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Characterization and Functional Analysis of ABSCISIC ACID INSENSITIVE3-like Genes from Physcomitrella patens Heather H. Marella*, Yoichi Sakata1*, and Ralph S. Quatrano2 Department of Biology, Washington University, 1 Brookings Dr., St. Louis, MO 63130, USA * These authors contributed equally to this work 2 Corresponding Author: Ralph Quatrano Address: Washington University, 1 Brookings Dr., Campus Box 1137, St. Louis, MO 63130 Email: rsq@wustl.edu Telephone: 314-935-6850 Fax: 314-935-8137 Email of authors: Heather Marella- hinkle@biology.wustl.edu Yoichi Sakata- sakata@nodai.jp.ac Running title: Physcomitrella patens ABI3
Key Words: ABI3, abscisic acid, Physcomitrella, ABI5, VP1, transcriptional regulation
Accession Numbers: PpABI3A-AB233419, PpABI3B-AB233420, PpABI3C-AB245516
Word Count: Total-9875 Summary-250 Introduction-1083 Results-1547 Discussion-1823 Experimental Procedures-1898 Acknowledgements-118 References-2259 Figure Legends-897 1 Present address; Dept. of Bioscience, Tokyo University of Agriculture Sakuragaoka, Setagaya-ku, Tokyo 156-8502 Japan
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SUMMARY
Although the moss Physcomitrella patens is known to respond to abscisic acid (ABA) by
activating gene expression, the transcriptional components involved have not been
characterized. Initially, we used the ABA-responsive Em promoter from wheat linked to
β-glucuronidase (GUS) to determine if ABI3/VP1, transcriptional regulators in the ABA
signaling pathway in angiosperms, were similarly active in the ABA response of P.
patens. We show by particle bombardment that ABI3 and VP1 affect Em-GUS
expression in P. patens in a manner similar to angiosperms. We also show the
involvement of ABI1 in the pathway, utilizing the abi1-1 mutant allele. We isolated
three ABI3-like genes from P. patens. Using an Em-like ABA-responsive promoter from
P. patens (PpLea1), we demonstrate that PpABI3A, only in the presence of ABA,
strongly enhances PpLea1-GUS expression in P. patens. PpABI3A also enhances ABA-
induced Em-GUS expression in P. patens. In barley aleurone, PpABI3A transactivates
Em-GUS but to a lesser extent than VP1 and ABI3. PpABI3A:GFP is localized to the
nucleus of both protonemal cells and barley aleurone indicating that the nuclear
localization signals are conserved. We show that at least a part of the inability of
PpABI3A to fully complement the phenotypes of the Arabidopsis abi3-6 mutant is due to
a weak interaction between PpABI3A and the bZIP transcription factor ABI5, as assayed
functionally in barley aleurone and physically in the yeast-two-hybrid assay. Our data
clearly demonstrate that P. patens will be useful for comparative structural and functional
studies of components in the ABA response pathway such as ABI3.
3
INTRODUCTION
The phytohormone abscisic acid (ABA) not only regulates processes occurring during
seed development (e.g. desiccation tolerance), but also controls processes associated with
responses to water stress during vegetative development of seed plants (e.g. stomatal
opening and closing). Since ABA is found in most land plants (Finkelstein and Rock,
2002), and has demonstrated physiological (Goode et al., 1993; Minami et al., 2003;
Minami et al., 2005; Werner et al., 1991) and molecular responses in non-seed plants
such as mosses (Knight et al., 1995), we have decided to take a comparative approach to
determine the evolution of this response pathway as well as the role of specific regulatory
proteins and protein domains that may be conserved.
Genetic approaches in Arabidopsis have been primarily responsible for
identifying several components involved in the ABA response pathway in seed plants.
Arabidopsis ABA-insensitive (abi) mutants were isolated by their ability to germinate in
the presence of ABA (Brocard-Gifford et al., 2004; Finkelstein, 1994; Koornneef et al.,
1984) and have been extensively characterized. The abi genes that have been cloned
revealed a diverse set of proteins including ABI1 and ABI2 which encode type 2C protein
phosphatases (PP2Cs) and are ABA signaling intermediates that act as negative
regulators (Gosti et al., 1999; Leung et al., 1994; Leung et al., 1997). The ABI3
(Giraudat et al., 1992), ABI4 (Finkelstein et al., 1998) and ABI5 (Finkelstein and Lynch,
2000) genes each encode a different type of transcriptional regulator, while ABI8
represents a novel plant specific protein (Brocard-Gifford et al., 2004). We will take a
comparative approach to characterize the ABA response in Physcomitrella patens and
will initially focus on the plant specific transcriptional regulator ABI3 from Arabidopsis,
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and its ortholog from maize VP1 (Giraudat et al., 1992; McCarty et al., 1991) and
determine if a similar regulator is found in P. patens.
ABI3/VP1-like genes have been found in various seed plants (Bobb et al., 1995;
Chandler and Bartels, 1997; Footitt et al., 2003; Hattori et al., 1994; Lazarova et al.,
2002; Rohde et al., 2002; Shiota et al., 1998), and regulate a set of proteins expressed
during the later stages of seed development. One such gene, the Em gene (Marcotte et
al., 1989), requires both ABA and ABI3/VP1 for expression (Bies-Etheve et al., 1999;
McCarty et al., 1991; Vasil et al., 1995). All of the ABI3/VP1-like genes cloned from
seed plants revealed highly conserved protein domains, designated A1, B1, B2 and B3,
starting from the N-terminal (Suzuki et al., 1997). In fact, maize VP1 can complement
the major phenotypes of the Arabidopsis abi3-6 mutant (Suzuki et al., 2001). The B3
domain has been shown to bind DNA in vitro (Suzuki et al., 1997), whereas the B1
domain is involved in the physical interaction with the bZIP transcription factor, ABI5
(Nakamura et al., 2001). The B2 domain has been shown to be responsible for the ABA-
dependent activation of ABA-regulated genes, like Em, through the ABA-responsive
element (ABRE) (Bies-Etheve et al., 1999; Ezcurra et al., 2000; Hill et al., 1996), and
facilitates the interaction with bZIP transcription factors (Hill et al., 1996) such as ABI5.
However, the mechanism by which this domain functions in seed plants remains to be
elucidated. Until recently, ABI3/VP1 was thought to function exclusively during seed
development, specifically as a component of the ABA signaling pathway involved in the
maturation and germination of seeds (Giraudat et al., 1992; McCarty et al., 1991;
Nambara et al., 1995; Nambara et al., 2000; Parcy et al., 1997). However, recent reports
have pointed out that ABI3 might have broader functions outside of the seed, such as
5
plastid development, flowering time, and outgrowth of axillary meristems (reviewed in
Rohde et al., 2000). These analyses also revealed a novel crosstalk between ABA and
auxin in seed germination and lateral root formation in Arabidopsis (Brady et al., 2003;
Suzuki et al., 2001). These data clearly indicate that ABI3/VP1 is not only involved in
the ABA-regulation of seed development and germination, but also has broader functions
in vegetative growth.
The ABA response pathway in the moss P. patens was demonstrated previously
using the ABA-responsive promoter of the wheat Em gene. The wheat Em promoter can
be activated by exogenous ABA in a transient assay using protonemal tissue as well as in
stable expression lines of P. patens (Knight et al., 1995). Also, the in vitro footprint of
proteins of P. patens on the Em promoter was identical to that of seed plants, indicating
that the moss transcriptional machinery recognizes the same promoter region as the seed
plant factors (Knight et al., 1995). This suggests that higher plants and P. patens share
common ABA regulatory components and as such will be suitable for a comparative
approach to elucidate the mechanism(s) involved and the evolution of the transcriptional
response to ABA. Furthermore, a homolog of the wheat Em gene from P. patens,
PpLEA1, has recently been described (Kamisugi and Cuming, 2005). Similar to the
wheat Em gene, the expression of PpLEA1 is highly inducible by ABA and is mediated
through an ACGT motif in the promoter, a common feature of ABA-inducible genes in
seed plants (Kamisugi and Cuming, 2005). This provides another comparative tool for
the study of the ABA response pathway in P. patens.
