tomato hydroxymethylglutaryi-coa reductase is required ... · development is characterized by a...

The Plant Cell, Vol. 1,181-190 February 1989, © 1989 American Society of Plant Physiologists

Tomato HydroxymethylglutaryI-CoA Reductase Is Required Early in Fruit Development but not during Ripening

Jonathon O. Narita and Wilhelm Gruissem 1

Department of Botany, University of California, Berkeley, California, 94720

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and the level of its mRNA have been determined at various stages of tomato fruit development. The HMGR reaction makes mevalonate, a necessary component in the synthesis of all isoprene containing compounds, such as sterols and carotenoids. A cDNA clone encoding the active site region of HMGR has been isolated from a tomato library derived from young-fruit mRNA. The clone hybridizes to a one- or two-copy fragment in high-stringency DNA gel blot analyses and detects an mRNA of approximately 3.0 kb. Both HMGR activity and mRNA levels are high in early stages of tomato fruit development, when rapid cell division occurs, as well as in the subsequent early stages of cellular expansion. In contrast, ripening fruit have very low levels of reductase activity and mRNA, even though large amounts of the carotenoid lycopene are synthesized during this period. Furthermore, in vivo inhibition of HMGR during early fruit stages disrupts subsequent development, whereas inhibition during later stages of fruit expansion has no apparent effect on ripening. We conclude that the pool of mevalonate responsible for the synthesis of phytosterols is synthesized primarily during the first half of tomato fruit development. In addition, the final period of fruit expansion and ripening is not dependent upon HMGR activity, but instead utilizes a preexisting pool of pathway intermediates or requires the use of salvage pathways in the cell.

INTRODUCTION

The enzyme 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase (HMGR) catalyzes the conversion of HMG-CoA into mevalonate (MVA) in the first committed step of the isoprenoid synthesis pathway in all eukaryotic cells. The NADPH-dependent reaction is required for cellular sterol synthesis and has been linked to the control of DNA replication in cells (Quesney-Huneeus et al., 1979; Brown and Goldstein, 1980). The enzyme is located on cytosolic membranes and accumulates on the endoplasmic reticulum in HMGR-amplified hamster cells (Chin et al., 1982). A second minor activity has been identified that co- sediments with mitochondrial membrane fractions (Brooker and Russell, 1975). An extensive body of information concerning HMGR exists in mammalian systems because this enzyme performs the rate-limiting step in the synthesis of sterols and is regulated by a variety of feedback controls (for review, see Sabine, 1983, and Priess, 1985). Accumulation of cholesterol and other end products results in a general decrease in HMGR protein and mRNA levels, whereas the substrates of the reaction, HMG-CoA and NADPH, affect HMGR activation by phosphatases.

To whom correspondence should be addressed.

In contrast, the accumulation of the immediate product of the reaction, mevalonate, has little direct effect on enzyme activity (Edwards et al., 1983; Chin et al., 1985; Feingold and Moser, 1986). In plants, the isoprenoid pathway is responsible for the synthesis of membrane sterols, carotenoids, some auxin hormones, the phytol moiety of chlorophyll, and other isoprene-containing compounds (for an overview, see Bach, 1987). The amount and variety of end products dependent upon this pathway is greater in plants than in animals. In addition, a third activity, associ- ated with the chloroplast membrane fraction, has been identified in plants (Brooker and Russell, 1975). The site of HMGR activity in plants is proposed to be the cytosolic membranes for sterol synthesis, and possibly the plastid membranes for carotenoid synthesis (Camara et al., 1983; Bach, 1987; Schulze-Siebert et al., 1987). Currently, it is unknown how plant HMGR controls and coordinates the synthesis of this diverse group of plant compounds or how three HMGR activities are regulated in separate compartments.

We are using tomato fruit as a model system to investigate the role of HMGR in the developmental process. The tomato fruit undergoes its complete developmental cycle in 5 to 6 weeks, is easily staged, and requires HMGR

182 The Plant Cell

products for membrane biogenesis in early stages and for carotenoid synthesis during ripening. For the following work we have divided tomato fruit development into three phases characterized by cell division (phase I), cell expansion (phase II), and fruit ripening (phase III). Pollination initiates phase I, in which rapid cell division accounts for most size increase in the early fruit stage. Phase II of fruit development is characterized by a period of cellular expansion and photosynthetic activity lasting approximately 3 to 4 weeks. This period ends at the "mature green" stage when the fruit has achieved its maximal size. Phase Ill of development is the fruit ripening period, during which the size of the fruit remains constant. This phase is characterized by the differentiation of chloroplasts into lycopene-containing chromoplasts, and by fruit softening (for review, see Varga and Bruinsma, 1986).

