Journal of Experimental Botany, Vol. 53, No. 371, pp. 1219–1221, May 2002
Cloning and expression of an ABSCISIC ACID-INSENSITIVE 3 (ABI3)
gene homologue of yellow-cedar (Chamaecyparis nootkatensis)
Galina Lazarova, Ying Zeng and Allison R. Kermode1
Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6
Received 15 November 2001; Accepted 21 December 2001
Abstract a dormancy-breaking treatment) (Ren and Kermode, 1999)
were pooled together for extraction of total RNA.
A homologue of the ABI3 gene was isolated from the conifer
Poly(A)qRNA was puriﬁed from total RNA using the
species, Chamaecyparis nootkatensis. The deduced protein of
PolyATtract2 magnetic-bead system (Promega, Madison,
794 amino acids exhibited sequence similarity to other VP1uABI3
WI, USA) and the poly(A)qRNA was used for cDNA
proteins within four regions. Expression occurs exclusively in
library construction with the ZAP express cDNA synthesis
seeds, with no detectable mRNA in leaves and roots. Unlike the kit from Stratagene (La Jolla, CA, USA) using the primer
homologues of angiosperms, CnABI3 may be encoded by more 59-(GA)10ACTAGTCTCGAG(dT)18-39 for reverse transcrip-
than one gene. tion. PCR was used to clone a partial ABI3uVP1-like gene
fragment from this cDNA library. Based on the sequences of
Key words: Abscisic acid, ABI3, seed dormancy, yellow-cedar. highly conserved regions of ABI3uVP1 proteins from other
species, a degenerate primer was designed (59-GTNTGG-
AAYATGMGNTAY-39 wfwdx) and used for PCR together with
ABI3uVP1 proteins are members of a large group of transcrip- an anchor primer, 59-GAGAGAACTAGTCTCGAG-39 (rev).
tion factors which act as intermediaries in regulating abscisic Products were cloned into a pGEM-T Easy vector (Promega,
acid (ABA)-responsive genes during seed development. Lesions Madison, WI, USA) and sequence analysis identiﬁed a clone
in the genes affect the later stages of seed development and having high homology to other ABI3uVP1 genes. A 32P-labelled
have adverse effects on many important processes such as probe using this cDNA as a template was synthesized for cDNA
reserve deposition, dormancy imposition and the acquisition of library screening. In total, six positive clones were obtained from
a tolerance of seed tissues to desiccation. ABI3 does not act in library screening and pBK-CMV phagemids containing the
isolation to control gene expression central to seed maturation inserts were excised by the in vivo method from the ZAP express
programmes, but rather acts in concert with various other vectors. All six clones were sequenced and compared with
transcription factors such as LEC1, LEC2 and FUS3 (Harada, ABI3uVP1 genes from other species. One of the clones, B11
2001); these interactions prevent the precocious activation of (designated CnABI3) contained the entire coding region for an
genes associated with germination and growth (Nambara et al., ABI3uVP1-like protein, as well as 516 bp of 59-untranslated
2000). Homologues of VP1uABI3 genes have been isolated from sequence and 300 bp of a 39-ﬂanking region. Based on sequences
several angiosperm species, and recently from a woody of clone B11 cDNA and VP1uABI3 genes of other species,
angiosperm (Rohde et al., 1998). PCR primers, 59-ATGGACCAACATGAATTGCG-39 (fwd)
Following dispersal from the parent tree, seeds of yellow- and 59-CGAGTCCACCAAGTCTAGAA-39 (rev) were designed
cedar (Chamaecyparis nootkatensis D. Don Spach) are dormant and PCR ampliﬁcations were carried out using genomic DNA.
and require several months of moist chilling before they will A single band product was obtained and directly sequenced
germinate. The dormancy mechanism of this conifer species is for analysis of intron positions and their sizes.
complex and ABA has been implicated as a key regulator CnABI3 encodes an ABI3uVP1-like protein of 794 amino
(Schmitz et al., 2002). The isolation of the ABI3 gene homologue acids with a predicted molecular mass of 88 kDa and isoelectric
from yellow-cedar (CnABI3) is reported here and its expression point of 5.08. The protein from yellow-cedar is the largest so far
at the mRNA level characterized. of the ABI3uVP1 family. Like other homologues, the CnABI3
Yellow-cedar seeds (clone 108) at mid-maturation were gene contains six exons and ﬁve introns, the latter having sizes
collected from the Mount Newton Seed Orchard (Saanichton, of 105, 113, 110, approximately 1000, and 142 bp, respectively.
