ISOLATIGN, CHARACTERIZATION AND ROLE 0F END‘OGENOUS AUXINS ANT) CYTDKININS IN SOUR CHERRY (Prunus‘ cerasus L cv. Montmorency) FRUIT EEVELGPMENT Thesis for the Degree or PIL D. MICHIGAN STATE UNIVERSITY MURRAY EDWIN HOPPING 1972 .- IIII1II IIIIITIIIIIIIIIIIIIIII 8IIIIII 'J LIBPARY L1 IIIII 93 01004 217 Michigan State University This is to certify that the thesis entitled I Isolation, Characterization and Role of Endogenous Auxins and Cytokinins in Sour Cherry (Prunus I cerasus L. cv. Montmorency) Fruit Development. I presented by Murray Edwin Hopping has been accepted towards fulfillment of the requirements for Ph - D. degree in We .:ZZé2M %9\y/‘\/§/(z// . \. ~ It 8 30- ' - 3 t In E q ‘S q 20 I- ' 2 \ o k \ 3 ID- - I k v v ' 1 ° . 0 7 I4 2| 28 3542 49 56637| DAYS AFTER ANTHESIS Figure 5. 55 Changes in levels of endogenous growth substances in seed and pericarp tissues, expressed as IAA equivalents/fruit, in relation to fruit develop- ment. IAA equivalents (-0-, -O-). Fruit weight (-V-) . 20 IS 5 0| 5 IAA EQUIVALENTS (ng - fruit) 5 A SEED - H O--O PERICARP 56 ISOPROPANOL: H20 (4H) HEXANE: WATER (9:0 0 7 l4 2| 28 354249566370 ° I L l l DAYS AFTER ANTHESIS N u g FRUIT WEIGHT (9) 57 .Amm tam musoflmv Aanmv kucz " mcmxmmm .Amm .sm musmfimv AHuvv Hmuc3 “ HOCMQOHQOmHH o.>H v.m h.m >.m o m cm o.HH H.vm mNH baa o NH mv h.H 0.x o o o 0 mm m.o m.v h.mmH h.m 0 «ma Hm mwoowumm mm mom ohm mmm 0 mm mm coma own «me mow em mm mv h.m >.¢ omam NmHH omm em mm men Homa owmm mamm OSHA mmva Hm comm Nam 3H a xaemao :Axse mHH :Axsm «HH cflxsa :Hx94 mammrpae “momav aflxsm Hopes Hmpoe Hmnpsmz amuusmz owcflod Houmm mama A.u3 who Emwm\mcv mucoao>flsom <¢H .Hhma can mood cw ucmfimoam>mo DHDHM mo mmomwm.ucmnwmwwo um mmsmmau mumoflnwm one comm mnumzo HDOm CH owwompoo.mc0HDmuucwocoo cflxsm mo comHHoQEOUII.H mamme 58 .Amv .dv musmwmv Aaumv umumB u mcmxmm N .Amv .dv musmflmv hauvv Hmum3 “ HOCMQOHQOmHH H.m m.H o.o m.m o H.v om m.m o.m o.mm m.om o N.m mv N.o o.H o o o 0 mm v.0 N.o H.m «.0 o h.m Hm mnmownmm o.m v.oa m.h o.o o 5.0 mm m.mH o.m o.HH m.oH v.0 5.0 we No.0 No.0 m.mH v.0H h.h m.o mm o.m o.m m.Hv H.Nm o.m N.HH Hm comm 3m 3H m a Aahmav cwxsd mHH cflxsm «HH cflxsc cflxsm mflmmnpcd Ammmav cwxsfl Hmvoa Hmuoe Hmuwsoz Hmnusmz cepwo< Hopwm whom ADAsAu\mcc mpamfim>flsam «4H .Hhma one moma cw pcmEmon>mc uwsum mo mommum Dcmumwmfio Dc mmsmmflp mwmoflumm can comm hnumso HDOm CH cowomump mHm>mH aflxsm mo cOmHHmmEOUII.m mqmda 59 Furthermore, bioassay of known amounts of IAA that had been processed through the extraction and fractionation procedure used to determine changes in auxin levels in 1969 showed that substantial losses occurred. Expression of the amount recovered as a ratio of the amount added gave values of 0.22, 0.18, and 0.23 for three replicates. Part of these losses can be attributed to the solubility of one solvent in another during partition chromatography (Figure 3A, B, Appendix A). Further losses have been reported to occur during chromatogram equilibration and development (Kefford, 1955). Accordingly, all Chromatograms involving quantitative determinations (Tables 1, 2) were equilibrated and develOped in a nitrogen atmosphere although recovery values were not obtained. Thus the differences between harvest dates are the only valid comparisons. High concentrations of acidic and neutral auxins (Figure 6B, 17) were located in seed extracts 21 days after anthesis (Table 1). Subsequent to the third week from anthesis all auxin levels, except for seed neutral IIA, Idecreased (28 and 42 days after anthesis) and then increased. This was also true in 1969 (Figure 4A). In all seed tissues analyzed (1971) the neutral IIB auxin was found to be the Inajor auxinic component. Total auxins in the pericarp (Table 1) decreased from a maximum at day 21 to a non- S HQ WGQkQ-flk >\\ kmmxxk term 1 bar; (21 1 1n alens nia Rf (O .l 1 Lu. W. nu _ mvumwunw Ina “w .1: _ .1_ . .nn Tam. 3 m 5. u 3 M . mmi§§u _nv .nn mw .nu nu 7, q‘ q d an 0.5 Rf 4M C 0 m A L. . 3 w w; ”M B n O . m m. m. m o E g3 .25 :5 be u last «god .3ng 3 Kurt ilt :ia l 62 from anthesis). No neutral auxin IIA was detected in pericarp tissues. When the above data are expressed in terms of IAA equivalents/fruit differences between years are evident (Table 2). The highest level of total seed auxin was found at 21 days after anthesis in 1971 rather than at 42 or 56 days (1969). Seed acidic auxin content rapidly fell to a low, and nearly constant level (anthesis + 28, + 42, + 56), while the level of neutral IIA and IIB auxin declined at a much slower rate. The pericarp fraction, however, had an initially low level of neutral IIB auxin (anthesis + 21, + 28) which increased by day 42 to a maximum and then declined. During the same period, the acidic auxin decreased from an initially high level and then increased with the later stages of fruit growth. Characterization of Seed Acidic Auxins Anthesis + 21 days.--Chromatographic separation of acidic substances and subsequent detection by bioassay revealed two peaks of activity (Figure 6A). The first <3f these (Rf 0.3 - 0.6, designated Acidic I) was active in both the A3393 first internode and curvature bioassays euui chromatographed with applied standards (IAA, IBA). 12mg second peak of activity (Rf 0.8 - 1.0, designated Acidic II) ‘vas active only in the straight growth test. 63 Preparation of a new sample (as described above), elution of Acidics I and II, and re-chromatography of the eluates in a basic solvent resulted in no further resolution of Acidic I (Figure 6B) but separated Acidic II into two substances which were inactive in the 53323 curvature test (Figure 6C). The peak of activity located from the Acidic I chromatogram (Rf 0.3 - 0.7, Figure 6B) chromatographed with applied standards (IAA, IBA). Since both standards ran together, the promotive activity at Rf 0.3 - 0.7 (Figure 6B) was again re-chromatographed in an attempt to further separate the active moieties. Replicate samples of Acidic I were prepared according to the method outlined above. One of these samples was re—chromatographed, in 3233, in acetonitrile : water (4:1) and bioassayed (Figure 7A). The other sample was divided and each half separately chromatographed in acetonitrile water (4:1). One of these latter Chromatograms was bioassayed directly (Figure 7B) to provide a replicate of that shown in Figure 7A. Re-chromatography in acetonitrile : water (Figure 7A) did not resolve Acidic I into further components. This xxesult withstood replication (Figure 7B). However, the peak of activity (Figure 7A, 7B) chromatographed with applied IAA anui IBA standards. In an attempt to determine if the active moiety was IAA-like or IBA-like, the remaining Chrcmatogram.(see above) was cut at the Rf of IAA (Rf 64 Figure 7.--Histograms depicting the Avena first internode bioassay response to acidic seed auxins (21 days after anthesis). A. Re-chromatography of 0.2 gram equivalents Acidic I (Rf 0.3 - 0.7, Figure 6B). Solvent system:- acetonitrile : water (4:1). Re-chromatography of 0.1 gram equivalents Acidic I (Rf 0.3 — 0.7, Figure GB). Solvent system:— acetonitrile : water (4:1). Re-chromatography of elute from Rf 0.35 - 0.47 (Figure 7A). Solvent system:- acetonitrile : water (4:1). Re-chromatography of eluate from Rf 0.47 - 0.70 (Figure 7A). Solvent system:- acetonitrile : water (4:1). Control value (——-). (D m 0 O .p. O N O O (D O 03 O A FIRST INTER/V005 ELOIVG‘Af/OIV— % 0F INITIAL LENGTH O N O 65 - IAA IAA +- l-—-l H t——1 IBA ' H )- b i * 3.5-[3750 35 IAA IPA MA {/79} B .___.IBA D ZIBA E fr E " If. ”’1 , l _I O 0.5 LO 0 0.5 LO 3.5Ll'350 Rf Rf 35 IAA Ing/ 66 0.35 - 0.47) and at the Rf of IBA (Rf 0.47 - 0.70) and both strips were individually eluted. Re-chromatography of these eluates in acetonitrile : water (4:1), and subsequent bioassay, showed that each component (Rf 0.35 - 0.47, Rf 0.47 - 0.70) yielded identical peaks (Figure 7C, 7D, respectively). This evidence strongly suggests that the peak of activity shown in Figure 7A, 7B is homogeneous and similar to IAA. Chromatogram strips, taken from the edge of the develOped streaks, were sprayed with Salkowski reagent. With the exceptions of Acidic II, all Chromatograms gave the intense red-purple reaction typical of IAA at those zones that would have been adjacent to detected growth promotive activity. Anthesis + 42 days.--A single, broad, peak of acidic growth promotive activity was located between Rf 0.0 - 0.4 when a relatively concentrated sample (4.5 gram equivalents) was chromatographed in isopropanol : water ( 9:1) (Figure 8A). Re-chromatography of an eluate of this zone in two further solvents resulted in single peaks of activity (Figure 8B, 8C) at the respective Rf's of IAA. The Rf of the promoter in Figure BB corresponded with that found earlier (Figure 2) for the seed fraction 28, 35, 42, and 49 days after anthesis. Similarly, the activity shown in Figure 8C occurred at the Rf of the active fraction found in the seed 21 days after anthesis (Figure 7A, 7B). 67 Figure 8.—-Histograms depicting the Avena first internode bioassay response to seed aCidic auxins (42 days after anthesis). A. B. 4.5 gram equivalents chromatographed in isopropanol : water (9:1). Re-chromatography of 1.5 gram equivalents Acidic I (Figure 8A, Rf 0.0 - 0.4). Solvent system:- isopropanol : water (4:1). Re-chromatography of 1.5 gram equivalents Acidic I (Figure 8A, Rf 0.0 - 0.4). Solvent system:- acetonitrile : water (4:1). Control value (-—). 68 |.O i Aim . D D n b h b L O O O 0 $55.3.qu Exits: k0 o\o In 20R @62qu MQQEQMK \<\ khkfix IAA Ing) C o 7 550 J. lAA (n9) IAA H 1 Ag. 05 Rf LO H 0.5 Rf IAA H H B p b r— p )— )- — , 0 20km “I TKQENQ .VSKtS K9 o\o l§QtF$rwu qfixk :5 us o\u I\6: 33.6 Nw MGSEx-flk \<\ kmgxxk 8O (Rf 0.3 - 0.6, Figure 13A) that chromatographed with IAN. When this latter promotive activity (Rf 0.3 — 0.6) was again re-chromatographed in hexane : water (upper phase), resolution into two fractions was achieved (Figure 13B). One fraction remained at Rf 0.0 - 0.1 while the second moved an appreciable distance to Rf 0.6 - 0.7. Neither fraction chromatographed with IAN nor reacted with chro— mogenic reagents (Salkowski, Ehrlich). Anthesis + 42 days.--Chromatography of the neutral seed auxins and subsequent bioassay resulted in a zone of weak promotion (Rf 0.5 - 0.6, Neutral I) (Figure 14A) and a zone of strong inhibition (Rf 0.8 - 0.9, Neutral II) similar to that shown previously (Figure 2, anthesis + 28, + 35, + 42, + 49 days, Figure 12A). When both fractions were re-chromatographed separately, only the Neutral II fraction exhibited a promoter peak at Rf 0.6 - 0.7 (Figure 14C). This promoter had been previously shown in the seed fraction 21 days after anthesis (Figure 13B). Characterization of Pericarp Neutral Auxins Anthesis + 42 days.--Chromatography of the neutral pericarp fraction (Figure 15A) yielded weak promotive zones at Rf 0.1 - 0.3 and Rf 0.7 and a zone of strong inhibition (Rf 0.8 - 1.0, Neutral II). Since the zone at Rf 0.5 - 0.7 had been previously shown to be weakly promotive (Figure 12A, 14A), the zone at Rf 0.5 - 0.7 (Neutral I, Figure 15A) was 81 Figure 13.--Histograms depicting the Avena first internode (open bars) and Avena curvature (shaded bars) bioassay response to seed neutral auxins (21 days after anthesis). A. Re-chromatography of 0.4 gram equivalents Neutral II (Figure 12A, Rf 0.8). Solvent system:- water. B. 0.3 gram equivalents Neutral II (Figure 12A, Rf 0.8) re-chromatographed in hexane : water (upper phase) after equilibration over water. Avena first internode bioassay control value (———). Avena curvature bioassay control value <—--). 