I IV : 7‘ j _ ‘sr: i. =:3;*§;i c? . fifl Jr '3‘ . c . L 55:*£i:53= ' r. ‘ - v“ ‘* mfi'fi‘tfi .3, f‘ 4n . w‘ ' par, , ‘ 1:3;éAr--'{r‘ I: - «Lt. 1c.;;‘ ‘.2; , .:«:.A .~ 1 ‘ .. t #15 fimzs t5 a v: ‘5 1, ‘1 .. n nnt '4 3 L .- ‘f 'al‘f‘ - ‘4“..I- _— - ii?” Ii i . . yaw—”A ‘1 I} vii-.1. A ~tawm [31"l 3 D. . :J; L“ .g‘fi'qfifll ‘ y’lv' " ks « K -3;.‘..'..; .A-..< AS..- "9'4? ‘22:. - I’I‘ :1 .11"le . ‘ . ‘- . ”v...; yA mfiwuw w*wwfifii , 31%;! b!" ', ‘ ‘W ‘- . ,9!» "z 2‘ ' .> - ‘l‘fm -‘ Z . 43v 31st.t%f3. ‘4’ fig , - w s - waf‘ifi. I . ' -49 - g; .. '- A au‘ruuy..." .:. :::.:::':::r.:: ~ ‘..!:L§$"‘l'5"“' . 33-- ‘ ,~ _A “ :4: 1 .~¢. n ‘ p“ , ‘- "T. .‘ £1.41 ~J~$v .t" “v . N. w L; , 5:2... - ethylene (Adams and Yang, 1979). The conversion of AdoMet to ACC and of ACC to ethylene is mediated by ACC synthase and ACC oxidase, respectively (for a review, see Kende, 1993). ACC synthase is a pyridoxal phosphate- dependent enzyme related to aminotransferases. It has been purified from a number of tissues, and numerous genes encoding ACC synthase have been cloned (Zarembinski and Theologis, 1994). ACC synthase is encoded by a multi-gene family in all plants that have been examined, and the members within the gene family are often differentially regulated in response to auxin treatment, wounding, and a variety of other stimuli (Kende, 1993). Sequence similarity of individual ACC synthase isoforms across species is often greater than within a species (Zarembinski and Theologis, 1994). ACC oxidase is related to dioxygenases, however, it differs from other dioxygenases in that it requires C02 as a cofactor, presumably being required for activation. Further, dioxygenases utilize a- ketoglutarate as a co-substrate by reducing it to succinate, whereas ACC oxidase does not require at-ketoglutarate for activity (Prescott, 1993). Many semi-aquatic plants possess the capacity to elongate upon submergence (Ridge, 1987). In many cases, this response is mediated by ethylene and also requires either auxin or gibberellin. Regnellidium diphyllum is a semi-aquatic fern that undergoes ethylene-mediated elongation when submerged (Musgrave and Walters, 1974; Cookson and Osborne, 1978). Elongation of Marsilea quadrifalia is also promoted by submergence (Karsten, 1888, quoted by Cookson and Osborne, 1978), but the 22 involvement of ethylene in this process has not been demonstrated with this fern. Cookson and Osborne (1978) also showed that the pathway of ethylene biosynthesis in Regnellidium may be different from that of higher plants. Their report was published prior to the full elucidation of the ethylene-biosynthetic pathway in higher plants and did not deal with the production of ACC and the enzymes of the ACC-dependent pathway. Because of the physiological response of Regnellidium to ethylene and the possible evolutionary implications of an alternative ethylene-biosynthetic pathway in lower plants, we have re-investigated ethylene biosynthesis in ferns. Our results show that both ACC synthase and ACC are present in Marsilea and Regnellidium. However, the capacity to convert ACC to ethylene is either lacking or present at low levels. Thus, a major part or all of the ethylene produced by these two ferns appears to be synthesized via an ACC- independent pathway. Studies done with aquatic plants, such as deepwater rice have shown that ethylene can mimic the effect of submergence on growth. A series of experiments were conducted to investigate whether the submergence response differed between Regnellidium and higher plants. The results demonstrated that Regnellidium exhibits a similar elongation response as higher plants, and this may serve as a usefirl condition for further studies on the ethylene biosynthetic pathway in this fern. Some of the results presented here have been published previously (Chemys and Kende, 1996). MATERIALS AND METHODS Plant material and growth conditions. Regnellidium diphyllum Lindm. was purchased from Maryland Aquatic Nurseries (J arretsville, MD, USA). Marsilea quadrzfalia L. was obtained from the Botany 23 Greenhouse Collection at Michigan State University. Both ferns were grown in pots containing wet soil under greenhouse conditions with supplementary light. For most experiments, leaflets were removed from fronds between 5 and 10 cm in height and with newly expanded leaflets. As control, Arabidapsis thaliana (L.) Heynh. (ecotype Columbia) was used throughout the study. Plants were grown under a photoperiod of 16 h light (250 pmol rn'2 s") and 8 h dark at 20 to 23°C. Lower leaves from 6- to 8-week-old plants were used in the experiments. Application of test substances. Leaflet discs, 1 cm in diameter, were cut from fronds of either Marsilea or Regnellidium, and about eight discs were placed, abaxial surface down, on filter paper that had been moistened with a solution of the inhibitor or water as control inside 25-m1 Erlenmeyer flasks. In the case of Arabidapsis, two or three leaves were placed, abaxial surface down, on filter paper. The flasks were then sealed, and 1 ml of the gas phase was removed at 4-h intervals for ethylene determination by gas chromatography. To study the requirement for iron, the leaf discs or leaves were incubated with 1,10-phenanthroline. To test for reversal of inhibition by 1,10- phenanthroline, the leaves or leaf discs were transferred into flasks with 0.2 M FeSO4. Aminoethoxyvinylglycine (AVG) was a gift of Hoffinann—LaRoche (Nutley, NJ, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA). Radiolabeling. Leaflet discs were placed, abaxial surface down, on filter paper moistened with 74 kBq of [U-“C]methionine (9.5 GBq/mmol, New England Nuclear, Boston, Mass, USA) or 37 kBq of [2,3-‘4c1Acc (3 GBq/mmol, CEN Saclay, Gif-sur-Yvette, France) in 1 ml 24 of distilled water. Ethylene was allowed to accumulate for 4 h, after which time 1 ml of the gas phase was removed to determine the ethylene concentration. The gas inside the flask was removed using a 60-ml syringe while being replaced with saturated M)2SO4 to allow complete removal by preventing formation of a vacuum inside the flask, or with air if the tissue was used for ACC analysis. The syringe was immediately capped, a l-ml gas sample was removed to determine losses of ethylene, and 0.4 ml of 0.2 M mercuric acetate in methanol was added to adsorb the ethylene. In the case of ACC assays, the gas in the vial was removed with a lO-ml syringe, and 0.2 ml of 0.2 M mercuric acetate in methanol was added to the syringe. The syringes were shaken in the cold for 2 h, following which the mercuric acetate was transferred to a scintillation vial to which 10 ml of scintillation fluid had been added. The yellow coloration caused by mercuric acetate was eliminated by adding a few drops of glacial acetic acid. The radioactivity was determined using a liquid scintillation counter. ACC determination and thin-layer chromatography. Approximately 1 g FW of leaflets was frozen in liquid nitrogen and ground using a mortar and pestle. ACC was extracted in 100 mM Na-phosphate buffer, pH 11.5, and the homogenate was centrifuged at 10,000g for 15 min. The supernatant was used directly for ACC determination according to Lizada and Yang (1979). To confirm that ACC was, in fact, present, leaflets from Marsilea and Regnellidium and leaves from Arabidapsis were frozen in liquid nitrogen and extracted with 80% methanol (2 ml/g FW). The extracts were centrifuged at 10,000g for 15 min. The supernatant was evaporated to dryness, and the resulting residue was resuspended in 50 pl 80% methanol. Ten to 20 pl of each extract were chromatographed on 0.2-mm cellulose glass plates 25 using n-butanolzacetic acidzwater (60:15:30 v/v/v) as solvent (Boller et al., 1979). The plates were divided into 1- or 1.5-cm zones that were removed and eluted with 100 mM Na-phosphate buffer, pH 11. The eluate was assayed for ACC according to Lizada and Yang (1979). Standard ACC (0.1 pmol) was chromatographed in parallel with the extracts, and, after removal of the zones with samples, the remaining part of the plates was sprayed with ninhydrin reagent to determine the Rf of ACC. AC C synthase assay. Approximately 2 to 3 g F W of leaflet tissue was ground in liquid nitrogen and extracted with 1 mI/g FW of buffer (100 mM Na-phosphate, pH 8.0, 5 mM DTT, 10 pM pyridoxal 5-phosphate). The slurry was centrifuged at 15,000g for 20 min at 4°C. The supernatant was dialyzed overnight against two changes of buffer (10 mM Na-phosphate, pH 8.0, 5 pM pyridoxal S-phosphate) at 4°C. The extract was concentrated to ca. 1/5 to 1/3 its original volume by placing the dialysis bag in dry polyethylene glycol 8000. For the assay of ACC synthase activity, 0.6 ml of the extract was combined with 100 pl of l M Na-phosphate, pH 8.0, and either 100 pl of 1 mM S-adenosyl-methionine (AdoMet) or H20 in a 9-ml tube, and the reaction mixture was shaken at 30°C for 1 h. The ACC produced was determined by the method of Lizada and Yang (1979). Submergence treatment. Regnellidium diphyllum plants with newly expanded leaflets, petiole lengths between 3 and 5 cm and containing a 2 cm portion of the rhizome, were used. A 2-g weight was attached to the rhizome, and the plant submerged in a 1 1 jar such that the leaflets were below the water surface. Leaflets from these plants were used for ethylene determination as described above. For ethylene treatment experiments, the plant with a 26 weight attached was suspended in a 1 l beaker such that the leaflets were held above the water surface. The beaker was placed inside a dessicator into which ethylene was injected. After each day, the dessicator was aerated and the ethylene replaced. Water was refilled as required. Leaflets from these plants were used for ethylene determination as described above. Cloning of a fragment of A CC synthase from Marsilea. For RNA isolation, fresh leaflets were fiozen in liquid nitrogen and ground to a fine powder using a morter and pestle. Frozen tissue was added to extraction powder [4 M guanidine thiocyanate, l M ammonium thiocyanate, 3 M sodium acetate, 0.5 % polyvinylpyrrolidine (MW 360, 000), 1% B-mercaptoethanol, 0.5% laurylsarcosine], and the homogenate placed at 65°C for 30 min. The homogenate was centrifirged at 10,000g for 10 min, and the supernatant extracted with chloroform/isoamyl alcohol (24: 1). The top phase was removed and two volumes of absolute ethanol were added. The nucleic acids were centrifuged at 10,000g for 10 min, and the pellet washed twice with 80% ethanol. The pellet was dissolved in cetyltrimethylarnmoniumbromide (CTAB) Buffer [1% CT AB, 10 mM EDTA-Naz, 0.70 M NaCl, 0.5% polyvinylpyrrolidine (MW 360,000), 1% B-mercaptopethanol], and heated at 65° C to assist in resuspension. Undissolved material was spun down at 10,000g for 10 min, and the supernatant extracted with CHCl3/isoamyl alcohol. The top phase was transferred to a clean tube and 1/ 10th volume of 10% CTAB (0.7 M NaCl, 10% CTAB) was added. The mixture was again extracted with CHCl3/isoamyl alcohol, and two volumes of CTAB precipitation buffer (1% CT AB, 50 mM Tris-HCl pH 7, 10 mM EDTA), was added to the supernatant. After 30 min at room temperature, the solution was spun (10,000g for 10 min), and the 27 pellet washed with 80% ethanol and resuspended in water. Reverse transcription (RT) was carried out on 4 pg total RNA using an oligo d(T) primer and MMLV reverse transcriptase (Gibco-BRL). The procedure followed was the same as described in the MMLV reverse transcriptase instruction manual. Five pl of the 20 pl RT reaction was used in PCR amplification. Degenerate primers HK62 (CTC GAA TTC ACC AAY CCN TCA AAY CCN YTR GG) and HK63 (CTC AAG CTT ACN ARN CCR AAR CTY GAC AT) designed against block 4 and block 6, respectively, of ACC synthase (Kende, 1993) and containing EcaRl (HK62) and HindIII (HK63) at the ends of each the primers were used in the amplification. Conditions used for the PCR reaction were as follows: 94 1 min, 40 1 min, 72 45 sec for 35 cycles. The PCR product was purified from an agarose gel, digested with HindII and EcoRl, and ligated into pBluescript. RESULTS Effect of exogenous AC C (in viva AC C oxidase activity). As a first test whether the biosynthetic pathway in ferns differs from that in higher plants, ACC was applied to leaflet discs of both Marsilea and Regnellidium, and ethylene production was monitored over a 24-h period. Because wounding did not increase ethylene production in either Marsilea or Regnellidium (results not shown), leaflet discs were used throughout the study to improve the uptake of ACC or other test compounds. Leaves of Arabidapsis, which produce ethylene via the ACC pathway, were used as a positive control in all experiments. Addition of 0.1 mM ACC increased ethylene production in Arabidapsis while concentrations of up to 1 mM had little, if any, effect in Marsilea and in Regnellidium (Figure 2.1; see also Table 2.2). 28 Eflect of inhibitors of the higher-plant ethylene biosynthetic pathway. Substances that are known to inhibit ethylene production in higher plants were tested for their effect on ethylene synthesis in Marsilea and Regnellidium. Aminoethoxyvinylglycine (AVG), a potent inhibitor of pyridoxal phosphate-mediated enzyme reactions, such as catalyzed by ACC synthase (Boller et al., 1979), strongly inhibited ethylene formation in Arabidapsis when applied at 0.1 mM. AVG up to 1 mM did not decrease ethylene formation in either Marsilea or Regnellidium (Figure 2.2). To address the question whether the inability of added ACC to increase ethylene production was a consequence of saturated ACC oxidase, a-aminoisobutyric acid (AIB), a competitive inhibitor of ACC oxidase (Satoh and Esashi, 1983), was applied. Concentrations of up to 5 mM AIB had no effect on ethylene production in Marsilea and in Regnellidium, but completely inhibited ethylene formation in Arabidapsis (Figure 2.3). E fleet of Iron. Ethylene production in higher plants is dependent upon ferrous iron due to the requirement of ACC oxidase for it as a cofactor (Bouzayen et al., 1991). To test whether iron had any effect in Regnellidium and Marsilea, leaf discs were incubated with 1,10- phenanthroline, a chelator of ferrous iron. This treatment resulted in inhibition of ethylene production in Arabidapsis, Marsilea, and Regnellidium. This inhibition could be reversed by incubation of the leaves or leaf discs on 0.2 M F eSO4 (Figure 2.4). Thus, despite the apparent absence of ACC oxidase in the ferns, iron is required for ethylene production. 