gibberellins and gravitropism in maize · plantphysiol. vol. 83, 1987 minimized, the presentation...

Plant Physiol. (1987) 83, 645-6510032-0889/87/83/0645/07/$0 1.00/0

Gibberellins and Gravitropism in Maize Shoots1ENDOGENOUS GIBBERELLIN-LIKE SUBSTANCES AND MOVEMENT AND METABOLISM OF[3H]GIBBERELLIN A20

Received for publication May 16, 1986 and in revised form October 23, 1986

STEWART B. ROOD*, PETER B. KAUFMAN, HIROSHI ABE2, AND RICHARD P. PHARISDepartment ofBiological Sciences, University ofLethbridge, Lethbridge, Alberta, Canada TIK 3M4(S.B.R.), Department ofCellular and Molecular Biology, University ofMichigan, Ann Arbor, Michigan48109 (P.B.K.), and Plant Physiology Research Group, Department ofBiology, University ofCalgary,Calgary, Alberta, Canada T2N IN4 (H.A., R.P.P.)

ABSTRACT

PHjGibberellin A20 (GA2o) of high specific radioactivity (49.9 gigabec-querel per millimole) was applied equilaterally in a ring of microdrops tothe internodal pulvinus of shoots of 3-week-old gravistimulated andvertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate)maize mutant, lazy (la). All plants converted the VIHlGA2. to IH]GAI-and IHIGA2v-like metabolites as well as to several metabolites with thepartitioning and chromatographic behavior of glucosyl conjugates of PHIGA,, IHIGA29, and V3HGAs. The tentative identification ofthese putativePHIGA glucosyl conjugates was further supported by the release of thefree IHIGA moiety after cleavage with cellulase. Within 12 hours of thePHIGA2o feed, there was a significantly higher proportion of total radio-activity in lower than in upper halves of internode and leaf sheath pulviniin gravistimulated normal maize. Further, there was a significantly higherproportion of putative free GA metabolites of [HJGA2., especially 1H1GA1, in the lower halves of normal maize relative to upper halves. Thedifferential localization of the metabolites between upper and lower halveswas not apparent in the pleiogravitropic mutant, la. Endogenous GA-likesubstances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endog-enous free GA-like substances in upper and lower leaf sheath andinternode pulvini halves were extracted, chromatographed, and bioas-sayed using the 'Tanginbozu' dwarf rice microdrop assay. Lower halvescontained consistently higher total levels of GA-like activity. The quali-tative elution profile of GA-like substances differed consistently, upperhalves containing principally a GA2o-like substance and lower halvescontaining mainly GA,-like and GA,9-like substances. Gibberellins A,(10 nanograms per gram) and A2. (5 nanograms per gram) were identifiedfrom these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are con-sistent with a role for GAs in the differential shoot growth that followsgravitropism, although the results do not eliminate the possibility thatthe redistribution of GAs results from the gravitropic response.

The involvement of endogenous growth substances in the

'Supported by Natural Sciences and Engineering Research Councilgrants U0286 to S. B. R., A-2585 to R. P. P., and National Aeronauticsand Space Administration Space Biology Program grant NAGW-34 toP. B. K.

2 Present address: Department of Plant Protection, Faculty of Agricul-ture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo183, Japan.

gravitropic growth response in higher plants has long been pos-tulated. While the involvement of IAA in the response of maizeand other plants is now well established (1, 2, 9, 20), the possibleinvolvement of GAs3 is less well documented. It is known,however, that the application of GA3 or GA4/7 will accelerateshoot upturning in grasses (9, 17), and upturning of lateralbranches of decapitated or intact conifers (see references cited inPickard [20]), and further, that differential localization of GA-like substances occurs between the upper and lower halves ofgravistimulated shoots of oats, sunflower and maize (17, 18, 21).

Gravistimulation of intact Avena plant shoots for 48 h resultedin differential levels of GA-like substances of different polarities(i.e. different GAs) in the top and bottom halves of both the p- Inode-pulvinus and internode above it (17). A similar differentiallocalization of [3H]GA4 and its metabolites was also noted (17)and the differences in free [3H]GA-like and [3H]GA conjugate-like metabolites indicated that differential metabolism and/ormovement was correlated with the gravitropic growth responsein oat shoots.However, studies following the movement of ['4C]GA3 (oflow

