Regulation of Glutamate-l-semialdehyde Aminotransferase Expression during Tomato (Lycopersicon esculentum Mill.) Fruit Development

• JOUR.AL OF Ii

J Plant Physiol. Vol. 155. pp. 41-47 (1999)

Plan. Ph,s.olo.,

http://www.urbanfischer.de/journals/jpp

© 1999 URBAN & FISCHER

Regulation of Glutamate-1-semialdehyde Aminotransferase Expression during Tomato (Lycopersicon esculentum Mill.) Fruit Development* GARY

F.

POLKING**,

David J. Hannapel, and

RICHARD

J. GLADON

Department of Horticulture and Interdepartmental Plant Physiology Major, Iowa State University, Ames, IA 50011-1100 USA Received November 7, 1997 . Accepted July 1, 1998

Summary

A full-length clone that encodes tomato fruit tissue (Lycopersicon esculentum Mill. 'Rutgers') glutamateI-semialdehyde aminotransferase (GSAT; EC 5.4.3.8) was isolated and characterized. Amino acid sequence analysis showed that the tomato GSAT clone exhibited a high level of homology to the amino acid sequences of corresponding GSAT proteins from other plant species. The primary structure of GSAT consists of a 481-amino acid precursor that includes a 46.7 kilo unit (kU), 437-amino acid mature protein and a transit peptide of 44 amino acids. Southern analysis showed that a single copy of the GSAT gene was present in the tomato genome. Northern analysis showed that the abundance of GSAT transcripts declined throughout tomato fruit development and ripening. GSAT protein content decreased dramatically by 25 days postanthesis, and GSAT protein was undetectable by day 45, the approximate beginning of chlorophyll loss and carotenoid synthesis. These results show that GSAT is regulated developmentally at the level of transcript accumulation. In addition, posttranscriptional regulation may occur through decreased translation or increased degradation of GSAT protein.

Key words: Fruit ripening, gene expression, glutamate-l-semialdehyde aminomutase, GSAM, GSAT, Lycopersicon esculentum (Mill. 'Rutgers'). Abbreviations: ALA = 5-aminolevulinic acid; ALAD = 5-aminolevulinic acid dehydratase; GSA = glutamate-l-semialdehyde; GSAM = glutamate-l-semialdehyde aminomutase; GSAT = glutamate-l-semialdehyde aminotransferase; SSC = 0.15 mol· L-I NaCl, 15 mmol· L-I Na-citrate; SSPE = 0.15 mol· L-I NaCl, 0.25 mol· L-1 NaH 2P0 4 , 25 mmol. L-1 Na2EDTA. Introduction

The later stages of tomato fruit development and ripening are characterized by a decrease in chlorophyll content and an increase in lycopene synthesis. Chlorophyll and heme biosyn-

* Journal Paper No. J-16869 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project no. 3189. ** Present address: Iowa State University, DNA Sequencing & Synthesis Facility, 1184 Molecular Biology Building, Ames, IA 50011-3260.

thesis occur via a common pathway from the first committed precursor, 5-aminolevulinic acid (ALA), to metal-ion insertion at protoporphyrin IX (Beale and Weinstein, 1990). In plants, algae, and certain bacteria, ALA is formed via a 5-carbon pathway in which the conversion of glutamate to ALA occurs via a three-step process. This process involves the ligation of glutamate to tRNAGLU by glutamyl-tRNA synthetase, the reduction of Glu-tRNAGLU (with the concomitant release of tRNAGLU) to glutamate-l-semialdehyde (GSA) by gluta0176-1617/99/155/41 $ 12.00/0