29 1200 - 300 800» 400 , E 0’ U. 24- Pb) 18’L :9 12 ~ g 6. '55 0 :E LIJ 200 ~ 100- 8 1’2 16 2‘0 24 Time (h) Figure 2.1A-C. Effect of l-arninocyclopropane-l-carboxylic acid (ACC) on ethylene production in Arabidapsis (A), Marsilea (B), and Regnellidium (C). Data points are the means of 4 observations from a representative experiment, and the error bars represent SE. 0 , control; I , 0.1 mM ACC; O, 1 mM ACC. This experiment was repeated four times with similar results. 30 4‘ Detection of A C C in ferns. The presence of ACC was detected in leaflets of both Regnellidium and Marsilea (Table 2.1) and confirmed by thin-layer chromatography (Figure 2.5). Zones of the tlrin- layer chromatograrns were scraped off and assayed. Those that yielded ethylene had Rf values corresponding to that of standard ACC (Figure 2.6). Radialabeling experiments. To confirm the conversion of methionine to ACC and to check whether methionine was converted to ethylene, radiolabeling experiments were performed. Little [”C]methionine was converted to [14C]ethylene by either Marsilea or Regnellidium (Table 2.2; see also Cookson and Osborne, 1978). To assess whether the small conversion observed occurred via an ACC-dependent route, [”C]methionine was applied together with [12C]ACC to leaflet discs. The addition of [12C]ACC should decrease incorporation of label into ethylene if the conversion occurs via ACC as an intermediate. This occurred in Arabidapsis but not in Marsilea and Regnellidium (Table 2.2). Further evidence in support of an ACC-independent pathway in ferns came from experiments where ['4C]methionine was fed to leaflet discs of Regnellidium, and the specific radioactivity of C-2 and C-3 of ACC was compared to that of ethylene. The specific radioactivity of the two carbon atoms of ACC that give rise to ethylene was close to 30-fold higher than the specific radioactivity of ethylene, indicating that the major part of ethylene was not derived from ACC (Table 2.3). In contrast, the specific radioactivities of C-2 and C-3 of ACC and of ethylene were approximately the same in Arabidapsis, as would be expected if ethylene were derived from ACC. 31 ‘_Ll. Um ' :9 20. 2 10. g i o :E “J 300- C 2001 100- o 0 4 8 12 16 20 24 Time (h) Figure 2.2A-C. Effect of aminoethoxyvinylglycine (AVG) on ethylene production in Arabidapsis (A), Marsilea (B), and Regnellidium (C). Data points are the means of 4 observations from a representative experiment, and the error bars represent SE. 0 , control; I , 0.1 mM AVG; O, 1 mM AVG. This experiment was repeated four times with similar results. 32 20. 10 60. B 40- Ethylene (nl g'1FW) 200 » 100- 02181421621124 Time (b) Figure 2.3A-C. Effect of a-aminoisobutyric acid (AIB) on ethylene production in Arabidapsis (A), Marsilea (B), and Regnellidium (C). Data points are the means of 4 observations from a representative experiment, and the error bars represent the SE. 0 , control; 6, 5 mM AIB. This experiment was repeated four times with similar results. 33 Ethylene (nl g"1=w h“) 12 16 20 24 28 Time (h) Figure 2.4. Ethylene production in the presence of the iron chelator 1,10-phenanthroline (PA) in Arabidapsis (A), Marsilea (B), and in Regnellidium (C). Transfer of the leaf discs or leaves to 0.2 M FeSO4 reversed the inhibition. The position of the arrow indicates the time at which transfer occurred. The experiment is representative of two similar experiments. 0, control; I , 0.2 mM PA; 0 , 2 mM PA. 34 Table 2.1. 1-arninocyclopropane-1-carboxylic acid (ACC) levels in Arabidapsis, Marsilea and Regnellidium. ACC was extracted from the tissue and measured by the method of Lizada and Yang (1979). The data represent the average of three experiments i SE. Sample ACC levels (nmol g" FW) Arabidapsis 0.28 i 0.05 Marsilea 0.12 i 0.09 Regnellidium 0.34 i 0.11 To further test whether ACC was converted to ethylene in fern leaflets and to verify the results of Figure 1, radiolabeling studies were carried out using [”C]ACC. Some conversion of [”C]ACC to [”C]ethylene occurred in both Marsilea and in Regnellidium but, compared to the conversion in Arabidapsis, it was low (Table 2.4). Extracts of Regnellidium leaflets and Arabidapsis leaves that had been labeled with [14C]methionine were chromatographed on thin-layer plates. The chromatogram was divided into ten zones, and the eluate of each zone was subjected to the ACC assay of Lizada and Yang (1979). The amount of ethylene produced from each zone, as well as the level of radioactivity associated with it, were determined (Figure 2.5). A region of the thin-layer chromatogram, which corresponded to the Rf of standard ACC, released ethylene in the Lizada-Yang assay and also exhibited a peak of radioactivity. When extracts of [”C]ACC-labeled leaflets were chromatographed, no radioactive metabolites were detected (results not shown). Presence of A CC Synthase. Because ACC is present in both ferns, we tested whether ACC synthase activity was present as well. Low but significant levels of ACC synthase activity could 35 __l Ethylene (nI) o 0.25 0.5 10.1751 1:0 Figure 2.5A-C. Thin-layer chromatography of extracts from the leaflets of Marsilea (A), leaves of Arabidapsis (B), and leaflets of Regnellidium (C). After completion of the chromatographic nm, the plates were divided into 1-cm zones which were scraped off and analyzed for ACC by the method of Lizada and Yang (1979). Ethylene evolved per l-cm zone is plotted against the Rf. The region of the chromatogram which yielded the highest level of ethylene in the ACC assay corresponded to the Rf of ACC (0.45) run as a standard on the plates and stained with ninhydrin (illustrated in the bar above the charts). Similar results were obtained in three experiments. 36 Table 2.2. Incorporation of radiolabeled methionine into ethylene. Leaves from Arabidapsis and leaflet discs from Regnellidium and Marsilea were incubated on filter paper moistened with 74 kBq of ['4C]methionine in 1 ml of water. In a separate experiment, the leaves or leaflet discs were incubated with ['4C]methionine to which unlabeled ACC (100 pM) had been added. All experiments gave similar data. Sample Total C2H4 [fiC]C2H4 (nl h'l g’l) (Bq nmol'l) Arabidapsisa 9.4 396 Regnellidium” 9.3 86 Marsileac 2.0 19 Arabidapsisb + ACC 64.3 86 Regnellidiumb + ACC 9.2 73 Marsileac + ACC 3.1 20 ‘ Average of 4 experiments b Average of 3 experiments ° Average of 2 experiments consistently be detected in leaflets of Marsilea and Regnellidium (Table 2.5). The inhibition of this activity by the addition of 0.1 mM AVG to the Regnellidium extract confirmed its identity as ACC synthase. Cloning of the gene for AC C Synthasefiom Marsilea. ACC synthase has seven regions that are highly conserved among species (Kende, 1993). Two such regions were chosen for design of degenerate PCR primers. These primers were used in RT-PCR to amplify a fragment of the predicted size, around 320 bp. The sequence of the fragment shared a high degree of identity, at the amino acid level, with other ACC synthases. An alignment of the deduced amino acid sequence of the cloned Marsilea ACC synthase gene fragment with several other ACC synthases is shown in Figure 2.7. Northern blots of RNA from Marsilea, Regnellidium, pea, and Arabidapsis using the cloned fragment as a probe were tried but there was cross-reaction with Arabidapsis and pea. It would therefore seem that in order to draw conclusions about Marsilea ACC synthase gene expression, the full-length sequence would need to be 37 Table 2.3. Determination of the specific activity of ACC and ethylene produced by Regnellidium and Arabidapsis. Leaflets from Regnellidium and leaves from Arabidapsis were placed on filter paper saturated with 74 kBq [14C]methionine in 25-ml Erlenmeyer flasks. After 4 h of incubation, the gas phase was transferred to a syringe, an aliquot of the gas phase was analysed for ethylene, and the ethylene was adsorbed to mercuric acetate. The leaves or leaflets were analyzed for ACC according to Lizada and Yang (1979). The ethylene produced during the ACC assay was transferred to a syringe, measured, and adsorbed to mercuric acetate. The radioactivity associated with the mercuric acetate was determined by scintillation counting. The data shown are from one experiment that was repeated with similar results. Sample ['“C]C2H4 [”C]ACC Ratio (kBq nmol'l) (kBq nmol") [”C]ACC / [”C]C2H4 Arabidapsis 329 385 l .2 Regnellidium 41 l 136 28 Table 2.4. Incorporation of radiolabeled ACC into ethylene. Leaves of Arabidapsis and leaflet discs fi'om Regnellidium and Marsilea were placed on filter paper containing 37 kBq [”C]ACC in 1 ml of water. The ethylene produced was adsorbed to mercuric acetate, and the radioactivity determined by scintillation counting. The data represent the average of three experiments. Sample Total C2H4 [”C]C2H4 (n1 h" g“) (kBq nmol") Arabidapsis 17.7 2.5 Regnellidium 9.4 0.20 Marsilea l .5 0.04 38 a -4 ’a m 99, a .3: .5 g “5 2 0 m (U o .9. =6 " '0 m (U (r n: "‘ r: E 1.5 5 v a) g 1 r: a) a) '5. .053: 5 ' 5 NJ Lu 0 .05 .35 .65 .95 .05 .35 .65 .95 0 Rf R: Figure 2.6A-C. Thin-layer chromatography of extracts from leaves of Arabidapsis (A,C) and leaflets of Regnellidium (B,D) which had been treated with [”C]methionine. After completion of the chromatographic run, the plates were divided into 1.5-cm zones which were scraped off and analyzed for ACC by the method of Lizada and Yang (1979). The radioactivity associated with ethylene (A,B) and the amount of ethylene evolved per 1.5- cm zone (C,D) is plotted against the Rf. The region of the chromatogram which yielded the highest level of ethylene in the ACC assay corresponded to the Rf of standard ACC. 39 Table 2.5. ACC synthase activity in Marsilea and Regnellidium leaflets and in Arabidapsis leaves. The data represent the mean of three experiments i SE. Sample C2H4 (pmol h'1 g'1 FW) Marsilea 2.1 i 0.5 Regnellidium 2.5 i 0.6 Arabidapsis 3.1 :l: 0.7 Regnellidium + heat not detected Regnellidium + 100 pm AVG 0.4 i 0.2 obtained and gene-specific probes designed. Eflect of Submergence. Previous studies have shown that Regnellidium, like rice, responds to submergence with petiole elongation. In rice, submergence results in increased ethylene synthesis. We tested whether the submergence of Regnellidium also results in increased ethylene synthesis. Submergence resulted in an increased growth rate; this increase in growth could be partially mimicked by application of ethylene (Figure 2.8). Leaf discs from plants that had been submerged had higher rates of ethylene evolution (Figure 2.9). To test whether this increased production resulted from synthesis or release due to accumulation following submergence, ethylene evolution of submerged leaflets was monitored at 25° and 4° C. Ethylene evolution decreased by 78% and 82% in submerged and control leaflets respectively, incubated at 4°C (Figure 2.9). As release of the entrapped ethylene would occur equally well at both 4°C and 25°C, these results indicate that at least part of the increased ethylene evolved from submerged leaflets is due to increased synthesis. 40 DISCUSSION The results support the notion that at least the major part, if not all, of the ethylene synthesised by Regnellidium and Marsilea is derived from a precursor other than ACC, despite the fact that the initial step of the pathway, namely the formation of ACC from methionine, is functional in both ferns. This conclusion is based upon the following results obtained with Marsilea and Regnellidium leaflets: (1) ACC failed to stimulate ethylene formation, (2) AVG and AIB failed to inhibit ethylene formation, (3) ACC and ACC synthase were present, (4) [”C]methionine was converted to [”C]ACC but [14C]methionine and [”C]ACC were poorly converted into ethylene, and (5) the specific radioactivity of ACC formed from [14C]methionine was much higher than the specific radioactivity of the ethylene evolved. In all of these experiments, leaves of Arabidapsis were used as positive controls and gave results that were consistent with the functioning of the ACC-dependent pathway. These results are consistent with those of Osborne et al. (1996). Two pathways of ethylene biosynthesis have been described in microorganisms (for a review, see Fukuda et al., 1993). In Escherichia coli, ethylene is synthesized from methionine via 2-keto-4-methylthiobutyric acid (KMBA). The enzyme that catalyzes this conversion has been purified and has been shown to be a NADHzFe3+ oxidoreductase. In the second pathway, present in Penicillium digitatum and in Pseudomanas syringae, ethylene is synthesised from or-ketoglutarate. The enzyme catalyzing this reaction has been purified, and the gene encoding it has been cloned. It is similar to higher-plant ACC oxidase in that it requires oxygen and Fe”, and is affected similarly by a number of inhibitors. The low level of conversion of [14C]methionine to [14C]ethylene in ferns 41 Tomato Pea Marsilea Rice Apple Cucumber Tomato Pea Marsilea Rice Apple Cucumber HFJHPHAH ISE§KS---EV IAE IBH-DTD vstIQ---BM VAErVBARGG- VAEEIBDRo-- ME LKBRSSE 1 1 ,trvrsLskrns Figure 2.7. Alignment of the deduced amino acid sequence of the 300 bp RT-PCR product obtained from Marsilea with ACC synthases from pea (AF 0164959), tomato (AB013100), rice (X97066), apple (U738l6), and winter squash (U37774). The amino acid shaded in black are identical in all sequences. Grey areas indicate similar amino acids in all sequences. 