82 350 3.5 IAA {/79} 87.5 r :5 o Nnmvxbk v8 n<§u Ana. 1 T 5...: H OR %/ mu ”7.” B H 0 F- p p . p b F - p p n . - b w m m o m w m m 0 $353qu Nfixkts k0 o\.. .l EQRVGEQQW WQQEQMK>Q kmmxxk 83 Figure l4.--Histograms depicting the Avena first internode bioassay response to seed neutral auxins (42 days after anthesis). A. B. 4.5 gram equivalents chromatographed in isopropanol : water (9:1). Re-chromatography of 1.5 gram equivalents Neutral I (Figure 14A, Rf 0.5 - 0.6). Solvent system:- hexane : water (upper phase). Re-chromatography of 1.5 gram equivalents Neutral II (Figure 14A, Rf 0.8 - 1.0). Solvent system:- hexane : water (upper phase). Control value (—-—). 84 :IIL. E M- 330 3.5 1, IAA (n9) 1 0.5 LO IAN H C I..r o 1 0 5 Rf |.O A E: 1.1" F3 J O 6 4 2 05 562.3 «335 “B u N last .626 d M855 3 33k B m H 0 p L . 0 O O O 2 O tkmzmq 121.53: K0 & I EOE 36?qu MQQ>E>N~ >\\ k mkxk L. 1.0 3 5 350 35 Rf Rf IAA IngI 85 Figure 15.--Histograms depicting the Avena first internode bioassay response to pericarp neutral auxins (42 days after anthesis). A. B. 12.0 gram equivalents chromatographed in isopropanol : water (9:1). Re-chromatography of 4.0 gram equivalents Neutral I (Figure 15A, Rf 0.5 - 0.7). Solvent system:— hexane : water (upper phase). Re-chromatography of 4.0 gram equivalents Neutral II (Figure 15A, Rf 0.8 - 1.0). Solvent system:- hexane : water (upper phase). Control value (———). 86 IAA {/79} um H 1.0 05 Rf A o s‘k b>>wq q vQK :5 k0 R 11%ka V$\Q\k 33,696 NQSSSNQS 5“.ka IAA Ing) 87 eluted from duplicate Chromatograms and re-chromatographed (Figure 15B). No promotive peaks were found. Re- chromatography of Neutral II in this latter solvent system (Figure 15C) again demonstrated growth promotive activity at Rf 0.5 - 0.7 similar to that found previously in seed fractions 21 and 42 days after anthesis (Figure 13B, 14C). ‘ Further Characterization of the Neutral Auxins Chromatography in two solvents.--When equal gram equivalents of the seed neutral fraction (42 days after anthesis) were chromatographed in two dissimilar solvents, separation into two growth promoting substances was achieved with the one (hexane : water) but not with the other (isopropanol : water) (Figure 16). Each of the two growth promoters separated by hexane : water were highly active in the first internode bioassay. The promoter at Rf 0.0 - 0.2 and at Rf 0.5 - 0.7 (Figure 16B) will hereafter be designated as neutral auxin IIA and neutral auxin IIB, respectively. To test the hypothesis that the zone of inhibition (Rf 0.9 - 1.0) from isoprOpan01V: water (9:1) Chromatograms was due to overlapping of the two promoters, the neutral phase was chromatographed in hexane : water and the zones Rf 0.0 - 0.2 (neutral IIA) and Rf 0.5 - 0.7 (neutral IIB) eluted. Each eluate (0.1 gram equivalents) was chromato- graphed individually, or in combination, in isopropanol water. Bioassay of the developed Chromatograms revealed 88 Figure 16.--Histograms depicting the Avena first internode bioassay response to seed neutral auxins (42 days after anthesis). A. B. Chromatography of 1.0 gram equivalents in isopropanol : water (9:1). Chromatography of 1.0 gram equivalents in hexane : water (upper phase) after equilibration over water. Control value (———). 89 350 IAN H 6 .. I__I_.L_I _- 3.5| I.O m J. p b n p b h b b 0 0 0 0 0 0 O 0 4. 2 8 6 4. 2 tkwkmu q‘tts “\Q ...\9 I \EQK>§ 595k . 750 /AA (n9) 35' Rf 90 weak growth promotive zones at Rf 0.9 i 0.1 for neutral IIA (Figure 17B) and Rf 0.7 i 0.1 for neutral IIB (Figure 17C) although the Rf's obtained are the reverse of what would be eXpected in terms of presumed polarities. Co- chromatography of neutral IIA and neutral IIB in isopropanol : water (Figure 17A) resulted in a single promoter peak at Rf 0.8 i 0.1 that overlapped the distri- bution of both auxins (Figure 178, 17C). Dilution curves.--The bioassay response to 0.1 gram equivalents of the crude neutral ether fraction (seed tissue, anthesis + 42 days) approximated that obtained with 100 ng IAA (Figure 18). At concentrations greater than 0.35 gram equivalents, growth inhibition occurred which would explain the zone of inhibition (Rf 0.8 - 0.9) on Chromatograms developed in isopropanol : water (9:1; 4:1) (Figure 2, 3, 8A, 12A, 15A, 17A) and the higher total auxin content shown in Tables 1 and 2. Although the bioassay response to 0.1 gram equivalents was approxi— mately equal to the maximum IAA reSponse, the two dilution curves deviate markedly at higher, or lower, concentrations suggesting that the biological response of the active endogenous moieties are quite different from IAA. In order to relate the dose-response curve obtained with the crude neutral ether fraction to neutral auxins IIA arufl IIB, the neutral ether fraction (42 days after anthesis) Was fractionated by paper chromatography (hexane : water) 91 Figure 17.--Histograms depicting the Avena first internode bioassay response to seed neutral auxins (42 days after anthesis). A. 0.1 gram equivalents of neutral auxin IIA (Figure 168, Rf 0.0 - 0.2) and neutral Auxin IIB (Figure 16B, Rf 0.5 - 0.7) co- chromatographed in isopropanol : water (9:1). B. 0.1 gram equivalents of neutral auxin IIA (Figure 16B, Rf 0.0 - 0.2) chromatographed in iSOprOpanol : water (9:1). C. 0.1 gram equivalents of neutral auxin IIB (Figure 168, Rf 0.0 - 0.2) chromatographed in isopropanol : water (9:1). Control value (———). Upper and lower 5% fiducial limits (---). 92 [A .w. /n. 9 M O . a I. N J .1 .HFJIHWI . I _ . . 15f _ OR . . . . . . A . _ . . T O r p I- F b O 0 W 3 2 m 0 xxx QEN .x .xYxk x>xx .uxQ o\.. 11 >6: ‘QEQNM MQQ>xxxNk >xx khmxxux C 111.1. --- -- o 1 L. 1.9 /AA (n9) LO 1 0.5 Rf I.O 1 05. Rf 33.1 B . . 0 P b — p L O O 0 O O 4 3 2 I xxkbxsflx .x‘x.xx>xx KS «n 1|?ka 36%be MQQ§QNx>xx .5“..ka 93 and the zones corresponding to neutral auxin IIA and IIB (Figure 16B) eluted. Eluates were combined to give a total of 0.1 and 0.5 gram equivalents of each auxin and then bioassayed. The mean responses (3 replicates) were 58.3% and —9.5%, respectively. These results fit the dilution curve for the neutral ether fraction (Figure 18) extremely well, suggesting that the response obtained is solely attributable to the presence of neutral auxins IIA and IIB. Factorial combination.--Two factors (neutral auxin IIA and neutral auxin IIB, eluted from Chromatograms developed in hexane : water) were combined at 5 levels (0, 0.05, 0.10, 0.25, 0.50 gram equivalents) in a ran- domized complete-block design with 3 replications. Each replicate was blocked to account for possible variation between successive trays of seedlings used for the Aygna straight growth bioassay. The dose-response curves for neutral auxin IIA (in the absence of neutral auxin IIB) and neutral auxin IIB (in the absence of neutral auxin IIA) were found to be nearly identical (Figure 19) although the maximum response was less than half that obtained when both auxins were assayed together (Figure 18). The similarity between curves could possibly be attributed to incomplete chroma- tographic development of a single substance. However, 94 Figure 18.--Dose-response curves obtained by Avena first; internode bioassay of the crude neutral etluer fraction (42 days after anthesis) and knowr: concentrations of IAA. Each value is the mean of 3 replicates. Control value (---). Figure l9.--Dose-response curves obtained by Avena first internode bioassay of neutral auXin IIA, neutral auxin IIB (seed tissue, 42 days after anthesis), and known concentrations of IAA. Each value is the mean of 3 replicates. Control value (---). 95 H NEUTRAL ETHER PHASE $1 60F o—o IAA A \ s - ; 40 - 9 1x g5 20- 3 h: k OFL- —————————————————————————— “.1 k l l 0.00| 0. OJ NEUTRAL 6' THE? PHASE (g. equiv.) l.75 I75 I75 875 IAA (I29) o—o NEUTRAL AUXIN A 30 P H NEUTRAL AUXIN a v—v IAA (x 0.5) \o é. 20 E § § IO 3 ‘“ 0 '1: k J. O l l NEUTRAL AUX/IV (9. equiv.) l.75 I 7.5 IAA (n9) - 1 1 0.05 0.10 ’ 0.25 0.50 1 175 96 re-chromatography of neutral auxin IIA, or neutral auxin IIB, in hexane : water did not result in further resolution (Figure 4, Appendix A) strongly suggesting that neutral auxin IIA and neutral auxin IIB are distinctly different entities. Addition of successive increments of one neutral auxin to fixed levels of the other was expected to result in an increased growth response until the optimal concen- tration (0.1 gram equivalents) had been reached, followed by a decreasing response as the concentration became supra- optimal. This expectation was realized only in part (Figure 5, Appendix A). Accordingly, the mean increase in section length (final length-initial length) for each treatment was calculated and the data so obtained were subjected to analysis of variance (Table 1, Appendix A). Since the preliminary analysis had indicated significant differences within treatments and interactions, the treat- ment (and interaction) degrees of freedom and sums of squares were partitioned into single components and retested for significance. The finding that all levels of neutral auxin IIB, in the absence of neutral auxin IIA, and all levels of neutral auxin IIA (except 0.5 gram equivalents), in the absence of neutral auxin IIB, were significantly different can be seen graphically in Figure 19. The significant interaction between the first level of neutral auxin IIA and all levels of neutral auxin IIB 97 (Table 1, Appendix A) is readily apparent when the bioassay response is plotted as a function of total gram equivalents applied (Figure 20). In each case the response is greater than would have been expected, suggesting a synergistic effect between the first level of neutral auxin IIA and any level of neutral auxin IIB. The interaction between 0.10 gram equivalents of neutral auxin IIA and 0.25 gram equivalents of neutral auxin IIB (a2b3, Figure 20) resulted in the lowest level of significance found (Table 1, Appendix A) and, as such, could represent a difference between replicates rather than a significant effect on the bioassay, peg s2, No reason can be advanced to explain the signifi- cant interaction between 0.50 gram equivalents of neutral auxin IIA and 0.10 gram equivalents of neutral auxin IIB (a4b2, Figure 20). The significant interaction between the highest levels of both auxins (a4b4, Figure 20) which yields the lowest growth response and the similarly signifi- cant interaction between a3bl (0.25 gram equivalents neutral auxin IIA and 0.05 gram equivalents neutral auxin IIB) which gives the highest response may be attributed to differences between minimum, and maximum, responses. gas-liquid Chromatography of the NeutraI’IIB Auxin The major growth promoting substance in the neutral ether fractions (seed and pericarp tissues) was shown to be I 98 Figure 20.--Dose-response curve obtained by Avena first internode bioassay of factorial combination of 5 levels of the neutral auxin IIA (0, 0.05, 0.10, 0.25, 0.50 gram equivalents) with 5 levels of the neutral auxin IIB (0, 0.05, 0.10, 0.25, 0.50 gram equivalents). All values are the mean of 3 replicates. Control value (--—). Unlabeled points not significantly different interaction values (Table 1, Appendix A). 