1992), rice (Zarembinski and Theologis, 1993), mustard. (Wen et al., 1993), ..... Pelargonium genomic DNA was cut with SacI, BsfXI,. BamHI, or EcoRV and ...
Plant Physiol. (1995) 109: 627-636
Identification and Characterization of cDNAs Encoding Ethylene Biosynthetic Enzymes from Pelargonium x hortorum cv Snow Mass Leaves’ Tzann-Wei Wang2 and Richard N. Arteca*
Department of Horticulture, The Pennsylvania State University, University Park, Pennsylvania 16802 have been identified from a variety of plants including zucchini (Sato and Theologis, 1989), winter squash (Nakajima et al., 1990), tomato (Van Der Straeten et al., 1990; Rottmann et al., 1991), apple (Dong et al., 19911, mung bean (Botella et al., 1992a, 1992b, 1993; Kim et al., 1992), carnation (Park et al., 1992), Arabidopsis tkaliana (Liang et al., 1992; Van Der Straeten et al., 1992), tobacco (Bailey et al., 1992), rice (Zarembinski and Theologis, 1993), mustard (Wen et al., 1993), orchid (ONeill et al., 1993), broccoli (Pogson et al., 1995b), and potato (Destéfano-Beltrán et al., 1995; Schlagnhaufer et al., 1995). Increases in ACC synthase activity during fruit ripening and flower fading and in response to exogenous signals, such as wounding, auxin and cytokinin, and ethylene appear to be based on increased levels of ACC synthase mRNA, although in-depth gene expression studies have been conducted only in a limited number of plant species. It has been shown using gene-specific probes that differential expression of two ACC synthase genes occurs in ripening and wound-induced tomato pericarp tissue (Olson et al., 1991).Similarly, tomato fruit, cell cultures, and hypocotyls express four ACC synthase genes that are differentially regulated during ripening, wounding, and auxin treatment (Yip et al., 1992). Expression of two ACC synthase genes in winter squash is also differentially regulated by auxin and wounding (Nakagawa et al., 1991). In carnation flowers wound-induced and senescence-related ACC synthase genes were differentially expressed (Park et al., 1992). Therefore, the ACC synthase multigene family may reflect the evolution of a group of proteins with different properties (e.g. K,) to effectively utilize AdoMet in different tissues during plant development or under externa1 stimuli (Rottmann et al., 1991). Early studies led to the isolation of a differentially expressed cDNA clone pTOM13, which coded for a 35-kD protein (Slater et al., 1985) and was correlated with increased ethylene synthesis during ripening of tomato fruits and following wounding of green fruits and leaves (Smith et al., 1986).More recently, it has been shown that pTOM13 is an ACC oxidase clone (Hamilton et al., 1990; Kock et al., 1991). ACC oxidase sequences have been identified from other species, such as avocado (McGarvey et al., 1990), carnation (Wang and Woodson, 1991), petunia (Wang and Woodson, 1992; Tang et al., 1993), apple (Dong et al., 1992),
Two Pelargonium 1-aminocyclopropane-1 -carboxylate (ACC) synthase cDNAs (GAC-7 and GAC-2) were identified and characterized. GAC-7 is 1934 bp long with a 1446-bp open reading frame encodirig a 54.1 -kD polypeptide. GAC-2 i s a 1170-bp-long ACC synthase polymerase chain reaction fragment encoding 390 amino acids. Expression of GAC-7 and GAC-2 together with a previously identified ACC oxidase (CEFE-7) was examined in different Pelargonium plant parts, and leaves were subjected t o osmotic stress (sorbitel), metal ion stress (CuCI,), auxin (2,4-dichlorophenoxyacetic acid [2,4-D]), and ethylene. GAC-7 expression was not detectable in any of the plant parts tested, whereas high levels of GAC-2 were expressed in the leaf bud, young leaf, young floret, fully open floret, and senescing floret. GAC-2 was expressed t o a lesser degree i n fully expanded leaves or roots and was undetectable in old leaves and floret buds. GEFE-1 was detectable at all leaf ages tested, in young and fully open florets, and in the roots; however, the highest degree of expression was in the senescing florets. GAC-1 was indoced by sorbitol. Both GAC-7 and GAC-2 were only slightly affected by CuCI, and induced indirectly by 2,4-D. GEFE-1 was highly induced by sorbitol, CuCI,, and 2,4-D. GAC-7, GAC-2, and GEFE-1 were unaffected by ethylene treatment. These results suggest that CAC-7 i s only induced by stress and that GAC-2 may be developmentally regulated, whereas GEFE-7 i s influenced by both stress and development.
