The Low-Oxygen-Induced NAC Domain Transcription
Factor ANAC102 Affects Viability of Arabidopsis Seeds
following Low-Oxygen Treatment1[W][OA]
Jed A. Christianson, Iain W. Wilson*, Danny J. Llewellyn, and Elizabeth S. Dennis
CSIRO Plant Industry, Canberra, Australian Capital Territory 2601, Australia
Low-oxygen stress imposed by field waterlogging is a serious impediment to plant germination and growth. Plants respond to
waterlogging with a complex set of physiological responses regulated at the transcriptional, cellular, and tissue levels. The
Arabidopsis (Arabidopsis thaliana) NAC domain-containing gene ANAC102 was shown to be induced under 0.1% oxygen within 30
min in both roots and shoots as well as in 0.1% oxygen-treated germinating seeds. Overexpression of ANAC102 altered the
expression of a number of genes, including many previously identified as being low-oxygen responsive. Decreasing ANAC102
expression had no effect on global gene transcription in plants but did alter expression patterns in low-oxygen-stressed seeds.
Increasing or decreasing the expression of ANAC102 did not affect adult plant survival of low-oxygen stress. Decreased ANAC102
expression significantly decreased germination efficiency following a 0.1% oxygen treatment, but increased expression had no
effect on germination. This protective role during germination appeared to be specific to low-oxygen stress, implicating ANAC102
as an important regulator of seed germination under flooding.
Transient waterlogging, which can impose lowoxygen stress on established plants, has been shown
to reduce yield in a number of crops, including cotton
(Gossypium hirsutum; Hodgson and Chan, 1982), wheat
(Triticum aestivum; Collaku and Harrison, 2002), barley
(Hordeum vulgare; Setter and Waters, 2003), maize (Zea
mays; Mason et al., 1987), and canola (Brassica napus;
Cannell and Belford, 1980). Even rice (Oryza sativa),
which is well adapted to growing partially underwater,
is adversely affected when the entire plant is submerged (Singh et al., 2001). As well as in growing
plants, waterlogging poses a significant problem to
seeds prior to emergence. Germination rates decline
dramatically as oxygen concentrations are reduced for
12 plant species, including Brassica vegetable species,
soybean (Glycine max), pea (Pisum sativum), wheat,
maize, and rice (Al-Ani et al., 1985). Soil-based experiments have also shown large impacts on seed viability
following 4 d of flooding stress in oat (Avena sativa),
triticale (x Triticosecale), wheat, and barley (Setter and
Waters, 2003). Declines in seed germination in a number of low-oxygen environments (1%215% oxygen)
1
This work was supported by CottTech, a research alliance
between the Commonwealth Scientific and Industrial Research
Organization, Cotton Seed Distributors, and the Cotton Research
and Development Corporation.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Iain W. Wilson ([email protected]).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.131912
1724
have also been observed in Brassica oleracea, a close
relative of Arabidopsis (Arabidopsis thaliana; FinchSavage et al., 2005).
The effects of low oxygen on seeds can vary between
plant species. For many species, a lack of oxygen
prevents germination. Some cereals, such as barley,
rely on limiting oxygen availability to embryos as a
mechanism for imposing and maintaining dormancy
(Benech-Arnold et al., 2006). In contrast, rice seeds are
able to germinate under full anoxia, and this capability
is dependent on ethanolic fermentation pathways
(Kato-Noguchi, 2001). However, very little is known
about the effects of low oxygen on seed viability in
Arabidopsis. Germination did not occur in wild-type
Arabidopsis seeds in an anoxic environment and was
severely reduced in those treated seeds following a
recovery period (Mattana et al., 2007). This impairment of germination was partially ameliorated by
overexpression of the rice transcription factor Mybleu
(Mattana et al., 2007). Arabidopsis germination has also
been found to be reduced significantly more in ALCOHOL DEHYDROGENASE1 (ADH1) mutants than in
wild-type plants following anoxic treatments (Jacobs
et al., 1988), which suggests a role for fermentative
metabolism in sustaining seeds under low oxygen.
Following perception of a lack of oxygen, changes in
gene transcripts, proteins, and metabolism rapidly
result. A set of about 20 anaerobically induced polypeptides, the majority of which were involved in glycolysis and fermentation, have been identified as part
of the low-oxygen response in a number of plant
species (Sachs et al., 1980; Kelley and Freeling, 1984a,
1984b; Dennis et al., 2000).
Functional approaches to studying the effects of low
oxygen have focused on adult plants and have involved
altering the expression of these enzymes of fermenta-
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ANAC102 Is Required for Seed Viability in Low Oxygen
tion and glycolysis. The genes SUCROSE SYNTHASE1
(SUS1) and SUS4, ADH1, and PYRUVATE DECARBOXYLASE1 (PDC1) have been shown to be vital in
Arabidopsis for tolerance to low oxygen, as knocking
out their function leads to a reduction in plant growth
or survival (Ellis et al., 1999; Rahman et al., 2001;
Ismond et al., 2003; Kursteiner et al., 2003; Bieniawska
et al., 2007). Overexpression of PDC, considered to be
rate limiting in ethanol fermentation, has been shown
to increase Arabidopsis survival (Ellis et al., 2000;
Ismond et al., 2003). Increased root survival was also
observed in LACTATE DEHYDROGENASE (LDH)overexpressing Arabidopsis lines (Dolferus et al.,
2008). Altering the expression of ALANINE AMINOTRANSFERASE, which controls the Ala fermentation
pathway, did not affect tolerance to low oxygen in
Arabidopsis (Dolferus et al., 2003; Miyashita et al.,
2007). While this candidate gene approach has yielded
some promising results, overexpression of transcription factors that can alter the expression of stress
response networks may have a greater impact. The
SUB1A ERF/AP2-type transcription factor in rice has
proven capable of increasing survival in fully submerged conditions by regulating the transcription of a
suite of genes associated with carbohydrate consumption, ethanolic fermentation, and cell expansion and by
leading to decreased growth, chlorophyll degradation,
and carbohydrate consumption (Fukao et al., 2006; Xu
et al., 2006).
Global gene expression studies in Arabidopsis have
revealed widespread and complex responses to low
oxygen that have typically found significant changes in
approximately 5% to 10% of all the genes assayed (Klok
et al., 2002; Liu et al., 2005; Loreti et al., 2005). Early
microarray experiments on low-oxygen responses in
Arabidopsis hairy root cultures (Klok et al., 2002)
identified known and putative transcription factors
whose expression was induced early in low-oxygen
stress, including the NAC domain-containing gene
ARABIDOPSIS NAC DOMAIN-CONTAINING PROTEIN102 (ANAC102; At5g63790).
NAC domain genes are a plant-specific class of
transcription factors with functions in development
and stress responses (for review, see Olsen et al., 2005).
Arabidopsis contains 105 genes with NAC domains,
which can be divided into two major groups and
further partitioned into 18 subgroups (Ooka et al.,
2003). ANAC transcription factors are characterized
by an N-terminal DNA-binding NAC domain composed of five subelements and a variable C-terminal
transcriptional activator region (Ooka et al., 2003). The
NAC domain was first identified and characterized in
the petunia (Petunia hybrida) NO APICAL MERISTEM,
ATAF1, ATAF2, and CUP-SHAPED COTYLEDON
genes (Souer et al., 1996; Aida et al., 1997). Numerous
members of the ANAC gene family have been shown to
be involved in response to stresses: ANAC019,
ANAC055, and ANAC072 are all up-regulated in response to drought, high salinity, and abscisic acid
(ABA), and overexpression of any one of ANAC019,
ANC055, and ANAC072 was shown to increase
drought tolerance in Arabidopsis (Tran et al., 2004). In
addition to modulating lateral root formation in Arabidopsis, AtNAC2 is also salt responsive (He et al.,
2005). The NTL8 gene has been shown to influence
flowering time under salt-stress conditions (Kim et al.,
2007). ANAC102 belongs to the ATAF subfamily of
NAC domain genes. Members of the ATAF subfamily,
ATAF1 and ATAF2, negatively regulate responses to
drought and wounding, respectively (Delessert et al.,
2005; Lu et al., 2007).
