DNA Sequence-The Journal ofSequencing andMapping, Vol. 5, pp. 41-49 Reprints available directly from the publisher Photocopying permitted by license only

0 1 994 Harwood Academic Publishers GmbH Printed in the United States of America

Multiple secondary plant product UDP- glucose glucosyltransferase genes expressed in cassava (Manihof escdenfa Crantz) cotyledons JANE HUGHES and MONICA A. HUGHES

Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK

Database Accession Nos. X77459 (pCGT1 ), X77460 (pCGT4), X77461 (pCGTZ), X77462 (pCGT5), X77463 (pCGTG), X77464 (pCGT7)

Six different putative UDP-glucose glucosyltransferase clones were isolated from a cassava cotyledon cDNA library probed with an Acc I-Bgl I I restriction fragment from a UDP-glucose flavonoid 3-0-glucosyltransferase from Anfirrhinum majus. The heterologous probe contained a glucosyltransferase consensus signature amino acid sequence which was also present in the cassava cDNA clones. Nucleotide and derived amino acid se- quences are presented for two of the clones. Northern analysis showed different patterns of expression for the six genes in de- veloping seedling tissues, indicating temporal and tissue-specific regulation. A comparative analysis was made of the six cassava clone derived amino acid sequences and other reported UDP- glucosyltransferase genes. Highly conserved residues in plant genes from three species allow redefinition of essential residues within the signature sequence for secondary plant product me- tabolism glucosyltransferase genes.

KEY WORDS cassava, cDNA, cotyledon, flavonoids, UDP-glu- cose glucosyltransferase

INTRODUCTION

Cassava (Manihor esculenta Crantz) is a major tropi- cal root crop with an estimated world annual pro- duction of 150 m i l l i on tonnes of fresh roots (Hershey, 1993). It i s the major crop plant of tropi- cal Africa, with average African per capita con-

Address for correspondence: Professor M.A. Hughes, Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH UK.

sumption at over 100 kg per annum (Hahn, 1989). The biology of cassava is seriously under-researched and there is considerable scope for the improve- ment of agronomic and quality characteristics.

Cassava i s cyanogenic, that i s hydrocyanic acid (HCN) i s released from all tissues following me- chanical damage. HCN release is brought about by the sequential action of a P-glucosidase and an a- hydroxynitrilase on two structurally related cyanoglucosides ( l inamarin and lotaustralin) (Poulton, 1990). These cyanogenic glucosides are synthesised in leaf tissue (including the cotyledons) and transported throughout the plant (Koch et a/., 1992). The cyanogenic glucosides are synthesised from two precursor amino acids (val ine and isoleucine) to form two unstable cyanohydrins ( 2 - hydroxy-2-methylpropionitrile and 2-hydroxy-2- methylbutyronitrile) which are glucosylated by a UDP-glucosyltransferase to produce the stable cyanoglucosides. Ungerminated seeds of cassava contain very small quantities of cyanoglucoside (1-1 0 nmolheed) but during germination rapid syn- thesis occurs in the hypogeal cotyledons so that after 10 days the seedlings contain about 8 p o l of cyanoglucoside per seed (unpublished data). Given the rapid synthesis of cyanoglucosides in the cotyle- dons, the cyanogen ic U DP-gl ucose gl ucosyltrans- ferase i s expected to be present in this tissue.

Glycosylation of a number of secondary plant compounds, including flavonoids, steroidal alka-

41

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I. HUGHES AND M. A. HUGHES 42

