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ASCORBATE PEROXIDASE6 Protects Arabidopsis
Desiccating and Germinating Seeds from Stress and
Mediates Cross Talk between Reactive Oxygen Species,
Abscisic Acid, and Auxin1[C][W][OPEN]
Changming Chen, Ilya Letnik, Yael Hacham, Petre Dobrev, Bat-Hen Ben-Daniel, Radomíra Vanková,
Rachel Amir, and Gad Miller*
Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel (C.C.,
I.L., B.-H.B.-D., G.M.); Laboratory of Plant Science, Migal Galilee Research Institute, Kiryat Shmona 12100,
Israel (Y.H., R.A.); Tel Hai College, Upper Galilee 12210, Israel (Y.H., R.A.); and Institute of Experimental
Botany Academy of Sciences of the Czech Republic, 16502 Prague 6, Czech Republic (R.V., P.D.)
A seed’s ability to properly germinate largely depends on its oxidative poise. The level of reactive oxygen species (ROS) in
Arabidopsis (Arabidopsis thaliana) is controlled by a large gene network, which includes the gene coding for the hydrogen
peroxide-scavenging enzyme, cytosolic ASCORBATE PEROXIDASE6 (APX6), yet its specific function has remained unknown.
In this study, we show that seeds lacking APX6 accumulate higher levels of ROS, exhibit increased oxidative damage, and display
reduced germination on soil under control conditions and that these effects are further exacerbated under osmotic, salt, or heat
stress. In addition, ripening APX6-deficient seeds exposed to heat stress displayed reduced germination vigor. This, together with
the increased abundance of APX6 during late stages of maturation, indicates that APX6 activity is critical for the maturation-drying
phase. Metabolic profiling revealed an altered activity of the tricarboxylic acid cycle, changes in amino acid levels, and elevated
metabolism of abscisic acid (ABA) and auxin in drying apx6 mutant seeds. Further germination assays showed an impaired
response of the apx6 mutants to ABA and to indole-3-acetic acid. Relative suppression of abscisic acid insensitive3 (ABI3) and
ABI5 expression, two of the major ABA signaling downstream components controlling dormancy, suggested that an alternative
signaling route inhibiting germination was activated. Thus, our study uncovered a new role for APX6, in protecting mature
desiccating and germinating seeds from excessive oxidative damage, and suggested that APX6 modulate the ROS signal cross
talk with hormone signals to properly execute the germination program in Arabidopsis.
Seed development and seed germination are two
critical phases in the plant life cycle. Dehydration and
rehydration during seed development or during germination are associated with high levels of oxidative
stress (Dandoy et al., 1987; Rajjou et al., 2012). Overaccumulation of reactive oxygen species (ROS) can cause
oxidative damage to a wide range of cellular components
and cause DNA damage, reducing the seed’s ability to
germinate (Bailly et al., 2008; Chen et al., 2011; Parkhey
et al., 2012). An optimal range of ROS levels is required
1
This work was supported by the Marie Curie ActionsInternational Career Integration Grant (grant no. 293999), the Israel
Science Foundation (grant no. 938/11), and the Czech Science Foundation (project no. 206/09/2062).
* Address correspondence to [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:
Gad Miller ([email protected]).
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Some figures in this article are displayed in color online but in
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www.plantphysiol.org/cgi/doi/10.1104/pp.114.245324
370
for successful germination (Bailly et al., 2008). Below this,
germination is suppressed (e.g. in dormant seeds), and
above it, cellular oxidative damage accumulates, delaying or inhibiting germination. This concept, termed the
oxidative window of germination, demonstrates the duality of ROS in seed physiology (Bailly et al., 2008). Experiments in rice (Oryza sativa), grasses, and Arabidopsis
(Arabidopsis thaliana), in which suppression of ROS
production inhibited germination, demonstrate the requirement of ROS for germination (Sarath et al., 2007;
Leymarie et al., 2012; Liu et al., 2012). It was further
suggested that ROS accumulation during a period of
dry storage following harvest, so called after-ripening,
acts as a key signal in changing proteome oxidation to
prepare the embryo for germination (Job et al., 2005;
Oracz et al., 2009). Arabidopsis nondormant seeds, in
which dormancy was alleviated by after-ripening or light
treatment, produced more ROS than dormant seeds
during imbibition (Leymarie et al., 2012).
The commitment of seeds to germination is determined during the seeds’ maturation on the mother plant,
with desiccation, accumulation of storage proteins, and
transcription of genes that are translated during imbibition (Rajjou et al., 2004, 2012; Finch-Savage and LeubnerMetzger, 2006; Finkelstein et al., 2008; Holdsworth et al.,
Plant PhysiologyÒ, September 2014, Vol. 166, pp. 370–383, www.plantphysiol.org Ó 2014 American Society of Plant Biologists. All Rights Reserved.
