Involvement of phytohormones in germination of dormant andnon-dormant oat (Avena sativa L.) seedsN

A. Poljakoff-Mayber1, I. Popilevski2, E. Belausov2 and Y. Ben-Tal2,*1Department of Botany, The Hebrew University, Jerusalem, Israel; 2Department of Ornamental Horticulture,The Volcani Center, P.O. Box 6, 50250 Bet Dagan, Israel; *Author for correspondence (e-mail:vcflow@agri.gov.il; phone: +972-3-9683357; fax: +972-3-9669583)Received 8 May 2001; accepted in revised form 1 August 2001

Key words: Abscisic acid, Dormant, Embryos, Endosperm, Gibberellins, Imbibition, Non-dormant, Oat seeds

Abstract

Oat seeds are susceptible to high temperature dormancy. Dormant grains do not germinate at 30 C unless af-terripened, dry, for several weeks. Isolated embryos of dormant grains do germinate, especially if GA3 is addedto the germination medium. ABA inhibits germination proportionally to the concentration applied and GA3 canovercome the ABA inhibitory effect. Measurements of endogenous ABA and several GAs revealed that the initiallevels of ABA in dormant and non-dormant grains were quite similar. But, endogenous ABA in non-dormantseeds almost disappeared within the first 16 h of imbibition, while the amount in dormant grains had decreasedby less than 24%. The level of GA19 in non-dormant seeds was higher, and GA19 appears to be converted toGA20 within the first 16 h. The GA20 was converted to GA1 at least during the first 48 h of the germinationprocess. Both phytohormones thus appear to be involved in the germination process of non-dormant seeds. ABAfirst declines, while GA1 is produced during the first 16 h of imbibition to allow proper germination. In dormantgrains the level of ABA remained high enough to prevent germination during at least a week and precursor GAswere not converted to GA1.

Abbreviations: GAs gibberellins, GA3 gibberellin A3, ABA abscisic acid, GC-MS-SIM gas chromato-graphy-mass spectrometry-selected ion monitoring, D dormant, ND non-dormant.

Introduction

Seeds might be expected to germinate when environ-mental conditions are adequate. Yet, often they do notbecause they are dormant. Seed dormancy is commonespecially in grasses but appears in many other spe-cies (Hilhorst and Karssen 1992; Bewley and Black1994). There are several forms of dormancy, whichare controlled by different genetic traits, environmen-tal conditions and biochemical balances betweenplant hormones (Hilhorst (1995, 1998); Wang 1996).High temperature dormancy is one type of dormancy

that prevails in barley, wheat and oat grains (Corb-ineau et al. 1991). Dormant grains of these cereals donot germinate at high temperatures (30 C) unless af-terripened in dry storage for several weeks. Thishigh temperature dormancy may be released if theembryos are separated from the endosperm-aleurone(Poljakoff-Mayber et al. 1990; Corbineau et al. 1991).Addition of ABA to the germination medium de-creases germination rates of oat grains proportionallyto the concentration of ABA applied. Simultaneousapplication of GA3 on top of the ABA can overcomethe inhibitory effect of ABA (Poljakoff-Mayber et al.1990). These findings indicated that both phytohor-mones are involved in the high temperature dor-mancy of oat seeds. However, whole dormant grainsdo not germinate even after GA3 is added to the ger-

N This article is dedicated to the memory of Prof.Alexandra Poljakoff-Mayber who initiated thisproject but passed away before its completion.

7Plant Growth Regulation 37: 716, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands.

mination medium. This phenomenon is well knownin many cereals (Simpson 1990; Corbineau et al.1991; Foley 1994) as well as in non-cereal seeds(Groot and Karssen 1992; Nicolas et al. 1996;Fernandez et al. 1997).

Endogenous ABA and GAs in dormant and non-dormant seeds have been measured in several species:Douglas fir (Bianco et al. 1994); barley (Green et al.1997; Wang et al. 1994); wheat (Lenton et al. 1994).However, these findings failed to explain the relation-ships between these two hormones and to relate themto the germination process. Some of these analysessuggested that high levels of endogenous ABA existin the endosperm-aleurone part of these grains andinterfere with the germination of dormant cerealgrains. But, if this was the case, removal of the en-dosperm-aleurone part of dormant grains should haveresulted in normal germination of dormant embryos.It has been shown by a number of studies that dis-sected embryos of dormant grains did germinate, andwhen they were treated with GA3 they germinatedeven faster (Lenton and Appleford 1991; Van Beckumet al. 1993; Wang et al. 1995; Visser et al. 1996; Wang1996). These authors suggested that the endosperm ofdormant cereal grains contained high levels of ABAand that this ABA had to be removed before germi-nation could take place.

