Download Cloning of a Rice cDNA Encoding a Transcription Factor

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.
(Received September 12, 1996; Accepted December 26, 1996)
Downloaded from http://pcp.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
Ubii.cas Ubi1.OsGAmyb
Chen, X., Wang, B. and Wu, R. (1995) A gibberellin-stimulated ubiquitinconjugating enzyme gene is involved in a-amylase gene expression in rice
aleurone. Plant Mol. Biol. 29: 787-795.
Christensen, A.H., Sharrock, R.A. and Quail, P.H. (1992) Maize ubiquitin genes: structure, thermal pertubation of expression and transcript
splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 20: 849-856.
Gerlach, W.L. and Bedbrook, J.R. (1979) Cloning and characterization of
ribosomal RNA genes from wheat and barley. Nucl. Acids Res. 7: 18691885.
Goldman, S., Mawai, Y.R., Tanida, I. and Wu, R. (1994) Studies of agibberellin-dependent binding protein related to the expression of ariceaamylase gene. Plant Sci. 99: 75-88.
Gubler, F. and Jacobsen, J.V. (1992) Gibberellin-responsive elements in
the promoter of a barley high-pi a-amylase gene. Plant Cell 4: 14351441.
Gubler, F., Kalla, R., Roberts, J.K. and Jacobsen, J.V. (1995) Gibberellinregulated expression of a myb gene in barley aleurone cells: evidence for
Myb transactivation of a high-pi a-amylase gene promoter. Plant Cell 7:
1879-1891.
Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T. (1994) Efficient transformation of rice (Oryza saliva L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271-282.
Hooley, R. (1994) Gibberellins: perception, transduction and responses.
Plant Mol. Biol. 26: 1529-1555.
Huang, N., Sutliff, T.D., Litts, J.C. and Rodriguez, R.L. (1990) Classification and characterization of the rice a-amylase multigene family. Plant
Mol. Biol. 14: 655-668.
Itoh, K., Yamaguchi, J., Huang, N., Rodriguez, R.L., Akazawa, T. and
Shimamoto, K. (1995) Developmental and hormonal regulation of rice
a-amylase (RAmylA)-gusA fusion genes in transgenic rice seeds. Plant
Physiol. 107: 25-31.
Karrer, E.E., Litts, J.C. and Rodriguez, R.L. (1991) Differential expression of a-amylase genes in germinating rice and barley seeds. Plant Mol.
Biol. 16: 797-805.
Lanahan, M.B., Ho, T.-H.D., Rogers, S.W. and Rogers, J.C. (1992) A gibberellin response complex in cereal a-amylase gene promoters. Plant Cell
4: 203-211.
Li, L., Qu, R., de Kochko, A., Fauquet, C. and Beachy, R.N. (1993) An
improved rice transformation system using the biolistic method. Plant
Cell Rep. 12: 250-255.
Ou-Lee, T.-M., Turgeon, R. and Wu, R. (1988) Interaction of a gibberellin-induced factor with the upstream region of an a-amylase gene in
rice aleurone tissue. Proc. Natl. Acad. Sci. USA 85: 6366-6399.
O'Neil, S.D., Kumagai, M.H., Majumdar, A., Huang, N., Sutliff, T.D.
and Rodriguez, R.L. (1990) The a-amylase genes in Oryza sativa: characterization of cDNA clones and mRNA expression during seed germination. Mol. Gen. Genet. 221: 235-244.
Schuurink, R.C., Vain, P.V. and Jones, R.L. (1996) Modulation of
calmodulin mRNA and protein levels in barley aleurone. Plant Physiol.
I l l : 371-380.
Skriver, K., Olsen, F.L., Rogers, J.C. and Mundy, J. (1991) cis-Acting
DNA elements responsive to gibberellin and its antagonist abscisic acid.
Proc. Natl. Acad. Sci. USA 88: 7266-7270.
Sutliff, T.D., Lanahan, M.B. and Ho, T.-H.D. (1993) Gibberellin treatment stimulates nuclear factor binding to the gibberellin response complex in a barley a-amylase promoter. Plant Cell 5: 1681-1692.