Sinapic Acid Ester Metabolism in Wild Type and a … · This is journal paper no. 15074 of the ... of this reaction is a 1-O-Glc ester that has a high free energy ... into methylene

Plant Physiol. (1 996) 11 2: 162.5-1 630

Sinapic Acid Ester Metabolism in Wild Type and a Sinapoylglucose-Accumulating Mutant of Arabidopsis’

Maike Lorenzen, Vicki Racicot, Dieter Strack, and Clint Chapple* lnstitut für Planzenbiochemie, Halle (Saale), Germany (M.L., D.S.); and Department of Biochemistry, Purdue

University, West Lafayette, Indiana 47907 (M.L., V.R., C.C.)

Sinapoylmalate is one of the major phenylpropanoid metabolites that is accumulated in the vegetative tissue of Arabidopsis tbaliana. A thin-layer chromatography-based mutant screen identified two allelic mutant lines that accumulated sinapoylglucose in their leaves in place of sinapoylmalate. 60th mutations were found to be recessive and segregated as single Mendelian genes. These mutants define a new locus called SNGI for sinapoylglucose accumulator. Piants that are homozygous for the sngl mütation accumulate normal levels of malate in their leaves but lack detectable levels of the final enzyme in sinapate ester biosynthesis, sinapoylg1ucose:malate sinapoyltransferase. A study of wild-type and sngl seedlings found that sinapic acid ester biosynthesis in Arabidopsis is developmentally regulated and that the accumulation of sinapate esters is delayed in sngl mutant seedlings.

The general phenylpropanoid pathway gives rise to a wide array of soluble metabolites in plants. In Arabidopsis these compounds include flavonoids and sinapic acid esters (Chapple et al., 1994). Flavonoid-deficient mutants have been identified based on the altered seed color of plants that are unable to make the flavonoid pigments that are found in the wild-type seed coat. Eleven of these tt (transparent testa) loci are known in Arabidopsis, and some of the genes that correspond to these loci have been cloned (Feinbaum and Ausubel, 1988; Shirley et al., 1992, 1995). In contrast, to date only one locus that affects sinapic acid ester biosynthesis has been identified. The fahl mutant is blocked at the step of the general phenylpropanoid pathway catalyzed by F5H (Chapple et al., 1992). Plants homozygous for thejahl mutation lack a11 sinapic acid esters. The identification of a T-DNA tagged allele of fahl has enabled the cloning of the gene encoding F5H, a Cyt P450- dependent monooxygenase (Meyer et al., 1996).

Sinapate esters play an important role in UV-B resistance in Arabidopsis (Landry et al., 1995); however, these metabolites are dispensable under laboratory conditions (Chapple et al., 1992). As a result, the sinapate ester biosynthetic pathway is an ideal candidate for genetic analysis. This

’ This research was supported by Division of Energy Bio- sciences, U.S. Department of Energy grant no. DE-FGOZ- 94ER20138, the Deutsche Forschungsgemeinschaft, and the Fonds der Chemischen Industrie. This is journal paper no. 15074 of the Purdue University Agricultura1 Experiment Station.

* Corresponding author; e-mail [email protected]; fax 1-317-496-1641 or 317-494-7897.

pathway has been examined in radish seedlings, in which sinapic acid is converted to sinapoylglucose by sinapic acid: UDPG sinapoyltransferase (Fig. 1; Strack, 1980). The product of this reaction is a 1-O-Glc ester that has a high free energy of hydrolysis (Mock and Strack, 1993), thus providing the necessary energy for the subsequent transacylation reaction. In this final reaction, sinapoylglucose acts as a sinapate donor in the reaction catalyzed by SMT (Strack, 1982), a vacuolar enzyme that generates sinapoylmalate (Sharma and Strack, 1985). The activity of these two enzymes estab- lishes the sinapic acid ester profile seen in mature leaves. In developing seeds, sinapoylglucose is transesterified by si- napoylg1ucose:choline sinapoyltransferase to form sinapoylcholine (Strack et al., 1983), which serves as a choline reserve in seeds. Following germination, hydrolysis of sinapoylcholine by sinapoylcholinesterase (Strack et al., 1980) provides choline for phospholipid biosynthesis (Strack, 1981) and sinapic acid for sinapoylmalate biosynthesis in the developing cotyledons (Strack, 1977). Despite the detailed biochemical understanding of this pathway, none of the genes in- volved has been cloned, and relatively little is known about how this pathway is regulated.

