Genes encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase in Phaseolus vulgaris L.: nucleotide sequence of cDNA clones and initial studies of expression

Plant Molecular Biology 18: 567-579, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium. 567

Genes encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase in Phaseolus vulgaris L.: nucleotide sequence of cDNA clones and initial studies of expression

Marc R. Knight i and Gareth I. Jenkins *

Departments of Biochemistry and Botany, University of Glasgow, Glasgow G12 8QQ, UK (* author for correspondence); t Present address: Institute of Cell and Molecular Biology, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH.

Received 12 June 1991; accepted in revised form 16 October 1991

Key words: Phaseolus vulgaris L., photoregulation, rbcS genes, ribulose 1,5-bisphosphate carboxylase/oxygenase

Abstract

The small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in the French bean Phaseolus vulgaris L. is encoded by a small gene family consisting of a minimum of three members. Three small subunit genes (rbcS genes) represented in a light-grown primary leaf cDNA library were charac- terised by sequencing two cDNAs which were full-length and one which was deficient in part of the sequence encoding the transit peptide. The cDNA clones are identical in their coding sequences, for both the transit peptide and the mature polypeptide, but divergent in their untranslated sequences. The derived amino acid sequence is very similar to that reported for other species, although the first amino acid of the mature polypeptide is isoleucine, which differs from the methionine found in all other higher plant rbcS genes. Surprisingly, one of the cDNA clones contains two introns, which are at positions conserved in rbcS genes from other species. It is concluded that this cDNA resulted from the cloning of an unprocessed transcript. Alternative polyadenylation sites are found for two of the genes. Expression of the rbcS genes in the primary leaves is stimulated by light, although transcripts can readily be detected in dark-grown leaves. Expression is also organ-specific, as in other species. The frequency of cDNA clones in the library indicates that the different genes show quantitative differences in expression and S 1 nuclease analysis suggests that individual rbcS genes are photoregulated.

Introduction

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is a bifunctional en-

zyme located in the chloroplast stroma in higher plants. Through its carboxylase activity it catal- yses the first step in carbon dioxide fixation in the Calvin cycle of photosynthesis and through a

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X57022, X59999 and X60000.

568

competing oxygenase activity it initiates the process of photorespiration [26]. The holoenzyme in higher plants consists of eight large subunit polypeptides of Mr 52 000-55 000 and eight small sub- units of M r 12000-15000. The large subunit is encoded and synthesised within the chloroplast whereas the small subunit is encoded in nuclear DNA and synthesised on cytosolic ribosomes as a higher molecular mass precursor [ 20 ]. The small subunit precursor has an N-terminal transit peptide which is required for transport of the precursor across the chloroplast envelope [20, 31 ]. Once the precursor is located within the chloroplast, the transit peptide is proteolytically removed [ 36 ] leaving the mature small subunit polypeptide available for assembly into the holoenzyme. This assembly process involves a chaperonin protein called the Rubisco subunit binding protein [21 ].

The nuclear genes encoding the small subunit precursor (rbcS genes) have been studied exten- sively with regard to their organisation, structure, evolution and expression [ 15, 32]. The rbcS genes in different species are normally present in small multigene families; for example, pea has five rbcS genes [13, 35], as does tomato [44], petunia has eight [ 17, 19] and Lemna has at least twelve [41]. Cucumber is unusual in that it is reported to have only one rbcS gene per haploid genome [25 ]. The structure of the rbcS genes is highly conserved within the coding sequence in a given species, more so in the region encoding the mature polypeptide than the transit peptide, but significant differences in amino acid sequence are found between species [ 15, 32]. Although the pattern of rbcS gene expression also varies between species, expression is usually stimulated by light [24, 28] and is tissue-specific, being greatest in tissues containing actively photosynthetic chloroplasts [42, 45]. Studies have shown that expression is controlled principally at the transcriptional level [23, 24, 27, 28] although other levels of control have been shown to be important in certain in- stances [4, 40]. In recent years much research has been concerned with defining the regulatory, cis-acting DNA sequence elements of the rbcS genes and the sequence-specific DNA-binding proteins that interact with them [24, 28].

Our present understanding of rbcS gene structure and expression has come from the study of cDNA and genomic clones of rbeS genes from several different species, and information from further species will enable a more complete pic- ture to be built up. We are interested in studying the rbcS genes of the French bean Phaseolus vulgaris L. because the primary leaves of this plant are well suited to studies of the photoregulation of gene expression in relation to leaf ontogenesis. In this paper we report the isolation and sequencing of three cDNA clones of the rbcS genes from P. vulgaris. We have investigated the number of copies of the genes in the genome and have un- dertaken initial studies of rbcS gene expression.