specific activity) or [3H]GA3 (high specific activity) in gravistim-ulated maize coleoptiles have produced variable results (30, 31).Part of this ambiguity may have arisen from the use of GA3, aGA not known to be native in maize. Although GA3 is structur-ally similar to GA,, the probable biologically active native GAcontrolling shoot elongation (19, 29), its metabolism, includingrapidity of conjugation, may differ from that of GA,. Also, themobility of [3H]GA1 (and probably of GA3) in maize is lowerthan that ofits monohydroxylated precursor, [3H]GA20 (SB Roodand RP Pharis, unpublished data). To investigate the dispositionand metabolism of GAs in gravitropically stimulated maize, itthus seemed reasonable to use a native GA of high specificradioactivity which has high mobility and which is converted tothe probable 'effector' GA (GA,) in maize shoot elongation (19,23, 29). The use of [3H]GA20 satisfied all of these criteria. The[3H]GA20 was applied after 48 h gravistimulation and conse-quently after initiation of the growth response.

In previous studies involving differential movement and/ormetabolism of plant hormones across gravistimulated shoots, ithas been difficult to control for the environmental differencesexperienced by the upper and lower portions of the shoot.Although studies are usually conducted under greenhouse orgrowth chamber conditions where temperature gradients are

3Abbreviations: GA, gibberellin; C/D R GA20, C/D ring-rearrangedGA20; GC-SIM, capillary gas chromatography-selected ion monitoring;HPLC-RC, HPLC-radioactivity counting; KRI, Kovats retention index;Rt, retention time.

645

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Plant Physiol. Vol. 83, 1987

minimized, the presentation of light is still directional. Conse-quently, phototropic responses may confound the study of grav-itropism (20). Further, the presence of a bench below the plant,as well as the pot in which the plant is growing, introduceadditional physical differences experienced by the upper andlower halves of the shoot. While sophisticated growth chamberdesigns can minimize some of these physical differences, analternate 'control' in gravitropism studies is also available;namely, the use of pleiogravitropic (prostrate) mutants. Such amutant exists in maize in the single gene recessive mutant, lazy(la) ( 16).The mutant, la, displays a prostrate growth habit after 2 to 3

weeks of normal growth (16), although the la phenotype issomewhat variable and can be modified by environmental con-ditions (SB Rood and PB Kaufman, unpublished data). Further,Corcoran (3) has recently suggested that la is not pleiogravitropicbut is, in fact, positively gravitropic. In either case, la displays avastly different gravitropic response than that of normal maize,and hence, it may be useful for comparisons in studies of thegravitropic growth response. While displaying the prostrategrowth habit, the upper and lower halves of the la shoot experi-ence physical conditions similar to those of the horizontallypositioned normal maize plant. However, the normal maizeshoot responds to this condition by negative gravitropic growthwhile la remains prostrate. Differences that may occur in growthsubstance movement and/or metabolism across the horizontalshoot in the la mutant may thus reflect differences in the envi-ronment or physical orientation, but not differences that causeor accompany negative gravitropic curvature. Thus, any differ-ences in the movement or metabolism of hormones between thegraviresponding normal maize and the pleiogravitropic la maizeare more likely to be related to the gravity-sensing/transducingmechanism.

Iftransport and differential metabolism ofexogenously appliedGAs of high specific radioactivity are physiologically represent-ative, similar differences should be observed in the localizationof endogenous GAs. Railton and Phillips (21) observed thatgravistimulation of excised maize coleoptiles increased the over-all level of diffusable GA-like substances and also led to higherlevels in the lower halves. Their report of a differential level ofGA-like substances from excised organs should also be reflectedin vivo if GAs are important in the gravitropic growth response.Thus, in the present study we have also investigated the influenceof gravistimulation on the level and distribution of endogenousGA-like substances in shoot tissue from intact maize seedlings.

MATERIALS AND METHODS

This study began in the summer of 1981, and subsequentexperiments were conducted in 1982 and 1984, building uponprevious results. In all experiments normal ('Stowell's Evergreen',a gift from Dr. R. Bandurski, Michigan State University) andlazy (initial seed was a gift from Dr. R. J. Lambert, DepartmentAgronomy, University of Illinois), Champaign-Urbana) (16)maize (Zea mays L.) plants were grown in glasshouse conditions(20-23°C day; 17°C night) at Ann Arbor, Michigan.Movement and Metabolism of I3HIGA2o in Normal and la

Maize. After the 3rd week of growth (at which time la plantsdisplayed a prostrate growth habit) 10.8 kBq [2,3-3H]GA2o ofhigh specific activity (49.9 GBq per mmol) (15) was applied in afive-point ring (each 10 ,l droplet containing 14.3 ng GA2o) perapplication point (applied with a micropipette after puncturing2 mm long holes in the internodal pulvinus with a teasing needle)to the shoots of la and normal plants. Normal maize plants wereplaced in the horizontal position 0, 24, 48 or 96 h prior to theaddition of [3H]GA20, and incubations with [1H]GA2o were al-lowed to proceed 6, 12, or 48 h as indicated in Tables I to III. Atharvest, tissue immediately apical to the site of [3H]GA20 appli-

cation was carefully dissected into upper and lower halves andfurther dissected into leaf sheath and internodal pulvinus seg-ments (9, 17). Tissue samples were frozen and lyophilized at theUniversity of Michigan and extracted at the University of Leth-bridge.

