cdna cloning of carrot (daucus soluble acid with wall physiol. (1994) 104: 1351-1357 cdna cloning of...

Plant Physiol. (1994) 104: 1351-1357

cDNA Cloning of Carrot (Daucus carota) Soluble Acid ,6-Fructofuranosidases and Comparison with the

Cell Wall lsoenzyme

Christoph Unger, Markus Hardegger, Susanne Lienhard, and Arnd Sturm*

Friedrich Miescher-lnstitut, Postfach 2543, CH-4002 Basel, Switzerland

Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid 8-fructofuranosidase (BF) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular BF). Using antibodies against isoenzyme I of carrot soluble BF, we isolated several cDNA clones encoding polypeptides with sequences characteristic of BFs, from bacteria, yeast, and plants. l h e cDNA-derived polypeptide of one of the clones contains all partia1 peptide sequences of the purified isoenzyme I and thus codes for soluble acid BF isoenzyme 1. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. lhese two soluble BFs, have acidic isoelectric points (3.8 and 5.7, respectively) clearly different from the extracellular enzyme, which has a basic isoelectric point of 9.9. Marked differences among the three nucleotide sequences as well as different hybridization patterns on genomic DNA gel blots prove that these three isoenzymes of carrot acid BF are encoded by different genes and do not originate from differential splicing of a common gene, as i s the case in the yeast Saccharomyces cerevisiae. All three carrot acid BFs, are preproenzymes with signal peptides and N- terminal propeptides. A comparison of the sequences of the soluble enzymes with the sequence of the extracellular protein identified, C-terminal extensions with short hydrophobic amino acid stretches that may contain the information for vacuolar targeting.

The acid PFs (EC 3.2.1.26, also called invertase) are a group of Suc-hydrolyzing enzymes, each of which exists as several isoenzymes. The isoenzymes have broad pH optima for activity between pH 4.5 and 5.5. The polypeptides have different pIs and accumulate either in the vacuole or in the extracellular space. In plants, acid PFs are considered to be important for growth, providing growing tissues with hexoses as a source of energy and carbon (ap Rees, 1974). It has also been suggested that acid PFs are involved in Suc partitioning (Eschrich, 1989), osmoregulation (Meyer and Boyer, 1981; Wyse et al., 1986; Perry et al., 1987), gravitropism (Wu et al., 1993), and the responses of plants to wounding (Matsushita and Uritani, 1974; Sturm and Chrispeels, 1990) and infection (Krishnan and Pueppke, 1988; Sturm and Chrispeels, 1990; Benhamou et al., 1991; Storr and Hall, 1992).

Although extracellular and soluble acid PFs have been cloned from various plant species (Sturm and Chrispeels, 1990; Arai et al., 1992; Klann et al., 1992; Elliott et al., 1993; Hedley et al., 1993), there has been no molecular character-

ization or comparison of the major isoenzymes from the same plant. These are the aims of the present study. The molecular properties of the different isoenzymes will, for example, clarify whether the different proteins originate from a common gene by differential splicing, as in the yeast Sacckaro- myces cerevisiae (Carlson and Botstein, 1982), or are encoded by distinct genes. The results may also help us to understand how the different isoenzymes are targeted to their different subcellular locations. However, the most interesting question is that of the specific roles of the different isoenzymes, and answering this question is the main goal of our future work.

As a model plant, we are using carrot (Daucus carota L. cv Nantaise), which has a tap root that stores mainly Suc and hexoses (Alabran and Mabrouk, 1973; Phan and Hsu, 1973). This carrot cultivar contains at least three isoenzymes of acid PF: an extracellular form ionically bound to the cell wall and two soluble acid PFs (isoenzymes I and 11), which are most likely located in the vacuole (Unger et al., 1992). The extracellular acid PF has been purified (Laurière et al., 1988) and cloned (Sturm and Chrispeels, 1990; Ramloch-Lorenz et al., 1993). Isoenzymes I and I1 of the soluble acid PF have been characterized (Laurière et al., 1988; Stommel and Simon, 1990; Unger et al., 1992), and isoenzyme I has been purified and partially sequenced (Unger et al., 1992). In this commu- nication, we report the cDNA cloning of isoenzymes I and I1 and a comparison of their sequences with that of the extracellular protein.

MATERIALS AND METHODS

Plant Material

Carrots (Daucus carota L. cv Nantaise) were grown in a field near Basel, Switzerland. Buds, flowers, green seeds, and mature seeds were harvested in the 2nd year of development. Carrot seeds were surface sterilized with absolute ethanol for 30 min and germinated on moist filter paper in the dark at 2OOC. Seedlings were harvested 7 d postgermination. Cells of D. carota, Queen Anne’s lace (wild carrot cell culture line WOOlC; Sung, 1976), were grown in Murashige and Skoog (1962) medium supplemented with 0.1 mg/L 2,4-D at 26OC in the dark. The suspension cells were transferred at weekly intervals into fresh Murashige and Skoog medium.