Ethylene production is observed in a11 higher plants, where it is involved in numerous aspects of growth, development, and senescence. The ethylene biosynthetic pathway was established by Adams and Yang (1979). Ethylene biosynthesis requires ACC synthase to convert AdoMet to ACC, which is then converted to ethylene by ACC oxidase. ACC can also be converted by ACC N-malonyltransferase (Kionka and Amrhein, 1984) to 1-(malony1amino)-cyclopropane-1-carboxylic acid, which is thought to be an inactive end product of ACC (Amrhein et al., 1981; Hoffman et al., 1982). In recent years the genes for various steps in the ethylene biosynthetic pathway such as AdoMet synthetase, ACC synthase, and ACC oxidase have been cloned in a variety of systems (Kende, 1993). ACC synthase sequences
’ Contribution No. 300 from the Department of Horticulture, The Pennsylvania State University. Present address: Department of Botany and Plant Sciences, University of California, Riverside, CA 92521. * Corresponding author; e-mail rich-arteca8agcs.cas.psu.edu; fax 1- 8.14 - 863- 6139.
Abbreviation: AdoMet, S-adenosylmethionine 62 7
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mustard (Pua et al., 1992), kiwifruit (MacDiarmid and Gardner, 1993),pea (Peck et al., 19931, mung bean ( E m and Yang, 1994), orchid (Nadeau et al., 1993; Nadeau and ONeill, 1995), geranium (Wang et al., 1994), and broccoli (Pogson et al., 1995a). There have been severa1 recent studies showing differential expression of ACC oxidase in tomato (Bouzayen et al., 1993), orchid (Nadeau et al., 19931, broccoli (Pogson et al., 1995a), and carnation (Tang et al., 1993). Although there have been a number of ACC synthase and ACC oxidase genes cloned in recent years, there are only a limited number of in-depth studies of differential expression patterns, and in these studies data for both genes are not typically presented. In this study we identified two cDNAs for ACC synthase, GAC-1 and GAC-2, from Pelargonium, one of the most ethylene-sensitive flowering plants (Nell, 1993). The expression of these two ACC synthase cDNAs together with an ACC oxidase (GEFE-1) (Wang et al., 1994) was evaluated in different Pelargonium plant parts and in response to stress-induced by osmotic changes (sorbitol) or metal ions (CuCl,), and the effects of ethylene or auxin (2,4-D) induction were evaluated in Pelargonium leaves.
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region of the major stems or branch shoots. The firsi leaf below the apical region is defined as a young leaf and adjacent to this leaf is the fully expanded leaf. The young leaf is small, light green, folded, and wrinkled, whereas the fully expanded leaf is dark green, not folded, and wrinkled. Branches are usually formed from small segments of the apical meristem isolated in the axil of the leaves (Adams, 1971). A green leaf under a newly produced branch may be defined as an old leaf. In this study, plant parts were frozen in liquid N, and stored at -80°C until analyzed unless specified otherwise. The inflorescence of Pelargonium may contain from 50 to greater than 300 individual florets depending on the general nutrition and age of the plant (Adams, 1971). Floret buds were the closed florets containing a calyx and a pedicel approximately 2 mm long (Fig. 1B). The age of the florets was determined by morphology. Young florets have open petals with closed stigmas, whereas fully open florets (Fig. 1C) have open stigmas that are ready for pollination (Fig. 1D). Senescing florets have dried stigmas and the petals have shattered. Cuttings obtained from stock plants as mentioned above were rooted in one-quarter-strength Hoagland solution (Hoagland and Arnon, 1938) for 7 weeks, after which the roots were frozen in liquid N, and stored at -80°C until analyzed.
MATERIALS AND METHODS Plant Preparation
Treatments to lnduce Ethylene Biosynthesis
Unrooted Pelargonium X hortorum cv Snow Mass cuttings were obtained from Oglevee Associates (Connellsville, PA) culture-virus-indexed stock. Three cuttings were rooted in a 15-cm-diameter standard plastic pot with growing medium on a mist bench in a greenhouse under a natural photoperiod and light levels at the Pennsylvania State University (University Park). The growing medium contained 184 g of calcitic lime (CON-LIME, Inc., Bellefonte, PA), 31.4 g of KNO, (Hummert, Inc., St. Louis, MO), 21.2 g of triple superphosphate 0-46-0 (available P,O,; Hummert), 83.4 g of 83% gypsum (Hummert), and 4.03 g of fritted trace elements No. 555 (W.R. Grace and Co., Fogelsville, PA) in 100 L of peat:perlite:soil (4:2:1). The medium was steam sterilized for at least 4 h before use. The pots were placed under a mist (6 s of mist every 6 min) lasting 20 h d-' during the first 3 d and gradually decreased (by 4 h per week) to 8 h d-l by the 4th week. After 4 to 6 weeks, the pots were transferred to a bench without mist. Plants were fertilized at every watering with 250 parts per million of nitrogen from a 15-16-17 (15 N-7.04 P-14.1 K) fertilizer (W.R. Grace). The pots were leached once a week with tap water. Fungicides and pesticides were used as needed. The greenhouse was vented when the day temperature reached 26°C and was kept at 18°C during the night. Following remova1 from the mist, plants were grown for 4 to 6 weeks prior to use.
