Identification and characterization of a proline-rich mRNA that accumulates during pod development in oilseed rape (Brassica napus L.)

Plant Molecular Biology 23: 1223-1232, 1993. © 1993 Kluwer Academic Publishers. Printed in Belgium. 1223

Identification and characterization of a proline-rich mRNA that accumulates during pod development in oilseed rape (Brassica napus L.)

Simon A. Coupe 1, Jane E. Taylor 1,3, Peter G. Isaac 2 and Jeremy A. Roberts 1., 1 Department of Physiology and Environmental Science, Faculty of Agriculture and Food Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK (* author for correspondence); 2 Nickerson Biocem Ltd., Cambridge Science Park, Milton Road, Cambridge, CB4 4GZ, UK; 3 Present address: Division of Biological Sciences, Institute of Environmental and Biological Sciences, Lancaster University, Lancaster, LA1 4YQ UK

Received 8 June 1993; accepted in revised form 24 September 1993

Key words: abscission, dehiscence, pod, proline-rich, shatter

Abstract

Pod development in oilseed rape (Brassica napus) culminates in a process known as dehiscence (shatter) which can result in the loss of seed before the crop is harvested. In order to investigate the biochemi- cal and the genetic basis controlling this process, a cDNA library was constructed from the dehiscence zone of developing pods. This resulted in the isolation of a cDNA clone (SAC51). The mRNA encoded by SAC51 had a transcript size of ca. 700 nucleotides and was found, by northern analysis, to accu- mulate preferentially in the dehiscence zone of the pod and in no other part of the plant analysed. The predicted polypeptide is rich in the amino acids proline (14.2~) and leucine (14.2 ~o). The sequence of the polypeptide has more than 40 ~o amino acid sequence identity with polypeptides isolated from carrot embryos, maize roots, soybean seeds and young tomato fruit. The function of these proteins is unknown. Genomic Southern analysis suggests that SAC51 is encoded by a single gene or small gene family. The role of the peptide in the development of pods of oilseed rape is discussed.

Introduction

Abscission is the process that causes the shedding of a range of plant parts, including leaves, flowers and fruit [33]. The process occurs at precise sites and involves coordinated cell wall breakdowns. Associated with cell separation is an increase in the activity of several hydrolytic

enzymes including fl-l,4-glucanase (cellulase, EC 3.1.2.4) [40] and polygalacturonase (PG, EC 3.2.1.15) [37, 38].

The process of pod dehiscence, or shatter as it is commonly termed, in oilseed rape shares a number of features with abscission. Degradation and separation of cell walls occurs along a dis- crete layer of cells, termed the dehiscence zone,

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X71618.

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and a localized increase in the activity of cellulase has been reported prior to the onset of dehiscence [23, 24]. This process is agronomically important because it may result in the premature shedding of seed before the crop can be harvested. Adverse weather conditions can exacerbate the process, resulting in greater than 50~o loss of yield [20].

Attempts to solve this problem over the past 20 years have focused on the breeding of shatter- resistant varieties. One approach is to introduce germplasm from related species by interspecific hybridization. Related species such as Brassica nigra, B. juncea and B. campestris have been used for this purpose [1, 17, 27] but plants resulting from these crosses are frequently sterile and lose favourable characteristics which have to be re- gained by back-crossing. This is both time- consuming and laborious. Other methods employed to alleviate the problem include chemicals, in the form of desiccants and pod-sealants. A common method to prevent seed loss by shatter, is the mechanical technique of swathing in order to get uniform desiccation of the crop and reduce wind-stimulated shattering in the upright crop.

The present investigation was initiated in order to study changes in gene expression that may accompany the process of pod dehiscence in an effort to isolate genes that may control the process.

Materials and methods

Plant material

Seeds ofB. napus cv. Rafal were grown [23] with the following modifications. Single seedlings were planted into 10 cm pots, and after vernalization, were re-potted into 21 cm pots. At anthesis, tags were applied daily to record flower opening. This procedure facilitated accurate age determination of each pod. Pods were harvested at various days after anthesis (DAA). The dehiscence zone which also comprises vascular regions and a replum that divides the seeds (see Fig. 1)was excised from the non-zone material and seed using a scalpel blade [24] and immediately frozen in liquid N2 and stored at -70 °C.

ZONE

NON ZONE

Fig. I . Diagrammatic representation of a transverse section through a B. napus pod showing the different parts used for RNA extractions. The asterisk denotes where the seeds were located.

