Floral expression of a gene encoding an E2-relatedubiquitin-conjugating protein from Arabidopsis thaliana

Plant Molecular Biology 26: 445-451, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 445

Short communication

Floral expression of a gene encoding an E2-relatedubiquitin-conjugating protein from Arabidopsis thaliana

Felicity Z. Watts 1, Neil Butt 1, Philip Layfield 1, Jesse Machuka 1, Julian F. Burke 2 and Anthony L. Moore 1 1Biochemistry Department and 2 Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Falmer, Brighton, East Sussex, BN1 9QG, UK

Received 6 September 1993; accepted in revised form 16 April 1994

Key words: ubiquitin, Arabidopsis, flower, senescense

Abstract

An Arabidopsis thaliana gene (UBC6) encoding a homologue to ubiquitin-conjugating enzymes has been isolated which is capable of encoding a protein of 183 amino acids of ca. 21 kDa. Northern analysis indicates that the gene is expressed in flowers, seeds and, to a somewhat lesser extent, in 10-day seedlings but not in mature leaves, callus and pre-flowering plants. This pattern of expression is confirmed using transgenic Arabidopsis plants containing a UBC6 promoter-GUS gene fusion construct. These plants display GUS activity in mature anthers prior to dehiscence, in developing embryos, sepals and the style after pollination.

Ubiquitination of proteins is a general process which has been observed in all eukaryotes stud- ied. It is involved in a number of cellular events including receptor modification [26], programmed cell death [30], DNA repair [16, 28], cell cycle progression in both Saccharomyces cerevisiae [ 12] and Xenopus [ 11 ] and protein degradation [ 13]. Evidence also suggests that ubiquitin may directly modify the structure and hence the function of some proteins. For example, in higher eukaryotes ubiquitin has been detected in asso- ciation with the histones H2A and H2B [41], Drosophila actin [ 1] and viral coat proteins [8].

The action of the ubiquitin degradation pathway has been reported in several plant species. Conditions in which this process has been observed include high-temperature stress in wheat [9], phytochrome degradation in oat [32], and

senescing leaves in oat [38]. Ubiquitin and polyubiquitin genes have been cloned from plants and, as in other eukaryotic species, are highly conserved [3, 5, 6, 14, 20, 39]. Additionally, a number of ubiquitin-conjugating enzyme genes have been isolated [33, 34, 37], but the precise roles of these plant enzymes have yet to be elucidated. In yeast, individual ubiquitin-conjugating enzymes have been shown to have a variety of different roles. For example, UBC1, UBC4 and UBC5 are involved in stress response [18], UBC2 (RAD6) is required for DNA repair [ 17], UBC3 (CDC34) is required for progression through the G1/S phase boundary in the cell cycle [12] while UBC10 (equivalent to PAS2) has a role in peroxisome biogenesis [40].

We report here on the isolation of the Arabi- dopsis UBC6 gene and the characterisation of the

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E Bc C N N S B H E

I II III IV V VI

2 0 0 b p

Fig. 1. Restriction enzyme map of the Arabidopsis UBC6 gene. Closed boxes, exon sequences; open boxes, introns; 1-VI, num- bers of exons; B, Bgl II; Bc, Bcl I; C, Cla I; E, Eco RI; H, Hind III; N, Nco I; S, Sal I.

expression of the gene using a combination of northern analysis and transgenic plants containing UBC6 promoter-GU S gene fusion constructs. The results presented here provide the first evidence for the tissue-specific expression of a ubiquitin-conjugating enzyme in plants.

The UBC6 gene (Fig. 1) was isolated during screens ofArabidopsis genomic and cDNA librar-

ies by hybridisation with yeast RAD gene probes [27] in experiments designed to identify Arabi- dopsis homologues to yeast DNA repair (RAD) genes. The putative UBC6 protein comprises 183 amino acids with a predicted molecular mass of 20785 Da. Comparison of the predicted protein sequence with the sequences in the databases indicates that the protein has high levels of sequence

ATUBC6

WHUBC4

ATUBC 1

UBC8

RAD6

CDC34

ATUBC 6

WHUBC4

ATUBC 1

UBC8

RAD6

CDC 34

ATUBC6

WHUBC4

ATUBC 1

UBC8

RAD6

CDC34

ATUBC6

WHUBC 4

ATUB C 1

UBC8

RAD6

CDC34

ATUBC 6

WHUBC4

ATUBC 1

UBC 8

RAD6

CDC34

CDC 34

Fig. 2. Alignment of the Arabidopsis

MASPSKRREMDMMI<LMMSDYKVDTV ....... NDDLQMFYVTFH .... GP 39

.S ....... ,..L ........... MI ....... ..GMHE.F.H..----..

