Effects of Gibberellins on Seed Germination of ...

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Experiments were carried out to explore the involvement of gibberellins (GAS) in the light-induced germination of Arabidopsis thaliana (L.) Heynh, using wild ...
Plant Cell Physiol. 36(7): 1205-121 1 (1995) JSPP 0 1995

Effects of Gibberellins on Seed Germination of Phytochrome-Deficient Mutants of A rabidopsis thaliana

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Young-Yell Yang Akira Nagatani 2, Yu-Ju Zhao Bong-Joong Kang Richard E. Kendrick and Yuji Kamiya

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Laboratory for Plant Hormone Function, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, 351-01 Japan Laboratory for Photoperception and Signal Transduction, Frontier Research Program, The Iristitute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, 351-01 Japan

Experiments were carried out to explore the involvement of gibberellins (GAS) in the light-induced germination of Arabidopsis thaliana (L.) Heynh, using wild type (WT) and phytochrome-deficient mutants (phyA, phyB and phyAphyB deficient in phytochrome A, B and A plus B, respectively). Seed germination of WT and phytochrome-deficient mutants was inhibited by uniconazole (an inhibitor of an early step in biosynthesis of GA, the oxidation of entkaurene) and prohexadione (an inhibitor of late steps, namely, 28- and 38-hydroxylation). This M GA,. The relative activity of inhibition was overcome by simultaneous application of GAS for promoting germination of uniconazole-treated seeds was GA4>GA, =GA9>GA,,. The wild type and the phyA and phyB mutants had an increased response to a red light pulse in the presence of GA,, GA,, GA9, GA,, and GA2, but there were no significant differences in activity of each GA between the mutants. Therefore, neither phytochrome A nor hytochrome B appears to regulate GA biosynthesis from GA,, to GA4 during seed germination, since the conversion of GA12to GA, is regulated by one enzyme (GA 20-oxidase). However, GA responsiveness appears to be regulated by phytochromes other than phytochromes A and B, since the phyAphyB double mutant retains the photoreversible increased response to GAS after a red light pulse. Key words: Arabidopsis thaliana - Germination

After breaking primary dormancy, seeds often require certain environmental conditions such as light, appropriate nutrients and a suitable temperature before germination takes place. Light is one of the most extensively studied seed-germination-stimulatory factors, but much still remains unknown about the steps between photoperception and germination. Since GA can replace the light requirement in several species, it has been proposed that phytochrome might act in one or more of the following ways: (i) by stimulating the synthesis of GAS; (ii) by increas-

- Gibberellins - Phytochrome.

ing the responsiveness to GAS within the seed; and (iii) by decreasing the level of an inhibitor of germination such as abscisic acid (ABA). In lettuce seeds, an increase in the level of GA9 was observed following exposure to light (Bianco and Bulard 1981). Recently, Toyomasu et al. (1994) found that red light (R) increased endogenous levels of GA,, which is active in the induction of seed germination in lettuce. However, it has been proposed in several species that light increases sensitivity to GAS (Taylorson and Hendricks 1976, Fredericq et al. 1983, Derkx et al. 1994). The germination of Arabidopsis seeds is under phytochrome-mediated photocontrol (Shropshire Jr. et al. Abbreviations: FR, far-red light; GAS, gibberellins; phyA, phytochrome A-deficient mutant; phyB, phytochrome B-deficient 1961). Phytochrome is encoded by a small multigene family mutant; prohexadione, 3,5-dioxo-4-propionylcyclohexanecarb- and at least five family members, called A, B, C, D and E oxylic acid; R, red light; uniconazole, (E)-1-(4-chloropheny1)-4,4- have been identified in Arabidopsis (Sharrock and Quail dimethyl-2-(l,2,4-triazol1-yl)-1-penten-3-01; WL, fluorescent 1989). Analysis of type-specific phytochrome-deficient muwhite light; WT, wild type. tants should reveal the functions of the different mem-

Y-Y. Yang et al.