To examine phenylpropanoid metabolism in Arabidop- sis, we have isolated Arabidopsis mutants that are defective in the synthesis of sinapoylmalate. Sinapate esters display a bright blue fluorescence under UV light, allowing the facile identification of these mutants by TLC of crude methanolic leaf extracts. In this paper we describe the characterization of a new Arabidopsis mutant that is named sngl for siaapoylglucose accumulator. Plants homozygous for the sngl mutation fail to accumulate sinapoylmalate but instead accumulate its precursor, sinapoylglucose.

MATERIALS AND METHODS

A11 experiments were performed using Arabidopsis thali- una L. Heynh. ecotype Columbia cultivated at a light in- tensity of 100 pmol m-’s-* at 23°C under a photoperiod of 16 h of light/8 h of dark. Homozygous sngl-1 plants used in biochemical experiments had been backcrossed to wild type and reselected from segregating F, populations six times to remove unlinked background mutations. Segrega- tion tests were performed using the Landsberg erecta ecotype.

Abbreviations: F5H, ferulate-5-hydroxylase; MS, Murashige- Skoog; SMT, sinapoylg1ucose:malate sinapoyltransferase; sngl, _ _ sinapoylglucose - accumulator.

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1626 Lorenzen et al. Plant Physiol. Vol. 11 2, 1996

OCHl

PHENVLALANINE ------ PAL CIH C3H OHT FW OMT o 4;: SlNAPlC ACIO "C

ShOlUlB -ó HZO 4 y

OCHs OCH,

o<o-&:H OCH, S I N W O Y L W T E

O

Figure 1. Sinapate ester biosynthesis. The enzymatic steps required for the conversion of Phe to sinapic acid are Phe ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), p-coumarate-3-hydroxylase (C3H), caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT), and F5H. The enzymes that are un ique to sinapate ester biosynthesis are sinapic acid UDPC:sinapoyltransferase (SGT), SMT, sinapoylg1ucose:chol ine sinapoyltransferase (SCT), and sinapoylcholinesterase (SCE).

Plants for mutant screening and characterization were grown in soil-less potting mixture. For plant material to be used in the analysis of sinapate esters and SMT activity during early seedling development, seeds were surface- sterilized for 30 min in a 2:l mixture of 0.1% Triton X-100 and household bleach. Seeds were rinsed thoroughly with sterile water and plated on Miracloth (Calbiochem) discs on modified MS medium (ammonia-free medium in which an additional 20.6 mM potassium nitrate was added in place of ammonium nitrate) (Murashige and Skoog, 1962) containing 1% SUC and 0.7% agar.

Genetic Methods

For mutant screening, leaves of 7600 individual M, plants from ethyl methanesulfonate-mutagenized populations were extracted for 30 min in 50 pL of 50% methanol. Five microliters of the extract was analyzed by silica gel TLC using a mobile phase of n-butano1:ethanol:water (4: 1:1, v/v). Putative mutants were retested by the same procedure in the next generation, and those that showed a heritable change in sinapate ester accumulation were backcrossed to wild type to remove unlinked background mutations. Homozygous sngl progeny were reselected from segregating populations by TLC analysis.

lsolation and ldentification of Sinapoylglucose

The isolation of the fluorescent compound that accumulated in the sngl mutant was accomplished by fractionation of extracts by reverse-phase chromatography, followed by crystallization of the putative sinapoylglucose. Briefly, 30 g of sngl leaf material was extracted for 20 min in 500 mL of 50% methanol. The extract was filtered through Miracloth and concentrated in vamo to 10 mL. The residual aqueous

phase was extracted twice with 5 mL of chloroform, and the aqueous phase was again reduced in vacuo to 2 mL. The aqueous residue was applied to two Fisher C,, reverse- phase cartridges, which were then each eluted with 2 X 1 mL of water, and then 1 mL each of 5,10,15,20,30,50, and 100% methanol. Samples of each fraction were analyzed by silica gel TLC using n-butano1:ethanol:water (4:1:1, v /v) as a mobile phase. The putative sinapoylglucose eluted in the 10 to 20% methanol fractions, which were stored overnight at 4"C, during which time a white crystalline precipitate formed. These crystals were separated from the supernatant and recrystallized twice from methanol to give a final yield of 2.9 mg of colorless needles.