42 submerged control ethylene + + + 16 14 i 12 b 10 h Peliole length (cm) 10.50300 Days Figure 2.8. Submergence response in Regnellidium. Regnellidium plants were submerged such that the leaflets were held below the surface of the water. Ethylene treatment was carried out as described in Materials and Methods. The data shown here are representative of three experiments. 43 submerged 25'C I control 25'C submerged 4'C 1:] control 4°C J _L 0" Ethylene (9" FW 11") Time (h) Figure 2.9. Ethylene evolution at 25° and 4° C from control (non-submerged) Regnellidium leaflets and from Regnellidium leaflets that had been submerged for three days. The data shown here are representative of two experiments. (Table 2.2; Cookson and Osborne, 1978) is evidence against the existence of the KMBA- dependent pathway in ferns. The second pathway does not seem to operate in ferns either as [U-“Cj glutamate added to leaflets was not converted to ethylene (results not shown). However, the pathway that does exist in ferns is similar to that in higher plants and bacteria in having a requirement for iron. A number of other ACC-independent pathways of ethylene synthesis have been reported. Copper-induced ethylene biosynthesis in Spiradela may be mediated by peroxidation of unsaturated fatty acids, e.g., linoleic acid (Mattoo et al., 1992). An ACC- independent pathway is also indicated in lichens (Lurie and Garty, 1991), sweet potato infected by Ceratacystisfimbriata (Hyodo and Uritani, 1984), stressed pine needles (Chen and Wellburn, 1989), and mung bean epidermal cells (Todaka and Irnaseki, 1985). Because of its simple chemical structure, there are many compounds that may be converted to ethylene through various chemical reactions. This makes identification of the precursor to ethylene difficult. The evolution of land plants required a series of changes in vascular development, and potentially in biochemical pathways. It is possible that one such change is the adjustment to differing water conditions and different gas concentrations, including oxygen. The acquisition of an ethylene biosynthetic pathway that was more highly regulated may be an event that is related to the transition fiom water to land. Semi- aquatic ferns may possess a means of sensing of the low oxygen concentrations that would be present when a plant is submerged; and this sensing may involve ethylene production but is not necessarily responsive to the same stimuli that would exist for a land plant. Regnellidium has a similar adaptation response to submergence as higher 45 plants, but appears to have a different pathway for synthesis of the hormone that is involved in this response. Another variation in the ethylene biosynthetic pathway in an aquatic plant is that of Patamagetan pectinatus which responds to submergence by stem elongation and contains ACC but does not produce ethylene (Summers et al., 1996). The submergence effect provides a useful physiological condition in which increased ethylene synthesis can be related to changes in ACC levels, ACC synthase activity, and potentially be useful in the purification of enzymes of the fern pathway. The presence of ACC synthase and the apparent absence or very low in viva activity of ACC oxidase in Regnellidium and Marsilea is interesting from an evolutionary standpoint. Because ACC synthases and arninotransferases are related and because phylogenetic analysis indicates that the polymorphism of ACC synthase genes arose prior to the divergence of monocots and dicots (Zarembinski and Theologis, 1994), it will be interesting to compare the ACC synthase gene(s) and proteins(s) of Regnellidium and Marsilea to those of higher plants. This is difficult to do only fiom the partial sequence of Marsilea ACC synthase that is from a region conserved in all ACC synthases. Obtainment of the full-length clone should be usefirl in this regard. Similarly, it will be interesting to compare the ethylene-forming enzyme(s) of these two ferns to those of fungi and bacteria and to ACC oxidase of higher plants. REFERENCES Adams DO, Yang SF (1979) Ethylene biosynthesis: identification of l-arninocyclopro pane-l-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc Natl Acad Sci USA 76: 170-174 Boller T, Hemer RC, Kende H (1979) Assay for and enzymatic formation of an ethylene precursor, 1-arninocyclopropane-1-carboxylic acid. Planta 145: 293-303 Bouzayen M, Felix G, Pech J C, Boller T (1991) Iron: an essential cofactor for the 46 conversion of l-arninocyclopropane-l-carboxylic acid to ethylene. Planta 184:244-247 Chen YM, Wellbum, AR (1989) Enhanced ethylene emissions from red and Norway spruce exposed to acidic mists. Plant Physiol 91: 357-361 Chemys J, Kende H (1996) Ethylene biosynthesis in Regnellidium diphyllum and Marsilea quadrtfalia. Planta 200: 113-1 18 Cookson C, Osborne, DJ (1978) The stimulation of cell extension by ethylene and auxin in aquatic plants. Planta 144: 39-47 Fukuda H, Ogawa T, Tanase S (1993) Ethylene production by micro-organisms. Adv Microbiol Physiol 35: 275-306 Hyodo H, Uritani I. (1984) Ethylene production in sweet potato root tissue infected by Ceratacystisfimbriata. Plant Cell Physiol 25: 1147-1152 Kende H (1993) Ethylene biosynthesis. Annu Rev Plant Physiol Plant Mol Biol 44: 283- 307 Lizada MCC, Yang SF (1979) A simple and sensitive assay for l-arninocyclopropane-l- carboxylic acid. Anal Biochem 100: 140-145 Lurie S, Garty J (1991) Ethylene production by the lichen Ramalina duriaei. Ann Bot 68: 3 l 7 -3 19 Mattoo AK, Mehta RA, Baker JE (1992) Copper-induced ethylene biosynthesis in terrestrial (Nicotiana tabacum) and aquatic (Spiradela aligarrhiza) higher plants. Phytochemistry 31: 405-409 Musgrave A, Walters J (1974) Ethylene and buoyancy control rachis elongation of the semi-aquatic fern Regnellidium diphyllum. Planta 121: 51-56 Osborne DJ, Walters J, Milborrow BV, Norville A, Stange LMC (1996) Evidence for a non-ACC ethylene biosynthesis pathway in lower plants. Phytochemistry 42: 51-60 Prescott AG (1993) A dilemma of dioxygenases (or where biochemistry and molecular biology fail to meet). J Exp Bot 44: 849-861 Ridge I (1987) Ethylene and growth control in amphibious plants. In: Plant Life in Aquatic and Amphibious Habitats, Crawford, R., ed. Blackwell Scientific Publications, Oxford, UK. pp 53-79 Satoh S, Esashi Y (1983) a-Aminoisobutyric acid, propyl gallate and cobalt ion and the mode of inhibition of ethylene production by cotyledonary segments of cocklebur seeds. Physiol Plant 57: 521-526 47 Summers JE, Voesenek LACJ, Blom CWPM, Lewis MJ, Jackson MB (1996) Patamagetan pectinatus is constitutively incapable of synthesizing ethylene and lacks l- arninocyclopropnae-l-carboxylic acid oxidase. Plant Physiol 111: 901-908 Todaka I, Irnaseki H (1985) Epidermal cells do not contribute to auxin-induced ethylene production in mung bean stem sections. Plant Cell Physiol 26: 865-871 Zarembinski TI, Theologis A (1994) Ethylene biosynthesis and action: a case in conser- vation. Plant Mol Biol 26: 1579-1597 48 Chapter 3 Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of ABA biosynthesis in avocado ABSTRACT Avocado (Persea americana) is a climacteric fruit that exhibits a rise in ethylene as the fruit ripens. This rise in ethylene is accompanied by a series of molecular and biochemical changes that cuhninate in a soft, flavorful fruit. One such change is a rise in abscisic acid (ABA), with the highest level occurring just afier the peak in ethylene production. ABA is synthesized from carotenoid precursors, which in leaves are present in large quantities in comparison with ABA, and thus are not limiting for ABA synthesis. The cleavage reaction produces xanthoxin, which can subsequently be converted into ABA via ABA-aldehyde. Much evidence supports the cleavage reaction as the regulatory step in ABA synthesis. This cleavage reaction is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED), the gene for which was first cloned from the va 4 Viviparous mutant of maize. Three genes encoding 9-cis-epoxycarotenoid dioxygenase cleavage-like enzymes were cloned from avocado fruit. Two genes, PaNCED] and PaNCED3, are strongly induced as the fruit ripens, and share approximately 60% identity at the amino acid level with the maize gene, VpI4. The other gene, PaNCED2, is constitutively expressed during fruit ripening and lacks a chloroplast signal peptide. It is, therefore, unlikely to be involved in ABA biosynthesis. All three genes were heterologously expressed, and the recombinant protein used to test for enzymatic function. Recombinant PaNCEDl and PaNCED3 were capable of in vitra cleavage of 9-cis-xanthophylls into xanthoxin and C25 epoxycarotenoids. Taken together, the results indicate that ABA 49 biosynthesis in fruit is regulated at the level of carotenoid cleavage. INTRODUCTION Fruit ripening involves a complex series of biochemical events in which the tissue undergoes programmed changes in texture, aroma, coloration, flavor, and firmness (Brady, 1987). Clirnacteric species, such as avocado (Persea americana), are characterized by the autocatalytic production of the ripening hormone ethylene and a ripening-related transient burst in C02 evolution (Biale and Young, 1981). In avocado, the increase in ethylene production is followed by an increase in abscisic acid levels (ABA) (Adato et al., 1976). While ethylene induces the synthesis of many genes involved in fruit ripening (Brady, 1987), it is not known whether the rise in ethylene is related to the increase in ABA in avocado. Further, the role that ABA plays in the ripening process is also unknown. Ripening avocado fruit produces high levels of ABA and thus provides an ideal system in which to study the regulation of ABA biosynthesis. ABA plays a role in adaptation to various stresses (e. g. cold and osmotic stress), and also during deve10pmental changes, such as seed germination and embryo development (Zeevaart and Creelman, 1988). The increase in ABA levels in water- stressed leaves can be prevented by both transcriptional inhibitors (Guerrero and Mullet, 1986), and translational inhibitors (Stewart et al., 1986), indicating that RNA and protein synthesis are necessary to mediate the drought-induced increase in ABA levels. ABA is synthesized fi'om carotenoid precursors that are present in relatively large quantities in most photosynthetic tissues in comparison to ABA (Norman et al., 1990). Biochemical (Zeevaart and Creelman, 1988), and genetic data (Rock and Zeevaart, 1991) have indicated that the cleavage of 9—cis-xanthophylls is likely the key regulatory step in 50 the ABA biosynthetic pathway. The cleavage of 9-cis-xanthophylls produces a C25 apocarotenoid and xanthoxin (Zeevaart, 1999). The xanthoxin can be subsequently converted into ABA via ABA-aldehyde. The enzymes that carry out these later conversions (xanthoxin into ABA-aldehyde and ABA-aldehyde into ABA) are constitutively expressed in leaves (Sindhu and Walton, 1988) and are therefore not limiting for ABA biosynthesis. Other steps in the ABA biosynthetic pathway, such as the conversion of zeaxanthin into violaxanthin catalyzed by zeaxanthin epoxidase, show little up-regulation during water stress of leaves (Burbidge et al., 1997a). This further supports the notion that another part of the ABA biosynthetic pathway must be regulatory. Continuation of the regulatory nature of the cleavage reaction was provided by the cloning of VpI4. vp14 mutants of maize are Viviparous and exhibit a defect in ABA biosynthesis (Tan et al., 1997). The derived protein sequence of Vpl4 is related to lignostilbene dioxygenases, bacterial enzymes that catalyze a double bond cleavage reaction analogous to the carotenoid cleavage reaction in ABA biosynthesis. Recombinant VP14 protein catalyzes the cleavage of 9-cis-xanthophylls into ABA (Schwartz et al., 1997) in a reaction that requires oxygen, ferrous iron, ascorbate, and a detergent for activity in vitra. Northern analysis of maize leaves showed that Vp14 is induced during wilting in parallel with the increase in ABA levels (Tan et al., 1997). In the time since the cloning of Vp14, a number of genes with sequence similarity to Vp14 have been reported (Watillon et al., 1998; Neill et al., 1998; Burbidge et al., 1997b), and a number of additional homologous genes are present in the database. Based upon the degree of sequence similarity of these genes with Vp14, it can be inferred that the encoded proteins catalyze reactions in which a double bond is oxidatively cleaved, 51 yielding two products with aldehyde groups at the site of cleavage. While not all of the homologous genes are necessarily involved in ABA biosynthesis, it would seem as though at least some are. In particular, the natabilis mutant of tomato is impaired in the ability to convert C40 precursors to xanthoxin, and hence natabilis mutants have a reduced ABA content (Parry et al., 1988) and exhibit a wilty phenotype. The cloned gene is highly homologous to Vp14 (Burbidge et al., 1999), and message levels of this gene are increased during leaf wilting. The nomenclature now used for designating genes that have homology to Vp14 is gine-gis-gpoxycarotenoid dioxygenase genes or NCED. The regulation of ABA levels in fruit has not previously been investigated. Labeling studies of ABA using 1802 have shown that the indirect pathway (i.e. synthesis from C40 carotenoids) of ABA biosynthesis in leaves is operational in both avocado and apple fruit (Zeevaart et al., 1989). Carotenoid levels in various fruits appear to be high enough so as not to be limiting for ABA biosynthesis, and therefore the cleavage of xanthophylls is probably the regulatory reaction in fruit. To test this, three Vp14 homologs were cloned from ripening avocado fruit, and their expression during the ripening process was monitored. Two of these genes (PaNCED! and PaNCED3) are induced in parallel as the hit ripens. A third gene, PaNCED2 exhibits constant expression both during fruit ripening and during the wilting of leaves, suggesting that it has a housekeeping role. The tissue-specific differences in expression of the NCED genes, and differences in the activities of the expressed proteins, may have implications for their in viva physiological role in regulating ABA biosynthesis. 