42

GARY F. POLKING, DAVID J. HANNAPEL, and RICHARD J. GLADON

my1 tRNA reductase, and the transamination of GSA to form ALA by glutamate-1-semialdehyde aminotransferase [glutamate-1-semialdehyde aminotransferase (GSAT); EC 5.4.3.8] (Jahn et al., 1992; Beale, 1991). In contrast, animals, yeast, and other bacteria form ALA for heme synthesis via a onestep condensation of glycine with succinyl coenzyme A that is catalyzed by ALA synthase (Leeper, 1985). With the exception of Euglena gracilis, which utilizes the ALA synthase route for the production of extrachloroplastic tetrapyrroles, and the 5-carbon pathway for the biosynthesis of chloroplastic tetrapyrroles, all photosynthetic organisms examined to date use only the 5-carbon pathway for all tetrapyrrole syntheses (Richards, 1993). Glutamate-1-semialdehyde aminotransferase, the final enzyme in the ALA synthesis pathway, is nuclear-encoded and located in the chloroplast stroma of higher plants (Wang et al., 1981). The enzyme exists as a functional homodimer in Synechococcus (molecular mass, 46 kU; Hennig et al., 1997), barley (subunit mass, 46 kU; Grimm et aI., 1989), pea (subunit mass 45 kU; Nair et al., 1991), and E. coli (subunit mass, 40 kU; nag et al., 1991). GSAT catalyzes the transamination of GSA to ALA in the presence of a pyridoxal-5'-phosphate (PLP) or pyridoxamine-5'-phosphate cofactor (Jahn et al., 1992). In higher plants, cDNAs that encode GSAT have been isolated from barley (Grimm et al., 1989; Grimm, 1990), soybean (Sangwan and O'Brian, 1993), Arabidopsis (!lag et al., 1994; Wenzlau and Berry-Lowe, 1995), tobacco (Hofgen et al., 1994), and tomato fruit tissue (Polking et al., 1995). Regulation of chlorophyll synthesis is not well understood, but it may be regulated at the level of ALA synthesis and protochlorophyllide reduction. ALA synthesis is regulated both by feedback inhibition by heme and protochlorophyllide (Beale and Weinstein, 1990), and by light, via phytochrome (Huang et al., 1989). Activiry, protein levels, and mRNA levels of NADPH : protochlorophyllide reductase, which catalyzes the light-requiring reduction of protochlorophyllide to chlorophyllide, decrease upon exposure to light, although these changes vary considerably among species (Beale and Weinstein, 1990). In addition to these primary control points, under higher light intensiry 5-aminolevulinic acid dehydratase (ALAD) may act as a control point in limiting chlorophyll synthesis via its competitive inhibition by 4,5-dioxovalerate, an intermediate in the synthesis of ALA (Kotzabasis et al., 1989). It also has been shown that ALAD activity increases during chloroplast development that occurs after illumination of etiolated radish cotyledons (Shibata and Ochiai, 1976) and that this increase in activity, as well as the increase in ALAD protein synthesis, was phytochromemediated (Kasemir and Masoner, 1975; Tchuinmogne et al., 1989). Chlorophyll synthesis and turnover are important aspects of tomato fruit development and ripening. Current once-over mechanical harvest of tomatoes causes a 10 to 30 % loss of production because green (immature) tomatoes are not usable. Insight into the expression and regulation of the genes involved in pigment synthesis and degradation and other ripening changes may provide a basis for achieving premature initiation of ripening changes in immature fruits. As a step toward understanding the regulation of GSAT and the role that GSAT may play in the process of chlorophyll synthesis

and degradation during fruit ripening, we report the characterization of a GSAT cDNA that is expressed in tomato fruit tissue.

Materials and Methods Plant Material

Tomato (Lycopersicon esculentum Mill. cv. Rutgers) plants were grown in an environmentally controlled greenhouse (20·C night/ 24·C day). Supplementary irradiance from high-pressure sodium lamps (P. L. Light Systems Canada Inc., Grimsby, Ontario, Canada) was used to provide a minimum 12 h daylength with a minimum irradiance of -300 ~mol. S-I. m- z at the plant canopy. Plants were grown from seeds in a rockwool hydroponic system and transferred to 30-cm azalea pots four weeks after seeding. The sterilized soil mixture was 2 parts field topsoil: 4 parts peat: 4 parts coarse perlite. Plants were trained to a single stem and grown for one fruiting cycle as described by McAvoy et aI. (1989). Plants were watered daily and maintained with 1O.7moIN· m- 3 from Cal-Mag Peters Excel® fertilizer (15-2.2-12.45; The Scotts Co., Marietta, GA). Flowers were harvested at anthesis, fruits at 10, 15, 25, 35, 45, and 55 days postanthesis, and leaf, stem, and root samples six weeks after sowing. All samples were frozen in liquid nitrogen and stored at -80·C until used for RNA or protein extraction. Additional leaf samples were lyophilized, ground into powder, and stored at -20·C until used for genomic DNA extraction. Construction ofHomologous GSAT Probe

