Gibberellins in Immature Seeds of the Gramineae - Chemotaxonomic considerations -

By YUTAKA MURAKAMI*

Department of Physiology and Genetics, Nationallnstitute of Agricultural Sciences (Yatabe, lbaraki, 305 Japan)

The Gramineae is a large family and is collected in the Kanto district from 1975 to recognized by its characteristic inflorescence. 1977 and listed in Table 1 with the date of A great deal of work on the family has been harvesting. Fifty g fresh weight of ears with done in various research ranges. In the field immature seeds were used for the extraction of biochemistry, synthesis of C.1-dicarboxylic of endogenous GAs. To check changes in the acids as the initial products of CO2 assimila­ quality of GAs during seed development, bar­ tion,2,12> sensitivity to the herbicide, isopropyl­ ley (HordeU1n vulgare cv. Ehimehadaka No. 1) N-phenyl carbamate1 > and the nature of re­ and rice (Oryza sativa cv. Ginbozu) serve carbohydrates,0,11> have been compared were cultivated in the artificial field of the within the Gramineae and discussed in re­ National Institute of Agricultural Sciences lation to systematics of the family. at Nishigahara in Tokyo. During the survey of endogenous GAs·•·lC· in higher plants, the present author0 > observed 2) Extraction and fractionation that GA activities were detected in extracts Extraction, fractionation, thin-layer chro­ of immature seeds of wheat, rye, and triticale matography, and bioassay were similar to by the Waito-C rice seedling assay. But no those reported in a previous paper.7 > clear GA activity could be found in those of Sample material was homogenized in 70% rice seeds, unless Tan-ginbozu rice seedlings aqueous acetone with a blender, kept for one were used as the assay .10 > Thus it was day at room temperature, and filtered. After of interest to examine whether endogenous the acetone was evaporated, the aqueous solu­ GAs of the immature seed of the Gramineae tion was adjusted to pH 2.5 with phosphoric could be related to any taxonomic scheme of acid and partitioned three times against ethyl the family. The survey was extended to many acetate. The combined ethyl acetate phase was of the Gramineae except the Bambu­ extracted thi'ee times with 1 M phosphate seae. buffer at pH 7. The buffer phase was adjusted to pH 2.5 with phosphoric acid and partitioned Materials and methods three times against ethyl acetate to give an acidic ethyl acetate fraction. After drying 1) Plant materials over anhydrous sodium sulfate, acidic ethyl The ears with immature seeds were taken acetate fraction was evaporated to dryness. from 100 species of the 71 genera. They were 3) Thin-layer chromatography * Present address: Department of Applied The evaporated extract was taken in a Physiology, National Institute of Agro­ small volume of acetone and subjected to TLC. biological Resources (Yatabe, Ibaraki, 305 Thin-layers of Silica gel H were used with Japan) the solvent system, isopropyl ether/ acetic acid ** Abbreviations: GA (s), gibberellin (s); GAn, gibberellin An; TLC, thin-layer (95 :5). The TLC plate was divided into 10 chromatography. equal zones between the starting line and the 242 JARQ Vol. 18, No. 4, 1985

Table 1. GA activities in the extracts from ears with immature seeds of the Gramineae

Subfamily, tribe, and species T-assaya) W-assayb) Date of harvesting Pharoideae Oryzeae Leersia sayanuka ++ 0 Sept. 26 Oryza sativa ++ 0 Sept. 2 Arundoideae Arundineae Arundo donax var. vesiculor + 0 Oct. 29 Cortaderia argentea + 0 Oct. 3 Moliniopsis japonica + 0 Sept. 2 P hragmites com1nunis + 0 Sept. 25 Arundinelleae Arundinella hirta + 0 Aug. 1 Glycerceae Glyceria ischyroneura ++++ 0 May 21 Phaenospermeae Diarrhena japonica ++ 0 Aug. 7 Pooideae Agrosteae Agrostis clavata var. nukabo +++ +++ May 14 A grostis palustris ++ + June 4 Alopecurus aequalis var. amurensis +++ ++ June 3 Anthoxanthum odoratum ++ 0 May 31 Arrhenatherum elatius +++ ++ May 31 Avena fatua ++ + May 29 Avena sativa ++ + June 3 B eck1n.annia syzigachne ++ 0 June 3 Calamagrostis arundinacea var. brachyt?-icha ++ 0 Sept. 7 Calamagrostis epigeios + 0 June 25 Calamagrostis pseudo-phragniites ++ 0 June 20 H olcus lanatus +++ +++ June 20 Lagurus ovatus ++ ++ May 26 P halaris aritndinacea +++ + May 29 P halaris canariensis +++ ++ May 17 PhleU1n pratense +++ +++ Aug. 24 Polypogon fugax + + ++ May 7 Trisetum bifidu1n ++ 0 May 21 Festuceae Brachypodiu1n sylvaticum +++ ++ July 22 Briza maa-ima ++ + May 25 Bromus catharticus + 0 May 29 Bro·nius japonicus ++ 0 June 4 Bromus remotiftorus + 0 Aug. 1 Cynosurus cristatus ++ ++ June 20 Dactylis glomerata +++ +++ Aug. 24 Festuca arundinacea +++ +++ June 4 Festu,ca elatior ++++ +++ June 4 Festuca myuros ++ ++ May 25 243

