Plant Physiol. (1992) 99, 263-2680032-0889/92/99/0263/06/$01 .00/0

Received for publication August 29, 1991Accepted December 10, 1991

Effects of Sulfur Nutrition on Expression of the SoybeanSeed Storage Protein Genes in Transgenic Petunia'

Toru Fujiwara*, Masami Yokota Hirai, Mitsuo Chino, Yoshibumi Komeda, and Satoshi NaitoDepartment of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113,Japan (T.F., M. Y.H., M.C.); and Molecular Genetics Research Laboratory, University of Tokyo, Hongo, Bunkyo-ku,

Tokyo 113, Japan (Y.K., S.N.)

ABSTRACT

The 7S seed storage protein (,6-conglycinin) of soybean (Gly-cine max [L]. Merr.) has three major subunits; a, a', and ft.Accumulation of the a-subunit, but not the a- and a'-subunits,has been shown to be repressed by exogenously applied methi-onine to the immature cotyledon culture system (LP Holowach,JF Thompson, JT Madison [1984] Plant Physiol 74: 576-583) andto be enhanced under sulfate deficiency in soybean plants (KRGayler, GE Sykes [1985] Plant Physiol 78: 582-585). Transgenicpetunia (Petunia hybrida) harboring either the a'- or j#-subunitgene were constructed to test whether the pattems of differentialexpression were retained in petunia. Petunia regulates thesegenes in a similar way as soybean in response to sulfur nutritionalstimuli, i.e. (a) expression of the ,-subunit gene is repressed byexogenous methionine in in vitro cultured seeds, whereas the a'-subunit gene expression is not affected; and (b) accumulation ofthe a-subunit is enhanced by sulfur deficiency. The pattem ofaccumulation of major seed storage protein of petunia was notaffected by these treatments. These results indicate that thismechanism of gene regulation in response to sulfur nutrition isconserved in petunia even though it is not used to regulate itsown major seed storage proteins.

Soybean is one of the major protein sources for human andagriculturally important animals. However, the amino acidcomposition of seed protein is not ideal because of its poorcontent of sulfur-containing amino acids. Soybean seeds con-tain two major seed storage protein components, namely, I lSprotein (glycinin) and 7S protein (,B-conglycinin). Glycinin,which consists of approximately 50% of total seed protein, isrelatively rich in sulfur-containing amino acids (1.8%). ,B-Conglycinin, the second abundant protein, is relatively poorin these amino acids (0.6%) (see ref. 28 for review). f3-Congly-cinin forms homo- or heterotrimers ofthree subunits, namely,a' (76 kD), a (72 kD), and /3 (53 kD). Among these, the ,B-subunit has an especially low sulfur content (10). By increas-ing the content of sulfur rich protein and decreasing the levelsof sulfur-poor proteins, one can improve the nutritional qual-ity of soybean.

' This work was partly supported by Grant-in-Aid for ScientificResearch on Priority Areas from the Ministry of Education, Science,and Culture ofJapan to M.C. (02261101), Y.K. (02261103), and S.N.(01622001 and 03262101).

Expression of the f3-conglycinin genes has been studiedextensively. All three subunit genes of the f3-conglycinin areexpressed specifically in middle to late stages of soybean seeddevelopment, although expression of each subunit gene isregulated differently in terms of timing and tissue specificityof expression (16, 22, 24, 25). The levels of expression arealso different. Copy numbers of the a'- and ,B-subunit genesare estimated to be at least three (18) and eight to 13 (30) perhaploid genome, respectively, whereas the levels of each pro-tein accumulated in soybean seeds are approximately thesame (16), indicating that the a'-subunit gene has a higherexpression level than the ,3-subunit gene per gene copy. Whenthese genes were introduced into transgenic tobacco or petu-nia plants, the pattern of expression was found to be similarto that in soybean plants in terms of the timing (5, 8, 9, 25),levels of accumulation (25), and tissue specificity (2, 5, 8, 9).These facts indicate that tobacco, petunia, and soybean havea common mechanism of seed-specific gene regulation.These genes are also known to respond differently to nutri-

