Reprintcd from Nature, Vol. 282, No. 5738, pp. 527-529. N ovcmber 29 1979

© Macmillan Joumals Ltd., 1979

N-terminal amino acid sequencing of prolamins from wheat and related species

Table 1

Species a nd geno me designations

Jean-Claude Autran*, Ellen J.-L. Lew, Charles C. Nimmo & Donald D. Kasarda Food Proteins Research Unit, Western Regional Resea rch Center, Agricultural Research, SEA, US D e pa nmc nt o r A griculture, Berkeley, California 94710

The gliadins of commun bread wheat (Tritic11111 aestiv11111 L.) make up the major storage protein fraction of gra in endosperm and comprise a complex mixture of proteins with similar amino acid compositions and properties 1 • Two-dimensional methods of electrophoresis2 •3 , in which separations are based mainly on net charge, separate gliadins of a single wheat variety into 30-46 components and there is considerable variation among the electrophoretic patterns of different wheat varieties 1 • The gliadins seem to be encoded by clusters of duplicated genes2 and may co nstitute a multigene famill. The fact that several purified gliadin components have closely similar N-terminal amino acid sequences5 •6 led us to consider the possibility that, despite the complexity of the mixture, gliadins have sutficient ho mology in their N-termina l sequences to allow estimation of the homology by sequencing the unfractionated mixture. Here, we provide evidence fo r two major groups of gliadins in wheat based on N-terminal seq uencing, show that similar groups of prolamins (a general tenn for equivalent proteins in other species) are found in related species of Tritic11111 and Aegilops that may ha\•e contributed genomes to polyploid wheats with A BD genome composition, and show thal only one of these groups is found in rye (Secale cereale L.). Wheat a nd o the r grain samples used a re listed in Table 1. T he prolam ins o f commo n wheat, Triric11111 111011ococcum, a nd rye were prepared from e ndosperm on ly (f'lour) whe reas those o f other sa mples, of which we had o nl y small a mo unts, were prepa red from d e-germed grain tha t was gro und in a Wiley mill. Prola mins were pre pa red acco rding to the procedure of Cha rbonnie r7 except that the final product was disso lved in 0.01 M acetic acid or 60 % c:thanol-wa ter a nd dial ysed thoroughly against dist illed wa te r. The small a mount of precipitate that formed (5 % or less o f the amount disso lved) was removed by centri fugation a nd the clear supern a ta nt was freeze-dried. Rye prolami ns were Jess so lu ble in water-about two -thirds of the preparatio n precipitated-and both the precipitate a nd· the supe rna ta nt were freeze-dried separately for use in sequencing. Yie lds of prolamins ranged from 3 to 8 % of starting m aterial. Electrophoretic patterns of typical preparatio ns, which ill ustra te the comp lexity of the mixtures, a re shown in Fig. l. Automatic sequencing8 was carried o ut o n a Beckman seque ncer mode! 8908 wit h DMAA pept ide p rogram 111 374 . E ight cycles we re conside red s utfi cie nt for our purposes. (One sample, T. monococcum, was seq uenced o nly through six cycles.) Residue iden tification was by gas chroma tography (GC) according to the procedure of Pisano et al.9 • In ail cases, only a few phenylthio hydanto in (PTH ) amino acids (some times o nly o ne) were ide ntified a t each cycle o f the Edma n degradatio n and recoveries were sufficien t to accoun t fo r 70-80% o f the protein sample when moisture conte nts (10 % ), probable seque nce r yie lds (65% assumed 10) and prola min molecul ar we ights (36,000 assumed 1) were taken into account. Typica l results a re given in Table 2. The major PTH a mino acids at each cycle usua lly co rresponded to the seque nce Va l-(Arg?)-Val-Pro-Val- Pro-GlnLe u- for ail species of Triticum and Aegilops except for the w heat variety 'Justin' and a n accession of Triticum urarru (G1876), w here it was the minor sequence. T his sequence cor• Permanent nddress: lnstitul Nn1iom1 J de ln Recherche Agronomique. Laborn toirc de Tech nologie des Blés Durs et du Ri z. 34060 Monlpcll ier. France.