Extraction, purification, and chromatography were conductedas previously described in detail (23, 25, 26). Briefly, sampleswere ground in cold (-20C) 80% aqueous methanol and ex-tracted for 24 h. After samples were dried in vacuo, extracts wereloaded onto glass fiber discs prior to stepwise elution (25) SiO2partition chromatography (5). Peaks ofradioactivity eluting fromsio2 columns were subsequently microfiltered and further chro-matographed on gradient-eluted reversed-phase C18 HPLC-RC(11), as were authentic standards of ['H]GA,, ['H]GA8, ['H]GA20, and ["C]GA29.For statistical purposes, the total radioactivities eluting at the

Rt of (a) ['HJGA20 plus ['H]C/D R GA20 (a possible metaboliteand/or product of workup [23]), (b) ['H]GA, plus ["C]GA29,and eluting in (c) the methanol wash fraction from the SiO2partition columns were bulked. Data from the replicates fromeach study (1981-three replicates; 1982-two replicates;1984- 10 replicates) were analyzed by two-way analyses of var-iance. In 1984 analyses, the replicate number of individual pairsof samples from a given plant were treated as one factor; arecommended procedure for evaluating paired comparisons (28)which allowed for the effective removal of the substantial varia-tion in uptake and metabolism across replicates.

Distribution of Endogenous GA-Like Substances. In the springof 1982, 3-week-old, nonradiolabeled, normal plants were grav-istimulated for 48 h, and subsequently, shoot segments contain-ing node-pulvini were dissected longitudinally into upper andlower halves. These were immediately frozen and lyophilizedand GA-like substances were extracted, purified, and bioassayed,as previously described (24, 26). Three replicate samples ofabout0.1 g dry weight each were extracted with methanol, and purifiedwith polyvinylpolypyrrolidone prior to stepwise-elution Sio2 par-tition chromatography (5, 25). The 26 fractions from the SiO2column of each extract were bioassayed using a modified (24)dwarf rice cv. 'Tan-ginbozu' microdrop method (13) at twodilutions (1/50, 1/100). Bioactive peaks (Fig. 2) were subse-quently chromatographed on reverse-phase C,8 HPLC (11) andagain assayed by the microdrop assay to further determine theirqualitative nature.To verify that GA, and/or GA20 were present in these tissues,

2.1 g (dry weight) oflower leafsheath pulvini tissue was extractedas above and an internal standard spike of 833 Bq [2,3-'H]GA2o(49.9 GBq per mmol) (15) and 833 Bq [1,2-3H]GAI (1.21 TBqper mmol, Amersham) was added during the initial extraction(e.g. 5.5 ng GA20 and 0.2 ng GA,). Subsequent purification andchromatography was as described for samples following the ['H]GA20 feed. [17, 17-2H]GAI and [17, 17-2HJGA2o were used asinternal standards, being simultaneously derivatized, coinjectedwith the appropriate HPLC fractions, and analyzed by GS-SIM(24). Six ions were monitored, three for the endogenous GA, twofor the [2H](d2)GA, and one (m/z 99) for a hydrocarbon standardwhich allowed for the calculation of the Kovats retention index.Ion abundances were corrected for the contribution from thedeuterated GAs, and the amount of carrier GA20 associated withthe [3HJGA2o spike was subtracted to determine the level ofendogenous GA20.

RESULTS AND DISCUSSIONMovement and Metabolism of IHIGA2o in Normal and la

Maize. For the first 2 weeks following seedling emergence therewere no phenotypic differences between la and normal maizeplants. When la plants at this early stage of development werepositioned horizontally they responded with negatively gravi-

646 ROOD ET AL.



GIBBERELLINS AND GRAVITROPISM

tropic shoot curvature similar to normal plants. At about 3 weeksafter seedling emergence, the la shoots began to bend over andassumed a prostrate growth habit. All applications of [3H]GA20were made 3 weeks following seedling emergence, at which time,all la plants displayed the prostrate growth habit (Fig. 1).