In order to assess this hypothesis we have mea-sured endogenous amounts of ABA and GAs in wholecaryopses, embryos and endosperm of dormant andnon-dormant oat seeds.

Materials and methods

Plant material

Avena sativa L. (cv. Moyencourt) grains, harvested in1994, were used. Grains exhibiting afterripening dor-mancy had been stored at 20 C and non-dormantgrains at room temperature (Corbineau et al. 1991).Husks were removed just before each experiment.Embryos were isolated by dissection with a sharpscalpel blade, with as little endosperm adhering aspossible (Poljakoff-Mayber et al. 1990). The remain-der of the grain after dissection is referred to as en-dosperm in future discussion. In most experimentsdry oat grains were dissected, but in some experi-ments whole caryopses were imbibed for varying pe-riods of time and then dissected. The two parts of thegrains were extracted immediately after dissection.

Some experiments were carried out with whole seedsthat were extracted at the end of the imbibition pe-riod.

Germination experiments

Usually, 25 caryopses or isolated embryos wereplaced in Petri dishes and were incubated in darknessat 30 C (Corbineau et al. 1986). Three replicateswere always used for every experiment. An embryo,or seed, was considered germinated when the radicleprotruded through the seed coat (Corbineau et al.1991).

For determinations of endogenous phytohormonestwo series of experiments were performed. (1) Drygrains were dissected before imbibition and embryos,endosperms and whole-dehulled caryopses were im-bibed for 16 h in separate Petri dishes before extract-ing ABA and GAs. (2) Caryopses were imbibed for16 h, then dissected and immediately extracted. Todetermine whether ABA had leached out from wholeor dissected grains into the imbibition medium, theamount of ABA in the medium in which the caryopsesor embryos were imbibed was determined. In theselatter experiments 3 groups of 250 grains or embryoswere imbibed to make sure that even small quantitiesof ABA were detected.

Extraction and purification of endogenous hormones(GAs and ABA)

Extraction and purification of ABA and GAs weredone as described by Koshioka et al. (1994a, 1994b)and Grnzweig et al. (1997) with minor modifica-tions. In preliminary experiments, samples werespiked with [3H]-GA3 (196 GBq mmol-1) (purchasedfrom Rotem Industries Ltd., Beer Sheva, Israel) forcalculating losses of GAs throughout the purificationprocedure. Briefly, whole peeled caryopses, isolatedembryos or endosperms were homogenized and ex-tracted twice with cold 80% aqueous methanol(MeOH). Standards of [2H6]-ABA, [17-2H2]-GA1,[17-2H2]-GA8, [17-2H2]-GA19 and [17-2H2]-GA20,kindly provided by Prof. L.N. Mander (AustralianNational University, Canberra, Australia), were addedto the aqueous MeOH extracts. Phenols and pigmentswere removed by n-hexane. Phytohormones were ex-tracted by acidic ethyl acetate (EtOAc). For betterpurification the samples were eluted by DEA col-umns, filtered through a 0.4 mm Teflon filter and re-

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duced to dryness in a vacuum concentrator before theHPLC step (Grnzweig et al. 1997).

HPLC

Samples were redissolved in absolute MeOH andloaded onto a LiChrosorb RP-18 column (10 mm i.d. 250 mm; Merck, Darmstadt, Germany). For elutionof the plant hormones, the program used by Edelsteinet al. (1995) was followed. A flow rate of 2 ml min1was maintained. The first 35 fractions of 2 min eachwere collected. During elution UV detection at 269nm for ABA was performed (Dumbroff et al. 1983).External standard of ABA (Sigma) was comparedwith the extracted endogenous ABA that contained aknown amount of 2H6-ABA on the basis of retentiontimes (Dring and Bachmann 1975). Eluted fractionswere dried in a Heto VR-1 Speedvac and kept at 20C until used.

