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Plant Cell Physiol. 38(3): 362-365 (1997) JSPP © 1997 Short Communication Cloning of a Rice cDNA Encoding a Transcription Factor Homologous to Barley GAMyb Frank Gubler', Robyn J. Watts 1 , Roger Kalla2, Peter Matthews1, Margaret Keys2 and John V. Jacobsen I>2 1 2 Co-operative Research Centre for Plant Science, PO Box 475, Canberra, ACT 2601, Australia CSIRO, Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia Key words: Aleurone — a-Amylase — Germination — Gibberellin — Myb — Rice. Over the last few years there has been a strong interest in identifying the biochemical and molecular steps involved in the gibberellin (GA) signal transduction pathway in cereal aleurone cells (Hooley 1994). Although the receptor and other components in the pathway leading to the expression of a-amylase and other hydrolytic enzymes remain to be identified, progress has been made in identifying transcription factors which are the target of the signalling pathway. GA-dependant DNA binding factors have been reported for rice and barley a-amylase gene promoters (Ou-Lee et al. 1988, Sutliff et al. 1993, Goldman et al. 1994). Recently, a Myb-like transcription factor, GAMyb, was cloned from barley and shown to be a transcriptional activator of an a-amylase gene in barley aleurone cells (Gubler et al. 1995). In this study, GAmyb gene expression was shown to be GA-regulated and the gene product was shown to bind to a GA-response element (TAACAAA) in the promoter (Skriver et al. 1991, Gubler and Jacobsen 1992). To establish whether GAMyb is a component of GA-signal transduction pathways in other cereal aleurone cells, we isolated the rice GAmyb homologue and investigated its role in a-amylase gene expression. Cloning of OsGAmyb cDNA— To isolate cDNA clones encoding OsGAMyb, a cDNA library made from Abbreviations: GA, gibberellin; GA3, gibberellic acid; GUS, /J-glucuronidase. The nucleotide sequence reported in this paper has been submitted to EMBL/GenBank/DDBJ Databases under the accession number X98355. 362 gibberellic acid (GA3)-treated embryoless-half grains (Chen et al. 1995) was screened with the partial genomic clone isolated by screening an IR36 rice genomic library (Clontech) with a barley 3' GAmyb probe at low stringency (2 x SSC, 0.1% SDS at 54°C). From a screening of 150,000 plaques, 23 positive clones were identified. Six plaques were picked and isolated for further analysis. The longest cDNA insert (2,373 bp) contained a single open reading frame extending between nucleotides 396 and 2,054 which codes for a Myb-like protein of 553 amino acids (calculated molecular weight 59 kDa). Comparison of sequences revealed 88% identity at the amino acid level between the i/vGAMyb and the protein encoded by the rice cDNA (Fig. 1). There is very high sequence identity between the DNA binding domains of both Mybs. Sequence comparison between the R2 and R3 repeats show high sequence identity (99%) indicating that both Mybs are likely to have very similar binding specificities. Sequence identity remains high downstream of the DNA binding domain up to and including a putative activator domain (amino acid residues 372 to 386 in the QsGAMyb open reading frame) (Gubler et al. 1995). Downstream of the activator domain, sequence identity decreases to 75%. Based on the high level of sequence identity, it appears likely that the rice cDNA encodes a homologue of the //vGAMyb. Rice genomic DNA