BC, Canada). Embryos and megagametophytes were excised All introns of the CnABI3 gene contain splice sites consistent
from the developing seeds, ﬂash frozen in liquid nitrogen and with the consensus sequence 59GT . . . AG39. Comparison of
stored at –80 8C prior to use. Mature seeds of seedlot 30156 the deduced amino acid sequence of CnABI3 to other ABI3uVP1
were obtained from the Tree Seed Centre in Surrey, BC, proteins by multiple-alignment (Fig. 1) indicates that the
Canada. Analyses were also conducted on leaves and roots of homologue of yellow-cedar has all four regions that are typically
15–20 cm seedlings; these were immediately ground to a ﬁne conserved. These include the three highly conserved basic
powder in liquid nitrogen and stored at À80 8C. regions: B1 (aa 261–327), B2 (aa 459–490) and B3 (aa 533–651).
Yellow-cedar seeds at different stages (mid-maturation, The core of B2, RKKR, is cited by several authors as a putative
maturity and mature seed subjected to different durations of nuclear targeting motif and is invariable in all angiosperm
To whom correspondence should be addressed. Fax: q1 604 291 3496. E-mail: email@example.com
ß Society for Experimental Biology 2002
1220 Lazarova et al.
Fig. 1. Alignment of the deduced amino acid sequence of Chamaecyparis nootkatensis ABI3 protein (CnABI3) with that of other VP1uABI3 proteins.
VP1uABI3 proteins (CnABI3 waj131113x; PtABI3, Populus trichocarpa ABI3 waj003165x; PvAlf, Phaseolus vulgaris ABI3-like factor wu28645x; ABI3,
Arabidopsis thaliana ABI3 wx68141x; VP1, Zea mays VP1 wm60214x) were aligned and compared with the computer programs Clustal W and Boxshade.
The four darkly shaded boxes correspond to the previously described regions of highest sequence homology: the acidic A1 region and the three basic
regions, B1, B2 and B3. The putative nuclear targeting signals (RKNR located in the B2 region, and RKRK of CnABI3, located in the B1 region) are
indicated by the solid dots above the sequence.
Cloning and expression of an ABSCISIC ACID-INSENSITIVE 3 gene 1221
homologues characterized so far. In the yellow-cedar protein, two bands only, instead of the three detected (Fig. 2C, predicted
this sequence of the B2 region is RKNR. However, a putative numbers of bands on Southern blot). Similarly, EcoRI digestion
nuclear targeting motif (RKRK) is found in CnABI3 at posi- should yield a single band (due to a site at 687 bp in B11). There
tion 325–328, within the B1 region. In the B3 region, the is no HindIII site, therefore only one band is expected instead
sequence identity is over 90% within a stretch of 119 amino of the two (or possibly three) that are evident. Figure 2C also
acids. There is a substitution of glycine (which is conserved in all shows predicted numbers of bands that would be consistent with
other homologues) with valine at position 593. The N-terminal a gene copy number of two and three copies. Sequence analysis
region contains the acidic domain (A1) that plays an important revealed no BamHI, EcoRI or HindIII sites within the introns
role in the function of these proteins as transcription activators; of the CnABI3 gene; the numbers of fragments on the Southern
this region shares a much lower degree of homology, which is blot are inconsistent with a single copy or multiple copies
typical of other ABI3uVP1 proteins (Fig. 1). (2 or 3 copies) of a single gene. Although inconclusive, the
Southern blot hybridization conducted under high stringency results are indicative of a gene family for CnABI3 in this
conditions exhibited multiple bands of varying intensities coniferous species, or perhaps some other gene(s) with high
for DNA samples digested with BamHI, EcoRI or HindIII homology to CnABI3 (e.g. FUSCA3; Leurssen et al., 1998). This
(Fig. 2A). Two BamHI sites occur at 1222 and 1236 bp in B11 is in contrast to the homologues of angiosperms, in which
(Fig. 2B); if CnABI3 is encoded by a single gene, there should be ABI3uVP1 is encoded by a single gene. A possible exception
may be the homologue of Phaseolus vulgaris, in which an addi-
tional band is revealed on Southern blots, although only under
lower stringency conditions (Bobb et al., 1995).