Bioassays

The HPLC fractions were first subjected to a micro-drop bioassay (Nishijima et al. 1992) with minorchanges (Grnzweig et al. 1997). Dwarf rice (Oryzasativa L. cv. Tan-ginbozu), a gift from Dr. M. Ko-shioka (National Research Institute of Vegetables, Or-namental Plants and Tea, Ministry of Agriculture,Forestry and Fisheries, Kusawa, Ano, Mie, Japan),was used. Nine seedlings were planted in small bea-kers containing 15 ml of 0.8% agar and incubated at30 C for 72 h under continuous light (80 mol m2s1; Osram Daylight L65W/10S). Eluted HPLC frac-tions were dissolved in 100 l of 95% aqueous etha-nol and 0.5 or 1 l of every fraction that had to betested was applied to seven nice similar seedlingswith a micro-syringe. Three days later, the length ofthe second leaf sheath was measured by an electroniccaliper (Mitutoyo, Tokyo, Japan).

GC-MS measurements

ABA identification and quantificationThe HPLC fractions containing ABA were combined,evaporated to dryness and methylated with excessethereal diazomethane (CH2N2 ca. 500 l). After me-thylation the residue was redissolved in 10 l di-chloromethane (CH2Cl2) and injected into a Hewlett-Packard 5890 gas chromatograph 5971a massspectrometer instrument (GC-MS) in splitless mode.The column used was a DB-1 capillary column (0.25

mm 30 m; J & W, Folsom, CA, USA) (Grnzweiget al. 1997) and the GC program (Dunlap and Guinn1989). The presence of ABA was verified by tracesof m/z 190, 162, 134, 125, 91 using full-scan GC-MS.For quantitative analysis of ABA by GC-MS-SIM, thecharacteristic ion pair (deuterated ion/protio ion) 194/190 was monitored. Additional characteristic ion pairs166/162 and 138/134 were also monitored to confirmthe identity of these compounds. The amount of en-dogenous ABA was calculated by comparing the ra-tio of the areas under the 190 peak and the 194 peakthat evolved from a known amount of deuteratedABA (Cohen 1982; Or et al. 2000).

Gibberellin identificationThe HPLC fractions exhibiting GA-like activity in thebioassay were combined into two groups, evaporatedand methylated with excess ethereal diazomethane for30 min. After redissolving in CH2Cl2 and drying,samples were trimethylsilylated (MeTMSi). The de-rivatized samples were dried and redissolved in 10 lCH2Cl2 and injected onto the GC column togetherwith 0.1 l of hydrocarbon standards (Gaskin andMacMillan 1991), added in order to determine theKovats Retention Indices (KRI) (Kovats 1958). Iden-tification of endogenous GAs was accomplished asdescribed (Grnzweig et al. 1997).

Gibberellin quantificationThe SIM mode was set for a dwell time of 100 ms.The following ions were monitored: for GA1/[2H2]-GA1 MeTMSi, m/z 506/508, 448/450; for GA8/[2H2]-GA8, m/z 594/596, 448/450; for GA19/[2H2]-GA19, m/z 434/436, 374/376; for GA20/[2H2]-GA20,m/z 418/420, 375/377. The endogenous concentra-tions of the above GAs were calculated based on thepeak area ratios of 506/508, 594/596, 434/436 and418/420, respectively, by reference to calibrationcurves (Grnzweig et al. 2000).

Statistical analysisMost of the experiments were performed at least 3times. Germination experiments always included 3replications of every treatment or condition tested.Phytohormones identification or quantification wasrepeated 45 times. In all statistical analyses of theresults standard errors were calculated and are pre-sented in each figure.

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Results

Endogenous levels of ABA in dormant and non-dor-mant dehulled oat caryopses are shown for dry grains,and after different periods of time during imbibition(Figure 1). ABA content in dry dormant grains wassimilar to the amount in non-dormant dry seeds (Fig-ure 1, time 0). However, the patterns of germinationas well as changes in ABA level during imbibition ofthe two types of grains were significantly different(Figure 1). ABA in non-dormant seeds declined grad-ually to 16.2% of its original level during the first 24h of imbibition when almost 100% germination wasobserved (inset Figure 1). A slight increase was laterobserved, possibly because at this stage ABA was al-ready extracted from developing seedlings.

When whole dormant grains were imbibed undernormal germination conditions their ABA levelsdropped only by about 24% during the first 16 h, and

remained at this level through the next 32 h (Fig-ure 1). Dormant grains had a maximal germinationlevel of 1.2% (inset Figure 1).