RNA isolation

All chemicals were molecular biology grade from either Sigma Chemical (Dorset, UK), or Fisons (Loughborough, UK). Total RNA was extracted using the polysomal extraction method [7], with the following alterations. The plant material was ground to a powder in liquid N2 and then in 10 volumes of extraction buffer (200 mM Tris- acetate pH 8.2, 200mM magnesium acetate, 20 mM potassium acetate, 20 mM EDTA, 5~o w/v sucrose; after sterilization 2-mercaptoethanol was added to 15 mM and cycloheximide added to a final concentration of 0.1 mg/ml). The super- natant was layered over 8 ml 1 M sucrose dis- solved in extraction buffer and centrifuged in a Kontron (Switzerland) TFT 70.38 rotor at 45000 rpm (150000 xg) for 2 h at 2 °C. The re- sultant pellets were resuspended in 500 gl 0.1 M sodium acetate, 0.1~o SDS, pH6.0, extracted with phenol/chloroform (1:1 v/v), and the total RNA precipitated. Poly(A) + RNA was isolated from total RNA extracted, from both the zone and non-zone tissue of 40, 45 and 50 DAA pods, using a Poly(A) Quik mRNA purification kit (Stratagene, Cambridge, UK) following the manufacturer's instructions, and then pooled. Total RNA was also extracted from leaves, seeds and roots [ 10] for use in northern analyses.

cDNA library construction and screening

A cDNA library was constructed using 5 #g poly(A) + RNA extracted from the dehiscence zone of pods prior to and during dehiscence. The library was constructed using the 2ZAP-cDNA synthesis kit according to the manufacturer's instructions (Stratagene). This resulted in the production of a library containing 1.2 x 106 re- combinants. Several plaques were picked at random and inserts excised in vivo [35]. The average insert size was 1 kb. Differential screening was performed using single-stranded cDNA probes synthesized from 1 #g poly(A) + RNA isolated from dehiscence zone or non-zone pod material. The probes were synthesized using the method of [26] and used to screen 40000 recombinant plaques by in situ plaque hybridization. Duplicate plaque lifts were obtained using Hybond N + membranes (Amersham, Aylesbury, UK) and were then treated, according to the manufacturer's instructions. The lifts were hybridized with [ ~32p ] dCTP radio-labelled probe at 65 °C overnight in 5 x SSPE (0.9 M sodium chloride, 0.05 M sodium phosphate, 5 mM EDTA), 5 x Denhardt's solution, 0.5~ SDS and 500/~1 of denatured salmon sperm (1 mg/ml). The final wash was at 65 °C in 0.1 x SSPE, 0.1~ SDS. Plaques hybridizing preferentially to zone probes were re- screened at densities of 50-100 plaques/plate. Chosen plaques were isolated from the plate and phagemids isolated using the in vivo excision procedure [35]. cDNA inserts were amplified by polymerase chain reaction (PCR). This procedure utilized oligos homologous to the T3, T7 bacteriophage promoters which border the cDNA to amplify the insert which was subsequently used for probes. Isolated plasmid was also used as a template for sequencing.

Northern blot analysis of RNA

Approximately 10 #g total RNA isolated from various parts of the oilseed rape plant were separated on a 1 x MOPS (3-[N-morpholino]pro- panesulphonic acid) buffer (20mM MOPS,

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5 mM sodium acetate pH 7.0, 1 mM EDTA), 1 ~/o agarose/6~o formaldehyde denaturing gel. The RNA was transferred onto a nylon membrane (GeneScreen, NEN/DuPont, UK)using capillary transfer. A radio-labelled probe was generated using 100 ng of insert from the plasmid pSAC51, using [~32p] dCTP (110 TBq/mmol, Amersham) and a Nick Translation Kit (Boehringer Mann- heim, Lewes, UK). Unincorporated label was removed by passage through a Sephadex G-50 col- umn. The blot was hybridized as described for plaque screening. The final wash was at 65 °C in 0.1 × SSPE, 0.1~o SDS and the blot exposed to Kodak X-AR5 films with intensifying screens at -70 °C.