Template DNA for use in polymerase chain reactions (PCR) was obtained by carrying out a mass in vivo excision and phagemid rescue of a young tomato (cv. VFNT Cherry) fruit AZAP®n eDNA library (obtained from Dr. Wilhelm Gruissem, U.c., Berkeley) according to manufacturer's protocols (Stratagene Cloning Systems, La Jolla, CA). Two primers, designated GP551 [5' GCA AGC ITT ACA TTG ACT ATG TCG GAT CAT GGG 3'] and GP552 [5' CGC TCG AGA AGT GTC AAA TCT GGA GIT ATT CC 3'] were designed based on available sequence information of known GSAT clones from barley (Grimm, 1990), soybean (Sangwan and O'Brian 1993), Arabidopsis (Ilag et aI., 1994), and tobacco (Hofgen et aI., 1994), and were synthesized at the DNA Sequencing and Synthesis Facility at Iowa State University. PCR amplification was carried out in a final volume of 50 ~L that contained 300 np of template DNA, 40 pmol of each primer, 1.5 mmol . L- MgCl z, 200 ~mol. L-1 of each dNTp, 1 x taq polymerase buffer A (Promega Corp., Madison, WI), and 2.0 ~ of taq polymerase and was overlaid with 50 ~L of mineral oil. The initial PCR cycle (5 min at 94 ·C, 1.0 min at 65 ·C, and 1.5 min at 72 ·C) was followed by 30 cycles (45 sec at 94·C, 1.0 min at 65 ·C, and 1.5 min at 72 ·C), and a final cycle (45 sec at 94 ·C, 1.0 min at 65 ·C, and 10.0 min at 72 ·C). The identity of the 0.63-kb PCR fragment was verified by automated dideoxy sequencing (Sanger et aI., 1977) performed at the Iowa State University DNA Sequencing and Synthesis Facility and compared with known GSAT sequences in GenBank by using the GCG program (General Computer Group, Inc., Madison, WI). eDNA Library Screening

The 0.63-kb PCR fragment was 3zP-labeled by usi~ nick translation, and it was used as a probe to screen the AZAPRn cDNA library from young (3 to 8 day postanthesis) tomato (cv. VFNT Cherry) fruits. Filters were prehybridized and hybridized in 50 % formamide, 6 x SSC (1 x SSC: 0.15 mol· L-1 NaCl, 0.015 mol. L-1

GSAT Changes During Tomato Fruit Development Na-citrate), 3.3 x Denhardt's solution (1 X Denhardt's: 0.02 % Ficoll, 0.02 % polyvinylpyrrolidone, and 0.02 % bovine serum albumin), 25 mmol· L-1 Na-phosphate buffer, pH 7.0, 0.115 mg· mL -1 (mg/ mL) salmon sperm DNA, and 0.4 % (w/v) sodium dodecyl sulfate (SDS) at 42 'c. Filters were washed once for 5 min in 2 X SSC at 23 ·c, 0.1 % SDS, twice for 20 min in 2 X SSPE (1 X SSPE: 0.15 mol· L-1 NaCl, 0.25 mol· L-1 NaH 2P04 , 25 mmol· L-1 Na2EDTA), 0.1 % SDS at 65 'C, and once for 10 min in 2 X SSPE, 0.1 % SDS at 65·C. Filters were exposed to X-ray film with an intensifYing screen at -80'C for 72 h. Twenty-five positive clones were selected and their pBluescript® SK- phagemids were excised according to manufacturer's protocols (Stratagene Cloning Systems, La Jolla, CAl. The plasmid that contained the longest insert (1.7 kb) was submitted for automated dideoxy sequencing (Sanger et al., 1977) at the Iowa State University DNA Sequencing and Synthesis Facility on both strands, using T3, T7, poly(T), and synthetic oligonucleotide primers. DNA sequences were analyzed by using the GCG program (General Computer Group, Madison, WI). The nucleotide sequence for the tomato GSAT cDNA clone has been submitted to the GenBank Nucleotide Sequence Database under the accession number L39279 (Polking et al., 1995).

Southern Analysis Genomic DNA was extracted from 'Rutgers' tomato leaves according to the method of Rogers and Bendich (1985). Ten micrograms of DNA were digested with appropriate enzymes, separated by electrophoresis on a 1.0 % agarose gel, denatured, and transferred to a nylon membrane with 25 mmol· L-1 Na-phosphate, pH 7.0 buffer. Membranes were prehybridized and hybridized at 42 ·C in either 30 or 50 % formamide, 6.0 X SSC, 3.3 X Denhardt's solution, 25 mmol . L-1 Na-phosphate buffer, pH 7.0, 0.4 % SDS, and 0.115 mg . mL -1 (mg/mL) salmon sperm DNA. The 32P-Iabeled 0.63 kb GSAT PCR fragment was used as a probe. Membranes were washed once for 5 min in 2 X SSPE, 23'C, and either twice for 15 min in 1.0 X SSC, 0.1 % SDS at 23'C (30 % formamide filters) or twice for 15 min in 0.1 X SSC, 0.1 % SDS at 65'C (50 % formamide filters), then exposed to film for autoradiography.