Under normal growth conditions, expression of the CnABI3
gene occurs exclusively in seeds of yellow-cedar, with no
detectable mRNA in leaves and roots of young seedlings
(Fig. 3). Expression in seeds is not conﬁned to mid-maturation
(Fig. 3), but is also detected in both the embryo and mega-
gametophyte of the mature (dormant) seed (data not shown).
In Avena fatua, expression of the VP1 gene is correlated with
the degree of embryo dormancy and may be important for
maintaining ABA-controlled metabolism in the imbibed seed
(Jones et al., 1997). The role of CnABI3 in maintaining
dormancy of yellow-cedar seeds is presently being examined.
This research was supported by a Forest Renewal BC grant and a
Natural Sciences and Engineering Research Council of Canada Strategic
grant awarded to ARK.
Fig. 2. (A) Southern blot hybridization of yellow-cedar genomic DNA. Bobb AJ, Eiben HG, Bustos MM. 1995. PvAlf, an embryo-speciﬁc acidic
The 32P-labelled cDNA probe was prepared from clone B11 by EcoRI transcriptional activator enhances gene expression from phaseolin and
and XbaI digestion which released a 2127 bp fragment containing 89% phytohemagglutinin promoters. The Plant Journal 8, 331–343.
of the CnABI3 gene coding region. Genomic DNA was digested with Harada JJ. 2001. Role of Arabidopsis LEAFY COTYLEDON genes in seed
BamHI (B), EcoRI (E) and HindIII (H). (B) Probe generated from B11 development. Journal of Plant Physiology 158, 405–409.
that was used for the Southern blot hybridization showing location of Jones HD, Peters NCB, Holdsworth MJ. 1997. Genotype and environment interact
BamHI and EcoRI sites. (C) Predicted numbers of bands on Southern to control dormancy and differential expression of the Viviparous 1 homologue
blot, if the CnABI3 gene of yellow-cedar is present as a single copy or as in embryos of Avena fatua. The Plant Journal 12, 911–920.
multiple copies of a single gene. No BamHI, EcoRI and HindIII sites Leurssen H, Kirik V, Herrmann P, Misera S. 1998. FUSCA3 encodes a protein
are present in the introns of the CnABI3 gene. with a conserved VP1uABI3-like B3 domain which is of functional importance
for the regulation of seed maturation in Arabidopsis thaliana. The Plant Journal
Nambara E, Hayama R, Tsuchiya Y, Nishimura M, Kawaide H, Kamiya Y, Naito S.
2000. The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase
transition from late embryo development to germination. Developmental
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Ren C, Kermode AR. 1999. Analyses to determine the role of the mega-
gametophyte and other seed tissues in dormancy maintenance of yellow cedar
(Chamaecyparis nootkatensis) seeds: morphological, cellular and physiological
changes following moist chilling and during germination. Journal of
Experimental Botany 50, 1403–1419.
Rohde A, Ardiles-Diaz W, Van Montagu M, Boerjan W. 1998. Isolation
Fig. 3. Northern blot analysis of total RNA isolated from yellow-cedar and expression analysis of an ABSCISIC ACID-INSENSITIVE 3 (ABI3)
seeds (at mid-maturation), and leaves and roots of young seedlings. homologue from Populus trichocarpa. Journal of Experimental Botany
Ten mg of RNA was loaded on each lane and the 32P-labelled probe was 49, 1059–1060.
the same as that in the Southern blot hybridization. Ribosomal RNA Schmitz N, Abrams SR, Kermode AR. 2002. Changes in ABA turnover and
(18S) was also probed after stripping the same membrane to verify equal sensitivity during termination of dormancy of yellow-cedar (Chamaecyparis
amounts of RNA loading. nootkatensis) seeds. Journal of Experimental Botany 53, 89–101.