Identification and functional analysis of two alternatively spliced transcripts of ABSCISIC ACID INSENSITIVE3 (ABI3) in linseed flax (Linum usitatissimum L.)
Identification and functional analysis of two alternatively spliced transcripts of ABSCISIC ACID INSENSITIVE3 (ABI3) in linseed flax (Linum usitatissimum L.)
Yanyan Wang 0 1 2
Tianbao Zhang 0 1 2
Xiaxia Song 0 1 2
Jianping Zhang 0 2
Zhanhai Dang 0 2
Xinwu Pei 0 1 2
Yan Long 0 1 2
☯ These authors contributed equally to this work. 0 2
0 Funding: The work was supported by the National Natural Science Foundation of China No.31371657) and the Agricultural Science and Technology Innovation Program in China. The funders had no role in study design , data collection and analysis
1 MOA Key Laboratory on Safety Assessment (Molecular) of Agri-GMO, Institute of Biotechnology, Chinese Academy of Agricultural Sciences , Beijing , China , 2 Crop Institute, Gansu Academy of Agricultural Sciences , Lanzhou , China
2 Editor: Keqiang Wu, National Taiwan University , TAIWAN
Alternative splicing is a popular phenomenon in different types of plants. It can produce alternative spliced transcripts that encode proteins with altered functions. Previous studies have shown that one transcription factor, ABSCISIC ACID INSENSITIVE3 (ABI3), which encodes an important component in abscisic acid (ABA) signaling, is subjected to alternative splicing in both mono- and dicotyledons. In the current study, we identified two homologs of ABI3 in the genome of linseed flax. We screened two alternatively spliced flax LuABI3 transcripts, LuABI3-2 and LuABI3-3, and one normal flax LuABI3 transcript, LuABI3-1. Sequence analysis revealed that one of the alternatively spliced transcripts, LuABI3-3, retained a 6 bp intron. RNA accumulation analysis showed that all three transcripts were expressed during seed development, while subcellular localization and transgene experiments showed that LuABI3-3 had no biological function. The two normal transcripts, LuABI3-1 and LuABI3-2, are the important functional isoforms in flax and play significant roles in the ABA regulatory pathway during seed development, germination, and maturation.
Abscisic acid (ABA) is an important hormone that regulates many aspects of plant growth and
development such as the synthesis of seed storage proteins and fatty acids[
], the promotion
of drought tolerance and dormancy in seeds, the suppression of seed germination, and the
transition from vegetative growth to reproductive growth [
]. Previous studies have shown
that exogenous ABA could suppress germination of immature embryos [4±6]. Many maize
mutants for ABA synthesis, including vp2, vp5, vp7, vp8, and vp9, have demonstrated that ABA
could suppress seed germination . In Arabidopsis thaliana and tobacco, ABA-synthesis
decision to publish, or preparation of the
mutants lost their dormancy characteristics, indicating that endogenous ABA could suppress
seed germination and promote seed dormancy [
In the ABA signaling pathway, four key regulatory genes, including LEAFY COTYLEDON1
(LEC1), LEC2, FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE3 (ABI3) [
partially functionally redundant in the regulation of seed maturation. Of these, ABI3 is highly
conserved among different plant species, including Arabidopsis, maize, rice, wheat, tomato,
and oat . In Arabidopsis lines over-expressing ABI3, expression of the seed-specific At2S3,
AtEM1, AtCRC, AtEM6 and AtSOM genes was induced by exogenous ABA, with expression in
the roots and seeds found to be more sensitive to ABA treatment[
ABI3 is a transcription factor belonging to B3 domain-containing gene family. Previous
studies have shown that ABI3 has four domains, A1, B1, B2, and B3, that are conserved in
different plants. The A1 domain is an acidic transcriptional activator; the B1 domain is a region
needed for interaction with specific bZIP transcription factors such as ABI5, bZIP10, bZIP25,
]; the B2 domain can bind to either ABA response elements or the G-box
element (CACGTG) and so could be involved in both transactivation or nuclear localization [
and the B3 domain has been shown to bind to the RY motif (CATGCA) in vitro[
Alternative splicing is a process that generates multiple proteins from single genes. For
eukaryotes, this process is not only an important post-transcriptional regulatory system, it is
also an essential mechanism for increasing transcriptome plasticity and proteome diversity. In
Arabidopsis, approximately 42% of transcripts are alternatively spliced, with the resulting
