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An mRNA encoding a response regulator protein from Brassica napus is up-regulated during pod development

An mRNA encoding a response regulator protein from Brassica napus is up-regulated during pod development
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  Journal of Experimental Botany, Vol. 50, No. 332, pp. 335–341, March 1999 An mRNA encoding a response regulator protein from Brassica napus is up-regulated during pod development Catherine A. Whitelaw 1 , Wyatt Paul 2 , Elizabeth S. Jenkins 1 , Vivien M. Taylor 1 andJeremy A. Roberts 1,3 1 School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough,Leicestershire LE125RD, UK  2 Nickerson BIOCEM Ltd., Cambridge Science Park, Milton Road, Cambridge CB44GZ, UK  Received 8 September 1998; Accepted 18 November 1998 Abstract chemical changes take place, thus preparing both seedsand pod for the event. Shatter eventually occurs as a A novel cDNA (SAC29) was isolated by differential result of a combination of factors including: the creation display from pod dehiscence zone tissue during a of tensions within the pod between the lignified valve study directed at identifying genes expressed during edge cells of the endocarp and the unlignified dehiscence pod development in oilseed rape ( Brassica napus L.). zone (DZ) cells, weakening of the DZ cell walls by SAC29 encodes a 136 amino acid putative peptide, hydrolytic enzyme activity and, ultimately, due to physical which shows homology to various prokaryotic and forces such as strong winds or harvesting machinery. eukaryotic response regulator proteins. The homolog- Pod development in B. napus can be segmented into ous regions are centred about the four highly three stages. In the first stage, which occurs 0–20 DAA, conserved amino acid residues of bacterial receivers the newly formed siliques, consisting of two seed- including the aspartic acid motif required for phos- containing carpels separated by a false septum and a phorylation and subsequent transduction of the initial replar region, grow to their full length of around 10 cm. signal.Northernblotanalysisof SAC29showedhybrid- The seeds begin to grow when the pods are virtually full ization to a 0.6 kb transcript which was most abundant size (Hocking and Mason, 1993). Between 10 and 20 in RNA extracted from the dehiscence zone of pods at DAA the cells in the replar region begin to di ff  erentiate 40 d after anthesis. The role of SAC29 as a component into replar cells, large valve edge cells and form a distinct in a signalling cascade regulating dehiscence of the region, 1–3 cells wide, comprising the DZ (Meakin and pod is discussed. Roberts, 1990 a ).The second stage occurs between 20 and 50 DAA. Key words: Brassica napus , dehiscence, pod shatter, From 20 DAA, in conjunction with termination of pod response regulator, signal transduction. elongation, secondary cell wall material is deposited inthe valve edge cells, and the replar cells become increas- Introduction ingly lignified. The DZ cells do not exhibit thickening of the cell wall. A progressive shrinkage and loss of organ-The production of seed is an important developmentalelles is apparent in the DZ cells from 40 DAA onwardsprocess in all higher plants. In oilseed rape ( Brassica and eventually these cells separate completely due to napus ) following abscission of floral parts, pods or siliqueshydrolysis of the middle lamella (Meakin and Roberts,are formed which contain 15–30 seeds. Around 50–70 d1990 a ). In the final stage of pod development, whichafter anthesis (DAA) the pods become susceptible tooccurs 50–70 DAA, the lignified cells undergo senescenceshatter, a process that serves to expel the mature seedsand the necessary tensions are created so that the desic-into the surrounding environment. In the days leading todehiscence, an array of anatomical, molecular and bio- cated pod, containing mature seed, eventually shatters. 