211 Gene, 145 (1994) 211-214

0 1994 Elsevier Science B.V. All rights reserved. 037%1119/94/$07.00

GENE 07996

Potato (Solanum tuberosum) invertase-encoding cDNAs and their differential expression

(h-Fructosidase; reverse transcription-polymerase chain reaction; single-stranded conformational polymorphism; apoplastic enzyme)

Peter E. Hedley, Gordon C. Machray, Howard V. Davies, Lindsay Burch and Robbie Waugh

Cell and Molecular Genetics Department, Scottish Crop Research Institute, Invergowrie, Dundee DO2 5DA, UK

Received by J.R. Kinghorn: 4 November 1993; Revised/Accepted: 13 January/21 January 1994; Received at publishers: 28 March 1994

SUMMARY

A full-length cDNA clone encoding a potato invertase (Inv) has been isolated. It is highly related (77% nucleotide identity) to a previously characterised potato cDNA clone encoding a putative extracellular Inv. These Znv genes encode a subfamily of apoplastic enzymes which are shown to be distinct, on the basis of sequence similarity, from the related subfamily of vacuolar enzymes. In order to differentiate between the expression of the two potato genes encoding apoplastic Inv, a single-stranded conformational polymorphism (SSCP) assay was developed for products generated by reverse transcription-polymerase chain reaction (RT-PCR) utilising primers designed to amplify both potato sequences. Using this approach, we have shown that these two identified Znv from potato are expressed in a tissue-specific and developmentally regulated manner.

INTRODUCTION

Invertases (EC 3.2.1.26; Inv) cleave the disaccharide sucrose to the hexose sugars glucose and fructose (Myrback, 1960) so contributing to a variety of meta- bolic and transport processes in plants. These enzymes, isolated from a wide spectrum of plants (e.g., Bracho and Whitaker, 1990; Weil and Rausch, 1990; Batta et al., 1991; Moriguchi et al., 1991; Ranwala and Masuda, 1991; Burch et al., 1992; Unger et al., 1992) show variation in

Correspondence to: Dr. G.C. Machray, Cell and Molecular Genetics

Department, Scottish Crop Research Institute, Invergowrie, Dundee

DD2 5DA, Scotland, UK. Tel. (44-382) 562-731; Fax (44-382) 562-426;

e-mail: cmggm@uk.ac.sari.scri

Abbreviations: aa, amino acid(s); bp, base pair(s); GCG, Genetics

Computer Group (Madison, WI, USA); Inv, invertase(s); Inv, gene

(DNA) encoding Inv; kb, kilobase or 1000 bp; nt, nucleotide(s); oligo,

oligodeoxyribonucleotide; ORF, open reading frame; pfu, plaque-

forming unit(s); RT-PCR, reverse transcription-polymerase chain reac-

tion; SDS, sodium dodecyl sulfate; SSCP, single-stranded conforma-

tional polymorphism.

SSDI 0378-l 119(94)00207-9

both peptide composition and subcellular localisation. Inv genes encoding mung bean (Arai et al., 1992) and tomato (Klann et al., 1992; Elliott et al., 1993) vacuolar enzymes, and carrot (Sturm and Chrispeels, 1990) cell- wall enzymes have been cloned. We have previously re- ported the cloning and characterisation of a partial cDNA encoding an Inv from potato (Hedley et al., 1993) which, on the basis of greater sequence similarity to the carrot cell wall enzyme, may also be associated with the apoplast. A related gene, with 53% nt identity to the apoplastic gene and encoding a potential vacuolar Inv, has also been cloned from potato (U. Sonnewald, per- sonal communication). We report here the characterisa- tion of a full-length cDNA sequence representing another member of this gene family in potato and demonstrate that plain Inv can be classified into sub-families based on sequence similarity.

Many plant genes are organised into complex multigene families. Distinction between the expression of closely related members of gene families has been facili- tated by the application of RT-PCR. One approach relies

212

upon the presence of regions of low homology between

genes for the design of gene-specific primers (Simpson

et al., 1993). A second approach uses restriction enzyme

digests to discriminate between RT-PCR products from

common primers (Bohl and Apel, 1993). The applicability

of both methods is sequence dependent. We have devel-

oped an alternative RT-PCR strategy which employs

primers common to both cDNAs for RT-PCR and

discriminates between products post-amplification using

a single-stranded conformational polymorphism (SSCP)

assay procedure. SSCP analysis has been used extensively

in human genetics to detect mutations within genes and

has been shown to be able to detect single point mut-

ations (Hayashi, 1991). Its use to differentiate between

the expression of the two closely related genes encoding

potato apoplastic Inv. revealing tissue-specific differences

in their expression, is described.

EXPERIMENTAL AND DISCUSSION

(a) cDNA characterisation

The complete nt sequence of cDNA clone pCD141 is

presented in Fig. 1. This 1952-bp cDNA has an ORF

extending from a putative start codon at nt 7 to a stop

codon at nt 1753, giving 6 bp of untranslated 5’ and

198 bp of untranslated 3’ sequence. When aligned with

the previously characterised potato Inr clone pCDl1 I

(Fig. 1) the nt sequence identity is 77%, suggesting that

pCD141 constitutes a different gene and not a second

41

11

41 11

41

11

41.