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Role of AP6 in Seed Physiology
2008). Therefore, the potential of seeds for rapid uniform
emergence and development under a wide range of field
conditions (i.e. seed vigor) greatly depends on the proper
execution of seed maturation- and desiccation-related
processes (Finch-Savage and Leubner-Metzger, 2006).
ROS also play a key regulatory role in the germination
program under favorable conditions (Sarath et al., 2007;
Liu et al., 2010; Bahin et al., 2011; Ye and Zhang, 2012).
Germination begins with the release of dormancy, which
is controlled by abscisic acid (ABA) and the activation
of GA, which control germination-promoting signals
(Finch-Savage and Leubner-Metzger, 2006; Finkelstein
et al., 2008).
Recent studies in Arabidopsis and barley (Hordeum
vulgare) have shown that hydrogen peroxide (H2O2) mediates the regulation of ABA catabolism, antagonizes ABA
signaling, and promotes GA synthesis and its germination
program (Liu et al., 2010; Bahin et al., 2011; Ishibashi
et al., 2012; Krishnamurthy and Rathinasabapathi, 2013).
Furthermore, dormancy release, in both dry and imbibed
states, has been associated with ROS production and the
specific oxidation of embryonic proteins, of fatty acids,
and of mRNA molecules (Job et al., 2005; Oracz et al.,
2007, 2009; Bazin et al., 2011). Protein carbonylation, the
most prevalent type of protein oxidation caused by ROS,
has been shown to target a specific set of embryo proteins and was suggested to be part of the dormancy
alleviation mechanism in sunflower (Helianthus annuus)
and Arabidopsis seeds (Job et al., 2005; Oracz et al., 2007,
2009). Recent studies also support interactions between
ROS, ethylene, cytokinin, and auxin in controlling seed
germination and early seedling development (Oracz et al.,
2009; Liu et al., 2010; Subbiah and Reddy, 2010; He et al.,
2012; Krishnamurthy and Rathinasabapathi, 2013; Lin
et al., 2013). All of these accumulated findings indicate
that ROS signals play key roles in seed development
and germination and demonstrate the diversity and
complexity of ROS function.
Because ROS metabolism and signaling are central
in dormancy and germination control, a tight regulation
is required to properly execute these programs while
avoiding oxidative stress. In Arabidopsis, there are over
150 enzymes dedicated to reducing ROS types, such as
H2O2, superoxide ions, and others, to their lesser reactive forms (Mittler et al., 2004; Miller et al., 2008, 2010).
Ascorbate peroxidases (APXs) comprise a small family
of nine enzymes in Arabidopsis that use ascorbic acid
(AA) as a substrate to reduce H2O2 to water (Mittler
et al., 2004; The Arabidopsis Information Resource 10).
Of the three cytosolic ascorbate peroxidases (cAPXs),
the functions of APX1 and APX2 are relatively well
established (Panchuk et al., 2002; Davletova et al., 2005;
Suzuki et al., 2013a). APX1 is the most abundant APX,
which functions in protecting cellular components,
including chloroplasts, from oxidative damage as well
as modifying cellular and intracellular ROS signals
(Davletova et al., 2005; Vanderauwera et al., 2011;
Suzuki et al., 2013b). In contrast, the APX2 expression
level is constitutively very low under normal conditions
but is highly induced in response to high temperatures
and increased light intensity (Panchuk et al., 2002;
Suzuki et al., 2013a). However, very little is known
about the expression of the third cAPX, APX6, and its
function is practically unknown. In this work, we identified the function of APX6 in seeds using two independent Arabidopsis knockout lines. Here, we show that
APX6 is important for protecting mature desiccating
seeds as well as germinating seeds from excessive oxidative stress and also functions in maintaining seed
vigor under stress conditions. In addition, we discovered
a novel interplay between ROS and ABA and between
ROS and auxin that could be interdependent.
RESULTS
Germination Phenotype of APX6-Deficient Mutants under
Favorable and Stress Conditions
To study the role of APX6 in Arabidopsis, three independent transfer DNA (T-DNA) insertion lines were
obtained from the Arabidopsis Biological Resource Center (Fig. 1A). Phenotypic evaluation of the identified homozygous lines indicated relatively slower and poorer
germination rates on soil as compared with the wild
type. The germination phenotype of apx6-1 and apx6-3
was consistent, and these lines were later shown in reverse transcription-PCR to be true knockout lines (Fig. 1B).
In contrast, the 39 untranslated region insertion line
apx6-2 showed an inconsistent germination phenotype
and showed APX6 expression similar to the wild type
(Fig. 1B). Therefore, only the apx6-1 and apx6-3 lines were
further characterized.
Following germination, under favorable conditions, the
seedlings and mature knockout plants were comparable
Figure 1. Gene structure and expression of APX6 in seeds. A, Gene
map of the APX6 gene model. Exons are represented by boxes (untranslated regions in gray and coding sequence in black) and introns by
the black line. The T-DNA insertions into the gene are shown as triangles. Arrowheads below represent primers (P1–P4) used for absence
verification of the APX6 transcript expression in B. B, Semiquantitative
PCR in dry seeds (DS) and in germinating seeds at 2 DAI. Col-0,
Columbia-0.
