Planta (1983)159:366-372

P l a n t a 9 Springer-Verlag 1983

Nutritional control of storage-protein synthesis in developing grain of barley (Hordeum vulgare L.) S. Rahman, P.R. Shewry, B.G. Forde, M. Kreis and B.J. Miflin Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ, UK

Abstract. Sulphur starvation of barley results in decreased accumulation of the 'sulphur-rich' Bhordein polypeptides, with little or no effect on the 'sulphur-poor' C hordeins. The populations of mRNAs for C hordeins and the two major subfamilies of B-hordein polypeptides have been assessed by in-vitro translations in a wheat-germ extract and by ' d o t ' hybridisations to 32p-labelled B- and C-hordein complementary-DNA clones. The results show that the relative changes in the rates of accumulation of the three groups of polypeptides are correlated with similar changes in the abundances of their respective mRNAs. In addition, the deficiency of sulphur also appears to increase the efficiency of translation of C-hordein m R N A and to decrease the efficiency of translation of the B-hordein mRNAs. Thus the results indicate that there may be two components to the effect of sulphur deficiency on hordein accumulation, one acting at the level of transcription, or m R N A degradation, and one at the level of translation. Key words: Complementary D N A clones - Endosperm (hordein synthesis) - Hordein - Hordeum (protein synthesis) - m R N A - Protein synthesis (in vitro).

Introduction The storage proteins of seeds are a complex mixture of components which provide a store of nitrogen and sulphur for germination (Miflin and Shewry 1981). In accordance with this dual funcAbbreviations: cDNA=complementary DNA; PAGE=poly-

acrylamide-gel electrophoresis; Poly(A)+RNA=polyadenylated RNA; SDS = sodium dodecylsulphate

tion, the storage proteins of many species can be classified into at least two groups which differ in the relative amounts of the sulphur-containing amino acids, cysteine and methionine. For example, in legumes the 11S, or legumin-type proteins, form the sulphur-rich group and the 7S, or vicilintype proteins, the sulphur-poor group (Derbyshire et al. 1976). In wheat and rye, the c~-gliadins and e~-secalins respectively are sulphur-poor whereas the other major groups of storage proteins are sulphur-rich (Miflin et al. 1983 a). In barley, the B and C hordeins together account for about 95% of the total alcohol-soluble storage proteins (Shewry et al. 1983 b). The B hordeins contain about 2.5mol % cysteine and 0.6 tool % methionine whereas C hordeins contain no cysteine and only a trace of methionine (Shewry et al. 1980b). A third hordein group, D hordein, accounts for less than 5% of the total (Shewry et al. 1983b) and has an intermediate content of sulphur-amino acids (Field et al. 1982). Sodiumdodecylsulphate polyacrylamide-gel electrophoresis (SDS-PAGE) of hordein from the cultivar Sundance shows that B hordein consists of two major doublets of MrS 35,000 (called B1) and 46,000 (B3) with minor bands between them (B2). In the Chordein region there is a major group of bands of M r 59,000 (C1) and a minor band of M r 72,000 (C2). The D hordein is a single band of M r 105,000. Further resolution of the component polypeptides can be achieved by two-dimensional gel electrophoresis (Rahman et al. 1982). Genetic analysis shows that although B, C and D hordein are complex mixtures of components, they appear to be coded for by single structural loci, called Hor 2, Hor 1 and Hor 3 respectively, which are located on chromosome 5 (Shewry et al. 1980, 1983a; Jensen et al. 1980). It has been proposed that these