52 MATERIALS AND METHODS Plant Material. Avocado fluit (Persea americana, cv. Lula) were kept at room temperature for up to 14 days in a tray moistened with wet paper towels and covered with Saran wrap to prevent dessication. Each day individual fluits were incubated in sealed containers, and alter a period of time, 1 m1 of the gas phase was removed for ethylene determination by gas chromatography. 0n various days during the ripening period, one avocado fluit was harvested by removing the rind and cutting it into small pieces that were then flozen in liquid N2 and stored at -80 °C to be used for subsequent analysis. Avocado seedlings were grown flom seeds of Lula under greenhouse conditions. Mature leaves flom the top of the plant were wilted to differing percentages of their flesh weight, up to a maximum 80%, using a pressure chamber (Boyer, 1995). After removal flom the chamber, the leaves were left in a moist plastic bag in the dark for 4 h, then flozen in liquid N2. Pea seeds (Pisum sativum var. Little Marvel) were supplied by Olds Seed Company (Madison, WI). Seedlings used for isolation of chloroplasts were grown at 25°C in a growth chamber for 9 to 12 days. Extraction and Purification of ABA and Carotenoids. ABA was extracted with acetone and purified as described by Zeevaart et al. (1989). A small amount of [3H]ABA was added to each sample to determine the percentage recovery. Quantification was by GC with electron capture detection (ECD) using endrin as an internal standard. Extraction and analysis of carotenoids were performed according to Rock and 53 Zeevaart (1991). Carotenoids were quantified by integration of the peak area for absorbance at 436 nm with a Waters 740 data module. A C25-apocarotenoid, trans- B- apo-8'-carotenal (F luka), was added to each sample as a standard so that percentage recovery could be calculated. Corrections were made for differences in extinction coefficient and for the differences in absorption at 436 nm and the maximal absorption for each carotenoid. RNA Extraction and Northern Analysis. Total RNA was extracted flom avocado mesocarp using a phenol-chlorofonn method (V anlerberghe et al., 1992). Leaf RNA was isolated by a method that is a combination of methods by Callahan et a1. (1989) and Ainsworth (1994). Tissue was ground in liquid nitrogen to a fine powder using a mortar and pestle. The ground tissue was transferred to a tube containing extraction buffer [100 mM sodium acetate, 500 mM NaCl, 50 mM Na2EDTA, 1.4% sodium dodecyl sulphate, 2% polyvinylpyrrolidone (Mr 40,000), 1% B-mercaptoethanol] and the homogenate was placed at 65°C for 30 min. Cellular debris was removed by centrifugation (10,000g, 10 nrin), and the supernatant was extracted with distilled phenol. After centrifugation (10,000g, 10 min), the aqueous phase was extracted with phenol/chloroform and re-centrifuged. The aqueous phase was extracted with chloroform and centrifuged (10,000g, 10 min). The aqueous phase was placed on ice, and 0.1 vol of 3 M Na-acetate (pH 4.8) was added. The pH was brought to 5 by the addition of glacial acetic acid. After 2 h on ice, the RNA was pelleted, and washed once with 80% cold ethanol. The pellet was resuspended in 2 ml of distilled water and precipitated overnight with 1/4 volume of 10 M LiCl. The RNA was pelleted by centrifugation (10,000g, 10 min), and washed with 80% cold ethanol. The pellet was 54 fi‘ d1 Pm resupended in ddH20 and RNA quantitated by absorbance at 260 and 280 nm determined. Northern analysis was carried out by electrophoresis of 30 pg of total RNA on 1.2 % (w/v) agarose gels containing 2.2% (v/v) formaldehyde according to Maniatis et a1. (1982) and transferred onto nylon membranes (Hybond N+, Amersharn). Hybridization was performed at 65°C using Church-Gilbert hybridization buffer (Church and Gilbert, 1984). Blots were washed first at low stringency (2x SSC, 0.1% SDS, room temperature - 2x, 15 min each followed by 15 min, 2x at 65°C), then at high stringency (additional wash 0.2x SSC, 0.1% SDS, 30 min at 65°C). Following hybridization and development of the autoradiograrns, blots were stripped in 0.2% hot SDS and re-probed. Genomic Southern Analysis. Avocado (Lula) genomic DNA was isolated flom leaves using an extraction method based on the detergent cetylrnethylarnmoniumbromide (Hulbert and Bennetzen, 1991). DNA digestion, gel and electrophoresis conditions were as described by Maniatis et a1. (1982). Low stringency and high stringency wash conditions were performed as for Northern analysis. Probe Synthesis and Labeling. Plasmids (pGEMTeasy) containing the full-length PaNCED genes as inserts were digested with either Natl (PaNCED3 and PaNCED!) or EcaRl (PaNCED2), electrophoresized on agarose gels, and inserts were purified using a Qiagen gel extraction kit. Purified probes were labeled by random-prime labeling (Gibco-BRL) and non- incorporated nucleotides removed by spin chromatography. For gene-specific probes, JZ205 and JZ225 (see Table 3.1 for a list of primers) were used to amplify a flagrnent corresponding to the 3' untranslated region of PaNCED3. The region amplified using 55 Table 3.1. Primers used in the amplification of 9-cis-epoxycarotenoid dioxygenase genes flom avocado fruit. Primer Sequence Gene Amplified Orientation JZ101 TTY GAY GGN GAY GGN ATG G NCED] S Ill 17 GCY TTC CAN AGR TCR AAR CA NCED] AS JZ108 ACR TAN CCR TCR TCY TC NCED2 AS JZ110 ATG ATN CAY GAY TTY GC NCEDZ S JZ120 GAG GCG GCA AGT GAT NCEDZ AS JZ121 CAG TTA TTG CGA AAT CAT GC NCED2 AS JZl47 AAA TGA GCT GTA TGA AAT GA NCED2 S JZ148 CAG TGG AAT CGT GAA AGA GAA NCED2 S JZ153 AAC GAA TCT GCG CAG GCA TTT CCG NCED2 S JZ161 AAC TTG AAA GCA GTA NCED2 S JZ162 GTT TAC GTA GTT TTA TTT TGC TCG NCED2 AS JZ184 ACG CCG GTT CCG CTG GAG CCG TCG NCED] AS JZl85 ACA TCG CGA GGA GGT GCC GGT TGA A NCED] AS JZl86 GAT TCA TGA CTT CGT CAT TAC TGA NCED] S 12187 TTC GTC ATA ATT CCA GAC CAG CAG NCED] S JZ200 CCA ACA ACC CAT TGC TCT TCT NCED] S JZ205 ATT ATA GAG AAC CAG CTA AGG TAC NCED3 AS JZ206 TTC CAC ACC TAA AAC AAA CAA ATT NCED3 AS JZ222 CCC GGG GAC GCA AGC CTA AT NCED] AS JZ223 TCG ACT CAC TGA GTC GCA NCED] S JZ224 TAC AAG CAG TGG AAG AAG GGA AGG NCED] AS JZ225 GAA CAA GAC CCT GAG ACT GAG NCED3 S JZ245 GTC CAA GGC GAA GGC CAG CAG TCC NCED3 AS JZ240 CAT TCC CTC GTC GGC GTT CAC CA NCED3 AS Z2 A T TTAT T AAT TA T T NED3 Y=C/T, N=A/G/C/T, R=A/G S= sense, AS=antisense 56 these primers extended flom position 1939 to 2239 bp, resulting in a PCR product of 300 bp. Gene-specific primers JZ223 and JZ224 (corresponding to position 1827 and position 2162 bp, respectively) were used to amplify a 335-bp gene-specific flagrnent flom PaNCED] . For PaNCED2, a 200-bp gene-specific flagrnent was amplified using primers JZ161 (position 1742 bp) and JZ162 (position 1942 bp). RT-PCR. RNA was extracted florn fluit as described above. The first strand cDNAs were synthesized by RT flom 4 pg total RNA isolated flom avocado fruit ripened for 8 days using MMLV reverse transcriptase (Gibco-BRL) and an oligo dT primer. These cDNAs were used as templates for RT-PCR using degenerate primers JZlOl and Ill 17 for the amplification of PaNCED] , and degenerate primers JZlO8 and JZl 10 for the amplification of PaNCED2. These primers were designed based on the conserved regions of the tomato NCED and maize Vp14 genes. Conditions for RT were as follows: 65°C, 5 min, followed by 45°C for 1 hr, followed by 75°C, 5 min. PCR amplification was performed as follows: 30 cycles of 94°C 1 min, 55°C 1.5 min, 72°C 1 min. Amplification of F ull-Length cDNAs by RACE -PCR. To obtain the firll-length nucleotide sequences for PaNCED2 and PaNCED] , RACE-PCR was performed using a kit according to the manufacturer's instructions (Gibco-BRL). The 5' ends of each of the genes were amplified using the following gene- specific primers: JZ184 (nested) and JZl85 (outer) for PaNCED] ; JZ121 (outer) and JZlZO (nested) for PaNCED2; JZZ43 and J2240 for PaNCED3. To amplify the 3' ends, the following gene-specific primers were used: JZl86 (outer) and JZ185 (inner) for PaNCED] ; JZl48 (outer) and JZ147 (inner) for PaNCED2. Plasmids resulting flom 57 cloning of the 3' end of PaNCED] were heterogeneous as judged by the hybridization signal strengths on Southern blots probed with PCR flagrnent JZ101/JZ117 of PaNCED] . Sequencing of these plasmids revealed them to be a portion of a new gene that was related to PaNCEDl. The new gene was designated PaNCED3, and 5' RACE (using JZZ40 and JZZ45) was used to obtain the full-length clone. Primers JZ200 (at the 5' end) and J2222 (at the 3' end) were used in end-to-end PCR to obtain the full-length PaNCED] . Primers JZ162 (at the 3' end) and JZ153 (at the 5' end) were used in end-to- end PCR to obtain the full-length PaNCED2. Primers JZ 206 (at the 3' end) and JZ250 (at the 5' end) were used to obtain the full-length PaNCED3. Cloning and DNA Sequencing. The PCR products corresponding to either partial flagrnents or the full-length genes were ligated into pGEMTeasy (Promega) and then introduced into Escherichia coli DHSa. Plasmids were sequenced using a DNA sequencer with either the -21M13 or M13 sequencing primers. Protein Expression and Purification. PaNCED] in pGEMTeasy was digested with Natl to clone into the Natl site of pGEX5-2 (Pharmacia). PaNCED3 was excised flom pGEMTeasy using Natl and cloned into pGEXS-l. PaNCED2 was cloned into the EcaRl site of pGEX5-2. The plasmids were transformed into BL21 cells. Overnight 4 ml cultures were inoculated into 200 ml of 2YT medium and grown at 37°C until the optical density reached 0.7. At that time, 200 mM IPTG was added, and the cultures transferred to a shaker at 25°C in the case of PaNCED2 and PaNCED3 or to 18°C in the case of PaNCED] . After 5 h for PaNCED2 and-3 , or 16 h for PaNCED], cells were harvested by centrifugation at 12,000g for 10 58 min, washed once with Tris-buffered saline (TBS), pH 7, centrifuged at 12,000g for 10 min, and resuspended in 10 ml TBS. One ml of 100 mg/ml lysozyme was added and the suspension was left on ice 30 min before being flozen overnight at -20°C. The extract was thawed the next morning, 0.1 M DTT was added, and it was sonicated using a probe sonicator (Sonifier 450, Branson) in 15 sec pulses for approximately 6 min total. The extract was separated into soluble and insoluble flactions. The soluble flaction was added to a 1 ml 50% slurry of Glutathione Sephadex (Pharmacia), and incubated 2 h at 8°C. At this time the mixture was centrifuged in a table top centrifuge, the beads washed 3x with 1x TBS, and once with Factor Xa buffer. One-half ml of Factor Xa buffer and 25 units of Factor Xa (Pharmacia) were added, the beads were shaken for 3 h at room temperature. At this time 5 pl of 20% Triton X-100 were added, and incubation was continued for another hour. The eluted protein was collected and flozen at -80°C. Assay of Enzymatic Activity of NCED. Assay of the enzymatic activity of PaNCED], 2, and 3 was performed as described by Schwartz et al. (1997). The cleavage reaction products were analyzed by HPLC on a pPorasil column (Waters) equilibrated with 90% (v/v) hexane and 10% ethyl acetate. The column was eluted with a linear gradient to 20% hexane and 80% ethyl acetate over 17 min. The xanthoxin and C25 products flom the cleavage of 9'-cis- neoxanthin and 9-cis-violaxanthin were collected and identified by mass spectrometry according to Schwartz et al. (1997). A standard curve of xanthoxin was constructed by injecting known quantities and integrating the peak areas. 