loids and cyanohydriris, occurs at the end of their biosynthetic pathway (Sun and Hrazdina, 1991; Stapleton et a/., 1992; Reay and Conn, 1974). The most commonly used sugar is glucose and the reac- tion i s catalysed by a UDP-glucose. glucosyltrans- ferase to produce a stable water soluble compound that is often transported to the vacuole (Hrazdina and Wagner, 1985; F’oulton, 1990). A number of secondary plant compound UDP-glucosyltrans- ferases from different species have been studied [Hrazdina, 1988; Heilemann and Strack, 1991; lshikura et a/ . , 1993; U l lmann et a/., 1993; Vellekoop et a/., 1993). Two higher plant UDP-glu- cose glucosyltransferase genes have been cloned and their sequences reported in the literature: the UDP-glucose flavonoid glucosyltransferase specified by the bronze locus in maize (Ralston et a/., 1988; Furtek et a/., 1988) and a related gene from barley (Wise et dl . , 1990). Given the reported specificity of these enzymes and the large number of potential substrates, a wide range of different glucosyltrans- ferases may be expected to occur within a single plant species (Harborne, 1988). Here we report the use of a heterologous probe derived from a dicotyle- donary plant, Antirrhinurn rnajus (Martin et a/., 1991 ), to identify glucosyltransferase clones in a cassava cotyledon cDNA library. We present the nucleotide sequence of two cDNA clones and a comparative analysis of the derived amino acid se- quences of six cassava cDNA clones with other re- ported gtucosyltransferase genes.

RESULTS & DISCUSSION

cDNA Library Construction and Screening A hgt 10 cDNA library made from mRNA extracted from the cotyledons of 10 day old light grown cas- sava seedlings (Hughes et a/., 1992) was screened with a heterologous probe constructed from a 0.9 kb Acc I-Bgl II restriction fragment from a flavonoid glucosyltransferase from Antirrhinum rnajus kindly provided by Ur. C. Martin, John lnnes Institute (Martin et a/. , 1991). The nucleotide and derived amino acid sequences of the Antirrhinurn flavonoid glucosyltransferase clone used as a probe in the ini- tial library screen have not been published. The de- rived amino acid sequence of this fragment contains a region with high homology to a proposed gluco- syltransferase consensus signature sequence (PROSITE, Bairoch, 1991). Six independent clones

were selected and subcloned into the plasmid vec- tor pCem SZf(-), and confirmed by restriction map- ping and sequencing to be different from each other. The derived amino acid sequences of all of these clones contain the proposed glucosyltrans- ferase signature.

Southern blot analysis of genomic DNA from 54 cassava accessions, digested wi th Eco R 1 and probed with the s ix putative glucosyltransferase cDNA clones, gave different sized fragments and different levels of polymorphism for each clone, confirming that they represent different genes (per- sonal communication, Dr. H.R. Haysom).

Sequence Analysis Restriction maps of the six putative glucosyltrans- ferase clones from cassava are shown in Fig. 1. Four clones do not contain the complete coding se- quence but the position of the predicted stop codons for each clone in relation to the glucosyl- transferase signature sequence indicates the con- served 3’ location of this proposed UDP-binding site (Hundle, 1992). There i s a conserved Eco RI site within the PROSITE region in five of the six clones, and an Ndel site in two of the clones. None of the other restriction sites is conserved.

The nucleotide sequences of the two longest clones, pCGTl and pCGT5, are presented in Fig. 2, together with the derived amino acid sequences. pCGT5 is considered to be full length, as the tran- script size estimated from Northern blot analysis is 1.6 kb (Table 1 ). This clone contains a polyA tail of 54 residues and the open reading frame shown pre- dicts a protein of 487 amino acid residues with a calculated M, of 54,379. pCGT1 has an open read- ing frame that predicts a similar sized protein of 449 amino acid residues with a calculated M, of 50,280, but does not appear to contain all of the 3’ non-cod- ing region of the 1.6 kb transcript (Table 1).