Previously, Poljakoff-Mayber et al. (1990) showedthat dissected embryos of Avena sativa L. (cv. Moy-encourt) did not exhibit afterripening, or high temper-ature dormancy. Rather, the dormancy was inducedby the presence of the endosperm-aleurone part of theseed. To pursue farther the fast disappearance of ABAfrom non-dormant seeds, we measured endogenousABA levels in embryos and endosperm in the twotypes of grains that were dissected before or after 16h of imbibition. These ABA levels were comparedwith ABA in embryos and in endosperm of dissectedgrains that were not imbibed at all (Figure 2). Sur-prisingly, ABA level in dry embryos of non-dormantseeds was similar to its level in embryos of dry dor-mant grains, while ABA in the endosperm was simi-

Figure 1. ABA in dormant (D) and non-dormant (ND) oat seeds during imbibition. Whole grains were imbibed for various times. ABA wasdetermined at the end of each imbibition period.

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lar and negligible in both dormant and non-dormantseeds (Figure 2).

In contrast, the ABA level in embryos of dormantgrains that were dissected after 16 h of imbibition re-mained high (Figure 2). While its level in embryosfrom grains dissected just before the beginning of theimbibition period, in both dormant and non-dormantembryos, decreased to very low levels. However, theABA amount in dormant embryos was still twice thatin non-dormant embryos. Again, levels of ABA inboth types of endosperm after 16 h of imbibition werevery similar and about half of that of dry endosperm(Figure 2). ABA leakage out of the embryos and en-dosperm during imbibition of dissected grains mighthave caused these results. This was checked by im-bibing a large number (250) of dormant and non-dor-mant caryopses or dissected embryos for 16 h andmeasuring the levels of ABA in the imbibition me-dium. There was essentially no leaching of ABA intothe medium (note the scale), nor were there apprecia-ble differences between dormant and non-dormantgrains, or between whole and dissected seeds (Fig-ure 3).

The GA levels of dormant and non-dormant dehul-led oat grains were assessed before imbibition and atdifferent points during imbibition. Purified extracts ofdormant and non-dormant oat caryopses, either fromdry, imbibed or germinating seeds, contained GAs be-longing to the early 13-hydroxylation GA pathway(Appleford and Lenton 1991; Lenton and Appleford

1991; Lenton et al. 1994). Analysis by GC-MS of theabove extracts provided evidence for the presence ofGA1 and GA8, and GA19 and GA20 in two differentfractions obtained from the HPLC. These GAs werethen quantified by GC-MS-SIM using deuterated in-ternal standards (Figures 4A,B and 5A,B). GA19 (thedirect precursor of GA20) in non-dormant seeds de-clined rapidly within the first 16 h (when they alreadyreached almost complete germination), while GA20increased continuously after a slow beginning duringthe first 8 h (Figure 4B). In dormant grains there wasa gradual decrease of GA19 over the entire 48 h (Fig-ure 4A), whereas GA20 in dormant grains decreasedduring the first 16 h and then began to accumulate(Figure 4B). Changes in the levels of GA20, a directmetabolite of GA19, in both types of grains resultedprobably from conversion of stored GA19. However,it must be kept in mind that after the first 16 h of im-bibition the non-dormant seeds are fully germinatedand we actually extracted and measured GAs in rap-idly developing seedlings. In contrast, imbibed dor-mant grains showed virtually no germination for 48 h(Figure 1, inset).

Gibberellin A1 is known as the major factor con-trolling shoot elongation as well as aleurone responsein very young seedlings of cereals (Boother et al.1991; Jacobsen et al. 1995). The GA1 content in non-dormant seeds increased markedly during the entire48 h of germination (Figure 5A). In contrast, the lev-els of GA1 in dormant grains remained constant dur-ing this period (Figure 5A). Changes in levels of GA8(a GA1 metabolite) were insignificant and fluctuatedbetween 30 and 50 pg seed1 (Figure 5B).

To further study the high temperature dormancyphenomenon, endogenous levels of GAs, in non-dor-

Figure 2. Endogenous ABA in embryos and endosperm of oatseeds. Seeds were dissected before or after 16 h of imbibition.Their ABA content was compared with ABA extracted from dryseeds immediately after dissection. Unit = an embryo or endospermfrom a single grain.

Figure 3. The amount of ABA in the media. ABA was extractedfrom media in which 250 grains or embryos were imbibed for 16h.