59 Binding and Chloroplast Import Assays. Intact pea chloroplasts were isolated flom 8- to 12-day-old pea seedlings and purified over a Percoll gradient as previously described (Bruce et al., 1994). Intact pea chloroplasts were reisolated and resuspended in import buffer (330 mM sorbitol, 50 mM Hepes/KOH, pH 8.0) at a concentration of 1 mg chlorophyll/ml. The plasmid containing the gene for the small subunit of ribulose bisphosphate carboxylase/oxygenase (prSS) (Olsen and Keegsfla, 1992) was linearized with PstI and transcribed with SP6 polymerase. The plasmid containing outer envelope protein 0M14 (Li et al., 1991) was linearized and transcribed using SP6. Plasmids containing the coding region for PaNCED] , PaNCED2, and PaNCEDS were linearized and transcribed with T7 RNA polymerase. PaNCED] , PaNCED2, and PaNCED3 were translated using a nuclease-treated rabbit reticulocyte system using the suggested protocol of the manufacturer (Promega). Likewise, the control plasmids pSS and OM14 were translated using a nuclease-treated rabbit reticulocyte lysate system. All proteins were radiolabeled using [3SS]-methionine. Binding and import reactions were adapted flom Young et al. (1999). Briefly, chloroplasts were pretreated with 6 pM nigericin for 10 min in the dark to deplete internal ATP levels. The precursor proteins [35S]-PaNCEDl , [3SS]-PaNCED2 and [358]- PaNCED3 were purified by gel-filtration according to the method of Olsen et al. (1989). All reactions contained intact chloroplasts corresponding to 25 pg chlorophyll in a final volume of 150 pl and 500,000 dpm of either [35S]-prSS, [3SS]-OM14, [35S]-PaNCEDl, [3SS]-PaNCED2 or [3SS]-PaNCED3. Either no ATP, 0.1 mM ATP for binding, or 1 mM ATP for translocation was added to each of the reactions. All reactions were incubated 60 for 30 min at room temperature. Intact chloroplasts were recovered by sedimentation onto a 40% (v/v) Percoll cushion. The pellets were resuspended in lysis buffer (25 mM Hepes-KOH, pH 8.0/4 mM MgCl2), and incubated on ice for 15 min. After ultracentifugation at 100,000g a total membrane and soluble flaction were obtained and solubilized in 2x SDS-PAGE sample buffer. All flactions were analyzed by SDS-PAGE (Laemmli, 1970), and autoradiography. Treatment of reactions with thermolysin was performed as described by Cline et al. (1984). For extraction of total envelope membranes, a large-scale import reaction was performed as described above, and flactionated using the method of Perry et al. (1994). Total envelope membranes were extracted with either high salt, sodium carbonate, or Triton-X100 according to the method of Tranel et al. (1995). RESULTS Cloning of NCED genes. A number of conserved regions are present in 9-cis-epoxycarotenoid dioxygenase genes (Burbidge et al. 1997b). Degenerate primers JZ101 and JZ117 were used to amplify an approximate 1.1-kb flagrnent flom day 8 cDNA. This new gene was designated PaNCED] . The full-length gene, obtained using RACE-PCR, contained an open reading flame of 1710 hp, with a 3' untranslated region of 377 base pairs, and a 5' untranslated region of 116 bp. The complete nucleotide sequence of PaNCEDl is presented in Figure 3.1. The predicted molecular weight of the protein is 63.1 kDa, slightly smaller than the predicted molecular weight of VP14. At the amino acid level, PaNCEDl was approximately 60% identical to VP14. Degenerate primers JZ108 and JZ110 (see Table 3.1) were used to amplify an 61 approximately 600 bp flagment flom cDNA of day 8 avocado fiuit. This gene was designated PaNCED2. The full-length cDNA, obtained using 5' and 3' RACE, contained an open reading flame of 1575 bp encoding a protein with a predicted molecular weight of 59.6 kDa. The 3' and 5' untranslated regions were 226 and 166 bp, respectively. The nucleotide sequence of PaNCED2, along with the position of the start and stop codons, and the primers used in the amplification reactions, is presented in Figure 3.1. In comparison to VP14 and the tomato homolog, the deduced amino acid sequence was truncated at the amino-terminus and thus appeared to lack a transit peptide for chloroplast targeting. Overall, the gene shared approximately 30% identity at the deduced amino acid level with VP14, LeNCEDl, and PaNCEDl. During each of the RACE procedures, Southern blotting of the minipreps corresponding to the 3' and 5' ends of the gene was performed to ensure that the newly amplified region cross-hybridized with the previously cloned portion. During the cloning of the 3' end of PaNCED! , it was noticed that the minipreps differed in terms of the strength of the hybridization signal when the fragment corresponding to JZ101/JZ117 was used as probe on Southern blots. These plasmids corresponding to the weaker signal on Southern blots were sequenced, and discovered to be a unique NCED gene. This gene, designated PaNCED3, was 60% identical at the amino acid level to Vp14 and LeNCED, and 67% identical to PaNCED! . PaNCED3 contained an open reading flame of 1878 hp, with 3' and 5' untranslated regions of 388 and 44 bp, respectively. The nucleotide sequence of PaNCED3 is shown in Figure 3.1. An alignment of the deduced protein sequences of the three avocado genes with Vp14 is shown in Figure 3.2. Some properties of the three avocado genes are summarized in Table 3.2. 62 Figure 3.1. Nucleotide sequences of the three avocado NCED genes. The start and stop codons are indicated in bold and are underlined. The positions of the gene specific primers used are indicated with arrows above (for sense primers), or below (for antisense primers) the nucleotide sequences. Nucleotide Sequence of PaNCEDl 1 53 105 157 209 261 313 365 417 469 521 573 625 677 729 781 833 885 937 989 1041 1093 1145 CACTGTAGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCGG JZ200 CCGCGGGAATTCGATTCCAKCKACCCATTGCTCTTCTCCCTCGTCATTGGAT CTCAGCTGTCCAATGACAACCATCAGACAAAAACCCAAAACCTTCACAATCC ACAGCTCTTTGCATTCCTCTCCTGTTCTTCACCTCCCCAAACTCCTCACTAC TACTACTACTCCTCTTCATGAGAAGAGCCAAAGAGAATTGGGCTTGATCTTG CAAGAGCCAAATAGGGCCAAGTGGAATTTTTTCCAAAGGGCTGCAGCTGTGG CCTTGGACACGGTGGAGGACTCCTTCATCTCCGGCGTGCTCGAGCGCCGCCA CCCACTTCCGAAGACCTCCGATCCGGCAGTCCAGATTTCCGGTAACTTCGCT CCGGTGGACGAGCACCCGGTGCAACACCACCTTCCCGTCTCCGGCCGCATCC CACGGTGCCTTGACGGCGTCTACCTGCGCAACGGCGCCAACCCACTCCTTGA GCCCGTAGCCGGCCACCACTTCTTCGACGGCGACGGCATGGTCCACTCCGTC AGCCTCCGACAGGGAACCGCCAGCTATGCCTGCAGGTTCACCGAGACCCATC GGCTAGTGCAGGAGCGTGCGATCGGCCGGCCGGTCTTCCCCAAGGCAATTGG CGAGCTCCACGGCCACTCCGGCATCGCCCGCCTCCTCCTCTTCTACGCTCGC ACTGCTACCGGCCTCGTCGACGGCTCCAGCGGAACCGGCGTCGCCACCGCTG GTCTGGTCTACTTCAACCGGCACCTCCTCGCGATGTCCGAGGATGACCTCCC ,‘ J2185 CTACCACGTCCGGGTCACCTCCTCTGGCGACCTCGAGACCGTTGGCCGGTTC GACTTCATGGGTCAGCTCAATTCCGCCATGATTGCCCACCCCAAGCTCGACC CGGCTTCGGGCGAGCTCTTCGCTCTCAGCTACAACGTCATCAAAAAGCCATT TCTCAAGTTCTTCAAATTCACCTCAGATGGAAAGAAGTCCCCAGACGTCGAA JZl86 ’ ATCCCCATTGATCAGCCCACCATGATTCATGACTTCGTCATTACTGAGAATT JZ187 TCGTCATAATTCCAGACGBGCAGGTGGTTTTCAAGCTCCAGGAGATGATCCG TGGTGGCTCTCCGGTCGTCTATGACAAGAAGAAAACCGCCCGGTTCGGAATT 1197 1249 1301 1353 1405 1457 1509 1561 1613 1665 1717 1769 1821 1873 1925 1977 2029 2081 2133 2185 CTATTGAAAACCGCTGCCGACTCGAACGGTCTGAGGTGGATTGACGCGCCGG ACTGCTTCTGCTTCCACCTCTGGACCGCCTGGGAAGAACCCGAAACCGACCA GGTCGTCGTCATCGGCAGCTGCATGACGCCGCCGGACTCGATCTTCAACGAA TCCAACCAGAGCCTGAAAAGTGTTTTGACTGAAATCCGGCTTAATTTGAAAA CCGGCCTGTCGAGCAGGAGGGAGATCGACCCATCAAGGCACTTGAATTTGGA GGTTGGGATGGTGAACCGGAACCGGCTCGGCAGAGGACCCGGTGTGTCGCTA TCTAGCCATTGCCGACCGTGGCCAAAGGTGTCCGGGTTCGCCAAGGTGGACC TCTCGACCGGGGAGGTGACCAAGTTTATCTACGGCGAACAGTGCTATGGCGG CGAGCCATACTTTGTGTCCAGAGATCCGGTGGCGCCGGAGGACGATGGGTAT GTTCTGTCGTTCATGCACGACGAGAAGACGGCGCGATCAGAGCTGCTGATCG TCAATGCCATTACCATGCAGCTAGAGGCCTCTGTGAAGCTCCCATCCAGGGT CCCCTATGGATTCCATGGGACTTTCATCAGTAGTAAGGACCTTGCAAATCAG GCCIEQGTCGACTCAGTGAGTCGCATCGGAGCCTATCTGCATTTCATCAATC J2222 ATAGCATCTTTTTAAAGGAGTGGAGETTATACCTGAGGGATATGATTAGGCT TGCGTCCCCGGGATCTCCTTCCTTAGATGACCCATTTCGGGTTTTTAGCTTT TGTCTCTCCGCCAGTTACCACGGCCAGTGTGAGAGAAACCAGCTTGAAGCTT TGGGTGTAGATAGGTTTGGAGCTCAGCTGTTTCTCTGTAATTCTTTTGTGTT GTGCATGAATTGGATGTAATGTATGTATGTAATTGGGAAAGTGAGGTCTCTG JZ224 GGATCCCCTTCCETTCTTCCACTGCTTGTATATATAATAAAAAGGCTACACC TTTCGCCCCAAAAAAAAAA 65 Nucleotide Sequence of PaN.2 1 53 105 157 209 261 313 365 417 469 521 573 625 677 729 781 833 885 937 989 1041 1093 1145 J2153 CCATTAACGAACGAATCTGCGCAGGCATTTCéfiTTTTTCAAGAAAAACAGAG AATAGAGAATACAGAGAGAGGGTGTGTGTTCATAATAAGTAGGGCATACACA CCAGACACTAGAAAAAAACTGAGAGGAGAAGAGGAAGAGGAAGAAACCAATC AGAGCAGACEQEQCAGAAGGAGAATGAGAAGAAGAAGATCGTGATCCGTGAT CCGAAGCCGACTAAGGGATCGTATCGCGATGGTGGACGCATGGAGAAATTGA TAGTGGGTCCATGCCTACTCTCCGGGAATTTCCCCCTGCGGGTCGAAGAAAC TCCCTGCGAGAATCTCCCCATCAAAGGTACCTCCCGGATTGCTTGGATGGGG GAGTTTGTTAGAGTGGGCCCGAACCCGAAGTTTGCTCCTGTGGCTGGATATC ATTGGTTTGACGGTGATGGCATGGTTCATGGAATGCGTATTAAAGATGGAAA AGCAACCTATGTTTCACGTTACGTGAAGACATCTCGCCTTAAACAAGAAGAG TATTTTGGAGGAGCAAAATTTATGAAGATTGGAGACCTTAAGGGCATGTTTG GGCTTTTTATGGTTAACATGCAAATGCAAATGCTTCGAGCAAAACTCAAAGT GCTGGACGTTTCATATGGAACTGGGACAGGCAATACAGCTCTAATATATCAT CATGGTAAACTATTGGCCCTTTCAGAAGCAGACAAACCTTATGTCCTTAAGG TTCTGGAAGATGGCGATCTTCAAACCCTTGGGATGTTGGATTATGACAAGAG ATTGTCTCATTCATTCACTGCTCATCCCAAGGTCGACCCATTTACTGATGAG .ATGTTACATTTGGGTATCTCCCATACACCATACTTAACGTACCGCGTTATAT CCAAAGACGGCATCATGCACGATCCAGTCCCCATAACAATACCAGAAGTCAT JZ121 GATGCKTGKTTTCCCKKTKKCTGAAAACTATGCAATCTTCATGGATCTTCCT TTGTACTTCCGACCGAAGAAAATGGTGAAGGGGAAACTTATCTTCCCATTTG .ATGCAACAAAGAAAGCTCGTTTTGGTGTACTACCACGATATGCAAAGGATGA .ACTTCAAATGAGATGGTTTGAGCTCCCAAATTGTTTTATCTTCCATAATGCT JZ120 .AATGCTTGGGAGGAAGGTGGTGAAATTGTTCTAflhCACTTGCCGCCTCCAGA 66 1197 1249 1301 1353 1405 1457 1509 1561 1613 1665 1717 1769 1821 1873 1925 JZ148 ATCCGGGCCTGGACATGGTCAGTGGAATCGTGAAAGAGNKGCTTGAAAATTT JZ147 CAAEKKTCACCTGTKTGEKKfiEAGGTTCGATATGAAAACTGGTGCTGCTTCT CAGAAACAATTATCGGTATCTGCTGTAGATTTTCCCCGGATCAATGAGTCTT [ACACAGGCAGGAAACAACGGTTTGTCTATGGAACCATACTCAACAACATTAC AAACGTGAAGGGCATCATCAAGTCTGATCTGCAGCTGAACCAGAGGGACGGA AATCGAAGCTCGATGTTGGAGGTAATAATCAAGGCATCTTTATCTTTCAGTT GGGACCCGGGGTTTGTTCCTCGGAAGCCTGGTGTCACTTCAGAAGAGGATGA CGGCTATTTGATATTCTTTGTACTGACCGAACGGACTGGAAAATCCGAAGTT AATGTAATCGATGCGAAAACAATGTCAGCTGATCCTGTTGCGATTGTGGAAC TGCCCCATAGAGTTCCATTTGGGTTTCTGCCCTTCTTTGTATCAGAGGAACA JZ161 ACTTCAACAACAGGCAAAGATCIééIKCTGCTTTCKKGTfitTGGCTGATATT .ACTGGTAGGAGTATTGACTGCATTGCCCAGGATGAGTAAAGAGTTTCTTATT GTCTGTAAGATGGAAGCCACTGATATAGGTACTGTATTCATATAGAACTATG AAGGGAAGGCTTGAGATTGTACTAAGATATATGATCTTGATTTGCTCGAGCA JZ162 gfiTKKKKCTACGTAAACTTTTGAGTTCCCAAAAAAAAAAAAAA 67 Nucleotide Sequence of PaNCED3 1 53 105 157 209 261 313 365 417 469 521 573 625 677 729 781 833 885 937 989 JZ250 CTGAGAGAGACAGCTCTTCAAATCCAATACTCTTCGAT GGCTACTCCTACTACTACTTGTGGGGCAGGTGATCTACTTCAAAATCCCAAA TTGCTCCCCATTTCAAAGAATCTCAGCCGTCCAAAAAACTTCATCATGCTAA AACACAACACCCCATTAATTCAGTGCTGCTCACATTCTCCTTCTTCTTCTTC TGCTGCTGTCCTTCATCTACCACCAAAGCAGCCGACAAAATCCAAACCGTCC .ATCAAGAAAGGAGAGAAATCGTCGACTCTCACTCCATCGATAGAGAAGAATC CTGGCAGCCATCAAGTGAAAACAGATCAATCGGGTCCGAACCGGGTCGGACC CAACTGGAACATTTTTCAACGGACTGCngg;$CGCCTTGGACGCGATCGAG GAGAAACTCATTGCTCGGGTGCTCGAGCGCCGCCACCCGCTTCCAAAGACCG CGGACCCGGAGGTTCAGATTGCCGGAAATTTTGCACCGGTCGCCGAGCATCC TGTACAGCACGGCATCCCCGTCGCCGGAAGAATTCCTCGCTGTCTCGACGGC GTCTACGTCCGCAACGGTGCCAACCCCTTGTTCGAGCCGATCGCCGGCCACC ATTTCTTCGATGGAGACGGGATGATCCACGCCGTCCGGTTCCGAAATGGGTC CGCGAGTTACTCTTGCAGGTACACCGAGACTCGGAGGCTGGTGCAGGAGCGC CAGCTCAGCCGGCCGATTTTTCCGAAGGCTATCGGCGAGCTGCACGGCCACT CTGGCATCGCCCGCCTCCTTCTCTTCTATACAAGAGGGCTGTTTGGGCTGGT GAACGCCgigégGGGAATGGGAGTAGCCAACGCCGGTTTGGTCTACTTCAAT CGCCGCCTCCTCGCTATGTCTGAGGATGACCTCCCCTACCACGTCCGCATCA CCCCGTCCGGCGACCTGAAGACCGTCGGACGACACGACTTCGACAACCAGCT CCGCTCCTCCATGATCGCCCACCCCAAGCTCGACCCAGAATCGGGCGAGCTC 1041 TTCTCCCTCAGCTACGACGTCGCCCGAAAGCCTTATCTGAAATACTTCCACT 1093 TCGCCCCCGACGGCTGGAAGTCGCCGGACGTCGAGATCCCCCTCGACAGGCC 1145 GACCATGATCCACGACTTCGCCATTACCGAAAACTTCGTCGTGATTCCCGAC 68 1197 1249 1301 1353 1405 1457 1509 1561 1613 1665 1717 1769 1821 1873 1925 1977 2029 2081 2133 2185 2237 2289 CAACAGGTGGTCTTCAAGCTAGAAGAGATGATAAGAGGGGGCTCTCCGGTCG TCTACGACAAGAACAAGACCTCCCGATTCGGAATTCTCCCGAAATACGCCCC CGACGCGTCGGAAATGATATGGGTCGACGCCCCGGACTGCTTCTGCTTCCAT CTCTGGAACGCGTGGGAGGAGCCGGAGTCCGGCGAGGTGGTGGTAGTCGGCT CGTGCATGACGCCGCCAGACTCAATATTCAATGAAAACGAGGAGAGCCTGAA GAGCATTCTAACGGAGATCCGGCTCAACACGAGGACAGGTGAGTCGACTCGC CGGACCATCATCGACCCGCAGAAGCCGTTGAATTTGGAAGCTGGGATGGTGA ACCGGAATCGTTTGGGGAGGAAGACCCGGTTCGCGTATCTTGCCATTGCAGA GCCGTGGCCGAAGGTGTCGGGTATCGCGAAGGTGGATCTTGGGACGGGGGAG GTGAACCGGTTTGTGTATGGGGAGAGGCAGTTCGGTGGAGAACCGTATTTCA TTCCGAGAGAGCCGAGTACGTCGGGACGAGAGGACGACGGGTATGTGGTGTC GTTCATGCATGACGAGAAGACGTCGAGGTCTGAGCTGCTTATCTTGAATGCA .ATGAATATGAGGTTGGAGGCGTCTGTGATGCTTCCTTCCAGAGTCCCATATG GATTTCATGGCACTTTTATTAGTTCCAGGGACCTTGCAAAACAAGCCEQQCA J2225 CAGATCGGAGAGAGGAACAAGACCCTGAGACTdKGTCGGAATCGTCTTCGTC TTCTTCTTCTTCTGATTATTATTAGTGTTGTTATTGTTGTTATCTTTTTTTA AGGGGAGATAATACCAGGGGGATGAGTGGAGCTACGTCCCCGGAGTCTTCTC TTTTGCTATTACAGGCCTTCTACGTTTGTGGGTTTTACGTGGTTTCTCTCTT J2206 CTTCTTCTGCTTCTTTAAATATCTTTAgHTTGTTTGTTTTAGGTGTGGAACC JZ205 AGCTCGAAGCTTGTTAGATTGTAGGTAGATTTGTA <— TAATTATTTTACTCTAGTGTGAATGAATTTATGATCTTACTAGTGGTTACTA TGCAAAGAAAAAAAAAAAAAAA 69 Southern Analysis. In order to assess the number of genes present in the avocado NCED family, Southern blot analysis was performed using the three full-length genes as probes (Figure 3.3A-D). High stringency washes of Southern blots probed with fiill-length probes of either PaNCED] (Figure 3.3A), or PaNCED3 (Figure 3.3B) revealed that there was little cross-hydridization of the two genes under these conditions. The hybridization signals seen on the autoradiogram corresponded to those predicted based on the restriction map for the two genes. Southern blots of PaNCED2 washed at low stringency produced several bands in each lane digested with different restriction enzymes (Figure 3.3C). Based on the restriction enzyme map of PaNCED2, the strong signals (two bands are present in the BamHI lane, two in the EcoRI lane and two in the Hind III lane), correspond to PaNCED2. The signals that cannot be explained from the sequence of PaNCED2 are likely due to cross-hybridization with related sequences in the genome. When the same blot was washed at high stringency (Figure 3.3D), several faintly hydridizing bands still remained, particularly in the lane of the HindIII digest. The fainter bands still visible after high stringency washing are likely to be due to related members of the NCED gene family. Low stringency washes of blots probed with either PaNCED] or with PaNCED3 also produced multiple bands (data not shown). Monitoring of the Ripening Process. Avocado fruits left at room temperature require, on average, about two weeks to become fully ripe. Some variation occurs between varieties (Biale and Young, 1971). Lula produced little ethylene until six days after harvesting, at which time there was a 70 PaNCED3 PaNCEDl Vpl4 PaNCED2 —MsnTmTCGAGDILQNPqET SKNLS EflMLKHNIP ssss _____________ S MQGIPIVSIHRHIPARsfii-Saixsusvars -AISSVPP!EC ———— H H H H PaNCED3 PaNCEDl Vp14 PaNCED2 PaNCED3 PaNCEDl Vp14 PaNCED2 PaNCED3 179 .. PaNCEDl 126 '-' Vp14 154 - PaNCED2 69 PaNCED3 238 PaNCEDl 185 Vp14 214 PaNCEDZ 127 PaNCED3 296 PaNCEDl 243 Vp14 272 PaNCED2 187 .»lEIrEF K‘H \L~\DVTru {err' PaNCED3 356 PaNCEDl 303 Vp14 332 PaNCED2 245 FSPDVEIE LI'PTMIHDFAITENFV IPDQQVVFhLELWIPbb>Lxd1Ur rSPDVEIE DQPTMIHDFEITENFV IPDQQVVFKLQEMIRI} E PSDDVEIE ES PTMIHDFAITENFV H PaNCED3 416 K ‘ iINVDAPDLFCFHLWNAWEEPLfiJEVV\EJQCMTEi PaNCEDl 363 EMRV’IDAPDCFCFHLWEAWEEPEIMJVVI‘ SCMTH Vp14 392 DASEMMNV PaNCED2 304 ' ‘ PaNCED3 471 at ‘ ~ ' RNRLGRYTREAiLAIAEFWLFYJL PaNCEDl 418 :cr 1' " ‘ ‘ "I31 .4 Vp14 447 PaNCED2 362 PaNCED3 531 PaNCEDl 477 Vpl4 507 PaNCED2 418 PaNCED3 581 :. PaNCEDl 525 2: Vp14 559 a 1 PaNCED2 476 a..., Figure 3.2. Alignment of the deduced amino acid sequences of PaNCED], PaNCED2 and PaNCED3 from avocado with VP14 from maize. Amino acid residues identical in at least three of the sequences to the VP14 sequence at a given position are indicated by black boxes. Grey boxes indicate amino acid residues that are conserved in at least three of the four sequences. The arrows indicate the regions that were used in the design of the degenerate primers. 