3 To whom correspondence should be addressed. Fax: + 44 115 951 6334. E-mail:© Oxford University Press 1999  336 Whitelaw et al. Molecular studies of the penultimate stage of pod role for SAC29, as a signalling molecule during the eventsleading to shatter is presented and discussed.development have revealed a spatial and temporal correla-tion between the up-regulation of a number of mRNAsand pod dehiscence in B. napus . These mRNAs encode apolygalacturonase (PG) (SAC66) (Jenkins et al  ., 1996;Peterson et al  ., 1996), a proline-rich protein (SAC51) Materials and methods (Coupe et al  ., 1993) and a protein with homology to Plant material oxidoreductases (SAC25) (Coupe et al  ., 1994). Further B. napus cv. Rafal plants were grown essentially as described in analysis of the expression of the PG following fusion of  Meakin and Roberts (1990 a ). Minor modifications to the a pod-specific Arabidopsis thaliana PG promoter to GUS, growth conditions are reported in Coupe et al  . (1993). At has revealed that reporter gene expression is restricted anthesis, the flowers were tagged so that the age of the resultingpods could be determined accurately in days after anthesis precisely to the layer of cells comprising the pod DZ in (DAA). At 20, 30, 40, 50, and 60 DAA the pods were collected transgenic B. napus Jenkins et al  . (1999). From 40 DAA, and dissected into DZ and non-zone (DNZ). The tissue was Meakin and Roberts (1990 b ) reported a progressive frozen immediately in liquid nitrogen and stored at − 70 ° C. increase in b -1,4-glucanase (cellulase) activity in the DZ, To obtain abscission zone (AZ) tissue, cotyledon AZ explants although, to date, there have been no reports of cellulase were dissected from 21-d-old B. napus cv. Rafal plants. Theexplants were supported in 1% (w /  v) agar (Oxoid) and exposed gene expression in these cells. to 10 m l l − 1 ethylene for 0, 24, 48, and 72 h then dissected into In plants, dehiscence and abscission are comparable AZ and non-zone (ANZ). The tissue was frozen immediately processes, in that they involve controlled degradation of  in liquid nitrogen and stored at − 70 ° C. cell wall material and cell separation in a distinct groupof cells. Both ethylene and indole-3-acetic acid (IAA) Differential display appear to be important regulators of the timing of the Di ff  erential display was performed essentially as described by abscission process (Gonzalez-Carranza et al  ., 1998), but Liang and Pardee (1992) using two base anchor primers and the role of these plant hormones in dehiscence is less random decamer arbitrary primers with minor modifications as clearly defined. The increase in cellulase activity has been detailed here. The template for first strand cDNA synthesis was shown to correlate with a rise in the production of  total RNA that had been extracted from 40 DAA pod DZ and ethylene, mainly from the seed, which peaks at around DNZ using the CTAB (hexadecyltrimethyl ammonium bromide)method (Hamilton et al  ., 1995). First strand cDNA copies of  40 DAA (Meakin and Roberts, 1990 b ; Johnson-Flanagan the RNAs were made in a 20 m l reaction containing 50 U and Spencer, 1994). However, Meakin (1988) could find M-MLV (Moloney-Murine Leukemia Virus) reverse tran- no direct evidence that ethylene hastened the dehiscence scriptase (50 U m l − 1 ) (Stratagene), 1 × M-MLV bu ff  er, 2.5 mM process. Chauvaux et al  . (1997) have reported a transitory dNTPs (Pharmacia), 1 m g total RNA (40 DAA DZ /  NDZ),30 U RNAse inhibitor (Promega) and 10 m M anchor primer peak in IAA at 40 DAA which declines in the days (5 ∞ -TTT TTT TTT TTT VV-3 ∞ ) (Genosys). The reaction preceding cell separation in the DZ. The decrease in auxin conditions were as follows: 65 ° C for 5 min, 37 ° C for 90 min was shown to correlate with an increase in cellulase and 95 ° C for 5 min. Following first strand cDNA synthesis, activity in the DZ. Moreover, treatment of the pods with 60 m distilled water were added and the samples were either the auxin mimic 2-methyl-4-chloro-phenoxyacetic acid used directly for PCR or stored at − 20 ° C. For PCR, 2 m lcDNA were used as template in a 20 m l reaction containing 1 × (4-CPA) resulted in a delay in cellulase activity and cell PCR bu ff  er, 1 mM MgCl 2 , 2 m M dNTPs (Pharmacia), 10 m M separation in the DZ. These authors have postulated that anchor primer (Genosys) (same as that used for first strand this decrease in IAA may be important in controlling the cDNA synthesis), 2.5 m M arbitrary primer (Genosys), 0.5 m l timing of pod dehiscence. 