tional stimuli. Holowach et al. (21) reported that, in their invitro culture system of immature soybean cotyledons, accu-mulation of the ,B-subunit protein was completely repressedwhen cotyledons were cultured in the presence of methionine,whereas the accumulation ofthe a- and a'-subunits were littleaffected. The suppression of the ,B-subunit accumulation isregulated at the mRNA level (11) and the repression is re-versible, i.e. accumulation ofmRNA coding for the ,l-subunitreinitiates after removal ofmethionine from the medium (20).Sulfur or potassium deficiency also affects the seed storageprotein composition of soybean (17). Under sulfur deficiency,more ,B-conglycinin accumulates than glycinins. Subunit com-position of /3-conglycinin is also affected, and there is athreefold increase in the accumulation of the ,8-subunit pro-tein, whereas the other two subunits are little affected.To understand the molecular mechanism(s) of nutritional

regulation of soybean seed storage protein gene expression,we attempted to build a system in which artificially introduced,3-conglycinin genes would respond to nutritional stimuli in asimilar way as in soybean plants. Transient expression afterdirect gene transfer and stable transformation could be usedto accomplish this purpose. We chose to build transgenicpetunia harboring f3-conglycinin genes using Agrobacterium-mediated gene transfer because it has been shown that the f3-conglycinin genes were expressed when introduced into pro-toplasts from various sources of nonembryonic origin and didnot show methionine or ABA responses (15). In this paper,

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Plant Physiol. Vol. 99, 1992

-o pBIN 1 9-oc'Hind I I I

-q JD 1 151 17 p Isubunit gene

D subunit gene

Hind IIIBamHI EcoRI

HpOSNPTII:tNOSo pB IN 19In n No ~~TDR

Ku1 LD

Figure 1. Plasmid maps. All steps of cloning are described in thetext. Transferred-DNAs of the final plasmids are shown. A singleHindlIl site is present in the coding region of the a'-subunit gene (2.8kb from BamHl site). Arrows indicate directions of transcription. LB,left border; RB, right border; pNOS, nopaline synthase gene pro-moter; NPTII, neomycin phosphotransferase 11 gene; tNOS, nopalinesynthase gene 3' end.

we report that the accumulation of the :-subunit protein intransgenic petunia plants responds to sulfur nutritional stim-uli in a similar way to that of soybean.

MATERIALS AND METHODS

Plasmid Construction

All procedures used for DNA manipulation were carriedout according to the standard procedures of Maniatis et al.(23). A 3.8-kb2 DNA fragment coding for the a'-subunit genewas isolated from the Gmgl7.1-A904 clone (9, kindly pro-vided by Dr. R.N. Beachy) by digestion with BamHI andEcoRI and was recloned into the corresponding sites ofpBINl9 (6) to yield pBIN 19-a' (Fig. 1). The a'-gene contains0.9 and 0.3 kb of its 5'- and 3'-flanking sequences, respec-tively, which have been shown to be sufficient to maintain a

high level ofexpression in both a tissue-specific and temporal-specific manner in transgenic plants (9). A 4.2-kb DNAfragment coding for the #-subunit gene was excised frompGmg91 (30; also provided by Dr. R.N. Beachy) by digestionwith HindIII and recloned into the same site of pBINl9 toyield pBINl 9-f3. The orientation of the ,-subunit gene inpBINl9-B3 is opposite to that of the a'-subunit gene inpBIN 19-a' (Fig. 1). The A-subunit gene has 1.1 and 1.2 kb,respectively, of its 5' and 3' regions. The tissue specificity andtemporal pattern ofexpression ofthis gene in transgenic plantshave been shown to be similar to those in soybean (8).

2Abbreviations: kb, kilobase; MGRL media, 1.75 mm sodiumphosphate buffer (pH 5.8), 1.5 mM MgSO4, 2.0 mM Ca(NO3)2, 3.0mM KNO3, 67 Mm Na2EDTA, 8.6 Mm FeSO4, 10.3 Mm MnSO4, 30 Mm

H3BO3, 1.0 Mm ZnSO4, 24 nM (NH4)6Mo7024, 130 nM CoC12, 1 AMCuS04.

Construction of Transgenic Petunia Carrying thea'- or ,-Subunit Gene

pBIN19-a' and pBIN19-#, as well as pBIN19, were intro-duced into Agrobacterium tumefaciens strain C58C1 RifR(pGV2260) (12) by triparental mating using pRK2013 (14) asa helper. The resulting A. tumefaciens strains were used toinfect petunia (Petunia hybrida cv VR hybrid) leaf discs.Transgenic plants were regenerated within 3 months ofAgro-bacterium infection according to the method of Naito et al.(25). Total DNA was isolated from leaves of each transgenicplant and subjected to Southern analysis. DNA extractionand Southern analysis were performed according to themethod of Naito et al. (25) except that fractionated DNA wastransferred onto nylon membranes (Gene Screen Plus, NewEngland Nuclear, Boston, MA). Random priming was usedfor radiolabeling the probes. Seed protein was extracted andthe a'- and ,B-subunit proteins were detected by westernblotting using rabbit anti-soybean 7S antibody (kindly pro-vided by Dr. R.N. Beachy) by the procedure of Naito et al.(25). In most cases, two SDS-PAGE analyses were run inparallel, one was subjected to western analysis and the otherwas stained with Coomassie brilliant blue R250.