It has been proposed that the final step i n the biosynthesis of flavonoid and cyanogenic gluco- sides, that i s the addition of glucose to the aglycone, takes place in the lumen of the endoplasmic reticu- lum prior to transport to the vacuole, and that the glucosyltransferases involved are loosely associated w i th the endoplasmic re t icu lum membrane (Hrazdina and Wagner, 1985). Sequestration into the endoplasmic reticulum depends upon an amino terminal signal sequence consisting of 13-30 amino acid residues with an uninterrupted stretch of 7 or 8 hydrophobic residues and a more polar carboxy ter- minal region that defines the cleavage site (von

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CASSAVA GLUCOSYLTRANSFERASE GENES 43

Hind 111 EcoRI Ndel pCGT1

pCGT2

pCGT4

X

Eco RI

~~ *

EcoRI EcoRI

Pstl EcoRI I

kb

1.4

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X * n n 1.7 pCGT5 -I

pCGT6

pCGT7

Nsi I Srp I Eco RI Nde I Nde I * n 1.3

Pst I Barn HI Kpn 1 ssp I 1 .o *

Figure 1 ipCGTl and pCGT5). *: stop codon. A: polyA tail. The PROSITE glucosyltransferase signature region is underlined.

Restriction site maps of cassava glucosyltransferase cDNA clones. pCGTl-7: cassava cDNA clones. x: first methionine codon

Heijne, 1988). Hydrophobicity plots (Kyte and Doolittle, 1982) and sequence analysis of the de- duced N-terminal amino acids suggest that the pre- dicted proteins of pCGTl and pCGT5 may have amino terminal domains with the properties of a sig- nal sequence. The potential cleavage sites are be- tween cysteine (1 8) and histidine (1 9) for pCGTl, and leucine (29) and glycine 130) for pCCT5.

Comparative Analysis of Derived Amino Acid Sequences The PROSITE (Bairoch, 1991 ) UDP-glucosyltrans- ferase signature sequence i s based upon a con- served domain of 44 amino acid residues located in the C-terminal region of three cloned genes, namely a UDP-glucose flavonoid 3-0-glucosyltransferase from Zea mays (Furtek et a/., 19881, a mammalian U DP-gl ucuronosy ltransferase (Dutton, 1 980), and an ecdysteroid UDP-glucose glucosyltransferase from the baculovirus, Autographa ca l i forn ica (O’Reilly and Miller, 1990). In addition to other mammalian glucuronosyltransferase clones, two fur- ther sequences containing the glucosyltransferase signature are recorded on the Swiss-Prot and NBRF-

PIR protein sequence databases, namely, a second plant flavonoid 3-O-glucosyltransferase from barley (Wise et a/., 1990) and a zeaxanthin glucosyltrans- ferase from the non-photosynthetic bacterium, Erwinia herbicola (Hundle et a/., 1992). The agly- cone substrates of this group of glucosyltransferase enzymes are diverse and the glycosylated products include anthocyanidins, the insect moulting hor- mone ecdysone, the steroid hormone bilirubin and the carotenoid pigment zeaxanthin. A factor com- mon to all the enzymes is that none is involved in primary metabolism.

The proposed PROSITE UDP-glucosyltransferase signature sequence is shown in Fig. 3 together with the homologous region of the derived amino acid sequences of the six putative glucosyltransferase clones from cassava. The equivalent region from the amino acid sequences of the flavonoid 3 - 0 gluco- syltransferase genes from maize and barley are in- cluded for comparison. Identical and equivalent amino acid residues are indicated in bold type. Of the twenty-three residues specified by Bairoch (7 991), seventeen correspond exactly with the plant gene sequences (=) and a further three contain a sin-

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46 J. HUGHES AND M. A. HUGHES

Table 1 dons

Summary of putative UDP-glucose glucosyltransferase cDNA clones from cassava cotyle-

Expression

Length Transcript size Seedlings

Clone kb kb Cotyledon Hypocotyl Root Leaves

pCGT1 1.4 1 .b + - + -

pCGT4 0.8 1.6 (+I (4 (+) (+I pCGT5 1.7 1.6 (+) (4 (-) (4 pCGT6 1.3 1.5 + - - + pCGT7 1 .o 1.7 + + + +

pCGT2 1.1 1.6 + - + +

Rrackets indicate (OW levels of transcript

pCGTl Pca2 pc-4 pcoT5

pcGT7 pCGT6

zm Hv

PROSITE

PSPG

l0LPQVAVLAH PASGGLVSHSQPlMS I LESIWFOVPVA'IWPMY~ WQVAVLAEPAI QG FVSECGRQNSVLPSLWQ ATWPMYIWQ WSPQVLILSBPAI OAF F T H C m S TLEGI SAGVPIVACPLFAEQ ~PQIH~EPSVdVFLSHCOPONSVIgSf TAQVPI IAWPIYAEQ OQAPQVAILEHPAIOGFVSEC~SILESIWFSVPSATWPLYATLO WLPQVEILEEAALQVFVTBCGPQNS ILESIV-I C R P F m Q