To identify the phenolic moiety of the crystallized compound, a sample of approximately 0.1 mg was subjected to base hydrolysis in 1 mL of 0.1 M NaOH at 37°C for 1 h. The mixture was then acidified with 3 mL of 1 M HC1 and extracted with 4 mL of diethylether. The ether phase was dried and redissolved in methanol for analysis by silica gel TLC using a mobile phase of to1uene:glacial acetic acid (2:1, v /v) saturated with water. The hydrolysis product was identified by comparison with authentic hydroxycinnamic acids (Sigma) visualized under UV light.

To determine whether the crystallized compound contained a sugar moiety, another 0.1-mg sample was hydrolyzed at 80°C for 3 h in 1 M H,SO,. The sample was then neutralized with 9 M NH,OH, reduced (1 mL of 2% sodium borohydride in DMSO, for 90 min at 40"C), acetylated (4 mL of acetic anhydride plus 250 pL of 1-methylimidazole as a catalyst, for 10 min at room temperature), extracted into methylene chloride, and analyzed (Reiter et al., 1993) using a gas chromatograph (model5890, Hewlett-Packard) equipped with a 30-m X 0.75-mm i.d. column (model SP-2330, Supelco, Bellefonte, PA) and flame ionization detection.

MS analysis of the crystallized compound was performed on the native compound or on its trimethylsilylated derivative following treatment with N,O-bis(trimethylsily1) trifluoroacetamide at 80°C for 10 min. Plasma desorption MS, chemical ionization MS, and electron impact MS were performed at the Purdue University Campus-wide MS Cen- ter (West Lafayette, IN).

Sinapic Acid Ester Analysis

Columbia wild-type or sngl plants that were grown in either potting medium or modified MS medium were har- vested, weighed, frozen in liquid nitrogen, and stored at -70°C until use. Sinapic acid esters were extracted into 50% methanol at a tissue:solvent ratio of 100 mg mL-' by briefly grinding and refreezing the tissue at -7OOC for 30 min in extraction solvent. Extracts were thawed and centrifuged briefly, and 20 pL of the supernatant was used for quantitation with HPLC.

HPLC

For HPLC analysis of plant extracts, sinapic acid esters and free phenolic acids were separated on a Microsorb-MV C,, column (Rainin, Woburn, MA), using a gradient from

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Sinapic Acid Ester Metabolism in Arabidopsis 1627

12% acetonitrile in 1.5% phosphoric acid to 48% acetonitrilein 1.5% phosphoric acid. Eluted compounds were detectedby their UV A320. Quantitation of sinapoylmalate in SMTenzyme assays was performed by HPLC on the same col-umn using an isocratic mobile phase of 20% acetonitrile in1.5% phosphoric acid. Compound identity was determinedby co-chromatography with authentic sinapate esters iso-lated from Arabidopsis (Chappie et al., 1992) or with au-thentic phenolic acids.

Enzyme Assays

Columbia wild-type or sngl tissue that was grown onmodified MS medium was extracted for 30 min in 5 vol-umes of 100 mM potassium phosphate buffer (pH 6.2)containing 20% (w/v) polyvinylpolypyrrolidone. The ex-tracts were centrifuged for 5 min at 10,000g and the super-natant was used for determination of SMT activity. Eachassay contained 25 /aL of 2 mM sinapoylglucose, 15 /xL of100 mM potassium phosphate buffer (pH 6.0), 5 ;uL of 1 Mmalic acid in potassium phosphate buffer (pH 6.0), and 5p,L of the protein extract. Assays were incubated for 60 minat 30°C, stopped by the addition of 50 fj.L of methanol, andstored at -70°C until analyzed by HPLC. Protein contentwas determined according to the method of Bradford(1976) using bovine y-globulin as a standard.

Malic Acid Determination

For the determination of malic acid content, tissue wasextracted for 30 min in distilled water (100 mg tissue mL"1)in a boiling water bath. The samples were ground, re-extracted for a further 10 min, and centrifuged. Fifty mi-croliters of the supernatant was transferred to a microfugetube containing 730 jjL of 100 mM 1-amino-isopropanolbuffer (pH 10.0), 100 fjL of 30 mM J3-NAD, 100 /xL of 0.5 ML-Glu, and 4 units of glutamate:oxaloacetate aminotrans-ferase. Reactions were initiated by the addition of 30 unitsof malate dehydrogenase. The increase of absorption wasmeasured spectrophotometrically at 365 nm, and the ratesobtained were compared with a standard curve generatedusing known concentrations of malic acid (Moellering,1974). All enzymes were purchased from Sigma or Boehr-inger Mannheim.

