Planta (1992)188:432-438

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9 Springer-Verlag 1992

cDNA cloning of an extracellular dermal glycoprotein of carrot and its expression in response to wounding Shinobu Satoh I *, Arnd Sturm 2, Tadashi Fujii 1, and Maarten J. Chrispeels 3 1 University of Tsukuba, Institute of Biological Sciences, Tsukuba, Ibaraki 305, Japan 2 Friedrich Miescher Institute, PO Box 2543, CH--4002 Basel, Switzerland 3 Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA Received 21 April; accepted 30 May 1992

Abstract. Suspension-cultured cells of carrot (Daucus carota L.) synthesize and secrete a glycoprotein that is normally found only in dermal tissues (epidermis, endodermis and periderm). This protein, previously called GP57, is now referred to as E D G P (Extracellular Dermal GlycoProtein). We purified sufficient quantities of E D G P to obtain amino-acid sequences on two internal tryptic peptides and screened a e D N A library of young carrot roots with antiserum to E D G P and with oligonucleotides corresponding to the peptides. Here we report the derived amino-acid sequence of EDGP. Sequence comparisons show that it has 40% amino-acid sequence identity with 7S basic globulin, a protein that is released when soybean seeds are soaked in hot water for a few hours. We suggest that these two proteins belong to a new family of dermal proteins. As far as we know, this is the first reported derived amino-acid sequence for protein that is specific to the epidermis and other dermal tissues. The level of E D G P m R N A is low in dry seeds, but increases rapidly in growing seedlings as they develop dermal tissues. The level o f m R N A is low in storage roots, but increases rapidly in response to wounding. The presence of E D G P in dermal tissues and its up-regulation in response to wounding indicate a role in the response of plants to biotic and-or abiotic stresses. An unusual feature of the amino-acid sequence of E D G P is that it contains a short motif, which is present at the active site of aspartyl proteases such as pepsin and chymosin. Key words: Daucus (glycoprotein) - Defense Epidermis (glycoprotein) - Glycoprotein (extracellular) - Wound response

Abbreviations: cDNA=copy

DNA; 2,4-D=2,4-dichlorophenoxyacetic acid; EDGP = extracellular dermal glycoprotein; 7SBG = 7S basic globulin * To whom correspondence should be addressed; FAX: 81 (298) 53 6614

Introduction The wall of the plant cell forms a protective barrier around the protoplast and contains a variety o f macromolecules that play important roles in resisting biotic and abiotic stresses. When tissues are wounded or invaded by pathogens, the cells that are near the site of such a stress strengthen their walls by the secretion of additional structural components such as hydroxyproline-rich glycoproteins (HRGPs), lignin or suberin (for reviews, see Bowles 1990; Dixon and Harrison 1990). In addition, enzymes needed for the biosynthesis of cell-walt components (e.g. the anionic peroxidase involved in suberin biosynthesis, see Espelie et al. 1986), enzymes such as ]3-glucanase or chitinase that can generate additional signals capable of eliciting further defense responses (see Bol et al. 1990 for review), or enzymes with metabolic functions, such as invertase (Benhamou et al. 1991) are secreted into the cell wall in response to such stresses. The genes that encode these enzymes or protein components of the wall have often been found to be induced by pathogen invasion, mechanical wounding, or both. These same genes are often highly expressed in suspensioncultured cells and calluses, as these cell systems are themselves derived from wounded tissues at the start of the tissue culture process. Macromolecules that are induced by biotic or abiotic stresses are sometimes present at low levels in all the cells - H R G P s for example - or, as in the case of suberin, they may be present at high levels in barrier tissues. Stress may cause a general induction throughout the tissue or a new barrier may be established, as in the case for suberization below the surface of a wound site. In a previous study (Satoh and Fujii 1988), we identified an extracellular glycoprotein (GP57, now called extracellular dermal glycoprotein or E D G P ) that accumulates in dermal tissues of carrots. We found high levels of this protein in the epidermis and endodermis of young roots, the periderm of mature roots, and the epidermis of petioles and leaves. In addition, this protein accumulates in the space between the embryo and the endosperm of de-