WXXQXXZLXHXXXXAF'LSXSGXXSXXXSLXXXLPLXXXPLLSDQ I I T T T T I I 1 IITE 7 1 V A A A AV V V W A iuI M G G G GM M M MMG

FF

WSPQIXILXHPSXGXF%SHXGWNSILESLXXSVPIXXXPLYADQ A V VM AA LVT A V M G I G V I FGE

F T V M M F

Figure 3 Comparison of derived amino acid sequences of eight plant glucosyltransferase genes with the proposed PROSITE glucosyltrans- ferasc signature sequence. Identical and equivalent amino acid residues are shown in bold type. PROSITE sequence comparison: conserved speciiied residues (=, +); conserved unspecified residues (:), pCTCl-7: cassava cDNA clones. Zm. flavonoid 3-0-glucosyltransferase from Zed mays (Furtek eta/., 1988). Hv: flavonoid 3-0-glucosyltransferase from Hordeum vulgare (Wise eta/., 1990). PROSITE: proposed gluco- 5vhransterase signature sequence (Bairoch, 1991 ), PSPG: consensus sequence for plant secondary product glucosyltransferase genes.

gle mismatch (+). Twelve of the twenty-one unspec- iiied residues (X) also have identical or equivalent residues in seven or eight of the clones in Fig. 3 (:). Only two of the remaining twelve residues show no dement o f amino acid conservation. Amino acid \equence homology between these eight plant clones decreases rapidly on both the N and C termi-

nus sides of this highly conserved region. The UDP- glucosyltransferase signature sequences of the 3 non-plant genes (Dutton, 1980; Hundle et a/., 1992; O'Reilly and Miller, 19901, are not conserved to the same extent as the plant genes. A modified PROSITE UDP-glucosyltransferase signature 5e- quence (PSPG) i s proposed for secondary plant

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CASSAVA GLUCOSYLTRANSFERASE GENES 47

product metabolism (Fig. 3). This sequence contains fourteen residues with identity compared with ten in the original PROSITE sequence and includes four conserved prolines.

Homology within the group of six cassava clones, and between these genes and the f i v e other recorded genes containing the glucosyltransferase signature was further investigated with PROSIS (Pharmacia) and MACAW (Schuler et a/., 1991 ) soft- ware. Homology between the plant and non-plant genes, confined primarily to the glucosyltransferase signature region, is confirmed by the more powerful amino acid sequence data analysis programme MACAW, which compares multiple sequences and defines regions of homology at different levels of stringency (data not shown). Fig. 4 shows the rela- tive similarity between each pair of sequences (in- cluding regions outside the signature sequence) based upon the percentage homology score deter- mined by the PROSIS homology search programme. A high level of homology i s found between the maize and barley flavonoid glucosyltransferase genes (73%). pCGT7 is more similar (43% and 42%) to these monocotyledon flavonoid biosynthesis genes than to the other five cassava clones and may therefore also represent a flavonoid glucosyltrans- ferase gene. Three of the cassava clones, pCGT1, pCGT2 and pCGT6 form another closely related group (60-67%). Partial amino acid sequences from potato solanidine UDP-glucose glucosyltransferase reported by Stapleton et a/ . (1992) did not contain

pCGTl pCGT2 pCGT4 pCGTS

pCGTl - 67 33 29 35 34 pCGT2 -

39 pCGT4 - pCGT5 - pCGT6 pCGT7 Zm H v Ac R l Eh

the PROS ITE glucos y I transferase signature sequence and no significant homology was found between these sequences and the cassava glucosyltransferase clone derived amino acid sequences.