spliced transcripts encoding functionally different or non-functional proteins[
example, one positive regulator of the ABA signaling pathway, SDIR1, has three alternative mRNA
isoforms, SDIR1-822, SDIR1-691, and SDIR1-666, with the three isoforms having different
RNA accumulation levels[
]. Previous studies have found that ABI3 is alternatively
spliced in different crops such as Arabidopsis[
], tomato [
]], rice [
3], and pea [
]4]. In tomato, two transcripts, SlABI3-F and SlABI3-T, were found in the
genome. SlABI3-F encoded a full-length amino acid, while SlABI3-T encoded a truncated
protein that lacked 30 amino acids. These two transcripts accumulated in the developing seeds
and were differentially expressed at different seed development stages. This suggested that the
alternative splicing resulting in these two transcripts was developmentally regulated. In wheat,
McKibbin et al. (2002) found that early seed germination before harvesting was caused by the
incorrect splicing of one alternatively spliced transcript, vp1 [
]. Furthermore, many
truncated OsVP1 transcripts were found in the rice genome in plants with the same phenotype
as the maize vp1 mutant [
]. In the dicotyledon Pisum sativum, many alternatively spliced
ABI3 transcripts, PsABI3-1–PsABI3-7, were discovered in the genome, with sequence analysis
showing that full-length PsABI3-1 included the basic domains B1 and B3 and was expressed
only in seeds [
Flax (Linum usitatissimum L.) is an economically significant self-pollinated crop in which
the stem fiber and seed oil can both be used commercially. The seed oil and protein content
are important for linseed flax; seed germination-related traits are, therefore, important in this
species. In this study, ABI3 was identified in flax, with a total of three transcripts, LuABI3-1–3
found in the genome. Sequence analysis revealed that one of the transcripts, LuABI3-3, was
alternatively spliced and retained a 6 bp intron. RNA accumulation analysis showed that all
three transcripts were expressed during seed development, while subcellular localization and
transgenic plant experiments showed that LuABI3-3 had no biological function. The two
normal transcripts, LuABI3-1 and LuABI3-2, were the predominant isoforms in flax and played
significant roles in the ABA regulatory pathway during seed development, germination, and
2 / 12
Materials and methods
Plants of the linseed flax cultivar Zhangya No.2 were grown in a greenhouse(24ÊC, 16h light/
8h dark). The leaves of seedlings were collected for DNA extraction. When the plants flowered,
siliques were collected 10, 20, 30, and 40 d after pollination (DAP); roots, stems, and leaves
were also harvested for RNA extraction. The Arabidopsis ecotype Col-0 was used for gene
transformation experiments. Nicotiana tabacum was planted for subcellular localization
RNA isolation and cDNA synthesis
The coding sequences (CDS) of the ABI3 transcripts were isolated from linseed flax cv. Zhangya
No.2. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) and DNase treated
(New England Biolabs) before approximately 2 μg of RNA was reverse transcribed with the
oligo-dT primers to obtain first strand cDNA using a cDNA synthesis kit (Applied Biosystems).
The sequence of Arabidopsis ABI3 (At3g24650) from the NCBI database was used as the
query to blast the flax genome sequence (https://phytozome.jgi.doe.gov/pz/portal.html). Two
homologs of ABI3, Lus10022820 and Lus10011888, were identified in the flax genome. Primers
ABI3F and ABI3R (Table 1), based on the two homologs, were used to isolate the full CDS of
flax ABI3. The polymerase chain reaction (PCR) products were cloned into the pEasy-T1
cloning vector (Transgene, China), sequenced, and analyzed using Vector NTI Advance 11
software. For each PCR product, five clones were sequenced by the Tsing Company, China.
DNA isolation and genomic sequence identification
As with the cDNA sequences described above, full-length genomic sequences were obtained
from linseed flax cv. Zhangya No.2. Total genomic DNA was extracted from seedling leaves
using the extraction method of Murray and Thompson (1980) [
]. To obtain genomic
sequences, PCR amplifications were performed using the total genomic DNA and the ABI3.g
F/ABI3.g R primers (Table 1). The primers were designed based on the transcript sequences.
After PCR amplification, the amplicons were cloned into the pEasy-T1 cloning vector,
sequenced, and analyzed using Vector NTI Advance 11 software.