S. Satoh et al. : Wound-induced dermal glycoprotein v e l o p i n g c a r r o t seeds. A l t h o u g h the f u n c t i o n o f E D G P is u n k n o w n , its l o c a t i o n in d e r m a l tissues i n d i c a t e s a defense function. L i k e a n u m b e r o f o t h e r p o t e n t i a l p l a n t defense p r o t e i n s , E D G P is also a b u n d a n t l y s y n t h e s i z e d a n d secreted b y s u s p e n s i o n - c u l t u r e d cells ( S a t o h et al. 1986). S t a r t i n g f r o m k n o w n a m i n o - a c i d sequences o f t w o E D G P p e p t i d e s , we o b t a i n e d a c o p y D N A ( c D N A ) f o r E D G P , a n d here we r e p o r t the d e r i v e d a m i n o - a c i d sequence for the entire p r o t e i n . T h e a m i n o - a c i d sequence s h o w s c o n s i d e r a b l e i d e n t i t y (40%) w i t h a p r o t e i n , 7S b a s i c g l o b u l i n ( 7 S B G ) , t h a t is r e l e a s e d w h e n s o y b e a n seeds a r e s o a k e d in w a r m w a t e r ( H i r a n o et al. 1992), a n d we suggest t h a t b o t h m a y be in d e r m a l tissues. T h e e x p r e s s i o n o f E D G P m R N A is r a p i d l y i n d u c e d a n d reaches a m a x i m u m 12 h after c a r r o t p h l o e m p a r e n c h y m a tissue is w o u n d e d ; e x p r e s s i o n is h i g h in suspens i o n - c u l t u r e d c a r r o t cells. A l l these results a r e c o n s i s t e n t w i t h a role for E D G P as a p l a n t defense p r o t e i n , b u t the f u n c t i o n o f the p r o t e i n r e m a i n s to be elucidated.

Materials and methods Plant materials and cell culture. Embryogenic callus of carrot (Daucus carota L. cv. US-Harumakigosun) was obtained from segments of one-week-old hypocotyls which were cultured on Murashige and Skoog's (MS) agar medium (Murashige and Skoog 1962) that contained 1 mg- 1-1 (4.5 9 10-6 M) 2,4-dichlorophenoxyacetic acid (2,4-D). Non-embryogenic callus was obtained from the same plant material as described before (Satoh et al. 1986). Cultures of both embryogenic and non-embryogenic callus were maintained by transfer at intervals of two weeks to fresh MS medium supplemented with 1 mg 91-1 2,4-D. Somatic embryos were produced by the transfer ofembryogenic cells to auxin-free MS medium as described before (Satoh et al. 1986). Carrot crown gall was induced by inoculation of axenic carrot seedlings with Agrobacterium tumefaciens C58C1 that harbored pTiB6S3 (a wild-type octopine Ti plasmid), and cultured on agar medium without hormones (Ishikawa et al. 1988). Seeds of carrot (D. carota L. cv. US-Harumakigosun), parsley (Petroselinum crispum cv. Setoparamaunt), soybean (Glycine max L. Merrill cv. Miyagishirome), tobacco (Nicotiana tabacum L. cv. Wisconsin 38), chinese cabbage (Brassica rapa var. pervidis cv. Komatsuna), squash (Cucurbita maxima Duchesne • C. moshata Duchesne cv. Shintosa-ichigou), maize (Zea mays L. cv. Golden Cross Bantam) were sown on vermiculite beds and grown at 25 ~ C under a daily regime of 16 h light (40 ~tmol photons - m - 2. s- 1)/8 h darkness. Mature carrot plants (D. carota cv. Kurodagosun) were grown in the field in Ibaraki, Japan. Woundin9 and chemical treatments of carrot root. Carrot roots were wounded as described by Sturm and Chrispeels (1990). Briefly, thin slices of phloem-parenchyma tissue from maturing storage tap roots of carrot were incubated in water on a rotary shaker at 25 ~ C. Tissues were harvested at the time points indicated and immediately frozen in liquid nitrogen. Control tissue (time point 0) was taken directly after slicing. Determination of amino-acid sequences of EDGP peptides. EDGP was purified from the culture medium of non-embryogenic callus of carrot as described before (Satoh and Fujii 1988). EDGP was digested with trypsin and the resulting peptides were separated by high-performance liquid chromatography (HPLC) on Vydac C18 and C s columns as described by Sturm et al. (1987), and applied to a gas-phase protein sequencer. The amino-acid sequences obtained were IleAlaLeuProSerGlnPheAlaSerAlaPhe and IleSerThrlleAsnProTyr.