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bers of this family. Shinomura et al. (1994) demonstrated that phytochrome B plays a principal role in seed germination. However, a promotive role for phytochrome A in germination has also been reported (Shinomura et al. 1994, Johnson et al. 1994). It is still not clear how the perception of light by phytochromes influences the biosynthesis of and/or responsiveness to GAS. We have investigated the structural requirements for GAS that can promote seed germination in Arabidopsis using endogenous GAS of Arabidopsis and two different inhibitors of the biosynthesis of GAS under different light conditions. We examined whether phytochrome A or phytochrome B might affect the responsiveness to and/or the biosynthesis of GAS during seed germination by using phyA and phyB mutants, as well as a phyA phyB double mutant.

Materials and Methods Plant materials-The alleles of Arabidopsis thaliana (L.) Heynh Landsberg erecta phytochrome null mutants used in the present study were phyA-201 (Nagatani et al. 1993, Reed et al. 1994), phyB-8-36 (Reed et al. 1993) and the corresponding double mutant (Reed et al. 1994). Seeds of the WT, the phyA, and phyB mutants and the phyAphyB double mutant were propagated in a temperaturecontrolled room (22 -+ 1"C) under continuous white fluorescent light (WL). Seeds of A. thaliana harvested in various seasons showed different responses to GASand light, which depend upon their level of dormancy (Derkx and Karssen 1994). To minimize this variation we propagated the seeds used in this study under controlled light and temperature conditions. After harvest, seeds were kept at 4+ 1°C in a plastic box with dry silica gel for at least two months to break primary dormancy. The proportion of seeds that germinate depends upon the batch of seeds, but we obtained consistent results as long as we used the same batch of

seeds during each experiment. Many factors influence seed germination, such as temperature and the concentration of nitrate in the germination medium. In our experiments we focused on the relationship between phytochromes and GAS. Chemicals-GA, and GA, were obtained from Kyowa Hakko Kogyo Co., (Tokyo, Japan). GA,, GA,, GA,, and GA2, were gifts from Prof. N. Murofushi (University of 1-(4-chloropheny1)Tokyo). Uniconazole-P, namely, (E)4,4-dimethyl-2-(1,2,4-triazol-l-yl)-l-penten-3-01 (S-3307D; optical purity, loo%), and prohexadione, namely, 3,5-di0x0-4-propionylcyclohexanecarboxylicacid (purity, 98%), were supplied by Sumitomo Chemical Co., Osaka, Japan, and Kumiai Chemical Industry Co., Tokyo, Japan, respectively. Germination tests and light treatment-Triplicate groups of 40 seeds were sown in 4.5-cm glass Petri dishes on a layer of filter paper and moistened with 1.2 ml of the test solution, and kept in darkness at 25°C until the light treatment. To determine germination frequencies, germinated and non-germinated seeds were counted 5 days after the start of the germination test. Protrusion of a radicle was taken as evidence of germination. Light sources-Red light (R) at 4.5 W mP2 was supplied from fluorescent tubes (FL20S.BRF; Toshiba, Tokyo) filtered through a red acrylic filter (Shinkolite A102; Mitsubishi Rayon, Tokyo). Far-red light (FR) at 3.5 W rn-, was supplied from far-red fluorescent tubes (FL20S.FR-74; Toshiba) and filtered through a far-red acrylic filter (Deraglass 102; Asahikasei). White light at about 4 W m-2 was from white fluorescent tubes (FL40SS. W37; National).

Results Responses to light-Table 1 shows the effects of light on germination of seeds of the WT and the phytochrome-

Table 1 Effect of light on percentage seed germination of the wild type (WT) and the phytochrome-deficient mutants of A. thaliana

White light

95k1

90+3

95+3

95k5

Darkness

36+ 1

40+4

51k1

2k1

3f1

2+1

2+1

4+2

R/FR a

" Seeds were imbibed in the dark in 1.2 ml of the test solution for 2 h, irradiated as indicated (R, red light (10 min); FR, far-red light (10 min)), and returned to the dark at 25OC for 5 d. All solutions contained 0.1% (v/v) ethanol. Results are the meansf SE for three independent determinations.