Table 1 (Continued) Subfamily, tribe, and species T-assaya) W-assayb) Date of harvesting ---- Lolium multi/forum +++ +++ July 14 Lolium teniulentuni +++ +++ May 19 Poa acroleuca ++++ ++ Apr. 29 Poa annua ++++ ++++ Oct. 26 Poa pratensis +++ ++ Apr. 21 Triticeae Agropyron ciliare ++ 0 June 3 Agropyron tsuksiense var. transiens ++ 0 May 29 Asperella longe-aristata +++ ++ June 20 Elymus mollis + + June 5 H orcleum, vulgare ++ + May 5 Secale cereale +++ + May 18 Triticum aestivum, ++ + May 5 Eragrostoideae Chlorideae Chloris gayana + 0 Sept. 12 Cleistogenes hackelii + 0 Sept. 14 Cynodon dactylon ++ 0 Aug. 9 Diplachne fuse a +++ ++ Aug. 25 Eleusine coracana +++ ++ July. 26 Eleusine indica ++ + Nov. 2 Eragrostis curvula ++ 0 June 1 Eragrostis poaeoides ++ 0 Aug. 13 Leptochloa chinensis ++ ++ Nov. 10 M ithlenb e1· gia curviaris tata + 0 Oct. 7 Mithlenbergia japonica + 0 Oct. 2 Spo1·obolus elongatus ++ + Sept. 15 Lappagineae Zoysia macrostachya ++ 0 Sept. 15 Zoysia japonica ++ 0 May 18 Isachneae Isachne globosa + 0 Aug. 10 Paniceae Digita1ici adscendens ++ 0 July 27 Echinochloa crus-galli ++ 0 July 29 Echinochloa critS-{Jalli var. frunientacea ++ 0 Aug. 6 Eriochloa villosa +++ 0 Aug. 4 Oplis1nenus unditlatif oliits ++ 0 Sept. 7 Panicu1n bisulcatum + 0 Sept. 14 Panicu1n maximu1n + 0 Sept. 12 Panicu1n miliaceuni ++ 0 Aug. 4 Paspalu1n dilatalwrn ++ + July 14 Paspalum distichum +++ ++ Sept. 26 Paspalum notatu1n +++ + Sept. 12 Pennisetum alopecu1·oides ++ 0 Sept. 30 Sacciolepis indica var. oryzetoru.m + 0 Sept. 28 Seta1ia chondrachne + 0 Oct. 4 Seta1ia itcilica ++ 0 Aug. 1 244 JARQ Vol. 18, No. 4, 1985 Table 1 (Continued) Subfamily, tribe, and species T-assaya) W-assayb) Date of harvesting Setaria viridis + 0 July 13 Andropogoneae A rthraxon hispi.dus + 0 Sept. 9 Cy1nbopogon tortilis var. goeringii + 0 Sept. 15 Eccoilopus cotulifer ++ 0 Aug. 24 H emarthria sibirica + 0 Aug. 29 lmperata cylindrica var. koenigii +++ 0 Apr. 15 I schaemum anthephoroides ++ 0 Sept. 15 Microstegiit1n japonicum ++ 0 Oct. 3 Microstegfa1n vimineum + + 0 Sep.t 26 Microstegium vimineum var. polystachyum + 0 Oct. 3 Miscanthus oligostachyus + 0 Sept. 9 Mi sccinthus sinensis ++ 0 Sept. 30 Phacelitrus latifolius ++ 0 June 25 Pseudopogonatherum quadrinerve + 0 Oct. 29 PseudopogonatherU1n speciosum + 0 Nov. 8 S01·gu111, bicolor + 0 Aug. 4 Sorgum halepense ++ 0 July 30 sibiricus ++ 0 Aug. 24 The1necla japonica + 0 Sept. 15 Maydeae Coix lacryma-jobi ++ 0 Aug. 9 Zea mays +++ 0 Aug. 19 --- - a) 'l'an-ginbozu rice seeding assay, C-3-hydroxy and C-3-deoxy GAs are detectable. b) Waito-C rice seedling assay, C-3-hydroxy GAs are detectable but not C-3-deoxy GAs. Relative GA activities: + + + + very high, + + + high, + + moderate, + low, O very low to inactive. solvent front (the first zone was further sub­ at 32°C under continuous light of about 5000 divided into two zones). The scraped Silica lux. After 3 days the length of the second gel of each zone was eluted with 50 % acetone, leaf sheath was measured with a ruler. The evaporated to dryness, and dissolved again in result was summarized as histograms, each 0.1 ml of 50 % acetone for bioassay. indicating the average of 5 test plants.