Here, we investigated the role of ANAC102 in plant
responses to low oxygen and provide evidence for a
role for this gene in transcriptional regulation during
low-oxygen response and as an important positive
regulator of seed viability under low-oxygen conditions.
RESULTS
ANAC102 Is Up-Regulated in Shoots, Roots, and
Imbibed Seeds in Response to 0.1% Oxygen
ANAC102 is significantly up-regulated at early time
points in hairy root cultures exposed to oxygen stress
(Klok et al., 2002). To confirm that ANAC102 is induced
in whole plants, 3-week-old Arabidopsis plants were
subjected to 0.1% oxygen for 0, 0.5, 2, 4, 8, and 24 h
(Fig. 1A). Both root and shoot tissues showed a similar
response, with increased expression detected at 0.5 h
and remaining high for at least 24 h (Fig. 1A). There
was no substantial difference between root- and shootspecific expression in untreated plants (1.2-fold). Lowoxygen induction of gene expression also occurred in
the presence of 10 mM cycloheximide (17.0-fold; data
not shown), demonstrating that no new protein synthesis is required for ANAC102 induction.
Several classical hypoxia-induced genes, such as
ADH, LDH, and AlaAT, exhibit increased expression in
germinating seeds following exposure to low oxygen
levels (Ricoult et al., 2005). Imbibed and stratified
seeds, both unstressed and subjected to 0.1% oxygen in
the light for a period of 6 d (no germination occurs
while the seeds are in 0.1% oxygen), were assayed for
ANAC102 and ADH1 expression. Both ANAC102 and
ADH1 expression in low-oxygen-stressed seeds was
increased over unstressed seeds (2.5-fold and 11.9fold, respectively), but the expression level of
ANAC102 and ADH1 in unstressed seeds was lower
than in unstressed adult tissue (3.2-fold and 2-fold
lower expression in seeds than in roots; Fig. 1B).
ANAC102 Is Expressed in Both Roots and Early
Rosettes, But Is Concentrated in the Root Cortex and
Root Caps
To characterize the spatial and developmental patterns of ANAC102 expression in Arabidopsis, lines
transformed with an ANAC102 promoterTGUS fusion
were generated (Fig. 2). GUS staining was clearly
visible in parts of the root system. In the aerial portion
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Christianson et al.
tional efficiency under low-oxygen conditions (BaileySerres and Dawe, 1996). As the ANAC102 promoterT
GUS construct used here does not contain the ANAC102
untranslated region sequences, it may be that the
GUS mRNA produced under low oxygen is translated
at decreased efficiency, resulting in lower amounts
of protein formed despite having higher levels of
transcript.
Arabidopsis Lines with Altered ANAC102 Levels Do
Not Show Any Gross Phenotypic Differences from the
Wild Type
Figure 1. Expression of ANAC102 in response to low oxygen. A, Time
course of ANAC102 expression subjected to 0.1% oxygen. Plants of
ecotype Col-0 in liquid MS medium were placed in chambers
containing 0.1% oxygen for 0.5, 2, 4, 8, or 24 h. Expression changes
were monitored in both root (black triangles) and shoot (white triangles)
tissues. Data are expression at different times relative to the 0-h time
point (6SE; n 5 9). B, Expression of ANAC102 and ADH1 in imbibed
seeds under ambient and 0.1% oxygen (6 d of exposure) relative to
3-week-old root tissue in ambient oxygen (6SE; n 5 9).
of the plant, faint staining was detected in older sepals
and leaves. The public Arabidopsis gene expression
database Genevestigator (Zimmermann et al., 2004)
largely corroborated the expression patterns observed
in the ANAC102 promoterTGUS fusion lines, indicating high expression in the lateral root cap, stele,
epidermal atrichoblasts, endodermis, and cortex. Public expression data also indicated that ANAC102 is
relatively highly expressed in the stem and sepal.
Sepal expression was observed in the ANAC102
promoterTGUS lines (Fig. 2D), but stem expression
may have been too low or diffuse to be observed. GUS
expression was not seen in imbibed seeds, but expression was visible in the radicle of seedlings at 2 d after
emergence (Fig. 2E). No induction of GUS expression
could be detected following treatment with 0.1% oxygen for 4 h, and the GUS signal appeared to be weaker
in the treated plants (Fig. 2, G and H). In contrast to the
visible GUS staining, quantitative real-time (QRT)PCR demonstrated that transcript levels of both the
native ANAC102 gene and the ANAC102 promoterT
GUS fusion gene were increased in these plants following a 4-h 0.1% oxygen treatment (Fig. 2I). It has
been observed that maize ADH1 5# and 3# untranslated regions are important for maintaining transla-
Two independent Columbia (Col-0) lines carrying
insertions in the second exon of the ANAC102 gene that
would be predicted to eliminate protein function were
obtained and designated KO-1 (SALK_030702) and
KO-2 (SALK_094437). ANAC102 mRNA expression in
these two lines was also found by both QRT-PCR and
subsequent microarray experiments to be between 3.5and 15-fold lower in KO-1 and approximately 15-fold
lower in KO-2 than in the wild type (Supplemental Fig.
S1). Also, two independent ANAC102-overexpressing
lines (OX-1 and OX-2) were generated in the C24
ecotype, as this ecotype is more susceptible to lowoxygen stress than Col-0 but has similar ANAC102
expression profiles (data not shown), potentially making it easier to detect any increase in tolerance derived
from ANAC102. Each OX line had 25- to 30-fold higher
expression of ANAC102 than the wild type in whole
3-week-old plants (Supplemental Fig. S1). No gross
phenotypic differences were observed between the
knockout or overexpressing lines and their respective
parental ecotypes grown under standard laboratory
conditions, except for a slightly lighter green leaf color
in the ANAC102-overexpressing lines. Lighter green
coloration was also observed when the closely related
ATAF2 gene (ANAC081; .92% amino acid similarity to
ANAC102) was overexpressed in C24 (Delessert et al.,
2005); however, none of the other ATAF2 overexpression phenotypes, such as increased leaf size, wrinkled
leaves, or increased biomass, were observed in the
ANAC102-overexpressing lines. Although ANAC102 is
highly expressed in root tips, there were no obvious
differences in root morphology or quantity (data not
shown).
Overexpression of ANAC102 Modifies Expression of
Downstream Genes, But ANAC102 Knockout Lines Do
Not Affect Global Gene Transcription in
Growing Plants
As ANAC102 is a putative transcription factor, the
global impact of overexpressing or knocking out
ANAC102 on the Arabidopsis transcriptome was examined using the Affymetrix ATH1 Arabidopsis
arrays. RNA extracted from both untreated and 4-h
low-oxygen-treated 3-week-old seedlings of the wild
type (Col-0), KO-1, and KO-2 lines was used for
microarray analysis. Neither comparisons between
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ANAC102 Is Required for Seed Viability in Low Oxygen
Figure 2. Tissue-specific expression, induction of ANAC102, and vital staining of low-oxygen-treated seeds. An ANAC102
promoterTGUS fusion line was constructed and plants were stained with 5-bromo-4-chloro-3-indolyl-b-glucuronic acid to
localize ANAC102 expression. A, Untreated whole plant. B, Untreated roots. C, Closeup of untreated roots. D, Untreated leaf
and developing flowers. E, Seedling at 2 d after germination. F, Vital staining of an Arabidopsis embryo that failed to germinate
following a 6-d treatment of 0.1% oxygen. The embryo was isolated from the seed coat prior to staining. Green tissue is alive, and
red-stained tissue is dead. C, Embryonic cotyledons; R, embryonic radicle. G, Untreated 3-week-old plant stained for 8 h. H,
Three-week-old plant treated with 0.1% oxygen for 4 h, stained for 8 h. I, Expression of native ANAC102 gene and
ANAC102TGUS fusion relative to ANAC102 expression in untreated plants. Black bars represent expression in untreated plants,
and hatched bars represent expression in plants treated with 0.1% oxygen for 4 h. Error bars represent 1 SE (n 5 9).
untreated KO lines and the wild type nor comparisons
between low-oxygen-treated KO lines and the wild type
showed any differences in gene expression at a fold
change cutoff of 1.5 and an adjusted P value of ,0.05,
save for ANAC102 itself, which was underexpressed in
KO lines in both circumstances (17- to 50-fold; Table I).