433 Synthesis and screening of cDNA. Total RNA was prepared from roots of 10-d-old seedlings of carrot as described by Prescott and Martin (1987) and was purified by successive lithium-chloride precipitations. Polyadenylated RNA was isolated by oligo(dT)-cellulose chromatography (Maniatis et al. 1982) and used to construct a eDNA library in kgtl 1 according to eDNA Synthesis System Plus and Cloning System protocols (Amersham International, Amersham, Bucks., UK). The non-amplified library was screened with rabbit anti-EDGP antiserum (Satoh 1990) according to Young and Davis (1983). Eight positive clones obtained from 2.5" 105 plaque-forming units were rescreened with two kinds of synthetic oligonucleotides which corresponded to the antisense sequences of the tryptie peptide sequences. ThrIleAsnProTyr [5'-TA(ACGT)GG(AG)TT(AGT)AT(ACGT)GT-3'] and IleAlaLeuProSerGlnPheAla [5'-GC(AG)AA(CT)TGI(CG)(AT) IGGIA(AG)IGCIAT-3']. Briefly, lambda DNA was cut with EcoRI and separated on a 1% agarose gel and then transferred to a GeneScreen Plus membrane (New England Nuclear, Boston, Mass., USA) and probed with the 3zp-end-labeled oligonueleotides at 30 ~ C in 6 x SSC, 1% sodium dodeeyl sulfate (SDS), 10% dextran sulfate sodium salt followed by washing with 6 • SSC (1 9SSC = 0.9 M sodium chloride containing 0.09 M sodium citrate, pH 7.0), 1% SDS at 35~ C or 45 ~ C for the shorter or longer oligonueleotides, respectively. One clone was positive for both oligonucleotides. Both DNA strands were sequenced by the dideoxynucleotide chaintermination method (Messing 1983). Gel blots analysis of RNA. Total RNA (10 ~tg/lane) from various tissues was separated on agarose gels containing formaldehyde (Maniatis et al. 1982), transferred to a GeneSereen Plus membrane and probed with the cDNA 32P-labeled by random priming (Maniatis et al. 1982) in 1 M NaC1, 1% SDS, 10% dextran sulfate (sodium salt) at 60 ~ C, followed by the washing with 2 x SSC, 1% SDS at 60 ~ C. In a low-stringency condition, both hybridization and washing were performed at 50~ C. For the wound-induction experiment, we stripped a blot previously probed with an invertase eDNA and an aetin eDNA (Sturm and Chrispeels 1990).

Results Cloning o f c D N A and sequence analysis o f E D G P . T o o b t a i n clones o f E D G P , we screened a )~gtl 1 l i b r a r y m a d e f r o m y o u n g c a r r o t r o o t s w i t h the E D G P antiserum. W e o b t a i n e d e i g h t p o s i t i v e clones in 2 . 5 . 1 0 5 p l a q u e - f o r m i n g units, a n d o n e o f these h y b r i d i z e d w i t h two o l i g o n u c l e o t i d e s r e p r e s e n t i n g t w o i n t e r n a l a m i n o a c i d sequences o f E D G P . T h e insert o f this clone was used to rescreen new l i b r a r i e s 0~gtl0 a n d )~gtl 1) t h a t were made from young carrot roots and non-embryogenic c a r r o t callus. I n a d d i t i o n , we s u b j e c t e d the l i b r a r i e s directly to the p o l y m e r a s e c h a i n r e a c t i o n u s i n g )~gtl0 a n d )~gtll p r i m e r s ( T a k a r a , T o k y o , J a p a n ) a n d the s a m e o l i g o n u c l e o t i d e s u s e d for screening. I n this way, we o b t a i n e d a n u m b e r o f clones a n d f o u r o f these were sequenced. T h e clones f o r m e d a n e s t e d set at their 5' ends, a n d Fig. 1 s h o w s the n u c l e o t i d e (1439 bases) a n d ded u c e d a m i n o - a c i d sequence o f the l o n g e s t c l o n e named EDGP-I. EDGP-I contains one open reading f r a m e s t a r t i n g at n u c l e o t i d e 1 a n d e n d i n g a t n u c l e o t i d e 1299 b e f o r e a T G A s t o p c o d o n . T h e o p e n r e a d i n g f r a m e e n c o d e s a p o l y p e p t i d e c h a i n o f 433 r e s i d u e s w i t h a c a l c u l a t e d m o l e c u l a r w e i g h t o f 45840. T h e t w o intern a l a m i n o - a c i d sequences o b t a i n e d f r o m E D G P a r e present in the d e d u c e d a m i n o - a c i d sequence as i n d i c a t e d b y the d o u b l e u n d e r l i n e s in Fig. 1, c o n f i r m i n g t h a t E D G P encodes EDGP of carrot.

434

S. Satoh et al. : Wound-induced dermal glycoprotein

~TACTTCACTTCAAATTACCTTATTTTCCCTACTCTTCATTTTCACCATCACTCAAGCTCAGCCATCTTTCCGACCATCCs

A~aThr~er~G~n-~j~Thre~f~e~Pbe~j~heThr~j~ThrG~nA~aG~nPr~erPheA~gPr~erA~LeuV~ GTTCCGGTGAAGAAAGATGC•TCGACGCTCCAATATGTGACCACGATCAACCAAAGAAC•CCTCTAG•G•CCGAAAATCT•GTTG••GAT •a•Pr••a•Lys•ysAspA•a•erThr•euG•nTyr•a•ThrThr••eAsnG•nArgThrPr••eu•a•SerG•uAsn•euValValAsp