99 0.. .mewav3 beM $3.06 00 Nd _ _.O _ 1 _ ___ Add _ 9 O N O 1.0 H.19IV37 7V/JINI :IO “/9 —NOI.IV.9N073 EUONHBJNI 153/Id 100 the neutral IIB auxin (Table l, 2). Consequently, this auxin was prepared for gas-liquid chromatography in the same manner as the acidic auxin even though the methylation step was assumed to be superfluous. Chromatography of the presumed TFA derivative on 3% SE - 30 at 140°C did not yield satis- factory resolution. Improved resolution was achieved at lower column temperatures. Injection at a column temperature of 120°C yielded major peaks at 3.10, 4.50, and 9.95 minutes and minor peaks at 6.10, and 8.50 minutes (Figure 21A). The retention time of standard IAA, at this temperature, was 9.70 minutes. Chromatography of the neutral IIB auxin on DC - 200 (column isothermal at 120°C) gave major peaks at 3.35, 4.85, and 10.87 minutes and minor peaks at 6.65 and 8.80 minutes (Figure 21B). The shoulder observed at a retention time of 9.45 minutes (SE - 30, Figure 21A) separated as a small peak (retention time, 9.15 minutes) on the DC - 200 column (Figure 21B). The retention time of standard IAA, at this temperature, was 10.70 minutes. Combined Gas-liquid Chromatography--Mass- §pectrometry of the Neutral IIB Auxin Chromatography of 8.0 ul of presumed TFA - neutral auxin IIB on 3% SE - 30 (column temperature 120°C) revealed three major peaks with retention times of 5.9, 17.7, and 20.4 minutes and three minor peaks at 3.4, 7.2, and 8.6 101 Figure 21.--Gas-liquid chromatographic resolution of the presumed trifluoroacetic ether of the neutral IIB auxin. A. Column packing - Gaschrome Q (60-80) coated with 3% SE-30; column temperature -120°C; column flow rate (N2) - 23 ml/minute. Retention time of TFA-Me-IAA, 9.70 minutes. Column packing - gaschrome Q (60-80) coated with 3% DC-200; column temperature -120°C; column flow rate (N2) — 42 ml/minute. Retention time of TFA-Me—IAA, 10.70 minutes. RELATIVE INTENSITY 102 1 1 1 1 1 1 1 1 1 1 1 I 2 5 4 5 6 7 a 9 10 II a A 1 1 1 1 1 1 L 7: 1 1 1 1 I 2 3 4 5 6 7 9 IO II I2 RETENTION TIME (MINUTES) 103 minutes (Table 2, Appendix A). The center of each major peak was introduced into the mass spectrometer. A further sample of TFA - neutral auxin IIB was chromatographed on 3% SE - 30 (column temperature 130°). Major peaks were located at 3.9, 10.5, and 11.9 minutes while minor peaks occurred at 2.3, 4.3 and 5.2 (doublet) minutes (Table 2, Appendix A). The center of the first two major peaks was introduced into the spectrometer. Spectra obtained from peaks at 5.9 (120°C) and 3.9 (130°C) minutes were identical. Expression of these reten- tion times relative to that of IAA showed that the peaks analyzed corresponded to that with a retention time of 4.50 minutes (Table 2, Appendix A). Spectra obtained from peaks at 17.7 (120°C) and 10.5 (130°C) minutes were also identical. These peaks were found to correspond to the peak at a retention time of 9.95 minutes (Table 2, Appendix A). The mass Spectrum of the compound with a retention time of 5.9 minutes (peak 2; Table 2, Appendix A) is shown in Figure 22. The molecular ion (M) is located at m/e 186 and the base peak at m/e 91. The absence of a fragment ion at m/e 69 (Figure 11A, 11B) strongly suggests that the TFA derivative was not synthesized. Similarly, the absence of an intense fragment ion at m/e 130 strongly suggests that the compound is non-indolic (Jamieson and Hutzinger, 1970). The base peak (m/e 91) is indicative of the tropylium ion; 104 Figure 22.--Mass spectrum of component 2 of the neutral auxin IIB complex. This component was resolved on 3% SE—30 (column temperature 120°C). The relative intensity (per cent of base peak) of each fragment was plotted as a function of its mass-charge ratio. Figure 23.--Mass spectrum of component 6 of the neutral auxin IIB complex. This component was resolvai on 3% SE—30 (column temperature 120°C). The relative intensity (per cent of base peak) of each fragment was plotted as a function of its mass-charge ratio. Figure 24.--Mass spectrum of component 7 of the neutral auxin IIB complex. This component was resolved on 3% SE-30 (column temperature 120°C). The relative intensity (per cent of base peak) of each fragment was plotted as a function of its mass-charge ratio. 105 —-17 40— 20— 1— 15 f _ _ _ 4M _ _ a 4 a m w m. w m. E Emzmzz. 9:3: 1— 2S 2O—I 180 140 100 m/e 106 the compound thus contains a benzene ring with at least one methylene group as a side chain. Other features of the spectrum include fragment ions at M - 31 (associated with the loss of a methoxy group), M - 64 and M - 79. The fragment ion at M - 64 shows the loss of m/e 33 from M - 155 suggesting the loss of a thiol group. Such a sug- gestion is reasonable in light of the large M + 2 contri- bution (5.65% of M). The fragment ion at M - 79 could result from the loss of methylmercaptan. Fragment ions at M - 121 and M - 147 are indicative of the loss of acetylene from the ring. The mass spectrum of the compound with a retention time of 17.7 minutes (peak 6; Table 2, Appendix A) is shown in Figure 23. This spectrum bears a strong resemblance to that found above (Figure 22). The molecular ion (M) is located at m/e 185 and the base peak at m/e 91. Again, the absence of fragments at m/e 69, m/e 130 strongly suggest that the TFA derivative was not synthesized and that the compound is non-indolic. The compound has a benzene ring (tropylium ion, m/e 91) with at least one methylene group as a side chain. Other features of the spectrum include fragment ions at M - 30 (associated with the loss of either the CH2 = NH; ion, the N = 0 ion or the fragment CHZO), at M - 64 (associated with the loss of a thiol group; M + 2 contribution, 5.15% of M), and at M - 120 and M - 146 107 (associated with the loss of acetylene from the ring). The fragment ion at m/e 30 (M - 155) is indicative of the N = 0 ion. The third mass spectrum obtained (peak 7; Table 2, Appendix A) exhibits a molecular ion (M) at m/e 185 (Figure 24). The base peak occurs at m/e 91 (associated with a tropylium ion). No fragment ions were found at m/e 130 or 69 strongly suggesting that the compound is non-indolic and did not form the TFA derivative. A fragment ion at M - 30 and the presence of a peak at m/e 30 is highly suggestive of the loss of the CH2 = NH: ion or the N = 0 ion) similar to that found above. However, few fragment ions occur between m/e 91 and m/e 155. In addition, the M + 2 contribution (10.81% of M) is very large suggesting a dithio group. Fragment ions at M - 120 and M - 146 are due to the loss of acetylene from the ring. Characterization of Seed Basic Substances Anthesis + 21 days.--Chromatographic separation of seed basic substances did not yield any distinct promotive activity in either bioassay (Figure 25A). Mindful of the possibility of supraoptimal concentrations, the zone between Rf 0.3 - 0.7 (Basic I) was eluted from duplicate chroma- tograms and the eluate re-chromatographed in water (Figure 25B). The weakly promotive zone at Rf 0.5 - 0.6 just exceeded the upper 5% fiducial limit (16.3%). Application 108 Figure 25.--Histograms depicting the Avena first interwuode (open bars) and Avena curvature (shaded balms) bioassay response to seed basic substances (21 days after anthesis). A. 0.3 gram equivalents chromatographed in isopropanol : water (9:1). B. 0.4 gram equivalents Basic I (Figure ZEflx, Rf 0.3 - 0.7). Solvent system:- watery Avena first internode bioassay control valxma (-——J. Avena curvature bioassay control valixa (zero). 109 A IAN IAA L, 3.5 I 350 r _ w .w. _ L In. w A , «Wm M W _ L.” £3. _ 3 2 3 o Mtbkcsmxbo m 5 0 1 q _ H nu. H 1' H _H m H .1 NH 0 W B mu 0 — — _ L p _ — b 0 0 0 0 O 0 O 0 3 2 I 3 2 I Ikmzmq qfixkxzx k0 Xlzoxkfibzoqm maoxfixmkzx kmtxnx 35 IAA (n9) Rf 110 of Salkowski or Ehrlich reagents did not reveal any chro- mogenic zones on either chromatogram. Anthesis + 42 days.--Chromatographic separation of the seed basic substances in isopropanol : water revealed a peak of activity at Rf 0.2 (upper 5% fiducial limit 31.2%) (Figure 26). Since this peak of activity was not revealed previously (Figure 25A) no further resolution was attempted. Characterization of Pericarp Basic Substances Anthesis + 42 days.--The pericarp basic ether phase showed negligible promotive activity when chromatographed in isopropanol : water (Figure 27). The zone at Rf 0.4 barely exceeded the upper 5% fiducial limits (31.0%). Since this activity did not correspond to that found previously (Figure 25A, 26) no further resolution was attempted. Characterization of the "Bound" Auxins Fractionation of the aqueous phase.--Column chroma- tography of the acidic aqueous fraction (Figure 1), prepared from seed and pericarp extracts (28 and 42 days after anthesis), and subsequent bioassay of individual fractions (Figure 28, 29) revealed two, weakly active, growth pro- motive substances. One of these substances (Fraction ninnbers 2 and 3, Figure 28) can be attributed to residual 111 Figure 26.--Histogram depicting the Avena first internode bioassay response to seed Basic substances (42 days after anthesis). 0.45 gram equivaleMx chromatographed in isopropanol : water (9:1). Control value (——-). Figure 27.--Histogram depicting the Avena first internode bioassay response to pericarp basic substances (42 days after anthesis). 12.0 gram equivalemw chromatographed in isoprOpanol : water (9:1). Control value (———). 112 1—4 L. 1.1 3.5 350 LG IAA IAN I-——-I H P p p n . b O 0 O O 0 4. 3 2 I Ikuzmnx 4.1.5xe k0 «a IZkaqmzoqm MQsztmkzx kmtxk .35 IAA (ng) Rf 3'50 TI 3.5 |.O L IAN H 0.5 IAA 1 _ 0 0 4. 3 . IkGZMq 4.2.2): nxo «a IZOxkcGzQIxm MQQZQMsz kmtxux O 11.. a m 2 p 0 0 35 IAA (ng) Rf 113 Figure 28.—-Histograms depicting the Avena first interxuode bioassay response to water-soluble substances (seed and pericarp acidic aqueous fraction, 28 days after anthesis) fractionated by silicic acid column chromatography. Control value (_) 0 Figure 29.--Histograms depicting the Avena first interTumde bioassay response to water-soluble substances; (seed and pericarp acidic aqueous fraction, 42 days after anthesis) fractionated by silicic: acid column chromatography. Control value (___) o FIRST INTERNODE ELONGA TION— x OF INI TIA L LENGTH FIRST INTERNODE ELONGATION—X OF INITIAL LENGTH 114 40- 20h IS 29 39 FRACTION NUMBER I00 90 75 so 25 H20 ACETONITRILE (%I LH 01! 11" .mfil IO l9 FRACTION NUMBER IOO 90 , 75 ACETONITRILE (96) 115 acidic auxin resulting from incomplete partition chroma— tography. The other, and more active, substance (fraction numbers 12 and 13) was located in both seed (Figure 28) and pericarp (Figure 29) extracts. The possible identity of this second substance is unknown. Changes in "bound" auxin levels during fruit development.--Chromatography and subsequent bioassay of ethyl ether-soluble substances released by base hydrolysis of the acidic aqueous phase (1971 harvests) revealed the presence of a highly active growth-promotive substance(s) (Rf 0.46 - 0.80, Figure 30) which did not chromatograph with either standard. Since the neutral auxins chroma- tograph with IAN in the solvent system used (water), this preliminary evidence suggests that the growth promoter(s) is different from those previously characterized. Moreover, seed promoter(s) levels exhibit dramatic quantitative changes with successive stages of fruit development. Similar changes are not so readily apparent in the peri— carp fraction. In addition to the zone of promotion, an intense zone of inhibition (Rf 0.0 - 0.33) was detected in pericarp fractions (21, 28, 42 days after anthesis). An equivalent zone of inhibition was not found in seed fractions nor in the pericarp fraction just prior to fruit maturity (56 days after anthesis). 