Following the [3H]GA20 feeds in 1981, 1982, and 1984, threediscrete regions of radioactivity eluted from the SiO2 partitioncolumns (Fig. 2). The first was a peak coincidental with the Rtofthe precursor fed, [3H]GA20. The second peak was coincidentalwith the Rt ofGA, and GA29, which are the principal metabolitesof GA20 in maize seedlings (8, 23, 29). The region in which [3H]GA8, another known metabolite (4) would elute, was also col-lected but contained little radioactivity. The third region ofradioactivity eluting from the SiO2 partition columns consistedof the methanol wash where GA glucosyl conjugates will elute( 12). These same three regions of radioactivity were observed inall 3 years from extracts of leaf sheaths and internodal pulvini ofvertical and gravistimulated normal plants, as well as la plants(data not presented).Although the use of high specific activity [3H]GA feeds is most

desirable from a physiological standpoint (e.g. it reduces thechance of artifacts due to substrate overloading), it does precludethe more definitive characterizations that can be obtained whensubstrate levels are increased, and/or stable isotope-labeled pre-cursors are used (8, 29). However, in maize, the major endoge-nous GAs of both tassel and vegetative tissue are now known, asare many of the metabolic conversions of native GAs, includingGA20 (4, 7, 8, 23, 29). Hence, our tentative identifications inextracts of la and normal maize shoots of [3H]GA20 and its majormetabolites ([3H]GAI, [3H]GA29, and [3H]GA glucosyl conju-gate-like substances) based on sequential SiO2 partition chro-matography, C18 HPLC-radioactivity counting, and cellulose hy-drolysis ofthe putative conjugates, are probably adequate for thediscussion ofhow gravistimulation affects the disposition of [3H]GA20 and its metabolites during the subsequent growth response.

In all 3 years there was a trend toward increased levels of totalradioactivity in the lower shoot segments (Tables I-III). Thispattern was statistically significant in the well-replicated 1984study (df= 1; MS = 447172; F = 6.03; P=0.035). However, thesame pattern for total radioactivity was observed for the pleio-gravitropic la leaf sheaths (Table I) and hence, the pattern maynot necessarily relate to the differential growth that normallyfollows gravistimulation.The proportions of radioactivity that were associated with the

precursor [3H]GA20, did not show systematic differences in topversus bottom segments. However, in all 3 years lower shoot

\

I;.

FIG. 1. Lazy (left) and normal (right) maize plants 21 d after seedlingemergence under greenhouse conditions in May.

[1982

20 -

C.) -

0 19840< 20-

GA20 GA1,29 MeOH

10 -

10 20

SiO2 FRACTION NUMBER

FIG. 2. Distribution of radioactivity from stepwise-eluted SiO2 parti-tion columns loaded with methanolic extracts from 3-week-old maizeshoots following [3H]GA20 feeds. 1981- extract from the upper leafsheath of la maize which was fed [3H]GA20 and harvested 24 h later. AnyGA glucosyl conjugates present would be elute in the methanol wash.1982- extract from the upper half of the leaf sheath from normal maizegravistimulated 48 h, fed [3H]GA20, and harvested 24 h later. 1984-extract from the upper half of a leaf sheath internode of a normal maizeplant gravistimulated 48 h, fed [3H]GA2o, and harvested 12 h later.Retention volumes of authentic GAs are shown above the 1984 elutionprofile.

segments contained higher proportions of the radioactivity thatwas associated with SiO2 region II (e.g. the principal putativefree GA metabolites, [3H]GA1 and [3H]GA29) (Tables 1-III). Thispattern of distribution for putative [3H]GA1 plus [3H]GA29 washighly significant for the 1984 experiment (df= 1; MS=324;F=11.24; P=O.010). Subsequent reversed-phase C18 HPLC ofthis region resulted in two peaks, the first peak (35% of theradioactivity) eluting at 21 min where authentic ['3C]GA29 elutesand the second (45% of the radioactivity) eluting at 26 min,coincidental with authentic [3H]GAI. The ratio of putative [3H]GA29 and [3H]GA, did not differ significantly in upper versuslower samples. For radioactivity eluting in SiO2 region II nodifferences in proportion of total radioactivity were observed inupper versus lower la samples (Table I). Thus, this pattern ofdifferential distribution of putative [3H]GA1 and putative [3H]GA29 does not simply result from the horizontal orientation ofthe maize plants.

Hydroxylation ofGA20 at the 3-f3 or 2-(3 position will produceGA, or GA29, respectively, and this would be a logical regulatorypoint in the GA metabolic pathway in maize. The effector GAfor shoot elongation in maize is thought to be GA, (19), whereasGA29 shows little biological activity in the dwarf maize (or other)bioassays (22). However, in the present study, no differences inthe ratio of [3H]GA,- to the [3H]GA29-like metabolites wereobserved in the gravitropically responding lower tissues, relativeto upper halves.