Four fully expanded leaves were treated by placing the base of the leaf petioles in 10 mL of 5 mM CuCl,, 1 mM 2,4-D, or 2 M sorbitol solutions in a 250-mL bottle. The bottles were then kept under 35 Fmol m-'s-* fluorescent light at 25°C and were sealed for 2 h before ethylene was measured. Ethylene and ACC were measured at 6,12, and 24 h for sorbitol treatments; 3, 6, and 9 h for CuC1, treatments; and 3, 6, 9, and 20 h for 2,4-D treatments. Ethylene treatments were accomplished by placing leaves in a bottle with 10 mL of distilled water as described above, sealing, and injecting a known amount of ethylene to a final concentration of 20 parts per million of ethylene for 14 h. Air was injected as a control, and samples were taken after 14 h. Ethylene measurements were determined by GC as described by Tsai et al. (1988). For ACC analysis, 1 g of leaf tissue was extracted in 1 mL of water. The extract was microfuged at 45008 for 15 min. Three hundred microliters of the supernatant were mixed with 0.5 mL of ice-cold 20 mM HgC1, in a 13- X 100-mm test tube and sealed with a serum cap, and 0.2 mL of an ice-cold mixture of 5% NaOCl and 15 M NaOH (2:1, v/v) were injected to convert ACC to ethylene. The tube was vortexed for 15 s, and ethylene in the test tube head space was determined by GC (Tsai et al., 1988).
Sampling Plant Parts
Pelargonium plants exhibit a high degree of branching (Fig. 1A).As the dominant shoot grows longer, the distance between the apical meristem and lateral buds increase (Adams, 1971). Leaf buds were collected from the apical
RNA Extraction
Total RNA was extracted according to the method of Maliyakal (1992) with minor modifications. Plant parts (including leaves, florets, and flower stalks, 15-20 g fresh weight) were ground to a fine powder in liquid N, and then homogenized in a 30-mL homogenization buffer containing 5 M guanidium thiocyanate, 1%PVP, 0.62% sodium
cDNAs Encoding Ethylene Biosynthetic Enzymes in Pelargonium
629
Figure 1. Terminology used for plant parts. A, Pelargonium plant and the leaves used for analysis. LB, Leaf bud (not shown); YL, young leaf; FL, fully expanded leaf; OL, old leaf. B, Pelargonium flower showing floret bud (FB) and young floret (YF). C, Fully open Pelargonium florets. D, The difference in stigma morphology between a young floret (YF) and a fully open floret (FF).
Sarkosyl, 0.2 M Tris (pH 9), and 1 % /3-mercaptoethanol for two 1-min homogenization periods. There was a 1-min break between the homogenizations to avoid any potential heat buildup. The homogenate was centrifuged at 3,000g and 4°C for 20 min. The supernatant was filtered through two layers of Miracloth (Calbiochem) in a polycarbonate tube and centrifuged at 47,000g for 30 min at 4°C. The supernatant was layered on a 10-mL 5.7 M CsCl cushion and centrifuged at 112,000g for 24 h at 4°C. After the sample was centrifuged, the RNA pellet was collected from the bottom of the tubes as described by Sambrook et al. (1989). Total RNA was extracted from the roots according to the method of Botella et al. (1992a). Roots were extracted by grinding 0.8 g (fresh weight) of tissue to a fine powder in liquid N2 and then shaking for 15 min in 5 mL of a mixture composed of 5 mL of 100 mM Tris (pH 8.0), 5 mM EDTA, 100 mM NaCl, 0.05% SDS, and 1% (v/v) 0-mercaptoethanol and 5 mL of prewarmed (to 60°C) phenol. An additional 5 mL of chloroform:isoamyl alcohol (24:1) were added to the mixture and shaken for 15 min. It was then centrifuged and
the aqueous phase extracted with 5 mL of chloroform: isoamyl alcohol (24:1). The nucleic acids were recovered by ethanol precipitation. The pellet was resuspended in 2 mL of water, and RNA was precipitated by adding 1 volume of 4 M LiCl and incubating at 4°C for 16 h. After the sample was centrifuged at 8000g for 30 min, the RNA pellet was then dissolved in 100 u.L of water, ethanol precipitated, and dissolved in water. cDNA Library Construction
RNA was extracted from Pelargonium leaves treated with 2 M sorbitol for 14 h as described above. Poly(A)+ RNA was purified using oligo(dT)-cellulose according to the method of Sambrook et al. (1989). Double-stranded cDNA was prepared using Amersham's cDNA Synthesis System Plus kit. A 0.8% agarose gel was run, dried, and autoradiographed to evaluate the size and quality of the cDNA. Amersham's cDNA Cloning System Agtll kit was used for preparation of the library. Recombinant phage DNA was packaged, and Escherichia coli strain Y1090 was infected.