Ratio of a-type to y-type seq ue nce from cycle 4

4

a / y Ra tio

T. aes1ivw 11 L. e m. The il., 'Scout 66. (ABD ) T. aes1ivw 11 L. e m. Theil ., ·Justin' (A BD ) T. dicoccoides Korn., 64-11 48 (AB ) T. dicocct1111 Schubl., Khapli emmer, 63-2356 (AB) T. boeoticum Boiss. e m. Schiem., G-2512 (A?) T. 111011ococcum L., Univ. Manitoba (A?) T. 11rart11 T um., G-315 1 (A?, B ?) T. 11rart11 Tum., G-1876 (A?, B?) A e. speltoides Ta usch, U. C., Davis (B?) A e. sqiw rrosa L., 0- 623 (D ) A e squarrosa L., 64-1154 (D) Secale cereale L., ·Frontier·

1.6 0.9 1. 1 1.3 2.2 1.3 1.1 0.7 1.4 1.9 1.9

responds to that of a -gliadins5 ·6 ; we shall refer toit as the a-type seque nce, altho ugh Bie tz et al. 6 fo und this seque nce also in a ,B -gli adin a nd a y-gliadin (y 1 -gliadin). Arg and His were no t de te rmined by our G C me thod. We assume Arg to be a n important a mi no acid at position 2 by a nalogy with the a -gliadin seque nce5 ·6 a nd identified it a t cycle 2 in two samples by liquid chroma tography. A second sequence correspondin g to Asn-(Me t/ lle)-GlnVal-(Val/ Asp)-Pro-Gln-Gly- was present in ail the samples of Triric11111 a nd A egilàps. This seq uence is si milar to the y 2 - a nd y 3 -gliadin seque nces described by Bie tz et al. 6 (Tabl e 2) ; we shall refe r to it as the y-type seq ue nce. It was the minor seque nce in most samples except fo r 'Justin · and T. urarru G-1876, w he re it was prese nt in amounts about equal to (or greater th an) the a mount of a -type seque nce. T. 11rar111 G-1876 , however, had o nly a trace amou nt of Asn at cycle 1; the major ami no acid was Val (40 nm) fo llowed by A la (17 nm). We usua ll y fo und both Me t a nd Ile at cycle 2, but our sa mple o f T. monococcum showed o nly Ile at cycle 2 whereas our sa mple of Aegifops spelroides showed on ly Met a t cycle 2. On the basis of N-terminal sequences, o ur res ults indicate two major ho mologous groups of pro lamins in the species of Triric11m and A egilops we examined. Because these species were fairly re presenta tive of the two genera, this may be true in general. Based on sequencer yie lds, we estimate tha t these two groups account for about 70-80% of o ur prolamin preparations. A fai rly clear difference between the a- type and y-type seque nces occurs at residue 4 , where Pro is found in the former a nd Val or Gin in the latter (Table 2). Pro a nd Val we re the only two ami no acids we ide ntified at cycle 4 fo r ail species of Triricum a nd A egi/ops with the exceptio n of one accession of T. urarru

(/ il b ~

c

J

d

g

w

" I

p

a 16

Fig. 1 Polyacryla mide gel e lectrophoresis of prolamins from ditfere nt species, alumin iu m lactate buffer, pH 3.2, migration from left ( + ) to righ t (-): a, T. 111011ococcu111: b, T. boeo1ic11111 (G-25 12);

c, T. 11rart11 (G-3 151 ): d, Ae. squarrosa (0-623 ): e, Ae. speltoides : (, T. dicoccoides (64- 11 48): g, T. aestiv11111 ("Scout 66"); li, S. cereale (" Frontier'). The o-, {3-, y- a nd w- regions correspond to the usua l assignments of electropho re tic mobilities fo r gliadin patte rns or co mmon wheats 1 •

--- -------

Table 2

Species

2

T. aestiv11m 'Scout 66 '

Val (97) Asn (37)

T. aestiv11111 'Justin ·

Val (55 ) Asn (51)

T. dicoccoides 64- 1148

Val (64) Asn (33)

A e. squarrosa 64- 1154

Val Asn Ala Asn Ala Leu Val Asn Pro

S. cereale 'Frontier· (soluble fra ction) a -gliadin 5 ' 6 6 Y2-gliadin

y 3 -gliadin6

Recovery of PTH amino acids for different species (nmol)

Asn Pro

(119) (26) (12) (59) (24) (20)