was digested with EcoRl, Hindlll and Xba\, and analysed by DNA gel blotting using a 3' OsGAmyb PCR product as a probe (from position 1,003 to 2,113) under moderate stringency conditions. The results shown in Fig. 2 show that the probe hydridized with only one band in each restriction digest indicating that the OsGAmyb gene is a single copy as found for the barley gene (Gubler et al. 1995). Analysis of OsGAmyb gene expression in response to GA—The effect of GA3 on OsGAmyb gene expression was monitored by incubating hydrated endosperm (de-embryonated grains) of rice in the presence and absence of GA3 for up to 12 h. RNA was extracted and analysed by RNA gel blot analysis using the 3' OsGAmyb PCR product. As shown in Fig. 3 the probe detected only a single band of 3.9 kb in size. The abundance of OsGAmyb mRNA levels increased rapidly in response to GA3. Within Downloaded from http://pcp.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016 A cDNA clone, OsGAmyb, which encodes a homologue to the barley Myb-like transcription factor, //vGAMyb, was isolated from a rice endosperm cDNA library. The clone was used to show that expression of the OsGAmyb gene in aleurone cells was stimulated by gibberellic acid and the gene product was shown to transactivate an aamylase gene promoter in transient expression analyses. Cloning of OsGAmyb cDNA 363 O S MYRVKSESDCEMIHQE.QMDSPVADDGSS .GGSPHRGGGPEJLKKGPWTSA 48 II Mil H V MYRVKSESDCEMMHQEDQMDSPVGDDGSSGGGSPHRGGGPPjU£KGEHI£A_50 EDAILVDYVKKHGEGNWNAVQKNTGLFRCGKSCRLRWANHLRPNLKKGAF 9 8 11! 111 j! i! I [ 11111 i 11! I! 1111111111! 11 i 111! 1! I! I! 111 EDAILVDYVKKHGEGNWNAVOKNTGLFRCGKSCRLRWANHLRPNLKKGAF 100 TAEEERLIIQLHSKMGNKWARMAAHLPGRTDNEIKNYWNTRIKRC QRAGL 148 1 III TPEEERLIIOLHSKMGNKWARMAAHLPGRTDNEIKNYWNTRIKRqQRAGL 150 PIYPTSVCNQSSNEDQQCSSDFDCGENLSNDLLNANGLYLPDFTCDNFIA 198 1111 - M I M ! 11 M I <. M 11:1 M 111 -1! I!: I M M ! ! 11 I II M I I I I I I - I I I = I I I I I I I - 1 I M! I I I I - 1 HI I I I ! I - f I I I I I I - : II I NSEALSYAPQLSAVSISSLLGQSFASKNCGFMDQVNQAGMLKQSDPLLPG 250 LSDTINGVISSVDQFSNDSEKLKQAVGFDYLHEANSTSKIIAPFGGALNG 298 M M I I I . : | Mill l i l M I M I h M I I MM II-IMI Ml I Ill-I LSDTINGALSSVDQFSNDSEKLKQALGFDYLHEANSSSKIIAPFGGALTG 300 SHAFLNGNFSASRPTSGPLKMELPSLQDTESDPNSWLKYTVAPALQPTEL 348 MMIIMMI---MIMM!MIIMIIIMIMMMIhllMI SHAFLNGTFSTSRTINGPLKMELPSLQDTESDPNSWLKYTVAPAMQPTEL 350 VDPYLQSPAATPSVKSECASPRNSGLLEELIHEAQTLRSGKNQQTSVISS 3 98 11111111 • 11111111111111111! 111 ^ 1111 -111! 1111 MM VDPYLOSPTATPSVKSECASPRNSGLLEELLHEAQGLRSGKNOOLSVRSS 400 SSSVGTPCNTTVLSPEFDMCQEYWEEQHPGPFLNDCAPFSGNSFTESTPP 448 lllhllhllhllllhlMMM- I I : : I I I I I I hI : I hI SSSVSTPCDTTWSPEFDLCQEYWEER LNEYAPFSGNSLTGSTAP 445 VSAASPDIFQLSKVSPAQSTSMGSGEQVMGPKYEP..GDTSPHPENFRPD 496 :II ! M I : M I ' I HI I I I • I = M I I I - t: i M l llll-lllhlll MSAASPDVFQLSKISPAQSPSLGSGEQAMEPAYEPGAGDTSSHPENLRPD 495 ALFSGNTADPSVFNNAIAMLLGNDLSIDCRPVLGDGIMFNSSSWSNMPHA 546 hlllllll-IIIMIIIMIMh-^hlhlllllh-l Mhlll AFFSGNTADSSVFNNAIAMLLGNDMNTECKPVFGDGIMFDTSVWSNLPHA 545 CEMS.EFK 553 hll III CQMSEEFK 553 Fig. 1 Alignment of the deduced amino acid sequences for OsGAMyb (Oryza saliva, Os) and Z/vGAMyb (Hordeum vulgare, Hv). The R2 and R3 repeats of the Myb DNA binding domain are boxed. A putative transcriptional activation domain is underlined. The OsGAmyb sequence data have been submitted to EMBL/GenBank/DDBJ as accession number X98355. The accession number for the HvGAmyb sequence is X87690. 