Petunia Pod Culture

Transgenic petunia plants were grown under fluorescentlight at 800,E m-2 s-' with a 16-h light/8-h dark photoperiodin a mixture of perlite:vermiculite:peat moss (1: 1: 1) at 22°C.MGRL media was used to water plants. Flowers were hand-pollinated and tagged on the days of flowering. Immatureseed pods of appropriate maturing stages were cut at thepetioles and surface sterilized in a 10-fold dilution of com-mercial bleach solution containing 0.01% (v/v) Triton X- 100with gentle agitation for 15 min followed by several rinseswith sterile water. After the sepals were removed from thesurface-sterilized pods, the pods were cut in halflongitudinallyalong the joint of the two carpels, then put on agar plates withthe cut surface facing the agar, and cultured under the sametemperature and light conditions as for the transgenic plants.The culture media were essentially the same as the basal andmethionine-supplemented media described by Holowach etal. (21) except that the media were solidified by 1% agar (Sho-ei, Tokyo, Japan). One-half of each seed pod was cultured onthe basal medium, and the other half was cultured on themethionine-supplemented (8.4 mM) medium. The period ofculture was 6 d for protein analysis and 4 d for mRNAanalysis. After each period of culture, seeds were collectedand immediately frozen in liquid nitrogen and stored at-80C until use. Total RNA extraction and northern analysiswere done as described by Naito et al. (25) except that nylonmembranes (Gene Screen Plus) were used. Protein extractionand its western analysis were also done according to themethod of Naito et al. (25).

Hydroponic Culture

Cuttings were made from the original transformant lineTFPh--l and were cultured in control or sulfur-deficienthydroponic solution under continuous white fluorescent light

d. InMTfI 0a-

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SULFUR NUTRITION AND SEED PROTEINS IN TRANSGENIC PLANTS

at 400 IE m-2 s-' and 22TC. The control hydroponic culturemedium was the MGRL medium. The sulfate-deficient hy-droponic culture medium had the same composition as theMGRL medium except that MgSO4 was replaced by an equalmolar concentration of MgCl2. Sulfate concentrations of thecontrol and sulfate-deficient media were 1.5 mm and approx-imately 20 AM, respectively. Mature seeds from these cuttingswere subjected to protein analysis as described above.

RESULTS

Construction of Transgenic Petunia Carrying thea'- or j-Subunit Gene

Several independent transgenic plants carrying each con-struct were recovered. Plant lines TFPhfl- 1, 2, 8, 13 wererecovered from transformation with pBIN 19-0l, TFPha'-l, 2,14, 16 with pBINl9-a', and TFPhBIN-2, 5, 7 with pBINl9.Total DNA was extracted from leaves of each plant line andwas analyzed by Southern hybridization after digestion withHindIll (Fig. 2A). All TFPhfl lines showed a 4.2-kb band thatcorresponds to the intact fl-subunit gene. For transformationwith pBIN19-fl, the copy number of the f-gene inserted was<10 copies per diploid genome. Among them, TFPhfl-8seemed to be a single-copy transformant. On the other hand,all TFPha' lines contained many copies of the trans-gene. AllTFPha' lines showed an intense 2.8-kb band in addition toseveral high mol wt bands. The 2.8-kb bands correspond tothe internal HindIII fragment containing the a'-subunit gene.The 3.6-kb band seen in the copy number control lanes

corresponds to pUC9 (vector of Gmgl 7.1-A904 clone) plusthe 3' end of the a'-subunit gene (1.0 kb). The number ofhigh mol wt bands represents the number of different lociwhere gene insertion occurred because there is no HindIII sitebetween the internal HindIII site of the a'-subunit gene andthe left border. Wild-type petunia DNA did not hybridize tothe probe (data not shown). Judging from the intensity of the2.8-kb bands and the numbers of high mol wt bands, >10copies were introduced to all TFPha' lines in at least severaldifferent loci.