Northern Analysis of Expression Northern blots prepared from total RNA extracted from cassava seedling cotyledons, hypocotyls and roots at 5 defined developmental stages were probed with the putative cassava glucosyltransferase clones. Stage 0 is the seed plus newly emerged radi- cle, at stage 1 the cotyledons are still enclosed in the seed coat but this is split, at stage 2 the cotyle- dons are free of the seed coat, at stage 3 the seedling is morphologically similar to stage 2 but has green cotyledons following transfer to the light and at stage 4 the seedling is approximately 10 days old and a small true leaf is present. Three distinct patterns of expression were found (Fig. 5). pCGT1 and pCGT2 have maximum expression in stage 2 cotyledons, with low levels in hypocotyls and in- creasing levels in roots throughout this period of de- velopment. pCGT6 is expressed primarily in stage 3 and stage 4 cotyledons with very low levels in hypocotyts and no measurable expression in roots. pCGT7 is expressed in all tissues at uniform levels at all stages of development (data not shown). pCGT4 and pCGT5 appear to represent rare transcripts compared with the other four clones because sig- nals on the Northern blots were always low. Further, three of the genes, pCGTl, pCGT2 and

pCGT6 pCGT7

60 31 60 29 31 35 35 32

35 - -

Zm Hv Ac R l Eh

33 43 23 18 25 35 36 23 30 28 32 31 26 27 38 34 32 29 26 19 26 31 22 25 17 43 42 24 28 21 - 73 29 30 26

- 26 24 37 - 23 29

- 25 -

Figure 4 Pairwise sequence comparison of glucosyltransferase cDNA clone derived amino acid sequences (PROSIS, YO homology). pCGT1-7: putative UDP-glucose glucosyltransferase clones from Manihot esculenta. Zrn: flavonoid 3-0-glucosyltransferase from Zed mays (Furtek et al., 1988). Hv: flavonoid 3-0-glucosultransferase from Hordeum vulgare (Wise et a/., 1990). Ac: ecdysteroid UDP-glucose glucosyltransferase from Autographa californica (O’Reilly and Miller, 1990). R1: UDP-glucuronosyltransferase from rat liver (Mackenzie, 1986). Eh: zeaxanthin glucosyltransferase from Erwinia herbicola (Hundle, 1992).

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48 J. HUGHES AND M. A. HUGHES

kb 0 1C 1H 1R 2C 2H 2R 3C 3H 3R 4C 4H 4R Probe

1.6 pCGT2

* pCGT6 (iic 1.5

Figure 5 Northern blot analysis of total RNA from developing cassava seedling tissues. Key to wells: Developmental stages; 0, radicle emerged. 1, cotyledons enclosed in split seed coat. 2, cotyledons emerged from seed coat. 3, as stage 2, cotyledons green following 24 hrs in light. 4, first true leaf present, approximately ten days after germination. The seedlings were transferred from the dark to 12 hour light conditions between stages 2 and 3. T: total seedling. C: cotyledon. H: hypocotyl. R: root.

pCGT6, were isolated more than once during the Ii- brary screens. Northern blot analysis of total RNA from young cassava leaf tissue showed that pCGT2, pCCT4, pCCT6 and pCGT7 are expressed in leaves whereas pCGT1 and pCGT5 are not (data not shown). Kuhasek ef a/. (1 992) showed that enzymes involved in flavonoid biosynthetic pathways i n Arcdidopsis are coordinately regulated by a devel- opmental timing mechanism during germination. Northern blot analysis of the putative glucosyltrans- terase genes reported here suggests both temporal and tissue-specific regulation of expression in devel- oping cassava seedlings.

The cyanogenic glucosides, linamarin and lotaus- tralin, are not synthesised in seedling roots (Koch et a/., 19923. pCGTl , pCGT2 and pCGT7 can therefore be eliminated as cyanogenic glucosyltransferase clones because they are expressed in roots. The low level of expression of pCGT4 and pCGT5 also make i t un l i ke ly that these clones represent the cyanogenic enzyme, which has a high level of activ- ity in seedling cotyledons. It remains possible that pCGT6 is involved in cyanogenesis, since it has high levels of expression in cotyledons, which are the pri- mary site of cyanoglucoside synthesis in seedlings.