Materials and methods

Plant material

Seeds of Phaseolus vulgaris L. cv. Tendergreen were obtained from Booker Seeds Ltd (Sleaford, Lincs.). Plants were normally grown in a peat- based compost in a plant growth room at 20- 22 °C, under a 16h photoperiod provided by warm white fluorescent tubes giving a photon flu- ence rate of 150-200 #mol m - 2 S - 1 (400-700nm). Growth of plants in absolute darkness was at the same temperature for 8 days. Where indicated, dark-grown plants were illuminated with contin- uous white light (as above) for 48 h.

Construction and screening of the cDNA library

The cDNA library was constructed by Dr J.V. Cullimore at the Department of Biological Sciences, University of Warwick. Poly(A) + RNA was isolated from light-grown primary leaves at the time when they were fully green and expand- ing. Double-stranded cDNA was prepared by standard techniques and inserted into the Pst I site of the plasmid vector pUC8 by homopolymer tailing [37]. The library was maintained in an Escherichia coli JM83 host. Putative rbcS cDNA clones were selected by screening the library by colony filter hybridisation using a 32p-labelled

probe consisting of the recombinant inserts of cDNA clones pSSU60 and pSSU160, which to- gether encode the pea rbcS coding sequence [2]. Clones which retained a positive hybridisation signal to the pea rbcS probe in subsequent rounds of screening were selected for further study. Plas- mid isolations followed the procedure of Birn- boim and Doly [6].

DNA sequencing

Defined restriction fragments of the recombinant inserts of the cDNA clones were ligated into bacteriophage vectors M13 mpl8 and mpl9 and single-stranded DNA templates were prepared by standard procedures [37]. Dideoxy DNA sequencing [38] was performed with either Klenow polymerase using an Amersham DNA sequencing kit or with Sequenase version 2.0 (U S B Corp.) following the supplier s' in structions. The sequencing reactions were primed with either commercial primers or oligonucleotides complementary to specific regions of the cloned inserts. Single track sequencing for initial characterisation of the cDNA clones was performed using only the A sequencing reaction primed with an 18-mer oligonucleotide (5 ' -AAGCTGCCTAT- GTTTGGG-3 ' ) complementary to a region internal to the P. vulgaris rbcS coding sequence (see Fig. 1).

Analysis of genomic Southern blots

P. vulgaris genomic DNA was isolated essentially by the method of Dellaporta et al. [ 18]. 10 #g of DNA was digested to completion with the appro- priate restriction endonuclease and run on a 0.8 agarose Tris/borate/EDTA gel [37]. The DNA fragments were capillary blotted onto nitrocellulose filter which was then baked for 2 h in vacuo at 80 °C. DNA probes were labelled by nick translation or using random primers to specific activities of approximately 109 cpm/#g [37]. Hy- bridisation was carried out in a solution containing 5 × SSC, 50~/o (v/v) deionised formamide, 1 x Denhardt's solution, 200/~g/ml denatured

569

sheared salmon sperm DNA, 50 mM HEPES pH 7.0 for 16 h at 42 °C. Critical washes were in 0.5 × SSC, twice, for 30min at 60 °C. After washing, the filters were exposed to X-ray film using an intensifying screen at -70 °C.

Studies of rbcS transcript abundance

RNA was extracted from P. vulgaris tissue by ho- mogenisation in a phenol-detergent mixture, followed by DNase treatment and ethanol precipi- tation essentially as described by Bennett et al. [3]. 15/ag samples of RNA were fractionated on a 1.4~o agarose-formamide gel and blotted onto nitrocellulose [3]. Quantitative dot blot analyses were carried out with 1 #g samples of RNA essentially as described previously [30]. Samples containing different amounts (0.01-5/zg) of RNA from light-grown primary leaves were loaded onto the filters as standards to ensure that the assay gave a linear, quantitative estimate of relative rbcS transcript abundance. RNA samples were denatured in 50~o (v/v) formamide, 6~o (v/v) formal- dehyde, 5 m M Sodium acetate, 1 mM Na2 EDTA, 20 mM MOPS pH 7.0 and loaded in trip- licate onto nitrocellulose or Hybond-N (Amer- sham) membrane in 10 × SSC using a manifold. Filters bearing RNA were hybridised to 32p_ labelled DNA probes at 42 °C essentially as described [3], and the critical washes were carried out in 0.1 × SSC, twice, at 55 °C. After washing, the filters were cut up and the radioactivity associated with each dot was determined using a scin- tillation counter. The values were corrected for non-specific binding of radioactivity by subtract- ing cpm associated with control dots either lack- ing RNA or containing rat liver RNA.