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PCR Production of ACC Synthase Probes for Library Screening
ACC synthase probes were produced using PCR and the primers OLE-2, OLE-4, and OLE-6 as described by Botella et al. (1992a). Pelargonium genomic DNA was used as a template, OLE-2 and OLE-4 primers were used, and the PCR parameters were 1 min of template denaturation at 94"C, 1 min of primer annealing at 48"C, and 2 min of primer extension at 72°C for 45 cycles. To produce cDNA probes, total RNA was extracted from the leaves of Pelargonium cuttings stored at 25°C for 4 d, which was then reverse transcribed using the OLE-4 primer (Botella et al., 1992a). The cDNA product was amplified with OLE-2 and OLE-4 primers. The PCR parameters were 1 min of template denaturation at 94"C, 1 min of primer annealing at 48"C, and 2 min of primer extension at 72°C for 45 cycles. The PCR products were further amplified by OLE-2 and OLE-6 primers under 1 min of template denaturation at 94"C, 1 min of primer annealing at 52"C, and 2 min of primer extension at 72°C for 45 cycles. A11 PCR fragments generated were subcloned and sequenced using standard protocols (Sambrook et al., 1989).
cDNA Library Screening and Subcloning DNA Fragments
Approximately 320,000 clones were screened using standard protocols (Sambrook et al., 1989). Filters were prehybridized at 42°C with a solution containing 6X SSC, 5X Denhardt's reagent, 0.1% SDS, 100 mg/mL denatured fragmented salmon sperm DNA, and 50% formamide for 4 h. Hybridizations were performed overnight at 42°C using 1 X 106 cpm/mL probe labeled with [a-32PldCTP by the random-priming method. Hybridized filters were first washed at room temperature in 2X SSC plus 0.1% SDS for 15 min and then two times in 2X SSC plus 0.1% SDS at 62°C for 15 min each and two times in 0.2 X SSC plus 0.1% SDS at 62°C for 15 min each. The dried filters were then exposed overnight at -80°C to Kodak XAR-5 x-ray film with two intensifying screens. Individual positive plaques were purified by severa1 rounds of plating and hybridization. The insert cDNA from positive plaques was excised with EcoRI and ligated into Bluescripts (SKt) (Stratagene). The ligation mixtures were used to transform E. cozi DH5a. Transformants were selected on LB plates containing ampicillin (50 pg/mL) and 5-bromo-4-ch~oro-3-indolyl-~-~galactoside (0.033%, w/v). The alkaline lysis method described by Sambrook et al. (1989) was used to isolate the plasmid DNA.
DNA Sequence Analysis
DNA was sequenced by the dideoxy chain termination method (Sanger et al., 1977). Sequenase version 2.0 (United States Biochemical) was used with universal and reverse M13-sequencing primers. DNA sequence analysis and percentage sequence identity were facilitated by the use of Lasergene applications (DNASTAR, Inc., Madison, WI).
