed and was
greater than that of OASA1 mRNA. Furthermore, transcription
of OASA2, but not that of OASA1, appeared to be
induced by chitin heptamer. This N-acetylchitohepalose has been shown to possess elicitor activity; it induces phytoalexin production (Yamada et al., 1993) and jasmonate synthesis (Nojiri et
al., 1996) by rice cells in suspension culture. Maximal induction of
OASA2 expression was apparent after exposure of cells to the elicitor for 2 h, a time course similar to that for induction of
chitinase and Phe-ammonia lyase genes (He et al., 1998; Nishizawa et
al., 1999). These results thus suggest that OASA2 is a
defense-related gene induced by this elicitor.
Induction of gene expression by defense-related stimuli appears
to be a characteristic that differentiates between the members of each
pair of genes that encode AS -subunits in higher plants. On the
basis of this criterion, OASA1, Arabidopsis ASA2,
and R. graveolens AS2 belong to the same family of
constitutively expressed genes, and OASA2, Arabidopsis
ASA1, and R. graveolens AS1 belong to the same
family of inducible genes (Niyogi and Fink, 1992; Bohlmann et al.,
1995, 1996). This classification is thus consistent with the
phylogenetic relations of these genes.
Transformed Rice Expressing Mutated OASA1 Transgenes
The sequence element Leu-Leu-Glu-Ser (LLES)
participates in feedback inhibition of microbial AS -subunits by Trp
(Matsui et al., 1987; Caligiuri and Bauerle, 1991; Graf et al., 1993). This element is highly conserved in AS -subunits of plants, and the
sequence surrounding the LLES element has been implicated in feedback
inhibition of tobacco ASA2 (Song et al., 1998). However, substitution
of Ser-115 or Ser-129 of Arabidopsis ASA1, which correspond to Ser-65
or Ser-76 of the LLESX10S element in yeast, by
Arg did not result in an increase in the Trp content of or in
resistance to 5MT in transformed rice calli (Y. Tozawa, H. Hasegawa, T. Terakawa, and K. Wakasa, unpublished data). In contrast, transformed rice calli expressing the OASA1(D323N) transgene
were resistant to 300 µM 5MT and exhibited a
marked (up to 180-fold) increase in the concentration of free Trp. The
AS activity of these transformed calli was also inhibited by only
approximately 50% in the presence of 100 µM
Trp. We, therefore, concluded that Trp-insensitive AS activity was
responsible for the high level of Trp accumulation in these transgenic
lines. Inheritance of 5MT resistance was demonstrated by germination
tests on medium containing 300 µM 5MT, and the
leaves of resistant progeny also manifested a high Trp content.
Transformed calli expressing OASA1(M340T) or wild-type
OASA1 transgenes were not resistant to 100 µM 5MT and did not exhibit an altered Trp
content or AS sensitivity to Trp, suggesting that overproduction of the
AS -subunit itself was not responsible for the observed changes in
feedback regulation or Trp synthesis in OASA1(D323N)
transformants. The mutation of the Asp residue at position 323 of OASA1
to Asn thus appears to render the enzyme insensitive to Trp and 5MT.
Transformed Rice Expressing an OASA2 Transgene
Asp-323 of OASA1 is well conserved in other plant AS -subunits,
with the exception of OASA2. OASA2 contains an Asn at the corresponding
position (residue 351), although the surrounding residues are virtually
identical to those of OASA1. This observation suggested that
overexpression of OASA2 might result in Trp accumulation. However, transformed calli expressing an OASA2 transgene did
not exhibit Trp accumulation or altered sensitivity of AS activity to
Trp. Thus, the Arg at position 351 of OASA2 is not sufficient to render
the enzyme insensitive to feedback inhibition.
Only some of the transformed callus lines expressing the
OASA2 transgene were resistant to 100 µM 5MT and showed an increased level of AS
activity in the absence of Trp. These two properties appeared related
in that callus line numbers 14-1, 46-2, and 49-1 showed increased AS
activity and were resistant to 100 µM 5MT, whereas line 25-2 exhibited a reduced level of AS activity and was
sensitive to 5MT. The increased level of AS activity apparent in some
of these lines in the absence of Trp might result from the increase in
gene dosage for OASA2. The occasional and moderate resistance to 5MT manifested by calli overproducing OASA2 might also be
attributable to the increase in the number of AS -subunit molecules
compensating for the presence of inhibitory molecules of Trp or 5MT at
low concentrations.