To examine the role of HMGR in tomato fruit development, we have assayed HMGR enzyme activities in all three phases, and we have cloned a cDNA for the tomato HMGR and used it to analyze the expression of the HMGR gene. These studies show that HMGR is active during periods of rapid cell division and membrane expansion in phase I and early phase I1. Furthermore, the enzymatic activity profile correlates well with the accumulation of mRNA for HMGR in the tomato stages tested, suggesting that HMGR expression in fruit is determined by the level of its mRNA, rather than by protein modifications that modulate enzyme activity. Interestingly, the synthesis of large quantities of the carotenoid lycopene in phase III tomatoes occurs in the absence of high HMGR activities. These results are supported by experiments in which the

drug mevinolin was used to inhibit in vivo HMGR activity in developing fruit. Thus, HMGR activity is necessary for early fruit development, but is not required for late phase II cellular expansion or for the accumulation of carotenoids during fruit ripening. These results and the role played by HMGR in tomato fruit development and ripening are discussed.

RESULTS

HMGR Activity Is Regulated during Tomato Fruit Development

HMG-CoA reductase activities were quantified from membrane extracts prepared from various tomato fruit stages. Based on differences in isolation procedures, pH optima, and salt requirements, the three plant HMGR activities from cytosolic, chloroplast, and mitochondria can be assayed independently (Brooker and Russell, 1975). The cytosolic form of HMGR was assayed in young leaves and in fruit at different phases of development. The results are listed in Table 1. All samples were tested with and without NADPH to ensure that the MVA production was cofactor- dependent. As expected, the preparations with the highest specific activity were phase I and early phase II stages (0.1-cm to 1.5-cm fruit), which have large sterol requirements due to cell division and expansion. The membrane extracts from late phase II and phase III stages (2.0 cm to 3.5 cm) did not show any significant level of HMGR activity.

Table 1. HMGR Activities during Tomato Fruit Development

Cytosolic Enzyme Plastid Enxzyme"

Tomato Tissue Size Specific Activity b %° Specific Activity %

cm

Leaf 1-2 36 11 1 2 Flower ovules 0.1 10 3 Fruit 0.2 300 94 Fruit 0.4 320 100 Fruit 0.5 47 100 Fruit 1.0 101 32 Fruit 1.5 37 12 Fruit 2.0 5 2 Fruit 3.0 1 0 Fruit d (turning red) 3.5 0 0 Fruit + mevinolin 0.4 3 1 Fruit, mature green 3.5 0 0 Fruit, turning red 3.5 0 0

a HMGR activity from isolated plastids. See "Methods" for details. Specific activity = nanomoles of MVA/hour/milligram of protein.

c Percentage of HMGR activity compared with the highest stage tested (0.4 cm). d These fruit were starting to show red color in the pericarp; they are sometimes referred to as "breaker" fruit.

HMG-CoA Reductase Expression in Tomato Fruit 183

Because a chloroplast-localized HMGR activity could be responsible for carotenoid synthesis in plants (Camara et al., 1983; Schulze-Siebert et al., 1987), we assayed the plastid form of HMGR in selected tomato tissues. The plastid HMGR activity was very high in the young fruit (15% as active as the cytosolic form), but was undetecta- ble in the mature green and red fruit, as shown in Table 1. Therefore, it appears that the increase in carotenoid synthesis during tomato fruit ripening occurs without a corresponding increase in either cytosolic or plastid HMGR activities. In summary, cell division and early cell expansion events coincide with high HMGR activities, unlike the later half of fruit development in which HMGR activities are very low.

Tomato HMGR cDNA Isolation

To examine how the expression of HMGR during tomato fruit development is controlled at the molecular level, we isolated a cDNA using a strategy based on identifying sequence similarities among previously cloned genes. HMGR genes have been isolated from hamster, human, yeast, and Drosophila (Chin et al., 1982; Luskey and Stevens, 1985; Basson et al., 1988; Gertler et al., 1988), as well as other mammalian and bacterial sources. The COOH-terminal half of the animal and yeast HMGR proteins contains the active site of the enzyme, whereas the NH4-terminal half is composed of membrane-spanning domains (Basson et al., 1988). Sequence similarities among HMGR genes from different organisms are evident only in the COOH-terminal portion of the enzyme. The yeast and the human HMGR proteins share 65% amino acid identity and show an additional 25% conservative amino acid substitution in the active site region (from amino acids 667 to 1025 for the yeast HMG-1, and amino acids 512 to 871 for the human sequence). Similar conservation exists between amino acids in the active site regions of Drosophila and hamster (Gertler et al., 1988). A particularly long stretch of homology, corresponding to amino acids 840 to 861 in the yeast HMG-1 gene, is found in all mammalian and yeast HMGR genes. We constructed an oligonucleotide complementary to this region that consisted of 50 residues of the sequence: 5'-CCTTCIATCCAGTTIA- TIGCIGCIGGTTTTTTGTCIGTGCAGTAGTTICC-3'.