3 / 12
LuABI3 expression analysis in flax tissues
Quantitative real-time PCR (qRT-PCR) analysis was used to analyze the RNA accumulation
patterns of the different LuABI3 transcripts. cDNA derived from siliques harvested 10, 20, 30,
and 40 DAP, and also from roots, stems, and leaves was used. Transcript-specific primers were
designed based on the transcript sequences (Table 1). The six bases ªTCTCAGº were added to
the 50 end of the LuABI3-3.1F primer to specifically amplify the LuABI3-3 fragment. Before
being used in qRT-PCR, the qRT-PCR primers were first checked using normal PCR
amplification and sequencing of the PCR products to confirm that the primers were
transcript-specific. qRT-PCR was conducted using the ABI7500 Fast Real-time PCR system (Applied
Biosystems). In our previous study, we used high-throughput sequencing technology to do
RNA-seq for four tissues in four developing stages and found Actin gene could stably express
in all the samples. So the LuActin (EU830342) gene used as a reference gene to normalize the
gene expression.The efficiencies of all target genes (LuABI3-1 to LuABI3-3) and Actin were
determined by using a validation method as Banik described [
]. The cDNA was serially
diluted (50, 25, 12.5, 6.25 and 3.125 ng) and each cDNA was amplified by real-time PCR with
the gene-specific primers using the SYBR green method. Each dilution was replicated three
times. The mean of three replications was used in determining the absolute value of the slope
of log(input amount) versus ΔCT. For each gene, three independent PCR reactions were
applied for each sample, and 2-44Ct method was used to calculate the gene relative expression
Subcellular localization of LuABI3
For subcellular analysis, the complete open reading frame of the three LuABI3 transcripts was
amplified using primers 35s-ABI3-GFP -InF (50-GACCGGTCCCGGGGGATCCATGGGAATCGA
CCCGTTT-30) and 35s-ABI3-GFP-InR (50-CCTTGCTCACCATGGATCCTCTGTATGTATCGA
GTTGTTGGA-30) that incorporated BamHI restriction sites at both ends of the product.
The amplified PCR fragments were cloned into the binary vector pCAMBIA1305-35s-GFP
to generate 35S::LuABI3-GFP fusion constructs. These constructs were transformed into
Agrobacterium tumefaciens strain EHA105 using the freeze±thaw method, and these transformed
Agrobacterium strains were infiltrated into the leaves of 4- to 6-w-old tobacco plants as
described by Sparkes et al. (2006). Microscopic analysis was performed 2±3 d post-infiltration
using the confocal laser scanning microscope ZEISS LSM 800 system.
Vector construction and gene transformation
To develop LuABI3 overexpression constructs, the CDSs of the LuABI3-1–3 transcripts were
cloned into the pBinGlyRed3 vector. The plasmids were double digested with the restriction
endonuclease EcoRI and XmaI and the framework was then ligated with the specific transcript
fragment so that LuABI3-1–3 expression was under the control of the CaMV 35S promoter.
The constructs were transformed to Agrobacterium strain EHA105 using the freeze±thaw
method. Arabidopsis Col-0 plants were then transformed using the floral dip method [
with untransformed Arabidopsis plants used as wild-type (WT) controls. Transgenic plants
were selected on MS medium supplemented with kanamycin.
Phenotypic screening and RNA accumulation analysis of transgenic plants
For phenotypic screening, approximately 200 WT and T2 homozygous transgenic seeds were
sown on 1/2 MS medium plates containing 2% sucrose and different concentrations (0, 0.3,
0.5, 1.0, 2.0, and 3.0 μM) of ABA. Three replicates were used for each line. All plates were kept
4 / 12
in a greenhouse under standard conditions (24ÊC day/18ÊC night; 16 h light/8 h dark). Plant
phenotypes were observed after 16 d growth. In addition to the phenotype screening, whole
tissue of transgenic Arabidopsis plants treated with 2 μM ABA was harvested for RNA
isolation. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) as previously
described. The expression of the seed germination-related genes AtEM1, AtEM6, and AtSOM
was analyzed in the transgenic Arabidopsis lines using primers listed in S1 Table and qRT-PCR
as previously described. The expression level was normalized to the Arabidopsis ACTIN
(At5g62690) control gene, and 2−ΔΔCt method was used to calculate the relative expression
values. Three biological replicates and two technical replicates were used for each gene.
Identification of LuABI3 coding and genomic sequences in linseed flax
PCR amplification, sequencing, and sequence analysis led to the identification of three LuABI3
CDSs. The CDSs were 2106 bp (accession number MF964255), 2124 bp (accession number
MF964256), and 2030 bp (accession number MF964257) long and were named LuABI3-1,
LuABI3-2, and LuABI3-3, respectively. Blast analysis showed that the LuABI3-1 sequence was
highly homologous to the known sequence Lus10022820, and LuABI3-2 and LuABI3-3 were
highly homologous to Lus10011888. Primers to amplify the genomic sequences of these
transcripts were designed according to the CDS. After PCR amplification and sequencing, two
corresponding genomic sequences were obtained with lengths of 2661 bp and 2681 bp,
respectively (accession number MF964253 and MF964254). Structural comparative analysis of the
genomic and transcript sequences showed that the LuABI3 gene had six exons and five introns;
both LuABI3-2 and LuABI3-3 had the corresponding genomic sequence were 2681 bp long.