We have started to dissect the ABA-regulated transcriptional mechanism in P.
patens by characterizing the structure and function of ABI3-like genes. We show by
6
particle bombardment that the seed plant proteins, ABI1, ABI3, and VP1 can affect Em-
GUS expression in P. patens. We successfully cloned three P. patens cDNAs encoding
ABI3/VP1-like genes and tested their activity in both P. patens and barley aleurone cells
using ABA-responsive Group 1LEA promoters from P. patens and wheat (Em).
PpABI3A enhances ABA-induced PpLea1-GUS and Em-GUS expression in protonemal
tissue and in barley aleurone cells, similar to the response elicited by ABI3/VP1.
However, PpABI3A cannot significantly enhance GUS expression in the absence of ABA
unlike ABI3/VP1. We also demonstrate that PpABI3A is able to function in certain
cellular and molecular functions both in protonemal tissue and in aleurone cells. We
show that at least a part of the inability of PpABI3A to fully complement the molecular
response in the Arabidopsis abi3-6 mutant is due to a weak interaction between
PpABI3A and the bZIP transcription factor ABI5, as assayed functionally in barley
aleurone and physically in the yeast-two-hybrid assay. Our data clearly demonstrate that
P. patens will be useful for comparative structural and functional studies of components
in the ABA response pathway such as ABI3.
7
RESULTS
ABA transcriptional regulation and signaling in P. patens is similar to that of higher
plants
In order to determine if the seed plant proteins ABI1, VP1 and ABI3 can act in the ABA
induced gene expression pathway, we tested their function in P. patens protonemal tissue.
Co-bombardment of the reporter Em-GUS with either Ubi-VP1 or Ubi-ABI3
demonstrated that both VP1 and ABI3 were able to transactivate Em-GUS expression,
four- and ten-fold respectively (Figure 1A). This response to VP1 and ABI3, in the
absence of ABA, is similar to the effect each has in the seed plant ABA response
pathway. In Arabidopsis and barley, ABI3 and VP1 characteristically act downstream
from ABI1 in the ABA signaling pathway (Brady et al., 2003; Casaretto and Ho, 2003).
Predictably, over-expression of the dominant negative allele of ABI1, 35S-abi1-1
(Armstrong et al., 1995), with Em-GUS completely repressed induction of Em-GUS
expression by ABA (Figure 1B). Furthermore, Ubi-VP1 could overcome the repression
effect of abi1-1 placing VP1 also downstream from ABI1 in the ABA signaling pathway
(Figure 1B). These results suggest the involvement of molecules in P. patens with the
same or similar functions as the regulatory ABI3/VP1 proteins from angiosperms.
8
Figure 1
Seed plant ABA response factors function in P. patens.
A) Maize VP1 and Arabidopsis ABI3 transactivate Em-GUS expression in P. patens.
Em-GUS and Ubi-LUC were co-bombarded into P. patens protonemal tissue with or
without the effector constructs, Ubi-VP1 or Ubi-ABI3, using 0.2µg of each construct.
Bars indicate the relative GUS activities ±SE after 48h incubation (n=4).
B) Em-GUS expression in P. patens is repressed by abi1-1 and restored by VP1.
Em-GUS and Ubi-LUC were co-bombarded into P. patens protonemal tissue with or
without the effector constructs, 35S-abi1-1 and Ubi-VP1, using 0.2µg of each construct.
Bars indicate the relative GUS activities ±SE after 48h incubation with (black bars) or
without (white bars) 10µM ABA (n=4).
0
30
60
90
120
150
180
GU
S/L
UC
+ VP1 Em +abi1-1 +abi1-1
GUSEm 35S 3’
Reporter
Effector
abi1-135S NOS 3’
VP1Ubi NOS 3’
Em +VP1 +ABI30
10
20
30
40
50
60
GU
S/L
UC
GUSEm 35S 3’
Reporter
Effector
VP1Ubi NOS 3’
ABI3Ubi NOS 3’
A B
9
Identification of ABI3/VP1-like proteins from P. patens
By searching public EST and genomic databases, we cloned three PpABI3 genes. The
PpABI3A gene encodes a 658 amino acid product, while the PpABI3B gene and PpABI3C
gene encode 515 and 539 amino acid products, respectively. The domain structure of
PpABI3A, PpABI3B, PpABI3C, AtABI3, and VP1 is relatively conserved (Figure 2A).
However, PpABI3B is missing the acidic activation and the serine-rich domains, while
PpABI3C contains a serine-rich region, but lacks the activation domain (Figure 2A). The
position of three of the introns of the B3 domain is conserved across all species, but
PpABI3C has all five of the intron positions found in the higher plant ABI3/VP1 (Figure
2A).
The amino acid sequence alignment of PpABI3A, PpABI3B, PpABI3C, AtABI3,
and VP1 reveals that the B3 domain, underlined in blue, is the most highly conserved
with 50% identity in all five sequences (Figure 2B). The B1 domain, underlined in red,
and the B2 domain, underlined in green, have 23% and 40% identity in the five proteins,
respectively (Figure 2B). The P. patens PpABI3 sequences are more closely related to
each other than the higher plant sequences and share 34% identity over their entirety.
PpABI3A, PpABI3B and PpABI3C share higher identity in the conserved basic domains;
B1 is 68% identical, while B2 and B3 are 60% and 64% identical, respectively. These
data strongly suggest that higher plant VP1/ABI3 and P. patens ABI3A, ABI3B, and
ABI3C originated from a common ancestor gene.
10
Figure 2
Comparison of the PpABI3 proteins with Arabidopsis ABI3 and maize VP1.
A) Schematic representation of PpABI3A, PpABI3B, PpABI3C, ABI3 and VP1 proteins.
Conserved basic regions (B1-B3) and the Serine-rich regions (S-rich) are indicated by
boxes. Asterisks indicate the position of introns.