Gibberellin-induced germination in A. thaliana

deficient mutants. Under continuous WL, more than 90% of WT and mutant seeds germinated. A 10-min FR pulse completely inhibited germination. A 10-min pulse of R induced germination of the WT and thephyA mutant but not that of the phyB mutant or the phyAphyB double mutant. This result is consistent with the results of Shinomura et al. (1994). We used seed batches with these types of germination characteristics for further studies of the effects of GAS and light. Efects of inhibitors of the biosynthesis of GA and of diferent GAS on seed germination-Uniconazole and prohexadione inhibited germination of the seeds of the WT and the phytochrome-deficient mutants. The minimum concentrations of uniconazole and prohexadione for complete inhibition of germination in WL were M and M, respectively (Fig. 1). To minimize the side effects of these inhibitors, we used these concentrations for further experiments. The inhibition of germination by these inhibitors was overcome for the most part by application of lo-' M GA, (data not shown).

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Since GAI, GA4, GA9and GA, have been identified as endogenous GAS in A. thaliana (Talon et al. 1990, Derkx et al. 1994), their biological activities were compared. GA9 and GA,o are the immediate precursors of GA, and GA,, respectively (Graebe 1987). Uniconazole inhibits the oxidation of ent-kaurene (Izumi et al. 1985). We performed the experiments under R, FR and R/FR treatments to examine the relationship between phytochromes and responsiveness to GA. Figure 2 shows germination of uniconazole-treated seeds of the WT and the phyA mutant with different doses of GAS and the response

+R + FR

-

GAg

Concentration (M)

Fig. 1 Effects of uniconazole and prohexadione on seed germination in white light of wild type (WT) and phytochrome-deficient mutants of A. thaliana. The results are expressed as relative percentage germination by dividing the number of germinated seeds treated with inhibitors by the number of germinating seeds on the control plate. Seeds were imbibed in 1.2 ml of the test solution. All solutions contained 0.1% (v/v) ethanol. Values are means + SE for three determinations. Arrows indicate the concentration of GA biosynthesis inhibitors used for further experiments.

Concentration of GAS (M)

Fig. 2 Reversal of percentage seed germination inhibition by uniconazole with various doses of GAI, GA4, GA9 and GAm for wild type (WT) and thephyA mutant seed of A. thaliana after red (10 min after imbibition for 2 h in darkness) or far red (10 min after imbibition for 2 h in dark) irradiation. Seeds were imbibed in 1.2 ml of the test solution. Concentration of uniconazole was M. All solutions, including controls, contained 0.1% (v/v) ethanol. Values are meansf SE for three determinations.

Y-Y. Yang et al.

o

10-8 10-7

10-6

10-5

Concentration (M) Fig. 4 Reversal of percentage seed germination inhibition by uniconazole with various doses of GAz4for the wild type (WT), and the phyA and phyB mutants of A. thaliana after red (10 min Seeds were after imbibition for 2 h in darkness) irradiation. imbibed in 1.2 ml of the test solution. Concentration of M. All solutions, including controls, conuniconazole was tained 0.1 % (v/v) ethanol. Values are means SE for three determinations.

+

Concentration of GAS (M)

Fig. 3 Reversal of percentage seed germination inhibition by uniconazole with various doses of GAI, GA4, GA9 and GAm for the phyB and phyAphyB double mutants of A . thaliana after red (10 min after imbibition for 2 h in darkness) or far red (10 min Seeds were after imbibition for 2 h in darkness) irradiation. imbibed in 1.2 ml of the test solution. Concentration of uniconazole was lo-' M. All solutions, including controls, contained 0.1 % (v/v) ethanol. Values are means SE for three determinations.

+

to a pulse of either R or FR. The inhibition by uniconazole was overcome by GA,, GA4, GA9 and GA20. Figure 3 shows the results of a similar experiment with seeds of the phyB mutant and the phyAphyB double mutant. In all experiments GA, had the highest activity and GA20 the lowest, while GA, and GA, had intermediate activities. Table 2 shows the photoreversibility of the inhibition of germination of uniconazole-treated seeds of the WT and the phytochrome mutants by GA4and GA, at lop5M. Responsiveness of seeds to the GAS was increased by an R pulse, not only in the WT, but also in each of the phytochrome-