Plant Physiol. Vol. 109, 1995
Northern Analysis
Total RNA was fractionated on a 1.5% agarose gel containing 2.2 M formaldehyde. After electrophoresis, RNA was transferred overnight to a nylon membrane by capillary transfer in 20X SSC overnight. RNA was fixed to the membrane by air drying for 1 h, followed by UV exposure as described in the Hybond-N nylon membrane (Amersham) handbook. Prehybridization and hybridization conditions were the same as described for the library screening. Hybridization was performed overnight at 42°C using 1 X 106 cpm/mL GAC-1, GAC-2, and GEFE-2 probes labeled with [32P]dCTPby the random-priming method. Membranes were then washed at room temperature in 2 X SSC plus 0.1% SDS for 15 min, in 2X SSC plus 0.1% SDS at 62°C for 15 min, and in 0.2X SSC plus 0.1% SDS at 62°C for 15 min. The blot was then exposed to Kodak XAR-5 x-ray film with two intensifying screens at -80°C for 24 to 48 h. Blots were stripped and rehybridized with a pea ribosomal gene (Jorgenson et al., 1982) to ensure that equal amounts of RNA were present in each lane. Genomic Southern Analysis
Genomic DNA (10 pg) was digested and run overnight on a 0.8% agarose gel at 22 V. DNA was then transferred to a nylon membrane by using the VacuGene XL (Pharmacia) alkaline vacuum method. Prehybridizaion and hybridization conditions were the same as described for the library screening. Membranes were then washed in 2X SSC plus 0.1% SDS at room temperature for 15 min, in 2X SSC plus 0.1% SDS at 65°C for 30 min, and in 0.1 X SSC plus 0.1% SDS at 65°C for 30 min. The blot was then exposed to Kodak XAR-5 x-ray film with two intensifying screens at -80°C for 24 to 48 h. RESULTS AND DlSCUSSlON
In this study we used Pelargonium genomic DNA as a template together with OLE-2 and OLE-4 (Botella et al., 1993) primers to isolate an ACC synthase fragment as a probe to screen a stress-induced hgtll Pelargonium cDNA library. Two ACC synthase fragments were isolated. The first, gGAC-1, is 1665 bp long and contains a 1182-bp coding region with two introns, 92 and 391 bp long. The second fragment, gGAC-2, is 1683 bp in length and has three introns, 88, 94, and 331 bp long. RNA-based PCR using RNA from the leaves of Pelargonium cuttings stored at 25°C in the dark for 4 d produced a fragment identical with gGAC-2 except for the absence of introns, and this fragment is designated GAC-2. Approximately 320,000 clones from the stress-induced Pelargonium X hortorum Agtll library were screened using either gGAC-1 or GAC-2 as the probe. When gGAC-2 was used, GAC-1 was isolated. GAC-I is 1934 bp in length with a 79-bp 5' untranslated region, a 409-bp 3' untranslated region, and a 1446-bp (482 amino acids) open reading frame encoding a 54.1-kD polypeptide with a predicted pI of 6.4 (Fig. 2). No corresponding full-length cDNA was isolated from the library using GAC-2 (Fig. 3).
c D N A s Encoding Ethylene Biosynthetic Enzymes in Pelargonium
tatcactactctcgcttctgagtgcctaattatttttqtccaagctctcaqtacgtacqtgttgtacgtgtttac~ta gATGGAGACAGAGCAACAGCTTCTGTCAAAGATTGCAACCAACGACGGACACGGCGAGAACTCCCCATATTTCGA
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OLE-2F L E - 5 AAATCAGCTTTCATCTGACTTGATTGRAGATTGGGTGATTGGGTGAGGTCCAACCCAG~GCCTCATCTGCACTCTTGAGGGAGT N Q L S S D L I E D W V R S N P E A S I C T L E G V
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GAGCAGAGGAAGGGGCGGGAGGTCACATTTGATCCCGACCGTGTCGTCATGAGCGGCGGAGCCACCGGAGCCAACGA
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Figure 2. GAC-I nucleic acid and corresponding amino acid sequences. Horizontal arrows indicate the direction of primers OLE-2, OLE-3, OLE-4, OLE-5, and OLE-6 (Botella et al., 1993). The active site is underlined, and the 1 1 conserved amino acid residues found in aminotransferases are boxed. Near the poly(A)+tail a
potential polyadenylation signal is underlined. Vertical arrows indicate location of introns.