The design of the oligonucleotide incorporated inosine residues at ambiguous third positions of codons and also utilized G:T pairing when necessary. The oligomer was end-labeled and used to screen a cDNA library prepared from young tomato fruit mRNA. Of the 200,000 plaques screened, a single clone was identified. Sequence analysis of this clone showed 92% pairing between the site of hybridization and the oligonucleotide probe. Mismatches were found at positions 36, 38, 43, and 48 of the oligomer. (A complete DNA sequence analysis of the tomato HMGR gene will be published elsewhere.)

Figure 1 shows the deduced amino acid sequence of the tomato HMGR active site compared with sequences from yeast, hamster, human, and Drosophila. The conserved domains identified previously in other HMGR sequences were also found in the tomato gene. To illustrate further the similarity of these genes, hydropathy plots were constructed for the tomato, yeast, and hamster sequences according to the rules of Kyte and Doolittle (1982), and are displayed in Figure 2. The homology of the tomato gene to the yeast HMG-1 gene (from amino acid 776 to 1025) is 62% at the nucleotide level. At the amino acid level, there is 67% identity plus 23% conservative replacements. This level of homology is comparable with that observed among previously identified genes (Basson et al., 1988; Gertler et al., 1988), and corroborates the biochemical similarities between the plant and animal enzymes (Garg and Douglas, 1983; Bach, 1987).

Tom F N k s S RFARLQG I QCA [AGKNLYMRFSCSTCDAMCMNNVS KGVQNVLDYI~S EY P - - DMDV IG So: NST - - H I Q T C L - DL I~1 R 1 T r - I .VEYS-KQ#WEEYGWKD Eq/VS [ tam D ' " "KIMVTH" RN Y[ QSKT • 1 - T E K A . L K I ~ E - C F P E - Q I [ A Hum D ' " " K L I I T S I - RN Y I QSRS • I - T E K A . S K L I ~ - y F p E - Q I L A Dm D ' " g kDC{ l lkHd PQ Y I V A I T r V a L R W P F A E F T L - I t F P - B Q I | S

T o m | SwCNFG.e, DKK PAAvNW I EGRGIRVV C F ~ I I KF33VVldO/LKTLrq AALV ~ T G S K P ~ G a [ S c V ] Y T l • S A - T PCD" "RK s D V S ' L V - L IA "V S Ham 9 Y T - " T C "V P k K " " " g " q ' F g ' M l d V IN "V " Hum V y T " " S C "V P A K ' " " g " q q ' E ' N I ' V I N "V " Dm L F C " k T c T S A A t I ' S " D A K t l N - C KL mG •

T o m I ~A;FNAItAsN 1VsAXr/LATGODPAQNVESShC l T ~ F , - - A V I ~ L I t I SVTMPS 1EVG TvGG S c V F A L T VFL 1. P 1~ N " L k - - E V - I X ; - R" "VS" " V i Ham ] y " 1 " ] Y ] C A G " " I. - - A S G P T N E Y ' ' C T n • I " Hum 1 Y " 1 " I Y I C A G " " L " ASGPTNE Y ' ' C T " " I " l ~ [ N " H • V F L T P T • s A -CWAI~S E Y m t C T " 1 V "

Tom G~OLASQSAC LNLLCVKGANRDA PCSNARla.AT I VAGSVLAGELS I M s A 1 SAGQLVI(SHNKyNR S c V EP G m DL R pHATA T RQ R ACA L cA LA. " H ' - O -TH Ham N [.P Q " OH O "CKDN ~ KQ R CGT N - - I A - " H " . R -V- Hum N LP Q " QM Q "CKDN E RQ R CGT M "" LA''H'-K - I " I ~ G PG S • gl4 R - I ~ T R 0 IO( Q CAT M "" I , V n s D v l K r - "

X i d e n t i t y ~ c o n s e r v e d o v e r a l l h o t a Q [ o ~ y YF~ST : 6 7 . 5 2 2 . 9 9 0 . 4 I L M ~ T I ~ : 6 1 . 8 3 0 . | 9 1 . 9 HLINAN : 6 1 . 8 3 0 . 9 9 2 . 7 BROFA3PH| I~ : 5 6 . 6 3 0 . 9 8 7 . 5

Figure 1. Comparison of HMGR Active Site Sequences.

The amino acid sequence of the tomato HMGR active site is compared with the corresponding sequences found in yeast (Sc, amino acids 776 to 1025, Basson et al., 1988), hamster (Ham, amino acids 621 to 870, Chin et al,, 1984), human (Hum, amino acids 622 to 871, Luskey and Stevens, 1985), and Drosophila (Din, amino acids 644 to 693, Gertler et al., 1988). Positions of identity in all five sequences are left blank. Positions conserved among four of the five genes are indicated by a ". ," with the mismatched amino acid shown in lower case type. Gaps are indicated by a "-," and were placed in sequences to maximize the alignment of the five genes. The boxed sequence is the region used in constructing the oligonucleotide for the library screen. The overall homologies of these regions compared with tomato are displayed in the table at the bottom. Conserved amino acids were assigned by the Fastp analysis of Wilbur and Lipman (1983).