3

Cycle of Edman degradation 4 5 Pro (75) Val (69) Val (47) Asp (28)

6 Pro (91 )

Pro (53) Val (63)

Val (41 ) Asp (24)

Pro (76) Val (72)

7 Gin (71 ) Ser (50)

8 Leu (64) Gly (37)

Pro (104)

Ser (56) Gin (20)

Leu (46) Gly (42)

Val (68) Asp (16)

Pro (109)

Ser (70) Gin (25)

Leu (59) Gly (41)

Pro (94) Val (47)

Val (53) Asp (9)

Pro (99)

Gin (43)

Leu (39) Gly (10)

Gin (95) Leu (78)

Val (35) Asn (15 )

Pro (59) Ser (57)

Ser (73) Gin (14)

Gly (53) Gin (43)

Arg lie

Val Gly Gin

Pro Val

Pro Pro Gin

G in Trp

Leu Gly Leu

Met

Gly Gin

Val Gin

Pro (48) Gly (4 1) Asn (15 ) Val Asp Val Gin Asp Val

Pro Gin

Trp Gin

Gly

*

Met (40) Ile (22)

*

Met (42) Ile (40)

*

Met (33) Ile (29) Ile Met Met Gin

*(39) (17) (97 ) (87)

Val Gin Leu Val Gin Leu Val Gin Leu Phe Val Gin

(65) (54) (12) (50) (39) (32) (63) (29) (21) (5) (91) (25)

Recoveries are reported o n a basis of 10-mg samples. * Arg is likely to be an important residue at position 2, but Arg and His were not determined byour GC mcthod : in addition , the quantitation of Asn and Gin was lcss accurate than that of the other amino acids. (G- 1876), which also l~ad a small amount of Leu at th is positio n. The ratio of Pro to Val a t cycle 4 may prov ide a m easure of th e relative amounts o f th e two groups of prote ins in a sample. These ratios a re listed in T able 1, a nd ran ge fro m 0.8 to 2 .2. They some tim es differe d for diffe rent va rie ties or sa mples of th e same spccies. For example, the two hexaploid bread wheats, ·scout 66' a nd ·Justin ', had ratios of 1.6 a nd 0.8, respectively. The large amount of a- type sequence in ·Scout 66' is consiste nt with the large amounts o f a-gliadins in this variety 1 1• In co ntrast, 'Justin ' is low in a-gliadins 11 • The prese nce of both a -type and y -type prolamins in species of Tri1ic11111 and A egilops indicates the close re lat io nship of these genera, a nd suppo rts the proposa i 12 of Mo rris and Sears that A egilops be incorpora te d into the genus Tri1ic11111. Both the water-soluble a nd the wa ter-insoluble prolam ins from 'Frontie r· rye differed from those of the other species e xamined in lacking the a -type seque nce, a ltho ugh we might not have de tected a seque nce co rresponding to al:Jout 10% o r less o f the mixture. T he seque nces of these rye prolamin pre parations were no t ide nti cal to o ne ano ther but both were fairi y close to the y -type found in species of Tri1icu111 6 a nd Aegilops. Althoug h mino r ami no acids were identified a t most cycles of the rye prola min degrad a tio n, these did no t correspond to kno wn gliadin sequences. The e lectropho re tic patte rns of rye prolamins a t pH 3 vary some what according to varie ty 13 , b ut th ey do no t show a significa nt amount o f staining in th e region of mobility correspond ing to a-gli ad insD. As we h::ive no ted above , however, the a- type of seque nce has been fo und for proteins w ith o the r e lectrophoretic mobiliti es6 , so the agreeme nt between o ur seque ncing results a nd the e lectro pho re tic pa tte rns may not be essenti a l. The prese nce o f o nly the y-type seque nce in our sample of rye prola m in may indica te that rye is d oser evo lutio narily tha n Triricwn a nd A egi/ops to the commo n a ncesto r tha t gave rise to the sub-tribe Triticinae 1 ~, which includes bot h wheat a nd rye, within the family Gramineae. Diffe rentia tio n m ay have included the developm e nt a nd amplificatio n of genes coding for a- type prola mins in Triricum a nd A egilops. This specu la tion is based on res ults for only o ne va ri ety of rye; furth e r work on additional varieties a nd types is needed to a llow its ge nera lisa tio n to the species as a who le. The great homology among the mixtures o f prola mins we have s tudied e na bles considerable seque ncing information to be obta ined from the m witho ut iso lating single protein compone nts. This has a lso been demonstrated 1 ~ for the zein Pnmcd