90

180

CTTGGAGG•CGGTTCTTGTGGGTTGATTGTGATCAAAATTATGTCTCATCCACG•ACCGTCCTGTTCGATGTAGAAC•TCTCAATGCTCC270 LeuG•yG••ArgPhe•euTrpVa•AspC•sAspG•nAsnTyrValSerSer•hrT•rArgPr•Va•ArgC•sArgThrSerGlnC•sSer CTGTCGGGGTCCATTGCATGCGGAGATTGCTTTAATGGACCCCGACCAGGGTGCAATAACAATACTTGC~TTTTCCTGAAAACCCC 360 Leu~e~G~y~er~eA~a~ysG~yAsp~ysPheAsnG~yPr~ArgPr~G~y~ysAsnAsnk~nThrCysG~y~a~hePr~G~uAsnP~ GTGATTAATACCGCTACT~GTGAGGTTGCCGAAGATGT~GTGTCGGTGGAGTCTAC~GACGGGTCGAGTTCAGGTCGGGTTG~ACT 450 ~a~I~eAs~ThrAlaThrGlyG~yGluV~lAlaGluAsp~al~alSerValGluSerThrAspGlySerSerSerGlyArgValValThr GTTCCACGTTTCATTTTTTCGTGCGCACCAACTTCTTTG•TTCAAAACTTGGCTAGTGGTGTAGTAGGCATGGCCGGATTGGGAAGGACT 540 V~Pr~ArgPhe~ePheSerCysA~aPr~Th~SerLeuLeuG~nAsnLeuA~a~erG~yVa~a~G~yMetA~aG~yLeuG~y~rgThr AGAATTGCACTTCCTTCGCAATTTGCTTCGGCTTTTAGCTTCAAGAGGAAATTTGCGATGTGTCTATCAGGATCCACCTCTTCGAACAGT 630 Arg~eA~aLeuPr~SerG~nPheA~SerA~aPhe~e~heLysA~gL~sPheA~a~tCysLeu~erG~y~erThr$er~erAsnSer GTCATAATCTTCGGAAATGATCCATACACGTTTCTCCCGAACATCATTGTCTCAGATAAAACACTCACCTACACAC~CTTGTTAACAAAT 720 ~a~e~ePheG~yAsn~spP~Ty~ThrPheLeuP~Asn~e~e~a~SerAsp~ysTh~LeuThrTy~ThrPr~LeuLeuTh~Asn CCAGTGAGCACATCCGCGACTTCTACACAAGGCGAGCCTTCGGTTGAGTACTTCATCGGAGTAAAATCTATTAAAATCAACTCTAAAATC 810 P~a~e~Thr~e~A~aThr~erTh~G~nG~yG~uP~Se~a~G~uTyrPheI~eG~yVa~LysSerI~eLysI~eAsnSe~LysI~e GTTGCCTTAAACACATCACTACTCTCCATAAGTAGTGCAGGACTCGGTGGAACAAAAATTAGCACAATCAATCCATACACAGTCCTGGAA 900 Va~A~aLeuAsnThrSerLeuLeuSer~eSer~erA~G~yLeuG~yG~yThrLysI~eSerThr~eAsnP~T~rThrV~LeuG~u ACCTCGATCTACAAGGCGGTGACAGAGGCTTTCATCAAAGAATCCGCAGCCAGGAATATAACACGAGTTGCATCAGTTGCACCATTCGGG 990 ThrSer••eTyrLysA•aVa•ThrG•uA•aPhe••eLysG•uSerA•aA•aArgAsnI•eTh•Arg•a•A•a•er•a•A•aPr•PheG•y GCCTGTTTTAGCACAGATAACATCTTGAGTACGCGATTAGGGCCATCAGTACCGAGCATTGATCTGGTG~TACAGAGCGAAAGCGTGGTC 1080 A~C~sPhe~erTh~AspAsn~eLeu~e~ThrA~gLeuG~y~r~er~a~Pr~Ser~eAspLeu~a~LeuGln~erG~u~er~a~a~ TGGACAATAACAGGGTCGAATTCAATGGTTTACATTAATGACAATGTGGTGTGTCTTGGAGTTGTTGACGGAGGGTCGAACTTGAGAACC 1170 TrpThr~eThrG~SerAsnSerMet~a~T~r~eAsnAspAsnVa~a~ysLeuG~a~a~AspG~yG~ySerAsnLeuArgThr TCAATTGTGATCGGAGGGCATCAGTTGGAGGATAATTTGGTGCAATTCGATCTGGCAACTTCGAGAGTGGGATTTAGTGGGACTCTGTTG 1260 ~er~e~a~e~yG~yHisG~nLeuG~uAspAsnLeuva~G~nPheAspLeuA~aThr~rA~g~a~G~yPhe~erG~yThrLeuLeu GGGAGCCGGACTACG•GTGCGAACT••AA••••ACCTCATGAACA•ATTTTACGGCTCAGCT•GCTG•GT•ATTTAG•CTGTGATGT•GC GlySerArgThrThrCysAlaAsnPheAsnPheThrSer