.ST.ARK.L.RDF.RLQQ.PPAGIS ....... GAPQDNNIMLWNAVIF..

.... L...HQ..LI ....... .,SM.E.H,K.L .... ..

.ST.AR..L.RDF.R.KE.APPGVS ....... ASP.PDNVMVWNAMII..

MSSRK.TASSLLLRQYRELTDPKKAIPSFHIELE..SNI.TWNIGVM-VLA

TDSLYQGGVWKIKVELPEAYPYKSPSVGFVNKIYHPNVDESSGAVCLDVI 89

K,.I ....... VR.,,T ......... I,.T .......... M..S ......

D.TPWD.,TF.LSLQFS.D..N.P.T,R..SRMF...IYA-D.SI...IL

K.TP,EN...RLH .... DN ....... I ...... F...I.IA..SI .....

A.TP.ED.TFRLLL.FD.E..N.P.H.K.LSEMF .... YA-N.EI...IL

E..I.H..FF.AQMRF..DF.FSP.QFR.TPA ..... VYR-D.RL.ISIL

N ............ QTWSPMFDLINVFESFLPQLLLYPNPSDPFNGEAASL 127

.- ........... . ........ V.I..V .............. L .......

Q ............ NQ...IY.VAAILT.-IQS..CD...NS.A.S...RM

.- ........... S .... LY .... IV.WMI.G..KE..G...L.N...T.

Q ............ NR,T.TY.VASILT.-IQS.FND...AS.A.V..,T.

HQSGDPMTDEPDAE .... VQTVES.LI.-IVS..ED..INS,A.VD..VD

LMRDRAAYELKVKEYCEKYAKPEEILSDDDDDDSMSEDGSDSDDDDDDE- 176

M...KN...N ....... R ..... D.SPEEEEEE.DE.LSDAEGY.SG..-

YSESKRE.NRR,RDVV.QSWTAD

QL..KKL,.E.I...ID...TK.KYQQMFGG.NDSDDSD.GGLQEE.SDS

FKDHKSQ.VKR...TV..SWEDDMDDM ...... DDDD.DDEA.

YRKNPEQ,KQR..MEV.RSKQDIPKGFIMPTSE.AYISQ.KL.EPESNKD

-IVGKADP 183

A.M.H...

DEDMDGTGVSSGDDSVDELSEDLSDIDVSDDDDYDEVANQ

MADNFWYDSDLDDDENGSVILQDDDYDDGNNHIPFEDDDVYNYNDNDDDD

ERIEFEDDDDDDDDSIDNDSVMDRKQPHKAEDESEDVEDVERVSKKI

UBC6 protein sequence with other UBC sequences. ATUBC6, this paper; WHUBC4, wheat UBC4 [33]; ATUBC1, Arabidopsis UBC1 [34]; UBC8, S. cerevisiae UBC8 [17]; RAD6, S. cerevisiae RAD6 [16]; CDC34, S. cerevisiae CDC34 [ 12]. Dashes have been inserted to produce the maximum alignment while a dot represents a match with the UBC6 protein. Numbering refers to Arabidopsis UBC6 amino acids.

identity with UBC proteins from a range of species. Alignment of the predicted amino acid sequence of the Arabidopsis UBC6 protein with wheat UBC4 [33], the S. cerevisiae proteins RAD6 [16], CDC34 [12] and UBC8 [17], and Arabidopsis UBC1 [34] is shown in Fig. 2. The UBC6 protein described here is most homolo- gous to the wheat UBC4 protein with 86~o homology and 69~ identity and is more homolo- gous to wheat UBC4 than it is to any of the Arabidopsis UBC proteins reported to date [ 10, 34]. The levels of homology to the S. cerevisiae RAD6 and CDC34 proteins are somewhat lower, being 75 ~o and 45 ~o respectively. All the aligned sequences possess the essential cysteine residue at position 85, which has been shown, by in vitro mutagenesis of wheat UBC1 and UBC4 genes, to be essential for the transfer of ubiquitin to the target protein [33].