11

41 11

41 11

41 11

41 11

41 11

41 11

41 11

41

11

41 11

41 11

41

11

41

11

41 11

TTAAAGATGGAGATTTTAAGRAGATCTTCTTCTCTTn;GGT......ATTTGTTGA~CTTCTCACAAAGTTTATA n: 114 ............... .GT~..A.~G......."..TTTC..".......T.......A.A.....CC.*A.~~ATC~.C~C~~ C . .*A".*~'*A'~~**."**T~CCA 105

T...............An;CCACGmACAAAGAAACCATAGTAGAAATTTAACT.TATTGATCAAATGT 'TGTAATAAGTTCCAC**TmGTGGTG'C*T.TTPG'~T~CC*CA'~CA=*TTGA"TTM3 T A * .* l .*.*+~"*~G~*A.*TAT.CT*T*.~.A.*~*GC.GAC~**~T*T~

CTTATTGAA'TTAAGAGAATGAGTATCT%GTGCAATTTCT'FMl'~ . ..T.C'T..C... +AT*..*A*.ATA'*...A'A.*G.*A~A.~.=***..

234 222

354 342

474

462

594 582

714 693

1674 1659

Fig. 1. Nucleotide sequence of potato Ina cDNA clone pCD141 (41; EMBL accession No. 222645) compared to that of pCDl11 (11; Hedley et al.,

1993). Total RNA was prepared from potato (cv. Cara) leaf (Schroder et al., 1988) and poly(A)+RNA isolated using Dynabeads mRNA purification

kit (Dynal). cDNA was synthesised from 5 ug mRNA (Timesaver TM cDNA synthesis kit, Pharmacia) and ligated into EcoRI-digested hgtl 1 arms.

Subsequent packaging (h in vitro packaging kit, Amersham) and amplification (Sambrook et al., 1989) yielded 2 x 10” pfu/ml from an original titre

of 1.5 x lo6 pfu. A heterologous Inc nucleic acid probe was synthesised from carrot first strand cDNA as described (Hedley et al., 1993), radiolabelled

with C”*P]dCTP (random primed labelling, Boehringer-Mannheim), and used to screen lo5 recombinant phage, according to manufacturers recommen-

dations (Hybond N +, Amersham), with washing at low stringency (2 x SSC/O. 1% SDS). Clones isolated after three rounds of screening were subcloned

into pUCl9 and nt sequences were determined on both strands using the Sequenase version 2.0 kit (US Biochemical, Cleveland. OH, USA) and

progressive ohgo design. Using this procedure we have previously isolated an Inu cDNA clone, pCDl11 (Hedley et al., 1993). The derived aa sequence

contained an incomplete putative signal polypeptide sequence which, by comparison to signal sequence from a carrot cell-wail Inv, suggested that

the cDNA encodes an apoplastic enzyme. Further screening of the same cDNA library has yielded a second and full-length clone, pCD141. Sequence

analyses were carried out using the GCG package (Devereux et al., 1984). Asterisks (*) represent nt identical to pCD141. Highlighted sequences

indicate position of primers used for RT-PCR. SSC is 0.015 M NaCI/O.O15 M Na,citrate pH 7.6.

allele of the same gene from this tetraploid potato variety. The nt sequence differences within the ORF, shown in Fig. 1, are evenly distributed with no extensive regions of dissimilarity between the two genes.

(b) Plant invertases The deduced aa sequence of pCD141 (582 aa; Fig. 2a)

corresponds to 66 kDa, including a full signal sequence which appears to be characteristic of apoplastic Inv. Pairwise comparisons of the entire aa sequence and pre- viously cloned plant Inv enzyme sequences are repre- sented in Fig. 2b by a dendrogram generated using the GCG PILEUP program. The pCD141-derived sequence is placed in a subfamily which includes the potato se- quence we have previously described and the carrot apo- plastic enzyme. This subfamily is distinct, based on sequence similarity, from a second group which includes all plant vacuolar Inv. Plant Inv may, therefore, clearly be divided into apoplastic and vacuolar subfamilies on a sequence basis alone, which may suggest that divergence of genes encoding the two different classes of enzymes has occurred prior to speciation.

Fig. 2. The aa sequence deduced from pCD141 and comparison to

those derived from cloned plant Inu genes. (a) Derived aa sequence from

pCD141 (nt 7 to 1753 in Fig. 1). (b) Dendrogram representing pairwise

comparisons of the deduced aa sequence of pCD141 (cd141) and those

of other plant Inv. Abbreviations used: cdl 11, potato apoplastic (Hedley

et al., 1993); carapo, carrot apoplastic (Sturm and Chrispeels, 1990);

potvac, potato vacuolar (U. Sonnewald, personal communication);

tomvac, tomato vacuolar (Klann et al., 1992; Elliott et al., 1993); mung-

vat, mung bean vacuolar (Arai et al., 1992). Scale represents percentage

aa similarity.