Protein was extracted from mature seeds of each line (pri-mary transgenic plants) and expression of the introducedgenes was analyzed by western blotting. As shown in Figure2B, transgenic petunia carrying the fl-conglycinin gene accu-mulated protein(s) that interacts) with anti-soybean 7S anti-serum, whereas none of the TFPhBIN lines had detectableprotein. In the case of TFPhfl lines, the protein had the samemobility as the fl-subunit in mature soybean seed (53 kD).However, in the case of TFPha' lines, proteins recognized byanti-soybean 7S antiserum had various sizes ranging from 55kD up to the size of the a'-subunit protein in soybean seed(76 kD). These results are consistent with previous reports (9,25), indicating that the a'-subunit protein is susceptible toproteolytic cleavage in transgenic petunia and tobacco seedsin which the most abundant breakdown product was 55 kD.

Judging from intensities of bands, copy numbers of trans-gene and levels of the a'- or f-subunit accumulation arecorrelated well. fl-Conglycinin genes have been reported tohave little chromosomal position effect (8, 9, 25). The results

Figure 2. Characterization of transgenic plants.A, Determination of copy number by Southernhybridization. Total DNA from transgenic petuniawas digested by Hindill and subjected to 0.7%agarose gel electrophoresis. Fractionated DNAwas transferred to a nylon membrane andprobed by 32P-labeled a' (BamHI-EcoRl frag-ment for TFPha') or fi (HindlIl fragment forTFPhfl) subunit gene fragment. Appropriateamounts of Hindlil digests of plasmid DNA(Gmg17.1-A904 in pUC9 for TFPha' lines andpGmg9l in pSP64 for TFPhfl lines) were usedto constitute copy number controls. W, Untrans-formed petunia. B, Detection of i-conglycinin intransgenic petunia seeds by western blotting.Seed protein was extracted as described in thetext, and 50 Isg of total protein was subjected to12.5% SDS-PAGE, followed by western blotting.The subunits of fl-conglycinin were then de-tected by rabbit anti-7S antiserum. The 55- and53-kD bands correspond to the a'- and p5-sub-unit, respectively. Total soybean (cv Toyosuzu)seed extract (0.5 and 2 gg of total protein) wasused as standard (SB lanes). W, Untransformedpetunia.

AProbe: P subunit gene

TFPha copy numbercontrol

1 2 8 13 W 1 2 5 1 0

4.2 kb-0* mas

B

55kD53kDa

Probe: a' subunit geneI copy number

TFPhc control1 2 14 16 1 2 5 10

-~ ~~~a

TFPhP TFPPah TFPhBlN | SB1 2 8 1 2 1412 5 7 W .s2 rg

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Plant Physiol. Vol. 99, 1992

obtained here are consistent with these observations, eventhough the vector used for transformation of petunia wasdifferent from that used in the previous studies. This indicatesthat the 13-conglycinin genes themselves behave in a chromo-somal position-independent manner.

Response of a'- and 13-Subunit Gene Expression inin Vitro Cultured Pods to Exogenous Methionine

Immature seed pods from TFPhfl- 1 at various stages (12,16, and 20 DAF) were cut longitudinally into halves and werecultured on solid media with or without methionine for 6 d.Protein extracts were analyzed by western blotting. As shownin Figure 3A, seeds cultured on the basal medium containedmore ,3-subunit protein than did seeds before culture, indicat-ing that the ,3-subunit protein accumulated during the cultureperiod. Profiles of petunia major storage proteins were rela-tively unchanged during the culture. Irrespective of the stagesof seed development, the accumulation of the 1-subunit pro-tein during culture was repressed when the immature seedpods were cultured in the presence ofmethionine. Methioninerepresses the ,8-subunit gene expression in petunia even atlater stages of development when the expression has alreadyinitiated. This fact is consistent with the results obtained insoybean, in which the repression has been shown to be inde-pendent of the developmental stage (20). The repression wasmost evident when pods at 16 DAF were used for culture.Based on this result, subsequent cultures were started at 16DAF.Two other transgenic lines carrying the p3-subunit gene

(TFPh13-8 and TFPhfl- 13) showed a similar response (Fig.3B). In contrast, TFPha'-l and TFPha'-14 did not respondto exogenous methionine. Profiles of petunia major seedproteins were similar irrespective of methionine treatment inall the lines tested. These results indicate that the mechanismof gene regulation in response to methionine is commonbetween soybean and petunia, although in petunia, this mech-

anism is evidently not used to regulate its endogenous majorstorage protein accumulation.