S1 nuclease analysis

A 32p-labelled, single-stranded DNA probe was prepared as described by Burke [10] from a single-stranded template of M13 mp19 containing the 227 bp Alu I-Alu I fragment from pPvSS1672 inserted into the Sma I site of the vector in the correct orientation to produce a

570

rbcS- 1 :

rbcS-2 :

I

C~AATAT AGC ~GAAAGAAGTAATTCAGAAGTP~ T G A q t ~ L ~ I ] 2 O O C C ( ~ ~ ~ 100

r~S-3: -*--*****--*--*************************************************************************

M A S S M I S S P A V T T V N R A G A G

101

r~S- I : ~ C ~ G T A ~ ~ T I V J ~ ~ ~ CCAAGCAGGAA~TGATATTACFIUCGTTGCC9%ACAA~

r~S-2: ............................................................... **************************

r~S-3: ****************************************************************************************************

A G M V A P F T G L K S L G G F P S R K M N N D I T S V A N N G G

201

r~S- 1 : ~GTGCAATC~GGT~GAA~TAT2q Aq~AGGAAAATGTGAAGATTI~q'I~AG'I'rAG~CC.hA~ATGATGG

r~S-2: ***************** .........

r~S-3: ***************** ........................................................................

R V Q C I Q

301

r~S- I : A A A ~ T G A ~ A G ~ A ~ C g k A G A A ~ G A ~ C I L ~ I T C G ~ F ~ C ( ] C ~ C A A C A A ~ G G A A G F ~

r~S-2: ........... *************************************************************************************

r~S-3: ............ *************************************************************************************

V W P T V G K K K F E T L S Y L P P L T K Q Q L A K E V

401 ~ 1 r~S-l: A ~ A C C ~ G G A A A G G A T C ~ G T G C T I ~ C G A A T I ~ ' ~ T T ~ V ~ A A ~ C o A A T T G A A A ~ A ~ 9 ~ T A ~ T

r~S-2: ************************************************** ................................

r~S-3: ************************************************** .................................. D Y L L R K G W V P C L E F F L E

501 r~S- 1 : GAATGAATGAATGAATC9kAq~Aq~GATGTT~b~Cqq~GA~q`TGAq~TI~-ATAGCATG~qq~TA~CA~C~T

r~S-2: ........................................................... ****************************************

r~S-3: ..... **************************************** H G F V Y R E H N K S P G

601 .Alul ~ Alul.

r~S- 1 : A ~ T G A T C ~ A A G G T A C I ~ C G A T ~ G A A C ~ V ~ C ~ A T ~ T C / 3 b ~ I G C ~ T I L ~ C ~ A C ~ ~

r~S-2: ****************************************************************************************************

r~S-3: **************************************************************************************************** y y D G R Y W T M W K L P M F G C T D S S Q V L K E L Y E A Q T A H

701 . • r~S-1: C~C~TGGqT~ATCC~ATCATTGC~TIq~ACAACGq~L~G~A~AGqqq<IATTGC~ACAACCCACCAGGCTA~ GTC~XLAAA

r~S-2: ****************************************************************************************************

r~S-3: **************************************************************************************************** P D G F I R ] I G F D N V R Q V Q C I S F I A Y K P P G Y

801 • - r~S-l: ATT]L~AAAACJkGTIL~A~CC(]ZACq~~TTAAA~'AAACqT~A~T]TT~C~qTTGTTGTC~GGATgA~

r~S-2: ******C*CITT***TGqTqL~FA*q'F*~CCAAACT**~*TTG*~ *TT**~**TCA*C**C~T~*~*G**qTC'I~**T**GGA~*~ • ***** * ** * ~ * * . * * ** * * * * * ** ** r~S-3: C ~qCA T ~ C C A ~ A C~ A CCGAA C~A~GCC AATCC ~G G ~G AAA ~ATT A ~ A~

200

300

400

5 0 0

600

700

8 0 0

900

901 Alul A]ul • • rbcS- 1 : AAAGCTAGAGCITAATCCL~ITA/~-oG~ATC4SAC~ Aq~TATI'T(IATATATqTAATGAA~CATC'7~A~'~T~~C

. ** * * * * . * ** ** * * * * * a ' m ~ m , . , c ~ c . ~ c ¢ ~ mc' IsT G *T C C CrSCA ~ A N W W ~ A A . . . . . . . . . . . . . . . . . . . . . . rbcS-21 • * * ** * * *** * *** * * * * ** * * *** * *

rbcS-3: q ~ ACATA GG q'ITA TATAT TATATATATA ATG C GC~ G ACC AT AC A TC~ CT~ AqTC TI~ ACT TI~

I000

1001 . . . . 1100

rbcS- 1 : A T C ~ q C ~ A A ~ A T C A T C ~ ' ; ~ i A T T C A ( ~ A A ~ ~ A G T A A G T A G A ~

rbcS-2 *CA*A*A****A~GAAA* * * * ** ** * * A AAA A A AA AAA --- rbcS-3 : T*T*TG*TA

1101 1 1 3 5

rbcS- I :

rbcS-2:

rbcS- 3 : ...............................