Compared with LuABI3-1, LuABI3-2 and LuABI3-3 had three insertions in exon 1 and exon 6
(Fig 1). We also identified LuABI3-3 transcripts with a 6 bp insertion in intron 3 when
compared with LuABI3-2, indicating that this 6 bp intron sequence was not correctly spliced after
transcription in LuABI3-3. We concluded, therefore, that LuABI3-2 and LuABI3-3 were
alternative transcripts formed by alternative splicing.
Based on these three transcript sequences, deduced protein sequences were determined
(Fig 1). The deduced proteins for LuABI3-1±3 were 701, 707, and 709 aa, respectively. Domain
analysis showed that the three proteins had all four of the ABI3 conserved domains, including
A1, B1, B2, and B3 (Fig 1).
Analysis of LuABI3 transcripts expression
qRT-PCR analysis was used to analyze the RNA accumulation pattern of these three
transcripts. In order to ensure the similar amplification efficiencies for all the transcripts relative
to the reference gene, the validation experiments were performed. The results showed that
the absolute value of the slope versusΔCT was <0.1, indicating that the amplification
efficiencies of Actin and all the LuABI3 transcripts were similar. Then the expression values of
all the three transcripts were calculated. It was found that the three transcripts expressed in
all the tissues examined, including roots, stems, leaves, and siliques at different
developmental stages (Fig 2). LuABI3-1 was stably expressed in roots, shoots, leaves, and siliques at
different developmental stages. LuABI3-2 and LuABI3-3 were stably expressed in roots, shoots,
and leaves. In the developing siliques, LuABI3-2 expression increased as development
progressed, with the highest expression observed 40 DAP, where its expression was 66-fold
higher than that of LuABI3-1. LuABI3-3 expression increased from DAP10 to DAP30 and
was stable from DAP30 to DAP40.
5 / 12
Fig 1. Deducing proteins comparison for LuABI3. The PST, B1, B2 and B3 represented the four domains of the
Subcellular localization of LuABI3
Transient expression studies in tobacco showed that constructs containing LuABI3-1 and
LuABI3-2 produced a fluorescent signal. As expected, the two gene products could be detected
in the cell nucleus, while the gene product of LuABI3-3 had no signal (Fig 3). This indicates
that LuABI3-1 and LuABI3-2 had normal gene function in linseed flax, while LuABI3-3 was
Phenotypic analysis of transgenic Arabidopsis plants
Both WT and transgenic plants grew normally in the ABA-free medium, and the transgenic
plants grew normally in both 0.3 μM and 0.5 μM ABA medium (Fig 4). When the ABA
concentration increased to 1 μM, the transgenic plants overexpressing LuABI3-1 and LuABI3-2
grew better than the WT plants, while for the LuABI3-3 transgenic plants, the leaves were
wrinkled and the plants were weaker than the control plants. With an ABA concentration of
Fig 2. RNA accumulation analyses of three transcripts of LuABI3. (A) Expression pattern of different transcripts in
the root (R), shoot (S), and leaf (L) of zhangya2. (B) Expression pattern of different LuABI3 transcripts at different
6 / 12
Fig 3. The subcellular localization results of LuABI3. (A) The subcellular localization result of LuABI3-1. (B) The
subcellular localization result of LuABI3-2. (C) The subcellular localization result of LuABI3-3.
2 μM, the leaves of the overexpressing LuABI3-1 and LuABI3-2 resulting plants were green and
plant growth was inhibited.
Conversely, the germinated WT plants become yellow and then gradually died, as did the
transgenic plants overexpressing LuABI3-3. With an ABA concentration of 3 μM, parts of the
transgenic plants overexpressing LuABI3-1 and LuABI3-2 survived, while all the plants
overexpressing LuABI3-3 and the WT plants died. These results showed that the optimal
concentration for the survival of LuABI3 transgenic plants was 2 μM and that LuABI3-3 did not function
in the ABA signaling pathway.
Fig 4. Phenotypic analysis of transgenic T2 Arabidopsis plants and WT treated with different concentrations of
ABA. For each of the vector, seeds of three transgenic lines were selected for sowed in the petri dishes, also the seeds of
WT plants were sowed. The numbers with black characters represented the three lines. The characters WT mean the
wild type. (A) Phenotypic analysis of 35s:LuABI3-1 and WT plants. The three transgenic lines were line6, line19 and
line32. (B) Phenotypic analysis of 35s:LuABI3-2 and WT plants. The three transgenic lines were line3, line19 and
line33. (C) Phenotypic analysis of 35s:LuABI3-3 and WT plants. The three transgenic lines were line5, line9 and line19.