In the case of soybean, it has been demonstrated that theeffect of methionine is at the mRNA level (11, 20). TotalRNA from 4-d-cultured immature seeds of TFPh13-l weresubjected to northern analysis to determine how methionineaffects mRNA accumulation (Fig. 4). Seeds cultured on thecontrol media had a higher level of the 13-subunit mRNAaccumulation than seeds before culture, indicating thatexpression of the p3-subunit message occurred during culture.Much less 3-subunit mRNA accumulated in immature seedstreated with methionine than in seeds cultured withoutmethionine.These results indicate that the effect of methionine on the

expression of the 1-subunit gene in petunia is similar to thatseen in soybean, in that accumulation of the a'-subunitprotein is not affected, whereas accumulation of 1-subunitprotein as well as level of mRNA coding for the 1-subunit isreduced.

Response of the 13-Subunit Gene to Sulfur Deficiency

Cuttings from TFPh1-l were cultured in the control orsulfur-deficient hydroponic solutions to see the effect of sulfurdeficiency on expression of the 1-subunit gene in petunia.TFPh13- 1 plants cultured under sulfur deficiency were smallerin size and developed fewer leaves than those cultured oncontrol media. Root mass of sulfur-deficient TFPh13- 1, how-ever, developed much better than control (data not shown).Proteins from mature seeds were subjected to western analysis.As shown in Figure 5, TFPh,- I accumulated severalfold more1-subunit protein in seeds when the plants were grown undersulfur deficiency. Coomassie blue staining patterns show thatthere is no obvious difference in the accumulation of majorpetunia seed storage proteins. This again indicates that petu-nia has a functional mechanism to regulate the 13-subunit genein response to sulfur deficiency and that the mechanism is

Figure 3. Effect of methionine on the a'- and ,B-subunit protein accumulation. A, Effect of methi-onine on expression of 13-subunit gene at differ-ent stages of seed development. Staged imma-ture pods of TFPhB-1 were harvested on the dayshown and cultured for 6 d. Top, Accumulationof the 1-subunit in cultured seeds detected bywestern blotting. Total protein (50 Mg) wasloaded on each lane. Total soybean (cv Toyo-suzu) seed protein (0.5 and 2.5 Ag) was used asa standard (SB lanes). Bottom, Patterns of pe-tunia seed storage protein by Coomassie stain-ing. Total protein (10 ug) was loaded on eachlane. B, Seeds before culture; C, seeds culturedon the control (no methionine) media; M, seedscultured on the methionine-containing media.The arrow indicates the 18-subunit protein. B,Effect of methionine in independent transgeniclines. Methionine effect was tested on differenttransgenic lines. Culture and analysis were doneas described in the text. Lanes are the same asin A.

A

DAF: 12 16 20B CM B C M B CM

S;B0.5 2 5

'p?:

*- _I

B

IF Phi:- 8 TFPh; , rr P"l-;l l.BMCBM B M B. VB

-. r:

o,oom as s

_minCf1t aJ nin

#x w"wtw -*V f ,^1-Ii w*w~

266 FWIWARA ET AL.

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SULFUR NUTRITION AND SEED PROTEINS IN TRANSGENIC PLANTS

SBB C M 1 5

10_

Figure 4. Effect of methionine on the (3-subunit mRNA accumulation.Total RNA was extracted from 4-d-cultured (starting at 16 DAP)seeds of TFPhfl-1, and 5 Ag of RNA was subjected to electrophoresis.Fractionated RNA was blotted onto a nylon membrane and probedby 32P-labeled (3-subunit gene fragment. B, Seeds before culture; C,seeds cultured on control (no methionine) media; M, seeds culturedon methionine-containing media. Total RNA isolated from immaturesoybean (cv Provar) cotyledon (1 and 5 jg) was used as a standard(SB lanes). The arrow indicates the position of the (3-subunit mRNA.

apparently not utilized to regulate its own major seed proteingenes.