184 The Plant Cell

Tomato

Yeast

Hamster

Figure 2. Hydropathy Plots of Tomato, Yeast, and HamsterHMGR Active Sites.

The hydrophobicity of each amino acid is averaged over a 20-position window according to the method of Kyte and Doolittle(1982). The entire sequence shown in Figure 1 is displayed foreach plot. The bar underneath the tomato plot shows the area towhich the oligonucleotide was constructed.

One or Two Genes Encode the Tomato HMGR

To determine the number of genes encoding the plantHMGR, the genome organization of HMGR in tomato wasanalyzed using DNA gel blot hybridizations. The existenceof HMGR activities in three compartments in plant cells(cytosolic, mitochondrial, and plastid) raises the possibilityof several HMGR genes encoding separate products. Al-ternatively, all three HMGR activities may be encoded bya single gene, which gives rise to the different formsthrough RNA processing events, or by protein modifica-tion. When total genomic DNA was probed with the cDNAcontaining the HMGR active site region, a simple patternof hybridization was obtained, as shown in Figure 3. Asingle EcoRI fragment of >30 kb (lane a'), a single Hindlllfragment of 4.2 kb (lane b'), and a single BamHI/Bglllfragment of 1.7 kb (lane c') were the prominent hybridizingspecies. Copy number reconstructions suggest that the

hybridizing species are present in one or two copies intomato. It is possible, however, that additional genes withlower nucleotide homology to this region may be presenton the tomato genome. Minor hybridizing species weredetectable at 9.2 kb and 8.2 kb (EcoRI); 9.2 kb, 6.4 kb,5.1 kb, and 3.4 kb (Hindlll); and 8.4 kb, 7.9 kb, and 3.4 kb(BamHI/Bglll). If other genes for HMGR are present on thetomato genome, the nucleotide sequence of the conservedactive site region must have diverged significantly from thegene that we have isolated. In mammalian systems, allHMGR genes studied are present as single copies (Rey-nolds et al., 1984; Luskey, 1987), whereas in yeast thereare two copies of HMGR that share 82% nucleotide ho-mology over the active site region. Although our resultsprovide evidence for one or two copies on the tomatogenome, we cannot at this time exclude the existence ofadditional HMGR genes.

a' b' c

0.56kb-

Figure 3. DNA Gel Blot of the Tomato Gene Hybridized with theHMGR cDNA.

Tomato root DNA (3 ^g/lane) was digested with EcoRI (lane a),Hindlll (lane b), or BamHI + Bglll (lane c). The blot was hybridizedwith the tomato cDNA, containing the active site region shown inFigure 1. The final wash was done in 0.2 x SSPE + 0.2% SDSat 65°C. The autoradiograph of the blot is shown in lanes a' toc'. Hindlll-digested \ DNA was used as a molecular weight markerand the band positions are designated on the left.


HMGR mRNA Levels Are Highest in Young TomatoFruit

To determine whether the HMGR activity profile at differentstages of tomato fruit development correlated with HMGRmRNA levels, we examined the accumulation of mRNA forHMGR using RNA gel blot hybridizations. Figure 4 showsthe signals obtained when the cDNA clone was used toprobe various tomato RNAs. The HMGR cDNA probehybridized to a transcript of approximately 3.0 kb. This issufficient to encode a protein of M, 90,000-100,000, whichcorresponds to the size of animal HMGR proteins (Liscumet al., 1985; Priess, 1985). The rapidly dividing and ex-panding young tomato fruit stages showed higher accu-

f g h

r iFigure 4. RNA Gel Blot of Tomato RNA Hybridized with theHMGR cDNA.

(1) Total RNA (5 ^g/lane) was run in lanes a to i, and poly(A+)RNA (0.5 ng/\ane) was run in lanes j to k. The tomato cDNAcontaining the active site region was used as the probe. RNA wasisolated from leaf (lane a), root (lane b), green tomato seedlings(lane c), etiolated tomato seedlings (lane d), tomato suspensionculture cells (lane e), tomato suspension cells grown in 50 ^Mmevinolin (lane f), young fruit (0.4 cm to 0.6 cm) (lane g), maturegreen fruit (lane h), red fruit (lane i), young fruit (lane j), leaf (lanek), and root (lane I). The HMGR-specific hybridization is the upperband. The lower band (darkest in the leaf total RNA lane) is achloroplast rRNA band, which is not detected following higherstringency washes (see Figure 5). The final wash was done at 0.5x SSPE + 0.2% SDS at 65°C. This was an 18-hr exposure ofthe autoradiograph.(2) A 1-week exposure of the blot shown in (1). On very longexposures of this blot, it appears that HMGR signal is detectablein all tissues.