Genomic DNA isolation and characterization

DNA was isolated, by miniprep procedure using a modified form of the extraction buffer described [ 8]. Young expanding oilseed rape seedlings were homogenized in a 3.8:0.6:0.6 mixture of TNE buffer (0.05 M Tris.HCl pH 7.5, 0.2 M EDTA, 0.1 M NaC1)/5 ~o SDS/1 mg/ml Proteinase K; to this solution was added sodium diethyldithiocar- bamate and sodium bisulphite to 0.4~o (w/v). The samples were incubated for 1 h at 37 °C and de- bris removed by centrifugation at 11 600 x g for 5 min. The eluate was extracted with equal volumes of phenol/chloroform (1:1 v/v) followed by chloroform alone. Nucleic acids were precipitated by the addition of 2.5 vols 95~o ethanol containing 5~o (v/v) 2 M sodium acetate, pH 5.5. The sample was mixed and immediately centrifuged at 11600 x g for 5 min. The resulting pellet was resuspended in 300#1 TE, 10#1 of RNaseA (10 mg/ml) added, and then incubated at 37 °C for 15 min before 300 #1 cetyltrimethylammonium bromide (CTAB) buffer (0.2 M Tris.HCl pH 7.5, 0.05 M EDTA, 2 M NaCI and 2~o w/v CTAB) was added before a further incubation at 60 °C for 15 min. After re-extraction with an equal volume of chloroform, DNA was precipitated with an equal volume of isopropanol at -20 ° C. Sub- sequent digestions by restriction endonucleases were carried out using 10/~g DNA as detailed in

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[36]. The DNA was separated on a 1 x TBE, 0.8 % agarose gel and transferred to GeneScreen Plus nylon membrane (NEN). The probe was made as described for northern analysis and the hybridization conditions were as described for plaque screening. The final wash of the membrane was at 65 °C in 0.1 x SSPE, 0.1~o SDS.

DNA sequencing

Plasmid DNA was isolated by the alkaline lysis method [30]. Supercoiled plasmid DNA was isolated as reported in the TaqTrack sequencing manual (Promega Southhampton, UK). Ca. 5 #g of denatured plasmid was sequenced using the chain-termination method [31] using S equenase v. 2.0 (USB, c/o Cambridge BioScience, UK). Compressions were resolved by performing the reactions at 70 °C using Taq DNA polymerase (TaqTrack, Promega). DNA sequences were analysed using the University of Wisconsin Ge- netics Computer Group (UWGCG) package [ 11 ] and the DNA Strider program [21].

Results

Isolation of cDNA clones by differential screening

cDNA clones of mRNAs accumulating preferentially in the dehiscence zones of developing pods

were identified using a differential screening strat- egy. This employed random-primed, radioactively labelled first-strand cDNAs generated from poly(A) + RNA, isolated from the pooled 40, 45 and 50 DAA samples of the pod containing the dehiscence zone and from adjacent tissue lacking this zone (see Fig. 1). Potential positives were confirmed by isolation and rescreening at lower densities. By this method 36 clones were isolated which on cross-hybridization could be grouped into 13 families. When screened with the insert from the clone designated pSAC51, 19 other clones were shown to hybridize (data not shown), indicating that this cDNA may encode an abun- dant mRNA. The insert from pSAC51 was ca. 700 bp in length.

SAC51 mRNA expression by northern analysis

Pods were harvested at 20, 40, 45, 50, 60 DAA and dehiscence zone (Z) and flanking non-zone (NZ) tissue isolated (see Fig. 1). Total RNA was extracted from these excised parts and from the seed, leaves and roots. Northern analysis revealed that the 700 bp insert from pSAC51 hybridised to a mRNA of about 700 nucleotides (see Fig. 2). At 20 DAA hybridization was detectable in both Z and NZ but only on prolonged exposure of the blot (see Fig. 2a). The mRNA was not detected in NZ tissues at later stages of development whilst

Fig. 2. Northern blot analysis of total RNA (10/~g) from B. napus. The northern blot was hybridized to radiolabelled SAC51 cDNA insert, a. 20 DAA pod samples after a 48 hour exposure illustrating signal in the non-zone sample, b. Later stages of pod development after a 15 hour exposure. Key: Z, zone; N, non-zone; DAA, days after anthesis; L, leaf; R, root; S, seed.