RNA was extracted from 3-week-old seedlings of the
wild type (C24), OX-1, and OX-2 lines and used for
microarray analysis. A total of 113 genes were upregulated more than 1.5-fold at an adjusted P value of
#0.05, and 98 genes were found to be significantly
down-regulated in the overexpressing line (Supplemental Table S1). Seventy-five of the up-regulated genes and
61 of the down-regulated genes have been identified
as being differentially regulated in other low-oxygen
microarray experiments (Klok et al., 2002; Branco-Price
et al., 2005; Liu et al., 2005; Loreti et al., 2005). Notably,
ADH1 and SUS1, which have long been recognized as
key components of the low-oxygen response in plants,
were up-regulated by ANAC102 overexpression (2.9fold and 1.6-fold, respectively; Table I).
Relative expression levels in adult plants of 11 genes
reported to be low-oxygen responsive and shown here
to be significantly affected by ANAC102 overexpression were compared by QRT-PCR in KO-1, OX-1, and
wild-type lines across six time points following exposure to 0.1% oxygen (Fig. 3; Supplemental Fig. S2). All
of the genes examined changed in response to low
oxygen levels, in agreement with previous reports.
Of the 11 selected genes that were identified on
the microarray as being up-regulated in OX-1, two
(At1g02850 [glycosyl hydrolase] and At2g43820 [UDPglucosyl transferase]) were shown by QRT-PCR to
have higher expression in OX-1 over all time points.
A further eight of these genes were more highly
expressed in OX-1 during the initial stages of lowoxygen exposure, after which expression in the wild
type increased to match that in OX-1 (Supplemental
Fig. S2). The one selected gene that was identified on
the microarray as being down-regulated in OX-1 was
found by QRT-PCR to be down-regulated in comparison with the wild-type line at most time points. Taken
together, overexpression of ANAC102 increased or
at least preinduced expression of some low-oxygeninducible genes and decreased or delayed the induction of others.
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Christianson et al.
Table I. Microarray-derived expression ratios of selected genes with differential expression between ANAC102 mutant lines and the wild type
Arabidopsis
Genome
Initiative No.
Gene Title/Description
Expression
Ratio in KO-1
Expression Ratio
in KO-1 (0.1%
Oxygen)
Expression Ratio
in KO-1 Seeds
(0.1% Oxygen)
P
Ratioa
P
Ratioa
P
Ratioa
P
,0.01
0.06
0.4
0.02
0.04
0.03
,0.01
1
5
,0.01
,0.01
,0.01
,0.01
0.67
0.86
1.2
0.57
1
1
1
1
0.53
0.48
0.99
0.74
0.9
0.9
1
0.9
0.85
0.63
1
0.68
0.11
0.11
0.9
0.01
0
0
0
0
2
2
0
2
,0.01
,0.01
0.01
0.01
0.94
0.81
0.58
0.66
1
1
1
1
1.1
0.89
1.2
0.71
0.96
0.93
0.9
0.9
0.96
0.93
1.1
0.77
0.76
0.6
0.45
0.09
1
1
1
0
2
4
3
3
,0.01
0.68
1
0.66
0.9
0.98
0.94
0
1
,0.01
0.86
1
1
1
0.9
0.41
1
4
,0.01
1.2
1
0.86
0.9
1.1
0.38
1
1
1
0.95
0.97
1.8
,0.01
0
2
Expression
Ratio in OX-1
Ratioa
Selected genes with differential expression in OX plants
AT5G63790 ANAC102 (Arabidopsis NAC
18
domain-containing protein
102), transcription factor;
similar to ATAF2
AT1G02850 Glycosyl hydrolase family 1 protein
8.6
AT2G04040 MATE efflux family protein
5
AT1G77120 Alcohol dehydrogenase (ADH)
2.9
AT2G43820 UDP-glucoronosyl/UDP-glucosyl
5.6
transferase family protein
AT1G26770 Expansin, putative (EXP10)
5.1
AT3G59140 ABC transporter family protein
3.4
AT5G43580 Protease inhibitor, putative
2.6
AT1G72680 Cinnamyl-alcohol
2.7
dehydrogenase, putative
AT2G30140 UDP-glucoronosyl/UDP-glucosyl
2.4
transferase family protein
AT1G68570 Proton-dependent oligopeptide
2.5
transport (POT) family protein
AT3G56710 sigA-binding protein
0.4
Selected genes with differential expression in KO seeds under
AT3G22370 Alternative oxidase 1a,
0.94
mitochondrial (AOX1A)
AT3G23150 Ethylene receptor, putative (ETR2)
1
AT1G11580 Pectin methylesterase, putative
0.91
AT1G03090 3-Methylcrotonyl-CoA
1.2
carboxylase 1 (MCCA)
AT2G40220 Abscisic acid-insensitive 4 (ABI4)
0.95
0.1% oxygen
0.81 0.86
Selected genes with differential expression in both ANAC102
AT3G46670 UDP-glucoronosyl/UDP-glucosyl
2.4
transferase family protein
AT5G61440 Thioredoxin family protein
2
AT2G02120 Plant defensin-fusion protein,
1.9
putative (PDF2.1)
AT4G27830 Glycosyl hydrolase family 1 protein 1.8
AT5G20830 Suc synthase/Suc-UDP
1.6
glucosyltransferase (ASUS1)
AT2G43590 Chitinase, putative
0.66
AT5G60950 Phytochelatin synthetase-related
0.55
AT1G02640 Glycosyl hydrolase family 3 protein 0.48
AT1G73480 Hydrolase, a/b-fold family protein
0.05
NAC
NAC
Strictb Consensusc
0.91
0.68
0.28
1.1
1.2
0.97
1
1
1
1.2
1.1
0.83
0.9
0.95
0.9
0.51
0.48
0.47
,0.01
,0.01
,0.05
0
0
1
0
3
3
0.85
1.2
1
1
1
0.6
,0.01
0
0
OX plants and 0.1% oxygen-treated ANAC102 KO seeds
0.01 0.87
1
0.94
0.96
0.58
0.01
0
0
0.02
0.01
1.2
1
1
1
0.91
0.95
0.9
0.98
0.47
2
,0.01
0.01
0
0
0
4
0.03
0.04
0.85
1
1
1
0.77
0.97
0.89
0.98
0.62
0.55
0.01
0.03
0
0
2
0
0.04
0.05
0.04
0.01
1.1
1.2
1.1
0.79
1
1
1
1
0.89
0.95
1.2
0.7
0.96
0.95
0.93
0.9
2
0.65
0.48
0.62
0.03
0.02
,0.01
0.02
0
0
0
0
0
1
2
2
a
Relative expression ratio of OX-1 or KO-1 lines compared with the wild type. For OX-1, the wild-type background is C24; for KO-1, the wild-type
b
background is Col-0. Expression ratios significantly different from the wild type are highlighted in boldface.
Number of occurrences of the strict
c
NAC domain-binding site motif TTNCGTA in the 1,000-bp upstream sequence of the given gene, as defined by Olsen et al. (2005).
Number of
occurrences of the strict NAC domain-binding site motif [TA][TG][TAGC]CGT[GA] in the 1,000-bp upstream sequence of the given gene, as defined
by Olsen et al. (2005).