111

pro te in s, the prola mi ns o f m aize (Zea mars). This homology is notable in co nsideration of the complexity of the mixtures. The prola mins of species of Tri1ic11111 a nd A egilops consist of man y compone nts that may ditfer in mo lecular weight as well as in charge ; prolam in pre pa ra tions from various species of these genera were resolved into at least four (usually more) bands by SOS electro pho resis (E. C. Cole, J . G. Fullin gton and D . D.K., unpublished results). Small differences in a mi no acid compositio n have been found for glia din components that differ in el ectrophoretic mobility (a luminium lactate buffer, pH 3.2) 1, and gliadin pro te ins co rresponding to particular bands of the pH 3.2 patte rns exhibited simple inhe ritance in crosses between w heat va rie ties t ha t differed in their electrophoretic patterns2 • Regardless of whethe r the complexity of glia dins a nd other prola min mixtures results fro m multiple genes, post-translatio nal modifications o r some other mecha nism, the high degree o f homo logy we found for their N-te rminal seque nces suggests that this region has some impo rtant fun ctio n and thus te nds to be co nserved. We than k B. L. Jo hnso n, J . G . Wa ines, P . Ma tte rn , V. A. J o hn son, C. O. Qua lset, J . D vorak, B. L. Jones, a nd R. Biete r fo r providing us with g ra in samples. J .-C.A. is a V isiting Scientist . Refe re nce to a company a nd/ or product na me by the D epartme nt is on ly for inform ation and does no t imply approval or recomme ndat ion to the exclusion of o thers that may also be suitable.

Rccc1vcd 2 Ju ly: accc p1 cd 20 Scp1c rnbcr 1979.

1. Knsn rda. D . D .. Bern ardin, J. E. & Nirnmo, C. C. in Adr.:nnuJ 1t1 C~real Srin•Ct." and 1fclinologr Vol. l (cd. Pomcrnnz, V.} 158-236 tAmcricnn A ssociai ion of Ccrcal Chcmim. SI Paul. Minncso10. 1976). 2. ~kchnm. O. K.. Kasardn. O. D. & Ouolsc1. C. O. Biorht111. Ge11c1. 16, 83 1 853 (1978). 3. Wn glc) . C. W. & Shcphcrd. K. W. A1111. N. Y. A rad. Sei. 209, 154- 162 (1973). 4. liood. L. Ftdn Proc. 35, 21 58- 2167 (1976). 5. Kasordn. O. O .• Da Rozo. O. A.. Ohms. J. I. Biocl1i111. hiopl1)'S. Arta 351. 290-29* (1974). 6. Bic1z. J . A .. Hucbnc r. F. R .. Snndcrson. J. E. & Wall. J. S. Certnl Cl1t111. 54, 1070-1083 11977). 7. Charbonnier. L. Cr. l1tbd. Séanc. A rad. Sei.. Ptiris 271 Ser. O. 20*2- 20*5 (1970). 8. Ed mnn. I'. & BcllJl. G . E11rJ. 8 1orlie111. 1, 80-91 11967). 9. Pisano. J. J .. Bronzc n . T. J. & Orewcr. H. B. Jr A11al)·1. Biodirm. 45. 43- 59 ( 1972). 10. Niull. H. in Tiie Pro1ti11s. Vol. Ill (cd. Ncuralh, H.) 179- 238 (Acndcmic. New York. 1977). 11. Plan. S. G .. Kasarda. O. O. & Ounlsc1. C. O . J. Sei. Fd A~ric. 25, 1555- 1561 (1974). 12. Morri§, R. & Sears. E. R. in \Vlrrat ami \Vlzrat lm provemt'm {cds Oui cnberry, K. S. & Rc ilZ. L. P.) 19- 87 (A mcrican Soc1CI) of Agronomy. Madison. Wisconsin. 1967). 13. Konorc,. V. G .. Ga,'filyuk. I. P.. Gubnrc\O. N. K.& Pcneva. T. I. Ctr