Plant Physiol. (1999) 119: 41-48

Arachidonic Acid Alters Tomato HMG Expression and Fruit Growth and Induces 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Lycopene Accumulation¹

Manuel Rodríguez-Concepción and Wilhelm Gruissem^*

Department of Plant and Microbial Biology, 211 Koshland Hall, University of California, Berkeley, California 94720-3102

	ABSTRACT

Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, we manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Our results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.

	INTRODUCTION

Isoprenoids are a structurally diverse group of compounds that are involved in many aspects of cell development. All of these compounds are derived from a common isoprenoid precursor, IPP (McGarvey and Croteau, 1995). It is generally assumed that in eukaryotic cells the irreversible reaction that converts 3-hydroxy-3-methylglutaryl CoA into mevalonic acid, eventually leading to the synthesis of the biologically active IPP unit, is a key regulatory step in the isoprenoid pathway. This NADPH-dependent reaction is catalyzed by the enzyme HMGR (EC 1.1.1.34). In mammals HMGR activity is tightly regulated at many levels, from the expression of the only gene encoding the enzyme to the stability of the protein and the modulation of the activity by phosphorylation or allosteric effectors. This multivalent regulation responds to the levels of cholesterol, the principal end product (Goldstein and Brown, 1990).

In addition to sterols, plant cells produce a large number of IPP-derived compounds, including carotenoids, phytoalexins, and other specialized terpenoids, growth hormones (gibberellins, ABA, and cytokinins), and the polyprenol substituents of dolichols, quinones, and proteins (Bach, 1995; Chappell, 1995a; McGarvey and Croteau, 1995) (Fig. 1). In contrast to what is known of mammalian cells, it is still controversial whether HMGR is a rate-limiting step in isoprenoid biosynthesis in plant cells (Chappell, 1995a, 1995b; Re et al., 1995). The recent discovery of an alternative pathway for isoprenoid production in plants (Eisenreich et al., 1996; Lange et al., 1998) also raises the question of whether HMGR activity is required for all types of isoprenoid end products. HMGR is encoded by a small multigene family in all plants that have been examined thus far, ranging from two genes in Arabidopsis (Enjuto et al., 1994) to four genes in tomato (Bach et al., 1991; S.M. Jenkins and W. Gruissem, unpublished data). Tomato (Lycopersicon esculentum) HMG1 is highly expressed during the early stages of fruit development, when sterol biosynthesis is required for membrane biogenesis during cell division and expansion (Narita and Gruissem, 1989). HMG2 expression is not detectable in young fruit, but is activated during fruit maturation and increases strongly during ripening, in parallel with the accumulation of lycopene (Gillaspy et al., 1993).

View larger version (43K): [in this window] [in a new window]   Figure 1. Isoprenoid pathway in plants. Plant growth regulators and some enzymes of the pathway are circled. HMGR and PSY are highlighted in gray because of their possible rate-limiting roles. The steps catalyzed by PDS, FTase, and GGTase are also indicated. FPP and GGPP are the main branch points of the pathway. The alternative Rohmer pathway for the production of IPP in plastids is also indicated.

Based on the specific expression patterns of HMG genes that are correlated with end-product accumulation in tomato, it could be argued that the corresponding HMGR isozymes regulate the isoprenoid pathway to produce specific end products. Therefore, changes in the expression level of HMG1 and HMG2 should most strongly affect only the accumulation of the corresponding type of end product. We report experiments in which we have manipulated HMG1 and HMG2 expression levels in tomato fruit by using the fungal elicitor AA, which represses HMG1 and induces HMG2 expression in tomato leaves, stems, and fruit discs (J.O. Narita, M. Rodríguez-Concepción, and W. Gruissem, unpublished data). Unlike other studies in which heterologous (Chappell et al., 1995) or homologous (Re et al., 1995) HMG genes were ectopically expressed in transgenic plants, we show that AA can be used to simultaneously repress HMG1 and induce HMG2 expression in the same tissue. The results show that the lower level of HMG1 transcripts in AA-treated young fruit is correlated with growth inhibition. In mature fruit AA strongly and prematurely induced expression of HMG2 and PSY1, as well as lycopene synthesis. Although this would be consistent with a role of HMG2 in carotenoid biosynthesis, we found that concomitant inhibition of HMGR activity by mevinolin did not affect lycopene accumulation.