Northern Analysis Total RNA was extracted from the appropriate tissue (Dix and Rawson, 1983), separated on a 1.4 % agarose gel that contained 5 mmol· L-1 methyl-mercury hydroxide (Thomas, 1980) and transferred onto a nylon membrane with 25 mmol . L-1 Na-phosphate buffer. Before blotting, gels were stained with ethidium bromide and ribosomal RNAs were visualized under UV light to confirm that equal amounts of RNA were loaded in all lanes. Membranes were prehybridized and hybridized at 65'C in 50 % formamide, 1.0 mol· L-1 NaCl, 10 % (w/v) dextran sulfate, 1.0 % SDS, and 0.1 mg· mL- 1 (mg/mL) salmon sperm DNA. The plasmid that contained the 1.7-kb GSAT cDNA insert was linearized with SmaI and used as a template to generate an antisense RNA probe using the Riboprobe® System (Promega Corp., Madison, WI) with T7 RNA polymerase. Membranes were washed twice for 5 min in 2 X SSC, 0.1 % SDS at 23 'C, twice for 15 min in 0.1 x SSC, 0.1 % SDS at 65 ·c, then exposed to film for autoradiography. For the steady-state level analysis of mRNA from tomato organs and developing fruits, experiments were replicated and three northern blots were performed. Blots presented in the results and discussion section are representative examples of those replicates.

Immunoblot Analysis Protein was isolated by grinding plant tissue in extraction buffer (25 mmol· L-1 NaH 2P0 4 , pH 7.0, 0.1 % diethyldithiocarbamate,

43

and 0.1 % NaHS0 3) and polyvinylpolypyrrolidone (100 mg/1.0 g sample). The extract was centrifuged at 20,000 Kn for 20 min, then lyophilized. The lyophilized sample was resuspended in H 20, and the protein concentration was determined by the Bradford method (1976). SDS extraction buffer (25 mmol . L-1 tris-HCl, pH 6.8, 1.6 % SDS, and 2.6 % ~-mercaproethanol) was added to a volume of sample that contained 75 Ilg of protein to achieve a final volume of 50 ilL (approximately 50 % SDS extraction buffer v/v). The mixture was boiled for 1.5 min, and then 0.1 x volume of loading buffer (0.05 % bromophenol blue, 25 mmol· L-1 tris-HCl, pH 8.5, and 50 % glycerol) was added. Seventy-five micrograms of protein per sample were loaded per lane and subjected to SDS-PAGE (12.50/0 acrylamide). For immunodetection, proteins were transferred to a nitrocellulose membrane as described by Towbin et al. (1979). Immunoblotting and staining were carried out as described by Hannapel (1990) by using a 1: 1,500 dilution of an antibody to barley GSAT (Grimm et al., 1989) isolated from rabbit. The second antibody, at a 1: 3,000 dilution, was goat anti-rabbit IgG-horseradish peroxidase conjugate (Biorad Laboratories, Inc., Richmond, CAl. The immunoblot presented in the results and discussion section is a representative example of one of the three replications.