RESULTS

Isolation of the sngl Mutant

A mutant screen of 7600 plants by TLC analysis of meth-anolic leaf extracts identified two mutants that lacked si-napoylmalate but accumulated a novel blue-fluorescentcompound with a higher RF value in partition chromatog-raphy (Fig. 2A). In crosses to wild type, the mutant phe-notypes segregated as single, recessive nuclear mutations(Table I). These two lines failed to complement one an-other, indicating that they are alleles of a novel locus thatwe have named the SNG1 (sinapoylglucose accumulator).Quantitative HPLC analysis of wild-type and sngl-1 leafextracts confirmed that sngl-1 plants completely lackedsinapoylmalate but contained an equivalent amount of a

wild type

sngl-1

10 15 20 25

retention time (min)

Figure 2. Characterization of the sngl mutant by TLC analysis (A)and HPLC analysis (B) of methanolic extracts from 3-week-old leavesfrom wild type and the sngl mutant. SC, Sinapoylglucose; SM,sinapoylmalate; o, origin; sf, solvent front.

UV-absorbent compound with a shorter retention time(Fig. 2B). Both the TLC RF value and the HPLC retentiontime of the unknown compound that accumulated in thesngl mutants indicated that the compound was sinapoyl-glucose.

Identification of the Compound Accumulated in thesngl Mutant

The unidentified fluorescent compound that accumu-lated in the sngl-1 mutant was purified by reverse-phasecolumn chromatography, followed by crystallization frommethanol. Analysis of the purified compound gave an RFvalue on TLC and a retention time on HPLC that wereidentical to those of an authentic sample of sinapoylglucosethat was isolated from seeds of Raphanus sativus. Basehydrolysis of the compound, followed by TLC analysis ofthe hydrolysis products, yielded a single blue-fluorescentspot that co-chromatographed with authentic sinapic acid,indicating that the unknown compound was an ester ofsinapic acid. Acid hydrolysis, followed by reduction andacetylation of the products, indicated the presence of Glc instoichiometric amounts relative to the mass of the un-known compound that was hydrolyzed. Plasma desorptionMS of the unknown compound gave a prominent (M + H)+

parental ion at an m/z ratio of 387, the expected mass forsinapoylglucose. Chemical ionization MS of the compoundafter treatment with N,O-bis(trimethylsilyl)trifluoroaceta-

Table I. Segregation of sngl-1 homozygotes in an Fj populationderived from a sngl-1 X Landsberg erecta cross

Expected values represent those predicted for inheritance of arecessive allele of a Mendelian gene. x2 = 0.11, P > 0.70.

Mutation Sinapate Ester Accumulated Observed Expected

SNG7sngl /sngl

SinapoylmalateSinapoylglucose

11340

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mide gave prominent peaks at 746 and 747, corresponding to the expected Mt and (M+H)+ for the trimethylsilylated derivative of sinapoylglucose (Linscheid et al., 1980). Major ions were identified with m / z values of 674 and 602, corresponding to the loss of one or two trimethylsilyl groups from the parent compound. Electron impact MS of the trimethylsilylated compound gave major ions at 674, 368, 296, and 279 as expected, based on the characterization of sinapoylglucose from R. sutivus (Linscheid et al., 1980). Taken together, these data clearly indicate that the sngl mutants accumulate sinapoylglucose.

Comparison of Wild-Type and sngl Mutant Biochemical Profiles

An initial characterization of the sngl mutant was made using plants that were the same age as those used in the mutant screen. As shown in Table 11, 3-week-old wild-type plants contain sinapoylmalate as their major hydroxycinnamic acid ester and a small pool of its precursor, sinapoylglucose. In contrast, the sngl mutant fails to accumulate sinapoylmalate, and accumulates an equivalent amount of sinapoylglucose in its place. When the malate pools of both genotypes were examined, no significant difference was found, suggesting that the lack of sinapoylmalate in the sngl mutant was not due to the absence of one of the substrates for the reaction catalyzed by SMT. Finally, SMT activity was measured directly in crude extracts of wild- type and sngl mutant leaf tissue. Whereas SMT activity was readily detectable in wild-type leaf extracts, it was undetectable in extracts of mutant leaf tissue. These data suggest that the defect in the sngl mutant is in the gene encoding SMT or in a gene the product of which is required for SMT expression.