Analysis of the genomic clone for potential regulatory sequences upstream of the coding sequence in the genomic clone identified three sequences (ACACGTT, TCACGTC and TCA- CGTT at -1035, -985 and -947 relative to the initiating ATG respectively) which resemble the G-box core [21] found in several light-regulated promoters [29] and in the abscisic acid response element [25]. There are also two elements (CAT- GAAAAATCCAG and CACGACTATTCTTG at -1407 and -310 relative to the ATG, respectively) which have a 7/8 match with heat shock elements (HSEs) [2] from other eukaryotes.

Southern analysis was performed using a 32p_

labelled Sal I-Hind III probe encoding the C- terminal portion of the UBC6 gene product. Under conditions of high stringency hybridisation a single positively hybridising band is observed in DNA digested separately with three different enzymes (Fig. 3). Northern analysis (Fig. 4) using a probe (32p-labelled Cla I-Sal I fragment) from the less highly conserved region at the 5' end of the gene, reveals the presence of UBC6- hybridising species in total RNA isolated from flowers but not in RNA isolated from leaves, in- dicating tissue-specific expression of the UBC6 mRNA. Two hybridising species are observed; this could be due to cross hybridisation with

447

Fig. 3. Southern analysis. Arabidopsis genomic DNA digested with the restriction enzymes Dra I (lane 1), Eco RI (lane 2) and Hind III (lane 3) was subjected to electrophoresis on a 0.7 % agarose gel, transferred to Hybond N and probed with 32p_ labelled Sal I-Hind III fragment encoding the C terminal portion of the UBC6 gene in 2 x S SC at 65 ° C, with washes under these conditions followed by a 15 min wash in 0.1 x SSC, 1% SDS. The 1 kb ladder (Gibco BRL) was used as size markers.

Fig. 4. Northern analysis. 10 /~g total RNA isolated from Arabidopsis selected tissues was subjected to electrophoresis on a 1% glyoxal phosphate gel, transferred to GeneScreen plus and probed with 32p-labelled Cla I-Sal I fragment. Hy- bridisation was carried out in 50% formamide at 42 °C. Lane 1, callus; lane 2, root; lane 3, mature leaves; lane 4, flowers; lane 5, pre-flowering plants; lane 6, 10-day seedlings; lane 7, seed.

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related transcripts although this is unlikely due to the hybridisation conditions used, or the UBC6 gene may give rise to two transcripts (both species are of sufficient size, 1.2 and 0.8 kb, to en- code a protein of 183 amino acids.

In order to identify more precisely where the UBC6 gene is expressed, and to ensure that the tissue-specific expression observed in Fig. 4 is due to transcription from the UBC6 promoter as op- posed to transcription from a closely related gene, transgenic Arabidopsis plants containing a UBC6 promoter-GUS gene fusion were created. Spe- cifically, the 1.3 kb promoter-containing Eco RI- Nco I fragment was cloned upstream of the Escherichia coli/~-glucuronidase (GUS) gene with the nos terminator from pBI201.3 [15], into the vector pJE188 [19]. The resulting plasmid, pJUG1, was introduced into Arabidopsis using standard methods [36]. Histochemical analysis of GUS expression in the F1 progeny from ten independently isolated transgenic plants indicates that GU S activity, which is not detected in young floral organs (Fig. 5a), begins to be detected in the vascular region at the top of the filament, imme- diately adjacent to the anthers (Fig. 5b and 5d), and in sepals (Fig. 5b and 5c). Very low levels of GUS are observed in developing embryos (Fig. 5e), and this increases (Fig. 5f) before de- clining as the embryos mature (Fig. 5h and Fig. 5i). After pollination GUS activity is particu- larly high in the style (Fig. 5g), extending into the vascular tissue of the developing seed pod (Fig. 5h). At the time of seed maturation low levels of GU S activity are observed at the abcission site (Fig. 5i).