1350

AGTTTATGACATTCCAGATATGTATTCGAGAATAAATTCGGG~TTTCTGTTATTTATCATGTCTTCTACTTAATAAAACA~AAAAAAAA 1439 Fig. I. Nucleotide and amino-acid scqucnces of E D G P , a c D N A of an extracellular dermal glycoprotein of carrot. Nucleotides are numbcred from thc firstbase of thc c D N A clone. The dashed lines denote potcntialpolyadcnylation signals.The deduced amino-acid sequence of E D G P is indicatcd below the nucleotide sequence. The doubt-underlined scqucnccs dcnotc thc amino-acid sequences deter-

mined by the automated Edman degradation of two tryptic peptide sequences of EDGP. The arrow and wavy underlines indicate the putative signal-sequence cleavage site and hydrophobic cluster, respectively. The underlined sequences denote potential Nglycosylation sites

EDGP-1 lacks a translation initiation signal (ATG) in the 5'-terminal region, but has 140 base pairs of 3'-untranslated sequence in which AATAAA, the consensus signal for polyadenylation, is present in two places (dash-

ed underlines). EDGP is an extracellular glycoprotein and its derived amino-acid sequence can be expected to have a signal sequence. The absence of an initiating methionine indicates that it is unlikely that EDGP-1 has

435

S. Satoh et al. : Wound-induced dermal glycoprotein

EDGP

1'

ATSLQITLFSLLFIFTITQAQPSFRPSALVV-PVIGKDASTLQYVTTINQRTPLVSE ~,~ .......

7SBG

1"

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~SILHYFLALSLSCSFLFFLSD~TPTKPINLVVLPVQNDGSTGLHWANLQKRTPLMQV

EDGP 56' NLVVDLGGRFLWVDCDQNYVSSTYRPVRCRTSQCSLSGSIACGDCFNGPRPGCNNNTCGV ,.~.~.

~.~.~.~

~.~,.

~...~

....

~ .~

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7SBG 61" PVLVDLNGNHLWVNCEQQYSSKTYQAPFCHSTQCSRANTHQCLSCPAASRPGCHKNTCGL

~GP 116' FPENPVlNTATGGEVAEDVVSVESTDGSS--SGRVVTVPRFIFSCAPTSLLO, N-LASGVV ...~

.....

~..~

.....

~.~.

~ . . ~ . ~ . ~ .

~.~.

~ ....

7SBG 121" ~TNPITQQTGLGELGEDVLAIHATQGSTQQLGPLVTVPQFLFSCAPSFLVQI(GLPRNTQ EDGP 173' G~GLGRTRIALPSOYASffSFKRKFAMCLSGSTSSNSVIIFGNDPYTFLPNIIVSDKTL ~,~...~.~.~.~

~...~.~.

~

..~,..~..~

~.

..

7SBG 181" GVAGLGHAPISLPNQLASHFGLQRQFTTCLSRYPTSKGAIIFGDAP . . . . NNMRQFQNO.D

EDGP238' TYTPLLTNPVSTSATSTQGEPSVEYFIGVI(SIKI-NSKIVALNTSLLSISSAGLGGT~IS ..

~

.~ . . . .

~

~

,

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....

~.

,~

.,.

~ . ~

7SBG 237" IFHDLAFTPL---TITLQGE. . . . YNVRVNSIRITQHSVFPLNKISSTIVGSTSGGTMIS A

Fig. 2. Comparison of the amino-acid sequence of E D G P with the amino-acid

EDGP 292' TINPYTVLETSIYKAVTEAFIKESAARNITRVASVAPFGA~FSTDNILSTRLGPSVPSID sequence of soybean 7S basic globulin .X, ~g, ~ , ~ . ~ g . . . . . . . .

~ ~

~ ....

~

.

~,~

7SBG 290" TSTPHMVLQQSVYQACTQVCAQQL--PKQAQVKSVAPFGLCFNSNKI . . . . . . NAYPSVD EDGP 352' LVLQ-SESVVWTITGSNSMVYINDNVVCLGVVDGGSNLRTSIVlGGHQLEDNLVQFDLAT 7SBG 342" LVMDKPNGPV~ISGEDLMVQAQPGVT~LGVMNGGMQPRAEITLGARQLEENLVVFDLAR EDGP 411' SRVGFSGTLLGSR-TTCAN-FNFTS ~ . .

~ ~.

..~.

~..

