Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10515-10518, September 1996 Plant Biology

The dwarf-i (dl) mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway (biological activity/GC-MS identification/metabolism) CLIVE R. SPRAY*, MASATOMO KOBAYASHI*, YOSHIHITO SUZUKI*, BERNARD 0. PHINNEY*, PAUL GASKINt, AND JAKE MACMILLANt *Department of Biology, University of California, Los Angeles, CA 90095-1606; and tlnstitute of Arable Crops Research-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, BS18 9AF, United Kingdom Contributed by Bernard 0. Phinney, May 29, 1996

ABSTRACT In plants, gibberellin (GA)-responding mu- linear and there is no evidence for a metabolic grid with the tants have been used as tools to identify the genes that control other pathways, as has been shown for cell-free preparations specific steps in the GA-biosynthetic pathway. They have also obtained from seeds of bean, cucumber, and pea (for review, been used to determine which native GAs are activeper se, i.e., see ref. 3). further metabolism is not necessary for bioactivity. We The biological significance of the metabolic studies in maize present metabolic evidence that the Dl gene of maize (Zea comes from the use of GA mutants that exhibit a dwarf mays L.) controls the three biosynthetic steps: GA20 to GA1, phenotype, yet respond by normal growth to applied GAs. GA20 to GA5, and GA5 to GA3. We also present evidence that Thus, the relative responses of the mutants to specific GAs three gibberellins, GA1, GA5, and GA3, have per se activity in together with information on the role of the genes in control- stimulating shoot elongation in maize. The metabolic evidence ling specific steps in the pathway have led to the conclusion comes from the injection of [17-13C,3H]GA20 and [17- that only a limited number of GAs in the pathway are active 13C,3H] GA5 into seedlings ofdl and controls (normal and d5), per se, i.e., they do not require further metabolism to be followed by isolation and identification of the 13C-labeled bioactive (for reviews, see refs. 10-12). metabolites by full-scan GC-MS and Kovats retention index. The purpose of the present study was to examine the For the controls, GA20 was metabolized to GA1, GA3, and GA5; metabolic steps, GA20 to GA5, GA5 to GA3 and GA20 to GA1 GA5 was metabolized to GA3. For the dl mutant, GA20 was not in relation to the dl mutation (blockage after GA20). To this metabolized to GA1, GA3, or to GA5, and GA5 was not end, [17-13C,3H]GA2o and [17-13C,3H]GAs were fed to dl metabolized to GA3. The bioassay evidence is based on dosage seedlings and to controls (normal and d5 seedlings) and the response curves using dl seedlings for assay. GA1, GA3, and metabolites from the feeds were analyzed by full-scan GC-MS. GA5 had similar bioactivities, and they were 10-times more In addition, the bioactivities of GA20, GA1, GA5, and GA3 were active than GA20. determined using dl seedlings for assay. The gibberellins (GAs) are tetracarbocyclic diterpenes that occur naturally in higher plants (1). There is continued interest MATERIALS AND METHODS in the biosynthetic origin of the GAs since some of them are GAs. The [17-13C,3H]GA2o (0.915 atoms 13C per molecule, known to act as native regulators controlling a range of growth 1.79 GBq/mmol) was synthesized as described by Ingram et al. responses, including seed germination, floral development, (13) and purified as described by Kobayashi et al. (14). and shoot elongation (for reviews, see refs. 2 and 3). [17-13C,3H]GAs (0.915 atoms 13C per molecule, 1.51 GBq/ All GAs are biosynthesized from trans-geranylgeranyl mmol) was synthesized by Fujioka et al. (5) and purified before diphosphate (GGDP) via ent-copalyl diphosphate (CDP) and use by HPLC on a LiChrosorb C-18 column. Both compounds the tetracyclic hydrocarbon, ent-kaurene. ent-Kaurene is se- were radiochemically pure; no chemical impurities were de- quentially oxidized to ent-7a-hydroxykaurenoic acid, which is tected by GC-MS. then rearranged to GA12-aldehyde and oxidized to GA12. At The unlabeled GA1, GA3, GA5, and GA20, used for the least three pathways diverge from GA12-aldehyde and GA12: bioassays, were 99% pure as determined by GC-MS. No GA (i) the early-non-3,13-hydroxylation pathway, (ii) the early-3- contaminants were detected. For bioassays, each GA was hydroxylation pathway, and (iii) the early-13-hydroxylation dissolved in (Me)2CO/H20 (1:1, vol/vol). pathway. The early-non-3,13-hydroxylation pathway and early- Plant Material. The dl and dS maize mutants were seg- 3-hydroxylation pathway were initially shown to be present in regants from CC5/L317 hybrid seed stocks. The Dl gene the fungus, Gibberella fujikuroi, and later in higher plants (for controls a step(s) late in the pathway, whereas the D5 gene review, see ref. 4). The studies with higher plants also showed controls a step early in the pathway (5). For each stock, the presence of the early-13-hydroxylation pathway, which is seedlings segregated in the ratio of 3:1 normal/dwarf. Seeds unique to higher plants (for review, see ref. 3) and is the main were soaked in H20 for 24 hr and grown in vermiculite/soil pathway in vegetative shoots of Zea mays (maize). Our evi- (1:1) in the University of California (Los Angeles) greenhouse. dence for the presence of the early-13-hydroxylation pathway Ten-day-old seedlings (1-leaf stage) were used for bioassay; in maize was based on the GC-MS identification from vege- 3-week-old seedlings (4-leaf stage) were used for metabolic tative shoots of the 10 gibberellins (GA12, GA53, GA44, GA19, studies. GA17, GA20, GA29, GA1, GA5, and GA3) (5, 6) and the Bioassay Studies. The dl seedlings were treated by adding placement of these GAs in a series of metabolic steps by 50 ,ul of the GA solution into the axil of the first unfolding isotope labeling studies (7-9). Except for the terminal steps, leaf. Ten days later, the lengths of the first and second leaf the early-13-hydroxylation pathway in maize appears to be sheaths were measured and averaged to give the response data.