deficient mutants. Germination of seeds of the WT and thephyA mutant induced by loF5M GA9 after an R pulse were slightly higher than those of the phyB mutant and the phyAphyB double mutant. This difference suggests that 3j-hydroxylation of GA9 to yield GA4 might be regulated by phytochrome B. If this difference is significant, application of GA24, which is the immediate precursor of GA, should have the same effects. However, as shown in Fig. 4, GA24 promoted germination of seeds of the WT, and of the phyA and phyB mutants to an equal extent after an R pulse M. Another GA precursor, ent-kaurenoic acid at (lo-' M), failed to induce germination of seeds of the WT or any of the phytochrome-deficient mutants used. Prohexadione inhibits a later stage in the biosynthesis of GAS, namely, the 28- and 3j-hydroxylation of GAS (Nakayama et al. 1990). The inhibitory effects of prohexadione were overcome only by GA, and GA, (Table 3).

Discussion The role of plant hormones in regulating seed germination is often considered to reflect a balance between the germination-promotive effects of GA and the inhibitory effects of ABA (Bewley and Black 1985). The roles of GA and ABA in the germination of Arabidopsis seeds were studied by Koornneef et al. (1982) who used double mutants with defects in the biosynthesis of GA and ABA. Nambara et al. (1991) used both ABA-deficient and ABA-insensitive

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Gibberellin-induced germination in A. thaliana

Table 2 Reversibility by GA, and GA, of inhibition by uniconazole of seed germination of the wild type (WT) and the phytochrome-deficient mutants of A. thaliana R

A. thaliana

GA4

FR

GA9

GA,

R/FR

GA9

GA4

GA9

Seeds were imbibed in the dark in 1.2 ml of the test solution for 2 h, irradiated as indicated (R, red light (10 min); FR, far-red light (10 min)), and returned to the dark at 25OC for 5 d. Concentration of each GA and uniconazole was lo-' M. All solutions contained 0.1% (v/v) ethanol. Results are the means+SE for three independent determinations.

mutants, as well as uniconazole, and they concluded that GAS are not necessary for seed germination, if the rate of biosynthesis of ABA is reduced. However, most of the mutants used were probably leaky and it therefore remains possible that there is a prerequisite for GA to achieve germination. The light signal-transduction mutant, detl, which no longer requires light for germination, still requires GA for seed germination (Nambara et al. 1991). Shinomura et al. (1994) suggested that the phytochrome B-signalling pathway that controls seed germination might be independent of the pathway in which the DETl gene product functions. Derkx et al. (1994) identified GA,, G& and GA9as endogenous GAS in seeds of WT and a GA-insensitive (gar) mutant of Arabidopsis. They also found that the levels of GA, were low (below the limit of detection) in the darkness, but when transferred to continuous WL they increased, suggesting that light might regulate the biosynthesis of GA,. The levels of GA, were low and did not change after various light treatments. The most active GA in the stimulation of germination of seeds of the WT and the phyAphyB double mutant was GA, (Figs. 2, 3). The relative biological activities of GAS in uniconazole-treated seeds were GA, >GA, =GA, >GA,. It has been reported that the class of growth retardants to which it belongs

also effects sterol (Hedden 1988) and ABA biosynthesis (Zeevaart et al. 1988). Nambara et al. (1991) used uniconazole for studying the seed germination of ABA muM of uniconazole tants of Arabidopsis. They used and found strong resistance to uniconazole in the ABA mutants and the complete reversal of the inhibitory action by GA in the WT, suggesting that these side effects do not interfere with GA-induced germination in Arabidopsis at the concentrations used. The actual concentration of uniconazole in the seed, was probably much lower but, in any case, we used a 10-fold lower concentration than in their study to avoid these side effects. Azole-type growth retardants, such as uniconazole, inhibit the degradation of ABA, especially conversion of ABA to phaseic acid (Zeevaart et al. 1988). This effect induces the accumulation, not a decrease in level, of ABA. To reduce endogenous levels of active GA, we used another type of growth retardant, prohexadione which inhibits 2-oxoglutarate dependent oxidases, among which 21- and 31-hydroxylases are the most susceptible (Nakayama et al. 1990). Both GA, and GA,, failed to induce seed germination in prohexadione treated seeds, a result that suggests that GA9 and GA, are not active in seeds and that they induce seed germination after conversion to 31-hydroxylated GAS. This'hypothesis