116 Figure 30.-—Histograms depicting the Avena first internode bioassay response to growth-promoting sub- stances released by base hydrolysis of seexi and pericarp aqueous phases (21, 28, 42, and 56 days after anthesis) . 1.0 gram equivalents of each phase was chromatographed in water; Control value (—) . FIRST INTERNODE ELONGATION — 96 0F INITIAL LENGTH 117 SEED TISSUE PERICARP TISSUE IAN IAA IAN IAA BOI- H H H H 60*- 40 20 h h _ . - b 60 4O 20 - J E11137 ANTHESIS + 2| DAYS C :[Ebnw _jLWlLLL] ANTHESIS + 2| DAYS _iiLl 40 20 A’NTHESIS + 28 DAYS 40 20 C Jfl‘m ’ H. ”’1 ANTHESIS +42 DAYS o 0.5 , 'I.o o 0.5 I.O ANTHESIS + 56 DAYS 118 In order to relate the levels of the "bound" growth promoter(s) to fruit growth, the peak area (Rf 0.46 - 0.80) above the upper 5% fiducial limit was converted to IAA equivalents by means of a standard curve and expressed as IAA equivalents per fruit. Comparison between auxins (free and bound) detected in seed and pericarp fractions (Figure 31) shows that during the fruit growth period 21-42 days after anthesis, the total quantities of free and bound auxin per seed decreased while the totals in the pericarp increased. Subsequent to this time, the levels of both free and bound auxins in the seed decreased further (Figure 31). The level of bound auxin in the pericarp, however, continued to increase even though the level of free auxin decreased. Discussion Seasonal Variation in Fruit Auxin Content The seed auxin content (1969 fruit harvest), on the basis of ng IAA equivalents/gram dry weight, increased from an initially low value to a maximum 21 days after anthesis (Figure 4). This increase in seed auxin from day 14 to day 21 can be correlated with the rapid growth to full size of the nucellus and integuments (Inoue, 1970). It is unlikely that the endosperm contributed auxinic factors to this peak since, at this time, it has only attained 3% of its eventual 119 Figure 31.--Seasonal levels of free and bound seed and. pericarp auxins. All free auxin values are: totals of acidic, and neutral auxins obtaiJned from fruit harvested 21, 28, 42, and 56 days; after anthesis, 1971. 40” 30- N O l 5 I 120 O-—O FREE AUXIN O—O BOUND AUXIN IAA EOUI VALE/V 7'5 (fig/frail} 01 o O N O I I0- PERICARP I J 2! 3'8 42 56 DAYS AFTER ANTHES/S 121 size. Subsequent to the completion of nucellus and integu- ment growth, the seed auxin content fell rapidly to less than 1 ng/gram dry weight (Figure 4). A second peak of seed auxin occurred between 35 and 42 days after anthesis. The exact magnitude, and location, of this auxin peak is unknown because complete separation of all growth promoting substances was not achieved prior to bioassay (Figure 2, 3). However, both the endosperm and embryo undergo rapid development during the period in which the seed auxin content increased dramatically (Inoue, 1970). By 45 days from anthesis, the embryo had reached its maximum size. Yet a third, but considerably smaller peak of auxin activity was found 56 to 63 days after anthesis. Such a peak does not correlate with any macroscopic changes in the seed. When similar determinations were made in 1971 a somewhat different result was obtained (Table 1). Although a major peak of seed auxin was shown to occur 21 days after anthesis, no dramatic decrease (day 28) and subsequent increase to a second peak (day 35 - 42) was found. Instead, the seed auxin content decreased to a minimum of 450 ng/gram dry weight and then slowly increased to a second maximum of 550 ng/gram dry weight 56 days after anthesis. These differences can largely be ascribed to differences between years in the onset of rapid endosperm and embryo growth 122 and to the differences in fractionation technique (individual chromatography of acidic and neutral auxins in a nitrogen atmosphere). The pericarp auxin content, however, did not neces- sarily mirror the seasonal fluctuations found in the seed (Figure 4). For example, no evidence was found to suggest that the rapid loss of seed auxin (21 - 28 days after anthesis) was due to movement into the pericarp. Instead, the pericarp auxin content increased at an almost steady rate until 42 - 49 days after anthesis and subsequent to this time, declined. Thus the pericarp auxin content during the third stage of fruit growth appears to be inversely associated with rapid cell enlargement although this rela- tionship is not tenable during the period of fruit maturation. Comparison of these results with those obtained in 1971 suggests that movement did occur from the seed to the fruit (Table l), at least during the initial stages of fruit growth. Again, the total pericarp auxin content during the initial stages of the third growth stage (42 - 56 days after anthesis, Table l) appeared to be inversely associated with rapid cell enlargement. Although computing auxin content in concentration terms (ng/gram dry weight, Figure 4) is a valid means of comparing auxin levels at various growth stages within and between seasons, such computations invoke a "hidden" 123 dilution factor of increasing fruit dry weight with suc- cessive growth stages. Since cell division in the pericarp is complete by 21 days after anthesis, computation of auxin content in terms of ng IAA equivalents/fruit would be more meaningful physiologically. Similarly, initial growth of the seed is solely due to the enlargement of the nucellus and integuments (Tukey and Young, 1939; Inoue, 1970). Growth of the endosperm and embryo does not begin until the nucellus and integuments have reached their maximum size. Thus, eXpression of IAA equivalents/ seed as a function of time from anthesis would more ac- curately reflect the auxin contribution from ovule tissues than would expression in terms of IAA equivalents/gram dry weight. Comparison of seed and pericarp auxin levels obtained from two seasons of fruit growth (Table 2) showed that the nucellus and integument contribution (day 21) was appreciable in relation to that associated with the initial stages of endosperm and embryo development (day 28). Subsequent to this time the total seed auxin level increased (day 42, 1969) although the level detected from isopropanol : water (4:1) Chromatograms is an underestimate (super-imposition of neutral auxin IIA and neutral auxin IIB, Figure 2). A similar increase was not found for seed tissue obtained from a second season of growth (1971). 124 The level of seed neutral auxin IIB (1971) declined from day 28 to day 56 at a slower rate than that found for either neutral auxin IIA or the acidic auxin. Since both neutral auxins were found to co-chromatograph in isopropanol : water (9:1) (Figure 17) and were poorly resolved in hexane : water (9:1) (Figure 3) no definitive evidence was obtained that would indicate a high level of seed neutral auxin IIB subsequent to day 42 (1969). However, a major peak of promotive activity was shown by histograms (56, 63 and 70 days after anthesis, Figure 3) that corresponds with the Rf value of neutral auxin IIB chromatographed in hexane : water (upper phase; Figure 16B). High levels of an unidentified neutral auxin have been found in peach seeds subsequent to the completion of embryo growth (Powell and Pratt, 1966). The changes in total pericarp auxin levels (1971) reflect those found for the seed with the exception that the auxin level at day 28 was below the limit of bioassay detection. The level of acidic auxin decreased from a maximum at the onset of fruit growth stage II to a minimum at day 28. At the onset of growth stage III (day 42) the level of acidic auxin was considerably lower than that found for the neutral auxin IIB. Both auxins were detected at day 56 at which time the rate of cell enlargement in the pericarp had begun to decline. In contrast to the acidic 125 and neutral IIB auxins, no neutral auxin IIA was found at any stage of fruit growth. Comparison of total free and bound auxins in seed and pericarp fractions (Figure 31) showed that the decrease in free seed auxin from 21 - 42 days after anthesis was paralleled by a decrease in bound auxin. If the bound auxin level is assumed to be a reflection of free auxin metabolism then an increase in bound auxin should have been detected at least during the period 28 - 42 days after anthesis. Such an increase was not found suggesting a loss of bound auxin from the seed fraction. The bound auxin level in the pericarp, however, increased from day 28 to day 56. This increase would be expected if the loss from the seed was due to movement to the pericarp. Furthermore, if the bound auxin is physiologically signifi- cant in fruit growth the observations by Tukey (1936b), that fruit growth was not impaired when seeds were destroyed after the onset of the third growth phase, could be explained. Characterization of Seed and Pericarp Auxins Paper chromatography of the acidic and neutral phases from seed tissues revealed one acidic and two neutral auxins (Figure 6, 7, 8, 13C, 14C and 16B). No evidence was obtained that the seed auxins were different from those detected in the pericarp (Figure 9, 15). 126 Schulte and Holm (1964) reported the isolation of one acidic, one neutral and one basic auxin from the developing seeds of sour cherry, although no data have ever been presented. In the persent case, the weak growth activity obtained in A3333 first internode bioassays of the basic phase (Figure 25, 26, and 27) and the absence of a curvature response in the Ayegg_curvature bioassay (Figure 25) were taken as evidence against the possibility of a basic growth promoter. The Rf of the acidic auxin did not always coincide exactly with that of the applied standard IAA (Figure 6A, 68, 7A, 7B, 8A, 8C, 9A, and 9B). However, separation of the growth promoting peak (Figure 7B) into two components, and re-chromatography of each component (Figure 7C, 7D) showed that the acidic auxin chromatographed with standard IAA. Moreover, application of Salkowski reagent always yielded a red-purple chromogenic reaction at the Rf of maximum biological activity. IAA has been tentatively identified in seed extracts obtained from other Prunus sp. Stahley and Thompson.