The promoter regions of all of the genes showing
differential expression in OX-1 were analyzed for evidence of conserved motifs. Analysis with the Athena
visualization tool (O’Connor et al., 2005), which can
identify transcription factor-binding motifs in promoter sequences, showed some enrichment for the
ABA-responsive element (ABRE)-like binding site motif (Shinozaki and Yamaguchi-Shinozaki, 2000) in the
up-regulated genes, with 34 of the 113 genes containing
at least one copy of this element. The genes found to be
down-regulated in OX-1 were found to be enriched for
an AGC box enhancer element identified from a tobacco (Nicotiana tabacum) class I b-1,3-glucanase GLB
gene (Hart et al., 1993), the LS7 promoter element
required for salicylic acid induction of Arabidopsis
PR-1 (Despres et al., 2000), and the JASE1 motif required for leaf senescence and jasmonic acid induction
of Arabidopsis OPR1 (He and Gan, 2001). As Athena
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ANAC102 Is Required for Seed Viability in Low Oxygen
Figure 3. QRT-PCR analysis of selected genes
up-regulated in ANAC102-modified expression lines. Changes in expression were monitored for a set of genes in 3-week-old
Arabidopsis plants from each of Col-0, C24,
KO-1 (white squares), and OX-1 (black
squares) lines. Plants were grown exposed to
0.1% oxygen in anaerobic chambers and in
the dark for the various times specified. Data
presented are expression log2-transformed ratios for each of KO-1 and OX-1 relative to their
wild-type lines, Col-0 and C24, respectively,
at each time point. An expanded version of
this figure, including expression data for Col-0
and C24, is available in Supplemental Figure S2.
does not identify NAC domain transcription factor
binding sites, the Toucan analysis tool, which can
perform searches for specified motifs, was used to
search for these sequences. Both the set of genes upregulated in OX-1 and the set of genes down-regulated
in OX-1 had higher than expected incidences of the
NAC-binding site motif: 37.8% to 48.7% of the promoters in each data set contained one or more instances
of the strict NAC core-binding site (TTNCGTA), and
90.9% to 96.5% of the promoters in each data set
contained one or more instances of the general consensus NAC-binding site ([TA][GT][TACG]CGT[GA];
Olsen et al., 2005; Table II; Supplemental Table S1). The
Web-based tool POBO, which compares the frequency
of a given motif in a set of promoter sequences with
a random set of Arabidopsis promoter sequences,
confirmed that both the strict and general consensus
NAC-binding sites were statistically overrepresented
in promoters of genes affected by ANAC102 overexpression (P , 0.0001; Kankainen and Holm, 2004).
Overexpression or Underexpression of ANAC102 Has
No Significant Effect on Adult Plant Tolerance to
Low Oxygen
To determine whether ANAC102 is important for
plant survival under low oxygen, both KO and OX lines
were subjected to severe low-oxygen stress. In five
separate experiments, plants were subjected to 0.1%
oxygen, with or without a 24-h 5% oxygen pretreat-
Table II. Promoter region analysis for NAC-binding sites in genes affected by ANAC102 expression
The 1,000-bp upstream regions for all of the genes identified as significantly affected by ANAC102 were retrieved from the Arabidopsis Information
Resource (www.arabidopsis.org).
Gene Seta
OX-1
OX-1
KO-1
KO-1
No. of Genes
NAC Motif Strictb
POBO T Statisticc
POBO P
NAC Consensusd
POBO T Statisticc
POBO P
113
98
94
113
48.7%
37.8%
38.3%
38.9%
46.5
49.7
41.7
43.6
,0.0001
,0.0001
,0.0001
,0.0001
96.5%
90.9%
89.4%
82.3%
83.1
34.0
88.1
36.6
,0.0001
,0.0001
,0.0001
,0.0001
up-regulated
down-regulated
up-regulated
down-regulated
a
b
The gene sets used can be found in Supplemental Tables S1 and S4.
The strict NAC-binding site motif is TTNCGTA, as defined by Olsen et al.
c
(2005).
The T statistic was calculated by the promoter analysis program POBO (Kankainen and Holm, 2004) and is a measure of the difference
between the frequency of the searched-for motif in a given sequence set as compared with a randomly generated background set of
d
sequences.
The consensus NAC-binding site motif is [TA][GT][TACG]CGT[GA], as defined by Olsen et al. (2005).
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Christianson et al.
Seed Viability Is Impaired in ANAC102 Knockout Lines
following a Low-Oxygen Treatment
Six months after harvest, seeds from each line either
overexpressing or knocked out for ANAC102 and their
parental wild types, collected from plants grown at the
same time in the same growth cabinet, were subjected
to 6 d of exposure to 0.1% oxygen in the light. During
the exposure to 0.1% oxygen, no germination occurred, in contrast to seeds exposed to the ambient
atmosphere, which reached maximum germination
within 2 to 4 d. Germination percentages in the normal
atmosphere were lower for seeds from C24 background plants (63%280%; Fig. 5A) than for seeds
from the Col-0 background plants (92%297%; Fig.
5A), which appears to be a feature of C24 germination
on unsupplemented agarose, as these seeds will all
Figure 4. Survival after exposure to 0.1% oxygen. Plants with T-DNA
insertions within the ANAC102 gene (KO-1, KO-2, and Col-0 background) and plants overexpressing the ANAC102 gene (OX-1, OX-2,
and C24 background) and their respective parental ecotypes were
subjected to low-oxygen treatments. A, Plants were treated with 0.1%
oxygen in the dark for 3 d. Survival percentages are averages over five
experiments (6SE; n 5 15). Data points with different lowercase letters
denote lines with survival percentages significantly different from each
other as determined by ANOVA and Tukey’s honestly significant
difference test (P , 0.05). B, Plants were treated for 1 d with 5%
oxygen, followed by 3 d with 0.1% oxygen. Survival percentages are
averages over three experiments (6SE; n 5 9).
ment, in the dark for 3 d and then scored for survival,
shoot weight, and root weight 1 week after removal
from the stress condition. Survival counts were variable
(Supplemental Fig. S3), and no significant differences in
survival or weight measures between either KO lines or
OX lines and their respective wild types were found in
experiments with or without a 5% oxygen pretreatment. As expected, however, there was a significant
difference in survival between the Col-0 ecotype lines
and the C24 ecotype lines in experiments in which no
5% oxygen pretreatment was used (Fig. 4; accumulated
analysis of deviance, 5 degrees of freedom [df]; deviance ratio 5 6.77; approximate F probability , 0.001).
Alteration of ANAC102 expression did not appear to
have any effect on recovery or growth of plants surviving a low-oxygen treatment. Plants that survived the
low-oxygen stress were measured for root and shoot
tissue fresh weights. No difference in root or shoot
weight could be observed between OX or KO lines and
their wild-type counterparts following 0.1% oxygen
treatment with or without 5% oxygen pretreatment
(Supplemental Table S2).
Figure 5. Effects on germination of various abiotic stresses. Seeds were
scored for germination at intervals up to 1 week. Data represent average
numbers of germinated seeds from three replicates containing 20 to 50
seeds each (6SE; n 5 3). Black symbols represent data for untreated
seeds, and white symbols represent treated seeds. Data points with
different lowercase letters denote lines with germination percentages
significantly different from each other (only differences between lines
within a treatment are shown) as determined by ANOVA and Tukey’s
honestly significant difference test (P , 0.05). A, Seeds treated with
0.1% oxygen for 6 d. Germination was scored after removal to ambient
oxygen levels. B, Seeds sown on medium containing 200 mM NaCl. C,
Seeds sown on medium containing 5% (w/v) mannitol. D, Seeds sown
on medium containing 15 mM ABA.