* Corresponding author; fax 41-61-697-39-76. Abbreviations: PF, P-fructofuranosidase; pI, isoelectric point. 1351

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1352 Unger et al. Plant Physiol. Vol. 104, 1994

Purification of Anti-BF IgC

Anti-25-kD IgG and anti-43-kD IgG were purified by affinity chromatography. For this purpose, purified soluble PF isoenzyme I (1 mg) was coupled to cyanogen bromide- activated Sepharose 4B (Pharmacia, Uppsala, Sweden) as described in the manufacturer’s protocol. The affinity gel was tumbled overnight with anti-25-kD or anti-43-kD antiserum (Unger at al., 1992) and washed successively with 5 column volumes of PBS (10 m~ Na2HP04 and 150 mM NaCl [pH 7.5]), PBS plus 0.1% Tween 20, borate buffer (0.1 M Na2B407, 0.1% Tween 20, and 1 M NaCl [pH 8.5]), and PBS again and then eluted with 4 column volumes of Gly/NaCl buffer (0.2 M Gly and 0.5 M NaCl [pH 2.21). The eluate was immediately neutralized with 1 M Tris-HC1 (pH 9), and proteins were precipitated with (NH4)2S04 [ l O O mL of eluate plus 25 g of (NH4)2S04]. Affinity-purified IgG was harvested by centrif- ugation and redissolved in 0.5 mL of PBS containing 0.1% sodium azide.

cDNA Synthesis and Screening Procedures

Total RNA from flower buds and 7-d-old carrot seedlings was prepared by the method described by Prescott and Martin (1987) modified by adding 17.5 mg of Polyclar AT (Serva, Heidelberg, Germany) per gram of tissue before grinding in liquid nitrogen. Poly(A)+ RNA was isolated by oligo(dT)-cellulose chromatography (Maniatis et al., 1989) and was used for cDNA synthesis (cDNA Synthesis System Plus, Amersham). After EcoRI linkers were added, the cDNA was ligated into the Xgtll vector and packaged into phages [2.5X 106 plaque-forming units/5 pg of poly(A)+ RNA]. The nonamplified expression libraries were screened according to the method of Young and Davis (1983) with both the anti- 25-kD and the anti-43-kD IgG. Five double-positive clones with slightly different restriction enzyme patterns were isolated from the flower bud expression library. Two identical clones were isolated from the seedling expression library, which reacted only with anti-25-kD IgG. Both strands of the cDNA clones were sequenced by the dideoxy- nucleotide chain-termination reaction (Messing, 1983).

Analysis of RNA and DNA

For RNA gel-blot analyses, 10 Wg of total RNA were separated on 1.2% agarose gels containing formaldehyde (Maniatis et al., 1989). Genomic DNA (Mettler, 1987) was

restricted with EcoRI or KpnI and separated on 0.7% agarose gels (5 pg/lane) (Maniatis et al., 1989). RNA and DNA gel- blot analyses were performed on nylon membranes (Hybond- N, Amersham) with probes 32P-labeled by random priming (Maniatis et al., 1989). Gene-specific 3’ fragments of the cDNAs for the extracellular and the two soluble acid PFs were used as probes (extracellular acid PF: EcoIr.I/EcoRI, 750 bp; isoenzyme I of soluble acid PF: SphI/EcoliI, 1400 bp; isoenzyme I1 of soluble acid PF: EcoRIIEcoRI, 1750 bp). Prehybridizations were done at 65OC in 6X SSC: (Maniatis et al., 19E19), 5X Denhardt’s solution (Maniatis et al., 1989), 100 pg/mL denatured calf thymus DNA, and 0.5% SDS. Hybrid- izations were carried out in the same buffer overnight at 65OC. ‘The blots were washed twice with 0 . l X SSC and 0.5% SDS at 65OC for 30 min.

Analysis of DNA and Protein Sequences

Computer-assisted analyses of DNA and protein sequences were carried out with the Beta version of the Genetics Com- puter Group Sequence Analysis Software Pac’kage (version 7.3-Unix, June 1993; University of Wisconsin, Madison, WI).

RESULTS AND DISCUSSION

Characterization and cDNA Cloning of Acid @Fs from Carrot

One insoluble extracellular and two soluble intracellular acid PFs have been characterized in the carrot cv Nantaise (Unger et al., 1992). The three enzymes hydrolyze SUC and, with less efficiency, raffinose (Table I). The three polypeptides are N-linked glycoproteins (Sturm, 1991; Unger et al., 1992) with broad acid pH optima for enzyme activity (Lau-

,rière et al., 1988) and marked differences in their pIs (Table I). Extracellular acid PF has a pI of 9.9 and is ionically bound to the cell wall (Laurière et al., 1988). The two soluble enzymes (isoenzymes I and II), with acidic pIs of 3.8 and 5.7, most likely accumulate in the vacuole (Unger et al., 1992).

The extracellular PF was purified from cell walls of suspension-cultured carrot cells (Laurière et al., 1988). A corresponding cDNA clone (Sturm and Chrispeels, 1990) and a genomic clone (Ramloch-Lorenz et al., 1993) have been isolated and characterized.