7 / 12
Fig 5. Expression analysis of three seed development-related genes in transgenic Arabidopsis plants. (A) Gene
expression analysis in Line19 (with the 35s:LuABI3-1 construct) and WT plants. For LuABI3-1 gene expression, the
expression value was higher in the transgenic plants than that in WT plants. Expression values of EM1, EM6 and SOM
in the ABA treated plants was higher than that in untreated plants. (B) Gene expression analysis in Line19 (with the
35s:LuABI3-2 construct) and WT plants. For LuABI3-2 gene expression, the expression value was higher in the
transgenic plants than that in WT plants.
Expression of seed germination-related genes in transgenic plants
As all the transgenic plants overexpressing LuABI3-3 died after ABA treatment, RNA
accumulation analysis was done using samples from transgenic plants overexpressing LuABI3-1 and
LuABI3-2 and the WT control plants (Fig 5). For the ABA-untreated plants, LuABI3-1 and
LuABI3-2 were expressed more highly in the transgenic plants overexpressing LuABI3-1 and
LuABI3 than in the WT plants, indicating that both transcripts were successfully integrated
into the Arabidopsis plants. In the plants treated with 2 μM ABA, expression of both LuABI3-1
and LuABI3-2 was lower than in the corresponding untreated plants. This indicated that
exogenous ABA negatively regulated expression of LuABI3. Then expression of three other seed
germination-related genes, AtEM1, AtEM6, and AtSOM were compared between the ABA
treated and untreated planted for both LuABI3-1 and LuABI3-2 vectors. The results showed
that the expression in treated plants were higher than that in the corresponding untreated
plants in overexpressing LuABI3-1 plants. And in the overexpressing LuABI3-2 plants, the
expression of the three genes didn't have significant difficience between treated and control
plants. This indicated that the expression of AtEM1, AtEM6, and AtSOM was positively
regulated by exogenous ABA, and the LuABI3-1 and LuABI3-2 may have sub-functions in
controlling the seed germination process.
As a diploid crop, flax underwent a whole-genome duplication event about 5±9 million years
ago, after its divergence from poplar and castor bean[
]. That means that there are many
duplicate genes in the flax genome. For example, Shivaraj et al. (2017) identified 51 aquaporin
genes in the flax genome, many of which were duplicate genes [
]. In the current study,
blast analysis of public flax genome data revealed the presence of two homologous LuABI3
genes, Lus10011888 and Lus10022820. Gene annotation showed that the CDS length of these
genes was 2130 bp and 1125 bp, respectively. Careful analysis revealed that the assembled
Lus10022820 CDS lacked the upstream sequence. Next, two actual LuABI3 genes were
identified in flax genome, with homology analysis showing that these two genes were associated with
Lus10011888 and Lus10022820, respectively. Structural analysis showed that, like the gene
structure of ABI3 in Arabidopsis, the two flax homologous genes had 6 exons and 5 introns,
including the A1, B1, B2 and B3 domains and the PST domain. These four domains have been
confirmed to be conserved in members of the ABI3/VP1 subfamily of the B3-domain protein
]. Comparison of the two genomic and three transcript sequences revealed that the
8 / 12
three transcripts could be divided into two groups, corresponding to the two genomic
Previous studies showed that alternative splicing commonly exists in ABI3 in different
crops. For example, two splicing isoforms, ABI3-α and ABI3-û, have been found Arabidopsis
], while SlABI3-F and SlABI3-T have been found in tomato [
]. We hypothesized,
therefore, that LuABI3-2 and LuABI3-3 were alternatively spliced transcripts, with LuABI3-3
retaining a 6 bp intron. The mechanism of splicing was, therefore, intron retention. Previous studies
have shown that there are four types of alternative splicing events in Arabidopsis: exon
skipping/inclusion, an alternative 5' splice site, an alternative 3' splice site and intron retention. Of
these, intron retention was the most frequent type, responsible for up to 40% of the
alternatively spliced transcript in the genome[
]. Many alternatively spliced transcripts in different
crops are non-functional, including SlABI3-T in tomato[
]. This lack of functionality is often
a result of non-functional protein isoforms, such as truncated proteins, formed from
alternatively spliced transcripts with frameshifts resulting in premature stop codons.