7SBG 402" SRVGFSTSSLHSHGVKGADLFNFANA

the entire coding region. The presence of a hydrophobic cluster (10 out of 14 amino-acids, wavy underline) near the start of the open reading frame indicates the presence of a truncated signal sequence. The putative signalpeptide cleavage site is shown by an arrow in Fig. 1. The location of this putative signal-peptide cleavage site conforms to the rules of von Heijne (1986) and is in a position similar to that found in the homologous soybean protein 7SBG (Kagawa and Hirano, 1989; see also below). The different EDGP clones mentioned above all had their 5' ends within the putative signal sequence. Four potential N-glycosylation sites (Asn-X-Ser/Thr) are present and indicated with underlines. The apparent molecular weight of the peptide portion of EDGP, which had been estimated to be 55000 after deglycosylation with trifluoromethanesulfonic acid (Satoh and Fujii 1988) is about 9000 larger than the eDNA-encoded polypeptide. This discrepancy may be explained by its hydrophobic amino-acid composition (50%) and high isoelectric point (pH 8.8 and 9.5; Satoh and Fujii 1988) which may affect the mobility of polypeptides on SDS-polyaerylamide gels. Amino-acid sequence identity with other proteins. The amino-acid sequence of EDGP was used to search the

(7SBG). The amino-acid sequences of the two proteins are in one-letter code and have been aligned by introducing gaps (---) to maximize identity. Asterisks and dots indicate identical residues and conservative replacements, respectively. Arrow indicates the putative signalsequence cleavage site of EDGP. Closed and open arrowheads indicate the sites of signal-sequence cleavage and posttranslational processing of 7SBG, respectively (Kagawa and Hirano 1989)

protein-sequence data bank (the European Molecular and Biological Laboratory Swissprot Library). Significant sequence identity (39.6%) was found with a soybean protein called 7S basic globulin precursor (7SBG) (see Fig. 2). The amino-acid sequence identity was distributed over the entire protein. The protein 7SBG is unusual in that it is released from soybean seeds when the seeds are immersed in hot (40-60 ~ water. It consists of two polypeptides of 27 kDa and 16 kDa that are linked together by disulfide bonds (Kagawa and Hirano 1989). The proteolytic processing site and the signal-peptide cleavage site have been identified and are shown as upward pointing arrowheads in Fig. 2. The signal-peptide cleavage site of 7SBG corresponds to the putative signalpeptide cleavage site of EDGP shown in Fig. 1 and Fig. 2 (downward pointing arrow), confirming our identification of the first 20 amino-acids of EDGP as a partial signal sequence. No post-translational cleavage was observed in EDGP (Satoh and Fujii 1988). The positions of cysteine residues are highly conserved between EDGP and 7SBG indicating the presence of similar tertiary structures for these two proteins. Of particular interest is the finding that a sequence motif found at the active site of aspartyl proteases of yeast (proteinase A) and mammals is also

436

S. Satoh et al. : Wound-induced dermal glycoprotein

7SBG

VTPTKPINLVVLPVQNDGSTGLHWANLQKRTPLMQVPVL~i~DLNGNHI~i~iNCEQQYSSKTYQA

EDGP

*. ~i~!. *. *. *. * . * * . 9 OPSFRPSALVV-PVKKDASTLQYVTTINQRTPLVSENLVVi~Li~iGRF~iDCDQNYVSSTYRP--9 , 9 . . . . . ** ,. ,. ** . . . . . ~i ~.. ~i~i~ ... 9

PROA/Y PEP/H PCHY/B PENPEP/P REN/H

. *

. ***

**..

*.

**

. . . . . .

****.

..

---

*i~!*.

GGHDV- PL T NYLNA-Q YYT D I T LGT PPQNFKV I L~i~i$ SN~!j~IP $N ECGSL ACFL H - - -

Fig. 3. Comparison of the amino-acid sequence of EDGP with the amino-acid sequences of 7SBG and aspartyl proteinases from various organisms. The N-terminal sequences of mature polypeptides of EDGP, 7SBG and Proteinase A from yeast (PROA/Y) (Woolford et al. 1986), and the 16 amino-acids in N-terminal active sites of human pepsinogen A (PEP/H) (Sogawa et al. 1983), bovine

- --

o F T V V Fii il'

iS S N

' - - -

- - E F T VL F!~iT~i'~$$ DF'|i~',~:P- - - - - T L NL NFi~ Ti~i!SA D~ji~ F - - - - - T F KVVF!~ITi~i~SS N:V:~iP - - -

prochymosin (PCHY/B) (Hidaka et al. 1986), penicillopepsin from Penieillium janthinellum (PENPEP/P) (James and Sielecki 1983) and human renin (REN/H) (Imai et al. 1983) are shown. Shadowed boxes indicate the conserved motif of DXGXXXLWV in the active sites

present in E D G P and in 7SBG (Fig. 3). This m o t i f has the sequence D X G X X X L W V and is present near the N-termini of E D G P , 7SBG and the aspartyl proteases.