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 20 * 40 * 60 * 80
--------------------------------------------------------------------------------
--------------------------------------------------------------------------------
--------------------------------------------------------------------------------
MKSLHVAANAGDLAEDCGILGGDADDTVLMDGIDEVGREIWLDDHGGDNNHVHGHQDDDLIVHHDPSIFYGDLPTLPDFP
-MEASSGSSPPHSQENPPEHGG--------------------DMGGAPAEEIGGEAADDFMFAEDT------FPSLPDFP
: -
: -
: -
: 80
: 53
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 100 * 120 * 140 * 160
-----------------------MVLLSSVEAYQQDRLGLLKRSEPLREP-----------------------------T
--------------------------------------------------------------------------------
--------------------------------------------------------------------------------
CMSSSSSSSTSPAPVNAIVSSASSSSAASSSTSSAASWAILRSDGEDPTPNQNQYASGNCDDSSGALQSTASMEIPLDSS
CLSSPSSSTFS---------SNSSSNSSSAYTNTAG-----RAGGEPSEP------------------------------
: 28
: -
: -
: 160
: 89
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 180 * 200 * 220 * 240
GLRSWIDIPLGLEDYRHCASTFACNKDDQNFDMRGLSRIGFPNPTINLEIQGIGDFYSQARSPVASYSSGTSNDCNSAGT
--------------------------------------------------------------------------------
-----------------------------------------------MVNQGSGDFSSQDGS-LPSCSSTSSERNQSSGS
QGFGCGEGGGDCIDMMETFGYMDLLDSNEFFDTSAIFSQDDDTQNPNLMDQTLERQEDQVVVPMMENNSGGDMQMMNSSL
ASAGEGFDALDDIDQLLDFASLSMPWDSEPFPGVSMMLENAMSAPPQPVGDGMSEEK-----AVPEGTTGGEEACMDAS-
: 108
: -
: 32
: 240
: 163
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 260 * 280 * 300 * 320
STVDSTEDASLSSYLMDGN--VRAMLKKVTVNEGVLNQTIQSLGGGEKGLKELMAYIMAWVKEQNGE-NCDKLFLSQNL-
---------------MDGN--VRAVLKKVTVNEDSLSQAIQALGGGEKGLKELIGHIMLWVKQHNGEMDYGKTSLSQKL-
GLGEG------CSLLMDDE--VRAMLKKVTVNEAVLNRAIQSLGGGEKGLKSLMGYIMIWVKQQKE----CKSARSNSG-
EQDDDLAAVFLEWLKNNKETVSAEDLRKVKIKKATIESAARRLGGGKEAMKQLLKLILEWVQTNHLQRRRTTTTTTNLSY
-EGEELPRFFMEWLTSNRENISAEDLRGIRLRRSTIEAAAARLGGGRQGTMQLLKLILTWVQNHHLQRKRPRDVMEEEAG
: 184
: 62
: 99
: 320
: 242
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 340 * 360 * 380 * 400
------EKLNPPQMNPTFGFAPSLANNIPTCNLTLGTAACDQRSDNLRRTFDTVQSSDDTQDLNQVIQCSKSRRLMVE-G
------EKLHPPQMNHALEFASGKGISGTVYNPNLASEGYDQRNEKSPCPFVRVQSSEDLQDMNHLFQCIKSRRLMGE-G
------EQSSGSPFNFSMLGAEAAAEAEAIV-LQHTSDAFQSLNGILQP--SATQSPVNLPDTQHQ-QRFKSRRLMMEDA
QQSFQQDPFQNPNPNNNNLIPPSDQTCFSPSTWVPPPPQQQAFVSDPGFG----YMPAP----NYPPQPEFLPLLES-PP
LHVQLPSPVANP-PGYEFPAGGQDMAAGGGTSWMP---HQQAFTPPAAYGGDAVYPSAAGQQYSFHQGPSTSSVVVN-SQ
: 257
: 135
: 169
: 391
: 317
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 420 * 440 * 460 * 480
EVDSSYRADAYPRQGTFLNARGSVSED-FHSTKLESNMTNPSPIDAVVLQGMSSELGTLTQQAPCSQFGLSFNPGQNELN
GLDINFPRDVHSGSGTFVDVRGSVPDV-LHFPSGGMGPRSPNHIGPAMLQGMPCELDIQRPLIPCAQAGLSFSGEHDGLS
EVDAGLKVDAGFCPSNFMAVGVPPGDYSMYMPAMAGPAVSPYMPRLNYDSGMQQPVCNMQSMIPSTTSQNSMQP----IE
SWPPPPQ------SGPMPHQQFPMPPTSQYN-QFGDPTGFNGYNMNPYQYPYVPAGQMRD----------------QRLL
PFSPPPVGDMHGANMAWPQQYVPFPPPGASTGSYPMPQPFSPGFGGQYAGAGAGHLSVAP----------------QRMA
: 336
: 214
: 245
: 448
: 381
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 500 * 520 * 540 * 560
RTLAASTRAARRNRLSRQRQSVQ---------------------QSHRR-SPTSGTTVTSGGCGLWSASPPALDFMNTRW
RIFTATTRAARRNQISRQRQSGL---------------------QSNSTFTPSNTVTAVSSECGLWSVAPPAWD-MSVGV
QTPAMATKAARRNRMARQRQSMM---------------------KQHAR--ATNQANPVSVGFWVWNGAPPAGGTKKTEI
RLCSSATKEARKKRMARQRRFLSHHHRHNNNNNNNNNNQQNQTQIGETCAAVAPQLNPVAT-----TATGGTWMYWPNVP
GVEASATKEARKKRMARQRRLSCLQQQRSQQLSLGQIQTSVHLQEPSPRSTHSGPVTPSAGGWGFWSPSSQQQVQNPLSK
: 394
: 272
: 302
: 523
: 461
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 580 * 600 * 620 * 640
NHPGTQMPL---------------------------GASVKSEKKGNTTDMLTFLLQKELRPSDVGNLGRIILPKKEAEV
FLSRTQSPS---------------------------CGPDISDPK-----MLTFLLQKELRPSDVGNLGRIILPKKEAEA
SHSGAQPAQ---------------------------GTAVNAEQKGRNMDSLTFLLQKELRPSDVGSLGRIILPKKEAEQ
AVPPQLPPVMETQLPTMDRAGSASAMPRQ-------QVVPDRRQGWKPEKNLRFLLQKVLKQSDVGNLGRIVLPKKEAET
SNSSRAPPSSLEAAAAAPQTKPAPAGARQDDIHHRLAAASDKRQGAKADKNLRFLLQKVLKQSDVGSLGRIVLPKKEAEV
: 447
: 320
: 355
: 596
: 541
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 660 * 680 * 700 * 720
HLPILALREGVSLLMEDFDSGYCWNIRYRFWPNNKSRMYLLENTGEFVKSHHLKEGDLLILYRN-EQGNYVLRGKKKVPS
HLPILALREGILLQMEDFDSGHCWKIRYWFWPNNKSRMYLLENTGEFVKSHRLEEGDLLVLYKI-QEGNYVLRAQKKVHS
HMPFLSMRGGVCIQVEDFDSGHIWNLRYRFWPNNKSRMYLLENTGDFVKSHRLVEGDLLIIYRS-QQGDYVMRGKKR-KT
HLPELEARDGISLAMEDIGTSRVWNMRYRFWPNNKSRMYLLENTGDFVKTNGLQEGDFIVIYSDVKCGKYLIRGVKVRQP
HLPELKTRDGISIPMEDIGTSRVWNMRYRFWPNNKSRMYLLENTGEFVRSNELQEGDFIVIYSDVKSGKYLIRGVKVRPP
: 526
: 399
: 433
: 676
: 621
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 740 * 760 * 780 * 800
ETRVAYGSQHRTAHSLACRFSEGIPNKIEESFKDRDFGGGLTETASGKLKIEVKAEPLVVQPSTDGDKFEDVMSNIGSSF
ESSGAPGSQQKNAYSLARDFPEA--SKTEEPLKEKDIVG-FAEWSCG----KVNEERNILVRDDDPFFKDDQKHKISSSF
QRLETESVKIQFAHSSARDSNEEHTSPKGNGLPMERLYG-GGPPEK-----FVKEEP--PNLDDDPFL-KDMMMSMGPSF
SGQKPEAPPSSAATKR---QNKSQRNINNNSPSANVVVA------------------------------SPTSQTVK---
PAQEQGSGSSGGGKHRPLCPAGPERAAAAGAPEDAVVDG------------------------------VSGACKGRSPE
: 606
: 472
: 504
: 720
: 671
PpABI3A :
PpABI3B :
PpABI3C :
ABI3 :
VP1 :
* 820 * 840 *
ATEG-PLQPLERFPSLNLDFPLDEIMAGIPKEEDNDLTIEPHMDSKVKTEPAG
SPE--PLQSLERFPSLSIDFPLDEMMP---WDDPDEFNTDSELAELVG-----
GSDATSLQPLERFPSLNLDFPLDEIMAVNPKTE---------LE---------
-----------------------------------------------------
GVRRVRQQGAGAMSQMAVSI---------------------------------
: 658
: 515
: 539
: -
: 691
A
B
B1 B2 B3S-rich
ABI3
720* * ** *VP1
691* * ** *
PpABI3A
* * ** 658
* * * 515
PpABI3B
PpABI3C
539* * ** *
11
B) Comparison of amino acid sequences of PpABI3A, PpABI3B, PpABI3C, ABI3 and
VP1. The alignment was made by the T-coffee program. Identical residues between
PpABI3A, PpABI3B, PpABI3C, and ABI3 or VP1 are shaded black and similar residues
are shaded gray. The Bl, B2, and B3 domains are indicated respectively by the red,
green, or blue underlines.
Does PpABI3A act as a transcriptional enhancer of ABA responsive promoters?
In order to address the question of whether the PpABI3 proteins from P. patens were
active, we utilized the ABA-responsive promoter of the P. patens Late Embyrogenesis
Abundant (PpLEA1) gene linked to GUS (Kamisugi and Cuming, 2005). Only PpABI3A
strongly enhanced Lea1-GUS expression in the presence of ABA (Figure 3A) comparable
to that of VP1 (Figure 3B). All three PpABI3 proteins had little or no activity in
activating GUS expression in the absence of ABA. Interestingly, when comparing the
level of enhancement between VP1 and PpABI3A with and without ABA, PpABI3
exhibits a ten-fold increase in GUS activity versus a two-fold increase with VP1 (Figure
3B). VP1, however, clearly shows an enhancement of the ABA response in the absence
of ABA (Figure 3B). These results clearly show that the PpABI3A gene can act as a
strong transcriptional enhancer of an ABA response from a P. patens promoter of an
ABA responsive gene.