GCTCATCGTCTTCTGTTTGGCCATCCCGGCGACGCTTTCCTTCTCCCATCTCCTTATTATCCAGCAAACGACCGTGA L I V F C L A N P G D A F L L P S m Y Y P A N D R D
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CTTGCAGTGGCGAACCGGAGCTCAGATCATTCCGGTGCACCCAGAGAGGC
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ACTAGAAAGATCATACGCACAAGCACAGAAAGCAACATARAGATTGGGTCGT-GGCGTGCTCTTAACCAACCCATCGAACCC
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TCTAGACACAATTCTGGACCGCGACACTCTCAAGAGCATCGTCAGCTTCGTCACCGACACAACATCCACCTAGTCAT
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CACCACGTGCAACCCCGACCTCATCCACATCGTGTACAGCCTGTCCAAGGACTTGGGCATGCCCGGGTTCCGCGTCGG 936 T T C N P D L I H I V Y S L S g- D L G M P G F ~ V f Y j
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GATCGTGTACTCATTCAACGACGACGTCGTATCCTGCGCACGGAAGATGTCGAGCTTCGGGTTGGTGTC~CCCAGAC I V Y S F N D D V V S C A R K M S S F G L V S T Q T GCAGCACCTTCTCGCAGCGATGCTATCCGACGACGTTTTCGTGGAGCGGTTCCTCGCGGAGCGGAGGCGGTTGGGGAG Q H L L A A M L S D D V F V E R F L A E R R R L G R
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GAGGCACGGCGTGTTCACGAAGGGCTCGAGGAGTTGGGGATTGGGTGTTTAAAGAGCAACGCGGGGCTCTACTTCTG R H G V F T K G L E E L G I G C L K S N A G L Y F W GATGGATTTGCGGAAGCTTCTAGAGAAGAGACGTTTGAGGCGGAGATGGTGCTGTGG~GGTGATTATTAATGAGGT li48 M D L R K L L E E E T F E A E M V L W K V i i N E V
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GAGGTTCATGGACAACCTTAACCTCAGGCTGAGCTTCTCGTCGCTAAGGTACGATGAGAGTGTGATGTTGTCGCCGCA R F M D N L N L.RL S F S S L R Y 3 E S V M L S P H CATATGGTGTCCACGCACTCGCCGCTTGTTCGTGCGAGAACAtaatgagcatgcac~tttttatttgctact?tta~ I
M
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taattaactaattaattgttatttgattg;gtg~gtgctgaatgttggattctttctttg~agaagagaaqctataggagat
g t t t t t a a c c a a t t a c c g t a g a t a t a t a t g c a g t g g a a t t a a g a a a a a t a g a ? ~ t ~ a a a t a t t a a t t c c a t g c a t a t =:716
tatgtaggaaggaattggtacatattttaqqqtttgctgatgttttctttcatcatgaattggtacatatttatg~tg ttcaaggctccaagtgatggatacatggaggattcatttgqatqcatgccttgcaaga?tcagcaa~ctttg~taatt1 8 7 2 a g t g t a t g g t t t g t g a t a a t a a ~ g a t t c t g t g t t g t t t t a a a a a a ~ ~ ~ a a 1934 aa~~~
-
A multigene family for ACC synthase genomic sequences has been reported for zucchini (Huang et al., 1991), tomato (Rottmann et al., 19911, Arabidopsis (Liang et al., 1992; Van der Straeten et al., 1992), mung bean (Botella et al., 1992a, 19931, and rice (Zarembinski and Theologis, 1993). We have identified two different genes coding for ACC synthase in Pelargonium leaves (Figs. 2 and 3). The ACC synthase active sites for GAC-2 and GAC-2 are SLSKDLGMPGFR and SLSKDMGFPGFR, respectively (Figs. 2 and 31, and are similar to other ACC synthase active sites that have been reported (Kende, 1993). It has been suggested that ACC synthase could be an aminotransferase (Huang et al., 1991), since it contains 11 of 12 amino acid residues conserved in aminotransferases (Huang et al., 1991); both GAC-2 and GAC-2 contain these conserved residues. To evaluate the similarities between GAC-2 and GAC-2 with other ACC synthases, DNA sequences were compared using the Clustal method with the PAM250 residue weight table in the MegAlign application of Lasergene. GAC-1 has 70 and 64% sequence identity with GAC-2 at the amino acid and nucleotide levels, respectively. When GAC-2 was compared with ACC synthases reported in
other species we found 49 to 67% sequence identity at the amino acid level and 40 to 53% sequence identity at the nucleotide level. GAC-2 has 47 to 74% sequence identity at the amino acid level and 45 to 66% sequence identity at the nucleotide level for ACC synthases found in other species. Pelargonium genomic DNA was cut with SacI, BsfXI, BamHI, or EcoRV and probed with GAC-2 (Fig. 4A); BsfXI, BamHI, or EcoRV was used to cut DNA that was probed with GAC-2 (Fig. 4B). When the restriction enzyme-digested DNA was probed with GAC-1 probe (Fig. 4A), bands in the BsfXI, SacI, BamHI, and EcoRV lanes were in agreement with our sequence data. However, three faint bands were observed in the SacI lane, which may have been due to cross-hybridization with closely related ACC synthase clones. When the restriction enzyme-digested DNA was probed with GAC-2 (Fig. 4B), the bands in the BsfXI and EcoRV lanes were in agreement with our sequence data. Although there is no BamHI site in GAC-2 (Fig. 4B), two bands were found in the BamHI lane and was due to a BamHI site found in the third intron of this gene. In a previous study a cDNA encoding ACC oxidase, designated GEFE-2, was identified (Wang et al., 1994).