35 S-dATP ( > 1000 Ci mmol − 1 ) (Amersham) and 1 U Taq DNApolymerase (5 U m l − 1 ) (Gibco BRL). The thermocycling Developmental processes such as pod dehiscence, which conditions were as follows: 40 cycles of 94 ° C for 30 s, 40 ° C for involve highly regulated and controlled expression of an 2 min, 72 ° C for 30 s followed by 72 ° C for 5 min. The PCR array of di ff  erent genes at a precise time and cellular products were fractionated on a 6% (w /  v) polyacrylamide /  7 M location, clearly require an intricate signal transduction urea gel. Following electrophoresis, the gel was dried at 80 ° C network. Recently, there have been reports of plant genes under vacuum for 1 h then exposed to X-ray film (BioMax-MR,Kodak) in a light-tight cassette for 48 h. The dried gel and coding for bacterial two-component-like proteins and are autoradiograph were aligned so that DNA bands obtained from therefore implicated in reception and transduction of  DZ RNA only could be cut out and the DNA eluted according external signals (Chang et al  ., 1993; Hua et al  ., 1995; to Liang et al  . (1995). The eluted PCR products (4 m l) were Kakimoto, 1996; Imamura et al  ., 1998; Urao et al  ., 1998; reamplified in a 40 m l reaction containing 1 × PCR bu ff  er,1 mM MgCl 2 , 20 m M dNTPs (Pharmacia), 10 m M anchor Brandstatter and Kieber, 1998; Sakakibara et al  ., 1998). primer (Genosys), 2.5 m M arbitrary primer (Genosys), and 2 U In this paper, the isolation of a plant cDNA (SAC29) Taq DNA polymerase (Gibco BRL) under the same thermo- encoding a putative individual response regulator protein, cycling conditions as those used for di ff  erential display PCR. the expression of which is closely correlated with The resulting PCR product was cloned into the TA Cloningvector (Invitrogen) and sequenced. dehiscence of fruit in B  . napus is described. A potential  Up-regulation of an mRNA from Brassica 337 cDNA library screening a response regulator protein from Halobacterium A full-length cDNA clone of SAC29 was obtained by screening salinarium (Rudolph et al  ., 1995). In E. coli  , CheY is a a B. napus pod DZ cDNA library (Coupe et al  ., 1993). The member of a superfamily of proteins involved in the library was plated at a density of 25000 recombinant plaques regulation of chemotactic events (Mutoh and Simon, per 13 cm plate, then transferred in duplicate to Hybond-N + 1986) and plays a similar role in H. salinarium (Rudolph nylon membrane (Amersham). The duplicate filters were probed et al  ., 1995). Figure 2 shows an alignment of the predicted with 32 P-labelled SAC29 PCR product. Hybridization andwashing were carried out according to the manufacturers’ amino acid sequence of SAC29 with various prokaryotic instructions. Plaques which hybridized to the probe after the and eukaryotic response regulator proteins. Included in first round of screening were subjected to PCR using T3 primer the alignment are the equivalent regions (receiver and a SAC29-specific primer, SAC29RL (5 ∞ -TGC ATA CAT domains) of the A. thaliana ETR1 (Chang et al  ., 1993) ACA CAC TTA GAC G-3 ∞ ). The plaques from which the and CKI1 (Kakimoto, 1996), in addition to the predicted largest PCR products were amplified were presumed to containa full-length copy of SAC29 and these were taken through two protein sequence of a recently isolated cDNA encoding a further rounds of screening. response regulator protein from A. thaliana (Imamura et al  ., 1998; Urao et al  ., 1998; Brandstatter and Kieber, Northern blot analysis 1998). For Northern blot analysis, 10 m g total RNA extracted from20, 30 40, 50, and 60 DAA DZ and DNZ were fractionated on Expression analysis a 1% (w /  v) agarose /  3% (v /  v) formaldehyde /  20 mM sodiumphosphate bu ff  er (pH 6.5) gel. In addition, 10 m g RNA extracted An antisense strand-specific riboprobe of the SAC29 full- from cotyledon AZ and ANZ which had been exposed to length cDNA hybridized to a 0.6 kb transcript which was 10 m l l − 1 ethylene for 72 h, and RNA from root, seed, flower, present specifically in RNA extracted from DZ tissue and leaf were included. The RNA was transferred to a nylonmembrane (Genescreen, NEN DuPont) and hybridized to an (Fig. 3). In a time-course of pod development, SAC29 antisense strand-specific riboprobe of SAC29. Hybridization can be detected at 20 DAA. Expression of this putative and washing conditions were performed according to the response regulator increases progressively at 30 DAA and manufacturers’ instructions. a peak in expression is observed at 40 DAA. SAC29transcript then decreases at 50 DAA and is completelyabsent at 60 DAA. In comparison to DZ, expression of  Results SAC29 in RNA extracted from DNZ tissue at the same Isolation of a cDNA encoding a putative response regulator  time points is minimal. The presence of SAC29 in leaf, by differential display root, seed, and flower RNA was not detected by Northernhybridization. To ascertain whether SAC29 plays a roleThe di ff  erential display technique was applied to a studyin abscission, RNA from ethylene-treated cotyledon AZfocusing on changes in gene expression during develop-and ANZ (stem) were also included on the blot. Therement of fruit in oilseed rape. Using the srcinal