	MATERIALS AND METHODS

Plant Material and Treatments

AA was purchased from Sigma. The AA stock solution contained 100 mg/mL AA in ethanol. The AA working solution was freshly prepared by diluting 5 µL of the stock solution to 100 µL, and contained 5% (v/v) ethanol, 5 mg/mL (16 mM) AA, and 50 mM Tris-HCl, pH 7.0. The control solution was the same but did not contain AA. Mevinolin (a gift from Merck, Darmstadt, Germany) was prepared by dissolving 4 mg in 150 µL of ethanol. After adding 225 µL of 0.1 M NaOH and incubating at 50°C for 2 h, the pH was adjusted to pH 7.5 with HCl, and water was added to 1 mL to give a 10 mM stock solution of active mevinolin. A solution containing 9 mM active mevinolin in the AA working solution was used for fruit treatment.

Treatments were performed by injecting the same volume of the corresponding solution to plant-attached young (4 µL) or mature-green (10 µL) fruit with a 25-µL syringe (Bio-Phore, Bio-Rad) through the bottom of every fruit. After the indicated times, fruits were collected from the plant, weighed, frozen in liquid nitrogen, and stored at 70°C until used for experimental purposes.

RNA Analysis

Twenty micrograms of total RNA was loaded in 1.2% agarose gels containing formaldehyde. After electrophoresis the nucleic acids were capillary transferred to nylon filters (Hybond N⁺, Amersham) and UV cross-linked with a Stratalinker (Stratagene). The membranes were prehybridized for at least 15 min at 65°C in PSE buffer (0.3 M sodium phosphate, pH 7.2, 7% [w/v] SDS, 1 mM EDTA). Hybrid-ization was carried out in the same solution containing probes randomly labeled with [³²P]dCTP using the Stratagene Prime-It kit. Specific probes were prepared from the 3 end cDNA fragments SpeI-HincII of HMG1 and BglII-HincII of HMG2. The probes for tomato PSY1 (Bartley et al., 1992) and FTA (Yalovsky et al., 1997) were made from the full-length cDNAs. The PDS (Giuliano et al., 1993) probe was made from a cDNA with the 3 end truncated 140 bp upstream of the stop codon. A 1.5-kb XmnI-EcoRI fragment from pHA2 for pea 18S rRNA (Jorgensen et al., 1987) was used as a probe for tomato 18S rRNA.

After hybridization for 12 to 16 h at 65°C, membranes were washed sequentially in washing buffer (1× SSC, 0.1% [w/v] SDS), once at room temperature and three times at 65°C for 20 min each. Exposure to film (BioMax MS, Kodak) was at 80°C with intensifying screens from 1 to 6 d. Quantification of the hybridization signals was achieved after reexposing the blots using a phosphor imager (Molecular Dynamics, Sunnyvale, CA).

Lycopene Measurement

HMGR Activity

	RESULTS

AA Causes a Decrease in HMG1 mRNA Levels and Inhibits the Growth of Young Tomato Fruit

View larger version (49K): [in this window] [in a new window]   Figure 2. Effect of AA on young fruit development. Young tomato fruits attached to the plant were injected with a control or AA solution. Groups of three to five fruits were collected after 3 d or 1 month and used for experiments. A, Fruit weight 1 month after treatment with different concentrations of AA. B, Fruit samples from the experiment shown in C. C, Fruit weight 3 d after injection of 4 µL of a control solution or 5 µg/µL AA solution. Columns represent means and bars represent SD values. D, RNA blot of total RNA samples from the fruit samples shown in C. The same blot was hybridized with gene-specific HMG1, HMG2, and 18S rRNA probes.

RNA-blot analyses revealed that the level of HMG1 mRNA was decreased in AA-treated fruit compared with control fruit (Fig. 2D). AA treatment also induced the ectopic expression of HMG2 in young fruit (Fig. 2D), a stage at which HMG2 transcripts are normally absent (Gillaspy et al., 1993). Our results are consistent with earlier reports and with our own observations (Choi et al., 1992; J.O. Narita, M. Rodríguez-Concepción, and W. Gruissem, unpublished data) that HMG1 and HMG2 expression are affected differently by AA. The reduction in HMG1 transcript levels and the concomitant growth inhibition after AA treatment suggest that full HMG1 expression is required during fruit development. Apparently, the growth inhibition cannot be reversed by the ectopic expression of HMG2.

AA Induces Lycopene Accumulation in Mature-Green Fruit

View larger version (24K): [in this window] [in a new window]   Figure 3. Effect of AA treatment on mature tomato fruit. Mature-green fruits were injected with either 10 µL of a control solution or 5 µg/µL AA solution while attached to the plant, and collected 3 d later. A, Proportion of control and AA-treated fruits at different stages of pigmentation (Gillaspy et al., 1993). B, Lycopene concentration in control and AA-treated fruits from three different experiments. Columns represent means and bars represent SD values from three independent experiments.