Figure 3. CAC-2 nucleic acid and corresponding amino acid sequences. Horizontal arrows indicate the direction of primers OLE-2 and OLE-6 used in PCR. The active site is underlined, and the 11 conserved amino acid residues found in aminotransferases are boxed. Vertical arrows indicate location of introns.
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TATTTTGATGGGTGGAAGGCTTACGACAACAATCCTTTCCATCTCACCCAAAACCCTCAAGGTGTCATCCAGATGGGC Y F D G W K A Y D N N P F H L T Q N P Q G V I Q H
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DNA was cut with Taql, Hindlll, Xhol, or Pstl and probed with GEFE-2 (Wang et al., 1994) (Fig. 5). Bands in the Xhol and Pstl lanes were as predicted; however, an additional band was observed in the Taql and Hmdlll lanes. The extra band found in each may have been due to cross-hybridization with a closely related ACC oxidase; however, the possibility also exists that additional copies of this gene may be present. Expression of ACC synthase and ACC oxidase during different developmental phases of the plant was examined. GAC-1 expression was not detectable in any of the plant parts at the stages of development tested (data not shown). Whereas high levels of GAC-2 were expressed in the leaf bud, young leaf, young floret, fully open floret, and senescing floret, GAC-2 was expressed to a lesser degree in fully expanded leaves or roots and was undetectable in old leaves and floret buds. GEFE-1 expression was detectable at all leaf ages tested, in young and fully open florets, and in the roots. However, the highest degree of expression was in the senescing florets, and no detectable expression was found in the floret bud (Fig. 6). GEFE-1 expression was higher in older florets (Fig. 6). It is possible that this is in response to pollination, since increased ethylene production is thought to be stimulated by compounds such as ACC or auxin in the pollen (Abeles et al., 1992). Another possibility is the stigmatic recognition of pollen and tissue damage associated with stigma penetration and pollen tube growth (Abeles et al., 1992). Stress-induced ethylene can be caused by mechanical wounding (e.g. cutting, bruising, radiation, and insect infestation), temperature (e.g. freezing, chilling, and high temperature), drought, flooding, or chemicals (e.g. fungal exudates, herbicides, metals, ozone, SO2, or other pollutants) (Abeles et al., 1992). Increases of ACC synthase activ-
ity are typically associated with increased levels of ACC synthase message (Kende, 1993). ACC oxidase is often thought to be constitutive, but there have been reports showing that the activity of ACC oxidase increases in some plants in response to internal or external factors, resulting in ethylene formation (Kende, 1993). GAC-1 expression was not detectable in the control during a time course; therefore, northern blots are not shown for the control in Figures 7 to 9. When Pelargonium leaves were treated with sorbitol,
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Figure 4. A, Southern analysis of Pelargonium genomic DNA digested with Sacl, BsfXI, SamHI, and FcoRV and hybridized with 32 P-labeled GAC-1 as a probe. B, Southern analysis of Pelargonium genomic DNA digested with SsfXI, BamHI, and fcoRV and hybridized with 32P-labeled GAC-2 as a probe. Molecular weight markers are noted on the left of each panel.
cDNAs Encoding Ethylene Biosynthetic Enzymes in Pelargonium
633
When Pelargonium leaves were treated with CuCl2/ there was an increase in ACC and ethylene above the control 3 h following treatment initiation, reaching a maximum at 6 h and followed by a decline 3 h thereafter. This is in agreement with previous studies in other species, which showed that CuCl2 is an effective promoter of ethylene production (Abeles et al., 1992). Both GAC-1 and GAC-2 exhibited an increase in expression 6 h following treatment initiation, whereas GEFE-1 showed a dramatic stimulation 3 h following treatment initiation and remained at a high level of expression for the duration of the experiment (Fig. 8).
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there was a stimulation in ACC and ethylene levels 14 h following treatment initiation, which continued to increase through 20 h. Expression of GAC-1 followed the same trend as ACC and ethylene production, whereas GAC-2 was unaffected. GEFE-1 expression was higher in the control at all times tested (Fig. 7). It has been reported that water stress induces ACC and ethylene production in a variety of plant species and is thought to be responsible for inducing leaf senescence (Abeles et al., 1992). Since GAC-1 and GEFE-1 are induced by water stress, it is possible that they are involved in the production of the ethylene responsible for leaf yellowing and petal shattering in Pelargonium. LB YL FL OL
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Figure 7. Expression of ACC synthase (GAC-1 and GAC-2) and ACC oxidase (GEFE-1) in Pelargonium leaves treated with 2 M sorbitol (Sbt); leaves treated with distilled water served as controls (Ck). A, Ethylene and ACC levels. B, Northern analysis with 10 /xg of total RNA, which were successively hybridized with GAC-1, GAC-2, GEFE-1, and a ribosomal probe.