S. Rahman et al. : Storage-protein synthesis in barley loci consist o f m u l t i g e n e families. E v i d e n c e in supp o r t o f this has b e e n r e p o r t e d f o r H o r 2 ( F a u l k s et al. 1981 ; F o r d e et al. 1981 ; Kreis et al. 1983) w h i c h c o n t a i n s at least t w o sub-families o f genes t h a t c o d e f o r the B I a n d B3 p o l y p e p t i d e s respectively in the c u l t i v a r S u n d a n c e (Kreis et al. 1983). T h e relative p r o p o r t i o n s o f s u l p h u r - r i c h a n d s u l p h u r - p o o r s t o r a g e p r o t e i n s p r e s e n t in the seed o f a n u m b e r o f l e g u m e s a n d cereals c a n be g r e a t l y i n f l u e n c e d b y n i t r o g e n a n d s u l p h u r n u t r i t i o n (Blag r o v e et al. 1976; R a n d a l l et al. 1979; W r i g l e y et al. 1980). I n barley, increases in n i t r o g e n fertili s a t i o n cause d i s p r o p o r t i o n a t e increases in the a m o u n t o f C h o r d e i n ( A n d e r s e n a n d K o i e 1975; K i r k m a n et al. 1982) a n d it h a s b e e n s u g g e s t e d t h a t this is d u e to c h a n g e s in the p r o p o r t i o n s o f available n i t r o g e n a n d s u l p h u r in the p l a n t r a t h e r t h a n to the a b s o l u t e a m o u n t o f n i t r o g e n itself. I n s u p p o r t o f this, S h e w r y et al. ( 1 9 8 3 b ) s h o w e d t h a t s u l p h u r deficiency d r a m a t i c a l l y d e c r e a s e d the a m o u n t o f B h o r d e i n . C h a n g e s in the p r o p o r t i o n s o f h o r d e i n p o l y p e p t i d e s also o c c u r d u r i n g d e v e l o p m e n t o f the c u l t i v a r S u n d a n c e , n o t a b l y a d e c r e a s e in the p r o p o r t i o n o f C h o r d e i n in the early stages o f a c c u m u l a t i o n f o l l o w e d later b y a n increase in the p r o p o r t i o n o f B I h o r d e i n ( R a h m a n et al. 1982). H o w the relative rates o f a c c u m u l a t i o n o f the different h o r d e i n c o m p o n e n t s in the e n d o s p e r m are altered in r e s p o n s e to n u t r i e n t availability is n o t k n o w n . I n o r d e r to o b t a i n f u r t h e r i n f o r m a t i o n o n this a s p e c t o f the r e g u l a t i o n o f e x p r e s s i o n o f the H o r loci, we h a v e s t u d i e d the effects o f s u l p h u r stress o n the p o p u l a t i o n s o f h o r d e i n m R N A s prese n t in d e v e l o p i n g grain.

Materials and methods Plant growth. Seeds of Hordeum vulgare L. cv. Sundance were planted in washed vermiculite with six plants per 17-cm pot. The pots were placed in an unheated glasshouse during spring 1981 and watered daily with nutrient solution by a drip-feed irrigation system. The basic nutrient solution contained (in g dm -3) 0.522KC1, 0.136KH2PO4, 0.472 Ca-(NO3)2-4H20, 0.410 Mg(NOa)2.6H20, 0.006 NaC1, 0.438 CaC12, 0.277 MgSO4-7H20 , 0.018 FeNA EDTA (ethylenediaminetetraacetate) and 0.5 cm 3 dm a of a trace element solution containing (in g d m 3) 0.419 Zn acetate, 3.044 H3BO 3, 0.240 Cu(NO3) 2" 3H20, 4.940 Mn(NO3)z.4H20 and 2.023 ammonium molybdate. For the sulphur deficient treatment, the MgSO,.7H20 was omitted. Ears were checked for anthesis every 3 d and harvested 14 and 30 d after the date when 50% of the ears examined had anthesed for a given treatment. Grain was also harvested at maturity. Protein extraction. Hordein was extracted according to the method of Shewry et al. (1983b), the only difference being that the residue after polysome extraction was used to extract hordein from the low-sulphur sample after 14 d.