mulations of HMGR mRNA than any of the other tissuestested (lanes g and j). When the fruit reached the fullyexpanded mature green stage (phase III, lane h), the levelof HMGR RNA was reduced significantly, and was reducedfurther at the red fruit stage (lane i). The hybridizationsignal obtained with RNA isolated from slowly growing orexpanding tissues such as leaf or root was very low (lanesa and b), but HMGR mRNA could be detected in thepoly(A+) fraction in these tissues (lanes j and k). RNAisolated from green or etiolated seedlings (lanes c and d)showed that HMGR mRNA levels drop when the seedlingsare exposed to light. This corroborates previous findingsof negative regulation of HMGR activity by phytochrome(Brooker and Russell, 1979). Finally, tomato suspensionculture cells were grown in increasing concentrations ofthe HMGR-specific inhibitor mevinolin. Suspension culturecells that were not treated with the drug (lane e) had alower mRNA signal than the mevinolin-resistant cells, sug-gesting that the inhibition was overcome by increasing thelevel of HMGR mRNA.

A more detailed study of the HMGR mRNA levels duringtomato fruit development is shown in Figure 5. RNA wasprepared from flower ovules (lane b) and from tomato fruitat various stages of development. During phase I, theHMGR mRNA abundance increases significantly in the firstfew days of cell division (0.2-cm stage, lane c), and remainshigh through the initial period of fruit expansion (phase II),until the fruit reaches the 1.2-cm stage (lane e). After the1.2-cm stage, the level begins to decline and remains verylow from the 2.5-cm stage (lane h) through the final fruitexpansion (3.0 cm to 3.5 cm, lane i) and ripening (lane j).Thus, during the cell division period of phase I when theneed for phytosterols is high, there is a large increase inthe HMGR mRNA level that is maintained throughout theperiod of rapid membrane biogenesis. However, at themature green stage, which immediately precedes the onsetof increased carotenoid synthesis, there is no detectableincrease in HMGR mRNA levels. The level of carotenoidpigments in tomato fruit has been estimated to increase100-fold, going from immature fruit (late phase II) to redripe fruit (phase III) (Raymundo et al., 1976). Since therewas no detectable increase in HMGR activity or mRNAduring this period, it appears that carotenoid synthesisduring phase III is not rate-limited by HMGR. Therefore,carotenoids made during ripening must rely on isopreneunits that have accumulated during earlier phases of fruitdevelopment.

The fruit stages that had the highest HMGR mRNAlevels also had the highest enzymatic activity (Table 1),suggesting that the mRNA level regulates the overall am-ount of HMGR activity in tomato fruit. Based on theseresults, we suggest that HMGR activity is determinedprimarily by the level of mRNA in developing tomato fruit.Our results do not exclude, however, that translational orposttranslational modification might play some role in mod-ulating HMGR activity in fruit.

186 The Plant Cell

a b c d e f g h i j

3.0kb-

would have no significant effect on carotenoid synthesisor tomato fruit development.

Fruit representative of the phase I injections are shownin Figure 6. Injections to 0.3-cm to 0.6-cm fruit inhibitednormal fruit expansion and delayed substantially the onsetof fruit ripening (Figure 6, 1 b, 2a). A nearly normal pheno-type was recovered by the co-injection of MVA, the prod-uct of the HMGR reaction (Figure 6,1 a, 2b). MVA alone,water, or salt solutions did not interfere with normal fruitdevelopment (Figure 6, 2c,3a, 3b).

The final size (after 3 months) of phase I fruit injectedwith mevinolin alone was 0.8 cm to 1.7 cm with the largest

Figure 5. Regulation of HMGR and mRNA Accumulation duringFruit Development.

Tomato fruit total RNA (5 M9/lane) was run in lanes b to j. RNAwas isolated from leaf (lane a), flower ovules (lane b), 0.2 cm fruit(lane c), 0.6-cm fruit (lane d), 1.2-cm fruit (lane e), 1.6-cm fruit(lane f), 2.0-cm fruit (lane g), 2.5-cm fruit (lane h), 3.0-cm fruit (lanei), and red fruit (lane j). The transcript size was estimated bycomparison to the ribosomal RNA bands, visualized on the sameblot in separate experiments. The final wash of this blot was in0.1 x SSPE + 0.2% SDS at 65°C.