Isoenzyme I of soluble acid PF was purified from the soluble protein fraction of mature carrot seeds, and partia1 peptide sequences were determined (a total of 96 amino acid

Table 1. Characterization of acid BF isoenzvmes in carrot ~

Soluble Acid @F Sclluble Acid @F lnsoluble Acid @F Characteristic lsoenzyme I lsoenzyme II Cell Wall Bound

Location Vacuole Vacuole Extracellular Molecular mass o n SDS-poly- 68 n.d.“ 63

acrylamide gels (kD) Pl 3.8 5.7 9.9 pH optimum for activity 5.4 5.4 4.5 Raffinose cleavage (relative 54 40 70

activity in %)

a n.d., Not determined.



Acid (3-Fructofuranosidases from Carrot 1353

CCTAGTCTATGCTATTTAATTTTCCATCCAATGGATACCACCATTTTCTCCCCTCTCGT MetAspThrTyrHisPheLeuProSerArg

AGAACTTCTTTTCACTTTCAACCTCAAGAAAATTGGATGAATGATCCTAATGGTCCCTTA ArgThrSerPheHisPheGlnProGlnGlnGluAsnTrpM~tA~nAspProAsnGlyProLeu

43 kD

60

1 2 0

180

240

300

360

420

480

5 4 0

600

660

720

7 8 0

8 4 0

900

960

1020

1 O80

TTG~TCGAGTTTAGATGATGACAGGCITGITTACTATGCATTGGGT*CTTATGACCCG 1 1 40 LeuL~~SerSe~LeuA~~AspAspArgH~5AspTyrT~rAlaLeuGlyThrTyrAspPro

ATAAATGAT~GTGGACACCCGATAACCCGGAGTTGGATGTGGGCATTG~GTTGAGATTG i 200 IleASnAspLysTrpThrPrORspASnProGluLeuAspValGlyIleGlyLeuArgLeu

G A T T A T G G C ~ T A T T A T G C A T C A A A A A C A T T ~ A T G A T C A A G A T ~ A G A A A G ~ G A C T A 1260 ASpTyrGlyLy~TyrTyrAlaSerLysThrPheTyrAspGlnAspLysGluArgArgLeu

CTTTGGGGTTGGATTGGTGAAAGCGACAA~GAAAGTACTGATCTTCTC~GGGTTCGGC~ 1 3 20 LeuTrpGlyTrpIleGlyGluSerAspAsnGluSerThrAspLeuLeuLysGlyTrpAla ********* - SerValGlnSerIleProArgThrValValPheAspLysLysThrGlyThrAsnIleLeu

CAGTGGCCAGTCAAGGA AG'hGA AAGCTTG AGA ITGAGA AGTTATGAA AkAATGATGTG 1 4 4 O GlnTrpProValLysGluValGluSerLeuArgSerArgSerArgS~rTyrGluIleA~nA~pVal

- TCTGTTCAGAGCATTCCAAGAACAGTGGTTTTTGACAAAA~ACTGGAACTAATATACTT i 380

GAGCTTAAACCAGGATCACTTGTGCCGCT~AAGATAAGCTCAGCAGCACAGTTAGATATA i 500 G1ULeULy~ProGlySerLeuValPlOLeuLySlleSeIAlaAlaGlnLeuA~pIle

GTCGCAAGTTTTGAAGTTGATGAAGAGGCATTTAAAAGAACATATGAAGCAGATGCGAGT i 560 ValAlaSerPheGluValAspGluGluAlaPheLysArgThrTy~GluAlaAspAlaSer

TACAPITTGCACTGCCAGTGAGGGTGCTGCTGGTAGAGGCATTTTAGGACCATTTGG~TT i 6 20 TyrAsnCysThrAlaSerGluGlyAl~AlaGlyArgGlyIleLe~GlyProPheGlyIle -********* C T G G T T C T T G C T G A T G A T C C A C T C T C C G A A T T A A C T C C G G i 680 LeUValLeuAlaAspAspProLeuSe~GluLeuThrProValTyrPheTyrIleAlaLy~

25 kD GGTGTTGATGGAAATGCAAAAACTTATTTCTGTGCTGATC~ATCAAGATCATCAACTGCT i 740 GlyValAspGlyAsnAlaLysThrTyrPheCysAlaAspGlnSerArgSerSerThrAla

TCTGATGTTGATAAAGAGGTTTATGGTAGTGATGTTCCCGTGCTCCCTGGTGAAAGCTTA 1800 SerAsPValAspLySG1UValTYrGlySerAspValProValLe~ProGlyGluserLeu

TCCATGAGATTATTGGTGGATCATTCCATTGTTGAAAGTTTTGCTCAAG~AGGTAGAACA 1 860 SesMetArgLeuLeuValASpHisSerIleValG~uSerPheA~aGlnGlyGlyAr~h~