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ANAC102 Is Required for Seed Viability in Low Oxygen
germinate on other media. Modification of ANAC102
expression appeared to have no effect on seed germination in the absence of stress, as there was no significant difference in germination between ANAC102 KO
or OX seeds and their respective wild types under
normal conditions. Following removal from 0.1% oxygen, OX-1, OX-2, and the wild type all showed a
reduction in germination percentage, with no significant differences in response between the lines (Fig. 5),
but there was a marked difference between the
ANAC102 knockout lines and the wild type. Following
the low-oxygen treatment, wild-type germination percentages were reduced to between 65.4% and 80.0% of
the untreated control, while the ANAC102 knockout
lines KO-1 and KO-2 had germination percentages
reduced to between 26% and 38% of their untreated
controls (Fig. 5A). Germination percentages of KO-1,
KO-2, and the wild type untreated and 0.1% oxygentreated seeds at 7 d after stress were found by ANOVA
to have highly significant treatment and line differences, with a highly significant interaction between
treatment and line (treatment, df 5 1, F 5 140.0662,
P 5 9 3 10211; line, df 5 2, F 5 13.4125, P 5 2 3 1024;
treatment 3 line, df 5 2, F 5 9.25, P 5 0.001; Fig. 5A).
To determine whether the 0.1% oxygen treatment
had killed or only imposed a secondary dormancy
(where seeds that were viable and nondormant remain
viable but refuse to germinate) in those seeds that had
not germinated, all of the ungerminated seeds from the
assay were placed back at 4°C for 1 week (486 seeds
total). Only a small proportion (2%) of the ungerminated seeds germinated following the cold treatment,
indicating that a secondary dormancy had not been
induced in these seeds (Supplemental Table S3). Another set of ungerminated seeds from a separate experiment were stained with tetrazolium salts (510 seeds
total); 95% of these seeds turned pink, indicating that
respiration was occurring (Supplemental Table S3).
Vital staining with propidium iodide and fluorescein
diacetate was performed on another set of low-oxygentreated, ungerminated seeds, consisting of five ungerminated seeds from each of KO-1, KO-2, and the wild
type. Endosperm tissues were all alive, but in all cases
save one, seeds from KO-1, a mixture of live and dead
cells were seen in the embryo (Fig. 2F). The remaining
KO-1 embryo appeared to be completely viable.
We further examined whether germination signals
may be blocked in these seeds using ATGA3OX2
(At1g80340) expression as a marker for germination.
In Arabidopsis, the biosynthesis of gibberellins is a
crucial step toward initiating germination (Koornneef
and Vanderveen, 1980; Nambara et al., 1991). One of
the final steps in the production of active gibberellin is
catalyzed by ATGA3OX2, expression of which increases in germinating seeds (Yamaguchi et al., 1998).
QRT-PCR assays of ATGA3OX2 expression between
wild-type and KO-1 lines treated with 0.1% oxygen
showed no significant reduction in ATGA3OX2 expression in the KO-1 lines (1.2-fold induction in KO-1
line; t test; two-tailed P 5 0.17).
To determine whether the decreased germination
observed under low oxygen in the KO lines was
specific to low-oxygen treatments or was a generalized
stress response, we subjected Arabidopsis wild-type
and mutant lines to other stresses during germination.
Salt and osmotic stresses were chosen for these experiments, as public data available at the Genevestigator
Web site indicated that ANAC102 expression is also
inducible by salt and osmotic stresses (Zimmermann
et al., 2004). Due to its role in inhibiting germination in
Arabidopsis, we also tested the ANAC102 mutant lines
for differences in ABA sensitivity during germination.
Although germination in KO lines was transiently
lower than in the wild type when subjected to 200 mM
NaCl, neither 5% mannitol nor 15 mM ABA appeared to
have a stronger effect on KO lines compared with the
wild type (Fig. 5, B–D). These results suggest that the
decreases in germination observed in KO lines are
likely to be specific to low-oxygen stress.
ANAC102 Knockout Lines Show Changes in Global
Gene Expression in 0.1% Oxygen-Treated Seeds
Affymetrix ATH1 microarrays were used to interrogate RNA samples derived from wild-type (Col-0) and
KO-1 lines to assess the impact on global gene expression of loss of ANAC102 function. In comparisons
made using 3-week-old plants under both ambient
atmosphere and 0.1% oxygen for 4 h, no genes other
than ANAC102 itself (which decreased 17- to 50-fold;
Table I) were found to have significantly different
expression (greater than 1.5-fold change between
KO-1 and the wild type, with an adjusted P value of
#0.05). In contrast, when KO-1 and wild-type imbibed
seeds were subjected to 6 d of 0.1% oxygen and
assayed for differences in gene expression, 94 genes
were found to be more highly expressed and 113 were
more lowly expressed in KO-1 than in the wild type
(greater than 1.5-fold change at an adjusted P value of
,0.05; Supplemental Table S4). As was the case in the
ANAC102 OX lines, a large proportion of the genes
with altered expression in the ANAC102 KO seeds
have previously been reported to be responsive to low
oxygen; 29 of the 94 up-regulated genes and 58 of the
113 down-regulated genes have been listed as having
significant expression changes following low oxygen
in one or more microarray studies (Klok et al., 2002;
Branco-Price et al., 2005; Liu et al., 2005; Loreti et al.,
2005). There was very little overlap between genes
with altered expression in ANAC102 OX lines in air
and genes with altered expression in ANAC102 KO
seeds in low oxygen, with only nine genes responsive
in both instances (Table I).
Promoters of genes with altered expression in KO-1
seeds under low oxygen were analyzed using the
Athena visualization tool (O’Connor et al., 2005). This
analysis identified at least one of the following related
motifs (all share the core sequence ACGTG): the ABRElike-binding site motif (Shinozaki and YamaguchiShinozaki, 2000), the ABRE-binding site motif (Choi
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Christianson et al.
et al., 2000), the ABF-binding site motif, the G-box motif
CACGTG (Menkens et al., 1995), the Z-box motif (Ha
and An, 1988), or a GA down-regulation motif (Ogawa
et al., 2003) in 60 promoters of the 94 ANAC102 KO-1
up-regulated genes. Analysis of the promoters of the
113 genes down-regulated in ANAC102 KO-1 seeds
identified 39 with the DREB1A/CBF3 or DRE core
motif found in many stress-inducible genes (Maruyama
et al., 2004). Searching promoter sequences with Toucan
(Aerts et al., 2003, 2005) revealed that 38.3% and 38.9% of
the ANAC102 KO-1 up-regulated and down-regulated
genes, respectively, contain the strict NAC-binding
domain in their promoters and 89.4% and 82.3% of
the ANAC102 KO-1 up-regulated and down-regulated
genes, respectively, contain the general NAC-binding
domain in their promoters (Table II; Supplemental Table
S4). The cis-element analysis tool POBO confirmed that
both the strict and general NAC domain binding sequences are overrepresented in the promoter sequences
at P , 0.0001.
Ten genes, including ANAC102, identified as differentially regulated between wild-type and KO-1 seeds
following 6 d of exposure to 0.1% oxygen, were assayed
via QRT-PCR to determine their relative expression
levels in wild-type, KO-1, and OX-1 lines at 4 d under
0.1% oxygen as well as 6 d (Fig. 6). All genes tested
Figure 6. Relative expression of selected genes in
seeds exposed to 0.1% oxygen for 4 or 6 d. Seeds
of each line were imbibed, cold stratified, and
then placed in a 0.1% oxygen atmosphere for 4 or
6 d. Expression ratios for selected genes are given
relative to expression levels in untreated wildtype (Col-0 for Col-0 and KO-1; C24 for C24 and
OX-1) imbibed seeds. Bars represent expression
ratios for Col-0 (black columns), KO-1 (dotted
columns), C24 (shaded columns), and OX-1 (diagonal lined columns; 6SE; n 5 9). Data points
with different letters denote lines with relative
expression ratios significantly different from each
other as determined by two-way ANOVA (line 3
time) and Tukey’s honestly significant difference
test (P , 0.05). Uppercase letters differentiate
between Col-0 and KO-1 at the different times,
while lowercase letters differentiate between C24
and OX-1 at the different times. No comparisons
were made between lines in the Col-0 background and lines in the C24 background.