The publication costs of this article were defrayed in part by page charge Abbreviations: GA, gibberellin; AE, acidic ethyl acetate soluble; NB, payment. This article must therefore be hereby marked "advertisement" in n-butanol soluble; NBE, ethyl acetate soluble-hydrolyzed neutral accordance with 18 U.S.C. §1734 solely to indicate this fact. butanol; KRIs, Kovats retention indices.

10515 Downloaded by guest on September 23, 2021 10516 Plant Biology: Spray et aL Proc. Natl. Acad. Sci. USA 93 (1996)

Five dosage levels were used for each compound. Each assay 60°C and, after a 2 min isothermal hold, was programmed at point is the mean of measurements from 10 plants (standard 100 min-' to 150°C and then at 30 min-' to 300°C (OV-1) or errors are shown as vertical bars when greater than the point 280°C (OV-1701) with a 10-min isothermal hold at the end of size). the program. The pressure of the helium carrier gas was 100 Metabolic Studies. Each labeled substrate was dissolved in kPa. The column effluent was led directly to the ion source of EtOH/H20 (1:1, vol/vol) and 2 ,ul was injected into the basal a VG Analytical (Manchester, U.K.) 70-250 computerized part (about 1 cm below the ligule of the first leaf) of individual GC-MS with a source temperature of 220°C and an interface seedlings. Shoots were harvested 12 hr after treatment. temperature of 280°C. The ionizing potential was 24 eV and For [17-13C,3H]GA2o feeds, 10 each of dl, d5, and normal the mass spectra were recorded at 1 Hz. seedlings were injected with 4983 Bq per seedling. For [17- The metabolites (Tables 1 and 2) were identified by full-scan 13C,3H]GAs feeds, five seedlings of each genotype were in- GC-MS. A mixture of n-alkanes (C16-C36, ca. 1-15 ng of each) jected with 1800 Bq per seedling. was coinjected with each sample to provide a KRI (17) for each After harvest, the shoots were frozen in dry ice and homog- GC peak. enized with a mortar and pestle. The powdered plant material was added to a 10-fold excess (vol/wt) of MeOH/H20 (4:1, vol/vol) and stored at -20°C for 24 hr. The mixture was RESULTS filtered and the solid residue was extracted a second time with After treatment with the substrates [17-13C,3H]GA2o and a excess 10-fold (vol/wt) of MeOH/H20 (4:1, vol/vol). The [17-13C,3H]GAs, fractions were recovered from normal, dS, two aqueous methanolic extracts were combined and concen- and dl seedlings. The information from these fractions is trated in vacuo to give an aqueous residue (60 ml) that was shown in Tables 1 and 2. Each metabolite was tentatively solvent fractionated as described by Fujioka et al. (5) to give identified from its HPLC retention volume and conclusively the acidic ethyl acetate soluble (AE) fraction. The AE fraction established by comparing full-scan GC-MS and KRI data with was purified as described by Kobayashi et al. (15) by successive unlabeled reference spectra (16). Typical GC-MS data are use of Bond-Elut C18 and Bond-Elut DEA columns, followed shown in Table 3. by HPLC on a column of Nucleosil 5 C18, followed by Nucleosil 5 N(CH3)2. Radioactive fractions from the HPLC Metabolism of [17-13C,3H]GA2o (Table 1). For