12
Figure 3 PpABI3A enhances ABA-regulated gene expression in P. patens.
A) The PpLea1-GUS reporter construct and each of the PpABI3 effector constructs were
introduced into P. patens protonemal tissue by particle bombardment. The protonemal
tissue was allowed to recover 24h untreated and then subsequently incubated with (black
bars) or without (white bars) 10µM ABA for 24h. Protein extracts were made from the
treated tissue for GUS and LUC assays. Bars indicate the relative GUS activities ±SE
(n=4).
B) The PpLea1-GUS reporter construct and each of the effector constructs were
introduced into P. patens protonemal tissue by particle bombardment. The protonemal
tissue was allowed to recover 24h untreated and then subsequently incubated with (black
bars) or without (white bars) 10µM ABA for 24h. Protein extracts were made from the
0
200
400
600
800
1000
1200
PpLea1-
GUS
PpABI3A PpABI3B PpABI3C
GUS/LUC
w/o ABA
10uM ABA
0
200
400
600
800
1000
1200
1400
PpLea1-GUS PpABI3A VP1
GU
S/L
UC
w/o ABA
10uM ABA
GUSPpLea1 NOS 3’
Reporter
Effector
PpABI3AUbi NOS 3’
VP1Ubi NOS 3’
GUSPpLea1 NOS 3’
Reporter
Effector
PpABI3AUbi NOS 3’
PpABI3BUbi NOS 3’
PpABI3CUbi NOS 3’
A B
13
treated tissue for GUS and LUC assays. Bars indicate the relative GUS activities ±SE
(n=4).
Using the Em promoter (Group 1 LEA from wheat), we observed a similar pattern
with PpABI3A, with an enhancement effect only when ABA is present in P. patens
(Figure 4A). However, neither VP1 nor ABI3 showed an enhancement of GUS activity
in combination with ABA (Figure 4A). Comparing these results with those observed
with the PpLea1 promoter, we see that the enhancement effect is much greater (ten-fold)
with the endogenous PpLea1 promoter (Figure 3B) than with the wheat Em promoter
(three-fold) (Figure 4A).
To determine if PpABI3A could function in higher plants, we analyzed the ability
of PpABI3A, VP1, and ABI3 to transactivate Em-GUS in barley aleurone cells (Figure
4B). Co-bombardment of Ubi-PpABI3A and Em-GUS resulted in a three-fold activation
of expression. Em-GUS is more strongly activated by VP1 and ABI3 (four- and thirteen-
fold, respectively) in barley aleurone cells.
14
Figure 4
PpABI3A functions as a transcriptional activator in P. patens and barley.
A) PpABI3A functions as a transcriptional activator in P. patens.
The Em-GUS reporter construct was introduced into P. patens protonemal tissue with
each of the effector constructs by particle bombardment. P. patens protonemal tissue was
incubated with (black bars) or without (white bars) 10µM ABA for 48h and protein
extracts were made for GUS and LUC assays. Bars indicate the relative GUS activities
±SE (n=4).
B) PpABI3A functions as a transcriptional activator in barley aleurone.
GUSEm 35S 3’
Reporter
Effector
PpABI3AUbi NOS 3’
VP1Ubi NOS 3’
ABI3Ubi NOS 3’
0
50
100
150
200
250
300
Em PpABI3A VP1 ABI3
GU
S/L
UC
-ABA
+ABA
0
10
20
30
40
50
60
GU
S/L
UC
Em PpABI3A VP1 ABI30
50
100
150
200
250
300
Em PpABI3A VP1 ABI3
GUS/LUC
BA
15
The Em-GUS reporter construct was introduced into barley aleurone cells with each of
the effector constructs by particle bombardment. Barley half seeds were incubated for
24h and protein extracts were made for GUS and LUC assays. Bars indicate the relative
GUS activities ±SE (n=4).
What higher plant functions are conserved in PpABI3A?
For analysis of the sub-cellular of localization of PpABI3A, a GFP fusion construct
(PpABI3A:GFP) was introduced into cells via particle bombardment. In both barley
aleurone cells and P. patens protonemal cells bombarded with PpABI3A:GFP, the GFP
signal localizes at the nucleus (Figures 5B and 5E). The AtABI3:GFP control also
localizes to the nucleus of both barley aleurone cells and P. patens protonemal cells
(Figures 5A and 5D). The GFP control shows a diffuse localization, in both the
cytoplasm and the nucleus (Figures 5C and 5F). These results demonstrate that the
localization of PpABI3A is consistent with its function as a transcriptional activator and
that the nuclear localization signals of PpABI3A and AtABI3 are recognized in both P.
patens and barley cells.
16
Figure 5
A PpABI3A:GFP fusion protein localizes to the nucleus in barely aleurone and P. patens
cells.
Constructs for the over-expression of AtABI3:GFP (A and D), PpABI3A:GFP (B and E),
and GFP (C and F) protein were delivered into barley aleurone cells (A-C) and P. patens
protonemal tissue (D-F) by particle bombardment. Images of transformed cells were
taken by confocal microscopy. The GFP signal and auto-fluorescence of chloroplasts are
shown by green and red color, respectively.
To determine if PpABI3A is capable of functioning in higher plants, the
Arabidopsis abi3-6 mutant, a severe allele of abi3 (Nambara et al., 1994), was stably
transformed with PpABI3A under the control of the Arabidopsis ABI3 promoter. As a
control, abi3-6 plants were also transformed with a PAtABI3-AtABI3 construct. The seeds
of abi3-6 plants are green due to a failure to degrade chlorophyll and appear shriveled up
because of the inability to undergo desiccation, as previously reported (Figure 6B)
(Nambara et al., 1994). In complementation lines expressing PAtABI3-PpABI3A the seeds
A
FED
CB
17
are brown (Figure 6C), just like Columbia wild type and the PAtABI3-AtABI3 control
(Figure 6A and 6D respectively). Interestingly, in the PAtABI3-PpABI3A complementation
lines, even though the seeds are brown, they are not desiccation tolerant (data not shown).
Figure 6
PpABI3A complements the green seed phenotype of abi3-6.
Seeds from mature siliques were harvested from (A) Columbia wild type, (B) abi3-6, and
the transgenic lines of (C) PAtABI3-PpABI3A/abi3-6 and (D) PAtABI3-AtABI3/abi3-6.
The germination of abi3-6 seeds is insensitive to ABA, however, ABA inhibits
germination at levels higher than 3µM in Columbia wild type. At 100µM ABA, 100% of
the abi3-6 mutant seeds germinate, while a 40% reduction in germination is seen in the
PAtABI3-PpABI3A complementation line (Figure 7A). The germination of the PAtABI3-
18
AtABI3 control is sensitive to ABA like Columbia wild type (Figure 7A). Therefore,
expression of PpABI3A can partially complement the ABA insensitivity of germination
of the abi3-6 mutant.
Figure 7
PpABI3A partially complements the abi3-6 mutant.
A) PpABI3A partially complements the ABA insensitivity of abi3-6 seed germination.
Seeds of Columbia wild type (red circle), abi3-6 (green square), PAtABI3-PpABI3A/abi3-6
(orange cross), and PAtABI3-AtABI3/abi3-6 (blue triangle) were plated on MS media
containing 0, 0.1, 1, 10, or 100 µM ABA and cold treated for 4 days at 4°C in the dark.
The plates were then moved to 22°C with a 16-hour light and 8-hour dark light regime.
The germination rate was measured after 4 days using at least 40 seeds per assay. Bars
indicate ±SE of three independent replicates.
B) PpABI3A partially complements the expression of AtABI3 regulated genes in abi3-6
seeds.