1

Inhibition of HMGR in Phase I Inhibits Normal FruitDevelopment

To investigate further the role of HMGR in fruit develop-ment, we examined the effect of blocking HMGR activityduring phase I and late phase II. Mevinolin is a specificinhibitor of HMGR that has been useful for assaying the invivo contribution of the enzyme in both plants and animals(Alberts et al., 1980; Bach and Lichtenthaler, 1983; Sabine,1983; Priess, 1985; Bach, 1987). To test the effect ofHMGR inhibition on tomato fruit formation, mevinolin wasinjected into fruit through the pedicel during phase I (0.3cm to 0.6 cm) or during late phase II (1.5 cm to 2.0 cm).This assay was chosen based on the fact that injection ofmevinolin into 12 0.4-cm tomato fruit 24 hours prior to thepreparation of the membranes resulted in 99% inhibitionof cytosolic HMGR activity (see Table 1). Apparently, thedrug is taken up and distributed in the fruit through thevascular tissue. Therefore, we wanted to examine thephenotype of HMGR-inhibited fruit when mevinolin wasadministered during early fruit stages of phase I. In addi-tion, we wanted to determine whether mevinolin couldblock carotenoid synthesis when applied after most of thefruit expansion was complete. Based on the in vitro enzy-matic studies and the hybridization results, we predictedthat mevinolin would impair or stop fruit development whenapplied during phase I. We also expected that injection ofmevinolin into fruit during late phase II or during phase III

Figure 6. Effect of Mevinolin Injection on Tomato FruitDevelopment.

(1) Tomato fruit injected with mevinolin (b) at the 0.3-cm to 0.4-cm stage and allowed to develop for 5 to 6 weeks. Fruit of 0.3-cm to 0.4-cm size injected with mevinolin + MVA (a) showedmore normal development. The black bar at the lower rightrepresents 1 cm.(2) Tomato fruit injected at the 0.3-cm to 0.4-cm stage withmevinolin (a), mevinolin + MVA (b), and MVA alone (c).(3) Control fruit injected at the 0.3-cm to 0.4-cm stage with water(a), and KCI (b). For details see "Results" and "Methods."


fruit (3, 1.5 cm to 1.7 cm) turning red or orange. These large fruit tended to be those that were the largest at the time of injection (0.5 cm to 0.6 cm). The fruit remained green and hard, although some did develop patches of red when picked and allowed to sit at room temperature for several weeks. The mevinolin + MVA-lactone-injected phase I fruit reached a final size of 1.4 cm to 2.8 cm and were all orange or red after 2 months. A few fruit from this set were under 2.0 cm, and they were the smallest (0.3 cm to 0.4 cm) fruit to be injected. The shape and color of these fruit were somewhat variable, with areas appearing to grow at different rates and greenish regions still visible in the ripe fruit. Shape deformations and color abnormali- ties were much more severe in fruit injected with mevinolin alone. All of the fruit injected with water, MVA, or KCI ripened normally. Furthermore, all of the 1.5-cm to 2.0-cm (phase II) tomatoes that were injected with mevinolin, or any of the other control solutions, ripened the same as the uninjected control fruit (not shown in Figure 6).

From these experiments, we conclude that HMGR is required early in fruit development, presumably for phytosterol synthesis, and that inhibition of the enzyme disrupts the normal sequence of events necessary for fruit formation. The timetable of developmental events between phase I and phase III is abnormal in mevinolin-injected fruit if the drug is applied during the period of high HMGR activity. Consistent with the results of the in vitro assays and mRNA studies, the application of mevinolin during late phase II, before the onset of phase III, has no discernible effect on fruit size or color. We conclude that the isoprene units utilized during the final stage of fruit expansion and color formation are not made de novo during those periods.

DISCUSSION

HMGR Is Required for Fruit Development but not for Carotenoid Biosynthesis during Fruit Ripening

We have shown that HMGR is highly expressed in tomato fruits, but only during periods of rapid membrane biogenesis (phase I and early phase II). The period of carotenoid biosynthesis during phase III of fruit ripening does not have a concomitant increase in HMGR activity. We conclude that the reaction catalyzed by HMGR is not the rate-limiting step in the late stages of fruit expansion or in the synthesis of carotenoids during ripening. To complement the enzymatic assays, we have looked at the levels of HMGR mRNA in developing tomato fruit. The mRNA for HMGR accumulates to high levels in phase I and early phase II stages of tomato fruit. Furthermore, the mRNA falls to very low levels before the completion of fruit expansion and does not show an increase during the period of fruit ripening. Thus, the mRNA profile appears to coincide with the enzymatic activity assays.

Finally, we have used the specific HMGR inhibitor mevinolin to determine how in vivo inhibition of reductase affects fruit development. In agreement with the developmental profiles of HMGR activity and mRNA, injection of mevinolin during the period of cell division inhibits subsequent fruit expansion and delays or abolishes phase II and phase III events. Injection of mevinolin during the later parts of phase II or during phase III, however, has no apparent effect on subsequent fruit expansion or ripening. We conclude that the level of HMGR gene expression parallels the phytosterol requirements for rapid cell division or expansion during tomato fruit development in phase I and early phase II. The final stages of fruit expansion (phase III) and carotenoid synthesis during ripening are not achieved by increasing the HMGR activity in those cells, but instead must utilize preexisting isoprene intermediates, or must rely on salvage pathways in the cell.