normal seed- 13C-labeled GA1, and were column were combined (Tables 1 and 2), based on radioactivity lings, GA3, GA5, GA8, GA29 and the retention times of known standards. Fractions from identified from the AE fraction and 13C-labeled GA1, GA8, the Nucleosil 5 N(CH3)2 column were analyzed by GC-MS. and GA29 were identified from the NBE fraction. From the d5 13C-labeled and were For the feeds of [17-13C,3H]GA2o only, the aqueous residues seedlings, GA1, GA3, GA5, GA29 iden- from the extractions of the AE fraction were processed as tified from the AE fraction and 13C-labeled GA1 and GA29 described (15) to give the n-butanol soluble (NB) fraction and from the NBE fraction. From the dl seedlings, 13C-labeled ethyl acetate soluble-hydrolyzed neutral butanol (NBE) frac- GA29 was the only metabolite identified from the AE and NBE tion. The NBE fraction was purified as described for the AE fractions; no GA1, GA3, or GA5 was detected using full-scan fraction. The selection of fractions for analysis was based on GC-MS at a detection limit of 50 pg. From all three genotypes, radioactivity and the retention times of known GA standards. [13C]GA2o was recovered in the AE fractions. Radioactive fractions from the Nucleosil 5 N(CH3)2 column Metabolism of [17-13C,3H]GA5 (Table 2). For normal anddS were methylated and trimethylsilylated for GC-MS as previ- seedlings, [13C]GA3 was identified in the expected HPLC ously described (5). Derivatized samples were chromato- fractions. For normal seedlings, the 13C content, determined graphed on a DANI-3800 gas chromotograph (DANI, Monza, by gas chromatography-single ion current monitoring on the Italy) using a vitreous silica wall-coated open tubular (WCOT) M+ ion cluster, was less than that in the applied substrate by column (25 m x 0.2 mm i.d.) coated (0.25 ,um) with OV-1 or dilution with endogenous GA3. For dl seedlings, no radioac- OV-1701. Samples were injected onto the column at 30°C in tivity was present in the expected HPLC fraction for [17- the Grob splitless mode and the injector purge was activated 13C,3H]GA3. Moreover, [17-13C,3H]GA3 was not detected in after 180 sec. The column temperature was taken rapidly to this fraction by full-scan GC-MS at a detection limit of 50 pg Table 1. Analysis of metabolites from feeds of [17-13C,3H]GA2o to normal, dl, and d5 maize seedlings

Seedling recovered C18-HPLC N(CH3)2- Radioactivity, (g fresh weight) Fraction radioactivity fraction HPLC fraction Bq Products* Normal AE 61.7 6-7 12 9 GA8 (27.0) 6-7 15-16 136 GA29 9-11 10-11 69 GA1 9-11 12 17 GA3, isoGA3 15-18 16-18 144 GA5, GA20 NBE 14.6 5-7 13-14 31 GA8 5-7 17-18 54 GA29 8-11 11-12 41 GAl dl AE 75.3 6-7 14-16 301 GA29 (10.8) 17-18 17-19 45 GA2o NBE 10.8 5-7 15 72 GA29 d5 AE 61.0 4-7 15-16 103 GA29 (10.4) 8-10 10-11 857 GAl 8-10 12 9 GA3 16-18 17-19 642 GA5, GA2o NBE 16.0 4-6 15 328 GA29 8-9 10 11 GA1 Typical identification data are shown in Table 3. *Identified by comparison with reference spectra and Kovats retention indices (KRIs) of unlabeled compounds (16). Downloaded by guest on September 23, 2021 Plant Biology: Spray et al. Proc. Natl. Acad. Sci. USA 93 (1996) 10517