0
20
40
60
80
100
120
0 0.1 1 10 100
ABA (µM)
% G
erm
inat
ion
Ubq10
Rab18
Oleosin2
Napin
CruciferinC
Em6
Em1
Colum
bia W
TP AB
I3-A
tABI
3/abi3
-6P AB
I3-P
pABI
3A/ab
i3-6
abi3-
6
A B
19
RNA was extracted from siliques 11 days after pollination of Columbia wild type,
PAtABI3-AtABI3/abi3-6, PAtABI3-PpABI3A/abi3-6, and abi3-6. RT-PCR was performed
using 0.5 µg of total RNA and the expression of various seed genes controlled by ABI3
was analyzed.
What molecular responses are not complemented by PpABI3A?
Since the PAtABI3-PpABI3A/abi3-6 lines show partial complementation of the abi3-6
phenotypes, we tested for the expression of several seed genes controlled by ABI3 to
determine if PpABI3A could properly regulate their expression (see Table 1). Semi-
quantitative RT-PCR was performed on RNA extracted from siliques of Columbia wild
type, PAtABI3-AtABI3/abi3-6, PAtABI3-PpABI3A/abi3-6, and abi3-6 harvested 11 days after
pollination. In abi3-6 mutant seeds, the expression of these genes is reduced (Figure 7B).
In the PAtABI3-PpABI3A/abi3-6 complementation lines, the expression level of
CruciferinC, Napin, Oleosin2, and Rab18 is complemented to near wild type levels
(Figure 7B). However, the expression of AtEm1 and AtEm6 is not complemented in the
PAtABI3-PpABI3A/abi3-6 siliques (Figure 7B). The gene expression in the PAtABI3-
AtABI3/abi3-6 line behaves similarly to Columbia wild type (Figure 7B). These data
suggest that PpABI3A is able to partially complement gene expression in the abi3-6
mutant.
Because PpABI3A appeared to have a defect in the complementation of ABI5
dependent gene expression (i.e. AtEm1 and AtEm6, see Table 1) in the abi3-6
background, we tested for a functional interaction between PpABI3A and barley ABI5
(HvABI5) in barley aleurone cells. It is known that ABI5 alone is not sufficient to
20
completely activate ABA-responsive promoters, but co-expression of ABI3/VP1 with
ABI5 compensates for the effect of ABA (Casaretto and Ho, 2003; Hobo et al., 1999). In
the case of the Em promoter in barley aleurone cells, HvABI5 alone also did not show
strong activation (1.5-fold) (Figure 8A). When HvABI5 was over-expressed with VP1 or
ABI3 in the absence of ABA, Em promoter activity was almost the same level with that
of ABA-induction (Figure 8A). In the presence of ABA, HvABI5 co-expressed with
either VP1 or ABI3 enhanced the Em-GUS transactivation. PpABI3A in combination
with HvABI5 also showed synergistic enhancement (11.5-fold) in the absence of ABA,
however, the activation level was far lower than that of ABA induction of the Em
promoter (Figure 8A). Also, co-expression of PpABI3A and HvABI5 in the presence of
ABA lacked the enhancement of Em-GUS transactivation (Figure 8A). These data show
that PpABI3A is capable of a basal interaction with HvABI5 on the transactivation of
Em-GUS, however it is not functionally equivalent to the interaction between ABI3/VP1
and HvABI5 in barley aleurone cells.
We performed yeast-two-hybrid assays to further test the physical interaction
between PpABI3A and HvABI5 in a more quantitative manner. In the nutrient-requiring
growth assay, the colony size of the yeast expressing AD-PpABI3A with BD-HvABI5 is
smaller than the control yeast expressing AD-AtABI3 with BD-HvABI5 (Figure 8B). In
the β-galactosidase assay, the yeast expressing AD-PpABI3A with BD-HvABI5 yielded
25% of the activity of the AD-AtABI3 control (Figure 8C). These data reveal that
PpABI3A is able to interact with HvABI5 although the interaction is weak.
21
Figure 8
PpABI3A interacts weakly with HvABI5.
A) PpABI3A interacts weakly with HvABI5 in barley aleurone cells.
The Em-GUS reporter construct was introduced into barley aleurone cells in various
combinations with each of the effector constructs. Barley half seeds were incubated with
(black bars) or without (white bars) 20µM ABA for 24h. Bars indicate the relative GUS
activities ±SE (n=4).
B) PpABI3A interacts weakly with HvABI5 in yeast-two-hybrid plate assay for analysis
of reporter genes, GAL2-ADE2, GAL1-HIS3. The diploids harboring indicated BD and
AD constructs were grown for one week on SD-LW medium lacking adenine and
histidine containing 35mM 3-AT (see Materials and Methods). Bar=50mm.
GUSEm 35S 3’ Ubi NOS 3’
Reporter Effector
PpABI3AUbi NOS 3’
VP1Ubi NOS 3’
ABI3Ubi NOS 3’
HvABI5
0
100
200
300
400
500
Em
HvABI5
PpABI3A
VP1
ABI3
PpABI3A+HvABI5
VP1+HvABI5
ABI3+HvABI5
GUS/LUC
!-g
ala
cto
sid
ase a
ctivity
(Mill
er
units)
0
50
100
150
200
250
pGAD AD-PpABI3A AD-ABI3
pGBD
BD-HvABI5
pGBD
BD-HvABI5
pGAD AD-PpABI3A AD-ABI3
A
CB
22
C) β-galactosidase activity of the diploids indicated in (B). Black bars represent the BD-
HvABI5 interaction and white bars represent the pGBD control. The individual data is
the average ± SD (n=3). pGBD (pGBTK) and pGAD (pGAD424) are vector controls.
[The differences between pGAD/ABI5 and PpABI3/ABI5, pGAD/BD-ABI5 and
ABI3/ABI5, and PpABI3/HvABI5 and ABI3/HvABI5 were significant (P<0.05, P<0.01
and P<0.01. respectively)]
23
DISCUSSION
ABA signaling pathway in P. patens
The wheat Em promoter was previously shown to respond to ABA in P. patens
protonemal tissue, similar to the response in seed plants (Knight et al., 1995). They
showed that the ABA response was dependent on the ABRE [two ACGT-boxes separated
by 46-bp (Marcotte et al., 1989)]. The activation of genes in seed plants by ABA required
not only the ABRE and its bZIP transcription factor but also the transcriptional activator
VP1. VP1 can transactivate the Em promoter in a transient assay in the absence of ABA
and further enhance expression in the presence of ABA (Vasil et al., 1995). Mutants of
VP1, and its ortholog from Arabidopsis ABI3, failed to accumulate the Em transcript
during seed development (Bies-Etheve et al., 1999; McCarty et al., 1991; Parcy et al.,
1994). In this study, we confirmed the results of Knight et al. (1995), and further showed
that both VP1 and ABI3 can transactivate this promoter in P. patens without exogenous
ABA but unlike the response in angiosperms, they failed to enhance expression with
ABA.
The activation of ABA-regulated genes in seed plants also requires the signaling
intermediate ABI1, which encodes a PP2C phosphatase and acts as a negative regulator of
ABA signaling in Arabidopsis. The dominant negative abi1-1 allele was shown to inhibit
the ABA signaling not only in Arabidopsis but other seed plants such as barley, rice, and
tomato (Carrera and Prat, 1998; Casaretto and Ho, 2003; Gampala et al., 2002; Grabov et
al., 1997; Hagenbeek et al., 2000). We showed that abi1-1 blocks the ABA induction of
Em-GUS expression in P. patens. In addition, this negative regulation was overcome by
24
the co-expression of VP1, showing that abi1-1 acts upstream of the transcriptional
activator VP1. The involvement of abi1-1 in the ABA signaling pathway in P. patens
and its relationship with VP1 appears identical to its role in seed plants (Gampala et al.,
2002). At least five ESTs show significant similarity to ABI1 and ABI2 in the P. patens
database (data not shown), allowing for future functional comparisons of this class of
phosphatases in ABA signaling. Hence, results using the ABA signaling intermediate
ABI1, and the transcription factors ABI3/VP1, demonstrate that at least these
components of the angiosperm ABA signaling pathway function in a similar manner in a
bryophyte.