It is interesting that lycopene synthesis during fruit ripening is not controlled by HMGR, since HMGR catalyzes the rate-limiting step in the synthesis of most of the abundant isoprene-containing end products in mammalian systems (Sabine, 1983; Priess, 1985). One explanation may come from the fact that red formation in tomatoes is not a required part of fruit ripening. Many lines of tomatoes exist that maintain green and functional chloroplasts while com- pleting the climacteric events leading to softening and conversions of starches to polysaccharides (see Rick, 1956). Indeed, most of the wild-type tomatoes isolated do not show lycopene deposition as a part of ripening. It is possible that the lycopene synthesis seen in cultivated tomatoes is a result of chloroplast differentiation.

In cherry tomatoes most mRNAs for photosynthetic proteins decrease by the middle of phase II (Piechulla et al., 1986). The photosynthetic apparatus continues to persist until phase III, after which the photosynthetic proteins disappear and lycopene becomes visible during chromoplast formation (Harris and Spurr, 1969; Piechulla et al., 1987). During this transition, chloroplast thylakoid membranes degrade even though de novo synthesis of membranes does not occur elsewhere in the cell. Thus, there must be a sizable release of sterols and other membrane components into the plastid cavity with no apparent sink to absorb them. It may be that the excess sterols and isoprene compounds produced during fruit ripening in cultivated tomatoes are converted to the insol- uble carotenoid lycopene to remove them from solution. This process would occur to varying extents irrespective of other climacteric events.

The Role of HMGR in Plants

HMGR activities have been found in a wide variety of plant species, including latex, pea, potato, radish, sweet potato, barley, spinach, carrot, catnip, soybean, and pepper (for overviews see Garg and Douglas, 1983; Kondo and Oba,

188 The Plant Cell

1986; Bach 1987). Taken together, work in both plant and animal systems has established that HMGR activities are highest in sterol-requiring cells and all HMGR enzymes have similar biochemical parameters. All eukaryotic HMGR activities convert HMG-CoA to MVA in a two-step reduc- tion using NADPH as a cofactor. The apparent Km values for substrate and cofactor bindings are within an order of magnitude for most of the purified animal and plant enzyme preparations. Despite the similarities to the animal enzyme, the plant HMGR could potentially have some unique prop- erties. Plant and animal cells differ in several types of isoprene-requiring compounds. Examples include the phytol moiety of chlorophyll; plant-specific carotenoids; plant hormones like gibberellic acid, abscisic acid, and zeatin; and the plant flavonoids (see Bach, 1986, 1987). Also, an HMGR activity has been localized to chloroplast membranes (Brooker and Russell, 1975; Camara et al., 1983; Schulze-Siebert et al., 1987), but the relationship between the cytosolic and plastid HMGRs is not currently understood.

The biochemical similarities between the animal and plant HMGR enzymes raise the question of whether the enzymes share any of the same forms of pathway regulation. The major feedback inhibition of mammalian HMGR is controlled by cholesterol accumulation, although there is evidence that nonsterol products of this pathway must also play a role in regulating enzyme activity (Brown and Goldstein, 1980; Beg et al., 1987). Plants do not have a single predominant sterol like cholesterol, nor do they have a single abundant end product that is produced by this pathway. Instead, plants have several abundant compounds (sterols, chlorophyll, carotenoids, phytoalexins, latex, and fiavonoids) that are dependent upon this pathway and that are critical for cellular function.

In mammalian systems, gross fluctuations in HMGR activity seem to be controlled by changes in enzyme synthesis and degradation. For example, cholesterol accumulation lowers the transcription rate of the HMGR gene, and HMGR mRNA stability is also affected (Chin et al., 1985; Clarke et al., 1985). Other more rapid forms of HMGR regulation in mammalian cells include enzyme activation by phosphorylation and protein modifications that modulate diurnal cycle changes in HMGR activity (Roitel- man and Shechter, 1984; Beg et al., 1987; Zammit and Easom, 1987). In plants the forms of HMGR regulation appear to be more complex (reviewed by Bach, 1987). Phytohormones and phytochrome modulate HMGR activities but it is unclear whether these are direct effects. Cholesterol and stigmasterol inhibit pea HMGR activities in seedlings, whereas the commonly occurring sitosterol has no effect. In addition, HMGR activity can be affected by a variety of mechanisms including herbicide treatment, fungal infection, and diurnal cycles. Our results indicate that HMGR activity is determined primarily by mRNA levels in tomato fruit. The more rapid forms of regulation probably play a minor role in this developmental system. It remains

to be seen, however, how HMGR is regulated in other organs and developmental stages of the tomato plant.