publishedwas no hybridization of SAC29 to cotyledon AZ or stemprotocol (Liang and Pardee, 1992) with minor modifica-RNA (Fig. 3). Moreover, Northern blot analysis of tions, a PCR product amplified from 40 DAA DZ cDNASAC29 with an ethylene-treated cotyledon AZ RNA time-only, when compared to 40 DAA DNZ cDNA PCRcourse also revealed no hybridization to transcriptsproducts, was eluted from the polyacrylamide gel,present during the course of abscission (data not shown).re-amplified, cloned and sequenced. Subsequently, the465 bp PCR product was used to screen a B. napus podDZ cDNA library (Coupe et al  ., 1993). Screening 50000 Discussion recombinant plaques resulted in the isolation of a full-length clone. The cDNA (SAC29) is 622 bp in lengthGenerally, bacterial two-component systems consist of a(including the poly A tail) and the largest open readingsensor protein and a response regulator protein. Theframe encodes a putative 136 amino acid protein (Fig. 1)sensor protein is located in the cell membrane and has awith an estimated molecular weight of 15 kDa.variable input domain protruding out of the cell, whichis responsible for detection of the environmental signal, Sequence comparisons and a transmitter histidine kinase domain projectinginto the cell. The cytoplasmic response regulator proteinHomology searches of SAC29 nucleic acid sequence,using the FastA program (Devereux et al  ., 1984) typically consists of a receiver domain linked to an outputdomain, however, these proteins are modular and canshowed no significant homology to sequences in theGenbank /  EMBL databases. However, a low but consist- exist in a variety of configurations. Once an externalsignal is perceived, the transmitter module undergoesent homology to bacterial two-component proteins wasobtained by comparing the predicted amino acid sequence autophosphorylation whereby a phosphoryl group fromATP is attached to the highly conserved histidine residue.of SAC29 to the OWL protein sequencedatabase (Bleasby et al  ., 1994). The highest identity (30%) was with CheY, The phosphate moiety is then transferred from the histid-  338 Whitelaw et al. Fig. 1. The nucleic acid and predicted protein sequence of SAC29 is shown (Genbank accession number AF057027). The full-length cDNA is622 bp including the poly A tail. The extent of the srcinal 465 bp PCR product isolated by di ff  erential display is shown by 3 . The first ATG codonis 20 bp from the 5 ∞ end of SAC29 and there is a stop codon (TGA) 408 bp downstream of the ATG triplet. This longest open reading frameencodes a putative protein of 136 residues. Shown in boxes are the conserved amino acids of bacterial receivers that are known to be required forphosphorylation, i.e. Asp-22, Asp-23, Asp-68, and Lys-119. The binding sites of primers SAC29FL and SAC29RL are underlined and a putativepolyadenylation signal is shown in bold (Proudfoot and Brownlee, 1976). ine residue to an aspartate residue located in the receiver a histidine kinase domain which is attached to a responseregulator, i.e. the sensor and response regulator aredomain of the cognate response regulator protein.Phosphorylation of the response regulator results in large- contained in one protein. A second ethylene receptorfrom A. thaliana is encoded by the ERS  gene (Hua et al  .,scale conformational changes and consequent alterationsin gene transcription (Parkinson and Kofoid, 1992). 1995). The structure of this gene is di ff  erent to ETR1 inthat it does not contain a receiver domain, therefore, byAn alignment of the deduced amino acid sequence of SAC29 with the bacterial response regulator proteins comparison to the bacterial systems the upstream com-ponent with which SAC29 interacts could exhibit a similarCheY (from H. salinarium ) (Rudolph et al  ., 1995) andSpo0F (from Bacillus subtilis ) (Kofoid and Parkinson, structure to the protein encoded by ERS  .It has been shown that the proteins encoded by ETR1 1988) (Fig. 2) reveals a resemblance with these singledomain receiver proteins. Each protein in the alignment and ERS  interact directly with CTR1, a Raf-relatedkinase, in the ethylene response pathway (Clark et al  .,contains the four highly invariant residues correspondingto Asp-22, Asp-23, Asp-68, and Lys-119 in SAC29, which 1998). This association is conceivable as the three-dimensional structure of the response regulator CheY ishave been shown, in bacteria, to play major roles inphosphorylation and subsequent signallling activities of very similar to the Ras protein with which Raf is knownto interact (Chang et al  ., 1993). The