(l959) extracted an acidic growth promoter from Halehaven peach seeds which possessed chromogenic (Salkowski and Ehrlich reagents) and Rf properties in four solvent systems similar to IAA. More convincing evidence has been presented by Powell and Pratt (1966) who prepared the methyl ester of 127 the acidic auxin found in extracts of Halehaven peach seeds and compared its fluorescence spectrum with that of authentic methyl indole-3—acetate. Gas-liquid chromatography of the TFA - methyl ester of the acidic auxin gave retention times identical to those obtained for IAA (Figure 10). Co-chromatography of the endogenous acidic auxin and standard IAA, yielded a single, homologous, peak on two dissimilar columns (SE - 30, DC - 200). Unequivocal evidence that the acidic auxin is IAA was provided by mass spectrometry (Figure 11). The neutral auxins, however, did not yield a chromogenic reaction with reagents specific for indoles (Salkowski, Ehrlich, 4-dimethylaminocinnamaldehyde) although both neutral auxins were active in the Aygna_first internode bioassay (Table l, 2, Figure l3, 14, 15, 16, 17, 19 and 20). Since both neutral auxin IIA and neutral auxin IIB co- chromatographed in isopropanol : water and water alone, it is not known if both auxins were responsible for the Avena curvature response obtained (Figure 12A, 13A). A zone of growth-promotive activity (Rf 0.7 - 0.9) has been demonstrated in extracts of peach, plum and sweet cherry chromatographed in isopropanol : water (4:1) (Stahley and Thompson, 1959; Ugolik and Nitsch, 1951; Pillay, 1965). This promotive zone resembles that found for sour cherry (Figure 2) although the latter was masked 128 by supraoptimal concentrations (Figure 12). Ugolik and Nitsch (1959) reported that this promotive activity was the only growth-promotive substance in the neutral ether phase. An analogous growth promoter was detected in eluates from silicic acid column chromatography of peach seed extracts (Powell and Pratt, 1966). Gas-liquid chromatography of neutral auxin IIB, however, yielded 3 major and 2 minor components (Figure 21). Since a limited quantity of this auxin was available, the entire sample was subjected to combined gas-liquid chromatography-mass spectrometry rather than being purified further. The mass spectra obtained from each of the three major components indicated that all three were chemically similar, although at this time, a molecular structure cannot be proposed for any one compound. THE CYTOKININS Introduction Cell division, both before and immediately after anthesis, accounts for much of the initial increase in fruit size in the sour cherry and many other fruits. This period of cell division is of limited duration and is sub- sequently followed by one or more periods of rapid cell enlargement. Study of the physiological mechanisms con- trolling this period of cell division led to the isolation of cell division stimulants (Goldacre and Bottomley, 1959; Letham and Bollard, 1961) and cell division inhibitors (Letham, 1963a). One of these stimulants was isolated from immature plum fruitlets and sweet corn seed (£23 mays L.) and identified as 6-(4 hydroxy-3 methyl but trans-2 enyl) amino purine (zeatin) (Letham, 1963a, 1963c). Subsequently, zeatin ribonucleoside and zeatin ribonucleotide were identified from immature sweet corn seed (Letham and Miller, 1965; Letham, 1966a, 1966b). A further cytokinin, (—)dihydrozeatin, was isolated in immature yellow lupin seeds (Koshimuzu, et al., 1967). Evidence for the occurrence of the ribonucleoside of this latter cytokinin has been recently presented by Krasnuk, et al.,(l97l). 129 130 Although a considerable research effort has been expended on the isolation and identification of naturally occurring cytokinins, the determination of levels in relation to fruit growth has received only cursory examination. Perhaps the most comprehensive investigation of this latter aspect has been conducted by Blumenfeld and Gazit (1970) A and Gazit and Blumenfeld (1970) on the avocado. Both seed 1 coat and embryo tissue extracts were found to contain high levels of cytokinin during their initial growth stages although the level fell as the rate of fruit growth slowed. Comparable activity in the mesocarp was apparent only after acid hydrolysis. The level of bound cytokinin was positively correlated with the rate of mesocarp cell division, and the rate of fruit growth. Materials and Methods Source of Material Fruit from mature, fifteen-year-old trees growing at the Horticulture Research Center in East Lansing were collected at weekly intervals from anthesis, quickly frozen in the field with dry ice, and stored at -25°C until lyophilized. 131 The General Method Extraction Lyophilized whole fruit were ground in a Wiley Mill to pass a 20 mesh screen. Ground material was extracted at 22°C with successive portions of 95% ethanol (30 minutes), 80% ethanol (30 minutes, 2 times); the extract being stirred constantly. Filtrates from each extraction were combined and stored in the dark at -25°C. Fractionation Partition chromatography.--The combined filtrates were evaporated to the aqueous phase under reduced pressure (flash evaporator, water bath temperature 30°C). The aqueous phase was adjusted to pH 3.0 and partitioned against ethyl ether until the aqueous phase was free of chlorophyll (Figure 32). The acidic aqueous phase was then adjusted to pH 6.5 and partitioned against 50 ml portions of butanol (4 times). The butanol was evaporated to near dryness under reduced pressure and the residue lyophilized to remove traces of butanol. The aqueous phase was similarly treated. Each lyophilized phase was solubilized in 95% ethanol and stored in the dark at -25°C. Paper chromatography.--Extracted butanol-soluble and water-soluble substances were further fractionated by ascending paper chromatography. Aliquots of the extracted 132 Figure 32.--Flow diagram showing the procedure for extraction and fractionation of butanol-soluble and water- soluble cytokinins. 133 CHERRY FRUIT Filtered. Concentrated. AQUEOUS PHASE Partitioned with ether, pH 3.0 (HCl) AQUEOUS PHASE ETHER PHASE Partitioned with butanol, pH 6.5 (NaOH) AQUEOUS PHASE BUTANOL PHASE Extracted with 95% and 80% ethanol. 134 substances were streaked on Whatman No. 3 paper, equili- brated over the solvent, and the solvent front developed to a distance of 20 cm. All papers were pre-washed in the developing solvent to remove potential growth inhibitory substances (Burnett, et al., 1965). Developed Chromatograms were dried in a cool air stream and stored in a nitrogen atmosphere at -25°C, in the dark. Detection The radish cotyledon bioassay.--Radish seed (Raphanus sativus L., cv. Vicks Scarlet Globe), screened for uniformity of size, were surface sterilized with 1% NaOCl for 10 minutes. Surface sterilized seed were washed with sterile distilled water (10 times) and sown on moistened filter paper in Petri dishes. All Petri dishes and filter paper had been previously autoclaved for 20 minutes at 15 lb/ square inch. Seeds were germinated, in the dark at 26°C (Letham, 1968) for 36 hours. At this time, etiolated cotyledon pairs had begun to separate. The smaller of the two cotyledons (the inner cotyledon) was excised, care being taken to remove all the hypocotyl. Cotyledons were held on sterile, moist, filter paper until their addition to test solutions. Chromatograms to be tested (4 cm wide) were cut into 10 equally sized strips, each strip being placed into a Petri dish (4.5 cm). Control strips were taken from 135 the area below the start line. All strips were moistened with 1.0 - 1.5 m1 of sterile distilled deionized water and 5 cotyledons of uniform size were laid on each paper strip. Known concentrations of kinetin were also bioassayed. In the latter case, 1.0 - 1.5 ml of standard was added to 2 x 4 cm strips of paper out from developed, "blank," Chromatograms. Petri dishes were randomly assigned to plastic trays lined with moist paper towels (and covered with clear plastic) and incubated at 25°C under 175 foot candles of continuous fluorescent light (Cool White). After 72 hours, cotyledons were removed from each Petri dish, blotted dry, and weighed. All treatment bioassays were replicated 3 times. Computation of response.--The mean fresh weight was computed from the three replicates of each treatment and the results expressed in histogram form as a function of chromatogram Rf. The soybean callus bioassay.--Zones of growth promo- tion, located on Chromatograms of the aqueous and butanol phases by the radish cotyledon bioassay, were cut from duplicate Chromatograms and prepared for the soybean (Glycine max [L.J Merrill, cv. Acme) callus bioassay. Each zone of growth promotion was subdivided on the basis of Rf, and the resulting 2 x 4 cm strips shredded by a paper cutter. Shredded strips were placed in Erlenmeyer 136 flasks (150 ml) and 50 ml of basal medium added (Miller, 5 10 1965). Standard kinetin solutions (1 x 10- M to l x 10- M, 10 ml) were added to 4 x 2 cm strips of "blank" developed chromatography paper, in 150 ml Erlenmeyer flasks, and taken to complete dryness by lyophilization. Subsequently, 50 ml of basal medium was added to each flask. Flasks were stoppered with cotton wool plugs, capped with aluminium foil, and autoclaved for 20 minutes at 15 lb/square inch. All treatments were replicated 3 times. Three pieces of soybean callus (each weighing approximately 7 mg) were aseptically transferred to the surface of the cooled medium. Flasks were incubated at 26°C with intermittent fluorescent light flashes (Cool White) for 42 days. At the completion of the incubation period, the 3 callus pieces from each flask were blotted free of adhering medium and weighed. Computation of response.--The mean fresh weight per flask was computed for each treatment. Treatment means were converted to kinetin equivalents by means of a standard curve. Changes in Cytokinin Levels During Fruit Development Ethanolic fruit extracts were fractionated into aqueous and butanol phases (Letham and Williams, 1969) and each phase chromatographed in isopropanol : water 137 (4:1) (Heide and Skoog, 1967). Developed Chromatograms were bioassayed by the radish cotyledon test. Growth- promoting zones detected by this bioassay (Rf 0.7 - 0.9, butanol phase; Rf 0.0 - 0.3, aqueous phase) were also tested for substances that would promote cell division in the soybean callus bioassay. Partial Characterization of Fruit Cytokinins I Ethanolic fruit extracts (21 days after anthesis) were fractionated into aqueous and butanol phases and each phase chromatographed in iSOpropanol : water (4:1). Zones of growth-promoting activity were eluted with 95% ethanol (2 times) and 80% ethanol (3 times) and eluates re- chromatographed in the following solvents (Nitsch, 1968; Letham and Williams, 1969):- 0.03 M boric acid adjusted to pH 8.4 with NaOH (Butanol I, II; Aqueous I) Water, pH 6.0 (Aqueous I, II) Ethyl methyl ketone saturated with 0.04 M boric acid (Butanol II) After development, Chromatograms were tested for growth promoting substances by the radish cotyledon bioassay. Results Changes in Cytokinin Levels During Fruit Development Chromatograms of the aqueous phase exhibited one major zone of growth promotion at Rf 0.0 - 0.3 (Figure 33). 138 Figure 33.--Histograms depicting the radish cotyledcni bioassay response to growth-promoting surr- stances detected in aqueous and butanol pflmases. Solvent system : isoprOpanol : water (4:10 . Control value (———0. Upper and lower 5% fiducial limits (---). A - Rf of adenine. Z - Rf of zeatin. COTYLEDON FRESH WEIGHT (mg) 139 AQUEOUS FRACTION BUTANOL FRACTION 4O IOO 80 IOO 80 60 40 40 I) A H O.l99 O.I99 0.389 .9. ANTHESIS + l4 DAYS 0499 A -_.. _ n- ”.