GTTATAACGTCACGAGTTTATCCAACCAGAGCAATTTACAGTGCAGCAA~AGTCTTTTTG 1 9 20 V a l I l e T h r S e r A r g V a l T y r P r o T h ~ A ~ g A l a I l e T y r S e r A l a A l a A r g V a l P h e L e u

TTTAACAATGCCACAGGAGTGAGTGTCACAGCGTCAGTGAAGGCATGG~*AATGG~TT~A 1 $80 PhrASnASnAlaThrGlyValSerValThrA~aSerValLysAlaTrpGlnMetAlaSer ********* ,

GCMCTCTCAAACCTTTCCCATTCGATCAGTTGTAACCTTGTA~TATAGCTACCCCAGTG 2040 AlbThrLeuLy~ProPheProPheAspGlnLeu

TGTACCTATCTTTCCTTGGGTCAACAAATAATTGAAGGACGAGAGTTTCGCCTGGCAGCA 2100

AATCGTGATTTATTGAAGGAACAAAATTTGGAAGTGATTCTATTATGAAGAGAGGTATTT 2 1 60

TTTATGATGGACTTGCAAGTTGCAACCAGAAGGTTAGAAAGAT~GGCTTGTCCCTATTGC 2220

ATATATGTATTGTATTCTTTCTTTTTTTTGATTATTTGTGTTGTATT~CAATCTGAGT 2280

GAAATTTGCAATATATAGTGAAGAGTGAAATTTGGATTAAAAAaAAA 2327

residues). Polyclonal antibodies against a deglycosylated N- terminal (43-kD polypeptide) and a C-terminal fragment (25- kD polypeptide) of the enzyme were raised and characterized (Unger et al., 1992). For the isolation of a cDNA clone for isoenzyme I of soluble acid PF, poly(A)+ RNA from flower buds was used for cDNA synthesis. For the isolation of a cDNA clone for isoenzyme 11, poly(A)' RNA was extracted from 7-d-old carrot seedlings (Unger et al., 1992). cDNA was synthesized and Xgtl 1 expression libraries were constructed. The libraries were screened with affinity-purified IgG (Young and Davis, 1983), and severa1 clones were obtained.

Nucleotide Sequences of the cDNA Clones for Soluble Acid /3F and Analysis of the cDNA-Derived Amino Acid Sequences

The five cDNA clones isolated from the flower bud expression library fall into three different classes (1, 2, and 3) with three identical cDNA clones belonging to class 1 and one clone each to classes 2 and 3. The class 2 and 3 clones code for very similar polypeptides (99.1% identity) with sequences containing a11 partial peptide sequences obtained from the purified protein (underlined in Fig. 1). These cDNA clones, therefore, code for isoenzyme I of soluble acid PF. Because the six amino acid residues by which the class 2 and class 3 polypeptides differ are not located in the peptide sequences detennined, it is impossible to decide which of the two clones is the template for isoenzyme I. The polypeptide encoded by the class 1 cDNA clones is 98.6 and 98.3% identical with the class 2 and class 3 polypeptides, respectively, and 4 of the 9, respectively, 11 -amino acid differences are located in two of the tryptic peptides sequenced. Because these differences, e.g. the replacement of Pro by Arg, probably have drastic effects on the physicochemical properties of the peptides, they most likely do not comigrate, and, therefore, it is likely that we missed them. Again, it is impossible to decide which of the clones is the template for the soluble acid PF polypeptide. In summary, the sequences detennined from the purified protein do not contradict the idea that a11 three mRNAs, which gave rise to the three cDNA classes, are templates for isoenzyme I.

The three classes of cDNAs differ in their nucleotide sequences by up to 36 single-base-pair exchanges, mainly in the third codon position (for details see Fig. 2 and sequences in the EMBL data base), and, therefore, the majority of the changes observed have no effect on the amino acid sequence. The differences suggest that the three corresponding genes

Figure 1. Nucleotide sequence of the class 2 cDNA for isoenzyme I of soluble acid PF. Nucleotides are numbered from the first base of the cDNA clone, excluding the EcoRl linkers. The deduced amino acid sequence in the three-letter code is indicated below the nucleotide sequence. The sequence underlined and marked "43 kD" indicates the N terminus of both t h e mature protein and the 43-kD polypeptide (Unger et al., 1992). The bold line marked "25 k D indicates the N terminus of the 25-kD polypeptide (Unger et ai., 1992). The two underlined but unmarked sequences are partial sequences of tryptic peptides generated from t h e 43-kD polypeptide. The sequences marked by asterisks (*) denote potential glycosylation sites (Asn-X-Ser/Thr).




may have originated by the following hypothetical cascade of events: (a) duplication of the ancestral gene for isoenzyme I, (b) slight divergence of the sequences of the two genes by base-pair exchanges generating the genes for the class 1 and class 3 cDNAs, and (c) homologous recombination between alleles of the genes for the class 1 and class 3 cDNAs, followed by a few additional base-pair exchanges in the sequence of the recombined gene (class 2). Because the analysis of DNA from wild carrot for isoenzyme I sequences also indicates the presence of different but highly related sequences (see below), these events most likely took place before the breeding of the cv Nantaise.