213

(c) RT-PCR assay of potato Zm gene expression Discrimination between products of expression of

highly related genes of equal size has been difficult using traditional methodologies such as Northern analysis. The design of gene-specific primers for RT-PCR, relying on the presence of short regions of low homology between sequences for the production of specific gene amplifica- tion products is one solution to the problem (Simpson et al., 1993). In the case of potato Ino cDNAs, such se- quences cannot readily be identified, or available se- quences are unsuitable (e.g., contain a high AT/GC ratio). Discrimination between equally-sized amplification pro- ducts derived from conserved primers has been achieved by restriction enzyme digestion (Bohl and Apel, 1993). This approach is limited to RT-PCR products which have variation within sequences for restriction enzyme sites. We have developed an alternative approach which discriminates between RT-PCR products on the basis of conformational differences resulting from sequence diver- sity. This approach, utilising a combination of RT-PCR and SSCP analysis has been applied to the characterisa- tion of the expression of the two closely related genes encoding potato apoplastic Inv. RT-PCR reactions were performed on total RNA from a variety of plant tissues, using oligos which would be expected to prime on mRNA from both genes (highlighted in Fig. 1). In positive con- trols first strand cDNA was substituted with plasmid DNA (pCDl11 or pCD141) in PCR reactions employing the same primers. We have previously demonstrated that using standard denaturing polyacrylamide gel electro- phoresis, single products of the predicted size (65 bp) were obtained from RT-PCR assays detecting mRNA in source and sink leaf, stem and to a lesser extent in tuber (Hedley et al., 1993). No detectable message was found in root. Plasmid controls also generated single products in PCR reactions. The two clones differ at only seven bp within the 65bp region between the PCR primers (Fig. l), but the amplification products of PCR from the two plasmids could be clearly distinguished when run on a SSCP gel (Fig. 3, lanes 6 and 7). When products from the indicated tissues were examined, expression in sink leaf (lane 2) could be related to the product from the cDNA cloned in pCDl11. A minor band representing lower levels of expression of this gene was also evident in source leaf (lane 1) and stem (lane 3). The major pro- duct found in all other tissues (lanes 1,3 and 5), including source leaf, could be ascribed to expression of the gene represented by pCD141. Genes encoding potato apo- plastic Inv are therefore under developmental and tissue- specific control, with a switch in their expression during leaf maturation. The role of the cell-wall Inv expressed predominantly in young leaf, like that of the sucrose bind- ing protein (Grimes et al., 1992) found in developing leaf

214

I 2 3 4 5 6 7

Fig. 3. SSCP assay of RT-PCR products derived from transcripts en-

coding apoplastic Inv in tissues from potato cv. Cara. Total RNA was

isolated from fresh potato tissue (Schroder et al.. 1988). with sink leaf

defined as being immature and unexpanded (< 20 mm, apex to petiole),

and source leaf being fully developed and expanded (> 80 mm, apex to

petiole). DNase treatment and RT-PCR reactions were carried out as

described (Hedley et al.. 1993). Briefly, 5 ug DNase-treated RNA, from

various tissues, was reverse transcribed using 0.5 ug 3’ primer (see Fig. I,

5’-ACGATTCCGGTGTAGAG) and first strand cDNA subjected to

PCR amplification with 1 mM 3’ primer and 1.5 x 1Oh dpm 3ZP-end-

labelled 5’ primer (see Fig. I. 5’-ACATGGTCCGGGTCAGC) with an

annealing temperature of 60’ C over 24 cycles. Plasmid controls substi-

tuted 10 ng plasmid DNA for first strand cDNA. RT-PCR reactions

(2 ~1) were electrophoresed on a 40cm 1 x MDE’” (Mutation

Detection Enhancement gel, AT Biochem) gel. which was run at 1.5 W

for I6 h at room temperature, dried and subjected to autoradiography.

RT-PCR products are from lanes: 1, source leaf; 2, sink leaf; 3, stem; 4,

root; 5. tuber. PCR products, using identical primers, are from plasmid

DNA controls, lanes: 6, pCD111; 7. pCD141.

of soybean, remains to be determined. This study also

demonstrates the potential of the combination of

RT-PCR and SSCP assay as a tool to differentiate be-

tween expression of closely related genes. This post-

amplification polymorphic assay procedure does not re-

quire full knowledge of sequence variation within mem-

bers of a given gene family and will discriminate between

expression products of genes from sub-families which do

not show sequence polymorphism within sites for any

particular restriction enzyme. In addition, the assay is

technically less demanding than other methods currently

available, because it requires simply the electrophoresis

of RT-PCR products through a modified gel matrix

under non-denaturing conditions. Furthermore, this

methodology provides a means to clone specifically pre-

viously uncharacterised sequences through amplification

of fragments excised from SSCP gels.

ACKNOWLEDGEMENTS

This research was supported by ECSA Research Ltd

and the EC under the ECLAIR program. H.V.D. and

R.W. are funded by the Scottish Office Agriculture and

Fisheries Department. We thank the SERC SEQNET at

‘Daresbury for access to computing facilities.

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