2 h incubation with GA3> OsGAmyb mRNA levels began to increase and continued to increase up to 12 h following treatment. In the absence of GA3, OsGAmyb mRNA levels decreased continuously. Very low levels of OsGAmyb mRNA were detected in endosperm of dry grains (data not shown) compared to hydrated grains, indicating that OsGAmyb mRNA accumulated in the aleurone cells during the 24 h hydration period but subsequently declined in the absence of GA3. The accumulation of a-amylase mRNA in response to GA3 is shown in Fig. 3. The increase in OsGAmyb mRNA levels in response to GA3 preceded the rise in a-amylase mRNA by about 6 h, which is similar to that found for HvGAmyb gene expression in barley aleurone cells (Gubler et al. 1995). Functional analysis of OsGAmyb—To test whether QsGAMyb is a transcriptional activator of an a-amylase gene promoter, barley aleurone cells were cobombarded Downloaded from http://pcp.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016 PIYPASVCNQSSNEDQQGSSDFNCGENLSSDLLNGNGLYLPDFTCDNFIA 200 NSEALPYAPHLSAVSISNLLGQSFASKSCSFMDQVNQTGMLKQSDGVLPG 248 364 Cloning of OsGAmyb cDNA 0 1 2 3 6 12h -GA O Kb LU X GAmyb + GA 7.26.4- 0 1 2 3 6 12h -GA 2.3- a-Amylase + GA Fig. 2 DNA gel-blot analysis of the OsGAmyb gene. Genomic DNA was isolated from 11 day old rice seedlings (var. Taipei 309) and digested with EcoRl, Hindlll and Xbal. After blotting, the digested DNA was probed with a 3' OsGAmyb probe (nucleotides 1,003 to 2,113) labelled with digoxigenin. The blots were washed at moderate stringency (0.5 x SSC, 0.1% SDS at 68°C). 0 1 2 3 6 12h -GA rRNA + GA with the yS-glucuronidase (GUS) reporter gene fused to the barley Amy32b a-amylase promoter (mlo22; Lanahan et al. 1992) and with GAMyb effector constructs (Fig. 4a). The effector construct consisted of the OsGAmyb cDNA fused to the constitutive maize ubiquitin promoter (Christensen et al. 1992, Zhongyi Li, unpublished data). Fig. 4b shows that the OsGAmyb effector was able to transactivate the aamylase promoter both in the presence and absence of GA3. The effector construct resulted in a 176-fold increase in GUS expression in non-GA3 treated aleurone cells, compared to an effector construct which had no cDNA insert. The increase in GUS activity in response to overexpression of the OsGAmyb cDNA was 3-fold higher than found in GA-treated aleurone cells bombarded without the OsGAmyb cDNA. To test whether the effect of the OsGAmyb cDNA was specific, aleurone cells were bombarded with the aamylase promoter reporter construct and an effector construct expressing the maize Cl Myb (data not shown). The maize Cl Myb failed to transactivate the reporter gene in transient expression analyses. These results indicate that the rice cDNA encodes a transcriptional activator of a barley a-amylase gene promoter. On the basis of high sequence identity, responsiveness to GA3 and functional evidence, we propose that the OsGAmyb gene encodes the orthologue to Z/vGAMyb. The above results indicate that a Myb-like protein plays an important role in the GA3 control of a-amylase gene expression in rice and barley aleurone cells. We propose that QsGAMyb transactivates a-amylase gene expression in rice Fig. 3 Effect of gibberellin on GAmyb and a-amylase gene expression in rice endosperm. RNA was isolated from rice endosperm half grains (var. Taipei 309) (Schuurink et al.1996) which had been hydrated in 10 mM CaCl2 overnight at 28 °C and then incubated in 10 mM CaCl2, 1 5 0 ^ g m r ' cefotaxime, 50 units ml"' nystatin, and either no hormone (control) or 10~6 M gibberellic acid (GA) at 30°C. After blotting, the RNA was probed with a gene specific 3' OsGAmyb probe (described in Fig. 2), a barley aamylase cDNA aa24 (Peter Matthews, unpublished data), and a wheat ribosomal DNA clone pTA250 (Gerlach and Bedbrook 1979). Numbers above each lane indicate hours (h) after the start of the treatment. 