Countering the trend towards increased levels of putative [3H]GA, plus [3H]GA29 in bottom halves was a trend towards de-

647




Table I. Percentage ofMethanol-Extractable Radioactivity Eluting at the Rt ofGA2o, GA, plus GA29, or inthe Putative GA Glucosyl Conjugate Fraction (Methanol Wash) from Step-Eluted SiO2 Partition Columnsa

Percent Total Putative PutativeTreatment and Tissue Radioactivity [3H]GA20 [3H]GAI [3H]GA

(Total Extractable) [3HJGA29 Conjugates

% oftotal extractable radioactivityGravistimulated normal maize (21763 Bq)Leaf sheathTop 38.7 60.2 22.7 17.1Bottom 53.1 52.9 31.7 16.0

InternodeTop 4.1 29.7 31.7 38.7Bottom 4.2 32.8 37.1 30.2

La Maize (16132 Bq)Leaf sheathTop 31.9 28.7 46.6 24.7Bottom 41.0 33.2 46.4 19.9

InternodeTop 13.7 10.9 47.7 40.5Bottom 13.3 14.1 43.9 41.5a The columns were loaded with extracts from shoots of 3-week-old normal and la maize plants, gravistim-

ulated for 96 h, fed (3H]GA20, and harvested 12 h later (1982 experiment).

Table II. Percentage ofMethanol-Soluble Radioactivity Eluting at the Rt ofGA20, GA, plus GA29, or in thePutative GA Glucosyl Conjugate Fraction (Methanol Wash) from Step-Eluted SiO2 Partition Columnsa

Time after Percent Total Putative PutativeAddition of [3H] Radioactivity [3H]GA20 [3PHaGAi Putat

GA20 ~~~(Total [3+ ConjugateGA20 extractable) [3H]GA29 Conjugates

% oftotal extractable radioactivity6h(1021 Bq)Top 37.2b 39.7 47.5 13.0Bottom 44.0 37.9 50.0 12.2

12 h (1672 Bq)Top 19.1 49.4 29.7 20.9Bottom 63.3 48.6 37.4 14.1

a The columns were loaded with extracts from shoots of 3-week-old normal maize plants gravistimulated for48 h, fed [3HJGA20, and harvested after 6 or 12 h (1982 experiment). b Only leaf sheath samples wereanalyzed since internode tissue was low in radioactivity. The remaining radioactivity was in the internodesand, hence, the sum of top and bottom halves is not 100%.

creased levels ofputative [3H]GA conjugates (Tables I-III). How-ever, although consistently observed across the 3 years, this trendwas not statistically significant in any single year (for 1984; df= 1;MS=87; F=2.62; P=O. 141).The methanol wash fractions from SiO2 which will contain

glucosyl conjugates of GAs, if present, were further resolved byC18 HPLC, yielding five principal peaks of radioactivity (Fig. 3).The largest peak (I) eluted just prior to free [3HJGA8 while peaksIV and V eluted just prior to [3HJGA, and [3H]GA20, respectively.Given that most glucosyl conjugates elute on reverse-phase C18HPLC just prior to their respective free GA moieties (1 1, 27),peaks I, IV, and V probably represent glucosyl conjugates ofGA8, GA,, and GA20, respectively (25). Consistent with thisconclusion, incubation with cellulase yielded ethyl acetate-solu-ble radioactivity which chromatographed on subsequent SiO2partition and HPLC columns coincidental with authentic [3H]GA, and [3H]GA8 (data not shown, but see Rood et al. [25]). Acontrol incubation with boiled cellulase did not yield significantethyl acetate-soluble radioactivity.

Thus, following [3HJGA20 feeds to gravistimulated normalmaize seedings, consistent trends toward higher levels ofputative[3H]GA, plus [3H]GA29 were observed in the bottom halves ofshoots for 3 years' experiments (Tables I-III), and this trend was

highly statistically significant in the 1984 experiment. Con-versely, consistent trends indicated that the levels and percentageofputative (3H]GA glucosyl conjugates were lower in the bottomhalves relative to upper halves.The cumulative result of these patterns are considered for the