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mant and dormant oat grains that had been dissectedbefore or after 16 h of imbibition, were determinedand compared with dry dissected caryopses that wereextracted immediately after dissection (Figures 6 and7). The GA19 content in dormant dry embryos washigher than in non-dormant dry embryos (Figure 6A).However, when the seeds were dissected after 16 h ofimbibition, GA19 levels in embryos and endosperm ofboth non-dormant and dormant seeds were higherthan for dry caryopses. Embryos of non-dormantseeds showed the greatest change in GA19 when im-bibed after dissection. When seeds were dissected be-fore imbibition, the level of GA19 was much smallerin both components of dormant grains (Figure 6A).

The GA20 levels in embryos and endosperm ofnon-dormant dry seeds were appreciably larger thanin dormant grains (Figure 6B). The GA20 levels inembryos and endosperm of non-dormant seeds cutafter 16 h of imbibition were markedly diminishedrelative to dry seeds, and both GA19 and GA20 wereundetectable when dissection of non-dormant seedswas performed before imbibition (Figures 6A and B).In contrast, the GA20 level in dormant embryos dis-sected after 16 h of imbibition increased appreciably,

while its level in dormant endosperm decreased (Fig-ure 6B). Embryos of dormant seeds cut before imbi-bition had GA20 levels similar to dry embryos (Figure6B).

The GA1 levels in embryos and endosperm of non-dormant dry seeds were slightly higher than in dor-mant dry grains (Figure 7A). When grains were dis-sected after 16 h of imbibition, the GA1 levelincreased appreciably in non-dormant embryos andendosperm (Figure 7A). However, almost no GA1was found in embryos or endosperm of dormantgrains dissected before or after the imbibition (Figure7A), as was the case for dry seeds (Figure 7A).Changes in GA8 levels were not notable for anytreated grains (Figure 7B).

Discussion

Intact dormant oat grains do not germinate when im-bibed in water or even in 106 M of GA3 for a wholeweek. However, when dormant caryopses were dis-sected before imbibition and the embryos imbibed,they germinated quite well (98%) within 24 h (in GA3solution germination was even faster; data not

Figure 4. Changes in GA19 (A) and GA20 (B) in dormant andnon-dormant seeds during imbibition. Whole seeds were imbibedfor various periods of time and then their GAs content was mea-sured.

Figure 5. Changes in GA1 (A) and GA8 (B) of dormant and non-dormant seeds during imbibition. Whole seeds were imbibed forvarious periods of time and then their GAs content was measured.

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shown). This implies that there may be something inthe endosperm-aleurone part of the seed that inter-feres with the normal germination of seeds possess-ing afterripening dormancy. Applied ABA is knownto inhibit germination of non-dormant cereals (Polja-koff-Mayber et al. 1990; Corbineau et al. 1991; Len-ton et al. 1994; Wang et al. 1995), while addition ofGA3 overcame this inhibition (Poljakoff-Mayber etal. 1990; Corbineau et al. 1991). This implies thatABA levels must be lowered in dormant seeds in or-der to allow germination. However, we found thatendogenous levels of ABA in non-dormant seedswere similar to (Figure 1, time 0 h), or higher thanthe ABA level in dormant grains (Figure 2, drygrains). The difference might stem from the fact thatin Figure 1 the amount of ABA measured was fromwhole grains, while in Figure 2 the ABA was ex-tracted from dissected embryos and endosperm.

Germination of non-dormant oat seeds was almostcompleted after 16 h. Within this period of time lev-els of ABA rapidly decreased by more than 80%.While in dormant grains the reduction of ABA duringthe first 16 h of imbibition was only 24% and ABAremained at this level for the next 32 h (Figure 1).Thus, this level of ABA is probably too high to allow

for oat seed germination (Poljakoff-Mayber et al.1990; Corbineau et al. 1991). Wang et al. (1995)found that dormant dry embryos of barley containedhigher levels of ABA than non-dormant embryos. Incontrast, we found that the initial level of ABA in dry,non-dormant embryos was higher (Figure 2). Thesedifferences may be due to species, or to the methodof analysis, e.g. dissecting, purification and the use ofELISA (Wang et al. 1995) as a measurement tool ofendogenous plant hormones as compared with ourusing GC-MS. Most importantly though, was themain finding of both studies that the level of ABAduring the imbibition period is reduced very quicklyin non-dormant embryos, whereas ABA remains highand constant in dormant imbibed seeds.

Although the endogenous level of ABA in non-dormant dry embryos was higher than in dormant em-bryos, this ABA almost disappeared during the first16 h of imbibition, whether the seeds were dissectedbefore or after imbibition. In contrast, for dormantgrains, if dissected after 16 h of imbibition, most oftheir ABA remained in the embryos. But, if isolateddormant embryos were imbibed, almost all the ABA

Figure 6. Endogenous levels of (A) GA19 and (B) GA20 in em-bryos and endosperm of oat seeds dissected before or after imbi-bition. n.d. = not detected.