0so dl E E cmJ 1201 *GA, + * GA3 *GAq .01 AGA2 OH

0

s0 0 A GA2H COZ HO' aH GA20 ) K GA3 f-J C 0 OH Of / N H ij 0.03 0.1 03 1 3 <~~~

Do ge (pag/pant) HO CGH H FIG. 1. Dose-response curves for GA1, GA3, GA5, and GA20 when GAI assayed on dl seedlings. Each point represents the average from measurements of 10 plants. Standard errors are shown as vertical bars FIG. 2. Major steps in the maize early-13-hydroxylation pathway when greater than the point size. subsequent to GA20, showing the three steps controlled by the DI gene. or by gas chromatography-single ion current monitoring at m/z 504 with a detection limit of 1 pg. information on the lack of metabolism of GA20 to GA5 and GA5 Bioassays. The dose-response curves of the dl mutant are to GA3 in the dl mutant leads to the reevaluation of the Dl gene shown in Fig. 2. GA1, GA3, and GA5 gave similar responses, in the control of GA biosynthesis in maize. We now conclude especially at the dosage levels of 0.1, 0.3, and 1.0 ,ug per plant, from the data that the Dl gene controls each of the three steps, respectively; GA20 showed less than 10% the activity of GA1, GA20 to GA1, GA20 to GA5, and GA5 to GA3 (Fig. 1). GA3, and GA5. The simplest explanation for the control of three steps by the DI gene is that the gene product is a multifunctional enzyme that catalyzes the 3J3-hydroxylation of GA20 to GA1, the DISCUSSION 2,3-dehydrogenation of GA20 to GA5, and 3,3-hydroxylation of We have previously shown that the dl mutant of maize blocks GA5 to GA3 with rearrangement of the double bond. All three the metabolism of GA20 to GA1, a late step in the GA- steps are mechanistically possible for a 2-oxoglutarate- biosynthetic pathway. The conclusion was based on bioassays dependent dioxygenase. There are precedents for the multi- (18), endogenous levels of GA20 and GA1 (5), and metabolic functionality of 2-oxoglutarate-dependent dioxygenases in GA studies with labeled substrates (7). In these early studies, no biosynthesis. Multifunctional GA 20-oxidases have been puri- evidence was obtained for the metabolism of GA20 to GA5. fied from cucumber endosperm (19) and cloned and expressed Using improved methods of purification and analysis, we from cucumber cotyledons (20) and from arabidopsis (21, 22). subsequently demonstrated the presence of endogenous GA5 Such multifunctionality is also illustrated by the GA 20-oxidase and GA3 in normal maize by full-scan GC-MS (6) and the from spinach that has been shown to catalyze each of the steps, metabolism of GA20 to GA5 and of GA5 to GA3 by gas GA53 to GA44 and GA19 to GA20 (23). Information on the basis chromatography-single ion current monitoring (9). The for the multifunctional control of the Dl gene will no doubt present results confirm our previous studies for the metabo- come from the cloning and expression of the maize Dl gene. lism of GA20 to GA1, GA20 to GA5, and GA5 to GA3 in normal There are dwarf mutants in plants other than maize that and d5 seedlings; in addition, no evidence could be obtained for block the conversion of GA20 to GA1, e.g., the le mutant of pea the metabolism of GA20 to GA1 in dl seedlings. The new (13), the ga4 mutant of arabidopsis (14, 24), and the dy mutant Table 2. Analysis of metabolites from feeds of [17-13C,3H]GAs to normal, dl, and d5 maize seedlings