The RNA accumulation results obtained in this study showed that LuABI3-1–3 were
expressed in a range of different tissues, including roots, stems, leaves, and developing seeds. It
is important for choosing suitable reference gene for qRT-PCR analysis to detect the RNA
accumulation. In previous studies, different researchers selected different gene as reference
gene in flax. For example, Huis found that GADPH and 2 TEF genes could be used as reference
genes for evaluating RNA accumulation values based on different analysis methods [
Fernart et al., selected_c3168 and c10916 as reference genes based on their micro array analysis
]. In the current study, we selected Actin gene as a reference gene because it was found that
Actin could stably express in four tissues from four developing stages by using high-through
put sequencing technology. After selecting reference genes, the PCR effiencies were detected
first to confirm the consistent PCR amplification for target genes and reference gene. The
qRT-PCR experiments showed that three transcripts had different expression patterns in
developing seeds, with LuABI3-2 having much higher expression than LuABI3-1. This suggests
that these two transcripts may have different sub-functions during seed development. This is
consistent with different homologous genes having sub-functions in regulating one specific
biological process, particularly in polyploid plants. For example, there are, generally, six
homologous genes in the genome of a polyploid crop plant such as Brassica napus compared
with the model plant Arabidopsis because of the polyploidization process; Zou et al. (2012)
identified six BnFLC homologs in B. napus genome. RNA accumulation experiments using
these homologs showed that each had distinct expression patterns in different organs at
different developmental stages [
]. Although the alternatively spliced LuABI3-3 transcript was
expressed in different tissues, the subcellular localization and transgenic plant experiments
showed that this transcript had no biological function.
ABI3 is a core regulator of the ABA signaling pathway. It has been confirmed that
exogenous ABA can mediate ABI3 degradation via several regulators, allowing seeds to germinate
]. For example, Gao et al., (2014) identified two wheat AIP2 genes that could negatively
regulate ABI3 and ABI5 in the ABA signaling pathway and were found to have important roles
in seed germination ]. To dissect the biological function of LuABI3, the expression of
LuABI3-1 and LuABI3-2 was examined in transgenic plants overexpressing LuABI3-1 and
LuABI3-2 and WT control plants. Their expression was consistently lower in ABA-treated
plants than in plants without ABA treatment. This result was consistent with previous studies
and suggests that the flax ABI3 genes are sensitive to exogenous ABA and that their encoded
proteins may be degraded with ABA treatment. Meanwhile, three seed germination-related
genes AtEM1, AtEM6, and AtSOM, were more highly expressed in the transgenic plants with
ABA treatment than without ABA treatment. Overall, these results demonstrate that the ABI3
9 / 12
genes LuABI3-1 and LuABI3-2 in linseed flax function in regulating seed germination and
dormancy and that the expression of these genes is dependent on ABA and independent of these
two LuABI3 genes.
S1 Table. Primer sequences for expression analysis of seed development related genes.
We thank Emma Tacken, PhD from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/
ac) for editing the English test of a draft of this manuscript.
Data curation: Xiaxia Song.
Formal analysis: Tianbao Zhang.
Methodology: Yanyan Wang.
Project administration: Xinwu Pei, Yan Long.
Resources: Jianping Zhang, Zhanhai Dang.
Validation: Xiaxia Song.
Writing ± original draft: Yanyan Wang.
Writing ± review & editing: Zhanhai Dang, Yan Long.
10 / 12
11 / 12
1. Finkelstein RR , Tenbarge KM , Shumway JE , Crouch ML . Role of ABA in maturation of rapeseed embryos . Plant Physiol . 1985 ; 78 ( 3 ): 630 ± 6 . PMID: 16664296; PubMed Central PMCID : PMC1064789 .
2. Giraudat J , Hauge BM , Valon C , Smalle J , Parcy F , Goodman HM . Isolation of the Arabidopsis ABI3 gene by positional cloning . Plant Cell . 1992 ; 4 ( 10 ): 1251 ± 61 . https://doi.org/10.1105/tpc.4.10.1251 PMID: 1359917; PubMed Central PMCID : PMC160212 .
3. Rock CD . Pathways to abscisic acid-regulated gene expression . New Phytologist . 2000 ; 148 ( 3 ): 357 ± 96 . https://doi.org/10.1046/j.1469- 8137 . 2000 . 00769 . x WOS : 000166496000002 .
4. Hoecker U , Vasil IK , McCarty DR . Integrated control of seed maturation and germination programs by activator and repressor functions of Viviparous-1 of maize . Genes Dev . 1995 ; 9 ( 20 ): 2459 ± 69 . PMID: 7590227 .
5. Leung J , Giraudat J . Abscisic acid signal transduction . Annu Rev Plant Phys . 1998 ; 49 : 199 ± 222 . https://doi.org/10.1146/annurev. arplant.49.1.199 WOS:000074266700010. PMID: 15012233
6. Finkelstein RR , Gampala SSL , Rock CD . Abscisic acid signaling in seeds and seedlings . Plant Cell . 2002 ; 14 : S15±S45 . https://doi.org/10.1105/tpc.010441 WOS:000176187500004. PMID: 12045268
7. LeonKloosterziel KM , Gil MA , Ruijs GJ , Jacobsen SE , Olszewski NE , Schwartz SH , et al. Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci . Plant J . 1996 ; 10 ( 4 ): 655 ± 61 . https://doi.org/10.1046/j. 1365 - 313X . 1996 . 10040655 . x WOS : A1996VN08700008 . PMID: 8893542
8. Marin E , Nussaume L , Quesada A , Gonneau M , Sotta B , Hugueney P , et al. Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana . The EMBO journal . 1996 ; 15 ( 10 ): 2331 ± 42 . PMID: 8665840; PubMed Central PMCID : PMC450162 .