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ANAC102 Is Required for Seed Viability in Low Oxygen
showed a greater response to low oxygen after 6 d at
0.1% oxygen as compared with 4 d. SUS1, a key lowoxygen-responsive gene, showed induction in lowoxygen conditions at both time points tested, but this
induction was significantly lessened in the KO-1 seeds.
For only one gene (AOX; At3g22370) was there a significant difference in expression between KO-1 and
Col-0 after 4 d in 0.1% oxygen (Fig. 6). Differences in
expression between KO-1 and Col-0 were much more
apparent in most genes after 6 d at 0.1% oxygen (Fig. 6).
Overexpression of ANAC102 also had a significant
impact on gene expression under 0.1% oxygen (Fig.
6). Surprisingly, overexpression of ANAC102 had a
similar impact on gene expression as reduced expression of ANAC102; that is, where gene expression was
decreased in KO-1 as compared with Col-0, it was also
decreased in OX-1 as compared with C24.
DISCUSSION
ANAC102 is not required for normal growth and
development, and its primary role in Arabidopsis is
very likely to respond to stress. Neither of the two
independent ANAC102 knockout lines showed any
apparent phenotype under nonstressed conditions,
including no global changes in gene expression.
Knockouts of the stress-inducible NAC genes ATAF2
(Delessert et al., 2005), ATAF1 (Lu et al., 2007), and
AtNAC2 (He et al., 2005) similarly showed no phenotype under normal growth conditions.
ANAC102 expression increases very early following
the imposition of low oxygen levels. The rapid response of ANAC102 to low oxygen coupled with the
ability to respond to low oxygen in the presence
of cycloheximide, which blocks protein synthesis,
suggest that ANAC102 responds directly to lowoxygen stress and does not require for induction
any upstream low-oxygen-responsive transcription
factors. ANAC102 has been classed as an unstable
transcript with a half-life of less than 60 min under
normal conditions (Gutierrez et al., 2002). The increase
in ANAC102 transcript levels under low oxygen may
be partially due to an increase in transcript stability
rather than an increase in transcription, although the
ANAC102 promoter is capable of driving an increase in
GUS transcript in response to low oxygen (Fig. 2I).
In contrast to lines overexpressing ATAF2 (Delessert
et al., 2005), AtNAC2 (He et al., 2005), or ANAC055
(Tran et al., 2004), overexpression of ANAC102 had
little effect on plant phenotype other than a mild
yellowing of the leaves. Overexpression of ANAC102
resulted in altered expression of 211 genes under
normal conditions. Most (96.5%) of the genes with
altered expression in OX-1 contained the general
consensus NAC-binding site, indicating that these
genes may be targets for ANAC102 binding. Both
genes up-regulated and down-regulated in ANAC102overexpressing lines had high frequencies of the NAC
domain consensus-binding sequence, suggesting the
possibility that ANAC102 can act bifunctionally as
both an activator and a repressor of transcription. This
type of bifunctionality has been observed in the
NFYA5 transcription factor, overexpression of which
affected the abundance of mRNA from a number of
genes containing the NFYA5 recognition site CCAAT,
inducing some and repressing others (Li et al., 2008).
Overrepresentation of the WRKY-binding motif has
also been observed in promoters of genes both induced and repressed by overexpression of WRKY70
(Kankainen and Holm, 2004). Two-thirds of the genes
induced or repressed in ANAC102-overexpressing
lines have been previously identified as being lowoxygen responsive (Supplemental Table S1), including
ADH1 and SUS1, which are known to be vital for
adult plant tolerance of low oxygen (Ellis et al., 1999;
Bieniawska et al., 2007). In contrast, ANAC102 KO
lines showed no transcriptional differences from the
wild type in normal and low-oxygen conditions.
Neither ANAC102 OX nor KO lines showed any differences in growth or survival when adult plants
were subjected to low-oxygen stress. The ANAC102
OX microarray results under normal conditions, and
QRT-PCR results under low-oxygen conditions suggest that ANAC102 has a role in modifying transcriptional responses to low oxygen, but the survival assays
show that ANAC102-mediated responses are not sufficient to protect the plants or, as is the case with ADH1
overexpression, were not rate limiting for survival
under low oxygen (Baxter-Burrell et al., 2002; Ismond
et al., 2003).
The ANAC102 KO microarray and survival assay
results demonstrate that ANAC102 function is not
required for transcriptional or phenotypic response
to low oxygen in adult plants. However, ANAC102 KO
lines had impaired germination after 0.1% oxygen
treatment, and comparisons between wild-type and
KO-1 seeds showed expression differences in 207
genes. Loss of ANAC102 did not alter germination in
unstressed seeds, nor was any effect on germination
observed under salt, osmotic, or ABA stress. This
indicates that although ANAC102 is not essential for
adult tolerance to low oxygen, it is important for
tolerance to low-oxygen levels during germination. A
possible explanation for the difference between adult
and seed low-oxygen phenotypes is redundancy of
ANAC102 gene function. A total of 23 other NAC
domain transcription factors also possess significantly
altered expression under low oxygen, and many have
high sequence similarity to ANAC102, particularly
ANAC002 (ATAF1) and ANAC032 (Ooka et al., 2003).
In adult plants, ANAC102 is highly expressed in the
lateral root cap (Fig. 2; Supplemental Fig. S4), as are
ANAC002 and ANAC032 (Supplemental Fig. S4).
ANAC102 is also expressed at relatively high levels
in the radicle; however, ANAC002 and ANAC032 are
not strongly expressed in this tissue (Supplemental
Fig. S4). Differences in tissue-specific expression between these highly similar genes may account for the
difference in adult survival/germination rates under
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Christianson et al.
low-oxygen conditions and the lack of transcriptome
results observed in the ANAC102 mutant lines in adult
tissue.
Very little is known about how Arabidopsis seeds
respond to low oxygen, and it is unclear how disruption of ANAC102 function compromises seed tolerance
to low oxygen. Two-thirds of the genes up-regulated in
stressed ANAC102 KO seeds have a core motif of ABAresponsive elements in their promoters, and the ABA
response gene ABI4 shows lower than wild-type expression in KO lines following exposure to low oxygen
(Table I; Fig. 6), implying a role for ABA in the
ANAC102-mediated response to 0.1% oxygen in seeds.
ABA and oxygen levels interact to regulate dormancy
in barley, where the glumella limits oxygen diffusion
through the barley embryo, resulting in increased
sensitivity to ABA (Benech-Arnold et al., 2006). However, in germination experiments on media supplemented with ABA, neither KO nor OX lines showed
any change from the wild type in ABA sensitivity.
Furthermore, the Arabidopsis seeds used in this work
were nondormant, and low oxygen did not induce a
secondary dormancy, as seeds that failed to germinate
after low-oxygen treatment did not germinate after a
dormancy-breaking cold treatment.
Vital staining of seeds that failed to germinate
indicated that portions of the embryo may have been
killed by the low-oxygen stress. This damage may
have been sufficient to prevent the radicle from being
able to break through the seed coat. An alternative
hypothesis is that mobilization of energy reserves is
affected in ANAC102 knockout lines in such a way as
to leave the seed with too little energy to be able to
germinate following return to normal oxygen concentrations. Interestingly, SUS1, a Suc synthase gene,
shows lower expression in ANAC102 KO-1 seeds than
in wild-type seeds (Table I; Fig. 6). SUS1 is induced
under low-oxygen conditions, is responsible for providing Glc units for glycolysis, and double knockouts of
SUS1 and the very closely related ASUS4 show reduced
root growth following flooding (Bieniawska et al.,
2007). The alternative oxidase gene AOX1 is more
highly expressed in ANAC102 KO-1 seeds under low
oxygen as compared with the wild type. AOX1 has
been proposed as a possible sensor for oxygen levels
(Rhoads and Subbaiah, 2007) and has been shown to
have its expression regulated by anoxia and reoxygenation in barley roots and rice seedlings, where it may
be protecting against oxidative damage or overreduction of the mitochondrial electron transport chain (Szal
et al., 2003; Millar et al., 2004). The ethylene receptor
gene ETR2 also shows reduced expression in ANAC102
KO-1 seeds, relative to the wild type, under low oxygen. ETR2 is induced under low-oxygen conditions, as
are ACC synthase and ACC oxidase genes (Klok et al.,
2002; Branco-Price et al., 2005; Liu et al., 2005; Loreti
et al., 2005; Peng et al., 2005). Ethylene production is
stimulated in a wide variety of plant species subjected
to low oxygen and may be responsible for triggering
responses such as aerenchyma formation, stem elon-
gation, and adventitious root growth in species such as
maize and rice (He et al., 1994; Rzewuski and Sauter,
2008). It is possible that the decreased seed viability
phenotype observed in the ANAC102 KO seeds is the
result of changes in one or a combination of these
low-oxygen response mechanisms. Decreased SUS1
expression may lead to decreased throughput in the
glycolysis pathway and lower energy levels in the
low-oxygen-stressed embryos. Or decreased ethylene
sensitivity brought about by decreased ETR2 expression may result in a weaker ethylene-mediated lowoxygen response in these seeds.