367 Hordein fractions were separated by SDS-PAGE by a modification of the method of Laemmli (Laemmli 1970; Forde et al. 1981). Quantification by scanning of gels stained with Coomassie Brilliant Blue R-250 was as described by Rahman et al. (1982). Isolation of polysomes and RNA. Membrane-bound polysomes were isolated as described by Matthews and Miflin (1980). Membrane-bound polysomal RNA (afterwards referred to as polysomal RNA) was obtained from membrane-bound polysomes by deproteinisation with phenol-chloroform following the method of Wienand and Feix (1978). Membrane-bound polysomal polyadenylated RNA (afterwards referred to as poly(A) +RNA) was prepared from polysomal RNA by chromatography using oligo-dT cellulose following the method of Bantle et al. (1976) as described in Matthews and Miflin (1980). Total RNA was prepared as described by Ralph and Bellamy (1964) using methoxyethanol to remove contaminants after the initial deproteinisation with phenol-chloroform. In-vitro translation. The RNAs were translated in a wheat-germ system supplemented with L-[4,5-3H]leucine (7.43 TBq/mmol) and L-[2,3,4,5-3H]proline (3.77 TBq/mmol) supplied by Amersham International, Amersham, Bucks., UK. The method of Roberts and Paterson (1973) was followed, except that the preincubation step was omitted. Incubations were treated as described in Forde et al. (1981) prior to separation by SDSPAGE. Gels were prepared for fluorography using salicylic acid (Chamberlain 1979) and exposed to preflashed film (Laskey and Mills 1975). The relative proportions of radioactive products were measured by scanning the fluorographs in an RIIC gel scanner (Beckman, High Wycombe, Bucks., UK) at 540 nm. Dot hybridisation. Dot hybridisation was performed following the method of Thomas (1980) using previously characterised (Forde et al. 1981) 32P-labelled complementary-DNA (cDNA) probes (Rigby et al. 1977). Nitrocellulose filters were washed in five changes of sterile distilled water for 1 h and then in one change of 20 x SSC (SSC : 150 mol m -3 NaC1, 15 tool m -3 trisodium citrate) for 2 h. Filters were then placed on a Hybridot apparatus (BRL UK Ltd., Cambridge, UK) and air dried untit no surface water was visible. The RNA samples were prepared by dilution into 0.2-cm 3 volumes and washed with three volumes of 20 x SSC. Filters were dried in vacuo for 2 h at 80~ C. Prehybridisation (4 h) and hybridisation (24 h) conditions were as described in Forde et al. (1981). At the end of hybridisation, the filters were washed for 4 h in 1 x SSC, 0.5% SDS, (500 cm3x 2). Filters were then dried and exposed to preflashed film in cassettes with intensifying screens. The intensities and areas of the spots were measured using a Quantimet Image Analyser Computer.

Results Hordein accumulation. H o r d e i n was extracted f r o m g r a i n f r o m e a c h o f the t w o s u l p h u r t r e a t m e n t s at three stages o f d e v e l o p m e n t . T h e s e stages w e r e ' e a r l y ' (the o n s e t o f h o r d e i n a c c u m u l a t i o n ) , ' l a t e ' (midway through hordein accumulation) and 'mat u r e ' (the fully d e v e l o p e d , d r y grain). T h e h o r d e i n f r a c t i o n s were s e p a r a t e d b y S D S - P A G E (Fig. 1 A) a n d the relative a m o u n t s o f the p o l y p e p t i d e s determ i n e d b y s c a n n i n g o f gels s t a i n e d w i t h C o o m a s s i e