Wang and Arteca
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Figure 8. Expression of ACC synthase (GAC-1 and C/4C-2) and ACC oxidase (GEFE-1) in Pelargonium leaves treated with 5 ITIM CuCI2 (Cu2+); leaves treated with distilled water served as controls (Ck). A, Ethylene and ACC levels. B, Northern analysis with 10 /j.g of total RNA, which were successively hybridized with GAC-1, GAC-2, GEFE-1, and a ribosomal probe.
It has been shown that auxins have the ability to stimulate ethylene production in the vegetative tissues of a variety of plant species (Abeles et al., 1992). Recently, a cDNA (AIM-1) encoding an auxin-induced ACC synthase was identified and characterized (Botella et al., 1992a). AIM-1 expression is rapidly induced and corresponds with the induction of ACC in mung bean. In this study we have shown that 3 h following the initiation of 2,4-D treatment there was a stimulation in ACC and ethylene production that reached a maximum at 9 h and a decline by 20 h. Neither GAC-1 nor GAC-2 expression corresponded with
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Ck Figure 9. Expression of ACC synthase (GAC-1 and GAC-2) and ACC oxidase (GEFE-1) in Pelargonium leaves treated with 1 HIM 2,4-D; leaves treated with distilled water served as controls (Ck). A, Ethylene and ACC levels. B, Northern analysis with 10 ^g of total RNA, which were successively hybridized with GAC-1, GAC-2, GEFE-1, and a ribosomal probe.
cDNAs Encoding Ethylene Biosynthetic Enzymes in Pelargonium
Anderson, 1989; Schierle e t al., 1989; Ievinsh e t al., 1990), is due t o a n increase i n ACC oxidase activity. In addition, it h a s been shown that in many species ethylene caused autoinhibition in vegetative tissues (Abeles e t al., 1992). GAC-1, GAC-2, and GEFE-1 expression were not affected in response t o exogenous application of ethylene (data not shown). It is possible that other members of t h e t w o families of genes are responsible f o r autocatalytic ethylene production or autoinhibition, but the possiblitiy does exists that Pelargonium leaves do not respond t o ethylene by producing m o r e or less ethylene. A n a r g u m e n t has been made that t h e five genes found i n tomato are functional, although large differences occur in their a m i n o acid sequences. This also a p p e a r s t o be t r u e for GAC-1 and GAC-2, which we have identified in Pelargonium.Both have only 70 and 64% similarities i n a m i n o acid a n d nucleotide sequences, respectively. Rottmann e t al. (1991) suggested that t h e ACC synthase polymorphism may reflect t h e evolution of a family of proteins w i t h different enzymatic properties (Km, pI, etc.) t o effectively utilize AdoMet in different tissues during plant developm e n t or under different stress, which appears t o be t h e case w i t h the t w o Pelargonium genes that we have identified. In conclusion, we have shown that GAC-1 expression is ind u c e d only by stress, whereas expression of GAC-2 m a y be developmentally regulated, a n d GEFE-1 expression is influenced by b o t h stress a n d development. Our future work will follow t w o directions. The first is t o pursue fundamental research t o better u n d e r s t a n d how ACC synthase and ACC oxidase a r e regulated in Pelargonium. The second, more applied direction will be t o use t h e information from this s t u d y for biotechnological application, which will be t o transfer antisense GAC-1, GAC-2, or GEFE-2 into Pelargonium tissues t h r o u g h t h e Agrobacterium transformation o r particle bombardment. By using antisense technology we now have t h e ability to reduce t h e production of ethylene in response t o stress, which provides us w i t h t h e opportunity t o i m p r o v e t h e quality of Pelargonium by reducing leaf yellowing and peta1 abscission d u r i n g shipping a n d storage. Received April 18, 1995; accepted July 8, 1995. Copyright Clearance Center: 0032-0889/95/109/0627/10. The EMBL, GenBank, and DDBJ accessions numbers for the sequences reported in this article are U17228 (gGAC-I), U17229 (GAC-I), U17230 (gGAC-2), U17231 (GAC-2), and U07953 (GEFE-2). LITERATURE ClTED
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