Fig. 1. Nucleotide sequences and derived amino acid sequences of three P. vulgaris rbcS cDNAs from plasmids pPvSS1672, pPvS $965 and pPvS S 191, representing three genes designated rbcS-1, -2 and -3 respectively. The nucleotide sequence is numbered from the 5' end of the longest cDNA (rbcS-l) and other sequences are aligned to this. Regions where corresponding sequence is absent are indicated by dashed lines; this includes the two regions of intron sequence present only in rbcS-1. Regions of sequence identical to rbcS-1 are indicated by asterisks. Recognition sequences of restriction endonucleases referred to in the text are un- derlined. Putative polyadenylation signals are boxed. The arrow indicates the point from which single-track DNA sequence was obtained to characterise the different cDNA clones.

probe complementary to rbcS transcripts. S 1 nuclease assays were performed using 5/~g total RNA and 5 ng single-stranded probe as described by Nagy et al. [34]. The products of the assay were fractionated on a 8 M urea, 6 ~o polyacrylamide gel and the protected fragments were visualised by autoradiography as described above. Size markers were provided by pUC8 DNA cut with Hpa II and end-labelled with 32p.

Results

Characterisation of rbc S cDNA clones

Forty-two P. vulgaris rbcS cDNA clones were selected from a light-grown primary leaf cDNA library by screening with a pea rbcS cDNA probe. Initial characterisation of the cDNA clones in- volved a detailed comparison of single-track DNA sequencing reactions primed from a site 141 bp 5' to the translation termination codon and extending to the poly(A) tails. This analysis revealed that the clones can be grouped into three classes based on differences in their 3' untranslated sequences. Of the 42 clones, 23 are designated type one (rbcS-1), 15 are type two (rbcS-2), and 4 are type three (rbcS-3).

571

The nucleotide sequences of the complete inserts of representative cDNA clones (pPvS S 1672, 965 and 191 representing rbcS-1, -2 and -3 respectively) were determined and are shown in Fig. 1. Two of the clones contain the complete coding sequence of the rbcS precursor along with 3' and 5' untranslated sequences and the other (pPvSS965) contains the sequence encoding part of the transit peptide, the entire mature polypeptide and the 3' untranslated sequences. The complete sequences indicate that the precursor contains a 57 amino acid transit peptide and a 123 amino acid mature polypeptide, which would give a precursor of M r 20 141 and a mature polypeptide of Mr 14472. The nucleotide sequences of the three clones are 100~o identical in the coding regions but diverge in the 5' and 3' untranslated regions. Comparison of the first 120 bp of the 3' untranslated sequences of the three rbcS cDNA clones reveals a homology of 68~o between pPvSS1672 and pPvSS965. Homology to pPvSS191 is considerably lower in this region (41~o and 4 7 ~ to pPvSS965 and pPvSS1672 respectively).

Alternative polyadenylation signals are present in cDNA clones of the rbcS-2 and rbcS-3 types (Fig. 1). Poly(A)tails start either 162 bp or 132 bp from the TAA translation termination codon in

Table I. Donor and acceptor sites for introns in the P. vulgaris rbcS-1 gene, a soybean rbcS gene [5], and the consensus for legumes, plants [9] and eukaryotes [33]. The consensus for legumes was derived from the 57 legume intron sequences given by Brown [9].

Donor - Intron - Acceptor

RbcS-1 1. CAG:GTAAGA 2. GAG:GTGAAT

Soybean rbcS 1. CAG:GTAAGA 2. GAG:GTCAAT

Legumes NAG:GTAAGT

Plants ACAG:GTAAGT

Eukaryotes ACAG:GT~AGT

AAATGAAATGAGTAG:G GATGTGTGTTGATAG:C

TATGCAAATTACTAG:G TGTGTTTGTATATAG:C

A A AA AC T TT~ T T . ~ TTT~TAG:G

A A A A A TTTT T I T T T T TGCAG:G

G G G G G

T T T T T T T T T T T , T . ~ C C C C C C C C C C C ~C AcJ:cJ

572

(a)

French bean Soybean Pea Petunia Potato Tobacco Tomato Wheat Rice Maize Lemna gibba Arabidopsis Radish Sunflower Larix Acetabularia Chlamydomonas

MASSMISSPAVT-TVNRAGA-GAGM---VAPFTGLKSLGGFP-SR .... KMNNDITSVANNGGRVQC (59) ************-*******-*--*---**************-** .... *T******I********* (55) ***-********-************---*************** ..... K*V*T****ITS*****K* (57) ************-******--****---***************** .... *Q*L****I*S******* (57) ************-*****--****---*****************---******************* (58) ***********-*****--****---***************** .... *Q*L****I*S******* (57) *****V**A*AA-*RSNVA--Q*S*---*********AAS**VTK---KNN*V****L*S*****R* (58)

MA***M-ASSATT ......... ****Q****TA*L*V** .... RSRGSLG**-S****IR* (46) MA*S*M-ASSATT ......... ****Q*SSPPPACRRPP .... SELQLRQRQH--***IR* (45) MA*T*MMASSATA ......... ****Q****TASL*VA* .... RSSRSLGN*-S****IR* (47)