1984 samples which represent the best-replicated harvest. Bottomhalves at h 12 contained 58.4% of the radioactivity whereas theupper halves contained 41.6%. There was also differential me-tabolism, lower halves having approximately 7% more putative[3H]GA, plus [3H]GA29 than upper halves. The net result ofdifferential distribution in total radioactivity and differentialmetabolism would be a 45% increase in the amount of putative[3H]GA, plus [3H]GA29 in the lower halves.Only the GA1 would be biologically active; the GA29 is low in

biological activity. Although the proportion of putative [3H]GA,relative to the putative [3H]GA29 was 45% versus 35% , respec-tively (based on analytical C18 HPLC-RC), this proportion maybe deceptively low in terms ofGA turnover. The major [3H]GAglucosyl conjugates are a putative [3H]GA8-glucosyl conjugate(Fig. 3; peak 1) and the putative [3H]GA,-glucosyl conjugate(s)(Fig. 3; peaks III and IV), with the putative [3H]GA29-glucosylconjugate making up only a minor percentage of the total radio-activity (Fig. 3; peak II). Assuming that putative [3H]GA, glu-

648 ROOD ET AL.




Table III. Percentage ofMethanol-Soluble Radioactivity Eluting at the Rt ofGA20, GA, plus GA29, or in thePutative GA Glucosyl Conjugates Fraction (Methanol Wash) from StepEluted Partition SiO2 Columnsa

Total % Total Putative PttvReplicate Radioactivity Radioactivity [3H]GA20 [3H]GA1 [3HJGA

inai(q) in Bottom + CnuaeinPair~3q) Half [3H]GA29 Cnuae

% oftotal extractable radioactivityTop

1 11.6 34.6 52.92 22.4 36.8 40.83 21.2 44.2 34.64 19.1 46.4 34.15 16.9 43.5 39.16 21.7 44.8 29.07 23.5 46.0 29.48 22.1 46.1 31.69 Sample lost10 19.4 28.5 51.7X 19.8 41.2 38.1

Bottom1 391 66.3(+) 9.4(-) 37.6(+) 52.6(=)2 198 56.3(+) 12.8(-) 44.8(+) 49.9(=)3 477 63.6(+) 31.5(+) 47.8(=) 25.8(-)4 456 50.5(+) 20.3(=) 49.1(+) 30.4(-)5 333 47.8(-) 9.0(-) 49.5(+) 40.1(=)6 474 53.2(+) 20.7(=) 45.0(=) 33.9(-)7 480 54.1(+) 20.6(-) 67.3(+) 11.7(-)8 530 58.8(+) 13.5(-) 58.2(+) 33.2(+)9 395 53.0(+) Sample lost10 539 80.7(+) 16.9(-) 48.1(+) 35.0(-)X 427 58.4 17.2 49.7 33.7

Summary for bottom halves 9/10(+) 1/9(+) 7/9(+) 1/9(+)1/10(-) 6/9(-) 0/9(-) 5/9(-)0/10(=) 2/9(=) 2/9(=) 3/9(=)

a The columns were loaded with extracts from shoots of 3-week-old normal maize plants gravistimulated for48 h, fed [3H]GA20, and harvested after 12 h (1984 experiment). The individual replicate values allow forcomparison of the paired top and bottom halves of shoots. The (+) or (-) after the bottom value indicaterelationship of bottom value to corresponding top value, (=) indicates a difference of less than 1.5%. Leafsheath and internodal pulvinus tissue was not separated for these samples.

cosyl conjugate(s) and the putative [3HJGA8 glucosyl conjugatehad as their precursor free [3H]GA1, then by implication, a verysignificant proportion (top, 50.1%; bottom, 50.5%) of the [3H]GA20 (and presumably, endogenous GA20) is being metabolizedto [3H]GAI. A much smaller proportion (top, 18.0%; bottom,20.9%) goes to GA29 and its conjugate.

In the present study the replication of trends across severalyears, and the statistical validation of the downward movementof total radioactivity and metabolite (putative [3H]GA1 and [3H]GA29) radioactivity indicates that GA movement within leafsheath pulvini and internodal pulvini (which are largely respon-sible for the negative gravitropic growth of normal maize shoots)is downward. The present results are thus consistent with earlierwork (17) where (a) a similar downward movement of [3H]GA4and its free [3H]GA-like metabolites occurred in the p-1 nodepulvinus and adjacent internode of intact oat plants gravistimu-lated for 48 h, and (b) a reciprocal differential localization in[3H]GA glucosyl conjugate-like substances also occurred betweenupper and lower halves. Further, increased downward movementof [3HJGA1 and its 3H metabolites in excised oat stem segmentswhich were gravistimulated for only 1 h has recently been noted(10).