Induction of HMGR Activity Is Not Required for AA-Induced Carotenoid Accumulation

View larger version (61K): [in this window] [in a new window]   Figure 4. RNA-blot analysis of gene expression in control and AA-treated mature fruit. Mature-green fruits were injected with 10 µL of a control solution or 5 µg/µL AA solution while attached to the plant. Groups of three to five fruits were collected and ground together after 6, 12, 24, 48, or 72 h. A, RNA-blot analysis of total RNA extracted from the different fruit pools. The blot was hybridized with a gene-specific HMG2 probe, and other probes from tomato PSY1, PDS, and FTA. An 18S rRNA probe was used to compare the RNA amounts loaded in each lane. B, Quantification of the steady-state levels of HMG2 and PSY1 mRNA. Open symbols, Control fruit; closed symbols, AA-treated fruit. The values shown are normalized with the 18S rRNA amounts and are expressed relative to the level detected in red, firm fruit.

HMGR activity also increased in AA-injected fruits (Fig. 5A). HMGR activity 24 h after injection with AA was about 5-fold higher compared with control fruits, but unlike the pattern of HMG2 mRNA accumulation, the highest HMGR activity was detected at 48 h. By 72 h after injection HMGR activity also decreased (Fig. 5A).

View larger version (20K): [in this window] [in a new window]   Figure 5. HMGR activity and lycopene concentration in control and AA-treated fruit. Fruits were treated as described in Figure 4 with 10 µL of a control solution (C), with an AA solution (AA), or with an AA solution containing 9 mM mevinolin (AA+MEV), collected after 24, 48, and 72 h, and used for determination of microsomal HMGR activity (A) and measurement of lycopene concentration (B). Columns represent means and bars represent SD values from three independent fruit samples.

To determine whether the increase in lycopene formation in AA-injected fruit was a consequence of increased HMGR activity, a specific inhibitor of HMGR activity, mevinolin (Alberts et al., 1980), was coinjected with AA into mature green fruit (see ``Materials and Methods''). The pattern of HMG2 mRNA expression in fruits injected with AA and mevinolin was identical to that observed in fruits treated only with AA (data not shown), but the microsomal HMGR activity was inhibited to undetectable levels (Fig. 5A). This inhibition of HMGR activity by mevinolin, however, did not abolish the AA-induced accumulation of lycopene. The pattern of lycopene accumulation in fruits injected with both AA and mevinolin was essentially identical to that observed in fruits injected with AA alone (Fig. 5B). Accumulation of lycopene began 24 h after injection, and after 72 h the lycopene amount in fruits injected with AA alone or with AA and mevinolin was 3-fold higher than in control fruits. We conclude from these results that the higher rate of carotenoid production in the chloroplast induced by AA does not depend on increased cytoplasmic HMGR activity.

AA Induces the Expression of PSY1, the First Committed Step in the Biosynthesis of Carotenoids

The next step in carotenoid biosynthesis is catalyzed by the plastid-localized enzyme PDS, which converts phytoene into -carotene (Giuliano et al., 1993). Unlike PSY1, however, PDS was expressed at similar levels in control and AA-injected fruits (Fig. 4). We also analyzed the transcript levels of the tomato FTA gene, which encodes the -subunit that is shared by cytoplasmic FTase and GGTase (Yalovsky et al., 1997). These enzymes modify specific proteins by covalent attachment of farnesyl or geranylgeranyl to a conserved Cys at their carboxy terminus (Fig. 1). Thus, GGTase could compete for GGPP if the cytoplasmic intermediate were also required for carotenoid biosynthesis. Although FTA is expressed in both control and AA-injected fruits, mRNA accumulation was not induced by AA (Fig. 4A), as was observed for PSY1. Our results are consistent with PSY1 catalyzing the committed step for the biosynthesis of carotenoids, and show that regulation of this step of the pathway may be key in controlling the production of carotenoids in plastids during fruit development.