Potential Utility of the OASA1(D323N) Transgene
Various feedback-insensitive AS -subunits cause Trp
accumulation in higher plants. The Arabidopsis mutants trp-5
and amt-1 manifest a free Trp content that is three and five
times, respectively, that of wild-type plants; this increase in Trp
accumulation is attributable to altered feedback regulation of the AS
-subunit encoded by ASA1 (Kreps et al., 1996; Li and
Last, 1996). Expression of the feedback-insensitive tobacco ASA2
protein in the forage legume A. sinicus resulted in a 1.3- to 5.5-fold increase in the amount of free Trp in roots (Cho et al.,
2000). Arabidopsis ASA1 and tobacco ASA2 are thought to contribute to
the synthesis of secondary metabolites, given that ASA1
transcription is inducible by wounding and the Trp insensitivity of
tobacco ASA2 is similar to that of R. graveolens AS1. In
contrast, expression of the Trp-insensitive AS -subunit encoded by
OASA1(D323N) induced up to an approximately 35-fold increase
in the amount of free Trp in rice leaves. The Trp content of calli and
leaves expressing OASA1(D323N) reached a maximum of 2,832 and 12,829 nmol per gram of fresh weight, respectively, whereas the
corresponding mean ± SE values for parental
variety Nipponbare were 34.8 ± 19.0 and 70.2 ± 49.5 nmol
per gram of fresh weight, respectively. Such high levels of Trp
accumulation support the notion that OASA1 is important in
Trp synthesis, and suggest that AS -subunits that contribute to Trp
synthesis may be more effective in inducing Trp accumulation than are
homologs whose primary function is the synthesis of secondary
metabolites. Analysis of transgenic plants expressing
feedback-insensitive forms of OASA2 is necessary to confirm this hypothesis.
Mutation of rice OASA1 appears to affect only Trp
accumulation, with no changes in the content of anthranilate or other
amino acids having been detected, even though anthranilate is the
immediate product of AS activity. The 5MT-resistant rice mutant MTR1
exhibits increased concentrations of not only Trp (87-fold increase in calli), but also Phe (8-fold increase in calli; Wakasa and Widholm, 1987). Chorismate mutase, which is subject to feedback control, is
stimulated by Trp (Gilchrist and Kosuge, 1980); however, transformed rice cells expressing OASA1(D323N) contained the same amount
of Phe as did control cells. Rice plants that manifest an increase only
in Trp content may prove beneficial for breeding.
Cultured rice cells and rice plants that accumulated Trp grew normally.
Although a tendency toward reduced seed fertility was apparent in the
OASA1(D323N) transgenic plants compared with control
Nipponbare plants, enough seeds to obtain progeny were produced by many
of the transformed plants. Given that nutritional improvement is an
important goal of gene manipulation in plants (Falco et al., 1995; Goto
et al., 1999; Ye et al., 2000), the OASA1(D323N) transgene
may prove useful for generating a crop with increased Trp content. The
free Trp content was also increased in mature seeds of many of the
OASA1(D323N) transgenic rice lines (Y. Tozawa, H. Hasegawa, T. Terakawa, and K. Wakasa, unpublished data).