Characterization of the PpABI3 proteins
Since VP1 and ABI3 were active in P. patens, we proceeded to isolate three ABI3-like
genes from P. patens: PpABI3A, PpABI3B, and PpABI3C. Three copies of ABI3 are
present in the P. patens genome as compared to one in angiosperms, and although smaller
than their seed plant counterparts, the overall structure of the PpABI3 proteins follows
the same pattern with the three basic domains: B1, B2, and B3. Variation in the structure
occurs primarily at the N-terminus, where the PpABI3 proteins have either a shorter
acidic activation domain (PpABI3A) or lack this domain (PpABI3B and PpABI3C). The
serine-rich region found in the seed plant orthologs is also found in PpABI3A and
PpABI3C. The intron-exon junctions between the angiosperm orthologs and PpABI3C
are perfectly conserved. In contrast, PpABI3A and PpABI3B only share three of the five
intron positions in the B3 domain, suggesting that these paralogs were lost in the seed
25
plant lineage. These observations strongly suggest that the VP1/ABI3 gene family arose
prior to the divergence between mosses and seed plants.
The B1 and B2 domains of ABI3 and VP1 are responsible for some unique
functions of these proteins (Bies-Etheve et al., 1999; Ezcurra et al, 2000; Hill et al., 1996;
Nakamura et al., 2001). For example, the Arabidopsis abi3-7 mutant has a point
mutation that converts an alanine into threonine in the B2 domain and prevents the
expression of AtEm1 and AtEm6 (Bies-Etheve et al., 1999). This alanine residue is
perfectly conserved not only in all known ABI3/VP1s from seed plants, but also in the
PpABI3 proteins. Similarly, the abi3-8 mutation alters a conserved leucine in B1, which
is possibly involved with ABI5 interaction (Nambara et al., 2002). However, this leucine
is not conserved in P. patens. Alignments such as these will target those amino
acids/domains for further analysis in determining their conserved function during the
evolution of land plants.
Functional analysis of PpABI3A
We demonstrate that like VP1/ABI3 in angiosperms (Gampala et al., 2002; Vasil et al.,
1995), PpABI3A functions in the ABA-response pathway as a transcriptional enhancer of
ABA-responsive promoters. When PpABI3A is over-expressed in the presence of
exogenous ABA, enhanced expression from both P. patens (PpLea1) and wheat (Em)
promoters is observed in P. patens protonemal tissue. This enhancement is similar to
what has been reported in angiosperms, when VP1 is over-expressed in ABA-treated
cells. PpABI3A also functions weakly (compared to ABI3 and VP1) as a transcriptional
activator of the Em promoter in barley aleurone cells. In fact, the length of the activation
26
domain of these three proteins is proportional to their enhancement of the ABA response.
We also showed that PpABI3A and AtABI3 localize to the nucleus of both barley
aleurone and P. patens protonemal cells. The fact that PpABI3A localizes to the nucleus
and enhances expression in both P. patens and barley aleurone cells indicates that the
core transcriptional regulatory properties of PpABI3A are at least partially conserved.
We also show that PpABI3A, but not PpABI3B and PpABI3C, enhances
expression from a P. patens ABA responsive promoter, PpLea1-GUS (Kamisugi and
Cuming, 2005). PpABI3A contains all the conserved domains found in the seed plant
orthologs, including the activation domain, while PpABI3B and PpABI3C lack the N-
terminal activation domain. All three ABI3 proteins from P. patens either lack the ability
or show weak transactivation in the absence of ABA. Interestingly, these results in
protonemal tissues are specific to PpLea1-GUS promoter, since all three PpABI3 proteins
are able to transactivate the Em-GUS promoter (data not shown). Hence, the
structure/function relationship of the ABI3 proteins from P. patens reflects not only their
domain structure but also their interaction with specific ABA-responsive promoters. This
will allow further understanding of this interaction by the use of domain swaps of the
PpABI3 proteins with the angiosperm orthologs, along with the construction of specific
promoter sequences.
With respect to the specific ABRE in each of these promoters, PpLEA1 contains a
single ACGT motif while the ABRE from angiosperms have either two ACGT motifs or
one ACGT motif and a coupling element (Kamisugi and Cuming, 2005; Shen et al.,
2004). While we demonstrate that the wheat Em promoter functions in an ABA-
responsive manner in both barley and P. patens, the same cannot be said of the PpLea1
27
promoter, which fails to express in barley aleurone cells (Kamisugi and Cuming, 2005).
These studies appear to indicate that the ABA-responsive promoter in P. patens
(PpLEA1) represents the ancestral state, while ABA-responsive promoters from
angiosperms (wheat Em) have additional factors/sequences involved in the response.
(Kamisugi and Cuming, 2005). A further comparative analysis of the cis-elements in the
wheat Em and PpLea1 promoters which are required in aleurone and protonemal cells for
the level of ABA-responsiveness will aid in our understanding of the evolution of the
response elements and the factors that bind to them.
Complementation of abi3-6
Given the transcriptional activation and nuclear localization properties of PpABI3A in
barley we further showed that PpABI3A could partially complement the Arabidopsis
abi3-6 mutant. Similar results have been reported for ABI3/VP1 orthologs from seed
plants; abi3-6 lines expressing maize VP1 were unable to properly express CruciferinC
(Suzuki et al., 2001) while the yellow cedar ABI3 failed to fully complement the ABA
sensitivity of germination (Zeng and Kermode, 2004). However, in both of these
complementation experiments, ABI3/VP1 expression was driven by the enhanced 35S
promoter, which is known to have weak promoter activity during seed development
(Benfey et al., 1990; Sunilkumar et al., 2002; Terada and Shimamoto, 1990). Hence, the
failure of these higher plant proteins to fully complement may be due to improper spatial,
temporal, or level of expression of ABI3 or VP1. In our hands, complementation of the
abi3-6 mutant using 35S-PpABI3A or 35S-AtABI3 showed no detectable
complementation of the abi3-6 phenotypes (data not shown). However, when the AtABI3
28
promoter drives expression of PpABI3A or AtABI3, AtABI3 fully complemented the
mutant while PpABI3A only partially complemented.
The seed color phenotype of the abi3-6 was fully complemented (from green to
brown) in lines expressing PpABI3A. However, full ABA sensitivity was not observed
in PAtABI3-PpABI3A/abi3-6 seeds, where only 60% of the seeds germinate on 100µM
ABA. Seeds from abi3-6 germinated at 100µM ABA, whereas seeds from wild type and
AtABI3 complemented lines did not germinate above 10µM ABA. This partial
complementation of ABA sensitivity by PpABI3A may be due to a failure of PpABI3A
to interact with the Arabidopsis ABA response factors, such as ABI5, since certain
conserved amino acids in the B1 domain are lacking. The PpABI3 proteins lack the
conserved leucine of the abi3-8 mutation, which alters the B1 domain and possibly
the interaction with ABI5 (Nambara et al., 2002).
Transcript levels of several ABI3-regulated, seed-specific genes suggest that this
may indeed be the case. Expression of AtEm1 and AtEm6, both of which require ABI5
for expression (Finkelstein and Lynch, 2000), are not complemented in the PAtABI3-
PpABI3A lines. However, other seed-specific genes that are not regulated by ABI5 are
fully complemented by PpABI3A, such as Napin and CruciferinC, which require another
member of the bZIP family (OPAQUE2-like) (Lara et al., 2003), and Oleosin2 and
Rab18 (see Table 1).
29
PpABI3A interaction with ABI5
In this study, we showed that PpABI3A and HvABI5 functionally interact in aleurone
cells but it was not functionally equivalent to the interaction of ABI3/VP1 with HvABI5
in the same cells. This conclusion is also supported by our demonstration of a weak
physical interaction between PpABI3A and HvABI5 in the yeast-two-hybrid assays;
AtABI3 and HvABI5 have a strong interaction even though one protein is from a dicot
and the other from a monocot. The B1 domain of AtABI3 is necessary for the interaction
with AtABI5 in yeast two hybrid assays (Nakamura et al., 2001) and now targets this
domain for functional comparisons of ABI5 interactions. Several sequences for bZIP
transcription factors are in the P. patens genome, at least two of which fall into the
ABI5 subfamily. This will enable a functional comparison of the interaction
between ABI3 and ABI5 in P. patens. In this same regard, ABI3/VP1 are able to
physically interact not only with ABI5 and OPAQUE2-related bZIP transcription factors,
but also other proteins such as 14-3-3 (Schultz et al., 1998), the transcription factor
CONSTANS, a protein involved in the cell cycle regulation, and an E3 ligase with a
RING motif (Jones et al., 2000; Kurup et al., 2000; Zhang et al., 2005). It will be
interesting to determine how the composition of the ABI3/VP1 transcriptional complex
varies and how this relates to the different functions of the members of the ABA
signaling networks in P. patens and higher plants.