A set of genes identified as differentially expressed
between KO and wild-type seeds at a single lowoxygen time point via microarray were assayed in both
OX and KO lines using QRT-PCR at two time points
under low-oxygen stress. For many of these genes (five
of nine assayed), lower levels of expression were
observed in both the OX and KO lines than in their
wild-type counterparts after 6 d of exposure to low
oxygen, raising the possibility that excess ANAC102
expression my also be somewhat detrimental to seed
responses to low oxygen. Although the differences
were found to be not statistically significant, both OX
lines did show lower germination rates than the wild
type following low-oxygen stress (Fig. 5A).
The increased expression of putative low-oxygenresponsive genes in ANAC102 overexpressors and the
reduced germination rates of ANAC102 knockout lines
in response to low-oxygen treatment suggest that
ANAC102 positively regulates the response to low
oxygen, while the lack of global gene expression
change at the adult plant stage in ANAC102 knockouts
and the lack of significant adult plant survival differences between ANAC102 OX and KO lines and the
wild type suggest that the role of ANAC102 may be
functionally redundant at the adult plant stage. Both of
these features are in direct contrast with the mode of
action of both ATAF1 and ATAF2, which negatively
regulate responses to drought and wounding, respectively, and for which a loss of function of either of these
genes results in a readily observable phenotype under
those stress conditions (Delessert et al., 2005; Lu et al.,
2007).
ANAC102 is important for the Arabidopsis response
to low-oxygen levels during germination. This work
illustrates that responses to environmental stresses, in
this case low oxygen, are dependent on developmental
stage and that mechanisms for tolerance can be different at different stages. ANAC102 is required for tolerance to low oxygen at the seed stage but not at the
early adult plant stage. The transcriptional responses
to low oxygen also differ between developmental
stages, since in KO lines no genes show differences
in transcript levels in adult plant microarrays but 207
genes show significant expression differences in seeds.
At the other end of the developmental scale, it has
recently been reported that 12-week-old Arabidopsis
plants form aerenchyma in response to waterlogging
stress (Muehlenbock et al., 2007). This response to
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ANAC102 Is Required for Seed Viability in Low Oxygen
waterlogging was not previously thought to occur in
Arabidopsis, as most prior research had been conducted on younger, rosette stage plants that do not form
aerenchyma. Taken together, these findings highlight
the different responses to stress at different developmental growth stages.
MATERIALS AND METHODS
Plant Material
from the data. The model assessed variation due to Arabidopsis line differences, blocked for variation between experiments. Predicted probabilities of
survival were compared between each pair of lines using the LSD of each
comparison at a significance level of 0.33% (5% after a Bonferroni correction).
QRT-PCR
All QRT-PCRs were performed in triplicate on either a Corbett Rotor Gene
(Corbett Life Sciences) or an Applied Biosystems 7900HT (Applied Biosystems) system. Product was detected by fluorescence of incorporated SYBR
Green. All data were normalized to the expression of At4g26410, which has
been identified as being highly stable over a broad range of conditions
(Czechowski et al., 2005) and has been found not to change expression in
response to low oxygen in microarray experiments we have conducted. Primer
sequences are listed in Supplemental Table S5. In all cases, relative expression
levels were calculated using the DDCt method of Pfaffl et al. (2002). Expression
levels in various experiments were analyzed using a two-tailed t test assuming
equal variances to identify statistically significant changes in expression levels.
Arabidopsis (Arabidopsis thaliana) lines carrying T-DNA insertions in
ANAC102 (At5g63790; SALK_030702 and SALK_094437) were identified using
the SIGnAL Web site (http://signal.salk.edu). The two insertion lines were
obtained from the Arabidopsis Biological Resource Center (Alonso, 2003).
Insertions were verified and homozygote lines selected using PCR according
to the protocols provided by the SIGnAL Web site. KO-1 is a derivative of
SALK_030702, and KO-2 is a derivative of SALK_094437. Both insertions are
within the second exon of ANAC102, and both segregated as single-locus
insertions. Ecotype Col-0 was used as the wild type in all comparisons with
the insertion mutant lines. Since C24 is more susceptible to low-oxygen stress
than Col-0, the ANAC102 coding sequence under the control of the cauliflower
mosaic virus 35S promoter was transformed into ecotype C24 to make it easier
to detect any increase in low-oxygen tolerance. Two independent, highly
expressing lines (as assayed by QRT-PCR), designated OX-1 and OX-2, were
chosen for use in these experiments. Ecotype C24 was used as the wild type in
all comparisons with the ANAC102 overexpression lines. A promoterTGUS
fusion line was created by amplifying 1,611 bp upstream of the ANAC102
transcription start site and cloning the amplicon into BamHI/HindIII-digested
pBI101, a binary plant transformation vector, and transformed into C24 by the
floral dip method.
All plants were grown on Murashige and Skoog (MS) medium with 3% Suc
and 0.8% agar. For seed germination experiments, seeds were plated on either
0.6% agarose in water or half-strength MS medium. Plants or seeds for all
experiments were kept in a growth room at 22°C with a day/night period of
16/8 h and fluorescent lighting levels of approximately 75 mE. For gene
expression and low-oxygen assays, plants were transferred to liquid MS with
3% Suc. To obtain seeds for germination experiments, plants were grown in
soil at 22°C under a 16/8-h day/night cycle, and all lines tested were grown at
the same time and in the same location to minimize environmental influences
on seed development and subsequent germination.
Low-oxygen treatments for microarray and QRT-PCR experiments were
carried out in the same manner as for the low-oxygen survival assays. Plants
were grown on solid MS medium for 3 weeks, to around the four- to six-leaf
stage, then transferred to liquid medium 1 d prior to stress. For the lowoxygen treatments, plants were place in 3.5-L anaerobic chambers (Oxoid) and
purged with a 0.1% oxygen/balance nitrogen gas mixture for 20 min, after
which plants were left in the 0.1% oxygen atmosphere in the dark. For
cycloheximide treatment, 3-week-old plants were moved to liquid medium
containing 10 mM cycloheximide for 1 h, medium was refreshed with new
medium plus 10 mM cycloheximide, and plants were low-oxygen treated for 4
h. After treatment, plants were flash frozen and ground in liquid nitrogen.
RNA was extracted using a Trizol buffer (Invitrogen) following the manufacturer’s instructions. For seed microarray and real-time PCR experiments,
seeds were plated on 0.6% agarose in water, allowed to imbibe for approximately 4 h, and then cold stratified at 4°C overnight. Following stratification,
plated seeds were allowed to equilibrate to room temperature, then placed in
the anaerobic chambers and purged with the 0.1% oxygen gas mixture for 20
min. Seeds were then left in the 0.1% oxygen atmosphere for 6 d. Following
treatment, seeds were frozen and ground in liquid nitrogen. RNA was
extracted using a hot borate method (Cadman et al., 2006).