The polypeptide encoded by the class 2 and class 3 cDNAs (Fig. 1; only the sequence of the class 2 cDNA is shown) has 661 amino acid residues with an M, of about 74,000. The calculated pl of the mature isoenzyme I is 4.5, which is in agreement with the experimentally determined pI (Table I). The presence and location of the N-terminal sequences of both the 43-kD and the 25-kD polypeptides in the cDNA- derived amino acid sequence (underlined and marked in Fig. 1) confirm that the two polypeptides characterized by Unger et al. (1992) are N- and C-terminal fragments of the mature protein. The fragmentation observed for isoenzyme I of carrot soluble acid PF has also been observed for acid PF from tomato (Yelle et al., 1991), melon (Iwatsubo et al., 1992), and mung bean (Arai et al., 1991). The sequence of the polypeptide encoded by a cDNA clone for the mung bean enzyme revealed that the heterodimeric form originates from cleavage of a single polypeptide, with the 30-kD moiety on the N- terminal side and the 38-kD moiety on the C-terminal side (Arai et al., 1992). The subunits of the mung bean heterodi- mer are not formed from the monomer during purification (Arai et al., 1991) and, like the subunits of the carrot heter- odimer, are tightly associated. It is interesting that the ratios between the full-length proteins from both mung bean and carrot and the respective polypeptide fragments are devel- opmentally regulated: In very young seedlings the full-length protein predominated, whereas with increasing seedling age the fragments were more abundant (Arai et al., 1991; the data for the carrot protein are not shown).

Both fragments of the purified carrot enzyme are N-gly- cosylated with high-Man- and complex-type glycans (Unger et al., 1992), and potential N-glycosylation sites were found in the respective cDNA-derived amino acid sequence (Asn- X-Ser/Thr; four sites in the N-terminal fragment and two in the C-terminal fragment, marked by asterisks in Fig. 1).

A comparison of the N-terminal sequence of mature soluble acid PF isoenzyme I (first sequence underlined in Fig. 1)

200 Figure 2. Pairwise comparison of t h e sequences of the different classes of cDNA ciones I I

with the sequence of the cDNA-encoded polypeptide predicts that the primary translation product has a 115-amino-acid leader sequence, which is far too long for a normal signal peptide (Von Heijne, 1983). Thus, the polypeptide translated from the mRNA for soluble acid PF isoenzyme I is probably a preproprotein with a signal peptide and an N-terminal propeptide. Similar protein structures have been identified for the extracellular acid PF of carrot (Sturm and Chrispeels, 1990) and soluble acid PF of tomato (Klann et d. , 1992) and mung bean (Arai et al., 1992).

The amino acid sequence derived from a piitative cDNA clone for isoenzyme I1 of soluble acid PF is shown in Figure 3. The open reading frame encodes a polypeptide of 651 amino acid residues with a calculatedM, of 72,700. In analogy to isoenzyme I, the polypeptide has a 100-amino acid leader sequence, again indicating that the primary translation product is a preproprotein. The calculated pI of the mature polypeptide is 5.7, which is in good agreement with the pI determined experimentally (Table I). In view of this good agreement and the close relationship of the sequences of the mature polypeptides of isoenzymes I and I1 (68% identity and 81% similarity), this cDNA clone most likely codes for isoenzyme 11.

Comparison of the cDNA-Derived Amino Aciid Sequences with Known Sequences of Plant Ai5d @Fs

The cDNA-derived amino acid sequences of isoenzyme I and isoenzyme I1 of soluble acid PF were compared with the sequence of the extracellular enzyme (Sturm and Chrispeels, 1990). The amino acid sequence of isoenzyme 1 is 64% similar (44% identical) and that of isoenzyme I1 is 62% similar (44% identical) to the sequence of cell wall PF. Figure 3 shows the sequence comparison with bars between individual amino acid residues indicating identity and colons marking conservative replacements. No significant homology was found among the leader peptides of the three polypeptides. Marked homologies were identified between the sequences of the mature proteins, including a "PF motif" (boxed in Fig. 3) and a conserved Cys residue (Fig. 3; residue 313, marked by an arrow). Both sequence elements were identified as constitu- ents of the known amino acid sequences of PFs from bacteria, yeast, and plants (Sturm and Chrispeels, 1990).

The regions of marked homologies comprise two large polypeptide domains (Fig. 3; residues 120-450 and 510-620) linked by a stretch of about 50 amino acid residues with very little homology among the three proteins (hinge domain). The two regions of high homology, e.g. of isoenzyme I of

6 0 0 1 O00 1400 1800 2200 I I I I I I I I I

for isoenzyme I of soluble acid @F carried out by the GapShow program of the Cenetics Com- puter Group Sequence Analysis Software Pack- age. The cDNA sequences are indicated by horizontal lines marked 1, 2, or 3, corresponding to the three cDNA classes. Each single mismatch is marked by a vertical bar. The num-

sequences in number of base pairs.