*****MV*T*AV-ARV*PAQT--N*---*GA*N*CR*SVA**AT* .... *A***LSTLPSS****S* (57) *****L**ATMVASPA ..... ****---**************** .... *A******ITS*****N* (55) *****L**A**VTSQL ..... ****---**************** .... *T*T****I*S*****S* (55) ***--****-**-************---*************** ..... *KA**FSTLPS******* (55) ****IMALSSTAAVAAV*APSKTGNSNV*SA******MAQ**S*KTMSNAGAEWEQKTTS**S**R* (67) ***I*MNKSV*L-SKEC*KP-L*TP---KVTLNKRGFATTIA-TK .... NRE ............... (42) **AVIAK*SVSA-A*A*PAR-S ........... SVRPMAALK-PA .... VKAAPVAAP*QANQ .... (46)

(b)

French bean Soybean Pea Petunia Potato Tobacco Tomato Wheat Maize Rice Lemna gibba Arabidopsis Radish Sunflower Larix

Acetabularia Chlamydomonas

10 20 30 40 50 IQVWPTVGKKKFETLSYLPPLTKQQLAKEVDYLLRKGWVPCLEFEL*HGFVYREHNK ********************************************************* ********************************************************* ********************************************************* ********************************************************* *********************************************************

********************************************************5 **********************************************-********* ***********************************************-*********

M****IE*I*************VED*LKQIE**APFQV******SKVGFVYREN*KS ***********************************************-********* ********************************************************* ********************************************************* ******************************************************** *********************************************-***********

10 20 30 40 50 60 MM**QP,NN*H***F*F*****DE*ISKQ***I*TNS*T*****AASDQAYAGNE*CIRMGPV MM**TP*NN*M***F*******DE*IA*Q***IVAN**I*****AEADKAYVSNESAIRFGSV

French bean Soybean Pea Petunia Potato Tobacco Tomato Wheat Maize Rice Lemna gibba Arabidopsis Radish Sunflower Larix

60 70 80 90 100 110 120 SPGYYDGRYWTHWKL*MFGCTDSSQVLKELYEAQTAHPDGFIRIIGFDNVRQVQCISFIAYKPPGY ****************************************************************** ****************************************************************** ****************************************************************** ****************************************************************** ****************************************************************** ****************************************************************** ************************************************************************ ******************************************************************* _********************************************************************** **************************************************************** ********************************************************************

****************************************************************5**** ************************************************************5***** *****************************************************************

70 80 90 100 110 120 130 140 Acetabularia AST*Q*N* ******** ******G****S* IQACTK*F**AY**LVC**AN**** ISG*LVHR**SATDYRLPADRQV Chlamydomonas *CL* * *N* ** ** ** ** ** ** R* PM** *R* IVACTK* F* *AYV*LVA** *QK* * * IMG*LVQR*KTARDFQPANKRSV

Fig. 2. Comparison of the derived amino acid sequence of P. vulgaris rbcS cDNAs with sequences reported for rbcS genes from other species selected from the EMBL database. Regions of amino acid sequence identical to P. vulgaris are indicated by asterisks. Where more than one sequence is reported for a given species, only one gene has been used for alignment, a, transit peptide; b, mature polypeptide.

rbcS-2 clones and either 251 bp or 166 bp from the TAA in rbcS-3 clones. The longer transcripts are encoded by 11 of the 15 rbcS-2 clones and 3

of the 4 rbcS-3 clones. All 23 rbcS-1 clones are polyadenylated at a site 281 bp from the TAA translation termination codon.

Unexpectedly, two regions of intron sequence are present in the rbcS-1 cDNA clone pPvS S 1672 (Fig. 1). The first region of 98 bp in length is located between the codons encoding amino acids 2 and 3 of the mature polypeptide and the second region is between the codons encoding amino acids 47 and 48 of the mature polypeptide. The positions of these introns are conserved with respect to the rbcS genes of other species. The sequences of the intron-exon boundaries show good conservation with those reported for the soybean rbcS gene SRS1 [5], the consensus sequence for legume genes (derived from ref. 9) and the consensus sequences reported for plant [9] and eukaryotic [33] genes in general (Table 1). We have not examined all the cDNA clones for the presence of introns, although sequencing of two other rbcS-1 cDNAs shows that they do not contain intron sequences. Moreover, preliminary restriction analysis suggests that few, if any, of the other rbcS-1 cDNAs contain intron sequences.

The derived amino acid sequence of the coding region of the P. vulgaris rbcS precursor polypeptide shows a high degree of homology with those reported for other species (Fig. 2). The greatest homology is found with the soybean SRS 1 coding sequence [5]; the homology is 89~o for the transit peptide and 86~o for the mature polypeptide. A much lower degree of homology is found with the pea rbcS amino acid sequence, particu- larly in the transit peptide. Of particular interest is the amino acid sequence at the junction between the transit peptide and the mature polypeptide. The P. vulgaris transit peptide terminates with cysteine, as in all other rbcS genes, but, as a result of a single nucleotide change, the mature polypeptide starts with isoleucine in contrast to the methionine found in all other higher-plant species studied to date.