	DISCUSSION

Expression of HMG1 and HMG2 Can Be Modulated by AA during Fruit Growth

HMGR Activity Is Not Required for Carotenoid Biosynthesis in Tomato Fruit

The in vivo inhibition of HMGR with mevinolin, however, did not prevent the AA-induced accumulation of carotenoids (Fig. 5B), suggesting that the strong induction in HMGR enzyme activity that we could assay was not necessary for carotenoid biosynthesis. Similarly, treatment of tomato fruit discs with mevinolin did not arrest the ripening and accumulation of carotenoids (J.O. Narita and W. Gruissem, unpublished data). In other experiments, inhibition of HMGR with mevinolin had little effect on the synthesis of chlorophyll and carotenoids, but prevented sterol biosynthesis and reduced growth (Bach and Lichtenthaler, 1983; Narita and Gruissem, 1989). It is possible that carotenoid synthesis in mevinolin-treated tomato fruit results from utilization of preexisting isoprenoid intermediates, or that a residual HMGR activity may be sufficient to sustain carotenoid biosynthesis. In view of the recent discovery of an alternative glyceraldehyde 3-phosphate/pyruvate pathway for IPP synthesis in the chloroplast (Rohmer pathway, Fig. 1; Eisenreich et al., 1996; Schwender et al., 1996; Lange et al., 1998; Lois et al., 1998), it is more likely that this pathway is responsible for carotenoid biosynthesis. This would explain why inhibition of HMGR activity does not affect carotenoid accumulation in tomato fruit. At the same time, it raises the interesting question of why HMG2 is induced to high levels during ripening.

Is There a Role for HMGR2 in Defense Responses?

Although it is known that wounding accelerates ripening and the production of carotenoids (Ulrich and Renac, 1950; Hugueney et al., 1996), we have shown that carotenoid biosynthesis can also be induced by AA, an elicitor of pathogen defense responses. Because HMGR enzyme activity is not required for lycopene accumulation, it is likely that this induction event is parallel to the activation of HMG2 expression. AA also induces PSY1 expression, suggesting that the parallel activation of the cytoplasmic isoprenoid and chloroplast carotenoid biosynthesis pathways may be part of a general defense mechanism.

AA Induces the Expression of PSY1, Resulting in Accelerated Carotenoid Accumulation

Our results are consistent with reports for transgenic tomato plants showing that modulation of PSY1 gene expression by ectopic expression or antisense RNA inhibition of PSY1 alone is sufficient to alter carotenoid levels (Bird et al., 1991; Bramley et al., 1992; Fray and Grierson, 1993). In addition, constitutive expression of PSY1 in transgenic tomato also causes dwarfism, possibly by redirecting GGPP from the gibberellin pathway (Fig. 1) to the synthesis of carotenoids (Fray et al., 1995). In contrast, inhibition of PSY1 expression leads to the accumulation of GGPP and FPP and elevated gibberellin levels during tomato fruit development (Bird et al., 1991; Fraser et al., 1995). PSY1 is likely a branch-point enzyme whose regulation may control GGPP allocation to carotenoid biosynthesis.

	CONCLUSION

The regulation of HMG1 and HMG2 expression in tomato is consistent with the theory that levels of the different HMGR isozymes in plants are modulated in response to specific developmental and stress signals. It is likely that this results in the channeling of mevalonic acid to specific end products of the isoprenoid pathway (Bach, 1995; Chappell, 1995a, 1995b). Other enzymes at important branch points in the isoprenoid pathway, such as IPP isomerase, FPP synthase, and GGPP synthase (Scolnik and Bartley, 1996), are also encoded by small gene families, although little is known about the expression of their genes. In addition to the differential regulation of genes for isozymes, compartmentation, channeling through multienzyme complexes or "metabolons" (Srere, 1987), or the regulation of the activity of other rate-determining enzymes may also operate to control the accumulation of specific isoprenoid end products. Although our results suggest that the control of cytoplasmic isoprenoid synthesis can be exerted at the level of HMG1 and HMG2 expression, the activity of their enzymes in tomato does not appear to be required for plastid carotenoid production. Instead, based on the induction of PSY1 expression before lycopene accumulation, it is likely that plastid PSY has an important role in channeling GGPP from the HMGR-independent Rohmer pathway (Schwender et al., 1996) into plastid carotenoid production. The results reported here and in other recent reports (Chappell et al., 1995; Hugueney et al., 1996; Lange et al., 1998) offer an interesting opportunity to investigate the regulation of isoprenoid synthesis in different cellular compartments in combination with genetic approaches to determine the role of specific enzymes in the pathways.

	FOOTNOTES

   Received August 11, 1998; accepted October 6, 1998.

	ABBREVIATIONS

Abbreviations: AA, arachidonic acid. FPP, farnesyl diphosphate. FTA, -subunit of FTase. FTase, farnesyl transferase. GGPP, geranylgeranyl diphosphate. GGTase, geranylgeranyl transferase. HMGR, 3-hydroxy-3-methylglutaryl CoA reductase. IPP, isopentenyl pyrophosphate. PDS, phytoene desaturase. PSY, phytoene synthase.

	ACKNOWLEDGMENTS

We are grateful to Drs. R.L. Jones, S.M. Jenkins, and J. Keddie for critical reading of the manuscript. We also thank Drs. G.E. Bartley for the PDS and PSY1 clones, S. Yalovsky for the FTA clone, J. Jelesko and S.M. Jenkins for advice, and the departmental greenhouse staff for excellent care of plants.

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