4) Bioassay Results and discussion Two dwarf rice, Tan-ginbozu and Waito-C, were used to assay GAs. Tan-ginbozu is known In the beginning, it was examined whether to respond to many kinds of GAs, while endogenous GAs of a species of the Grami­ Waito-C responds to limited kinds such as neae are always active or inactive to the C-3-hydroxy GAs. 8> Such specificity between elongation of Waito-C seedlings during its the two dwarfs was used to identify the GAs seed development. Forty ears of barley and found in the extracts. those of rice were harvested at their several When the second leaf emerged from the growth stages after anthesis for the extrac­ first leaf, eluates were applied as a single tion of GAs. 1 µl droplet to the surface of each coleoptile Fig. 1 shows results of barley ears. GA with a micropipette. Test plants were grown activities detected by the Tan-ginbozu assay 245

G/>q ...... GA3Sland . ( ng} 3 May 5 May 19 -1 -0.1 :f~f~ -o -1 -0-1 -0

.'.f~,f~U• May 9 May 24 _, ~ -0-o. 1 :t I~ I I I I I I I I I I t I I I I I I I I I I I -1 ~ -0-1-o 0 - :t I~ t I e t I f e I I I t I I I I I I I I I I I £. en May 14 Jun. 1 -1 ~ '.f -o-0.1

-1 W-assay -0-1 ~ -o :l~t ' I p e • I I I e ' I 0 RF 0-5 1.0 0 RF 0-5 1.0

Fig. 1. Histogl'ams showing GA activities of extracts taken from barley ears at different stages of r ipening The acidic ethyl acetate fractions were separated with TLC using isopropyl ether/acetic acid (95 : 5) as the developing solvent. Eleven eluates were tested on Tan-ginbozu rice seedlings (T-assay) or Waito-C rice seedlings (W-assay). This legend also applies to Fig·. 2-Fig·. 7. were low in the ears of May 5, 7 days after its ripening stages whether the ea1· extract is anthesis, and those of June 1 at the mature active or inactive on the Waito-C assay. stage. However, GAs were detected by Waito­ The occurrence of endogenous GAs in the C seedlings as well. Fig. 2 shows results of extracts from ears with immature seeds are rice ears. At any ripening stage GA activities summarized in Table 1. The system used by could not be found in the histograms when Tateoka13 > is employed here for classification Waito-C seedlings were used as the assay of the Gramineae. The table was reproduced plant, while clear GA activity was detected on the basis of the histograms showing GA by Tan-ginbozu rice seedlings. This activity activities, some of which are shown in Fig. 3 is due to GA rn as already reported. 6 ,1 0 > Thus to Fig. 7. it is a characteristic nature independent of The extracts of 2 species of the subfamily 246 JARQ Vol. 18, No. 4, 1985

dwarf rice seedlings. The histograms of 6 species of the Agrosteae and those of the Festuceae are shown in Figs. 3 and 4, re­ Sep. 2 ( 5 days alter anthesis) GA3Sland spectively. Every species in these figures has ( ng) -0.1 a clear and high GA activities by both rice : t_~~~_.___.__Ta~s:a~y...... -0 seedlings. This means that these species con­ tain higher concentrations of C-3-hydroxy -0.1 GAs such as GA 3 in immature seeds. As -o shown in Table 1, no C-3-hydroxy GA was :r~0 0.5 1.0 detected in 3 species of Calamagrostis, 3 species of Bromus, and 2 species of Agro­ Sep. 10 py1·on. All species surveyed in the genera -0.1 Ji'est1.wa, Lolium and Po