368

S. Rahman et al. : Storage-protein synthesis in barley

Fig, 1A-H. Analysis by SDS-PAGE of hordein fractions and in-vitro translation products of RNA fractions from endosperms of sulphur-stressed and unstressed barley. A SDS-PAGE of hordein fractions. B-D Products of in-vitro translation of polysomes, polysomal poly(A) +RNA and total RNA, respectively. F~H Quantitative determinations of relative amounts of Cl, B3 and BI polypeptides in the hordein fractions, polysome products, poly(A) +RNA products and totalRNA products, respectively. Regions of histograms: white, C1; black, B3; hatched, B1. Results are the mean of duplicate scans of three gels (for E) or two autoradiographs (F-H). The SDs were about 3.0% of the mean values, a, Low-sulphur early; b, lowsulphur late; c, low-sulphur mature; d, high-sulphur early; e, high-sulphur late; f, high-sulphur mature material

Brilliant Blue R-250 (Fig. 1E). The relative amount of C hordein in the total hordein fraction decreased during development in both the sulphurstressed and unstressed grain. However, in the sulphur-stressed grain, C hordein accounted for 90%, 80% and 80% of the total hordein at the early, late and mature stages, respectively, compared with 40%, 30% and 20% in the unstressed grain. In the unstressed grain, the ratio of B1 and B3 hordein increased from 1:1 at the early stage to 1.8:1 at maturity. In the sulphur-stressed grain, however, this ratio remained at about 1 : 1 throughout development. Thus the sulphur deficiency has a more marked affect on the accumulation of B1 polypeptides than on B3 polypeptides. Extraction of polysomes and RNA. Membranebound polysomes and the p o l y ( A ) + R N A derived from them were extracted from the early and late stages of endosperm development. The yields per unit fresh weight are shown in Fig. 2. Sulphur stress led to a large reduction in the amount of extractable membrane-bound polysomes and poly(A) § RNA. This is consistent with the sharp reduction in the amount of protein in the mature grain. The amounts of polysomes and polysomal R N A present in the late stage were less than in

A

B

300

LL

6.o

u_

U3

200 ~.

c o


B3 > C. However, the amounts of B/and B3-hordein protein which are present per unit weight of the mature seed of stressed plants are only about 8% and 16%, respectively, of those in the controls (calculated from Fig. 1 E and the absolute amounts of hordein per g of the mature seeds as presented in Shewry et al. (1983 b)). There-

S. Rahman et al. : Storage-protein synthesis in barIey

fore, in the case of B-hordein mRNAs, translation would seem to be less efficient in sulphur-stressed plants than in the controls, presumably because the availability of methionine and cysteine is limiting. The less efficient translation of B-hordein mRNAs, and more efficient translation of C-hordein mRNAs, in stressed plants explains the discrepancy noted in Fig. 1 between the relative abundances of C, B3 and BI proteins in vivo compared with the products of in-vitro translation. Alterations in rates of initiation and elongation have been observed in mammalian cell cultures following changes in the nutritional status (Vaughan et al. 1971; Pain and Clemens 1973; Lodish 1976). We consider that these results indicate that there are two components to the effect of sulphur stress on the composition of the hordein fraction, one operating at the translational level and the other controlling the abundances of the respective mRNAs. There is, therefore, a possibility that the transcription of genes encoding the 'sulphur-rich' and 'sulphur-poor' storage proteins is differentially regulated by the availability of sulphur amino acids. However, the present results do not provide proof of such control mechanisms because the rates of turnover of the mRNAs and their translation products have not been studied. With regard to the hordeins, previous work (Shewry et al. 1979) has shown that they are not subject to turnover and the rate of accumulation can be assumed to reflect the rate of synthesis. Although it might be argued that sulphur-stress could change this, it seems unlikely that this would occur once the proteins had passed to the protein bodies. Measurement of transcription rates is technically difficult and has not yet been achieved for cereal endosperms. It is possible that these rates might changes as a result of sulphur stress since the lower efficiency of translation of B-hordein mRNA leaves a larger proportion of the mRNA unattached to ribosomes and more susceptible to ribonuclease attack in the cytoplasm. Chandler et al. (1983) have also recently shown that reduced synthesis of legumin in sulphur-stressed pea seeds is associated with reduced levels of legumin mRNAs, and suggested that this was a major regulatory effect of sulphur status. These results also show that sulphur stress must be added to the list of conditions under which there is differential regulation of the expression of the two sub-families of genes at the H ot 2 locus. Previously work has shown that the relative abundance of the B1 and B3 mRNAs (and the proteins coded by them) are differentially affected by allelic variation (Kreis et al. 1983), endosperm develop-