-JJ-_-.I.L - l ANTHESIS + 28 DAYS 0.1093 H o 0.5 LO 140 This promotive activity was found in all fruit extracts. Chromatograms of the butanol phase, however, exhibited a major zone of growth promotion at Rf 0.7 - 0.9 (Figure 33) for the early harvest dates (7, l4, and 21 days after anthesis) and a second major promotive zone (Rf 0.5 - 0.7) for all subsequent harvest dates. Expression of mobility of either promoter as a function of adenine mobility [Rf promoter Rf adenine :] resulted in values in the range of 1.55 i 0.05 for all Chromatograms. The close similarities in relative Rf values strongly suggest that growth promotion . was due to one substance. Minor growth-promotive activity was detected at Rf 0.4 - 0.7 (aqueous phase) and at Rf 0.0 - 0.2 (butanol phase). Since gibberellin has been reported to cause slight increments in cotyledon weight over the concentration range 0.5 — 50 mg/liter (Letham, 1968), 4 levels of kinetin (0, 21.5, 215, 2150 ng/Petri dish) were combined with 4 levels of gibberellic acid (0, 3.46, 34.6, 346 ng/Petri dish) in a factorial design and treatment combinations subjected to the radish cotyledon bioassay (Figure 2, Appendix B). With the radish cultivar used in this study, an appreciable increase in cotyledon weight was found in the absence of kinetin. At low levels of kinetin (21.5 ng/Petri dish) all levels of gibberellin slightly increased the bioassay response although at higher levels of kinetin (215, 2150 141 ng/Petri dish) the higher concentrations of gibberellin markedly increased cotyledon weight. Histogram peaks (Rf 0.0 - 0.3, aqueous phase; Rf 0.7 - 0.9, butanol phase 7-21 days after anthesis, Rf 0.5 - 0.7, butanol phase 28-42 days after anthesis) exceeding the upper 5% fiducial limit were converted to kinetin equivalents per 0.1 gram dry weight or per fruit by means of a standard curve (Figure 1, Appendix B). A new standard curve was errected for each bioassay. Similar computations were made using data from the soybean callus bioassay. Comparison of kinetin equivalents detected from the same fruit extract, but by different bioassays (Table 3) showed that the cotyledon bioassay was more sensitive to the aqueous promoter (Rf 0.0 - 0.3, Figure 33) than was the callus bioassay. The opposite was true for the butanol promoter (Rf 0.8 i 0.1, Figure 33). The aqueous promoter (as detected by radish cotyledon bioassay) was initially at a high concentration (day 7) which declined (day 14) and then increased (day 21). The increase in aqueous promoter concentration at day 21 was not found for the soybean callus bioassay (Table 3). Sub- sequent to day 21, the concentration of aqueous promoter decreased (day 28) and then increased (day 42). The increase in butanol promoter concentration during the first three weeks of fruit growth (Table 3) was shown by 142 .cmmmmmMOHn no: Hopoeonm H I o.oa>H I o.hmm mv HI m.HOH HI H.vm mm o.nmm m.mmH o v.Hm Hm ¢.m >.m H.o v.m ¢H v.m H.H v.0 o.HH h ApHsum\mcv I o.mm> I o.o>m mv HI m.m> HI o.mm mm o.mm>H o.mme o o.mmH Hm m.mMH o.Hom m.~ o.Hm vH m.omH o.vm >.mm o.oom 5 .us who .08 00H\mc mommmowm mommMOHm hmmmmon wommMOHm mHmmcpc< moHHmo coomHmuoo msHHmo coomeuoo Hopwo mama Hmuofioum Hocmuom Hmuoaoum msomsom mucmHm>Hsom cHumcHM .ucmfimon>mo uHsnm mo mommum ucwnmmmwc um wstum munmco snow CH oopomamo mHm>mH cam mGOHumuucmocoo GHconumo mo comHummEooII.m mamas 143 both bioassays. Subsequent to day 21 the concentration decreased (day 28) and then increased to a maximum (day 42). Expression of aqueous and butanol promoter concen- trations in terms of ng kinetin equivalents/fruit (Table 3) did not alter the direction of promoter change, only the magnitude. Peaks of promoter activity were located at day 42 (aqueous promoter) and days 21 and 42 (butanol promoter). Partial Characterization of Fruit Cytokinins The water-soluble cytokinins.--Chromatographic separation of the aqueous phase (21 days after anthesis) and subsequent detection by the radish cotyledon bioassay revealed a major zone of growth promotion (Rf 0.0 - 0.3, Aqueous I) and a minor zone of growth promotion at Rf 0.5 - 0.8 (Aqueous II) (Figure 33, 34A). Re-chromatography of Aqueous II, eluted from a duplicate chromatogram, resulted in further separation into two components (Rf 0.3, 0.8 - 1.0); (Figure 3A, Appendix B). Since Aqueous II could be due to butanol-soluble cytokinin remaining in the aqueous phase from incomplete partition chromatography, the Rf's of thetnw>promotive components in Aqueous II were compared with reported Rf values for zeatin and zeatin ribonucleoside. The expected Rf of zeatin, based on that of adenine, would be Rf 0.59 (Heide and Skoog, 1967). No activity was located at this Rf (Figure 3A, Appendix B). The expected Rf of 144 Figure 34.-“Histograms depicting the radish cotyledon bioassay response to the growth promoters in the aqueous phase (21 days after anthesis) . A. Chromatography of 0.14 gram equivalents of the aqueous phase in isopropanol : water (4:1). B. Re-chromatography of 0.18 gram equivalents of Aqueous I (Figure 34A, Rf 0.0 - 0.13). Solvent system:- water (pH 6.0). C. Re—chromatography of 0.12 gram equivalents of Aqueous I (Figure 34A, Rf 0.0 - ().3). Solvent systemr—0.03M boric acid adjtnsted to pH 8.4 with NaOH. Control value (———). A - Rf of adenine. Z - Rf of zeatin. 145 A A DINO l 1 2|!) _IL 1 2I 2l50 I.O [I Kinetin (ng/ 23 C H H I7 I720 Lo 0.5 n b p P 3:: SEN... swath >33 58 - O r L i O o O O O O _LLI O 9 8 7 6 5 I.O 0.5 AH r i F F n b . h . O O o o o 9 8 7. 6 5 35 tags: $54.4 >63 8:8 0 Kinetin (fly) Rf Rf 146 zeatin ribonucleoside would be Rf 0.75 (Miller, 1967; Gupta and Maheshwari, 1970) yet the observed promotive activity occurred at a higher Rf (0.8 — 1.0) (Figure 3A, Appendix B). Re-chromatography of Aqueous I in water (Figure 34B) resulted in a single promoter peak at Rf 0.7 - 0.9. However, re-chromatography of Aqueous I in 0.03M boric acid (Figure 34C) yielded poor results with the largest peak of activity occurring at Rf 1.0. Zeatin ribonucleoside has a reported Rf of 0.86 - 0.91 in this solvent (Tegley, et al., 1971; Krasnuk, et al., 1971) but this cytokinin would be expected to partition into butanol (Letham and Williams, 1969) suggesting that the major aqueous cytokinin is zeatin ribonucleotide or some closely related compound. The butanolésoluble cytokinins.--Fractionation of the butanol phase by ascending paper chromatography (7, 14, and 21 days after anthesis, Figure 33) revealed zones of weak growth promotion at Rf 0.0 - 0.2 (Butanol I), Rf 0.4 - 0.6 (Butanol II) and a major zone of growth promotion at Rf 0.7 - 0.9 (Butanol III). Re-chromatography of Butanol I (Figure BB, Appendix B) and subsequent detection by the radish cotyledon bioassay revealed two weak promoter zones at Rf 0.3 and 1.0 similar to the results obtained when Aqueous I was chromatographed in the boric acid solvent (Figure,34C). Incomplete partitioning would account for residual zeatin ribonucleotide in the butanol 147 phase. Similar re-chromatography of Butanol II revealed a promoter peak at Rf 0.9 - 1.0. Such behavior is sug- gestive of zeatin ribonucleoside (Miller, 1967). Re-chromatography of the major butanol phase com- ponent (Butanol III, Figure 35A) in the boric acid solvent system yielded several zones of activity between Rf 0.2 - 0.7 (Figure 35B). The reported Rf of zeatin in this solvent is 0.49 - 0.58 (Tegley, et al., 1971). More convincing evidence was obtained when Butanol III was chromatographed in methyl ethyl ketone saturated with boric acid (Figure 35C). In this instance, the major peak of activity chro- matographed with standard zeatin. Zeatin ribonucleoside would be expected to move only slightly from the start line (Letham and Williams, 1969). Moreover, standard zeatin chromatographed with Butanol III in the iSOprOpanol water (4:1) solvent system (Figure 33). This evidence suggests that the major cytokinin in the butanol phase is zeatin or a closely related compound. Discussion Seasonal Variation in Fruit Cytokinin Content The butanol-soluble cytokinin extracted from whole fruits (Figure 33) increased from an initially low value (day 7) to a peak of activity at 21 days after anthesis (Table 3). This change was detected by both cotyledon and 148 Figure 35.--Histograms depicting the radish cotyledon bioassay response to the growth promoters in the butanol phase (21 days after anthesis). A. Chromatography of 0.19 gram equivalents of the butanol phase in isopropanol : water (4:1). B. Re-chromatography of 0.37 gram equivalents of Butanol III (Figure 35A, Rf 0.7 - 0.9L Solvent system:- 0.03M boric acid adjusum to pH 8.4 with NaOH. C. Re-chromatography of 0.25 gram equivalents of Butanol III (Figure 35A, Rf 0.7 - 0.9L Solvent system:- ethyl methyl ketone saturated with 0.04M boric acid. Control value (———). A - Rf of adenine. Z - Rf of zeatin. 149 ./ a. H” mm _Hn _nm _ rug m mK ./ a. m o /n. I ‘5 n! _m5m I H ”my a _2 .m .I_U 15...] O K . OR - a ILL _HI I AH d b 15%”! AH w _H o C o _H _ o o. p p p _ n L I O 0 0 O O O a 9 8 7 6 ‘5 4 35 grams. tame... SE. 58 r 5 f AH IOR B . O p p . _ P L O O 0 O O O 9 8 7 6 5 4 35 .369: :85 393.36 150 callus bioassays. Moreover, the direction of the increase was not altered when data were expressed on a per fruit basis (Table 3). However, levels of water-soluble cytokinin (Figure 33), measured by the cotyledon bioassay did not correspond with those measured by the callus bioassay (Table 3). In this regard, no water-soluble cytokinin was found in fruit extracts (day 21) by the callus bioassay suggesting that either the cotyledon bioassay is more sensitive to water-soluble cytokinin, or that the chroma- togram zone bioassayed contains further substances that would be promotive in the cotyledon bioassay, e.g., gibberellins (Figure 2, Appendix B), or inhibitory in the callus bioassay. Gibberellin A32 has been shown to account for most of the gibberellin-like activity in seed and pericarp extracts of peach and apricot (Yamaguchi, et al., 1970; Coombe, 1971). Since the chromatographic location of this highly polar gibberellin would be close to the origin in isoprOpanol : water (4:1) gibberellin could account for much of the "aqueous cytokinin" activity at days 21, 28, and 42 in the cotyledon.bioassay.* However, re-chromatography of the water-soluble cytokinin (Figure 34B, 34C) revealed at least one inhibitor that co—chromatographed with the cytokinin in isoprOpanol : water (4:1). The relationships between the inhibitory substance and the callus bioassay 151 or between presumed GA32 and the cotyledon bioassay are unknown. The butanol-soluble cytokinin decreased between days 21 and 28 and then increased (day 42) (Table 3). Since reasonable agreement was found between bioassays during the period 7-21 days after anthesis the fluctuations between days 21-42 are assumed to be representative of changes in cytokinin concentration, peg s2, and not due to gibberellin. Although cytokinins have been isolated from several Prunus sp, determination of seasonal variations has only been attempted for the plum. The level of cytokinins in plum fruitlets increased to a maximum 15 days after anthesis. This increase was correlated with the onset of rapid cell division (Letham, 1963a, 1964). Water-soluble and butanol— soluble cytokinins isolated from the endosperm and embryo of peach (Powell and Pratt, 1964) could be detected long after cell division in the pericarp had ceased. A similar cytokinin contribution from the developing seed would explain the increase in concentration, and level, from 28-42 days after anthesis (Table 3). Partial Characterization of Fruit Cytokinins Re-chromatography of the butanol—soluble sour cherry cytokinin (Figure 35) in two solvents which would separate zeatin from its ribonucleoside yielded a single peak of activity which chromatographed with authentic 152 zeatin. In this regard, the butanol—soluble cytokinin is either zeatin or some closely related compound. Evidence for a water-soluble cytokinin (Figure 34) is clouded by the possibility that gibberellin could have stimulated radish cotyledon growth. GENERAL DISCUSSION The development of the sour cherry fruit occurs in three distinct stages. Fruit growth during stage I is principally by cell division in the pericarp followed by a brief period of cell enlargement. The rate of cell enlargement, however, slows during the period 21-28 days after anthesis and continues at a low rate until the onset of growth stage III (about day 42 from anthesis). During growth stage III the rate of cell enlargement in the mesocarp dramatically increases and is maintained at almost a constant rate until just before the fruit matures (63-70 days after anthesis). At anthesis, the number of cells in the radial direction through the fruit has been found to be in range of 14-17 for the endocarp and 22-24 for the mesocarp (Tukey and Young, 1939; Inoue, 1970). Subsequently, both tissues undergo a period of cell division (until 21 days after anthesis) which increases the cell number in the endocarp to 27-30 and 29-32 in the mesocarp. No cell division has been reported later than the end of growth stage I (Tukey and Young, 1939; Inoue, 1970). 