DISCUSSION

Studies of plant responses to nutritional stimuli are impor-tant because adequate nutrition is essential for proper growthof plants. The understanding of plant nutrition has a greatpotential for agricultural application. For this reason, anenormous amount of research has been conducted mainly byphysiological approaches. Changes in nutritional conditioncause a wide variety of physiological responses as well aschanges in patterns ofgene expression. Mainly because of thecomplex nature of responses, it is still not very clear howchanges in nutritional condition are perceived and how signalsare transmitted and transduced in plants.The effect of sulfur nutrition on accumulation of seed

storage proteins is one of the ideal models to reveal theunknown links between plant nutrition and gene expression.Sulfur deficiency increases the level of sulfur-poor proteins inmany plants including pea (26), lupin (7), wheat (33), barley(27), maize (3), cowpea (13), rape, and sunflower (29) inaddition to soybean. Under limited sulfur availability, plantsmaintain overall levels of seed storage protein by accumulat-ing more storage proteins of low content of sulfur-containingamino acids and less of those with high content (19). In thecase of pea, it has been shown that changes in the accumula-tion of seed proteins in response to sulfur deficiency areregulated at both the transcriptional and posttranscriptionallevels (4). The fact that similar responses have been observedamong a great diversity of plant species that have differentsets of storage proteins suggests that this response is universaland may be essential. On the other hand, little is knownconcerning methionine regulation of storage proteins amongvarious plant species. Soybean is the only example describedso far.

In this report, we demonstrate that expression of the (3-subunit of fl-conglycinin in transgenic petunia is repressed byexogenous methionine in in vitro pod culture, whereas it hasno effect on accumulation of the petunia major storage pro-

teins and the a'-subunit, which does not respond to methio-nine in soybean. This indicates that petunia has a functionalmechanism to regulate soybean storage protein synthesis inresponse to exogenous methionine in a way similar to that ofsoybean, although it seems that petunia does not use thismechanism to regulate its own major seed protein synthesis.The effect ofexogenously applied methionine has been shownto be at the level ofmRNA accumulation in soybean. In ourin vitro culture of transgenic petunia pods, addition of methi-onine repressed accumulation of the (3-subunit mRNA, indi-cating that the mechanism is similar in soybean and inpetunia. It has been shown that levels of free methionineremain relatively constant in a wide range ofsulfur availability(see ref. 1 for review). The methionine content ofseed declinesduring seed development of soybean, which may be respon-sible for the late initiation ofthe (3-subunit accumulation (3 1).Our observations may suggest that methionine content ofpetunia seeds also declines in the course of seed maturationand this is the cause of the difference of timing of expressionof the a'- and (3-subunit gene in transgenic petunia seeds (25).We also showed that petunia has a mechanism to control

the accumulation of soybean seed storage protein in responseto sulfur deficiency. The fact that the trans-gene is regulatedin a similar way as it is in soybean indicates that not only doplants show similar responses to sulfur deficiency but also themechanism of regulation of seed storage protein compositionis common. Again, petunia does not seem to use this mech-anism to control its own major seed storage protein synthesis.Spencer et al. (29) showed that pea legumin, which is knownto be down-regulated by sulfur deficiency in pea, accumulatedto a reduced level under sulfur deficiency in transgenic to-bacco seeds, whereas no change is observed in the tobacco

C -S SB C -Si.11d~... w "

SB1 5

Westernstaining

Figure 5. Effect of sulfur deficiency on accumulation of the (3-subunitprotein in transgenic petunia seeds. Total protein was extracted frommature seeds of TFPhB-1 cultured under either control medium (C)or sulfate-deficient medium (-S) and analyzed by western blotting(right). Total petunia protein (50 gg) was loaded except for totalsoybean (cv Toyosuzu) seed extract (1 and 5 gg, SB lanes). Left,Coomassie staining patten of petunia seed protein. Total protein (10jsg) was loaded onto each lane. The arrow indicates the position ofthe (3-subunit protein.

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Plant Physiol. Vol. 99, 1992

storage protein accumulation. These facts indicate that plantsshare mechanisms of both up-regulation and down-regulationof storage protein synthesis in response to sulfur deficiencyand that these mechanisms are present even in species thatdo not have storage protein genes to be regulated by thesemechanisms.

It is widely accepted that dicot plants have common mech-anisms of gene regulation. Most of the dicot genes put stablyin heterologous dicot hosts retain their patterns of expression,i.e. organ specificity, temporal pattern of expression, andinducibility by environmental stimuli (see ref. 32 for review).Our findings indicate that plants also share common mecha-nisms of gene regulation in response to sulfur nutritional

stimuli. This allows the identification of cis-acting element(s)in the genes responsible for these responses using transgenicplants. These experiments will help us to understand themolecular mechanism(s) of gene regulation in response tonutritional stimuli, which is one ofthe most important aspectsof plant nutrition.

ACKNOWLEDGMENTS

We are grateful to F. Nishigai for technical assistance and HalPadgett for critical reading of the manuscript.

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