30
EXPERIMENTAL PROCEDURES
Plant material
P. patens subspecies patens (Gransden) was used as the wild type strain. The strain was
maintained as previously described. Briefly, protonemal tissue were ground with a
PowerGen 125 homogenizer (Fisher Science, Hampton, NH) and inoculated onto
cellophane overlaid on 0.7 % agar plates of PpNH4 medium (1.8 mM KH2PO4, pH7.0,
3.4 mM Ca(NO3)2, 1 mM MgSO4, 45mM FeSO4, 0.22 mM CuSO4, 0.19 µM ZnSO4, 9.9
µM H3BO3, 2 µM MnCl2, 0.23 µM CoCl2, 0.17 µM KI, 0.1 µM Na2MoO4, 2.7 µM
ammonium tartrate). The tissue was cultured at 25°C under the cycle of 16 hours light
(90 µmol m-2s-1) and 8 hours dark.
Barley seeds (Hordeum vulgare cv Himalaya) from 1998 harvests at Washington
State University in Pullman were used in all experiments.
Arabidopsis thaliana ecotype Columbia was used as the wild-type. The abi3-6
mutant was a gift of Dr. Peter McCourt. Plants were grown in a mixture of 60% Redi-
Earth and 40% vermiculite at 22°C under a 16-hour light and 8-hour dark light regime in
a growth chamber. Seeds were surface sterilized for 5 minutes in 95% ethanol, dried, and
plated on MS media containing 0.8% agar and subjected to 4 days of cold-treatment in
the dark before being moved to a growth chamber at 22°C under a 16-hour light and 8-
hour dark light regime.
Cloning of PpABI3 cDNAs
The B3 domain of ABI3 from Arabidopsis was used as a query sequence for the BLAST
program (tblastn) (Altschul et al., 1990) against Physcobase (http://moss.nibb.ac.jp/)
31
(Nishiyama et al., 2003) to retrieve cDNAs encoding the B3 domain protein. As a result,
three independent cDNA clones were identified as candidates. From the sequence
information of the contigs, we set up primers to amplify the full-length cDNAs. The
primers to amplify the cDNA clone pphn12b05, which we named PpABI3A, are 5’-ATG
TGA GAG TGA GCC TGC AG-3’ and 5’-TCC ACC CCT GTA ACA GAA AA-3’ for
5’ and 3’ end, respectively. PCR of P. patens against the full-length cDNA library, a gift
from Dr. M. Hasebe (Nishiyama et al., 2003) was performed with KlenTaq LA
(Clontech, Mountain View, CA) and the supplied buffer with 25 µM each dNTP and
3.5mM MgCl2 by 35 cycles of 96°C for 1 minute, 56°C for 1 minute, and 68°C for 2
minutes. The DNA fragment was subcloned into pGEM-T Easy (Promega, Madison,
WI) and subjected to the DNA sequencing using the M13 forward and reverse primers
and Big Dye Terminator mix (Applied Biosystems, Foster City, CA). The amino acid
sequence was analyzed using DNASIS software (Hitachi Software Engineering, Tokyo,
Japan).
PpABI3B was cloned from a partial EST sequence (Frank et al., 2005) using 5’
and 3’ RACE. RNA was extracted from P. patens gametophore tissue using the RNeasy
Kit (Qiagen, Valencia, CA) for use with the 5’ and 3’ RACE kit (Invitrogen, Carlsbad,
CA) to amplify the remaining sequence. Three sequential rounds of 5’RACE were
required to reach the 5’ end of the PpABI3B mRNA. The full-length coding sequence of
PpABI3B was amplified by RT-PCR and cloned into the pCR 2.1-TOPO vector
(Invitrogen, Carlsbad, CA). The DNA sequence was checked using M13 forward and
reverse primers with the Big Dye Terminator mix (Applied Biosystems, Foster City, CA).
32
PpABI3C was obtained by a BLAST search (tblastx) (Altschul et al., 1997) of the
P. patens JGI raw sequences in Physcobase (Nishiyama et al., 2003) using the
Arabidopsis ABI3 coding sequence from the start codon through the B2 domain (bases 1-
1401) as the query. This search identified P. patens sequences with B1 and B2 domains
that did not match to either PpABI3A or PpABI3B. We assembled a contig from the
available genomic sequences (ASYA169080.bl, ASYA 384419.gl, AXOS216104.xl, and
AXOS216373.yl) and used this to design primers for 5’ and 3’ RACE based on predicted
amino acid conservation with the other PpABI3 proteins. One round of 3’RACE and two
rounds of 5’RACE were required to obtain the full cDNA. RNA for the RACE reactions
was extracted from protonema treated with 10µM ABA for two hours using the RNeasy
Kit (Qiagen, Valencia, CA) for use with the 5’ and 3’ RACE kit (Invitrogen, Carlsbad,
CA). The coding sequence of PpABI3C was amplified by RT-PCR and cloned into the
pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA). The DNA sequence was checked
using M13 forward and reverse primers with the Big Dye Terminator mix (Applied
Biosystems, Foster City, CA).
Amino acid alignments were created by the T-coffee program (Notredame et al.,
2000).
DNA constructs
The effector constructs of Ubi-PpABI3A, Ubi-PpABI3B, Ubi-PpABI3C, Ubi-VP1 and
Ubi-ABI3 were made as follows. The coding sequence of PpABI3A, PpABI3B,
PpABI3C, VP1 and ABI3 were amplified by PCR from the cDNAs, and subcloned into
pGEM-Teasy (Promega, Madison, WI) or pCR 2.1-TOPO vector (Invitrogen, Carlsbad,
33
CA). Accuracy of the amplified fragments was checked by sequencing, then each
fragment was placed downstream of the maize Ubi1 promoter (Christensen and Quail,
1996), followed by the terminator of nopalin synthetase gene. The GFP fusions were
generated by PCR to add GFP to the C-terminal end as a translational fusion in the
respective Ubi-ABI3/PpABI3A-nos vector. Em-GUS [pBM113KP, (Marcotte et al.,
1989)], Ubi-LUC [pAHC18, (Bruce et al., 1989)], Ubi-HvABI5 (Casaretto and Ho, 2003),
Ubi-GFP (Zentella et al., 2002), PpLea1-GUS (Kamisugi and Cuming, 2005) and 35S-
abi1-1 (Armstrong et al., 1995) were described previously.
Transient assays
In principle, DNA delivery to barley aleurone cells was performed as described
previously (Shen et al., 1993). Two µg of each reporter constructs (Em-GUS and Ubi-
LUC) and effector constructs were used to prepare tungsten particles for three shots.
Bombarded seeds were treated with or without 20µM ABA for 24 h at room temperature.
Transient transformation of P. patens by particle bombardment was carried out as
described (Bezanilla et al., 2003). We used 0.8 µg of each reporter construct (Em-
GUS/PpLea1-GUS and Ubi-LUC) and effector construct to prepare gold particles for four
shots. One-week old protonemal tissue was used for bombardment, and then incubated
on PpNH4 agar media with or without 10µM ABA for 48 h under the conditions
described above.
Four barley seeds or moss protonemal tissue were ground in extraction buffer
(Lanahan et al., 1992) and cell debris was spun down. GUS and luciferase activities were
measured as described (Lanahan et al., 1992). GUS activity was normalized by the
34
luciferase activity and represented as relative GUS activity (GUS/LUC) ± SE. All the
experiments consisted of four replicates.