Analysis ofrbcS genes in genomic DNA

To obtain an estimate of the number of rbcS genes present in the P. vulgaris rbcS gene family we hybridised Southern blots of genomic DNA, digested to completion with restriction endonu-

573

cleases, to specific rbcS cDNA probes. As can be seen in Fig. 1, each of the P. vulgaris rbcS genes represented in the cDNA library contains a single Eco RI site (in the sequence encoding amino acids 43 and 44 of the mature polypeptide). Hence a probe containing sequences from this site to the 3' end of the gene should hybridise to genomic fragments containing the 3' portion of at least one rbcS gene. Such a probe is the approximately 700 bp fragment of pPvSS1672 extending from the internal Eco RI site to the Eco RI site in the polylinker of the cDNA clone. As shown in Fig. 3, the pPvSS1672 Eco RI-Eco RI probe hybridises to three bands on the Southern blot of genomic DNA digested with Eco RI. One of the bands has a stronger signal than the other two, probably because this fragment contains non-coding sequences cognate to the rbcS-1 gene from which the probe was derived. Hybridisation of the probe to Southern blots of genomic DNA digested with Barn HI or Hind III, which do not cut within the coding sequence of the rbcS genes represented in the library, reveals three or four hybridising bands. As discussed below, these data suggest that there are relatively few rbcS genes in P. vulgaris genomic DNA.

Fig. 3. RbcS sequences in P. vulgaris genomic DNA. P. vulgaris genomic DNA was digested to completion with either Hind III (lane 1), Eco RI (lane 2) or Bam HI (lane 3), run on an agarose gel and blotted onto nitrocellulose. The Southern blot was hybridised to a 32p-labelled Eco RI-Eco RI fragment from pPvSS1672 extending from the Eco RI site in the coding sequence to the Eco RI site in the polylinker adjacent to the 3' end of the cDNA. Size markers are indicated on the right.

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expression of the P. vulgaris rbcS genes in primary leaves is regulated by light. Expression is readily detected in RNA from dark-grown primary leaves, but increases substantially when the plants are transferred to white light for 48 h.

The relative expression of the rbcS genes in different organs was measured by quantitative dot blot hybridisation using the above probe (Ta- ble 2). The results from several experiments indi-

Fig. 4. Photoregulation of rbcS gene expression in P. vulgaris primary leaves. Total RNA was isolated from the primary leaves of either dark-grown plants (lane 1) or dark-grown plants transferred to white light for 48 h (lane 2). The RNA was fractionated on an agarose-formamide gel, blotted onto nitrocellulose and hybridised to the pPvSS 191 cDNA.

Expression of rbc S genes

Expression of the rbcS genes was investigated initially by hybridisation of the complete cDNA insert of pPvSS191 to northern blots of P. vulgaris total RNA. This probe is expected to hybridise to the coding regions of transcripts of all rbcS genes expressed. Figure 4 shows a northern blot of total RNA from primary leaf tissue harvested from plants either grown in complete darkness or grown in darkness and then illuminated with white light for 48 h. The probe hybridises to transcripts of approximately 800-1000 nucleotides, which is consistent with the size expected from the sequence data. In addition, Fig. 4 shows that

Table 2. Relative levels of rbcS gene expression in different organs of P. vulgaris. Expression was measured by quantitative dot blot hybridisation using 1 #g total RNA and the 32p_ labelled insert of pPvSS 191 as a probe.

Tissue Relative rbcS expression (%)

48 h illuminated primary 100 leaves

Dark-grown primary leaves 14 Cotyledons 5 Roots 7 Stems 5 Pericarp 8

Fig. 5. SI nuclease analysis of P. vulgaris rbcS transcripts. Total RNA was isolated from the primary leaves of either dark-grown plants (lane 1) or dark-grown plants transferred to white light for 48 h (lane 2). 5/~g RNA was hybridised to a 32p-labelled, single-stranded probe complementary to rbcS transcripts derived from the 227 bp Alu I-Alu I fragment of pPvSS1672 containing 114 bp of coding sequence and 113 bp of 3' non-coding sequence. After digestion with S 1 nuclease the products were resolved on a sequencing gel. The approximate sizes of protected fragments revealed by autoradiography are indicated.

cate that expression in dark-grown primary leaves is approximately 14~ of that found in primary leaves illuminated for 48 h in white light. Expres- sion is detected in other organs, although at lower levels than in dark-grown primary leaves.

In order to investigate the expression of individual rbcS genes in primary leaf tissue we have used a S1 nuclease protection assay. A single- stranded DNA probe was generated from a defined 227 bp Alu I-Alu I fragment of pPvSS1672 which extends from 114 bp from the end of the coding sequence to 113 bp into the 3' untranslated region. This probe was hybridised to total RNA from primary leaf tissue of dark-grown or illuminated plants, the products were digested with S 1 nuclease and then resolved on a dena- turing polyacrylamide gel. As shown in Fig. 5, this analysis reveals protected fragments of approximately 225, 125 and 115 bp. The approximately 225 bp protected fragment (cognate to rbcS-1 RNA) is considerably more abundant in RNA from illuminated as opposed to dark-grown leaves, although it can be detected if 10 #g RNA from dark-grown tissue is used (data not shown). The abundance of the smaller protected fragments, which are interpreted to represent transcripts of rbcS-2 and -3 genes (see Discussion), also shows an obvious light-dark difference, and again these fragments are detectable in RNA from dark-grown tissue.