1

III IIIII I I II I I I I I I I I I I I I I I I II I I I

1 I I I I I I I III IIIII I II I I I I I I I II I I I I I I II I I I

2

bers above the lines indicate t h e length of the I I I I I I I I I 1



SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

SI1

SI

cw

Acid 0-Fructofuranosidases from Carrot 1355

4.0 MEHPITISHYTPLPDGEHSPSLT .... TTNTAEQSSRRRSLTF . . . VLLF .... MDTYHFLPSRDLEHASSYTPRPDSPETRHEPDPDRSKTNRRPIKIV ...................................... MGVTIRNRNYDH

: I : I I I I I I : I : 1 1 1 : :

: I 1

SSILAACLVMGTMVLF ............... PNSGNEAVEKSTVVPEETV SSVLLSTLILSFV1FLLVNPNVQQVVRKKVSKNSNGEDRNKASKSP.EML

GSL . . . PFLQSLLAILLVTTTTLHI . . . . . . . . . . . . . . . . . . NGV.EAF

I I : I I : : : : : : : I I I : I I I : : I : :: I : : : I 1 1 :: : I :

8? 120 EVAPRGVAEGVSMKSFRRPALNAEPPANFPWNSNVLSWQRSSFHFQPNQN

GPPSRGVSQGVSEKSFR ....Q ATAEPSYPWTNDMLSWQRTSFHFQPQEN

HEIHYNL.QSVGAENVK .... QV . . . . . . . . . . . . . . . HRTGYHFQPKQN

: : I I I : I I I I l l l I : : : I1 : : l l l l l I I I I I I : I : I : / : : 1 : l l : : l l l l : I

160

- 200

PVAMVTDHWYDVNGVWTGSATILPDGQIVMLYTGSTNES ... VQVQNLAY PFAMQPDQWYDINGVWTGSATVLPDGKIVMLYTGDTDDL . . . VQVQNLAY EPAIFPSKPFDKYGCRSGSATILPGNKPVILYTGIVEGPP~VQVQNYAI

I I1 l : l l l : l l I l I I l l l : l l l l l l l l l l l 1 : : l l l l l l l l I : I : I I : l l l l : l l : I I : I I I l :: I I I I I I

240 PADPSDPLLIEWVKYPGNPVLVPPPGIDFKDFRDPTTAWRTPEGKWRLII

PANLSDPLLLDWIKYP@NPVMFPPPGIGSTDFRDPTTRWIGRDGKWRITI

PANLSDPYLRKWIKPDNNPLVVANNGENATAFRDPTTAWLDKSGHWKMLV

I I : I I I I I : : I : I I I : I l I : I I I I I : I I I I I I I I I : I I I I : I I I I I I I I I I I I : I ) : : : I I I I I I I O I : : : I I : : :

280 i 320 . I

GSKLNKTGISLVYDTVDFKNFTLLDGVLHAVHGTGMWECVDFYPVSKFGE

GSKVNKTGISLMYKTTDFITYELLDNLLHAVPGTGMWECVDFYPVSVTGS

GSKRNRRGIAYLYRSKDFIKWTKAKHPIHSQANTGMWECPDFFPVSLKGL

l l l : l l l l l l l : l I I I 1 1 1 : I111 l l l l I I I l l l l l l l I I I I I : I I : I : I I I : : : I : I I I I I I I I : I l I : I

360 NGLDTSFDGVGVKHVMKASLDDDRNDYYAIGTYDPVSGKWVPDNPELDVG

NGLDTSVNGPGVKHVLKSSLDDDRHDYYALGTYDPINDKWTPDNPELDVG

NGLDTSVTGESVKHVLKVSLDLTRYEYYTVGTYLTDKDRYIPDNTSVDGW

400 IGLRYDYGIYYASKTFYDSNKKRRVLWSWIKETDSEISDVRKGWASVQGI

IGLRLDYGKYYASKTFYDQDKERRLLWGWIGETDSESADLLKGWASVQSI

AGLRYDYGNFYASKTFFDPSKNRRILWGWRNESDSTAHDVAKGWAGIQLI

I I I I I I : I I I I I I : I I I I I I I : I I I I : I I I I I : : I 1 I I I I I I I I I I I I I I I I I : I I I I I I I I I I : I 1 : I I I I : : I I I : I

I I I I I I I I I I I I I I I I : I I I : I I : I I l l l l l I : I l l l 1 l l : I I I I I I I : l l l l l l : l l : l l : l l l l I I1 I : I I I I : : I I

l l l : : l l / / I / : / / / ~ / 1 1 : I1 : : : l l : I : : I 1 l : l 1 ) ) : : ) : : I ) ) : / : I 1 1 : : : I I ) ) : I