Microscopy
Confocal microscopy was performed using barley aleurone tissue or protonemal tissue of
P. patens placed in water on a glass slide and covered with a cover slip. A Leica
Confocal System TCS SP2 (Mannheim, Germany) was used for imaging GFP signal with
an argon laser.
Light microscopy of Arabidopsis seeds and siliques was performed using an
Olympus SZX12 system (Melville, NY).
Arabidopsis Complementation
The vector pCHF3 (Jarvis et al., 1998), a gift from Michael Neff, which contains the
CaMV 35S promoter and pea RBCS terminator, was used to express PpABI3A or ABI3
in Arabidopsis. The abi3-6 mutant plants were transformed by Agrobacterium
tumefaciens strain GV3101 using the floral dip technique (Clough and Bent, 1998).
Transformants were obtained by selection on MS plates containing kanamycin (50 mg/L).
35S-ABI3 and 35S-PpABI3A plants in the T3 generation, which were segregating 3:1 for
kanamycin resistance, failed to show any complementation of the abi3-6 phenotypes.
Therefore, we removed the CaMV 35S promoter from pCHF3 and replaced it by EcoRI
and BamHI digestion with the first 1752 bp of the Arabidopsis ABI3 promoter, which we
amplified by PCR from the genomic sequence of the At3g24650 locus. The primers used
to amplify the ABI3 promoter were 5’ GAATTCCGTGTACGTTTAGGTGGCATG-3
35
and 5’-GGATCCAGAAGACTCTCTCTTTCTTTC-3’. The PAtABI3-PpABI3A and the
PAtABI3-AtABI3 constructs were then transformed into the abi3-6 mutant as described
above. We obtained four independent PAtABI3-PpABI3A complementation lines and seven
of the PAtABI3-AtABI3 lines, which had a single T-DNA insertion as determined by
kanamycin segregation. Seed color was scored after the siliques had turned yellow,
approximately 18 days after flowering.
ABA germination assay
Seeds were harvested at 15 days after flowering from Columbia wildtype, abi3-6, PAtABI3-
PpABI3A/abi3-6 and PAtABI3-AtABI3/abi3-6 complementation lines and surface sterilized.
Seeds were plated on MS media containing 0, 0.1, 1, 10, or 100 µM ABA and cold
treated for 4 days at 4°C in the dark. The plates were then moved to 22°C with a 16-hour
light and 8-hour dark light regime. The germination rate was scored after 4 days using at
least 40 seeds for each assay condition. The assay was performed in triplicate.
RT-PCR analysis
RNA was extracted from 11-day-old siliques using RNAqueous kit with the Plant RNA
isolation aid (Ambion, Austin, TX). 500 ng of total RNA was first treated with DNaseI
(Amplification grade, Invitrogen, Carlsbad, CA) for 15 minutes at room temperature.
The enzyme was inactivated by heating at 65°C for 10 minutes in the presence of 2.5 mM
EDTA. The RNA was used for making cDNA by the ThermoScript RT-PCR system
(Invitrogen, Carlsbad, CA). The RNA was first pre-incubated with oligo(dT)20 and
dNTPs at 65°C for 5 minutes, then placed on ice. The cDNA Synthesis Mix was added
36
to the RNA and incubated at 55°C for 1 hour. The reaction was terminated by heating at
85°C for 5 minutes and then RNaseH was added and incubated at 37°C for 20 minutes to
remove the RNA template. PCR was carried out using 2 µL of the cDNA with 0.4 µL of
Taq polymerase (Invitrogen, Carlsbad, CA) in a 50 µL reaction. The primer sequences
are AtEm1 (5’-CGTCAAAGCAACTGAGCA-3’ and 5’-TCCATCGTACTGAGTCCT-
3’); AtEm6 (5’-TGGCGTCTCAACAAGAGAAG-3’ and 5’-
TCTCCGGTGCTAAGACCA-3’); Napin (5’-GCAATCACAACACCTAAGAGCT-3’
and 5’-CTGGATTGGAATGGTCCGTG-3’); CruciferinC (5’ -
AGCTCAGCAATCTCCTCGTT-3’and 5’-TTTCCGGCCAAATGGAACAC-3’); Rab18
(5’-ATCCAGCAGCAGTATGACGAGT-3’ and 5’-ACCGGGAAGCTTTTCTTGATC-
3’); Oleosin2 (5’-AGGTCAGTATGAAGGTGACGGTG-3 and 5’-
TGCTCAGGCACAGTTCTCCTT-3’); Ubiquitin10 (5’-
AAGATCCAGGACAAGGAGGTATTCCT-3’ and 5’-
CATAACAGAGACGAGATTTAGAAACC-3’). The cycling parameters for AtEm1 and
AtEm6 were 35 cycles of 94°C for 30 seconds, 52°C for 45 seconds and 72°C for 1
minute. The cycling parameters for Ubq10, Oleosin2, and Rab18 were 21 cycles of 94°C
for 30 seconds, 58°C for 45 seconds and 72°C for 1 minute. The cycling parameters for
Napin and CruciferinC were 28 cycles of 94°C for 30 seconds, 56°C for 45 seconds and
72°C for 1 minute. Ten µL of each PCR reaction was run out on a 1.5% agarose gel.
37
Yeast Two Hybrid
Plasmid construction
For constructs used in yeast two hybrid experiments, the entire coding sequence of
HvABI5, ABI3 and PpABI3A were amplified by PCR. The HvABI5 fragment was cloned
into the multi-cloning site of pGBTK (Clontech, Mountain View, CA) in frame with
GAL4 binding domain sequence (BD-HvABI5), and fragments of ABI3 and PpABI3A
were cloned into the multi-cloning site of pGAD424 (Clontech, Mountain View, CA) in
frame with GAL4 activation domain sequence (AD-ABI3 and AD-PpABI3A).
Yeast two hybrid assays
Media was prepared as described (Sambrook and Russell, 2001). Yeast two hybrid
assays were carried out as described before (James et al., 1996). In this system three
different reporter genes, GAL2-ADE2, GAL1-HIS3 and GAL7-LacZ are available. A host
strain PJ69-4A-a was used for BD constructs, whereas PJ69-4A-AD-a was used for AD
constructs. Mating was carried out by co-incubation of a strain harboring the BD
construct and a strain harboring the AD construct. The diploid strains were selected on
SD media without leucine and tryptophan (SD-LW), and used for analyses of the
interaction between the transcription factors. For the plate assay, SD-LW medium
without histidine and adenine (SD-LWHA) with 35mM 3-aminotriazole (3-AT) was used
for culture. For the quantification assay, GAL4-BD driven β-galactosidase gene
expression was analyzed using ONPG (O-nitrophenylgalactoside) as the substrate
according to the protocol described at
http://www.fhcrc.org/labs/gottschling/yeast/Bga37.html.
38
ACKNOWLEDGEMENTS
We thank Dr. Peter McCourt for the abi3-6 seeds, Dr. Andrew Cuming for the PpLea1-
GUS construct, and Dr. Tuan-hua David Ho and Dr. Jose Casaretto for providing DNA
constructs of Ubi-HvABI5, Ubi-GFP, and Ubi-LUC. We thank Jana Hakenjos, Julie
Thole and Paul Klueh for their technical assistance and Drs. Ho and Casaretto for their
help with the particle bombardment of barley aleurone cells. We thank Yoshinori Murai
and Takahiro Kawato for their technical assistance with the yeast-two-hybrid
experiments. Some of the sequence data were produced by the U.S. Department of
Energy Joint Genome Institute http://www.jgi.doe.gov/. This work was supported
by a JSPS Postdoctoral Fellowship for Research Abroad to Y.S. and by The Danforth
Foundation/Washington University to R.S.Q.
39
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45
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46
Table 1. AtABI3 regulated genes grouped by co-regulator for RT-PCR analysis.
* Not ABI5
Co-Regulator Gene Reference
ABI5 AtEm1
(At3g51810)
Finkelstein and Lynch, 2000
AtEm6
(At2g40170)
OPAQUE2-like bZIPs CruciferinC
(At4g28520)
Lara et al., 2003
Napin
(At4g27160)
Unknown* Oleosin2
(At5g40420)
Finkelstein and Lynch, 2000
Rab18
(At5g66400)
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