Expression of EDGP-mRNA in different organs and culture conditions. The accumulation o f E D G P can be modulated in tissue culture by the inclusion o f 2,4-D in the culture medium. To check the effect o f culture conditions on the level o f E D G P m R N A , total R N A was extracted f r o m cultured cells and seedlings, and the levels of E D G P - m R N A were determined by probing R N A gel blots with E D G P . As shown in Fig. 4A, a strong signal was obtained with non-embryogenic callus cultured with 2,4-D, a weak signal with embryogenic callus cultured with 2,4-D, and no signal with somatic embryos cultured without hormone. A strong signal was also obtained with R N A extracted from carrot crown-gall cultures growing on a hormone-free m e d i u m (data not shown). Together, these data indicate that the level o f m R N A m a y be related to the presence of auxin (exogenously or endogenously). However, it is equally plausible that the level of expression is correlated with the degree of disorganized growth. When growing in a disorganized manner (callus, suspension culture, crown gall) the level o f expression is high, but when growing as an organized culture (embryos in suspension) the level o f expression is low. To determine if E D G P is expressed in seedlings, we extracted R N A f r o m the root and shoot portions of 1 0 - d - o l d seedlings. The results show a more intense signal with root R N A as opposed to shoot R N A (Fig. 4A). This m a y reflect the development of dermal tissues of the root. During seedling growth, the E D G P m R N A gradually increased over a 4-d period f r o m an undetectable level in dry seeds (Fig. 4B). This m a y again be a reflection of the rapid development o f the root early in seedling growth. The level of E D G P m R N A was found to be very low in developing carrot fruits (data not shown), yet substantial amounts of E D G P accumulate in the space between the embryo and the endosperm (Satoh and Fujii 1988). Thus, E D G P m a y be slowly synthesized and secreted t h r o u g h o u t seed development. Wound-induction of EDGP mRNA. The synthesis of several extracellular carrot proteins, such as extensin and

Fig. 4A, B. Accumulation of EDGP-mRNA in various cells and tissues (A) and seeds and seedlings (B) of carrot. Total RNAs (10 pg/lane) extracted from somatic embryos (S), embryogenic callus (E), and non-embryogenic callus (N), or the roots (R) and the upper parts (U) of 10-d-old seedlings; RNA from dry seeds (0), or seedlings at 1 (1), 2 (2) and 4 (4) d after sowing, were separated on formaldehyde-containing agarose gels and were transferred to nylon membranes. The blots were probed with EDGP-I

[3-fructosidase (invertase) is induced by wounding and their m R N A s accumulate as a result of wounding. To check whether E D G P m R N A also accumulates in response to wounding, we extracted R N A from carrot slices and subjected it to R N A gel-blot-analysis.

S. Satoh et al. : Wound-induced dermal glycoprotein

437

ing and pathogen invasion often lead to the synthesis and accumulation of proteins with similar functions. In this paper, we report the derived amino-acid sequence of an extracellular dermal glycoprotein (EDGP) that is found in dermal tissues of carrot plants, accumulates in the space between the embryo and the endosperm in mature carrot seeds and is secreted into the culture medium of suspension-cultured cells (Satoh and Fujii 1988).

Time After Wounding (hours)

Fig. 5. Time course of E D G P - m R N A accumulation in carrot tap root in response to wounding. The slices of phloern-parenchyma tissue of storage tap root were incubated in water for 0 (0), 1 (1), 3 (3), 6 (6), 12 (12), 18 (18), 24 (24) and 36 (36) h, and then subjected to RNA-gel-blot analysis as described in Fig. 4

In phloem parenchyma tissue slices of storage tap root of carrot incubated in water, EDGP-mRNA accumulation started at 1 h after slicing. The level of EDGP m R N A reached a maximum at 12 h after slicing (Fig. 5) followed by a slow decline, whereas the level of actinm R N A was not changed. The blot shown in Fig. 5, is the same blot shown in Fig. 5 of Sturm and Chrispeels (1990). The blot was stripped and re-probed with our EDGP probe. The actin controls from this same blot probed with an actin cDNA are shown in Sturm and Chrispeels (1990). This pattern of wound-induction is quite similar to that of 13-fructosidase m R N A (Sturm and Chrispeels 1990)! In this carrot-slice system, accumulation of the m R N A for phenylalanine ammonia-lyase was very rapid and reached a maximum at 3 h after wounding. In contrast to the [3-fructosidase mRNA, the accumulation of EDGP-mRNA was not enhanced by infection of carrots by Erwinia earotovora (soft-rot disease; data not shown).