Discussion

The present analysis of rbcS eDNA clones dem- onstrates that P. vulgaris expresses at least three highly homologous rbcS genes. The coding sequence of two of the genes is identical for the whole transit peptide and the mature polypeptide, while the coding sequence of the third gene is identical over the region sequenced. Members of rbcS gene families in other species generally show high degrees of homology, and 100~o identity in the nucleotide sequence throughout the coding region has also been reported for particular members of the tomato rbcS gene family [44]. In those species studied to date, including tomato, the

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greatest similarity is seen between rbcS genes that are closely linked in the genome [15, 32]. Such linkage has been reported in several rbcS gene families. In pea, for example, all five genes are linked at the same locus [35] and in tomato three of the five genes are linked [44]. In view of their identical coding sequence, it is to be expected that the P. vulgaris rbcS genes are linked, and recent observations are consistent with this hypothesis; hybridisation of the complete pPvSS 191 insert to Southern blots of P. vulgaris DNA digested to completion with Pst I, Xho I and Sal I shows only a major high-molecular-weight band, suggesting that all the rbcS genes are present on a common, large fragment (N.A.R. Urwin and G.I. Jenkins, unpublished). Our observations are in agreement with genetic analysis [46] which indicates that individual P. vulgaris genotypes express one type of rbcS polypeptide and that the rbcS locus be- haves as a single heritable character in crosses between cultivars.

The Southern blot hybridisation data presented in Fig. 3 indicate that P. vulgaris has a minimum of three different rbcS genes. The presence of the internal Eco RI site in the three genes represented in the eDNA library facilitates investigation of the number of gene copies and the simplest interpre- tation of the hybridisation analysis of the Eco RI genomic digest is that only three genes are present. However, it is possible that genomic fragments hybridising to the probe contain two rbcS genes arranged 'tail to tail'. Moreover, we cannot ex- clude the possibility that additional genes not containing Eco RI sites are present in the genome, although any such genes must be expressed at a very low level in illuminated leaf tissue otherwise they should have been represented among the 42 eDNA clones examined in this study. The hybridisation analysis of the Bam HI and Hind III genomic digests is consistent with the presence of a small gene family but further studies are required to determine exactly how many genes are present. In addition, experiments should be un- dertaken to investigate whether additional rbcS genes are expressed significantly in non-leaf tissues or in leaf tissue at other stages of development. RbcS genes which account for less than 1 ~o

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of the total transcripts in leaf tissue have been reported, for example, in petunia [ 17].

The P. vulgaris genes diverge in their 3' and 5' untranslated sequences, as do rbcS genes from other species, including those with the highest coding sequence homology. Multiple polyadenylation sites are also found in other species, for example, in petunia [16] and soybean [39]. The polyadenylation signal of rbcS-i is presumed to be the hexanucleotide AATAAT, which closely resembles the eukaryotic consensus AATAAA [7]. The rbcS-2 and -3 genes contain two and one copies respectively of the hexanucleotide AAT- GAA, which is likely to be the polyadenylation signal as it is identical to that found in four of the pea rbcS genes [22]. The first polyadenylation signal in rbcS-3 is not obvious from the sequence. The complexity of polyadenylation in rbcS genes and plant genes in general is discussed by Shirley et al. [39].

The presence of introns in one of the cDNA clones is very unusual. However, the location of the introns is consistent with the locations reported for rbcS genes in other species [15, 32]. For instance, two introns at these positions are present in the rbcS genes of the legumes pea [22] and soybean [5]. Genes with three introns are found in certain rbcS genes in members of the Solanaceae, and single introns are present in rbcS genes of certain monocotyledonous species [15, 32]. In addition to the locations of the introns, the splice junction sequences reported here resemble those found in other rbcS genes and in plant genes in general. Therefore there is no reason to suppose that the additional sequences in the pPvSS 1672 cDNA clone are anything other than genuine intron sequences. We have not detected introns in any of the other rbcS cDNA clones so it is unlikely that P. vulgaris RNA contains an unusually large population of unprocessed rbcS transcripts. Indeed, a larger transcript is not detected by northern blot analysis. Thus it seems most likely that the observation represents a low probability event, namely the fortuitous cloning of an unprocessed transcript.

The derived amino acid sequence of the P. vulgaris rbcS precursor polypeptide is highly

homologous to the sequences reported for other species. The sequence 6~YYDGRYWTMWKLP- MFG76, which is found in most higher plant rbcS genes [15] is conserved in P. vulgaris. However, one feature of the P. vulgaris rbcS genes which distinguishes them from those of all other species studied to date is the change in the first amino acid of the mature polypeptide from methionine to isoleucine. This substitution is unlikely to make a significant difference to the structure of either the precursor or the mature polypeptide. All three types of P. vulgaris rbcS gene exhibit this amino acid substitution and, since the precursors must presumably be efficiently transported into the chloroplast and processed to the mature size, the processing enzyme must be able to recognise and cleave the altered site.