The wheat UBC4 gene (to which the Arabidop- sis UBC6 gene shows the greatest homology) was originally described as having greatest homology to the S. cerevisiae UBC2 (RAD6) gene [33].

However it has subsequently been shown that the wheat UBCI gene is more similar to yeast UBC2 gene than is the UBC4 gene, although neither of these wheat genes can functionally substitute for the RAD6 gene in a S. cerevisiae rad6 mutant [34]. Since both theArabidopsis UBC6 and wheat UBC4 genes show relatively low levels of homology to any of the reported yeast gene sequences it is difficult to make any inferences from what is known about the S. cerevisiae proteins, as to the role(s) of these Arabidopsis proteins. The presence of the HSE and G-box related elements may sug- gest that expression of the gene is induced under certain conditions e.g. increased temperature or, by analogy with chalcone synthase [29], by UV light. The Arabidopsis homologues to yeast UBC4 and UBC5 are not strongly induced by heat shock [10], unlike their yeast counterparts [31 ]. These results, in conjunction with the fact that Arabi- dopsis polyubiquitin genes are only weakly induced by heat shock [4], has led to the sugges- tion that ubiquitin may have a lesser response in the stress response of Arabidopsis than in yeast [10]. Analysis of the promoter of the Arabidopsis heat shock gene HSPI8.2, using promoter-GUS gene fusions has shown basal levels of expression in leaves, sepals and carpels with increased expression in all tissues following heat shock. We have not investigated the response to heat, but the location of the GUS activity in transgenic plants described here suggests that expression of the gene is tissue specific as well as stage dependent. While the elements at - 1035, -985 and -947 share homology to ABA-responsive elements [25], the patterns of GU S activity obtained with the UBC6 promoter-GUS gene fusion differ from those obtained in similar experiments using abscisic acid- responsive sequences from the Em gene of wheat in transgenic tobacco [24]. Alternatively the gene

Fig. 5. GUS activity in transgenic Arabidopsis containing a UBC6 promoter-GUS gene fusion construct. Transgenic Arabidopsis plants were obtained by transformation with a construct containing the Eco RI-Nco I fragment from the UBC6 promoter cloned adjacent to the Escherichia coli GUS gene. Figures are arranged in approximately temporal order with respect to embryo development. All figures, except a, are from flowers after pollination, a, flower; b, petal and anthers; c, cryostat section of sepal; d, cryostat section of anther; e, hand section of maturing embryos; f, as e, but at slightly more mature stage; g, stigma and style at a more mature stage than f; h, maturing embryos at same stage as g; i, maturing embryos at more mature stage than h; j, cryostat section of seed. Magnification of the sections is as follows: a, b, c and f, x 100; e, g, i and j, × 200; d and h, × 400.

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may be induced by UV radiation, as is the DNA repair UBC gene, RAD6, from S. cerevis&e [23].

Ubiquitin has previously been described to have a role in leaf and flower senescence [7, 38]. In floral senescence, a process occurring after pollination, an increase in ubiquitination of a sub- set of total proteins has been observed. Thus a potential role for the UBC6 protein may be in the targeting of specific proteins either for destruction or modification as part of the mechanism of floral senescence. A number of senescence-related genes have previously been identified and some of these have been shown to be regulated by ethyl- ene [22]. To date we do not know whether expression of the UBC6 gene is regulated by ethyl- ene, however promoter analysis and the use of antisense constructs should elucidate more fully the role of the UBC6 protein.

Acknowledgements

We would like to thank Dr Judith Harmey and Ms Anna Clarke for help with DNA sequencing. We acknowledge the use of the computer facili- ties and Seqnet programs at the Daresbury labo- ratory. The research was supported by project grants from the AFRC (PMB 85/506 and 85/ 518), and J.M. was supported by a grant from the British Council. A.L.M. and F.Z.W. would like to acknowledge the receipt of travel grants from the Royal Society.

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Floral expression of a gene encoding an E2-relatedubiquitin-conjugating protein from Arabidopsis thaliana