Developmentally Regulated Sinapate Ester lnterconversion

Radish seedlings exhibit a developmentally regulated interconversion of sinapic acid ester pools (Strack, 1977). To evaluate whether the same interconversions occur in Arabidopsis and to determine how the sngl mutation im- pacts on phenylpropanoid metabolism during early seedling development, the levels of sinapic acid esters (Fig. 3A), free malic acid (Fig. 3B), and SMT activity (Fig. 3C) in wild-type and sngl Arabidopsis were measured during the first 8 d of seedling development. These experiments were conducted on seedlings grown on modified MS medium that contained nitrate as its sole nitrogen source because ammonia has previously been shown to inhibit sinapoylmalate accumulation and decrease SMT activity in radish seedlings (Dahlbender and Strack, 1984; Strack et al., 1986).

The dry seeds of both wild type and the sngl mutant contain approximately 250 pmol of sinapoylcholine and 50 pmol of sinapoylglucose, indicating that the sngl mutation does not affect the accumulation of sinapate esters in seeds (Fig. 3A). In wild-type seeds, sinapoylcholine levels de- clined during the first 3 d after germination as the compound was hydrolyzed, presumably by an enzyme that is similar to the sinapoylcholinesterase previously described in radish (Strack et al., 1980). Two to 3 d after imbibition there was a transient increase in sinapoylglucose levels. This increase was followed by a decline in the sinapoylglucose content on d 4 through 8 and a dramatic increase in sinapoylmalate. The increase in sinapoylmalate was corre- lated with an increase in both seedling malate content (Fig. 3B) and SMT activity (Fig. 3C). This pattern of interconversions is very similar to the changes seen in radish cotyledons during germination (Strack 1977,1982). In addition to sinapate ester interconversions, de novo synthesis of sinapic acid apparently occurs between d 5 and 8, since the levels of sinapic acid esters in 8-d-old seedlings (approximately 1000 pmol seedling-l) are significantly higher than those seen in dry seeds (300 pmol seed-l).

In the sngl mutant, as in wild type, sinapoylcholine hydrolysis begins within 1 d of imbibition and is complete between d 3 and 4 (Fig. 3). The decline in the sinapoylcholine content is matched by an increase in the sinapoylglucose pool, but transesterification of sinapoylglucose to form sinapoylmalate does not occur in the mutant. The failure of the mutant to accumulate sinapoylmalate does not appear to be due to an alteration in malate availability, since the sngl mutant seedlings show a time course of malate accumulation that is identical to that seen in wild tYPe.

In contrast to wild type, the sinapoylglucose accumulated by the sngl seedlings remains at a near-constant level between d 2 and 6 and begins to increase toward the levels seen in mature plants (Table 11) only after d 7. It is interesting that the altered kinetics of sinapate ester accumulation in the sngl mutant can be visualized in vivo under UV light. Likefukl mutants (Chapple et al., 1992), sngl mutant seedlings exhibit a red-fluorescent phenotype that is due to the suppression of sinapate ester accumulation in young seedlings. In contrast to fuhl seedlings, this phenotype disappears in more mature plants once the level of sinapoylglucose increases.

DI SC U SSI O N

Compared with the number of studies that have focused on the biosynthesis, molecular biology, and function of flavonoids in Arabidopsis (Feinbaum and Ausubel, 1988;

Table 11. Biochemical characterization of 3-week-old wild-type and sngl- 1 plants SMT Activity Plant Sinapoylmalate Sinapoylglucose Total Sinapate Esters Malate

pmol mg- ' 2 SD pkat mg-' protein i SD

Columbia 644 -C 44 6 4 ? 13 708 2 54 2980 i 193 1 1 2 ? 1.0 sng 1 n.d." 670 2 86 670 2 86 2760 2 1695 n.d.

a n.d., Not detectable.