Seedling recovered C18-HPLC N(CH3)2- Radioactivity, (g fresh wt) Fraction radioactivity fraction HPLC fraction Bq Products* Normal AE 85 9-10 12-13 667 GA3 (26.4) 11-13 14-15 10 16-17 16-18 1087 GA5 11-13 18-19 8 dl AE 88 9-10 11-13 0 (8.4) 11-13 14-15 13 16-17 16-19 2630 GA5 11-13 18-20 5 dS AE 78 9-10 12-13 41 GA3 (9.1) 11-13 14-16 9 16-17 16-19 2290 GA5 11-13 19-20 9 Typical identification data are shown in Table 3. *Identified by comparison with reference spectra and KRIs of unlabeled compounds (16). Downloaded by guest on September 23, 2021 10518 Plant Biology: Spray et al. Proc. Natl. Acad. Sci. USA 93 (1996) Table 3. [17-13C,3H]GA20 and [17-13C,3H]GAs metabolism: Representative KRI and GC-MS data for MeTMSi derivatives of 13C-metabolites and 12C-standards Compound KRI* Characteristic ions (m/z and relative intensities) GA1 (standard) 2669 m/z 193 207 235 313 375 376 377 416 448 491 506 Int. 8 23 6 9 9 14 12 5 18 9 100 [13C] GA, (metabolite) 2273 m/z 194 208 236 314 376 377 378 417 449 492 507 Int. 18 28 12 16 15 21 16 7 21 9 100 GA3 (standard) 2697 m/z 221 311 341 355 370 414 445 460 475 489 504 Int. 13 14 8 11 24 6 12 9 12 7 100 [13C] GA3 (metabolite) 2697 m/z 222 312 341 356 371 415 446 461 476 490 505 Int. 13 39 25 27 38 9 9 7 13 12 100 GA5 (standard) 2485 m/z 193 207 208 239 275 299 343 357 370 401 416 Int. 44 55 42 11 15 58 22 24 8 21 100 [13C] GA5 (metabolite) 2483 m/z 194 208 209 240 276 300 344 358 371 402 417 Int. 39 30 32 8 12 46 21 29 12 19 100 GA8 (standard) 2818 m/z 194 207 238 268 375 376 448 504 535 579 594 Int. 8 18 13 5 3 6 14 2 6 4 100 [13C] GA8 (metabolite) 2820 m/z 195 208 239 269 376 377 449 505 536 580 595 Int. 14 50 21 9 10 10 19 5 6 5 100 GA29 (standard) 2687 m/z 193 207 235 291 303 375 389 447 477 491 506 Int. 7 39 10 8 20 15 8 6 3 11 100 [13C] GA29 (metabolite) 2688 m/z 194 208 236 292 304 376 390 448 478 492 507 Int. 15 43 7 6 31 12 12 9 3 11 100 *KRI obtained using OV-1 GC column. of rice (14, 25). For each mutant, the position of the block was 5. Fujioka, S., Yamane, H., Spray, C. R., Gaskin, P., MacMillan, J., determined by bioassay and metabolic studies. However, it is Phinney, B. 0. & Takahashi, N. (1988) Plant Physiol. 88, 1367- unlikely that the three genes, Le, GA4, and Dy, have the same 1372. function as the Dl gene of maize, since normal seedlings of pea 6. Fujioka, S., Yamane, H., Spray, C. R., Katsumi, M., Phinney, (13), arabidopsis and B. O., Gaskin, P., MacMillan, J. & Takahashi, N. (1988) Proc. (14), rice (14) do not metabolize GA20 Natl. Acad. Sci. USA 85, 9031-9035. to GA5, and GA5 is metabolized to GA3 in normal and le 7. Spray, C., Phinney, B. O., Gaskin, P., Gilmour, S. J. & MacMil- seedlings of pea (26). In addition, no evidence has been lan, J. (1984) Planta 160, 464-468. published for the presence of endogenous GA5 and GA3 in 8. Kobayashi, M., Gaskin, P., Spray, C. R., Phinney, B. 0. & seedlings of pea, arabidopsis, and rice. MacMillan, J. (1996) Plant Physiol. 110, 413-418. Previously we had proposed that GA1 was the only endog- 9. Fujioka, S., Yamane, H., Spray, C. R., Phinney, B. O., Gaskin, P., enous GA with per se biological activity for GA-regulated MacMillan, J. & Takahashi, N. (1990) Plant Physiol. 94, 127-131. shoot elongation in maize (18). This conclusion was based on 10. Ross, J. J. (1994) Plant Growth Regul. 