153 154 The level of butanol-soluble cytokinin (Table 3) increased during the period of cell division and reached a maximum at the cessation of cell division (21 days after anthesis). The increase in this cytokinin paralleled the increase in pericarp cell division until day 21 and, as such, can be positively correlated with this period of cell division. However, the cessation of cell division does not appear to be related to a low level of butanol- soluble cytokinin at day 28 (Table 3). Letham (1963a) reported that cell division inhibitors could be separated from cell division stimulants by parti- tioning aqueous fruit extracts against ethyl acetate or ethyl ether at low pH. The inhibitors partitioned into the organic phase. When the ethyl ether phase (Figure 32) from cherry fruits was chromatographed, (isopropanol water [4:1]) and bioassayed (radish cotyledon) strong inhibitory activity was revealed at Rf 0.4 - 1.0 (21 days after anthesis). Insufficient evidence, however, was obtained to determine if this inhibitory activity was associated with the cessation in cell division. EXpression of plum fruitlet inhibitor content in relation to fruit develOpment showed that the level of inhibitors increased during the period of rapid fruitlet cell division and 155 reached a maximum at the time of cell division cessation (Letham, 1963a). The decrease in avocado mesocarp cell division has also been correlated with increased levels of an inhibitor (Gazit and Blumenfeld, 1970). Thus, the balance between promoter and inhibitor levels must be considered; high cytokinin levels in conjunction with high inhibitor levels could result in small increments of net growth, and thus account for the observed cessation of cell division when butanol-soluble cytokinin levels were high (day 21). The level of water-soluble cytokinin (Table 3, callus bioassay) decreased during the period of cell division and, as such, probably plays no role in fruit cell division. Cell division in the avocado mesocarp has been positively correlated with the level of water- soluble, "bound" cytokinin (Gazit and Blumenfeld, 1970). This cytokinin was inactive in the soybean callus bioassay prior to acid hydrolysis. Since the water-soluble cytokinin from sour cherry fruits was active in the soybean callus bioassay, pg£_§g, it is unlikely that it is a similarly "bound" cytokinin. Furthermore, the magnitude of the decrease in water-soluble cytokinin (Table 3) and concomitant increase in butanol-soluble cytokinin suggests that the latter did not arise from the loss of the former. 156 Rapid cell enlargement in both the mesocarp and endocarp began 10 days after anthesis (Inoue, 1970) and ceased 18 days later. Both the concentration and level of fruit auxin was shown to be low during the period 7-21 days after anthesis (Table 1, 2) even though the level of seed auxin increased dramatically (day 21, Figure 2, 3). The low level of auxin detected in the pericarp during this initial period of cell enlargement could be the result of utilization. Seed extracts from other Prunus sp. fruits have been shown to contain high levels of growth substances active in straight growth tests during the period of active nucellus and integument development (Stahly and Thompson, 1959; Ugolik and Nitsch, 1959; Powell and Pratt, 1966). Similar findings have been reported for black currant and grape fruits (Wright, 1956; Nitsch, et al., 1960). Yet in each of the above fruits the rate of cell enlargement in the pericarp began to decline about the time when the auxin associated with the development of the nucellus reached its maximum. In two fruits (black currant and grape) the rapid fall in the auxin content has been correlated with the onset of growth stage II (Wright, 1956; Nitsch, et al., 1960). In the present study, the level of seed auxin associated with nucellus and integument development decreased from a maximum at day 21 to a non-detectable 157 level at day 28 (Figure 4, 5). In a second season of growth, the level of total auxin associated with nucellus and integument development also declined (Table 1, 2) and this decline was more dramatic for the acidic auxin than for either of the neutral auxins. It is improbable that the decrease in seed auxin content is associated with the onset of growth stage II since the level in the pericarp was found to increase throughout the period of reduced cell enlargement (Figure 4, 5; Table 1, 2). It is not known, however, if this increase in pericarp auxin level is due to movement from the seed. Rapid development of both the endosperm and embryo during growth stage II has been correlated with high auxin levels in a number of other fruits (Stahly and Thompson, 1959; Crane, et al., 1959; Pillay, 1966; Powell and Pratt, 1966) although studies on the fate of this auxin have never been attempted. The control of cell enlargement during growth stage II could be due to one or more inhibitors specific to cell enlargement that conceivably would increase during growth stage I and be maintained at a high level during growth stage II. Subsequent to growth stage II the inhibitor(s) level would be expected to decrease. This presumed inhibition could be of the type shown by factorial combination of neutral auxins IIA and IIB (Figure 18, 19, 20). ’This hypothesis is not tenable 158 since neutral auxin IIA was not detected in any pericarp fraction inVestigated (Table l, 2). However, no evidence was obtained on the interaction between neutral auxin IIB and acidic auxin I. The presumed inhibition could also result from the interaction between one or more inhibitors and either acidic auxin I or neutral auxin IIB or from the interaction between inhibitor(s) and both auxins. However, no evidence was obtained that showed that ethyl ether-soluble inhibitors occurred in extracts of sour cherry pericarp tissue (Figure 2, 3, 9, 15, 27). However, these findings are tempered by the fact that the Ayega_first internode bioassay is less sensitive to inhibitory substances, such as abscisic acid, than is the A3223 coleoptile bioassay (Nitsch and Nitsch, 1956) or the wheat coleoptile bioassay (Luckwill, 1952). Yet, when the pericarp aqueous phase was subjected to base hydrolysis a highly potent ether- soluble inhibitor(s) of the Ayeng first internode bioassay was released (Figure 30). No similar inhibitory activity 'was located from the seed hydrolysate. Furthermore, the inhibitor(s) was present in pericarp extracts during growth stage II until the onset of growth stage III. No inhibitory activity was found in pericarp extracts at day 56 suggesting that this inhibitor(s), if released by enzynatic hydrolysis during growth stage II, could account for the observed lack of mesocarp cell enlargement. 159 With the onset of growth stage III the seed auxins increased from a low level (Figure 5) and reached a maximum 56-63 days after anthesis. This increase was not observed in a second season of growth (Table 2). The level of total pericarp auxin, however, decreased from day 42 to day 56 (Figure 5, Table 2) and this decrease can be attributed to a fall in neutral auxin IIB (Table 2). Subsequent to day 56 the level of pericarp auxin increased (Figure 5) although confirmatory evidence of this increase was not obtained in a second season of growth (Table 2). Correlations between increased seed auxin levels and the period of rapid cell enlargement during growth stage III have been sought by several investigators. Such evidence has been found only for peach (Powell and Pratt, 1966). As a result of this considerable body of negative evidence, Crane (1964) concluded that "no relationship has been proven between the levels of growth substances in developing seeds and fruit growth." However, the period of rapid cell enlargement immediately prior to fruit maturation has been shown to be independent of the seed and its attendant hormone levels for apple, peach, and sour cherry (Tukey, 1936b; Abbott, 1958; Southwick, et al., 1962). If the seed hormone content plays no part in cell enlargement during 160 growth stage III, then the pericarp auxin content sub- sequent to day 42 (Figure 5, Table 2) must have arisen either from translocation of auxin from vegetative organs to the fruit or from synthesis in the pericarp. Meristematic tissue located in shoot and root apices and in lateral buds has long been associated with the synthesis of growth-promoting substances. Transloca- tion of these substances to developing fruits could provide the necessary hormonal supply for continued cell enlargement during the period of final fruit swell. Examination of root exudates and xylem sap from a number of herbaceous plants has revealed the presence of cytokinins (Kende, 1964) gibberellins (Lang, 1970) and auxins (Nitsch and Nitsch, 1965). Similar investigations of xylem sap from fruit trees has not received commensurate attention although the xylem sap from apple branches has recently been shown to contain a cytokinin and an unidentified substance active in the Ayena_mesocotyl bioassay (Luckwill and Whyte, 1968). Examination of apple branch xylem sap at different times during the growth season showed that the levels of both substances was initially high during the spring and declined during the early summer to a low, but constant, level. Evidence, albeit indirect, against the hypothesis that final fruit swell is dependent on growth-promoting substances translocated from vegetative organs to the 161 fruit has come from in yitrg studies. Nitsch (1951) showed that tomato flowers, excised 2 days after pollination, could be grown to maturity under sterile conditions on a simple medium of mineral salts and sucrose. The validity of these experiments should be questioned since root formation on pedicels occurred. However, Nitsch (1967) reported that sweet cherry fruits grown on a medium con- taining mineral salts, sucrose, and two amino acids (L- glutamine, L-asparagine), did not reach a size comparable to those grown in the field, although they did undergo the normal color changes indicative of a maturing fruit. Triiodobenzoic acid has been shown to inhibit polar auxin (IAA) transport in various coleoptile and her- baceous stem sections (McCready, 1966; Winter, 1968; Hejnowica and Tomaszewski, 1969). Application of TIBA in lanolin to sour cherry pedicels for various