Sinapic Acid Ester Metabolism in Arabidopsis 1629

A - Figure 3. Developmental changes in sinapate ester content (A), malate content (B), and SMT activity (C) in seedlings of wild-type (filled symbols) and sngl (open symbols) Arabidopsis: O, O , sinapoylcholine; O, W , sinapoylglucose; A ,

tivity. Symbols and error bars represent mean values L SE.

wild type wild type

1200

1 O00

7- 800

a 0.10

0 0.05

0.00

20 m 5 600 - O] 'p 15

p 10 g 200

A, sinapoylmalate; V , 'I, malate; *, SMT ac- 400 - -

- + o J o + O 1 2 3 4 5 6 7 8 O 1 2 3 4 5 6 7 8

J J :: 3 1200 1000

; q-----J 35 800

'ii 600

400

E E 20

10

O O 200 5

O 1 2 3 4 5 6 7 8 O 1 2 3 4 5 6 7 8

seedling age (days)

Shirley et al., 1992, 1995; Li et al., 1993; Lois, 1994; Lois and Buchanan, 1994), the biosynthesis of sinapic acid esters has received a minimal amount of attention (Chapple et al., 1992; Mock et al., 1992). The identification and characterization of the sinapic acid ester-deficient f a h l mutant dem- onstrated the importance of sinapoylmalate in UV-B resistance (Landry et al., 1995) and led to the cloning of the gene encoding F5H (Meyer et al., 1996). Additional Arabidopsis mutants that are affected in sinapate ester biosynthesis will help to further elucidate the biosynthesis, regulation, and roles of these compounds. The sngl mutant identifies the second locus that is required for the accumulation of the wild-type profile of sinapic acid esters in Arabidopsis.

The nature of the defect in the sngl mutant can be inferred from severa1 pieces of evidence. First, the mutant accumulates sinapoylglucose, the substrate for the reaction catalyzed by SMT, in place of the SMT reaction product sinapoylmalate. Second, malic acid is accumulated to normal levels in the mutant, suggesting that the second substrate for the SMT reaction does not limit the progress of this reaction. Third, SMT activity is below detectable levels in the sngl mutant. These data indicate that the sngl locus encodes SMT or a regulatory protein that is required for SMT expression.

Although it is likely that the sngl mutation affects the structural gene encoding SMT, it has previously been re- ported that growth of radish seedlings on ammonia- containing medium resulted in a drastic inhibition of leaf malate accumulation and a decrease in extractable SMT activity (Dahlbender and Strack, 1984; Strack et al., 1986). These data support the possibility that the defect in the sngl mutant is in a malate- or ammonia-responsive SMT regulatory gene. Growth of Arabidopsis seedlings on ammonia-containing medium resulted in a 20-fold reduction in malate content, a 50% reduction in SMT activity, and a reduction in sinapoylmalate content (M. Lorenzen and C. Chapple, unpublished results). The magnitude of these responses, however, is inconsistent with the phenotype of the sngl mutant, and it is more likely that the defect lies within the structural gene for SMT. It is interesting to

note that protein extracts from R. sat ivus contain four SMT isoforms; the two major forms have pIs of 5.75 and 5.90. Extracts of Arabidopsis contain only one SMT with a pI of 5.15 (M. Lorenzen and D. Strack, unpublished results). The presence of a single SMT would be consistent with the hypothesis that SMT activity is eliminated in the sngl mutant of Arabidopsis by mutation of a single gene.

Germinating Arabidopsis seedlings exhibit a series of sinapate ester interconversions that are similar to those previously described in radish and Brassica rapa (Strack, 1977; Mock et al., 1992). These interconversions require a complex set of overlapping control mechanisms regulating the genes encoding sinapoylcholinesterase, sinapic acid UDPG:sinapoyltransferase, and SMT during the first 8 d of germination. Sinapate ester biosynthetic genes may also be regulated in concert with those encoding enzymes of the general phenylpropanoid pathway. The coordinate regulation of these genes may control the onset of de novo synthesis of sinapate esters approximately 6 d after germination (Fig. 3A), and a perturbation of this regulation may explain the slowed accumulation of sinapate esters in the sngl mutant. A more detailed understanding of this regulation will clearly require the cloning of the structural and regulatory genes that act in this pathway. The availability of the sngl mutant will permit the cloning of one of these genes, potentially that encoding SMT. SMT has previously been shown to be a vacuolar enzyme (Sharma and Strack, 1985). In concert with the sngl mutant, the cloning of the SMT gene will provide useful tools for investigating vacuolar protein targeting.

ACKNOWLEDCMENT

We would like to thank Dr. Klaus Herrmann for his careful review of this manuscript.

Received May 29, 1996; accepted September 9, 1996. Copyright Clearance Center: 0032-0889/96/ 112/1625/06.




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Sinapic Acid Ester Metabolism in Wild Type and a … · This is journal paper no. 15074 of the ... of this reaction is a 1-O-Glc ester that has a high free energy ... into methylene