Table 3 Reversal of percentage seed germination inhibition by prohexadione with various doses of GA,, GA,, GA, and GA, in white light for the wild type (WT) and the phytochrome-deficient mutants A. thaliana

Treatment

WT

Control Prohexadione Prohexadione GA, Prohexadione GA, Prohexadione GA, Prohexadione GA,,

+ + +

+

PhYA

P~YB

PhYAPhYB

95+3 2fl 50+3 95k3 10+3 2+1

Seeds were imbibed in 1.2 ml of the test solution. Concentration of each GA was M. Prohexadione was M. All solutions, including controls, contained 0.1 % (v/v) ethanol. Results are the means + SE for three independent determinations.

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is consistent with the result that 38-hydroxylated GAS are active in shoot elongation in Arabidopsis (Talon et al. 1990). The level of GA, in seeds was 4 to 6 times lower than that of GA, (Derkx et al. 1994). Since the apparent activity of GA4was higher than that of GA,, GA, appears to play a principal role in the GA-induced germination of seeds of A. thaliana. Since it has been proposed that 38-hydroxylation of GAS is regulated by phytochromes in shoots of peas (Campell and Bonner 1986) and cowpeas (Garcia-Martinez et al. 1987), we used GA,, GA4, GA9, GAXIand GA24 in our study. After an R pulse, seeds of thephyA mutant, unlike those of the phyB mutant germinated at high frequency. Therefore, Shinomura et al. (1994) suggested that induction of seed germination of A. haliana is regulated principally by phytochrome B. Germination of the WT and the phyA mutant induced by GA, after a R pulse was slightly higher than that of the phyB mutant and the phyAphyB double mutant. However, this difference was not significant when we used GA,,, which is the immediate precursor of GA,. These results suggest that metabolism of GA, to GA, is not regulated by either phytochrome A or B. In Arabidopsis (Phillips et al. 1994) and pumpkin (Lange et al. 1994), oxidation of C-20, namely, conversion of GA,, to GA,, is regulated by GA 20-oxidase. This enzyme is also a Zoxoglutarate dependent oxygenase. Also, the WT and the phytochrome-deficient mutants exhibited a similarly enhanced response to an R pulse in the presence of GA,. This result suggests that responsiveness to GA, is not regulated by either phytochrome A or B. We conclude that the responsiveness to GA and the conversion from GA12to GA, in Arabidopsis, is not regulated by phytochrome A or B. Possible roles for other members of the phytochrome gene family still exist and await further investigation. The reason for the low percentage of germination of seeds of thephyB mutant after an R pulse is unknown. One explanation is that phytochrome B might regulate an early step(s) in the biosynthesis of GAS during seed germination. We tested a precursor of early GA biosynthesis, entkaurenoic acid. However, it did not induce the germination of WT or any of the phytochrome-deficient mutants studied at lov5M suggesting that there was a problem with its uptake. We have recently cloned kaurene synthetase A from Arabidopsis (Sun and Kamiya 1994). Since the production of kaurene synthetase A is quite low in the WT, we could not show if light regulates kaurene synthetase A. Kaurene synthetase A is localized in chloroplasts and uses geranylgeranyl pyrophosphate as a substrate, which is also a substrate of carotenoid biosynthesis. Another explanation of the reduced germination in thephyB mutant is that phytochrome B would regulate the levels of ABA and thus the depth of dormancy of the seeds during development (Toyomasu et al. 1994). However, distinction between these possibilities will require analysis of levela of endoge-

nous GAS and ABA during the development and germination of seeds by gas chromatography/mass spectrometry. In conclusion, the WT and the phytochrome A-deficent and B-deficient single and double mutants examined here exhibited an elevated response to an R pulse in the presence of active GAS. This result suggests that an unknown R-absorbing photoreceptor, presumably a phytochrome that is neither phytochrome A nor phytochrome B, might regulate the responsiveness to GAS. We found no evidence for regulation of the conversion of GA,2 to GA, by either phytochrome A or B during germination. The authors thank Dr. S. Swain for his critical comments on the manuscript.