METHODS

Plant Material

The tomato cultivar used was VFNT cherry LA1221. All seedlings tested for HMGR activity were grown in "Magenta" boxes on 1 x MS salts in complete darkness for 7 days prior to 12 hr of light. All other tomato tissues were obtained from greenhouse-grown plants. Phase I lasts approximately 7 to 10 days in VFNT, and fruit range in size from 0.1 cm to 0.6 cm at this stage. Phase II fruit are approximately 0.6 cm to 3.5 cm. Tomatoes were harvested from greenhouse-grown plants between 10:00 AM and 12:00 noon. Tomato tissues used for enzymatic assays were harvested onto ice and prepared immediately. Tissues used for RNA preparations were placed immediately into liquid nitrogen and stored at -80°C until needed.

Hybridization Probes and Conditions

The synthetic oligonucleotide was kindly supplied by Gerard Zu- rawski, DNA-X, Palo Alto. The cDNA library was constructed and supplied by Yehiam Salts in our laboratory. Briefly, poly(A+) RNA was isolated from 1-week-old tomato fruit and cloned into Xgtl 0, and approximately 150,000 plaques were isolated and amplified. Library screens were done with 50 pmol to 100 pmol of end-labeled oligonucleotide (specific activity of 5 to 7 x 106 cpm/pmol). Hybridizations were done in 6 x SSPE, 5 x Denhardt's, 0.4% SDS, 100 #g/ml degraded herring sperm DNA, at 42°C for 24 hr to 48 hr. Washes were in 5 x, 2 ×, and 0.5 x SSPE + 0.2% SDS twice each at 48°C for 30 rain each. Plating, blotting, washing, and prehybridizations were all done as outlined in Maniatis et al. (1982). DNA was isolated from root tissue by the procedure of Bendich et al. (1979). RNA was isolated according to the procedure of Barkan et al. (1986). DNA gel blot and RNA gel blot hybridizations were done as reported previously (Sugita et al., 1987). The probes for DNA and RNA gel blots were made by random priming with hexamers (specific activity of 1 to 2 x 109 cpm/#g).

HMGR Extract Isolation

Fresh tissue was harvested between 10:00 AM and 12:00 noon. The grinding and isolation buffers were those used for pea (Rus- sell, 1985). Tomato tissues were ground with buffer in a mortar and filtered through muslin. Extracts were stored in 50% glycerol, 0.1 M KPO4 (pH 6.9) + 12.5 mM DTT at -20°C in a frost-free freezer. Plastid extracts were prepared as outlined by Russell (1985), and by Brooker and Russell (1975). Piastid membranes were assayed immediately after the pelleting of the membranes. The extract typically lost 25% of its activity upon storage for 1 week. Most data points were assayed on the same day as the extract isolation. Protein determinations were performed using the standard Bio-Rad protein assay kit, with lysozyme as the standard.


HMGR Assay

HMGR was assayed as in the microassay reported by Russell (1985). Silica gel was scraped off of both the upper and lower halves of the microscope slides and counted in Aquasol scintilla- tion liquid. The unit definition is 1 nmol of HMG-CoA converted per hour. Specific activity is defined per milligram of protein. Data points are the average of two to three independent preparations of tissue.

Fruit Injections

Mevinolin was kindly supplied by A.W. Alberts, of Merck Sharp & Dohme Research Laboratories (Rahway, N J). The water-soluble lactone form was made as described (Kita et al., 1980). A 5 mM solution was used for fruit injections. Four-microliter aliquots of mevinolin were injected into the fruit via the pedicel. Twenty fruit were injected in the 0.3-cm to 0.6-cm size range and 10 to 15 fruit were injected in the 1.5-cm to 2.0-cm range. Four microliters of a solution of 5 mM mevinolin and 0.5 M MVA-lactone (made by mixing 2 M MVA with an equal volume of 0.1 M KOH and then neutralizing with HCI) was also injected into 12 0.3-cm to 0.6-cm fruit and 6 1.5-cm to 2.0-cm fruit. Control injections with 0.5 M MVA-lactone (4 #1, 10 fruit), water (4 #1, 10 fruit), and 0.5 M KCI salt (4 #1, 10 fruit) solutions were also performed on 0.3-cm to 0.7-cm fruit. No more than one fruit per set of injections was lost due to mechanical damage during injection.

ACKNOWLEDGMENTS

We thank Thianda Manzara, Alice Barkan, and Lew Feldman for their helpful comments and reading of the manuscript. We also thank Kris Callan for assistance in the tomato tissue culture portion of this work. This work was funded by a U.S. Department of Agriculture grant to W.G.

Received December 6, 1988.

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DOI 10.1105/tpc.1.2.181 1989;1;181-190Plant Cell

J O Narita and W Gruissemduring ripening.

Tomato hydroxymethylglutaryl-CoA reductase is required early in fruit development but not

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