Distribution of GA-Like Substances. Both the leaf sheath andnode-pulvinus tissues were relatively rich in GA-like substances

(Table IV, Fig. 4). Four principal regions of GA-like activitywere eluted from the SiO2 partition columns. The first regionwas coincidental with [3H]GA20 (Fig. 4), and the third regioneluted coincidentally with [3H]GA, and GA19, (Fig. 4) (24).Gibberellins A1, A19, and A20 are native to maize (7, 8).Subsequent chromatography by reverse-phase C18 HPLC (11)

and bioassay on 'Tan-ginbozu' dwarf rice (data not presented)confirmed that the GA-like activity that eluted from the initialSiO2 partition columns also eluted at the Rt values ofGA,, GA19and GA20 (11, 24). Data from serial dilutions after HPLC indicatethat the concentration of GA,9-like substance was about twicethat of the GA1-like substance.Both GA, and GA20 were identified from lower leaf sheath

pulvini tissue based on (a) the occurrence of GA-like substanceswhich co-chromatographed with the respective authentic [3H]GA on sequential SiO2 partition and analytical reverse-phase C18HPLC, and (b) on data from subsequent GC-SIM using theauthentic [17,17-2H2]GA1 and GA20 as internal standards (24)(relative ion intensities [percentage] for: standard [2H](d2)GA,:M+ 508 [100], 493[9], 450[14] KRI = 2717; and maize putativeGA,: 506[100], 491[20], 448[20], KRI = 2717; standard [2H](d2)GA20: M+ 420[100], 405[12], 377[68], KRI = 2559; andmaize putative GA20: 418[100], 403[28], 375[143], KRI = 2559).Using the [3H]GA data to determine recovery efficiencies and

649




20-

10I-~~~ ~~~~~~~~~~I

0

O Q > BOTTOM

~ ~ GA9GAI GA20

10 20 30 40

HPLC RETENTION TlIME (min)

FIG. 3. Distribution of radioactivity from a gradient-eluted reversed-

phase C18 HPLC column loaded with the putative [3HJGA glucosyl

conjugate fraction (e.g. methanol wash) from step elution SiC)2 partition

columns (Fig. 2). The Rt values of authentic free [3HJGA8, [3HJGA1, and

P3HIGAso are shown above the elution profile. GA glucosyl conjugates

elute to several min before the free GA moiety (25, 27).

the internal standard [3HJ(d2)GA for quantitation, levels of en-

dogenous GA1 and GA20 were determined to be 9.6 and 4.9 ng/

g (dry weight), respectively.

The concentrations of endogenous GA-like substances were of

similar magnitude in leaf sheath and internode-pulvini (Table

IV). In the internode-pulvini, concentrations (total) in the bot-

tom halves were about four times higher than in the top halves

(Table IV). Further, in both leaf sheath and internode-pulvini,

top and bottom halves differed in the qualitative distribution of

GA-like activity (Table IV, Fig. 4). Upper halves contained a

large peak of GA-like activity at the Rt of GA20 (SiC)2 partition

column fractions 3-8) and relatively small peaks of GA1- and

GA19-like activity (SiC)2 partition column fractions 15-22). In

contrast, lower halves contained much lower levels of bioactivity

at the Rt of GA20 and higher levels of activity in the more polar

regions (Table IV, Fig. 4). Since GA1 is probably the effector GA

for shoot elongation in maize (19), the comparisons of these

polar regions may be particularly important (Table IV, SiO2fractions 15-22, Fig. 4). The concentration of GA-like activityin these fraction groupings was about 3 to 5 times higher inbottom leaf sheath halves and about 100 times higher in bottomhalves ofinternode-pulvini (Table IV), both relative to top halves.The presence ofGA, in these tissues was confirmed by GC-SIM(see above), although analysis by bioassay after HPLC indicatedthat GA19 was also present in this SiO2 grouping and was respon-sible for about two-thirds ofthe GA-like activity in SiO2 fractions15-22. Significant amounts ofGA-like activity were also detectedby the microdrop assay in the methanol wash fractions, whereGA glucosyl conjugates will elute (12) (SiO2 fractions 23-26,Table IV). This suggests the presence ofGA glucosyl esters sincethe microdrop assay will detect free GAs and GA glucosyl estersbut not GA glucosides (14).