9. Kroj T , Savino G , Valon C , Giraudat J , Parcy F . Regulation of storage protein gene expression in Arabidopsis . Development . 2003 ; 130 ( 24 ): 6065 ± 73 . https://doi.org/10.1242/dev.00814 PMID: 14597573 .
10. To A , Valon C , Savino G , Guilleminot J , Devic M , Giraudat J , et al. A network of local and redundant gene regulation governs Arabidopsis seed maturation . Plant Cell . 2006 ; 18 ( 7 ): 1642 ± 51 . https://doi.org/ 10.1105/tpc.105.039925 WOS:000238960500009. PMID: 16731585
11. Zhang XR , Garreton V , Chua NH . The AIP2 E3 ligase acts as a novel negative regulator of ABA signaling by promoting ABI3 degradation . Gene Dev . 2005 ; 19 ( 13 ): 1532 ± 43 . https://doi.org/10.1101/gad. 1318705 WOS:000230334600004. PMID: 15998807
12. Park J , Lee N , Kim W , Lim S , Choi G. ABI3 and PIL5 collaboratively activate the expression of SOMNUS by directly binding to its promoter in imbibed Arabidopsis seeds . Plant Cell . 2011 ; 23 ( 4 ): 1404 ± 15 . https://doi.org/10.1105/tpc.110.080721 PMID: 21467583; PubMed Central PMCID : PMC3101561 .
13. McCarty DR , Hattori T , Carson CB , Vasil V , Lazar M , Vasil IK . The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator . Cell . 1991 ; 66 ( 5 ): 895 ± 905 . PMID: 1889090 .
14. Ezcurra I , Wycliffe P , Nehlin L , Ellerstrom M , Rask L . Transactivation of the Brassica napus napin promoter by ABI3 requires interaction of the conserved B2 and B3 domains of ABI3 with different cis-elements: B2 mediates activation through an ABRE, whereas B3 interacts with an RY/G-box . Plant J . 2000 ; 24 ( 1 ): 57 ± 66 . https://doi.org/10.1046/j. 1365 - 313x . 2000 . 00857 . x WOS : 000089935800006 . PMID: 11029704
15. Suzuki M , Kao CY , McCarty DR . The conserved B3 domain of VIVIPAROUS1 has a cooperative DNA binding activity . Plant Cell . 1997 ; 9 ( 5 ): 799 ± 807 . WOS:A1997WZ63100012. https://doi.org/10.1105/tpc. 9.5.799 PMID: 9165754
16. Filichkin SA , Priest HD , Givan SA , Shen RK , Bryant DW , Fox SE , et al. Genome-wide mapping of alternative splicing in Arabidopsis thaliana . Genome Res . 2010 ; 20 ( 1 ): 45 ± 58 . https://doi.org/10.1101/gr. 093302.109 WOS:000273249500006. PMID: 19858364
17. Xiao LW , Tang X , Q. , LiXia Yu Y Y. L. , Yan B . Alternative splicing of SDIR1 gene in Arabidopsis thaliana (Cruciferae) . Acta Botanica Yunnanica 2010 ; 32 ( 2 ): 141 ± 6 .
18. Bies-Etheve N , da Silva Conceicao A , Giraudat J , Koornneef M , Leon-Kloosterziel K , Valon C , et al. Importance of the B2 domain of the Arabidopsis ABI3 protein for Em and 2S albumin gene regulation . Plant molecular biology . 1999 ; 40 ( 6 ): 1045 ± 54 . PMID: 10527428 .
19. Gao Y , Liu J , Zhang Z , Sun X , Zhang N , Fan J , et al. Functional characterization of two alternatively spliced transcripts of tomato ABSCISIC ACID INSENSITIVE3 (ABI3) gene . Plant molecular biology . 2013 ; 82 ( 1 ±2): 131 ± 45 . https://doi.org/10.1007/s11103-013-0044-1 PMID: 23504452 .
20. McKibbin RS , Wilkinson MD , Bailey PC , Flintham JE , Andrew LM , Lazzeri PA , et al. Transcripts of Vp-1 homeologues are misspliced in modern wheat and ancestral species . Proceedings of the National Academy of Sciences of the United States of America . 2002 ; 99 ( 15 ): 10203 ±8. https://doi.org/10.1073/ pnas.152318599 PMID: 12119408; PubMed Central PMCID : PMC126648 .