'The expression data determined by Northern blot analysis is summarised in Table 1 together with de- tails of cDNA clone and transcript sizes. The multi- plicity of glucosyltransferase genes with different developmental profiles expressed in a single tissue icotyledons) of young cassava seedlings demon- strates the complexity of this group of enzymes and suggests the production of individual glucosyltrans- ierase proteins with specific functions in the metab- olism of secondary plant products.

MATERIALS AND METHODS

Plant material Cassava seeds from the plant CM1223-11 were supplied by Dr. C. Hershey, CIAT, Cali, Colombia, and grown as described in Hughes et a/., 1992.

Selection of cDNA clones A cDNA library was constructed in the hGTlO vector using Not I/Eco RI adaptors, with mRNA extracted from cotyledons as de- scribed in Hughes et al., 1992. The library was initially screened with a heterologous probe derived from a 0.9 kb Acc I-Bgl II re- striction fragment from a flavonoid 3-0-glucosyltransferase cDNA clone from Antirrhiniurn majus (Martin et a/., 1991~. A second screen was carried out using a putative 1.1 kb cassava glucosyltransferase cDNA clone selected during the first screen. Probes were radiolabelled with a Random Primed DNA Labelling Kit (Boehringer-Mannheim). Standard procedures were used to prepare replica filters and Southern blots (Samhrook et a/., 1989). Filters were washed at 60°C at low stringency: twice tor ten min- utes in 4X SSC, 0.1% SDS and twice for ten minutes in 2 X SSC, 0.1% SDS (20X SSC is 0.3M sodium citrate, 3M sodium chloride pH 7.0). Selected cDNA inserts from recombinant phage were subcloned into the Not I site of the plasmid vector pGEM 5ZF(--) (Promega).

DNA sequencing Both strands of the cDNA clones were independently sequenced by the dideoxy chain termination method (Sanger, 1977) using a Sequenase Version 2 Kit (United States Biochemical Corporation). Double-stranded DNA was sequenced directly from the pCEM SZf(-) plasmid using standard universal forward and reverse primers (USB). Overlapping deletions were generated where possible using a Nested Deletion K i t (Pharrnacia-LKB), Subcloned restriction fragments or specific synthetic 17-mer oligonucleotide primers were used where nested deletions were unobtainable.

Computer analysis DNA sequence data was analysed with DNASIS software from Pharmacia. Analysis of the derived amino acid sequences was performed with the following programmes: PROSIS (Pharmacia), PROSITE (Bairoch, 1991), MACAW [Schuler ef a/., 1991). Homology searches were carried out with the CenBank and EMBL DNA sequence databases, and the NBRF-PIR and Swiss Prot protein sequence databases (May, 1992).

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Northern blot analysis of RNA Total RNA was extracted from plant tissues by the guanidine thio- cyanate method of Broglie et a/. (1984). 10 pg of total RNA per sample was separated on 1.5% agarose gels in the presence of formaldehyde, then transferred to Hybond N membranes (Amersham). Northern blotting and hybridisation were carried out by standard procedures (Sambrook eta/., 1989). Probes were prepared from cDNA clones as above. Filters were washed at 42°C at high stringency: twice for ten minutes in 2X SSPE, 0.1% SDS, once for fifteen minutes in 1X SSPE, 0.1% SDS, and twice for ten minutes in 0.1 X SSPE, 0.1 % SDS (20X SSPE is 3.6M NaCI, 0.2M NaP04 pH7.7, 0.02M EDTA).

ACKNOWLEDGEMENTS

We wish to thank Kathleen Kelly and Martin Fletcher for technical assistance.

Dr. J . Hughes i s supported by the EC-funded Casanova Project, grant no. ECSTD3 TS3*-CT9Z- 01 08.

(Received 10th lanuary 1994)

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