receiver domain of response regulators.By comparison to bacterial two-component-like ETR1 (and CKI1) is attached to the sensor and istherefore membrane-bound which means that anotherproteins, SAC29 encodes the receiver domain of aresponse regulator. At present, no potential partner histid- cytoplasmic element must be present for successful signaltransduction. Although this could be a role for SAC29,ine kinase has been identified for SAC29, but by analogyto the bacterial systems and from recent reports of being an individual response regulator, it is unlikely asthe phosphoryl group needs to be passed to anotheradditional eukaryotic two-component kinases, there islikely to be one. The A. thaliana genes ETR1 and CKI1 histidine residue before phosphorylating an aspartateresidue as in the yeast osmosensing signal transductionhave been implicated in the response of plants to thephytohormones ethylene (Chang et al  ., 1993; Schaller and system (Posas et al  ., 1996). Another potential functionfor SAC29 is that it may initiate a MAP kinase cascadeBleecker, 1995) and cytokinin (Kakimoto, 1996) respect-ively. Both proteins constitute a variable N-terminus and (Morgan et al  ., 1995). This could lead to changes in the  Up-regulation of an mRNA from Brassica 339 Fig. 2. An alignment of the predicted amino acid sequence of SAC29 with those of certain prokaryotic and eukaryotic receiver domains is shown.The prokaryotic response regulators included are CheY ( H. salinarium ) (Rudolph et al  ., 1995) and Spo0F ( B. subtilis ) (Kofoid and Parkinson,1988). The eukaryotic receiver domains included srcinate from ETR1 ( A. thaliana ) (Chang et al  ., 1993) and CKI1 ( A. thaliana ) (Kakimoto, 1996).In addition, the A. thaliana response regulator ARR (also known as ARR5 (Imamura et al  ., 1998); ATRR2 (Urao et al  ., 1998); IBC6 (Brandstatterand Kieber, 1988)) is included. Residues that are known to be required for phosphorylation of bacterial receivers are boxed whilst completelyconserved residues are indicated by an asterisk. In addition, the residues that are common to SAC29 and at least three of the six sequences in thealignment are shaded. Gaps have been introduced into the sequences to permit optimal alignment. The comparison was generated using the Pileupprogram (Devereux et al  ., 1984). Although DNA-binding ability of SAC29 has not beenstudied, the possibility that SAC29 acts directly as atranscription factor cannot be disregarded.The close correlation of SAC29 expression to poddehiscence, a natural event in the life cycle of this plant,is clearly di ff  erent to that of the recently reported Arabidopsis and maize cDNAs coding for responseregulator proteins, in that these transcripts are expressedmost abundantly in roots (Imammura et al  ., 1998; Urao et al  ., 1998) and in response to changes in temperature(Urao et al  ., 1998), cytokinins (Brandstatter and Kieber, Fig. 3. A Northern blot containing 10 m g RNA extracted from DZ and 1998; Taniguchi et al  ., 1998; Sakakibara et al  ., 1998) and DNZ (20, 30, 40, 50, and 60 DAA), cotyledon AZ and non-zone ANZ nitrate (Taniguchi et al  ., 1998; Sakakibara et al  ., 1998). (stem) (72 h exposure to 10 m l l − 1 ethylene), leaf (L), root (R), seed(S), and flower (F). The blot was hybridized to a 32 P-labelled antisense There are a number of potential roles for SAC29 in strand-specific riboprobe of SAC29, then stripped and re-probed with dehiscence. It is highly expressed in the DZ of  B. napus a rDNA probe corresponding to the 3 ∞ end of  B. napus 25S rRNA to pods in the days leading to shatter and could therefore show equivalent loading of RNA. itself be regulated by phytohormones such as ethylene orauxin, both of which reach a peak at around 40 DAAexpression of genes involved in pod dehiscence such as,(Meakin and Roberts, 1990 b ; Johnson-Flanagan andup-regulation of genes encoding PG, cellulase, senescence-Spencer, 1994). Once induced, this protein probablyrelated proteins and down-regulation of genes coding forinteracts with an upstream histidine kinase which, havingproteins involved in cell wall biosynthesis.autophosphorylated in response to an, as yet, unknownApart from the conserved residues in the receiverexternal elicitor, transfers a phosphoryl group to SAC29.domain, SAC29 contains no sequences homologous toOnce phosphorylated, SAC29 may undergo conforma-sequences in the Genbank /  EMBL databases, suggestingthat SAC29 does not encode a transcription factor. tional changes allowing it to interact either directly with
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