10/ig total RNA was loaded per lane. aleurone via the TAACAAA box which is highly conserved in the promoters of barley, wheat and rice a-amylase genes (Huang et al. 1990). a-Amylases in rice are encoded by a multigene family. Members of one subfamily, RAmyl are highly expressed in aleurone cells following germination and also respond to GA (O'Neil et al. 1990, Karrer et al. 1991). Recent studies of the promoter of the RAmyl A gene in transgenic rice has shown that the cis-acting elements required for GA-regulated expression are located in a region of the promoter (between —232 and +31) which contains the TAACAAA box (Itoh et al. 1995). We would predict that OsGAMyb transactivates RAmyl genes by binding to the TAACAAA box as has been demonstrated for a barley a-amylase gene (Gubler et al. 1995). It is of interest to note that another early gibberellin re- Downloaded from http://pcp.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016 3.7" Cloning of OsGAmyb cDNA a. Ubiquitin promoter Nos 3' Ubi.cas Ubiquitin promoter OsGAmyb Nos 3' 365 The authors would like to thank Xiongfong Chen and Ray Wu (Cornell University) for the generous gift of the rice aleurone cDNA library. We would also like to thank John Rogers (University of Missouri) and Zhongyi Li (CSIRO, Canberra) for supplying the mlo22 construct and maize Ubi expression cassette (pLZUbilcas) respectively. Ub1.OsGAmyb References b. Fig. 4 Transactivation of barley Amy32b a-amylase promoter by QsGAMyb in barley aleurone cells. The GAMyb effector construct was made by inserting the OsGAmyb cDNA containing the entire open reading frame into the multicloning site in the maize Ubil expression cassette, pLZUbicas. The expression cassette consists of the maize ubiquitin gene promoter fused to a multicloning site and Nos 3' terminator (Zhongyi Li, CSIRO, Canberra). The reporter construct, mlo22, consists of the barley Amy32b promoter fused to a GUS reporter gene (Lanahan et al. 1992). Intact aleurone cells were cobombarded with the reporter construct and effector constructs and incubated with no hormone (control) and gibberellic acid (GA) as described by Gubler et al. (1995). Preparation of extracts and assays of GUS activity have also been previously described (Gubler et al. 1995). sponse gene has been identified in rice aleurone (Chen et al. 1995). The expression of a gene encoding ubiquitin-activating enzyme was shown to respond within 1 h of GA application. It has been proposed that this enzyme is responsible for the derepression of Osamy-c (a-amylase) gene expression. The relationship between these two factors in regulating a-amylase gene expression remains to be determined. Future investigations will involve investigating biochemical and molecular mechanisms which regulate the expression and function of GAmyb in cereal aleurone cells. The ability to transform rice with high efficiency (Li et al. 1993, Hiei et al. 1994) will enable manipulation of GAmyb expression in rice aleurone and to test its function in vivo. 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