440 PRTILFDPKTGSNLLQWPVEEVNKLRLNKTVFE.NVEINTGAVLPLE1GS

PRTVVFDKKTGTNILQWPVKEVESLRSRSYEID.DVELKPGSLVPLK1SS

PRTLWLD.PSGKQLMQWPIEELETLRGSKVKFSRKQDLSKGILVEVKG1T

480 GSQLDITAEFEVDKESLERVQETN . . . . EVYDCKNNGGSSGRGALGPFGL AAQLDIVASFEVDEEAFKGTYEAD . . . . ASYNCTASEGAAGRGILGPFGI AAQADVEVTFSFKSLAKREPFDPKWLEYDAEKICSLKGSTVQGGVGPFGL

520 560

: I I I I I 1 1 1 1 I : 17: I : ) : I I I I I I I I I : / / I / : I I : : : : : I 1 : I I I I :

LILADKDLSEQTPVYFYIAKGSGGNLRTFFCADHSRSSKAVDVDKEIYGS

LVLADDPLSELTPVYFYIAKGVDGNAKTYFCADQSRSSTASDVDKEVYGS

LTLASEKLEEYTPVFFRVFKAQNTH.KVLMCSDATRSSLKEGLYRPSFAG

l : l l l I / / l l l l l l l l l l : I 1 : l : l l l l : l l l l I l l l l l : l l I I ( ( : 1 1 ( ( / : I : 1 : : : 1 : I 1 ( ( 1 :: : :::

600 ..VVPVLRGEKLTMRILVDHSIVESFSQGGRTCITSRVYPTKAIYNNAKV

..DVPVLHGESLSMRLLVDHSIVESFAQGGRTVITSRVYPTRAIYSAARV

FVDVDLATDKKISLRSLIDNSVVESFGAKGKTCISSRVYPTLAVYENAHL

I I I I : I I I I l : l l l l l I I I I I I I I I I I 0 l I I I I : I I I ( : I I I : : : I : I l : l : l : l l l l : I : I I I I I I I I I : I I : :

640 FLFNNATEARIIASLNIWQMNTA.QRQTHFADLV1

FLFNNATGVSVTASVKAWQMASATLKPFPFDQL.. I I I I I I I : : l l : 1 1 1 I : I : I

: : l l l : : : I I I YVFNNGSETITVENLDAWSMKKPLRMN . . . . . . . .

soluble acid PF, are predicted to form P-sheets (secondary structure prediction of proteins by Chou and Fasman with the PepPlot program of the Genetics Computer Group Se- quence Analysis Software Package; data not shown). It is interesting that the hinge region connecting the two domains contains the cleavage site between the 43-kD and the 25-kD polypeptide of isoenzyme I (between amino acid residues 491 and 492; marked in Fig. 3 by an arrowhead).

A schematic comparison of the amino acid sequences of the cDNA-encoded soluble acid PFs with the sequence of the cell wall-bound enzyme is shown in Figure 4. Because of the high homology between the sequences of the two isoenzymes for soluble acid PF, their common protein structure is shown. AI1 three acid PFs from carrot are preproenzymes with signal peptides and propeptides located at the N terminus (see above and Sturm and Chrispeels, 1990). The signal peptides are required for entry into the ER and, thus, into the secretory pathway (Blobel, 1980). The function of the N-terminal propeptides is unknown, but it may play a role in the regulation of enzyme activity and/or in the folding and the stability of the polypeptides (Winther and SBrensen, 1991). In contrast to the sequence of the cell wall enzyme, the sequences of the soluble acid PFs include short C-terminal extensions (Phe- Pro-Phe-Asp-Gln-Leu in the case of isoenzyme I and His- Phe-Ala-Asp-Leu-Val-Ile in the case of isoenzyme 11) (Fig. 3). Indirect evidence suggests that the two soluble enzymes are located in the vacuole (Unger et al., 1992). Therefore, in analogy to studies of barley lectin (Bednarek and Raikhel, 1991) and tobacco chitinase (Neuhaus et al., 1991), the C- terminal extensions may contain the infonnation for vacuolar targeting. This hypothesis is supported by the presence of short hydrophobic amino acid stretches (Phe-Pro-Phe in isoenzyme I and Leu-Val-Ile in isoenzyme 11) in the extensions of both soluble acid PFs, which may form the critica1 core of the vacuolar sorting signal (Bednarek and Raikhel, 1992).

The sequences of the three carrot enzymes were also compared with the sequences of other known plant acid PFs, and a phylogenetic tree (dendrogram) was generated (Fig. 5 ) . The amino acid sequence of isoenzyme I of carrot soluble acid PF is closely related to the sequence of acid PF from tomato (Klann et al., 1992), whereas the sequence of isoenzyme I1 has highest homology to the sequence of acid PF from bean (Arai et al., 1992). The sequence of carrot cell wall PF (Stunn

Figure 3. Comparison of the cDNA-derived amino acid sequence of isoenzyme II of soluble acid PF (s l l ) with the amino acid sequences of isoenzyme I (SI, sequence derived from the class 1 cDNA) and of extracellular acid PF (cw). The amino acid sequences are in the one-letter code and have been aligned by introducing gaps (. . .) to maximize identity. Vertical bars indicate identical amino acid residues, and colons indicate conservative replacements. The boxed region represents a peptide domain that is conserved in all pFs (PF motif). The arrow (1) marks a Cys residue that is also conserved in ali PFs and that seems to be important for the catalytic activity (Sturm and Chrispeels, 1990). The arrowhead between the amino acid residues 491 and 492 of isoenzyme I (SI) marks the site at which the full-length polypeptide is cleaved into the 43-kD and the 25-kD polypeptides.