15, 193-206. early studies before GA3 and GA5 had been identified as native 11. MacMillan, J. & Phinney, B. 0. (1987) in Physiology of Cell in maize shoots. Since GA1, GA3, and GA5 have relatively high Extension During Plant Growth, eds. Cosgrove, D. J. & Knievel, with D. P. (Am. Soc. Plant Physiol., Rockville, MD), pp. 156-171. bioactivities, compared GA20, and each of the three steps 12. Reid, J. B. (1993) J. Plant Growth Regul. 12, 207-226. leading to GA1, GA5, and GA3 from GA20 are blocked, it is 13. Ingram, T. J., Reid, J. B., Murfet, I. C., Gaskin, P., Willis, C. L. concluded that each of the three GAs is active per se. (An & MacMillan, J. (1984) Planta 160, 455-463. interesting corollary of theper se activity of GA5 means that a 14. Kobayashi, M., Gaskin, P., Spray, C. R., Phinney, B. 0. & 3f3-hydroxyl group is not an absolute requirement for GA- MacMillan, J. (1994) Plant Physiol. 106, 1367-1372. activity, at least for stem elongation of maize.) 15. Kobayashi, M., Gaskin, P., Spray, C. R., Suzuki, Y., Phinney, While we find GA5, GA1, and GA3 to have similar activities in B. 0. & MacMillan, J. (1993) Plant Physiol. 102, 379-386. promoting the stem elongation of dl seedlings, Brian (27) re- 16. Gaskin, P. & MacMillan, J. (1991) GC-MS ofthe Gibberellins and ported relative activities of GA5 (9%), GA1 (33%), and GA3 Related Compounds: Methodology and a Library ofSpectra (Can- (100%). However, in a subsequent review (28), Brian et al. tocks Enterprises, Bristol, U.K.). 17. Gaskin, P., MacMillan, J., Firn, R. D. & Pryce, R. J. (1971) interpreted these data as showing that GA5, GA1, and GA3 were Phytochemistry 10, 1155-1157. all highly bioactive on dl seedlings. Thus, the significance of the 18. Phinney, B. 0. & Spray, C. R. (1982) in Plant Growth Substances relative activities of GA5, GA1 and GA3 is still an open question 1982, ed. Wareing, P. F. (Academic, London), pp. 101-110. that may only be resolved by isolation of the GA receptor and 19. Lange, T. (1995) Planta 195, 108-115. determination of the binding properties of the GAs. 20. Lange, T., Hedden, P. & Graebe, J. E. (1994) Proc. Natl. Acad. Sci. USA 91, 8552-8556. We gratefully acknowledge financial support from the National 21. Xu, Y., Li, L., Wu, K., Peeters, A. J. M., Gage, D. A. & Zeevaart, Science Foundation Grant MCB-9306597 and from the Department of J. A. D. (1995) Proc. Natl. Acad. Sci. USA 92, 6640-6644. Energy Grant DE-FG03-90ER20016 (B.O.P.). IACR receives grant- 22. Phillips, A. L., Ward, D. A., Uknes, S., Appleford, N. E. J., aided support from the Biotechnological and Biological Sciences Lange, T., Huttly, A. K., Gaskin, P., Graebe, J. E. & Hedden, P. Research Council of the United Kingdom. (1995) Plant Physiol. 108, 1049-1057. 23. Wu, K., Li, L., Gage, D. A. & Zeevaart, J. A. D. (1996) Plant 1. MacMillan, J. & Suter, P. J. (1958) Naturwissenschaften 45, Physiol. 110, 547-554. 46-47. 24. Talon, M., Koornneef, M. & Zeevaart, J. A. D. (1990) Proc. Natl. 2. Graebe, J. E. & Ropers, H. J. (1978) in Phytohormones and Acad. Sci. USA 87, 7983-7987. Related Compounds-A Comprehensive Treatise, eds. Letham, 25. Murakami, Y. (1968) Bot. Mag. (Tokyo) 81, 33-43. D. S., Goodwin, P. B. & Higgins, T. J. V. (Elsevier, Amsterdam), 26. Poole, A. T., Ross, J. J., Lawrence, N. L. & Reid, J. B. (1995) Vol. 1, pp. 107-204. Plant Growth Regul. 16, 257-262. 3. Graebe, J. E. (1987) Annu. Rev. Plant Physiol. 38, 419-465. 27. Brian, P. W. (1966) Intern. Rev. Cytol. 19, 229-266. 4. Hedden, P., MacMillan, J. & Phinney, B. 0. (1978) Annu. Rev. 28. Brian, P. W., Hemming, H. G. & Lowe, D. (1964) Ann. Bot. Plant. Physiol. 29, 149-192. 28, 369-389. Downloaded by guest on September 23, 2021