Agr<>Slis clavata var. nvkab<> Lagvrvs <>Va /vs

-1 2 -0.1 :fL::_ ~~ -o 1 - I I I 1 l . I I -1 -o-01 :t~:t~0 0.5 1.0 0 0.5 1.0 E u Arrhenalhervm elativs Phalaris ,anariensis .c.· "' - 1 ~2~~ -s.1 -:0 1 l I I • I ~ - 1 W-assay ,:, [ W-assay n -0.1 ~-~21 d-~ -o 0 o.'s o · o's 1'.o 0 Phlevm pra ltn~ Holws /anatvs .t:. r:f~'.f~ - 1 1 - 1 - I I I - 1 ~ n W-assay 2 n_- - - - WW--asassasayy - • :l~-~ lt!--~ -o-0.1 0 RF 0.5 1.0 0 RF 0.5 1.0

Fig. 3. Histograms showing GA activities of extracts taken f1:om ears with immature seeds of the tribe Agrosteae

those of the Andropogoneae are shown in 3 species of the genus Paspalum,, P. dilatalum, ( Fig. 6 and Fig. 7. respectively. The histograms P. distichu1n, and .P. notata1n, contain C-3- of the Andropogoneae in Fig. 7 show that the hydroxy GAs, which are active on both dwarf GA activity appeared at the R,,. value corre­ rice seedlings. From geographical distribu­ sponding to GArn, tion, the Panicoideae is a group of grasses usu­ The Gramineae is subdivided into two ally found in the tropical and warm regions. contrasting subfamilies, the Festucoideae Non-C-3-hydroxylated GA such as GAio is the (Pooideae) and Panicoideae on the basis of major GA of this group. The Pooideae defined inflorescence characteristics."> In this taxo­ by Tateoka is a group of grasses in cool and nomic system, the Panicoideae contains the temperate regions. Most of the members of tribes, Arundinelleae. Isachneae, Paniceae, this group contain C-3-hydroxy GA which is Andropogoneae, and Maydeae, defined by Tate­ active in both Tan-ginbozu and Waito-C seed­ oka. In the Panicoideae of this system, only lings. The present study indicates that there 248 JARQ Vol. 18, No. 4, 1985

Brachypodium sylvaticum Festuca arundinacea

-1 -0,1 -o

"' Cynosurus cristatus -2 .c"' - 1 "' 2 t T- assay :f'--'-~--Lo-l iu_m_m.._ul-t1-- ·t1-or_:_m_,_-~ ~ -0.1-o t,~~~~~ -2 -1 ,:, ~21[~ -o-0,1 - 0I I 05' 1~' '.[~0 05 1~ 0 Oacty/is glomerata Poa pratensis

-1 -0.1 - 0

-'-0,1 -o

Fig. 4. Histogt·ams showing GA activities of extracts taken from ears with immature seeds of the tribe Festuceae

is a difference of metabolic pathway of GA species belonging to 26 genera. This is due between the Pooideae and the Panicoideae. to the fact that Tan-ginbozu seedlings re­ spond to many kinds of GAs, while Waito-C Conclusion seedlings respond to limited kinds such as C-3-hydroxy GAs. In conclusion, most of the Endogenous GAs in immature seeds of 100 members of the Pooideae contain GAs hy­ species belonging to 71 genera of the Grami­ droxylated at C-3 in immature seeds. How­ neae were studied by techniques based on TLC ever major GAs of immature seeds of the and bioassay on dwarf rice. GAs were de­ Panicoideae are not hydroxylated at C-3. tected in all species by using Tan-ginbozu rice seedlings. When another dwarf variety, Wait?-C was useµ, GAs were detected in 37 249

CynodM dac/ylon Eragrostis ~oidt'S -1 2 T· •ss•y r I -assay ~2~=8·1 g f r- I I • 1 _ I I I 2 r w.... . y ~-1~ 2[~=8·1 I - I I f O 0.5 1.0 . 0 0.5 1.0 "' -3 .. Oip/achM fvsca Ltpto<:h/oa ch;nt>nsis .. -1 -0.1 'i! ~ ~ -o NI :r ~ =81 I :r I I I

Fig. 5. Histograms showing GA activities of extracts taken from ears with immature seeds of the tribe Chlo­ rideae