371

ment (Rahman et al. 1982) and a mutant 'regulatory' gene (Miflin et al. 1983b). We are grateful to Julian Franklin for growing the plants. The work was partly supported by European Economic Community grant No. 470. The senior author is indebted to Dr. J.G. Vaughan for advice and discussion.

References Andersen, A.J., Koie, B. (1975) N fertilization and yield response of high lysine and normal barley. Agron. J. 67, 695-698 Bantle, J.A. Maxwell, I.H., Han, W.E. (1976) Specificity of oligo (dT) cellulose chromatography in the isolation of polyadenylated RNA. Anal. Biochem. 72, 413 427 Blagrove, R.J., Gillespie, J.M., Randall, P.J. (1976) Effect of sulphur supply on the seed globulin composition of Lupinus angustifolius. Aust. J. Plan Physiol. 3, 173-184 Chamberlain, J.P. (1979) Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate. Anal. Biochem. 98, 132-135 Chandler, P.M., Higgins, T.J., Randall, P.J., Spencer, D. (1983) Regulation of tegumin levels in developing pea seeds under conditions of sulphur deficiency: rates of legumin synthesis and levels of legumin mRNA. Plant Physiol. 71, 4%54 Derbyshire, E., Wright, D.J., Boulter, D. (1976) Legumin and vicilin, storage proteins of legume seeds. Phytochemistry 1, 3-24 Faulks, A.J., Shewry, P.R., Miflin, B.J. (1981) The polymorphism and structural homology of storage proteins (hordein) coded by the Hor 2 locus in barley (Hordeum vulgare L.). Biochem. Genet. 19, 841-858 Field, J.M., Shewry, P.R., Miflin, B.J., March, J.F. (1982) The purification and characterization of homologous high molecular weight storage proteins from grain of wheat, rye and barley. Theor. Appl. Genet. 62, 329-336 Forde, B.G., Kreis, M., Bahramian, M.B., Matthews, J.A., Miflin, B.J., Thompson, R.D., Bartels, D., Flavell, R.B. (1981). Molecular cloning and analysis of cDNA sequences derived from poly A+RNA from barley endosperm. Identification of B hordein-related clones. Nucleic Acids Res. 9, 6689-6707 Jensen, J., Jorgensen, J.H., Jensen, H.P., Giese, H., Doll, H. (1980) Linkage of the hordein gene loci Hor I and Hor 2 with the powdery mildew resistance loci Ml-k and Ml-a on barley chromosome 5. Theor. Appl. Genet. 58, 27-31 Kirkman, M.A., Shewry, P.R., Miflin, B.J. (1982) The effect nitrogen nutrition on the lysine content and protein composition of barley seeds. J. Sci. Food Agric. 33, 115 127 Kreis, M., Rahman, S., Forde, B.G., Shewry, P.R., Miflin, B.J. (1983) Sub-families of hordein mRNA encoded at the Hor-2 locus of barley. Mol Gen Genet 191,201-206 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the heads of bacteriophage T4. Nature (London) 227, 680 685 Laskey, R.A., Mills, A.D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 Lodish, H.F. (1976) Translational control of protein synthesis. Annu. Rev. Biochem. 44, 39-72 Matthews, J.A., Miflin, B.J. (1980) In vitro synthesis of barley storage proteins. Planta 149, 262-268 Miflin, B.J., Shewry P.R. (1981) Seed storage proteins: genetics, synthesis, accumulation and protein quality. In: Nitrogen