References Bewley, J .D. and Black, M. (1985) Seeds: Physiology of Development and Germination. Plenum Press, New York. Bianco, J. and Bulard, C. (1981) Influence of light treatment on gibberellin (G&, GA7 and GA9) content of Lactuca sativa L. cv. Grand Rapids achenes. 2.Pflanzenphysiol. 101:189-1 94. Campell, B.R. and Bonner, B.A. (1986) Evidence for phytochrome regulation of gibberellin Azo3/3-hydroxylationin shoots of dwarf (lele) Pisum sativum L. Plant Physiol. 82: 909-915. Derkx, M.P.M. and Karssen, C.M. (1994) Are seasonal dormancy patterns in Arabidopsis thaliana regulated by changes in seed sensitivity to light, nitrate and gibberellin. Anal. Bot. 73: 129136. Derkx, M.P.M., Vermeer, M. and Karssen, M. (1994) Gibberellins in seeds of Arabidopsis thaliana: biological activities, identification and effects of light and chilling on endogenous levels. Plant Growth Reg. 15: 223-234. Fredericq, H., Rethy, R., van Onckelen, H. and de Greef, J.A. (1983) Synergism between gibberellic acid and low Pfr levels inducing germination of Kalanchoe seeds. Physiol. Plant. 57: 402-406. Garcia-Martinez, J.L., Keith, B., Bonner, B.A., Stafford, A.E. and Rappaport, L. (1987) Phytochrome regulation of the response to exogenous gibberellins by epicotyls of Vigna sinensis. Plant Physiol. 85: 212-216. Graebe, J.E. (1987) Gibberellin biosynthesis and control. Annu. Rev. Plant Physiol. 38: 419-465. Hedden, P. (1988) The action of plant growth retardants at the biological level. In Plant Growth Substances. Edited by Pharis, R.P. and Rood, S. pp. 323-332. Springer-Verlag, New York. Izumi, K., Kamiya, Y., Sakurai, A., Oshio, H. and Takahashi, N. (1985) Studies of site of action of a new plant growth retardant (E)-1-(4-chloropheny1)-4,4-dimethyl-2-(1,2,4-triazol1-yl)-1-penten-3-01 (S-3307) and comparative effects of its stereoisomers in a cell-free system from Cucurbita maxima. Plant Cell Physiol. 26: 821-827. Johnson, E., Bradley, M., Harberd, N.P. and Whitelam, G.C. (1994) Photoresponses of light-grown phyA mutants of Arabi-

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throughout Arabidopsis development. Plant Cell 5: 147- 157. Sun, T.P. and Kamiya, Y. (1994) The Arabidopsis GA, locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis. Plant Cell: 6: 1509-1518. Sharrock, R.A. and Quail, P.H. (1989) Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory photoreceptor family. Genes Dev. 3: 1745-1757. Shinomura, T., Nagatani, A., Chory, J. and Furuya, M. (1994) The induction of seed germination in Arabidopsis thaliana is regulated principally by phytochrome B and secondarily by phytochrome A. Plant Physiol. 104: 363-37 1. Shropshire, W., Jr., Klein, W.H. and Elstad, V.B. (1961) Action spectra of phytomorphogenetic induction and photoinactivation of germination in Arabidopsis thaliana. Plant Cell Physiol. 2: 63-69. Talon, M., Koornneef, M. and Zeevaart, J.A.D. (1990) Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of semidwarf ga4 and ga5 mutants. Proc. Natl. Acad. Sci. USA 87: 7983-7987. Taylorson, R.B. and Hendricks, S.B. (1976) Interactions of phytochrome and exogenous gibberellic acid on germination of Larnium amplexicaule L. seeds. Planta 132: 65-70. Toyomasu, T., Yarnane, H., Murofushi, N. and Inoue, Y. (1994) Effects of exogenously applied gibberellin and red light on the endogenous levels of abscisic acid in photoblastic lettuce seeds. Plant Cell Physiol. 35: 127-129. Zeevaart, J.A.D., Gage, D.A. and Creelman, R.A. (1988) Recent studies of the metabolism of abscisic acid. In Plant Growth Substances. Edited by Pharis, R.P. and Rood, S. pp. 233-240. Springer-Verlag, New York. (Received February 13, 1995; Accepted July 11, 1995)