Figure 7. Endogenous levels of (A) GA1 and (B) GA8 in the em-bryos and endosperm of oat seeds dissected before or after imbi-bition.

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disappeared as occurred in non-dormant embryos(Figure 2).

Thus, some factor present in the associated en-dosperm-aleurone tissues appears to inhibit enzy-matic breakdown or metabolism of ABA in embryosof dormant oat seeds. This factor is not present innon-dormant oat seeds. Or, vice versa, a certain en-zyme in the alurone-endosperm of non-dormantgrains is activated by imbibition and breaks down theABA in these embryos.

The disappearance of ABA could not be due tomechanical leakage, because no difference was de-tected between the amounts of ABA leached out fromdormant or non-dormant whole seeds. However, theamount leached from whole seeds was smaller thanthe amount leached out from dissected embryos,likely due to the large cut adjacent to the embryos thatallowed more leaching. At any rate, this leaching isnegligible because it was less than 1% of the ABAextracted from embryos (Figure 3 compared with Fig-ures 1 and 2). At this stage it was already evident thatthe original level of ABA in the endosperm of bothtypes of grains was very small. So, there was no pointin measuring ABA leakage from the endosperm too.

Another factor controlling seed germination isGAs. Dormant embryos germinated when GA3 wasadded to the Petri dishes (data not shown). The bio-synthetic pathway of GA1, which together with GA3is the major growth-active GA in cereals, begins withGA12-aldehyde and after several oxidative steps con-tinues as GA19 GA20 GA1 GA8 (Heddenand Croker 1992; Lenton et al. 1994). We identifiedand measured only the last two precursors of GA1,GA19 and GA20, as well as GA1 itself and its metab-olite GA8. The level of endogenous GA19 in non-dormant seeds was twofold higher than in dormantgrains (Figure 4A, time 0 h). However, this GA19 wasdepleted within the first 16 h of germination in non-dormant seeds (Figure 4A, time 16 h), being rapidlyconverted to GA20 (Figure 4B, time 16 h).

Thus, it appears that germinating seeds produce denovo GA19 which results in more GA20 (Figures4A,B). In contrast, dormant grains that do not germi-nate have lower original levels of GA19 which furtherdecline during the next 48 h, presumably because node novo GA19 is produced during this period of time(Figure 4A). The GA20 level in dormant grains wasreduced quickly during the first 16 h and then beganto increase at a very slow rate (Figure 4B). The rea-son might be that some biological activity during thefirst 16 h resulted in limited conversion of GA20 to

GA1 and to GA8. The later rise in GA20 is a result ofthe continuous reduction of GA19 (Figure 4A). Therewas essentially no change in GA1 levels of dormantgrains (Figure 5A) and therefore they could not ger-minate, while in non-dormant seeds the level of GA1began to increase immediately upon imbibition andkept increasing through 48 h (Figure 5A). Similar re-sults were presented by Kobayashi et al. (1995) ingerminating barley seeds. The fact that the level ofGA19 was high in non-dormant embryos dissected af-ter 16 h of imbibition (Figure 6A) implies that denovo synthesis of GA19 was occurring in these ger-minating seeds. No such increase was observed indormant grains. The relatively high level of GA20 innon-dormant embryos (Figure 6B) implies that theywere ready to germinate and with imbibition theGA20 was rapidly converted into GA1 (Figure 7A).As noted above, the reduction of GA19 and GA20 indormant grains and embryos after the imbibition wasnot reflected in subsequent production of GA1 (Fig-ures 4, 5, 6 and 7A).

In conclusion, two separate processes appear to beoccurring simultaneously to facilitate germination ofoat seeds. (1) The endogenous level of ABA stored inthe dry seeds is being reduced to a very low levelupon imbibition, and (2) stored GA19 has to be con-verted rapidly to GA20, and then to GA1. All theseconversions have to occur in this order within the first16 h of imbibition. Both (1) and (2) occur in non-dormant oat seeds or in isolated embryos of dormantgrains. During the first 16 h of imbibition ABA islowered and GA1 coincidentally accumulated, pre-sumably triggering germination. However, for dor-mant grains this did not happen, even after a week.ABA was not lowered sufficiently and GA1 did notaccumulate and therefore germination did not occur.

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