GA3St•l1d. Echinochloa crus -gal/i Piispalum no/,1/um Ing) ...... 1

Erioch/0,1 vi1/os a PtMisttum alop«uroi'dts -1

t~ I I ::t~C: :t "'2[ -- w.aSS,l)' =81 0 1 - I ~(~ 0er::::::::: 0.5 =1.0 0 0.5 1.0

£ Panicum miliact um Sttari~ it-allca - 1 r'.t~·., ~t~. -o--,-0.1 -0.1 : ._[~"-----,,'~W~·-••_•_•y--:' :[ - o 0u:=::= RF 0.5 ~ ==1.0

Fig. 6. Histograms showing GA activities of extracts taken from ears with immature seeds of the tribe Paniceae 250 JARQ Vol. 18, No. 4, 1985

Eccoilopvs cotvllftr Phact/11r11s /atlfo/ivs -1 ~r~ -o- 0. 1 2[~ :r ~~w_-·_~_•r~- -o-0. 1 1 - ' I 1 E O 0~ LO 0 0.5 10

~ lmp,,rala cylindric~ var. ko'tnigii Sorghvm bicolor

"' T-usay ~ T-ass•t ~3f~- 2 2 -0.1 ~1 ~ 1cc3:i-·c== - o w. .uuy w• .,,ni,y =SI ·]: [ ~ :r c..rc-:. --r. -: 0 0 0.5 1.0 0 0.5 1.0

.c: llchatmum anthephoroides Spodiopogon sibiricvs a, -I C [ - -o-0.1 ~: ~ :r I~ w-~uwy W-ass,y ~------; -0- o.1 ~ r :[ ~ 0 RF 0.5 1.0 ORF 0.5 1.0

Fig. 7. Histograms showing GA activities of extracts taken from ea1·s with immature seeds of the tribe Andro­ pogoneae

6) Murakami, T.: Studies on comparative physi­ ology of sugars, oligosaccharides and acid References soluble nucleotides in the Gramineae. Bull. Na.t. Inst. Agr. Sci. Series D, 26, 223-262 1) Al-Aish, M. & Brown, W. V.: Grass germi­ (1975) [In Japanese with English sum­ nation responses to isopropyl-phenyl carba­ mary). mate and classification. Amer. J. Botany, 7) Murakami, Y.: New rice seedling test for 45, 16-23 (1958). gibberellins- Microdrop method. J ARQ, 5 (2), 2) Downton, W. J. S. & Tregunna, E. B.: Car­ 5-9 (1970) . bon dioxide compensation-its relation to 8) Murakami, Y.: Dwarfing genes in rice and photo-synthetic carboxylation reactions, sys­ their relation to gibberellin biosynthesis. In tematics of the Gramineae and leaf anatomy. Plant growth substances 1970. ed. Carr, Can. J. Botany, 46, 207-215 (1968). D. J., Springer, Berlin, Heidelberger, New 3) Hitchcock, A. S.: Manual of the grasses of York, 166- 174 (1972). the United States. Second edition revised by 9) Murakami, Y.: Comparison of gibberellin in Agnes Chase. Vol. 1. Dover Publications, developing ears of triticale, wheat and rye. New York, 13-26 ( 1971) . Chem. Reuulcition of Plants, 15, 90-99 ( 1980) 4) Jones, D. F., MacMillan, J. & Radley, M.: [In Japanese). Plant hormones. III. Identification of gib­ 10) Murakami, Y.: Endogenous gibberellins in berellic acid in immature barley and imma­ the panicle of rice plants determined by the ture grass. Phytochem., 2, 307-314 (1963). rice seedling bioassay. JARQ, 17, 149-153 5) Kurogochi, S. et al. : Identification of gib­ (1983). berellins in the rice plant and quantitative 11) Ojima, K. & Isawa, T.: Physiological studies changes of gibberellin A,o throughout its life on carbohydrates of forage plants. II. Char­ cycle. Plantci, 146, 185-191 (1979). acteristics of carbohydrate compositions ac- 251

cording to species of plants. J. Jpn. G1·assl. the Gramineae. P1·oc. Crop. Sci. Soc. Jpn., Sci., 13, 39-50 (1967) [In Japanese with Eng­ 40, 12-20 (1971) [In Japanese with English lish summary]. summary]. 12) Takeda, T. & Fukuyama, M.: Studies on the 13) Tateoka, T.: The explanation of the Grami­ photosynthesis of the Gramineae. I. Differ­ neae. Meibundo, Tokyo, 89-109 (1959) [In ences in photosynthesis among subfamilies Japanese]. and theil' relations with the systematics of (Received for publication, September 26, 1984)