71 E a Z ‘8 72‘ a: S 8 8 e s a: A °° “‘ "‘ B 4kb u - g 9kb . 31:19-17 - 2.5kb . .' . u 6 '5 11(1)i . 1.5kb - . -9 . PaNCED] PaNCED3 E § § E .. t:: C E 8 s D E “5 E on Lu t 83 § 5 IOkb «- Skb .~’ . 2.5kb “f - lkb "’ PaNCED2 PaNCED2 Figure 3.3A-D. Southern blot analysis of genomic DNA isolated from leaves of avocado, cv. Lula. Each lane contained 30 ug of DNA digested with the indicated restriction endonucleases. A) Analysis of PaNCED]. The blot was probed with a random-primed labeled probe (2.2 kb) corresponding to the fiill-length gene and washed at high stringency (see Materials and Methods). PaNCED] contains a restriction site for EcoRI and BamHI, but not for EcoRV. B) Analysis of PaNCED3. The blot was washed at high stringency. PaNCED3 has two restriction sites for EcoRl, which are approximately 600 bp apart, and does not have restriction sites for either Hind III or PstI. C,D) Analysis of PaNCED2. The blot was probed with the fiill-length gene (1.9 kb) and washed under low stringency. After development of the autoradiogram, the same blot was washed further under high stringency (D). The two strong signals present in the BamHI lane are consistent with the restriction map of PaNCED2, as are two of the signals seen in the HindIII digest, and the strong signal of approximately 5 kb in the EcoRI digest. 72 massive increase in ethylene production (Figure 3.4A). This autocatalytic ethylene production is typical of climacteric fruit and other senescing tissue (Brady, 1987). By day 10, when ethylene production had declined, the fruit had a sofi texture and fiuit maturation was complete. In fruit ripened for six days, ethylene production had peaked while ABA levels remained at a low level. By day l 1, four days following the peak in ethylene production, ABA levels had reached 30-fold higher levels compared to the level in unripe fi'uit. Because the enzyme involved in producing ABA would already be present by day 11, an earlier time point, namely, day 8 mm, was chosen as the RNA source used in RT-PCR. Northern Blot Analysis of the Three Genes. In maize, there is an increase in transcript level of Vp14 in leaves subjected to wilting (Tan et al., 1997). It was hypothesized that, as in water-stressed leaves, the increase in ABA levels during fruit ripening may also be accompanied by an increase in the mRN A levels of the NCED genes. Northern analysis of PaNCED2 showed that it remained fairly constant in expression during the ripening process (Figure 3.4B). For analysis of PaNCED] and PaNCED3, the same blot was stripped and reprobed with gene specific probes based on the 3' non-coding region of each of the genes. For PaNCED3, the gene specific probe was amplified using primers JZ225 and 12205. The PCR fragment produced using these primers corresponds to the region fi'om position 1939 bp to position 2239 bp. For PaNCED], a gene specific probe was amplified using primers JZ223 and JZ224. This product corresponds to the region extending from 1827 to 2162 bp. Control experiments revealed that each of these probes did not cross-hybridize with the other two genes (data not shown). 73 Both PaNCED] and PaNCED3 were barely detectable until 8 days after harvesting. At this time, mRNA levels of both PaNCED] and PaNCED3 increased in a similar fashion reaching the highest levels at day 10, and falling again as the fruit became very sofi (12 days). Since this increase in message levels precedes the increase in ABA levels, both PaNCED] and PaNCED3 can be viewed as possible cleavage enzyme genes. To test whether the avocado genes cloned from fruit were up-regulated during wilting of leaves, Northern analysis was performed on turgid avocado leaves, and leaves that had been wilted to 95, 88, and 80% of their fresh weights. As a result of the dehydration, ABA levels increased approximately 10-fold in leaves that lost 20% of their water content (Figure 3.5A). While PaNCED3 was undetectable under any of these conditions, PaNCED] increased dramatically in response to water loss (Figure 3.5B). PaNCED2 remained fairly constant under the same conditions. Chloroplast Import Assays. Carotenoids are found associated with the photosystem H light-harvesting complex of the thylakoid membrane, and in much smaller quantities, with the chloroplast outer membrane (Siefermann-Harms et al., 1978). Any enzyme that utilizes carotenoids as substrates must, therefore, be imported into the chloroplast. An example of this is zeaxanthin epoxidase, which has been shown to be targeted to the thylakoid membrane (Marin et al., 1996). Proteins destined for the chloroplast are synthesized as precursors with a serine/threonine-rich domain lacking in acidic amino acids at the amino terminus (V onHeijne et al., 1989). Within the chloroplast, the signal peptide is cleaved by a protease, and the protein targeted to the thylakoid membrane (Keegstra and Cline, 1999). Sequence analysis indicated the presence of a targeting domain in the deduced 74 12 J- +ABA .2w {Ethylene I 10- A .200 E A u 8 8- g 3 150 g a 6- o .100 4- e 2_ .50 o - - - . 'o 0 2 4 6 8 10 12 Figure 3.4A-B. Changes in ABA, ethylene levels, and in PaNCED], 2 and 3 transcripts accumulation during the course of fruit ripening in avocado. A. Analysis of ABA and ethylene levels plotted as a function of days of ripening. B. Northern analysis of NCED gene expression in the same fruit. Total RNA (30 pg per lane) was isolated fiom fruit, electrophesized and blotted onto nylon membranes. The same blot used for analysis of PaNCED] was stripped and reprobed with probes against PaNCED2 and subsequently PaNCED3, and 17S rRNA . PaNCED] and PaNCED3 increased as the fruit ripened while PaNCED2 remained constant. The specific probes used for the PaNCED genes are described in Materials and Methods. The 17S rRNA probe was used as a loading control. 75 amino acid sequences of both PaNCED] and PaNCED3, and the absence of such a domain in PaNCED2 (Emanueffson et al., 1999). Therefore, the avocado NCEDs were tested for in vitro import into isolated pea chloroplasts. Chloroplastic proteins carrying a transit peptide require ATP for their binding and translocation (Olsen et al., 1989). Radiolabeled PaNCED] was incubated with pea chloroplasts either in the absence of ATP or at low ATP levels (Figure 3.6A, lanes 1 to 8). Following fractionation of the chloroplasts into pellet and stromal fractions, PaNCED] was found in the pellet fraction. When ATP concentrations were raised to levels that are conducive to translocation of bound precursors (Olsen et al., 1989), [35$]PaNCEDl and chloroplasts were fractionated, PaNCEDl was not found in the stromal fraction, and did not undergo a decrease in molecular weight. Thus, PaNCED] was not imported and associated with the outer membrane (Figure 3.6A, lanes 9 to 12). In contrast, the small subunit of ribulose bisphosphate carboxylase/oxygenase (prS S), a representative stromal-targeted protein used as a control, was found in the stromal fraction (Figure 3.6B, lanes 7-10). Further, OM14 (outer envelope membrane protein of 3 kDa (Li et al., 1991)), a representative outer envelope protein used as a control, behaved in an identical fashion to PaNCED] (Figure 3.6B, lanes 1-4). PaNCEDl fractionated with the pellet fraction as opposed to the stromal fraction and did not undergo a decrease in size, indicating that no processing had occurred. When chloroplasts from the import assay of PaNCEDl (with the bound PaCEDl), were treated with thermolysin, (indicated by 'post' in Figure 3.6A, lanes 1-12), no protected protein fi'agments bands were observed. This suggests that if, in fact, PaNCED] is inserted into the membrane, it is oriented such that it is accessible to the added protease 76 6 I 4.5 8 a g 3 -1 1.5 - 0 I I ‘l 0 5 10 15 20 Water Loss Perceriage B 0 5 12 20 % H20 Loss NCED1 . NCED2 NCED3 1 7S rRNA Figure 3.5A-B. Accumulation of ABA (A), and of PaNCED] , PaNCED2, and PaNCED3 transcripts (B) in response to wilting of avocado leaves. RNA blot hybridizations were carried out with total RNA (30 pg per lane) isolated from leaves that had been wilted to increasing percentages of their fresh weights. The leaves were wilted using a pressure bomb for approximately 15, 30, and 50 min to achieve water losses of 5, 12 and 20%, respectively. After this time, the leaves were incubated in the dark for 4 h, and then frozen in liquid nitrogen. The specific probes used for the NCED genes and the probe (17S RNA) used as a control are described in "Materials and Methods". 77 and thus likely faces the cytosolic side. This result is identical to that found with OM14, which is also not protected by post-thermolysin treatment of import reactions (Figure 3.6B, lanes 1 to 4), and contrasts to prSS, which is protected from thermolysin treatment afier import (Figure 3.6B, lanes 7-10). To determine whether the bound [3SS]PaNCED1 was peripherally or integrally associated with the outer membrane, chloroplasts were subjected to NaCl and sodium carbonate extraction afier incubation with PaNCED]. Such treatments, which remove peripheral membrane proteins, were ineffective in extracting PaNCED] from the membrane, suggesting that it was at least partially assembled within the outer membrane of the chloroplast (Figure 3.6C). This behaviour is identical to that of OM14, used as a control (Figure 3.6C). Chloroplast proteins carrying a transit peptide require one or more protease susceptible surface components (e. g. a receptor) (Friedman and Keegstra, 1986). To address the requirements of protease-susceptible surface components, chloroplasts were pretreated with thermolysin prior to an import assay. After thermolysin treatment, chloroplasts were repurified over Percoll, washed twice with import buffer containing 4 mM ATP, and incubated with PaNCEDl. PaNCEDl could still insert into the outer membrane, indicative that no proteinaceous components (labeled as 'pre' in Figure 3.5A, last two lanes) were required for insertion. This result is identical to OM14 (Figure 3.6C, lanes 5 and 6), which also does not require receptors to mediate its insertion into the chloroplast envelope (Li et al., 1991). Chloroplast import experiments similar to those described for PaNCED] were performed on PaNCED2. Under no or low ATP concentrations, PaNCED2 was bound to 78 Figure 3.6A-C. Chloroplast import analysis of PaNCEDl , 2, and 3. Isolated pea chloroplasts were incubated with radiolabeled in vitro translation products, with either no (-) or low (0.1) ATP concentrations for binding, or under high (4.0) ATP concentrations for translocation. As control, prSS and OM14 were used. TP= total translation product. (A) Import reactions for PaNCEDl, PaNCED2, and PaNCED3 under each of the ATP concentrations were separated into pellet (P), and stromal (S) fractions. Samples in lanes 3, 4, 8, 9, ll, 12 were treated with thermolysin after import, as indicated by a "+" above the lanes. Chloroplasts used for import in the final two lanes (1 3 and 14) were treated with thermolysin prior to import to discern the requirement for protein receptors on the surface of the chloroplast (indicated by "pre" above the lanes). (B) Import analysis of a representative soluble protein (prSS), and an integral membrane protein (OM14). Under import conditions, radiolabeled mSS was found in the stromal fraction (S) and hence, was resistant to post-thermolysin treatment of the import reaction (lanes 7 to 10). OM14 was associated with the pellet fraction (P) following import, and was sensitive to thermolysin treatment of the import reaction (lanes 1 to 4). Pre- therrnolysin treatment of the chloroplasts used in the import reaction, did not affect the binding of OM14 to the chloroplast membrane (lanes 5 and 6). The import of prSS (lanes 11 and 12), was greatly reduced under the same conditions. (C) Extraction of the bound proteins from the chloroplast pellet fraction. Radiolabeled precursor proteins were incubated with chloroplasts under import conditions. The import reaction was scaled up over the standard reaction, as decribed in the Materials and Methods. After the import reaction, intact chloroplasts were re-purified and extracted with 100 mM sodium carbonate, or with 100 mM sodium chloride, or with 1% Triton X- 100 and separated into supernatant (S) and pellet (P) fractions. Extraction of a representative integral, outer membrane protein (OM14) is included. 79 Post. Pre. I — 0.1 4.0 fl - (mM) Mg-ATP l _ + H _ + H — + 1 I + l Thormolysln “8‘6” TP P S P S P S P S P S P S P 3 ~ — 51- a - -' "‘ PaNCED 1 ~.‘ 80“ —' --— - .— 51— PaNCED 2 8°- ii .. -- “"" M 51 _ PaNCED 3 1234567891011121314 — + I + |Thermolysln 80 uffer NaCl Nazca; Triton S - so - I "" "" '" , PaNCED1 c _ I“. ~ a he. *3 V PaNCED 2 I PaNCED 3 12345678 81 the chloroplast membrane (indicated by lanes 1, 3, 5, 7, 9, and 11 marked 'P' in Figure 3.6A), but was not imported, as indicated by lack of protein in the stromal fraction. When ATP concentrations were raised, import still did not occur (Figures 3.6A, lanes 9-12). Instead, PaNCED2 remained associated with the pellet fraction, presumably in the outer membrane. After the import reaction, PaNCED2 was accessible to thermolysin (Figure 3.6A, lanes 4, 8, 12, indicated by a"+” above the lanes), and hence, faces the cytosolic side of the chloroplast. Extraction of chloroplasts containing the bound PaNCED2 with NaCl and sodium carbonate, or solubilization of the chloroplasts membranes with Triton X-100 was ineffective at releasing the protein from the membrane (Figure 3.6C). Thermolysin treatment of chloroplasts prior to import did not affect the insertion of PaNCED2, indicating that protein receptors are not required to mediate insertion. The same import experiments were performed with PaNCED3, and similar results were obtained compared to PaNCEDl, PaNCED2, and OM14. These can be summarized as follows: 1) Association of PaNCED3 with chloroplast membranes in the absence of ATP (Figure 3.6A, lanes 1 to 4), and failure to be imported into the stroma when ATP concentrations were raised (Figure 3.6A, lanes 9-12), 2) Degradation of PaNCED3 following treatment of import reaction with thermolysin (Figure 3.6A, lanes 3, 4, 7, 8, 11, 12), 3) Insertion of PaNCED3 into the membrane was unaffected by pre-treatment of chloroplasts with thermolysin (Figure 3.6A, lanes l3, l4), 4) Insensitivity of PaNCED3 to washes of the chloroplast membranes with either NaCl or sodium carbonate, or to solubilization of the membranes with Triton X-100 (Figure 3.6C). 