The Trp biosynthetic pathway also provides indole compounds. For
example, anthranilate is a precursor for a class of phytoalexins referred to as avenanthramides in oats (Ishihara et al., 1999). The
biosynthetic pathway for 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and
2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which contribute
to defense against insects and microbial pathogens, branches off
the Trp pathway and utilizes indole as a direct intermediate in maize
(Frey et al., 1997; Melanson et al., 1997). The presence of
phytoalexins and defense compounds derived from the Trp pathway in the
Gramineae suggests the importance of studying coordinated regulation of
primary and secondary metabolism by the two AS -subunit genes in
monocotyledonous species. In addition to Trp, manipulation of other
aromatic compounds such as alkaloids and auxin might also prove to be
possible as a result of analyses of transgenic rice plants expressing
OASA1(D323N) or OASA2.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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Plants
Rice (Oryza sativa cv Nipponbare) seeds or plants
were used for callus induction and isolation of RNA or DNA. Seed calli
were induced from scutella by incubation for 3 weeks in the dark at 28°C on 2N6 medium supplemented with 2,4-D (2 mg L1),
casamino acids (1 g L1), and 0.2% (w/v) Gelrite
(Wako Chemicals, Osaka). Total RNA was isolated from calli
subcultured on 2N6 medium. For elicitor treatment, calli were
subcultured in R2 liquid medium (Ohira et al., 1973) as described
previously (Urushibara et al., 2001). Transformed and control calli
were subcultured at intervals of 3 weeks on 2N6 medium with or without
hygromycin (40 mg L1). All cultures with the exception of
primary seed cultures were maintained in a culture room at 28°C with
16 h of light per day, and regenerated rice plants were grown in a
culture room or greenhouse at 30°C under natural light conditions.
Isolation of cDNA
Standard recombinant DNA techniques were performed
basically as described (Sambrook et al., 1989). To prepare a probe for library screening we performed PCR with an Arabidopsis cDNA library and
the oligonucleotide primers ASA1-a (5'-CATATGTCTTCCTCTATGAAC-3') and ASA1-b (5'-GGATCCTCATTTTTTCACAAATGC-3'), which were based on
the Arabidopsis ASA1 genomic DNA sequence (GenBank accession no. M92353). The PCR product was purified with a Nick spin column
(Pharmacia Biotech, Piscataway, NJ) and was used to prepare a
fluorescein isothiocyanate-labeled probe with an enhanced
chemiluminescence random prime labeling system (Amersham,
Buckinghamshire, UK). A rice cv Nipponbare cDNA library described
previously (Tozawa et al., 1998) was screened with the labeled probe.
Purified phage DNA from isolated clones was digested with appropriate
restriction enzymes, and the resulting DNA fragments were subcloned
into the Bluescript phagemid vector (Stratagene, La Jolla, CA) and were sequenced with the use of fluorescein isothiocyanate-labeled primers, an AutoRead sequencing kit (Pharmacia Biotech), and an ALF II DNA
sequencer (Pharmacia Biotech). To recover the 5' region of the
OASA2 ORF, we performed reverse transcription-PCR with
primers based on genomic DNA sequence analysis. Analysis of amino acid sequences was performed with CLUSTAL W software (Thompson et al., 1994).
Isolation of Genomic DNA Clones
Genomic DNA was prepared from rice seedlings as described
previously (Tozawa et al., 1998). The DNA (15 µg) was digested with various restriction enzymes and was subjected to Southern-blot hybridization with the probe used for isolation of cDNA clones. Genomic
DNA digested with EcoRI was subjected to agarose gel
electrophoresis and fragments of approximately 6.0 to 6.5 kb were
recovered from excised gel slices with a Gene Clean Kit (BIO 101, Vista, CA). The DNA fragments were ligated into the
ZAPII/EcoRI/CIP cloning vector (Stratagene), and
the resulting genomic DNA library was screened with the probe used for
cDNA screening. The DNA fragments prepared from isolated clones were
digested with restriction enzymes and subcloned into a phagemid vector
for sequencing.
RNA Gel-Blot Hybridization
Total RNA was prepared as previously described (Tozawa et al.,
1998) from cv Nipponbare or transgenic rice plants. For sampling of RNA
from elicitor-treated cells, chitin heptamer,
N-acetylchitohepalose (1 µg mL1), was
added to rice calli in suspension culture (He et al., 1998). For the
detection of OASA1 and OASA2 transcripts,
digoxigenin-labeled antisense RNA probes were prepared with a DIG RNA
labeling kit (Boehringer Mannheim, Basel). A 710-bp fragment of
OASA1 cDNA (nucleotides 503-1,212) amplified by PCR
with primers 5MT-1 (5'-ACCGCTGCCTCGTCAGGGAGGACG-3') and RAS4
(5'-CTCAAAACGCTGGCTTAAGAC-3') was cloned into the pCRII vector
(Invitrogen). A 695-bp HindIII-EcoRV
fragment of OASA2 cDNA (nucleotides 898-1,592) was
subcloned into the pSPT18 vector (Boehringer Mannheim). The
corresponding antisense RNA probes were then synthesized by in vitro
run-off transcription with T7 RNA polymerase and digoxigenin-labeled
UTP. RNA gel-blot hybridization was performed as described previously
(Tozawa et al., 1998).