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*M A S R T K S F L A I AGAGGAATTTAACA ATG GCT TCA AGA ACG AAA AGC TTT TTA GCC ATT F LV I L N I L F C T T I S A Y

TTC TTG ATT CTG AAC ATC CTT TTC TGC ACA ACA ATC TCT GCC TAC G N c G c p

GGT AAC TGC GGT TGC CCT x K p ~ ~

CAT AAG CCA AAA CCT AAC T p S P V T

ACC CCT AGC CCT GTC ACA L G V C A N

CTA GGA GTC TGC GCC AAC L G K P P V

CTT GGG AAG CCA CCT GTG L A D L "E A

CTT GCT GAT CTT GAA GCC A N I L G I

GCT AAC ATC CTT GGG ATC L L L N V C

CTG CTT CTC AAT GTT TGT C *Z

TGC

s ~ K P K P D P S TCT CCC AAG CCA AAA CCT GAC CCC TCC P K P K P T P T P

CCT AAA CCC AAA CCA ACC CCA ACT CCA A K C P R D A L

GCC AAA TGC CCT AGA GAC GCT CTT AAA V L S G L L N I T

GTG CTC AGC GGT CTA CTC AAC ATC ACC K P C C T L I K G

AAG CCA TGT TGC ACC CTC ATC AAA GGA A A C L C T A L K

GCG GCT TGT CTT TGC ACC GCG CTT AAG N L N I P I S L S

AAC CTG AAC ATC CCT ATC TCA CTC AGT S K K V P P G F Q

AGC AAA AAG GTT CCC CCT GGT TTC CAA

TAA TCAAGATTATAATTATACAACCACCACTGGATGTCAACATATATACTTCTT

ii 47 26 92 41

137 56

182 71

227 86

272 i01 317 116 362 131 407 146 452 147 509

GTTTGGATAGACAAGATAATATATGTAATATAGATTCTGTAGTATTTCTGTGTGTTTAT 568

GTATGAATTGTATGTGTGTGTATGTGATTTCTACAACTCTAAACTTCACATTTGTTTTT 627

ATTTTGTTCTCTTAATTATATATACAGTCACAGGGGTGTTGTTGTACTGGTTGTTGTTT 686

A A A T T A A T A A A T A A T A T G T T T A A T A C T G ~ 745

AAAAAAAAAAA 756

Fig. 3. Nucleotide and deduced amino acid sequence of SAC51 cDNA. The initiation and termination codons are indicated by a single asterisk. The site of cleavage of the signal peptide is arrowed at amino acid residue 13. Pro-X and DALK sequences of interest are double-underlined and a possible glycosylation site is underlined. A putative polyadenylation signal is also underlined.

it accumulated in the dehiscence zone tissue to a maximum at 60 D A A (Fig. 2b). The transcript could not be detected in the leaves, seeds or roots. The c D N A SAC51 is likely to be full-length because the m R N A transcript size was similar to that of that c D N A insert size.

SAC51 sequence and amino acid analysis

Both strands of the cDNA were sequenced and the result is shown in Fig. 3. The cDNA is 756 bp in length, including the poly(A) tail. The largest open reading frame (ORF) is 441 nucleotides in length beginning at position 15 (A) and ending at position 455 (C). The deduced protein sequence of 147 amino acids has a calculated molecular mass of ca. 15 kDa and is rich in proline (14.2Yo), leucine (14.2 ~o) and lysine (10.2~o). At the end of the nucleotide sequence there was a large poly(A) tail that encompassed 42(A) nucleotides. A

prominent feature of the SAC51 deduced amino acid sequence was the arrangement of the proline residues into the repeated motif 'Pro-X' (double- underlined in Fig. 3). The sequence also contains a potential glycosylation site denoted by the motif 'N-X-S or T' [ 18] and a possible polyadenylation site [ 16 ].

188

3 3

I i 0 0

- I - -I - 2 ' -2

188

Fig. 4. Hydropathy profile of the cDNA clone SAC51 deduced amino acid sequence. The profile was computer generated according to [19]. A window of 11 consecutive amino acids of the predicted SAC51 protein was plotted against the amino acid number using the DNA Strider program [21].