... N-terminalsignal peptlde pTOpept|dBC-terminalextension

mature protein

Figure 4. Schematic comparison of the amino acid sequencesdeduced from the cDNAs for carrot extracellular acid /3F (cw) andisoenzymes I and II of carrot soluble acid /3F (si,II). The matureproteins share 64% similarity and 46% identity.

Kpnl

B

and Chrispeels, 1990) shares high homology with the se-quence of an acid /3F from potato with a putative extracellularlocation (Hedley et al., 1993), and both sequences are clearlydistinct from the sequences of the other plant acid /3Fs.

Genomic DNA Gel Blot

DNA gel-blot analyses (Southern, 1975) were performedto determine the number of genes that correspond to theisolated cDNA clones. DNA from leaves of the carrot cvNantaise (Fig. 6A) and from suspension cells of wild carrot(Fig. 6B) was digested with EcoRl or Kpnl. The DNA gel blotswere probed with gene-specific fragments of the cDNAs forisoenzyme I or isoenzyme II of soluble acid /SF. The hybridi-zation patterns were compared with the hybridization pattern

- potato-cw

- carrot-si

- carrot-Ill

Figure 5. Phylogenetic tree for plant acid /3Fs. The dendrogram wasgenerated by comparison of the known nucleotide sequences ofplant acid /3F by the PileUp program of the Genetics ComputerCroup Sequence Analysis Software Package, potato-cw, Acid ,3Ffrom potato with a putative extracellular location (Hedley et al.,1993); carrot-cw, extracellular acid j3F from carrot (Sturm and Chris-peels, 1990); tomato-s, soluble acid /3F from tomato (Klann et al.,1992); carrot-si, isoenzyme I of soluble acid /3F from carrot;bean-s, soluble acid 0f from bean (Arai et al., 1991); carrot-sll,isoenzyme II of soluble acid (IF from carrot.

CW si Sll CW Bl Sll

Figure 6. DNA gel-blot analysis of sequences of extracellular /3Fand the two isoenzymes for soluble acid /3F in the carrot genome.A, Cenomic DNA from the carrot cv Nantaise (5 /ig/lane) wasdigested with EcoRl or Kpnl. The fragments were separated byagarose gel electrophoresis and blotted before hybridization with32P-labeled gene-specific fragments of the cDNAs for extracellular/3F (cw) and isoenzyme I (si) and isoenzyme II (sll) of soluble acid0f. B, Cenomic DNA from wild carrot (5 jig/lane) was treated andprobed as described above.

obtained with a gene-specific fragment of the cDNA codingfor carrot cell-wall 0F. The cDNA fragments hybridized toonly a few restriction fragments specific for each probe,indicating the presence of single- or low-copy-number genesfor each of the isoenzymes. These specific hybridizationpatterns in addition to the marked differences between thenucleotide sequences clearly show that the three isoenzymesof carrot acid 0F are encoded by different genes and do notoriginate from differential splicing of a common gene, as inthe yeast Saccharomyces cerevisiae (Carlson and Botstein,1982).

The pattern of four bands obtained by hybridizing thecDNA fragment of isoenzyme I to EcoRI-digested DNA fromthe carrot cv Nantaise (Fig. 6A), compared to the single bandof the cDNA fragment of isoenzyme II hybridized to thesame genomic DNA, coincides with the restriction patternsof the three cDNA classes for isoenzyme I: the sequences ofthe cDNA classes 2 and 3 both contain an internal EcoRl site(at positions 1616 and 1581, respectively), whereas the se-quence for the class 1 cDNA has none. Only one class ofcDNA was isolated for isoenzyme II; this contains no internalEcoRl site and gives rise to the single band on the DNA gelblot (Fig. 6A).

ACKNOWLEDGMENTS

We thank Ortun Mittelsten Scheid, Jiirg Bilang, and Patrick Kingfor critical reading of the manuscript. www.plantphysiol.orgon July 8, 2018 - Published by Downloaded from

Copyright © 1994 American Society of Plant Biologists. All rights reserved.


Acid P-Fructofuranosidases from Carrot 1357

Received October 25, 1993; accepted December 14, 1993. Copyright Clearance Center: 0032-0889/94/104/135 1/07. The EMBL/GenBank/DDBJ accession numbers for the sequences

reported in this article are X75351, X75352, X75353, X67163, and M58362.

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cdna cloning of carrot (daucus soluble acid with wall physiol. (1994) 104: 1351-1357 cdna cloning of...

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