Complementation Analysis
Escherichia coli strain B666, which is deficient
in TrpE, was used for complementation tests. The
NcoI-BamHI fragment of
OASA2 cDNA clone OASA2 number 1, which lacks the 5'
region of the ORF, was cloned into the same sites of the pTrc99A vector
(Pharmacia Biotech), giving rise to pTASA2NB. For construction of an
expression vector for full-length OASA2, an
NcoI site was created by PCR with OASA2 number 1 as
template and the primers 2FNCO (5'-CGCCATGGAGTCCATCGCCG-3') and 5MT-4
(5'-GCTCCTGGGGATCTGCATAGGATC-3'). The PCR product was cloned into the
pCRII vector. Clones harboring the OASA2 cDNA fragment,
the incorporation of which results in the creation of an
ApaI site at the 5' side, were isolated and sequenced.
The ApaI-ApaI fragment of a selected
clone (the second ApaI site is located within the
OASA2 cDNA sequence) was excised and substituted for the
ApaI-ApaI fragment of pTASA2NB, yielding
the full-length OASA2 expression vector pTASA2F. This
vector was introduced into B666 cells by the standard transformation
method (Sambrook et al., 1989). The growth tests for each transformant
were performed on M9 plates.
Construction of Binary T-Vectors
T-vectors were constructed from pIG121-Hm (Ohta et al., 1990;
Hiei at al., 1994). A DNA fragment containing the sequence from the 5'
end of the nopaline synthase gene (nos) promoter to the 3'
end of the -glucuronidase gene intron was removed from pIG121-Hm and
was replaced with an artificial DNA fragment containing multiple cloning sites, yielding pYT8C-Hm (Urushibara et al., 2001). The maize
ubiquitin 1 gene (Ubi1) promoter (Toki et al., 1992;
Cornejo et al., 1993) and test gene fragments were inserted into this multicloning region to generate high-level expression vectors (Fig. 4).
PCR-mediated site-directed mutagenesis for construction of mutated
OASA1 cDNAs was performed with the primers OsAsN2
(sense, 5'-GTACATTTGCTAACCCCTTTGAGG-3') and OsAsC1 (antisense,
5'-CAAAGGGGTTAGCAAATGTACGC-3') for pUASA1D, and OsAsC3 (sense,
5'-CCTAGTCCTTATACGGCCTATCTACAG-3') and OsAsN3 (antisense,
5'-CTGTAGATAGGCCGTATAAGGACTAGG-3') for pUASA1M. The cauliflower
mosaic virus 35S-hygromycin phosphotransferase (HPT)-3'nos
(Ohta et al., 1990) was included as a selection marker in each vector
construct. The vectors were introduced into Agrobacterium tumefaciens EHA101 (Hood et al., 1986).
Transformation
Calli induced from seeds during a 3-week incubation were
transferred to 2N6, and, after 1 week, were harvested for
transformation, which was performed as described previously (Hiei et
al., 1994). Selection and plant regeneration were performed as
described (Urushibara et al., 2001).
Assay of 5MT Resistance
All transformed callus lines were tested for 5MT resistance on
2N6 solid medium containing 0, 100, or 300 µM 5MT.
Sensitivity to 5MT was assessed on the basis of the ratio of the growth
after 3 weeks of calli cultivated on medium containing 100 or 300 µM 5MT to that of calli cultivated in the absence of 5MT.
Prior to the test, calli were cultured on 2N6 plates without hygromycin or 5MT for 14 d, after which nine pieces of tissue (10-20 mg per piece) were transferred to each plate. The sensitivity of seedlings to
5MT was tested by allowing husked seeds to germinate on Murashige-Skoog medium (Murashige and Skoog, 1963) without hormones but containing 300 µM 5MT. Seedling growth was examined after incubation for 2 weeks.