Distribution of EDGP in the plant kingdom. Because a homolog of EDGP had been reported in soybean seed (7SBG), EDGP may be widely distributed in the plant kingdom. Therefore, total R N A was prepared from the roots of various plants and subjected to RNA-gel-blot analysis in a low-stringency condition using EDGP-1 as a probe. A strong positive signal was found for parsley, a weaker signal for tobacco, and very weak signals for soybean, squash; maize and Chinese cabbage (data not shown). It appears therefore, that other plants contain homologs of EDGP, but how widespread this protein is in the plant kingdom needs to be further investigated. Discussion

The cell wall is the first line of defense of the plant against pathogens, and plants respond to biotic and abiotic stresses by strengthening or modifying the wall. Wound-

Regulation of EDGP. Expression of EDGP is low in somatic embryos, as well as in developing seeds (data not shown) and fully developed seeds. The level of m R N A greatly increases as seedlings grow and form epidermal tissues, or when storage roots are wounded and lay down a new protective layer. Expression is also high in suspension-cultured cells. The high expression in cultured cells is probably best explained by considering that suspension cultures are derived from calluses which themselves usually originate from a wounded surface. Thus, a number of wound-induced proteins, such as invertase (Bacon et al. 1965), hydroxyproline-rich glycoproteins (HRGP; Chrispeels et al. 1974) and peroxidase (Shannon et al. 1971) are also expressed at high levels in response to wounding and in calluses. The walls of cultured cells are rich in extensin (HRGP) (Lamport and Northcote 1960) and invertase (Lauri~re et al. 1988) and the culture medium is rich in peroxidase. The same expression pattern appears to be followed by EDGP. The proteins mentioned above, and a number of other proteins that are induced by wounding or pathogen invasion are part of the general stress response of plant cells. When organized growth is induced in disorganized callus or suspension cultures, there is a drop in the expression of these wound-induced proteins. Thus, the shoots that grow out of a lettuce callus are low in HRGP, although the callus itself has a high level of this protein (Chrispeels and Sadava 1974). We have found that when 2,4-D is withdrawn from an embryogenic carrot cell culture, the clumps form somatic embryos and these embryos have very low levels of extracellular invertase (data not shown). Similarly, the expression of EDGP is low in these somatic embryos (Fig. 4). Moreover, the expression of EDGP is non-embryogenic callus is much higher than that in embryogenic callus. It appears, therefore, that the expression of EDGP is not so much governed by auxin, as we thought initially (Satoh and Fujii 1988), but by the degree of organization of the tissue. Other hormones (gibberellic acid, kinetin, abscisic acid, ethylene) and salicylic acid also did not induce EDGP. Neither did heavy metals such as CuC12, CdC12 or 0 . 3 M NaC1 induce EDGP expression (data not shown). Unique features of the derived amino-acid sequence. The derived amino-acid sequence of EDGP has at least two interesting features. First, there is considerable aminoacid identity with 7SBG, a protein that is released when soybean seeds are briefly soaked in warm water. This property of 7SBG indicates that it may also be a dermal protein and we suggest that EDGP and 7SBG are members of a new family of proteins present in plants. Of particular interest is the presence in EDGP of an aminoacid sequence motif found at the active site of a num-

438 b e r o f secreted a c i d p r o t e a s e s o f the a s p a r t y l - p r o t e i n a s e type. A s i l l u s t r a t e d in Fig. 3, this sequence m o t i f is D T G X X X L W V f o r the a c i d p r o t e i n a s e s a n d D L G X X X L W V for E D G P . I n all cases, this m o t i f is f o u n d n e a r the a m i n o t e r m i n u s o f the proteins. Very little w o r k has been d o n e o n the a s p a r t y l p r o t e i n a s e s f r o m p l a n t s , b u t r e c e n t evidence indicates t h a t a n e x t r a c e l l u l a r a s p a r t y l p r o t e i n a s e p a r t i c i p a t e s in the d e g r a d a t i o n o f p a t h o g e n e s i s - r e l a t e d p r o t e i n s in t o m a t o ( R o d r i g o et al. 1989, 1991). W h e t h e r E D G P c a n f u n c t i o n as a n a s p a r t y l p r o t e i n a s e in the e x t r a c e l l u l a r space o f p l a n t cells r e m a i n s to be d e t e r m i n e d . T h e s u b s t i t u t i o n o f a leucine residue for a t h r e o n i n e r e s i d u e in the D T G t r i p e p t i d e casts s o m e d o u b t o n this possibility. S o m e viral p r o t e i n a s e s have D S G at their active site, b u t n o n e is k n o w n with a D L G motif. Supported by a contract from the United States Department of Energy (Energy Biosciences) (to M.J.C.) and a Grant-in-Aid for Special Research on Priority Areas (01660002, Cellular and Molecular Basis for Reproductive Processes in Plants) from the Ministry of Education, Science and Culture, and by the Fund from Basic Research Core System of Science and Technology Agency, Japan (to S.S.).

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