82 Table 3.2. Comparison of Avocado NCED genes and their Encoded Proteins. PaNCED1 PaNCED2 PaNCED3 Total Message Length (bp) 2203 1967 2310 3' untranslated region (bp) 377 226 388 5' untranslated region (bp) 116 166 44 Total Coding Bases (bp) 1710 1575 1878 Number of Amino Acids 569 524 625 Predicted MW (kDa) 63.1 59.6 69.7 % Identity to VP14 63 29 61 (at amino acid level) Isoelectric Point 7.4 9.0 8.2 Induction during Yes No Yes Ripening Induction during Yes No No Wilting Predicted Chloroplast Yes No Yes Transit Peptide‘ Localization” OMc OM° OMc In vitro ability to Yes - No Yes produce xanthoxin ‘Based on the predictions of ChloroP (Emanuelsson et al., 1999) l’Based on the results of in vitro chloroplast import experiments ° O.M. = outer membrane of chloroplast 83 Assay of Enzymatic Activity of the NCED Protein Products. The results of Northern analysis and sequence homology to Vp14 supported a role for both PaNCED1 and PaNCED3 in ABA biosynthesis. In contrast, PaNCED2 is constitutively expressed, and is less similar to VpI4. Therefore, PaNCED2 seemed unlikely to be involved in ABA biosynthesis. To test whether the protein products of these genes could catalyze xanthoxin formation in vitro, all three genes were expressed as recombinant proteins fused to glutathione-S-transferase (GST). Although somewhat insoluble, the recombinant proteins were purified to homogeneity and used to assay for carotenoid cleavage. Both recombinant PaNCED1 and PaNCED3 cleaved 9-cis- violaxanthin and 9'-cis-neoxanthin to produce xanthoxin and a C25 epoxycarotenoid (Figure 3.7). The reactions exhibited both protein (Figure 3.7A) and substrate (Figure 3.7B) dependency. T rans-isomers of violaxanthin and neoxanthin were not cleaved, consistent with the results of the VP14 assays, and with the required configuration for cis-ABA synthesis (Schwartz et al., 1997). The identity of xanthoxin (Figure 3.8), and the C25 compounds produced from either neoxanthin or violaxanthin was confirmed by mass spectrometry. Under the same assay conditions used for PaNCED1 and PaNCED3, PaNCED2 did not cleave either the cis or the trans isomer of either violaxanthin or neoxanthin. Analysis of Carotenoid Composition of Ripening Avocado Fruit. The carotenoid composition of fruit ripened for varying lengths of time was analysed to determine whether decreases in the levels of specific xanthophylls corresponded to increases in ABA. The carotenoids were identified on the basis of their acid-catalyzed shift in the absorption maxima. Lutein and lutein epoxide were the most 84 abundant carotenoids in unripe fruit, with levels remaining high in relation to the other carotenoids in fruit ripened for 10 days (Table 3.3). Between days 1 and 6, there was an increase in lutein epoxide, violaxanthin, neoxanthin and violaxanthin. As ripening continued (days 9 and 11), levels of these carotenoids decreased. The substantial decrease in neoxanthin that occurred between fruit ripened for 9 and 11 days is consistent with the increase in ABA levels that occurred during that time, but the two quantities cannot be related to one another on a 1:1 stoichiometric basis. Table 3.3. Quantification of carotenoids from avocado fruit ripened for 1, 6, 9, and 11 days. Carotenoids were extracted from 1 g of fruit and purified using HPLC. The concentration of each carotenoid is expressed on a ug/g FW basis. Data are the mean of 4 measurements :1: one SD. Carotenoid Days of Ripening (ug/g FW) 1 6 9 11 Lutein 2.65 d: 0.74 2.87 i 0.56 6.49 :t 0.85 4.07 i 0.76 Lutein Epoxide 2.88 i 0.61 2.82 9; 0.64 1.68 i 0.54 1.41 d: 0.43 Antheraxanthin 1.34 :t 0.33 1.36 d: 0.41 1.45 i 0.40 0.93 i 0.36 All-trans-Violaxanthin 1.13 i 0.28 1.79 :t 0.47 1.23 :1: 0.31 0.60 i 0.23 9-cis-Violaxanthin 0.56 i 0.19 0.78 i 0.26 0.36 a: 0.13 0.27 :t 0.14 9'-cis-Neoxanthin 2.39 i 0.73 3.75 i 0.97 0.94 d: 0.36 0.74 :1: 0.38 DISCUSSION It is now becoming clear that under both developmental changes (fi'uit ripening) and physiological changes (wilting), ABA biosynthesis is regulated at the level of cleavage of C40 carotenoid precursors into xanthoxin. This paper is the first demonstration of the up-regulation of NCED genes during fruit ripening. The data support the circumstantial evidence derived from a variety of studies that implicated the cleavage reaction as the governing step in increasing ABA levels both in development and during wilting. 85 1.2 .0 co Xanthoxin (nmol) p A .0 o Protein (pg) Xanthoxin (nmol) 0.0 . . o 4 8 12 9-cis-violaxanthin (nmol) Figure 3.7A-B. (A) Increase in xanthoxin formed from either 9-cis-neoxanthin (A) or 9- cis-violaxanthin (0) as a function of PaNCED1 (—) or PaNCED3 (---) protein concentration. Assays contained 6 nmol of substrate. (B) Xanthoxin formed by PaNCED1 and PaNCED3 as a function of 9-cis-violaxanthin concentration. The xanthoxin and C25 epoxycarotenoids produced in the in vitro reaction were analyzed by HPLC and identified by mass spectrometry. 86 dam—023 3 @3383 cggxu—oSéYa v5 =35x3$n3do mo omega—o 95> S com 28 _QmUZum .«o 03:33.... a ma Eficaxooeécra momma 92:83 803 880% cam—Em .36 “an 28 as .88 .§ 8 22 8838828 8; Ea .28: 5:382 .5 526 E a» 83.58% 8 6:88.80 85.58% 3.2: $5. cam—02am common—58 66.5 S 3 cog—88 cggxu—oSéBd .«o omega—o Ea...“ couscous 565.3% co aggro—c 3535085 05 .«o 83.5% 9.32 in 0.53m Nxz omm eon emm sum am” so" am . an m-~ m-~ m m 1 _ m _ L . JL 1_ , . m 1 n. N _ _ H . 1 mm . L m m H _ mm .om L __ Fm v n m m m. . A N " mm 7 - mwfi rev me“ . . «mg as“ c 1 ms .06 4 . .mm T a N 1 CZO~UP"‘)U CIDCDQCOU 87 The increase in ABA that occurs as avocado fi'uit ripens is substantial. However, the role that this increased ABA has in the fruit is not known. Many changes occur during ripening and the potential interactions between these processes are numerous (Brady, 1987). Presumably, ABA acts in hit either by gene activation or by having a more direct effect, for example, by alteration of membrane properties. To determine the role that ABA plays in fruit, a system more amenable to transformation, such as tomato, may be useful. Antisense expression of the cleavage enzyme gene(s) may cause lowering of ABA levels, and the resulting effects on gene expression could be monitored. The 30- fold increase in ABA makes avocado a suitable fruit to study ABA biosynthesis. ABA mesocarp has also been used for other studies of ABA physiology (Lee and Milborrow, 1997) In maize, Vp14 is part of a multi-gene family (Tan et al., 1997). This is also the case in avocado. PaNCED1 and PaNCED3 are 60% identical at the amino acid level with Vp14 and the tomato homolog, LeNCEDI (Burbidge et al., 1999). Analysis of the sequence similarity of homologous sequences present in the database suggests that a large family of NCED genes exist. The genes can be grouped according to their identity to each other. For example, maize, bean, tomato, and avocado NCED] and -3 share approximately 60% identity at the amino acid level to each other. PaNCED2 is approximately 60% identical to an Arabidapsis sequence, called AtNCEDI (Neill et al., 1998), but only 30% identical to the aforementioned sequences. It would seem plausible that genes with 60% identity or greater may have the same fimction while those with less identity catalyze different reactions. The only proteins with demonstrated functions are the two avocado proteins described here, maize VP14, the bean protein, and lignostilbene 88 dioxygenase. The results of studies of the notabilis mutant (the mutant allele of LeNCEDI), would suggest that the tomato gene product catalyzes the same reaction (Burbidge et al., 1999). The regions that are conserved among all of the protein sequences are likely part of the active site, and may be involved in cofactor binding. Site-directed mutagenesis would be usefiil in determining the function of the conserved residues. The firnction of PaNCED2 and similar Vp14 homologs in other systems is not known. In Arabidapsis, a PaNCED2 homolog called AtNCEDI (60% amino acid identity) is moderately induced by rapid dehydration of leaves (Neill et al., 1998). We did not find up—regulation ofPaNCEDZ during dehydration of avocado leaves. In addition, PaNCED2 lacks a chloroplast targeting signal. It should be noted that proteins destined for the outer membrane of the chloroplast are not synthesized with an N-terminus extension, and possess a different import pathway in comparison with proteins destined for the thylakoid membrane (Keegstra and Cline, 1999). Since carotenoids are present in the envelope (Siefermann-Harms et al., 1978), a putative NCED need not be imported into the chloroplast for it to serve its function. Results of the in vitro import assay suggested that PaNCED2 is associated with the chloroplast outer membrane. However, immunolocalization will be necessary to confirm this. Import analysis into isolated pea chloroplasts demonstrated association of all three proteins with the chloroplast envelope fi'action. The interpretation of in vitro import experiments is complicated by the fact that the precursors utilized are from fruit, and the system used for import is pea chloroplasts. There is some evidence, for example, that import pathways for leucoplast proteins differ from those of chloroplasts (Wan et al., 89 1996), and therefore one may predict that proteins in fruit may have a different import pathway into chromoplasts. However, avocado fi'uit differs from fruits such as tomato in that chloroplast structure is maintained and distinct thylakoid membranes are still present (Milborrow, 1974). Further, PaNCED1 is expressed in leaves, and therefore is not a fruit- specific. Another consideration in in vitro import experiments is that the properties and folding characteristics of proteins affect their interaction with the translocation machinery. For example, hydrophobic proteins may insert into membranes randomly. Misfolded proteins may not have amino acid residues that are normally involved in interaction with a receptor protein exposed. Therefore, the results of the import experiments would need to be confirmed by other methods before drawing the conclusion that the three avocado proteins are localized to the outer membrane. To resolve the localization of the three proteins in vivo, antibodies designed against the three avocado proteins would need to be produced and immunolocalization and fiactionation experiments performed. The double bond present in both lignostilbene and in violaxanthin and neoxanthin is a common feature found in terpenoids, phytoalexins and many other natural products. Many of the biochemical pathways for these compounds occur in the cytoplasm. It is, therefore, possible that protein products of genes such as PaNCED2 have no involvement in ABA biosynthesis or in carotenoid breakdown. The immunological detection mentioned above, as well as studies with reporter genes, such as the green fluorescent protein, may be useful in determining the localization of the enzymes with unknown function. Screening of T-DNA insertion lines of Arabidapsis may also be useful in making deductions about firnction. 