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1 SAC51 MASRTKSFLA DC2.15 MGSKNSASVA pZRP3 MAPK .... VA

51 SAC51 KPTPTPTPSP DC2.15 ~ Y P S A pZRP3 PVVPTP.SSH

101 SAC51 GLADLE~.AAC DC2.15 GLVNLEII~VC pZRP3 GLVDLDLI~3.~

50 IPLILNILFC TTISAYGNCG CPSPKPKPDP SHKPKPNPKP LFFTLNILFF ALVSSTEKC ........ PDP .YKPKPKPTP LFLALSLLFA ATAHGCE ............ P NCSGPVVPTP

i00 VTAKCPRDI%L KLGVC~NVLS GLLNITLGKP PVKPC~ELIK • .GK~P~ KLGV~.~DVLN LVHNVVIGSP PTLP~SLLE SHGRCPIDI%L KLKV~LG L...VKVGLP QYEQ~'*PLLE

149 L~'TALEANIL GINI~IPISL SLLLNVQSKK VPPGTQC.. LC'TIIEANIL GKNLNLPIAL SL~QGKQ VPNGFECT. L~EIIKkNVL GIHLNVPLSL NFILNNCGRI CPEDFTCPN

Fig. 5. Comparison of amino acid sequences deduced from nucleotide sequences of the following cDNAs: SAC51, oilseed rape pods; DC2.15, carrot embryos [2]; pZRP3, maize roots [ 15]. The sequences were computer aligned using the Pileup and Pretty programs in the UWGCG package [11 ]. Common amino acids are in bold.

The hydropathy plot (Fig. 4) of the peptide indicates that the protein has several distinct domains. The protein has a hydrophobic amino terminus of 30 amino acids, characteristic of a membrane spanning cleavable signal sequence [13]. The site of cleavage of this signal peptide was calculated to be at amino acid 13. The signal sequence is followed by a hydrophilic region and a further hydrophobic region extending to the carboxy terminus.

Analysis of this sequence utilizing the U W G C G programs revealed the protein to share significant sequence identity with several protein s; 56~o with a carrot cDNA (DC2.15) representing a mRNA that accumulates during somatic embryogenesis [2]; 41 ~ with a cDNA (pZRP3) that represents a mRNA localised to cortical cells in maize roots [15]; it also had significant sequence identity with a soybean seed protein [25] and a maize embryo protein [ 12]. A comparison of the sequences of the carrot and maize proteins with that deduced from the nucleotide sequence of SAC51 is shown is Fig. 5. The deduced amino acids share similar compositions and hydropathy plots. They also have the 'Pro-X' domain within the first 50 amino acids and a conserved 'DALK' sequence of amino acids.

genomic DNA digested with Eco RI, Hind III and Barn HI (Fig. 6). The probe hybridized to several fragments ranging in size from 1 kbp to 5 kb. The sequence has an internal restriction site for Hind III at nucleotide 32.

Genomic Southern analysis of SAC51

The 756 bp insert ofpSAC51 was used as a probe for hybridization to Southern blots of B. napus

Fig. 6. Genomic Southern blot analysis of B. napus DNA probed with the pSAC51 cDNA insert. DNA was digested using the following restriction enzymes; E, Eco RI; H, Hin- d III; B, Barn HI. The position of Hind III digested lambda DNA size markers are also indicated.

Discussion

Pod dehiscence is a phenotype that is difficult to measure accurately and as a result the precise start of the process cannot readily be assessed. Cellulase activity increases in the dehiscence zone from 35 DAA (data not shown) and precedes the first visible signs of cell wall breakdown by 15-20 days [24]. Therefore mRNA extracted from different developmental stages of pod development (40, 45 and 50 DAA) were pooled in order to increase the chances of obtaining mRNAs that are preset prior to, and during, the process of dehiscence.

As the pods develop on an oilseed rape plant they undergo growth and expansion [22] which stops at 20 DAA. After this time the pods do not increase in size, but the seeds expand and fill. During the seed-filling stage the pods dry out and lose chlorophyll [22]. Accompanying these changes is the process of pod dehiscence [23], which becomes visible to the naked eye at 50- 60 DAA, but the process causes changes to cells as early as 35 DAA [22].

The aim of the work described in this paper was to identify changes in gene expression that accompany pod development and dehiscence. A cDNA library was produced from material containing dehiscence zones at a time when dehiscence was occurring and this library was subsequently screened in order to isolate mRNAs that are zone- and developmentally specific.

We have isolated a cDNA clone (SAC51) cor- responding to a mRNA that preferentially accumulates in the dehiscence zone of the developing pod prior to dehiscence (Fig. 2). In addition SAC51 mRNA is detectable in pod tissues at the early stages of development. The presence of SAC51 mRNA in the non-zone RNA at 20 DAA cannot be fully explained, but one possible ex- planation is that the polypeptide encoded by SAC51, which may be involved in the regulation of pod dehiscence, may have another role in earlier stages of pod development. Interestingly, the time course of cellulase activity in oilseed rape pods reveals that elevated levels of the enzyme are present in young pods. Enzyme

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activity then declines from 20 DAA before specifically rising again at 35 DAA in dehiscence zone tissue [24].