lengths of time during growth stage III (Figure 1, Appendix C) did not curtail cell enlargement in the mesocarp. Although these results could be taken as evidence that cell enlarge— ment during growth stage III is independent of an external auxin source no experimental evidence as to the inhibitory effect of TIBA on auxin transport through phleom and xylem tissues is currently available. The auxin extracted from pericarp tissues during growth stage III (Figure 4, 5; Table 1, 2) could have arisen either from synthesis or from hydrolysis of bound 162 auxin. No evidence has ever been presented for synthesis of auxin in pericarp tissues during the final stages of fruit growth. In contrast, bound auxins (Ueda and Bandurski, 1969), gibberellins (Lang, 1970), cytokinins (Gazit and Blumenfeld), and abscisic acid (Koshimizu, et al., 1968) have been detected in seed and fruit of a wide range of plant species. Hydrolysis of these bound hormones during final fruit swell could result in appreci- able levels of the free hormone. Comparison between the levels of free and bound auxin detected in sour cherry seed and pericarp tissue (Figure 31) showed that the level of free and bound auxin in the seed decreased as the level of free and bound auxin increased in the pericarp. The parallel decrease of free and bound auxin in the seed would argue against conversion of the free to the bound (Barendse, et al., 1968) unless the bound were lost from the seed. The increase in the level of pericarp bound auxin would suggest that this were the case. However, these results are confounded in so much as the auxin released by hydrolysis was chromato- graphically distinct from either of the neutral auxins or the acidic auxin (Figure 30). It is not known if this auxin was active in the Ayena first internode bioassay per se, or was metabolised to an active form within the Ayena_sections. Since base hydrolysis of the bound auxin in corn kernels yielded free IAA (Ueda and Bandurski, 1969) 163 hydrolysis may have been incomplete. Thus, the bound auxin levels depicted in Figure 31 may be an underestimate of the total present in the sour cherry pericarp. The inverse association found between the level of bound auxin in the pericarp and that in the seed suggests that the developing seed is the source of this pericarp auxin. Moreover, the level of bound auxin in the seed was found to be highest at a time when the levels of free auxin in the seed were at a maximum (day 21, Figure 31). This maximum in seed bound auxin content correlates with the development of the nucellus and integuments (Inoue, 1970). Destruction of specific seed tissues by application of maleic hydrazide to developing strawberry achenes showed that if application were delayed until the third day after anthesis, the receptacles were able to develop and ripen even though all achenes were devoid of viable embryos (Thompson, 1963). If the maleic hydrazide was applied prior to the third day from anthesis, however, development of the treated receptacles was prevented. This effect was shown to be due to the effect of maleic hydrazide on seed development, not to an effect of the chemical on receptacle development, peg s3. Treatment with 2-naphthoxyacetic acid partially overcame the effect of aborted achenes. Thompson (1963) concluded that the seed tissue directly controlling receptacle growth could be the nucellus, 164 since maleic hydrazide application inhibited the develop- ment of the nucellus when applied prior to the third day from anthesis but not when applied subsequently. Tukey (1936b) showed that mechanical destruction of developing sour cherry and peach seeds during growth stage I resulted in an abrupt check to fruit growth and eventual fruit abscission. This effect has also been observed following maleic hydrazide application to apricot fruits prior to pit hardening (Crane and Nelson, 1970), strongly suggesting that some factor associated with the developing nucellus and integuments plays a role in the prevention of premature fruit abscission. As seed destruction was successively delayed during growth stage II, higher percentages of treated fruits persisted and developed like untreated fruits (Tukey, 1936b; Crane and Nelson, 1970). Thus, development of fruits from various Prunus sp. is dependent on a factor associated with the development of the nucellus and integuments. In the present study, the bound auxin located in seed tissues is suggested to result from metabolism of the free seed auxin. Furthermore, the maximum level of loound seed auxin has been correlated with the development of the nucellus and integuments. If this bound auxin rmoved from the seed to the pericarp and there was hydrolyzed to the free form during growth stage III, the low level of pericarp auxin found during this time 165 and the rapid rate of pericarp cell enlargement could be explained. Moreover, the finding that sour cherry fruit growth is dependent on the presence of a developing seed until the end of growth stage II (Tukey, 1936b) could also be explained, since the bound auxin must be mobilized from the seed to the pericarp. In conclusion, correlations between the levels of any one free seed hormone and successive stages of fruit growth (Crane, 1964) should be questioned since the levels of free auxin in sour cherry pericarp tissue at different stages of fruit develOpment bore no relation to concomitant levels found in the seed. A further factor is introduced when the presence of bound hormones were considered. Since Baldev, et al.,(l965) showed that AMO-1618 application to pea pods growing on a defined medium inhibited seed gib- berellin synthesis by 87% while only inhibiting seed weight by 16%, high seed hormone levels could be the result of seed development rather than its cause. If accumulating hormones were converted to bound forms as a de-toxification mechanism and then mobilized to the fruit, growth substances would be available for subsequent fruit growth. Such a hypothesis would explain many of the negative correlations found for seed hormone content and the later stages of fruit growth. Furthermore, if the auxins found immediately after anthesis in naturally 166 parthenocarpic fruits (Gustafson, 1939a; Nitsch, et al., 1960; Coombe, 1961) were incorporated into bound forms which underwent hydrolysis during later fruit development, fruit growth in the absence of a seed could be explained. SUMMARY Changes in seed and pericarp auxin content were determined in developing sour cherry (Prunus cerasus L.) fruits. Although changes in free seed auxin content were correlated with the development of the nucellus and integuments, endosperm, and embryo, no correlation was found between the changes in free seed auxin content and any stage of fruit development. The pericarp auxin content was found to be approximately two orders of magnitude lower than that found in the seed. No cor— relation was found between the level of free pericarp auxin and growth stage II or III, or with changes in the level of free seed auxin. The level of bound seed auxin was found to parallel the changes in the level of free seed auxin. The level of pericarp bound auxin, however, was inversely associated with the levels of both free and bound auxin found in the seed. Changes in fruit cytokinin content were also determined in developing sour cherry fruits. The period of rapid cell division in the pericarp could be correlated 167 168 with the level of butanol-soluble cytokinin. The cessation of cell division in the pericarp, however, could not be correlated with decreased levels of this cytokinin. Characterization of free auxins in seed and pericarp tissues revealed both acidic and neutral substances that were highly active in the A3323 first internode and curva— ture bioassays. The acidic auxin was chromatographically and chromogenically similar to indole-3-acetic acid. This tentative identification was confirmed by gas-liquid co- chromatography with standard IAA and by combined gas-liquid chromatography-mass spectrometry. Repeated chromatography of the neutral auxin resolved the growth promotive activity into two components (neutral auxins IIA and IIB). Neither of these auxins chromatographed with standard indole auxins nor did they yield chromogenic reactions with reagents specific for indolic compounds. A3323 first internode bioassay of these neutral auxins, either alone or in combination, showed that the concentration range over which they were stimulatory to cell enlargement was extremely narrow, suggesting that both auxins are non-indolic. Moreover, combination of low levels of neutral auxin IIA with all levels of neutral auxin IIB yielded a bioassay response that was signifi— cantly greater than the reciprocal combination of low levels of neutral auxin IIB with all levels of neutral auxin IIA. The bioassay response to all other combinations 169 of these neutral auxins was indicative of additivity of the one auxin with the other. Gas-liquid chromatography of neutral auxin IIB resulted in the resolution of three compounds, none of which chromatographed with standard indole auxins. The non-indolic nature of these compounds was confirmed by combined gas-liquid chromatography-mass spectrometry. The mass spectra obtained from the three compounds exhi- bited many common fragment ions suggesting that all could be isomers. Insufficient evidence is presently available to assign a structure to these compounds. No consistent growth-promoting activity was detected in basic ether fractions of either seed or pericarp tissues. A similar absence of growth-promoting substances was found for the aqueous fractions. Base hydrolysis of such aqueous fractions, however, resulted in the release of an ether- soluble auxin that was chromatographically distinct from either the acidic or neutral auxins. Characterization of substances active in both the radish cotyledon and soybean callus bioassays revealed a Ibutanol-soluble cytokinin that possessed chromatographic pr0perties similar to that of zeatin. A further cytokinin, insoluble in butanol but soluble in water, was chromato- Igraphically similar to zeatin ribonucleotide. 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Residues from extraction and fractionation procedures chromatographed in isopropanol water (4:1). B. Residues from extraction and fractionation procedures chromatographed in hexane : water (9:1). Control values (—) . Upper and lower 5% fiducial limits (-——) . 187 __ __fi __ __ __ __ __ n. _n ._ _ __ ._ ._ h. __ _n __ _ l _ __ _ _ _ __ __ _ _. _ .— ._ u. _ —. _ A n. B Z: _, F t . _ P . P O O 0 0 0 0 0 3 2 I 3 2 I tkmimq d‘xk $5 .46 ..\o I ESE 30%qu NQQEQWK ‘5 khtxk I.O 0.5 Rf A mi 188 Figure 3.--Histograms depicting the Avena first internode bioassay response to hexane and aqueous phases obtained from partition chromatography of seed and pericarp tissues (28 days after anthesis) . A. 0.3 gram equivalents of hexane phase developed in isopropanol : water (4:1) . B. 0.3 gram equivalents of acidic aqueous phase develOped in isopropanol : water (4 :1) . Control values (-—). Upper and lower 5% fiducial limits (--—) . ., 0. . .-—..T I u _ _ . ._. . . . . . _ . , . . . _ . _ F . . . ‘ . _ A _ A . _ m _ n I m u . 15.:1 no. . . . _ 00” 1 _ - - - . . . u u _ . . . . . . . . . . _ . . _ _ _ . . _ . .. . A n B — b o .I p F - p _ p p _ 0 0 0 0 0 0 3 2 l O w 3 2 l O tkwkhq Ext 25 as ..\o I >6C3$