Results and Discussion

The nucleotide sequence of the tomato GSAT clone consists of 1,699 bp, including 24 bp of 5' -untranslated sequence, / 1,443 bp of coding region, and 232 bp of 3 -untranslated sequence, including a 35-bp poly(A) tail. A single open reading frame encodes 481 amino acids (Fig. 1). A putative cleavage site, I-R-M-.lJ.-T (Fig. 1), for the tomato chloroplast transit sequence was identified by comparison with the experimentally determined cleavage site of barley (Grimm et al., 1989; Grimm, 1990), the homologous areas of tobacco (Hofgen et al., 1994) and soybean (Sangwan and O'Brian, 1993), and the conserved features of other chloroplast transit peptide cleavage sites (Gavel and Von Heijne, 1990; Von Heijne et al., 1989). Cleavage at this position would produce a transit peptide of 44 amino acids, and a mature protein of 437 amino acids with a molecular mass of 46,669 units. The calculated mass is in accordance with those of barley and Synechococcus (46 kU, Grimm et aI., 1989), and pea (45 kU, Nair et al., 1991), but it is somewhat greater than those of Chlamydomonas reinhardtii (43 kU, Jahn et al., 1991) and E. coli (40kU, Ilag et aI., 1991). The putative transit peptide has features common to known chloroplast transit peptides, including high serine and threonine content, a relatively low abundance of acidic amino acids (Gavel and Von Heijne, 1990), and an overall positive charge (Schmidt and Mishkind, 1986). The tomato transit peptide was 70 % homologous to that of tobacco, and it showed lower homology to those of soybean (30 %) and barley (36 %). In contrast, the mature peptide of tomato shows a high degree of homology to that of tobacco (95 %, Hofgen et al., 1994), soybean (82 %, Sangwan and O'Brian, 1993), and barley (82 %, Grimm, 1990). All sequences showed high homology to each other in the region believed to be involved in binding the pyridoxal phosphate cofactor (Fig. 1, positions 319-326; Grimm et al., 1991). In addition, all species contained the lysine residue (Fig. 1, position 322) that has been identified in a number of aminotransferases as the binding site for pyridoxal 5'-phosphate

44 tomato tobacco soybean barley E. coli tomato tobacco soybean barley E. coli tomato tobacco soybean barley E. coli tomato tobacco soybean barley E. coli tomato tobacco soybean barley E. coli tomato tobacco soybean barley E. coli

GARY F. POLKING, DAVID]. HANNAPEL, and RICHARD]. GLADON

•

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Fig. 1: Comparison of the deduced amino acid sequence of tomato GSAT with those of tobacco, soybean, barley, and E. coli. Identical amino acids are indicted by asterisks (*). Gaps, indicated by periods (.), were used to optimize the alignment. The arrow indicates the approximate cleavage point of the transit sequence. The cofactor binding site is indicated by [- -p- -J. The lysine residue in the cofactor binding site (described in the text) is indicated in bold at position 322.

(PLP) (Grimm et aI., 1991). Proof of the involvement of this lysine residue in binding PLP was obtained by using sitedirected mutagenesis (Hag and Jahn, 1992). Southern analysis was performed by hybridizing the 0.63 kb GSAT PCR fragment to total genomic DNA from tomato leaves (Fig. 2). The hybridization pattern was similar under both low and high stringency conditions (data not shown), and this suggested that the GSAT gene was present as a single copy in the tomato genome. This is in contrast to the two distinct eDNA clones of GSAT in tobacco (Hafgen et al., 1994), soybean (Frustaci et al., 1995), and Arabidopsis (Ilag et al., 1994; Wenzlau and Lowe-Berry, 1995). The presence of two clones in tobacco may be attributable to the amphidiploid composition of tobacco (Hafgen et al., 1994). However, for Arabidopsis, two clones of GSAT exist, but they may have problems hybridizing with the probes from one another. Considering the low stringency conditions used for the Southern analysis, the high homology of the two tobacco nucleotide sequences to each other (96.4 %), and the high homology of these clones to the tomato GSAT clone (88.2 %

and 89.1 %), it seems unlikely that a second copy of the GSAT gene exists in tomato. Differential expression of GSAT mRNA was examined by northern blot analysis. Transcripts accumulated to the greatest level in leaves and stems (Fig. 3 A). Transcript accumulation was less in flowers and lowest in roots (Fig. 3A). Similar variation in the level of GSAT mRNA expression in leaves and roots was observed by Sangwan and O'Brian (1993). In Arabidopsis, however, accumulation of GSAT transcripts was essentially the same in all organs (Hag et al., 1994). These differences may be due to collection of samples at different stages of development or to species differences. Levels of GSAT transcripts decreased throughout tomato fruit development and ripening (Fig. 3 B), and transcript levels from 55-day fruits were comparable to those of roots (Fig. 3A and 3 B). The presence of GSAT mRNA in roots and ripe fruits can be explained by the involvement of GSAT in the biosynthesis of nonchlorophyllous porphyrins like heme (Beale and Weinstein, 1990). Immunoblot analysis showed that barley GSAT antibody reacted with a protein band that had an approximate molec-

GSAT Changes During Tomato Fruit Development

E

H B

45

I

23.1~

Fig. 4: Immunoblot analysis using barley GSAT antibody against total protein extracted from tomato fruit pericarp tissue harvested 10, 15, 25, 35, 45, and 55 days postanthesis. Seventy-five micrograms of protein were added per lane. Arrow indicates approximate predicted mass of mature GSAT protein of 46 kU based on prestained molecular mass markers.