Genomic Southern analysis (Fig. 6) shows that SAC51 may be encoded by a single or a small family of genes, but the presence and/or arrangement of introns has yet to be determined. The presence of weakly hybridizing bands in the Hind III-digested DNA may be due to an internal restriction site in the sequence. Additional restriction sites may also be present in the genomic sequence of SAC51. B. napus (n = 19)is an amphidiploid resulting from a natural interspecific hybridization event between B. oleracea (n= 9) and B. rapa (n= 10) and the resulting genome is likely to contain 2 copies of any gene, one from each parental genome [39].

The deduced protein sequence of SAC51 has several features that are worth discussing (Fig. 3). One area of the protein is rich in proline arranged in the form of a repeated motif 'Pro-X'. A further feature of the protein is a 'DALK' sequence of amino acids that is conserved in the other proteins exhibiting sequence identity [2, 15, 29] and may be important. Saul et al. [32] have identified a bacterial cellulase (encoded by the celB gene) that contains repeats of the sequence Pro-Thr, and it would be intriguing if the proline-rich repeats in the sequence of SAC51 formed a catalytic domain of a cellulase necessary for cell wall breakdown and dehiscence. However, Saul et al. [32] conclude that the exo- and endo-glucanase catalytic domains are located at the two ends of the celB encoded protein and that the Pro-Thr domain lacks enzymic activity.

Other characteristics include defined hydrophobic and hydrophilic domains (Fig. 4) and a putative hydrophobic membrane spanning cleavable signal peptide. The SAC51 deduced amino acid sequence has significant sequence identity to other characterized proteins whose functions are also unknown (Fig. 5). These proteins are from different plant species and from different plant organs; carrot embryos [2] and young maize roots [15]. The SAC51 protein also has significant sequence identity with a protein of unknown function from immature tomato fruit [29]. Sequence

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alignment analysis shows the homology to be with proline residues and with the hydrophobic domain particularly with the cysteine residues, but also with a short sequence of amino acids (DALK). There are also other proteins that have significant homology with SAC51 which have been isolated from soybean seeds [25]. Further analysis is necessary to determine whether the protein encoded by SAC51 is related to seed development. The soybean cDNA described [25] encodes a protein that is secreted, and raises the possibility that the seed could be a potential sink for this peptide. Given that the SAC51 protein is likely to be secreted and that the seed attach- ments to the pod occur in the region of the dehiscence zone, the peptide may have a role in seed development although, interestingly, no SAC51 mRNA can be detected by northern analysis in the seed itself.

The processes of abscission and dehiscence in- volve the breakdown of cell walls. The cell walls of a plant are composed of cellulose, hemi- cellulose, pectic compounds, proteins, suberin, lignin and water [3]. The protein component can consist of many structural proteins as well as enzymes [3], but can be grouped into three main types; hydroxyproline-rich glycoproteins (HRGPs) [4, 43]; glycine-rich proteins (GRPs) [9] and lastly proline (or hydroxyproline)-rich proteins (PRPs) [14, 42]. All these proteins are characterized by basic repeat motifs that are different for each type; Ser-(Hyp)4 for HRGPs, (Gly-X)n for GRPs and Pro-Pro-Val-X-Y for PRPs. The proline-rich protein encoded by SAC51 shows no significant identity to any of the aforementioned groups [3]. The proline-rich proteins that do show homology to SAC51 have all been characterized since the last review was written [ 3] and, given the different proline repeat motif 'Pro-X', they may be a new sub-group of proline-rich proteins, although the amount of proline they contain is much smaller. Despite these differences, SAC51 and the other proteins with which it shares homology, do have elements that link them to the other groups of proline-rich proteins. The SAC51 deduced amino acid sequence has one glycosylation site, as does the maize

protein [15], but the tomato [29] and carrot [2] proteins do not.

Many of the proline-rich proteins that have been characterized have been isolated from tissue capable of growth and cell expansion, and they may have a role in cell wall formation and structure [3]. Others are produced in response to wounding [44] and light [34]. The SAC51 mRNA is present and increases in abundance when the pod has stopped growing and the protein trans- lated from this may be involved in some other process, for example, pod dehiscence.