Amino Acid Analysis
A third or fourth foliage leaf was harvested from each rice
plant grown in a greenhouse. Leaves of regenerated plants from transformed calli with the OASA2 gene were harvested
53 d after transferring to a pot. Leaves of parental variety
Nipponbare were harvested 58 d after germination. Leaves of
progenies of transgenic plants with the OASA1 (D323N)
transgene or the OASA1 transgene were harvested 55 d after transferring to a pot from selection medium containing 40 mg
L1 hygromycin for 2 weeks. Leaves of cv Nipponbare were
harvested 55 d after transferring to a pot from germination medium
without hygromycin for 2 weeks. Transformed calli and cv Nipponbare
callus were precultured for 2 weeks on 2N6 medium. Cell extracts were prepared from approximately 500 mg of fresh leaf or 1 g of callus, as previously described (Wakasa and Widholm, 1987), and free amino acids were analyzed with an amino acid analysis system (PICO
TAG, Waters, Milford, MA).
Assay of AS Activity
AS activity was assayed as described by Niyogi et al. (1993),
but with several modifications. Cell extracts were prepared from 1 to
2 g of fresh calli. The concentration of glycerol in the grinding
buffer was reduced from 60% to 30% (w/v). Ground frozen callus powder
was added to 2.5 mL of grinding buffer containing 75 mg of
polyvinylpyrrolidone (Sigma, St. Louis), and the mixture was then
subjected to vigorous vortex mixing to ensure complete suspension. The
suspension was then centrifuged (15,000g for 10 min at
0°C) to remove insoluble material. The resulting supernatant (2.5 mL)
was applied to a PD-10 column (Pharmacia Biotech) that had been
equilibrated with column buffer (Niyogi et al., 1993), and proteins
were eluted with 5 mL of column buffer. Gln-dependent AS activity was
assayed in a reaction volume of 1 mL, including 500 µL of column
eluate. The reaction was performed at 32°C for 0 and 30 min and was
terminated by the addition of 100 µL of 1 N HCl. The
anthranilate produced was then extracted into 4 mL of ethyl acetate and
was quantified with a spectrofluorometer (FP-777, JASCO, Tokyo;
excitation, 338 nm; emission, 440 nm). The protein concentration of the
column eluate was measured with a protein assay kit (Bio-Rad, Hercules,
CA) to calculate the specific activity of AS.
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ACKNOWLEDGMENTS |
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We thank Roy H. Doi for critical reading of the manuscript, Erin Yoshida and Makiko Kawagishi-Kobayashi for helpful discussions, and Satoko Miyauchi for technical assistance.
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FOOTNOTES |
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Received January 3, 2001; returned for revision March 7, 2001; accepted April 26, 2001.
1 This study was supported by the Ministry of Agriculture, Forestry, and Fisheries of Japan for the Development of Next Generation Recombinant DNA Techniques.
2 Present address: Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan.
3 Present address: National Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518, Japan.
* Corresponding author; e-mail kwakasa{at}affrc.go.jp; fax 81-298-38-8484.
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LITERATURE CITED |
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TOP
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
MATERIALS AND METHODS
LITERATURE CITED
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genes encode tryptophan-sensitive and tryptophan-insensitive isoenzymes specific to amino acid and alkaloid biosynthesis.
Plant Physiol
111: 507-514[Abstract]-methyltryptophan resistance in amt-1, a mutant of Arabidopsis thaliana with altered tryptophan metabolism.
Plant Physiol
110: 1159-1165[Abstract] subunit.
Plant Cell
5: 1011-1027-glucuronidase (GUS) reporter gene containing an intron within the coding sequence.
Plant Cell Physiol
31: 805-813
factor in rice and its tissue- and light-dependent expression.
Nucleic Acids Res
26: 415-419-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm.
Science
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|---|---|---|---|
-Subunit Genes
OASA1 and OASA2. Tryptophan Accumulation in
Transgenic Rice Expressing a Feedback-Insensitive Mutant of
OASA1
) as well as calli of
transformant number W2 expressing OASA1 (
), and
transformant numbers D17 (
) and D13 (
) expressing
OASA1(D323N) were assayed for AS activity in the presence of
various concentrations of Trp. B, Untransformed calli (
), 25-2 (
) were assayed for AS activity. Data are expressed
as picomoles of anthranilate produced per minute per milligram of
protein, and are means of triplicates from a representative
experiment.