21. Fan J , Niu X , Wang Y , Ren G , Zhuo T , Yang Y , et al. Short, direct repeats (SDRs)-mediated post-transcriptional processing of a transcription factor gene OsVP1 in rice (Oryza sativa) . Journal of experimental botany . 2007 ; 58 ( 13 ): 3811 ±7. https://doi.org/10.1093/jxb/erm231 WOS: 000251506300027 . PMID: 18057047
22. Gagete AP , Riera M , Franco L , Rodrigo MI . Functional analysis of the isoforms of an ABI3-like factor of Pisum sativum generated by alternative splicing . Journal of experimental botany . 2009 ; 60 ( 6 ): 1703 ± 14 . https://doi.org/10.1093/jxb/erp038 WOS: 000265524400015 . PMID: 19261920
23. Murray MG , Thompson WF . Rapid isolation of high molecular weight plant DNA . Nucleic Acids Res . 1980 ; 8 ( 19 ): 4321 ± 5 . PMID: 7433111; PubMed Central PMCID : PMC324241 .
24. Banik M , Duguid S , Cloutier S. Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation . Genome . 2011 ; 54 ( 6 ): 471 ± 83 . https://doi.org/10.1139/g11-013 WOS:000291994900004. PMID: 21627464
25. Clough SJ , Bent AF . Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana . Plant J . 1998 ; 16 ( 6 ): 735 ± 43 . PMID: 10069079 .
26. Wang ZW , Hobson N , Galindo L , Zhu SL , Shi DH , McDill J , et al. The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads . Plant J . 2012 ; 72 ( 3 ): 461 ± 73 . https://doi. org/10.1111/j. 1365 - 313X . 2012 . 05093 . x WOS : 000310343200010 . PMID: 22757964
27. Shivaraj SM , Deshmukh RK , Rai R , Belanger R , Agrawal PK , Dash PK . Genome-wide identification, characterization, and expression profile of aquaporin gene family in flax (Linum usitatissimum) . Scientific reports . 2017 ; 7 : 46137 . https://doi.org/10.1038/srep46137 PMID: 28447607; PubMed Central PMCID : PMC5406838 .
28. Finkelstein RR , Gampala SS , Rock CD . Abscisic acid signaling in seeds and seedlings . Plant Cell . 2002 ; 14 Suppl: S15 ± 45 . https://doi.org/10.1105/tpc.010441 PMID: 12045268; PubMed Central PMCID : PMC151246 .
29. Sugliani M , Brambilla V , Clerkx EJ , Koornneef M , Soppe WJ . The conserved splicing factor SUA controls alternative splicing of the developmental regulator ABI3 in Arabidopsis . Plant Cell . 2010 ; 22 ( 6 ): 1936 ±46. https://doi.org/10.1105/tpc.110.074674 PMID: 20525852; PubMed Central PMCID : PMC2910958 .
30. Reddy AS , Marquez Y , Kalyna M , Barta A . Complexity of the alternative splicing landscape in plants . Plant Cell . 2013 ; 25 ( 10 ): 3657 ± 83 . https://doi.org/10.1105/tpc.113.117523 PMID: 24179125; PubMed Central PMCID : PMC3877793 .
31. Huis R , Hawkins S , Neutelings G . Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.) . BMC plant biology . 2010 ; 10 : 71 . https://doi.org/10.1186/ 1471 -2229-10-71 PMID: 20403198; PubMed Central PMCID : PMC3095345 .
32. Fenart S , Ndong Y-PA , Duarte J , Rivière N , Wilmer J , van Wuytswinkel O , et al. Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray . BMC genomics . 2010 ; 11 : 592 ±. https://doi.org/10.1186/ 1471 -2164-11- 592 PMC3091737. PMID: 20964859
33. Zou X , Suppanz I , Raman H , Hou J , Wang J , Long Y , et al. Comparative analysis of FLC homologues in Brassicaceae provides insight into their role in the evolution of oilseed rape . PloS one . 2012 ; 7 ( 9 ): e45751. https://doi.org/10.1371/journal.pone.0045751 PMID: 23029223; PubMed Central PMCID : PMC3459951 .
34. Gao DY , Xu ZS , He Y , Sun YW , Ma YZ , Xia LQ . Functional analyses of an E3 ligase gene AIP2 from wheat in Arabidopsis revealed its roles in seed germination and pre-harvest sprouting . Journal of integrative plant biology . 2014 ; 56 ( 5 ): 480 ± 91 . https://doi.org/10.1111/jipb.12135 PMID: 24279988 .