Recently, several proline-rich proteins have been isolated and characterized from flowering tissues [5, 6, 28, 41]. These may be a further sub-group of proline-rich proteins and it is not known if these proteins have a structural role or some other function. Future work will aim to es- tablish the role of SAC51 in pod development, especially in situ hybridisation studies to localize where SAC51 mRNA is expressed.

Acknowledgements

The authors wish to thank Dr Steve Picton, Dr Colin Watson and Dr Isaac John, for all their help and technical advice. Thanks also to Dr Joe Bowman at Nickerson Seeds Ltd., for the gift of oilseed rape seed.

References

1. Agnihotri A, Shivanna KR, Raina SN, Lakshmikumaran M, Prakash S, Jagannathan V: Production of Brassica

napus x Raphanobrassica hybrids by embryo rescue: an attempt to introduce shattering resistance into B. napus.

Plant Breed 105:292-299 (1990). 2. Aleith F, Richter G: Gene expression during induction of

somatic embryogenesis in carrot cell suspensions. Planta 183:17-24 (1990).

3. Cassab GI, Varner JE: Cell wall proteins. Annu Rev Plant Pysiol Plant Mol Biol 39:321-353 (1988).

4. Chen J, Varner JE: Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33 kDa protein. Proc Natl Acad Sci USA 82:4399-4403 (1985).

5. Chen CG, Mau SL, Clarke AE: Nucleotide sequence and style-specific expression of a novel proline-rich gene from Nicotiana alata. Plant Mol Biol 21:391-395 (1993).

6. Cheung AY, May B, Kawata EE, Gu Q, Wu H: Char- acterization of cDNAs for stylar transmitting tissue- specific proline-rich proteins in tobacco. Plant J 3: 151- 160 (1993).

7. Christoffersen RE, Laties GC: Ethylene regulation of gene expression in carrots. Proc Natl Acad Sci USA 79: 4060- 4063 (1982).

8. Clarke BC, Moran LB, Appels R: DNA analysis in wheat breeding. Genome 32:334-339 (1989).

9. Condit CM, Meagher RB: A gene encoding a novel glycine-rich structural protein of petunia. Nature 323: 178-181 (1986).

10. Dean C, van den Elzen P, Tamaki S, Dunsmuir P, Bedbrook J: Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small sub- unit multi-gene family. EMBO J 4:3055-3061 (1985).

11. Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12:387-395 (1984).

12. Estanyol JM, Avila RL, Puigdomenech P: A maize embryo-specific gene encodes a proline-rich and hydrophobic protein. Plant Cell 4:413-423 (1992).

13. von Heijne G: A new method for predicting signal sequence cleavage sites. Nucl Acids Res 14:4683-4690 (1986).

14. Hong JC, Nagao RT, Key JL: Developmentally regulated expression of soybean proline-rich cell protein genes. Plant Cell 1:937-943 (1989).

15. John I, Wang H, Held BM, Wurtele ES, Colbert JT: An mRNA that specifically accumulates in maize roots delineates a novel subset of developing cortical cells. Plant Mol Biol 20:821-831 (1992).

16. Joshi CP: Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis. Nucl Acids Res 15:9627-9640 (1987).

17. Kadkol GP, Halloran GM, MacMillan RH: Inheritance of silqua strength in Brassica campestris L. I. Studies of F 2 and backcross populations. Can J Genet Cytol 28: 365-373 (1986).

18. Kornfield R, Kornfield S: Assembly of asparagine-linked oligosacchaddes. Annu Rev Biochem 54:631-664 (1985).

19. Kyte J, Doolittle RF: A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105- 132 (1982).

20. Macleod J: Harvesting. In: Oilseed Rape Book. Cam- bridge Agricultural Publishing, Cambridge (1981).

21. Marck C: 'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers. Nucl Acids Res 16: 1829-1836 (1988).

22. Meakin PJ: The physiology of bud abscission and pod shatter in oilseed rape (Brassica napus L.). Ph. D. thesis, University of Nottingham, UK (1988).

23. Meakin PJ, Roberts JA: Dehiscence of fruit in oilseed rape (Brassica napus L.). I. Anatomy of pod dehiscence. J Exp Bot 41:995-1002 (1990).