The expression of rbcS genes in P. vulgaris is organ-specific as in other species and is regulated by light. We have used dot blots to estimate the relative abundance of the rbcS transcripts in different RNA samples because they are more quantitative than northems or S 1 assays. Expression is less strictly photoregulated than in, for example, pea [29], in that transcripts can readily be detected in dark-grown leaf tissue, but it is more strict than in barley [ 1] and cucumber [25]. The expression in certain non-leaf tissues (except pericarp) also appears to be somewhat greater than in pea [14], although it is less than in the dark- grown primary leaves. Several observations indicate that the extent of rbcS expression is related to the degree of leaf and plastid development [12, 42, 45]. Studies of the det mutants of Arabi- dopsis [ 12] demonstrate that plastid development and the expression of genes encoding chloroplast proteins, including the rbcS genes, is closely linked to leaf development, which in the wild type but not the mutants is dependent on light. The P. vulgaris primary leaves are well developed in dark-grown plants [8], certainly more so than in pea, and they have well developed etioplasts [8, 47]. These observations are therefore consistent with the higher degree of rbcS transcript accumulation in the dark-grown primary leaves compared to some other species. A study of plastid development in non-leaf tissues might

show a further correlation with rbcS transcript levels.

The individual rbcS genes of P. vulgaris show quantitative differences in expression, again as found in the rbcS gene families of other species [ 15, 32]. In petunia, for example, at least a hun- dred fold difference is seen in the transcript levels of the least and most expressed genes [17]. The representation of clones of different types in the cDNA library provides an indication of the relative levels of expression. The data for P. vulgaris indicate that the rbcS-1 gene accounts for approximately 55~o of the transcripts in illuminated primary leaves, rbcS-2 accounts for approximately 36~o and rbcS-3 approximately 9~o.

S 1 nuclease protection assays have been used by other workers to provide information on the expression of individual genes in a gene family, for instance for the different rbcS genes of pea [22]. The data in Fig. 5 provide initial information about the expression of the different P. vulgaris rbcS genes. The protected fragment of approximately 225 bp in Fig. 5 is likely to result from hybridisation of the rbcS-1 probe to its cognate transcripts (predicted size 227 bp). The approximately 125 bp fragment is similar to the predicted size (130 bp) of the fragment that would result from hybridisation of the probe to either rbcS-2 or -3 transcripts. Transcripts of these two genes cannot be distinguished using this probe as the initial point of divergence in the 3' untranslated sequence is the same for both. The 115 bp fragment is not predicted from sequence data but is unlikely to be derived from a separate rbcS gene because it would be expected, based on the abundance of the fragment, that such a gene would be represented in the cDNA library. It is more likely that the 115 bp fragment is an artefact of the S1 assay, resulting from S 1 nuclease digestion of the RNA-DNA duplex at an AT-rich region near to the terminus of the duplex, as reported by other workers [22]. This is an inherent feature of the S 1 assay [11 ]. Sequence data show that the cDNA clones pPvS S 191 and pPvS $965 both have a 7 bp AT-rich region starting 9 bp upstream from the theoretical end of the duplex. If this region was susceptible to attack by S 1 nuclease then a 121 bp

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protected fragment would be predicted, correlat- ing with the approximate fragment size observed.

The S 1 data indicate that rbcS-1 and one or both of the rbcS-2 and -3 genes are photoregulated. Although the 225 bp fragment representing transcripts of the rbcS-1 gene shows a greater difference in signal in RNA from dark-grown ver- sus illuminated leaves than the 125/115 bp fragments corresponding to the rbcS-2 and -3 genes, there is no differential photoregulation of the genes of the magnitude reported for the tomato rbcS genes [43]. Unfortunately, because of the similarity of the 3' untranslated sequences of the P. vulgaris rbcS genes, the expression of the different genes cannot be distinguished using a single probe in an S 1 assay. In addition, the presence of artefactual bands in the assay complicates in- terpretation of the data, and quantification of the relative signals becomes difficult. For this reason we have initiated work using gene-specific oligonucleotide probes to investigate further the quantitative and qualitative expression of the individual P. vulgaris rbcS genes.

Acknowledgements

We are indebted to Dr Julie Cullimore for gener- ously allowing us access to the P. vulgaris cDNA clones. G.I.J. thanks Anne-Marie Clarke and James Jardine for excellent technical assistance and the Agricultural and Food Research Council for the support of this research. M.R.K. thanks the Science and Engineering Research Council for a postgraduate studentship.

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Documents

Genes encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase in Phaseolus vulgaris L.: nucleotide sequence of cDNA clones and initial studies of expression