Previous studies have also indicated that increased levels ofGA-like substances (present endogenously or applied exoge-nously) are associated with gravitropic upturning in shoots ofAvena (10, 17) peanut (6), and sunflower (18). Additionally, itwas reported (21) that diffusion ofGA-like substances is fourfoldgreater from lower halves of gravistimulated excised maize co-leoptiles than from upper halves. Thus, results from a numberof plant systems are consistent with the present finding of in-creased levels ofGA-like substances in the lower halves of gravi-responding shoots.A causal role for GAs (and specifically GA,) in the control of

shoot elongation in maize is probable (19). The net downwardmovement ofGAs (Tables I-IV), and the differential metabolismin upper versus lower halves (Tables I-111) may thus be at leastin part, responsible for the increased accumulation of endoge-nous GA-like substances in the lower halves. Increased amountsof GA, and one or more its precursors, in lower shoot halveswould be expected to result in its continued or increased elon-gation, thereby causing negatively gravitropic growth. Thus, theobserved differences in movement and metabolism of [3H]GAs,and an increase in the level of endogenous GA-like substancesprovide a logical rationale for at least part of the hormonalregulation of the differential growth response of maize shootsfollowing gravistimulation. However, GAs are not the only hor-mone thought to be involved in gravitropism. The differentialmovement and metabolism of IAA with gravistimulation hasalso been well-documented for grass shoots, including maize (1,2, 20). Gravistimulation also results in the differential movement

Table IV. Concentrations ofEndogenous GA-Like Substances in Upper and Lower LeafSheaths plus Node-Pulvini of3- Week-Old Maize Seedlingsa

SiC2 Partition Column Fraction(Elution Region of Authentic GAs)

Tissue2363-8 9-14 15-22 23-26(GA20) (GA53) (GA,19) conjugates) Total

ng GA3 equivalents g-' dry weightLeaf sheathTop 129 19 4 6 158

(82) (12) (3) (4)Bottom 30 58 13 29 131

(23) (44) (10) (22)InternodeTop 46 16 1 17 80

(58) (20) (1) (21)Bottom 90 152 112 27 380

(24) (40) (29) (7)a Measurements were made 48 h after horizontal positioning, as determined by bioassay in serial dilution

on dwarf rice, 'Tan-ginbozu.' Values in parentheses indicate percentage of total GA-like activity in fractiongrouping.

650 ROOD ET AL.




4t14 V G2 A3G1Gl

°13 I, _- N2'l- X i f

E

x IV~~~~~~~~~~~~~~~~~~~~~~~~I

z

x1 210 20

PUFG14 . E GA-like5 GAn r

III

Lu 13 FI

U.12 Im

10

10 20 10 20

PARTITION COLUMN FRACTION NUMBER

FIG. 4. Elution profile of endogenous GA-like substances from 3-

week-old maize plants 48 h after horizontal positioning, as determinedby microdrop bioassay in serial dilution on dwarf rice cv 'Tan-ginbozu.'The sio2 partition columns were loaded with the acidic, ethyl acetate-soluble fraction of methanolic extracts from upper and lower leaf sheathpulvini, and internodal-pulvini. Average values of GA-like substancesfor each region from the SiO2 partition column for the three replicatesfrom each sample are provided in Table IV. The Rt values of authenticGAs which are known to be native in maize are shown in the bottomright figure. Gibberellin glucosyl conjugates would elute in fractions 23-26 (methanol wash), if present.

and/or localization of several other substances which are notnormally considered 'hormonal' (e.g. glucose and calcium [20]).Current evidence suggests that gravitropic growth responses inthe shoot are achieved through a differential balance of promo-tory (and perhaps inhibitory) hormones, only one of which maybe GA. The relative roles and importance of GAs and IAA aspromotory hormones, and the possible roles ofABA and ethylenein regulating the gravitropic response in maize shoots requiresfurther investigation.A final note must be made. The present study confirms that

changes in both the metabolism and/or movement of [3H]GA20and its metabolites (including putative [3H]GA,) and the distri-bution of endogenous GA-like substances (including GA,) occur

during the differential growth that occurs after gravistimulationof maize shoots. However, whether the differential GA distribu-tion is a cause of or a result of gravitropic upturning is stillunclear. The observation that exogenous GAs can accelerategravitropic upturning (9, 17) and the general theory that GA,controls shoot elongation in maize (19) are consistent with aregulatory role for GAs in elongation growth following gravistim-ulation. However, it will also be important to establish whenasymmetries in levels for GAs are first established and to comparethese asymmetries with the early time course of the uptuning ofthe shoot.

Acknowledgments-The authors thank Drs. R.I. Greyson and D. Waldren(University of West Ontario) for increasing the lazy seed. The capable technicalassistance of Mrs. Karen Larsen (University of Lethbridge) and Mr. Bruce Twitchin(Research School of Chemistry, Australian National University), and the helpfuladvice of Dr. David Pearce (University of Calgary), are gratefully acknowledged,as is a gift of ['3C]GA29 from Prof. J. MacMillan (University of Bristol).

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