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24. Meakin P J, Roberts JA: Dehiscence of fruit in oilseed rape (Brassica napus L.). II. The role ofceU wall degrading enzymes and ethylene. J Exp Bot 41:1003-1011 (1990).

25. Odani S, Koide T, Ono T, Seto Y, Tanaka T: Soybean hydrophobic protein. Isolation, partial characterization and the complete primary structure. Eur J Biochem 162: 485-491 (1987).

26. Picton SJ, Gray JE, Barton SL, AbuBakar U, Lowe A, Grierson DG: cDNA cloning and characterization of novel ripening related mRNAs with altered patterns of accumulation in the ripening inhibitor (tin) tomato ripening mutant. Plant Mol Biol, in press (1993).

27. Prakash S, Chopra VL: Reconstruction of allopolyploid Brassicas through non-homologous recombination: intro- gression of resistance to pod shatter in Brassica napus. Genet Res Camb 56:1-2 (1990).

28. Roberts MR, Foster GD, Blundell RP, Robinson SW, Kumar A, Draper J, Scott R: Gametophytic and sporo- phytic expression of an anther-specific Arabidopsis thaliana gene. Plant J 3:111-120 (1993).

29. Salts Y, Wachs R, Gruissem W, Barg R: Sequence coding for a novel proline-rich protein preferentially expressed in young tomato fruit. Plant Mol Biol 17:149-150 (1991).

30. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

31. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 (1977).

32. Saul DJ, Williams LC, Grayling RA, Chamley LW, Love DR, Berquist PL: CelB: a gene coding for a bifunctional cellulase from the extreme thermophile Caldocellum saccharolyticum. Appl Envir Microbiol 56:3117-3124 (1990).

33. Sexton R, Roberts JA: Cell biology of abscission. Annu Rev Plant Physiol 33:133-162 (1982).

34. Sheng J, Jeong J, Mehdy MC: Developmental regulation and phytochrome mediated induction of mRNAs encod- hag a proline-rich protein, glycine-rich proteins and hydroxyproline-rich glycoproteins in Phaseolus vulgaris L. Proc Natl Acad Sci USA 90:828-832 (1993).

35. Short JM, Fernandez JM, Sorge JA, Huse WD: 2ZAP: a bacteriophage 2 expression vector in with in vivo excision properties. Nucl Acids Res 16:7583-7600 (1988).

36. Stacey J, Isaac PG: Restriction enzyme digestion, gel electrophoresis and vacuum blotting of DNA to nylon membranes. In: Isaac PG (ed) Methods in Molecular Biology Protocols for Nucleic Acid Analysis by Non- radioactive Probes, vol. 20. Humana Press, Totowa, NJ, in press (1993).

37. Taylor JE, Tucker GA, Lasslett Y, Smith CJS, Arnold CM, Watson CF, Schuch W, Grierson D, Roberts JA: Polygalacturonase expression during leaf abscission Of normal and transgenic tomato plants. Planta 183: 133- 138 (1990).

38. Taylor JE, Webb STJ, Coupe SA, Tucker GA, Roberts

1232

JA: Changes in polygalacturonase activity and solubility of polyuronides during ethylene stimulated leaf abscission in Sambucus nigra. J Exp Bot 44:93-98 (1993).

39. U N: Genomic analysis of Brassica with special reference to the experimental formation ofB. napus and its peculiar mode of fertilization. Jpn J Bot 7:389-452 (1935).

40. Webb STJ, Taylor JE, Coupe SA, Ferrarese L, Roberts JA: Purification of fl 1.4 glucanase from ethylene-treated abscission zones of Sambucus nigra. Plant Cell Environ 16:329-333 (1993).

41. Wright SY, Sumer MM, Bell PJ, Vandim M, Greenland

AJ: Isolation and characterization of male flower cDNAs from maize. Plant J 3:41-49 (1993).

42. Wyatt RE, Nagao RT, Key JL: Patterns of soybean proline-rich proteins gene expression. Plant Cell 4: 99- 110 (1992).

43. Zheng-Hua Y, Varner JE: Tissue specific expression of cell wall proteins in developing soybean tissues. Plant Cell 3:23-37 (1991).

44. Zhou J, Rumeau D, Showalter AM: Isolation and characterization, of two wound-regulated tomato extensin genes. Plant Mol Biol 20:5-17 (1992).

Documents

Identification and characterization of a proline-rich mRNA that accumulates during pod development in oilseed rape (Brassica napus L.)