Low-Oxygen Survival Assay
Microarray Analysis of Gene Expression
Low-oxygen survival assays were performed as outlined previously (Ellis
et al., 1999), except that argon was not used to flush the anaerobic chambers
and plants were not subjected to anoxia but rather to mild hypoxia (5%
oxygen, balance nitrogen) and/or severe hypoxia (0.1% oxygen, balance
nitrogen). Briefly, plants were grown to the four- to six-leaf stage on solid
medium and then transferred to liquid medium for 1 d prior to treatment.
Immediately prior to treatment, plants were transferred to liquid medium that
had been sparged with either 5% oxygen, for pretreated plants, or 0.1%
oxygen, for nonpretreated plants. Plants were then placed in 3.5-L anaerobic
chambers (Oxoid) and purged with either 5% or 0.1% oxygen at a flow rate of
approximately 10 L min21 for 20 min. Pretreated plants were left in 5%
oxygen, in the dark with gentle shaking, for 24 h and then purged with 0.1%
oxygen and left for a further 3 d. Nonpretreated plants were purged with
0.1% oxygen and left in the dark with gentle shaking for 3 d. Nontreated plants
were placed in anaerobic chambers but left exposed to the ambient atmosphere and left in the dark with gentle shaking for 3 d. Following treatment,
plants were given fresh liquid medium and allowed to recover on an orbital
shaker in normal growth cabinet conditions for 1 week before survival scoring
and weight measurements.
Survival data from five independent experiments were pooled and analyzed for differences between lines in their response to low oxygen. The lowoxygen survival data were not well approximated by the assumption of
normality. Consequently, these data were analyzed with a binomial generalized linear model with a logit link. The dispersion factor was initially fixed at
1, but the analysis then flagged a large number of large standardized
residuals, so the data were reanalyzed, allowing the dispersion to be estimated
Whole plant or seed RNA was sent to the Australian Genome Research
Facility for labeling and hybridization to Affymetrix Arabidopsis ATH1
genome arrays (22,500 probes). In unstressed microarray experiments, two
biological replicates were used for the wild type, KO-1, and OX-1 and one
replicate was used for KO-2 and OX-2. As no differences were found between
KO-1 and KO-2 or between OX-1 and OX-2, data presented here are from
KO-1 and OX-1 comparisons only. At 3 weeks old, five plants of each line from
each replicate plate were bulked, flash frozen in liquid nitrogen, and ground.
RNA was extracted using a Trizol buffer (Invitrogen) following the manufacturer’s instructions. For low-oxygen stress microarrays at both plant and seed
stages, at least two biological replicates of the wild type and KO-1 were used.
For experiments on seeds, seeds were plated on 0.6% agarose in water and
low-oxygen treated as described above. Following treatment, seeds were
frozen and ground in liquid nitrogen. RNA was extracted using a hot borate
method (Cadman et al., 2006).
Resulting signal data were analyzed using the limma Bioconductor package in R. Array data were normalized using the EXPRESSO function (Gautier
et al., 2005). A robust multichip average was calculated using quantile
normalization, background correction, and the median polish method as
recommended by Bolstad et al. (2003) and Irizarry et al. (2003).
Low-Oxygen Treatments of Arabidopsis Seedlings
and Seeds
Promoter Analysis
Promoter regions of selected genes were screened for common motifs.
Three different Web-based analysis tools were used: Athena (O’Connor et al.,
Plant Physiol. Vol. 149, 2009
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Copyright © 2009 American Society of Plant Biologists. All rights reserved.
Christianson et al.
2005), Toucan (Aerts et al., 2003, 2005), and POBO (Kankainen and Holm,
2004). In each case, the 1,000-bp upstream regions of selected genes were
used for analysis. For the POBO analysis (Kankainen and Holm, 2004),
Arabidopsis_thaliana_clean was used as the background organism sequence set,
the number of sequences to pick out was set to equal the number of input
sequences, and number of samples to generate and sequence length were both
set to 1,000.
Supplemental Table S1. Genes identified with significant changes in
expression in OX-1 adult plants.
Germination Assays
Supplemental Table S4. Genes identified with significant changes in
expression in low-oxygen-treated KO-1 seeds.
Seeds of each line to be tested that had been harvested at the same time
from plants grown in the same environmentally controlled growth room were
imbibed in 1.5-mL microfuge tubes for approximately 2 h at room temperature. Twenty to 50 seeds of each line were then plated out approximately 0.5
cm apart on 0.6% agarose and then placed at 4°C for 1 week. For low-oxygen
treatments, seeds were placed in 3.5-L anaerobic chambers and either purged
with 0.1% oxygen at a flow rate of 10 L min21 for 20 min or exposed to the
ambient atmosphere. Seeds left exposed to ambient air were scored for
germination daily. Seeds treated with 0.1% oxygen remained in sealed
anaerobic chambers at 0.1% oxygen for 6 d, during which time no seed
germination occurred. Following removal from 0.1% oxygen, seed germination was scored daily. For the other stress treatments, seeds were plated on
half-strength MS medium containing 200 mM NaCl, 5% (w/v) mannitol, or 15
mM ABA. In all cases, germination was scored at intervals for 1 week. In all
cases, seeds were counted as germinated when the radicle penetrated the seed
coat. Data on the proportion of seeds germinated at 7 d from three replicate
experiments were analyzed with a one-way ANOVA blocked for replicate
experiments to detect significant differences between the lines. Arabidopsis
lines were grouped on the basis of germination percentage using Tukey’s
honestly significant difference.
Supplemental Table S5. Sequences of primers used.
Seed Staining
For GUS staining, plant material to be stained was immersed in a solution
composed of 3 mM 5-bromo-4-chloro-3-indolyl-b-glucuronic acid, 10 mM
EDTA, 100 mM NaPO4 buffer, pH 7.2, 0.3% Triton X, 500 mM ferricyanide,
500 mM ferrocyanide, and 10% methanol, vacuum infiltrated, and incubated at
37°C until color developed. Following staining, plant material was washed in
distilled water and cleared with two washes of 70% ethanol. Ungerminated
seeds from the low-oxygen germination assays were assayed for viability
using two vital staining methods, one using tetrazolium salts that turn from
colorless to pink in the presence of cellular respiration (Cottrell, 1947), and the
other utilizing a combination of propidium iodide, which cannot permeate
living cells, and fluorescein diacetate, which can, and is cleaved by cytoplasmic esterases to yield fluorescein (Jones and Senft, 1985). For tetrazolium
staining, seeds to be tested were scored with a 21-gauge needle to break the
seed coat. These seeds were then placed on filter paper soaked with a 1%
solution of tetrazolium salts and incubated in the dark overnight. For vital
staining with propidium iodide/fluorescein diacetate, embryos were dissected from seeds and both embryos and seed coats were stained with 10 mg
mL21 propidium iodide and 0.5 mg mL21 fluorescein diacetate for 5 to 10 min
before observation with a fluorescent microscope.
Microarray data from this article were submitted to the public National
Center for Biotechnology Information Gene Expression Omnibus database
(GEO accession no. GSE14420).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Expression levels of ANAC102 in knockout and
overexpressing lines.
Supplemental Figure S2. Expanded version of Figure 3 showing expression of selected genes over a time course in each of Col-0, C24, KO-1,
and OX-1.
Supplemental Figure S3. Survival after exposure to 0.1% oxygen for five
independent experiments.
Supplemental Figure S4. Heat map of gene expression for 23 low-oxygeninduced NAC genes.
Supplemental Table S2. Average root and shoot weights for Arabidopsis
lines following a 2-week recovery from low-oxygen stress treatments.
Supplemental Table S3. Germination of low-oxygen-treated seeds following cold treatment and seed mortality as determined by tetrazolium
staining.
ACKNOWLEDGMENTS
We thank Jun Yang for technical assistance and Donna Bond for providing
cDNA samples.
Received October 30, 2008; accepted January 23, 2009; published January 28,
2009.
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