90 Two avocado genes, PaNCED1 and PaNCED3, encode proteins that are capable of in vitro synthesis of XAN, the precursor of ABA. Evidence for the in vivo role of PaNCED1 and 3 in ABA biosynthesis is indicated by the correlation of mRNA levels of these genes with endogenous ABA levels. The chloroplast localization of PaNCED1 and PaNCED3 is also consistent with suppositions about the site of ABA synthesis (Zeevaart and Creelman, 1988). In vitro, PaNCED1 and PaNCED3 appear to be indistinguishable in terms of their substrate preference; both utilize violaxanthin more effectively than neoxanthin. Thus, ABA biosynthesis in fruit appears to be redundant, in the sense that two genes appear to encode proteins with identical functions. However, it should be emphasized that in vivo factors such as transport and degradation of ABA are also important in regulating levels. In addition, the in vivo accessibility of enzyme to the substrate, and the isomerization of violaxanthin into neoxanthin, may also be determinants of why two enzymes exist. It will be interesting to determine whether the presence of two very similar enzymes is typical of other plants as well, and if it occurs in organs other than fruit. The carotenoid data (Table 3.3) indicate that there is a substantial decrease in neoxanthin as the fruit ripens. When expressed on a molar basis, the amount of ABA at any stage of ripening exceeds the amounts of both violaxanthin and neoxanthin. For example, late in ripening (day 11), the sum of the molar amount of violaxanthin and neoxanthin present is 2.7 nmol versus 49.2 nmol of ABA (derived from the data presented in Figure 3.4A and Table 3.3). This indicates that the carotenoid pool must turnover more rapidly than the ABA pool. In tissues such as light-grown leaves and in fruit, the ratio of carotenoids to ABA is very high, making it difficult to demonstrate 91 correlations between decreases in xanthophylls and increases in ABA. In roots (Parry et al., 1992) and in dark-grown, fluridone-treated leaves of Phaseolus vulgaris (Li and Walton 1990; Parry and Horgan, 1991 ), there is a 1:1 correspondence between xanthophylls cleaved and ABA + ABA catabolites synthesized. The decreases in both violaxanthin and neoxanthin that correlate with increases in ABA leves in fruit are consistent with their proposed role as ABA precursors, despite the lack of a 1:1 stoichiometric ratio. Multi-gene families ofien encode genes with related functions, but in cases where the function is the same, differential regulation ensures that distinct genes are activated in response to different environmental and other stimuli. A good example of this is the ACC synthase gene family (Zarembinski and Theologis, 1994). The fact that NCED genes are part of a family can perhaps be construed as an indication that sensitive mechanisms are needed to regulate the amount and location of the ABA synthesized. In this regard, zeaxanthin epoxidase, an enzyme with both a structural and photoprotective role, does not exist as a gene family (Marin et al., 1996). For ABA to serve its role in drought response, a rapid signaling mechanism must be in place; perhaps the easiest way to achieve this is to have a specific signaling pathway to turn on the appropriate gene. Differential regulation implies that different signal transduction pathways are activated that allow gene expression in response to the specific stimuli. Ultimately, the distinction between which genes are induced in response to a given stimulus lies in the promoter region. In avocado, two cleavage enzymes are present: both are induced during fruit ripening but only one of the genes is induced by water stress. Analysis of the promoter region of these two genes should reveal whether a dehydration response 92 element (DRE), as is found in many osmotic-responsive genes (Shinozaki and Yamaguchi—Shinozaki, 1997) is also present in PaNCED] . Promoter elements for genes that are up-regulated during fi'uit ripening include those that have an ethylene-responsive box (e. g. Itzhaki et al., 1994), those that have ripening-specific elements (e.g. Atkinson et al., 1998; Deikman et al., 1998), and others in which no previously characterized regulatory elements are apparent (e.g. Beaudoin and Rothstein, 1997). Comparison of the promoters of PaNCED1 and PaNCED3 may reveal which, if any, common cis-acting elements are present, and deletion analysis should indicate the role(s) that these elements play in regulating gene expression during fruit ripening and drought stress. It will also be interesting to determine whether other developmental cues, such as seed germination and embryo development, induce the expression of a novel NCED, and if overlap exists between the signal transduction pathways that lead to the expression of wilt-related versus developmentally-regulated NCED genes. REFERENCES Adato I, Gazit S, Blumenfeld A (1976) Relationship between changes in abscisic acid and ethylene production during ripening of avocado fruits. 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Plant Physiol 121: 237-243 Zarembinski TI, Theologis A (1994) Ethylene biosynthesis and action: a case in conservation. Plant Mol Biol 26: 1579-1597 Zeevaart JAD, Creelman RA (1988) Metabolism and physiology of abscisic acid. Annu Rev Plant Physiol Plant Mol Biol 39: 439-473 Zeevaart JAD, Heath TG, Gage DA (1989) Evidence for a universal pathway of abscisic acid biosynthesis from 18O incorporation patterns. Plant Physiol 91: 1594-1601 Zeevaart (1999) Abscisic acid metabolism and its regulation. In: Hooykaas PJJ, Hall MA, Libbenga KR (eds) Biochemistry and Molecular Biology of Plant Hormones. Elsevier Science pp 189-207 97 Chapter 4 Identification of Cytochrome P450 monooxygenase genes using differential display ABSTRACT The rates of both biosynthesis and metabolism determine ABA levels. In many tissues, a major metabolic route of ABA is to the unstable intermediate 8' hydroxy- abscisic acid (8' OH-ABA), which rearranges to form phaseic acid. The enzyme that catalyzes this conversion, ABA 8'-hydroxylase, is a cytochrome P450 monooxygenase. Much evidence shows that ABA 8'-hydroxylase is regulated at the transcriptional level. Addition of exogenous ABA to suspension cultures can stimulate catabolism into phaseic acid. A modified differential display approach was used to isolate cytochrome P450 monooxygenase genes that are differentially expressed in response to ABA treatment, and hence represent candidate genes for ABA 8'-hydroxylase. Differential display was carried out on RNA isolated from untreated suspension cultures and from cultures that had been treated with ABA, using degenerate primers designed against conserved regions of cytochrome P450 monooxygenase genes. Several bands were excised, reamplified, and cloned. These bands were tested by Northern analysis. Some of the cloned genes were not differentially expressed in response to ABA treatment and therefore, represented false positives. Some of the differentially expressed genes were not cytochrome P450 monooxygenases. Possible strategies to isolate the gene encoding ABA 8'-hydroxylase are discussed. 98 INTRODUCTION ABA levels are regulated by the relative rates of biosynthesis and degradation. Large increases in ABA levels occur during wilting of leaves and during fruit ripening (see Chapter 3). In both of these cases, the increase in ABA is controlled by the rate of xanthophyll cleavage (see Chapter 3). Depending upon the tissue, ABA can be deactivated through two processes: catabolism and conjugation (Zeevaart, 1999). Catabolism occurs through oxidation of the 8' methyl group of ABA to form 8'-hydroxy- ABA (8'-OH-ABA), which is unstable and rearranges to form phaseic acid (PA). In addition to oxidation, conjugation of ABA by glycosylation or esterification may also be important in governing ABA levels in some tissues (Zeevaart, 1999). The pathway of ABA metabolism into phaseic acid via the unstable 8’-OH-ABA intermediate is shown in Figure 4.1. ~88 \ —- ~08 \ -- «on \ —* ~08 \ 0 0021+ 0 cow 0 coal 1* coat ABA ecu-ABA PA "0 DPA Figure 4.1. Pathway for the catabolism of ABA into phaseic acid (PA). The reaction of ABA into PA via 8'-OH-ABA is catalyzed by ABA 8'-hydroxylase, which is a cytochrome P450 monooxygenase. Much evidence indicates that the enzyme that mediates the conversion of ABA into PA (ABA 8'-hydroxylase) is a cytochrome P450 monooxygenase. Cytochrome P450 monooxygenases are membrane-bound heme proteins that catalyze reactions in which two reducing equivalents from NADPH or NADH are transferred to the P450, and in which molecular oxygen is cleaved with one oxygen being incorporated in the substrate 99 while the other is reduced to water. Binding of the heme is through a cysteine residue situated between 15% from the carboxy terminus. A diagnostic test for cytochrome P450 involvement in a reaction is inhibition by carbon monoxide (CO), followed by reversal of the inhibition by irradiation with blue light (Bolwell et al., 1994). The overall reaction is shown below: RH + 02 + NADPHH” —> ROH + H20 + NADP+ A myriad of diverse cellular reactions involve the incorporation of an oxygen atom into the substrate. Reflecting this, cytochrome P450 monooxygenases are encoded by a highly divergent gene superfamily containing over 450 known cytochrome P450 sequences distributed among 65 gene families (Nelson, 1999). There are over 100 plant P450 cDNA/ genomic DNA sequences that have been completed and that are distributed among 26 of these P450 gene families (Winkler et al., 1998). These sequences and other information about this class of enzymes can be found at the following web address: http://drnelson.utmem.edu/nelsonhomepagehtml). Numerous plant P4503 have been cloned and/or biochemically purified. Some of the functions that have been ascribed to these P4508 include certain steps in gibberellin biosynthesis, brassinosteroid biosynthesis, flavonoid biosynthesis, and herbicide detoxification (Schuler, 1996). However, for the majority of sequences, no function has been ascribed. A systematic attempt to assign fitnctions to the Arabidapsis P450 sequences is being carried out by Winkler et al. (1998). In this approach, T-DNA mutants tagged in various P450 genes are being isolated, and examined for phenotypic effects. The following lines of evidence support the notion that ABA 8'-hydroxylase is a cytochrome P450: 1) Incorporation of one 180 from 1802 into PA (Creelman and 100 Zeevaart, 1984; Zeevaart et al., 1989), 2) Addition of the cytochrome P450 inhibitor tetcyclacis to Xanthium leaves prior to rehydration prevented PA accumulation (Zeevaart et al., 1990), 3) A cell-free system of Echinocystis lobata that was capable of converting ABA into PA was inhibited by carbon monoxide, and required oxygen and NADPH. (Gillard and Walton, 1976), 4) In stressed and subsequently rehydrated Xanthium leaves, CO reduced the accumulation of PA (Creelman et al., 1992), 5) A cell-free system prepared from maize suspension cultures catalyzed the conversion of ABA into PA. The reaction required oxygen and NADPH, and displayed blue light reversibility of CO inhibition (Krochko et al., 1998). Transcriptional induction as a result of treatment with substrate is a common mechanism by which many cytochrome P450 monooxygenases are regulated. Specifically, in the case of ABA catabolism, several reports of induction of ABA 8'- hydroxylase by ABA exist: 1) Pretreatment of barley aleurone layers with ABA resulted in an increased conversion of [3H]ABA into [3H]PA (Uknes and Ho, 1984), 2) Potato and Arabidapsis suspension cultures pre-treated with 50 uM (i)-ABA exhibited an increased rate of [2H6]PA formation from [2H6]ABA (Windsor and Zeevaart, 1997), 3) A cell-free system from the embryonic axis of chickpea seedlings germinated in ABA had enhanced [”C]ABA to [14C] PA conversion (Babiano, 1995), 4) Maize suspension cultures treated with (+)-ABA exhibited 8'-hydroxylase activity while untreated cultures had no activity (Cutler et al., 1997). As cytochrome P4503 are membrane bound, and, in the case of ABA 8'- hydroxylase, of low abundance, biochemical purification appeared problematic. Therefore, the difference in activity observed in substrate-treated versus untreated 101 samples served as a basis to identify candidates for ABA 8'-hydroxylase. A molecular approach was undertaken involving differential display of untreated and ABA- treated materials and using degenerate primers designed against conserved regions of P450 genes. These regions used for primer design include the fingerprint heme-binding domain, present in all P4508, and two regions further upstream. These primers were used together with anchored oligo d(T) primers normally used in differential display. The use of degenerate primers in differential display in order to isolate specific classes of genes has been reported previously (Joshi etal., 1996). Recently, a similar approach was used successfully to isolate differentially-expressed cytochrome P450 monooxygenase genes from elicitor-treated soybean suspension cells (Schopfer and Ebel, 1998). MATERIALS AND METHODS Plant Material. Five— to seven-day-old Arabidopsis thaliana or potato suspension cell cultures were maintained at 25°C in the dark. For treatment of the cultures, (i)-ABA was added to a final concentration of 50 pM. At various times after the addition of ABA, aliquots of the culture were removed, filtered, and washed with fresh medium. The tissue was frozen at -80°C for later use in RNA isolation. RNA isolation and Northern analysis. RNA was isolated by the method of Vanderlerberge et al. (1992). For a description of cDNA synthesis, see differential display and cloning (next section of Materials and Methods). Northern analysis was carried out by electrophoresis of 30 ug total RNA on 1.2 % (w/v) agarose gels containing 2.2% formaldehyde according to Maniatis et al. (1982). RNA gels were blotted onto Hybond H)r membranes (Amersham). 102 Inserts to be labeled as probes were excised from the pGEMTeasy vector (Promega) using EcoRl. The DNA was purified using a Qiagen gel extraction kit and labeled with 32F using random-prime labeling (Gibco-BRL). Prehybridization and hybridization of the membranes were performed at 65°C using Church-Gilbert buffer (Church and Gilbert 1984). The membranes were washed twice in 2x SSC, 0.1% (w/v) SDS at room temperature, followed by two washes in 0.2x SSC, 0.1% (w/v) SDS at 65°C. Diflerential Display and Cloning. RNA used as the treated sample in the differential display was isolated from cultures that had been treated with (i)-ABA for 6 h using the method described by Vanderlerberge et al. (1992). The method of Liang et al. (1993) was used for differential display with modifications as described in the GenHunter manual. All reagents used in the reverse-transcription and PCR reactions, with the exception of Taq DNA polymerase (Gibco-BRL), the degenerate primers (described below), and the (33P)dATP, were from the GenHunter differential display kit. The main difference employed in the differential display technique employed here compared with the methods described by GenHunter is that the arbitrary primers normally used were replaced by degenerate primers that had been designed against conserved regions of cytochrome P450 monooxygenase genes. The sequences and relative positions of these three degenerate primers are shown in Figure 4.2. Each primer was used (at a concentration of 2 pM) in conjunction with the 3 anchored oligo d(T) primers (in this case oligo d(Ti 1). Conditions for reverse transcription were as follows: 65°C, 5 min; 37°C 60 min (after 10 min at 37°C, 1 ul MMLV reverse transcriptase was added to each tube), 75°C 5 min. 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