372 and carbon metabolism, pp. 195-248, Bewley, J.D., ed. Martinus Nijhoff, The Hague Miflin, B.J., Field, J.M., Shewry, P.R. (1983a) Cereal storage proteins and their effects on technological properties. In: Seed proteins, pp. 255-319, Daussant, J., Mosse, J., Vaughan, J., eds. Academic Press, London Miflin, B.J., Rahman, S., Kreis, M., Forde, B.G., Blanco, L., Shewry, P.R. (1983 b) The hordeins of barley: developmentally and nutritionally regulated multigene family of storage proteins. In: Proc NATO/FEBS workshop on structure and function of plant genomes, Cifferi, O., Dure, L., eds. Plenum Publishing Inc., New York (in press) Pain, V.M., Clemens, M.J. (1973) The role of soluble protein factors in the translational control of protein synthesis in eukaryotic cells. FEBS Lett. 32, 205-212 Rahman, S., Shewry, P.R., Mifiin, B.J. (1982) Differential protein accumulation during barley grain development. J. Exp. Bot. 33, 717-728 Ralph, R.K., Bellamy, A.R. (1964) Isolation and purification of undegraded ribonucleic acids. Biochim. Biophys. Acta 87, 9-16 Randall, P.J., Thomson, J.A., Schroeder, H.E. (1979) Cotyledonary storage proteins in Pisum sativum. IV. Effects of sulfur, phosphorus, potassium and magnesium deficiencies. Aust. J. Plant Physiol. 6, 11-24 Rigby, P.W., Dieckmann, M., Rhodes, C., Berg, P. (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase 1. J. Mol. Biol. 113, 237-251 Roberts, B.E., Paterson, B.M. (1973) Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheatgerm. Proc. Natl. Acad. Sci. USA 70, 2330-2334 Shewry, P.R., Faulks, A.J., Picketing, R.A., Jones, I.T., Finch,

S. Rahman et al. : Storage-protein synthesis in barley R.A., Miflin, B.J. (1980a) The genetic analysis of barley storage proteins. Heredity 44, 383-389 Shewry, P.R., Field, J.M., Kirkman, M.A., Faulks, A.J., Miflin, B.J. (1980b) The extraction, solubility and characterization of two groups of barley storage polypeptides. J. Exp. Bot. 31,393-407 Shewry, P.R., Finch, R.A., Parmar, S., Franklin, J., Miflin, B.J. (1983a) Chromosomal location of Hor 3 a new locus governing storage proteins in barley. Heredity (in press ) Shewry, P.R., Franklin, J., Parmar, S., Smith, S., Miflin, B.J. (1983b) The effects of sulphur deficiency on the amino acid and protein composition of barley grain. J. Cereal Sci. 1, 21-31 Shewry, P.R., Pratt, H.M., Leggatt, M.M., Miflin, B.J. (1979) Protein metabolism in developing endosperms of high lysine and normal barley. Cereal Chem. 56, 110-117 Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA, 77, 5201-5205 Vaughan, M.H., Pawlowski, P.J., Forschhammer, J. (1971) Regulation of protein synthesis initiation in HeLa cells deprived of single essential amino acids. Proc. Natl. Acad. Sci. USA 68, 2057-2061 Wienand, U., Feix, G. (1978) Electrophoretic fractionation and translation in vitro of poly (rA) containing RNA from maize endosperm. Evidence for two mRNAs coding for zein protein. Eur. J. Biochem. 92, 605-611 Wrigley, C.W., DuCros, D.L., Archer, M.J., Downie, P.G. and Roxburgh, C.M. (1980) The sulphur content of endosperm proteins and its relevance to grain quality. Aust. J. Plant Physiol. 7, 755-766 Received 10 May; accepted 19 July 1983

Nutritional control of storage-protein synthesis in developing grain of barley (Hordeum vulgare L.).

Sulphur starvation of barley results in decreased accumulation of the "sulphur-rich" B-hordein polypeptides, with little or no effect on the 'sulphur-...
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