2.0~

O.5~

Fig. 2: Southern hybridization of tomato genomic DNA with the 0.63 kb tomato GSAT PCR fragment. Ten micrograms of DNA were digested with EeoRl (lane E), HindIII (lane H), or BamHI (lane B). Lane I contains 10 pg of the GSAT PCR fragment, which, relative to the mass of genomic DNA per lane, is equivalent to 3 copies of the insert.

L

5

R

F

A

10 15 25 35 45 55

B Fig. 3: Northern blot analysis of the steady-state levels of GSAT mRNA in: (A) tomato leaf (L), stern (S), root (R), and flower (F) tissue; and (B) 10-, 15-, 25-, 35-, 45-, and 55-day postanthesis tomato fruits. Fifteen micrograms of total RNA were loaded per lane. Both filters were hybridized with a 32P-labeled RNA probe synthesized from linearized GSAT template DNA.

ular weight of 46 kU in fruits (Fig. 4, see arrow). Barley GSAT antibodies also reacted with a protein of -46 kU from tomato leaf and stem, but it was not detected in roots (data not presented). The 46 kU band corresponded to the observed molecular mass of barley GSAT (46kU; Grimm et aI., 1989) and pea (45kU; Nair et al., 1991), and to the predicted molecular mass of tomato (46.7kU). Barley GSAT antibody also reacted with a protein band of approximately 60 kU that may represent the unprocessed form of GSAT. It is conceivable that this higher molecular mass band could represent cross-reactivity with another developmentally regulated tomato protein. This higher molecular mass band also was detected in immunoblots of tobacco (Hofgen et aI., 1994). Changes in the intensity of both of these bands throughout fruit development and ripening showed that GSAT is relatively abundant in young (10- and 15-day) fruits, much less abundant in 25- through 45-day fruits, and undetectable in fully-ripe (55-day) fruits (Fig. 4). These data indicated that GSAT protein levels decreased throughout development and ripening. These results also showed that steady-state levels of both GSAT transcripts and protein decreased throughout fruit development. Similar developmental regulation of three other chlorophyll synthesis genes [ALA dehydratase (ALAD), porphobilinogen deaminase (PBGD), and NADPH: protochlorophyllide reductase (NPCR)] has been demonstrated in pea leaves by He et ai. (1994) where transcript levels of all three genes decreased in older tissues. At the protein level, NPCR was lowest in the oldest leaves, whereas ALAD remained consistently high (He at al., 1994), and this indicates that control of expression of these genes may be regulated at the posttranscriptionalievei. The changes in GSAT transcript and protein levels closely parallel the gradual decreases in both chlorophyll a and chlorophyll b content of tomato fruits from 15 days postanthesis through ripening (McMahon et al., 1990), suggesting that GSAT is involved in the regulation of chlorophyll synthesis in tomato fruits. The observed early down-regulation of GSAT agrees with previous findings that several other photosynthesis-specific genes are inactivated within two weeks after pollination (Piechulla et al., 1986). Taken together, these results suggest that the observed photosynthetic activity that occurs, at least through the breaker stage of fruit development (Piechulla et aI., 1987), is due to the stability of existing

46

GARY E POLKING, DAVID J. HANNAPEL, and RICHARD J. GLADON

chlorophyll and photosynthetic enzymes, rather than to de novo synthesis of these products. In addition to developmental regulation, GSAT expression also may have a lightregulated component, although this regulation was not addressed in this study. Light regulation of transcript levels of both GSAT and of the enzyme that immediately precedes it in the porphyrin synthesis pathway, glutamyl-tRNA reductase, has been demonstrated in Arabidopsis (Hag et al., 1994). He et al. (1994) also observed modulation of expression of chlorophyll synthesis genes by light, but they concluded that the primary regulation of these genes was developmental. In our study, in which a constant 12 h darkl12 h light photoperiod was used, both GSAT mRNA and protein accumulation decreased as tomato fruits developed and ripened, indicating that GSAT has a strong developmental component to its regulation. Acknowledgements

We thank Dr. Bernhard Grimm (Carlsberg Laboratory, Copenhagen, Denmark) for providing us with the GSAT antibody and Dr. Wilhelm Gruissem (Univ. of Calif, Berkeley, USA) for providing us with the tomato fruit cDNA library. We gratefully acknowledge partial funding for this research from a grant from the Iowa State University Research Foundation, Inc.

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