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Chen et al.
to wild-type plants, exhibiting similar growth rates and
mature plant sizes (Supplemental Fig. S1).
Germination of freshly harvested nonstratified apx6-1
and apx6-3 seeds on soil pellets resulted in a dramatic
delay and reduced level of germination, measured as
the appearance of cotyledons (Fig. 2A). Four days after
imbibition (DAI), only approximately 5% of the mutants were germinated, compared with 50% of the wild
type. The germination rate of apx6 mutants continued
steadily and slowly, reaching less than 45% germination
at 8 DAI (Fig. 2A). Stratification treatment (48 h at 4°C)
dramatically improved the germination rate, although it
did not completely reverse it (Fig. 2B). At 5 and 6 DAI,
germination was 63% and 85% relative to the wild type,
respectively (P , 5 3 1026 and P , 0.01, respectively). In
contrast, germination on plant growth medium (0.53
Murashige and Skoog [MS] medium and 0.8% agar) did
not result in apparent differences in the rate of germination (radicle emergence) between the wild type and
apx6 mutants (Fig. 2C). This delayed-germination phenotype on soil and the lack of it on MS medium was
consistent.
Soil pellets are relatively hyperosmotic compared
with MS media and the high-humidity environment inside the plates. This and the results in Figure 2 pointed us
to the possibility that APX6’s function might be in protecting germinating seeds under low water potentials. To
test this hypothesis, we germinated the wild type and
apx6 mutants on MS medium supplemented with sorbitol (100 and 200 mM) and NaCl (50 and 100 mM; Fig. 3).
Both apx6 lines showed severe inhibition of germination
in both treatments compared with the wild type. These
results clearly suggest that APX6 protects germinating
seeds from oxidative stress impediments that accompany osmotic stress.
We then evaluated the antioxidative impact of APX6
in seeds by comparing ROS levels and oxidative damage
accumulated in the dry seeds of apx6 and the wild type
(Fig. 4). The levels of H2O2 and superoxide radicals
measured in dry seed extracts were 28% to 35% and 20%
to 25% higher, respectively, in both apx6 lines compared
with the wild type (Fig. 4, A and B). In addition, the
higher levels of ROS in the mutants were correlated with
oxidative damage, as demonstrated by increased accumulation of carbonylated proteins (Fig. 4C). A general
peroxidase activity assay showed reduced activity in
dry seeds of apx6-1 (Supplemental Fig. S2), which is in
agreement with the elevated level of H2O2 and increased
oxidative damage. We further measured AA and total
glutathione in dry seeds of the wild type and apx6-1
(Supplemental Fig. S3). The level of total glutathione
accumulated in wild-type seeds was approximately 25%
higher on average compared with apx6-1. The level of
AA in apx6-1 was 5% lower and the level of dehydroascorbate (oxidized AA; DHA) was 25% higher on average compared with the wild type, although these
differences were not significant. However, the ratio of reduced to oxidized AA was significantly higher in the
wild type (P , 0.05, Student’s t test). Seeds of apx6
mutants were further tested for germination under oxidative stress on MS agar containing elevated concentrations of the superoxide-generating agent Paraquat. The
apx6 lines showed increased sensitivity, as demonstrated
by the dose-response-delayed germination of the mutant
relative to the wild type (Fig. 4D), indicating that
the apx6 lines are more sensitive to oxidative stress.
Taken together, the results presented in Figure 4 and
Supplemental Figure S3 suggested that there is a higher
oxidative level in dry apx6 than in the wild type.
Expression Profile of cAPXs in Maturing and
Germinating Seeds
A large degree of redundancy is assumed for the
ROS-gene network; however, some degree of specialization is expected (Mittler et al., 2004). This redundancy
Figure 2. Germination phenotype of apx6 mutants under favorable conditions. A and B, Germination rates of freshly harvested
seeds on soil pellets (A) and following stratification treatment at 4˚C for 48 h (B). The images above were taken 6 DAI and 4 d
after stratification (DAS) as indicated. The germination rate was scored as cotyledon emergence. C, Germination rate on 0.53
MS agar. Germination was scored as radicle emergence. The images above were taken at 48 HAI. SD values represent averages
of eight replicates of 50 seeds. Col-0, Columbia-0. [See online article for color version of this figure.]
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Role of AP6 in Seed Physiology
Figure 3. Germination assays of freshly
harvested seeds under hyperosmotic
conditions: 0.53 MS medium supplemented with NaCl or sorbitol. SD values
represent averages of eight replicates of
50 seeds. Col-0, Columbia-0. [See
online article for color version of this
figure.]
explains why the inhibition of germination in both
the wild type and apx6 mutants was not acute. A
Genevestigator developmental expression graph of all
nine Arabidopsis APXs revealed that, in seeds, only APX6
shows an increase while the levels of all others decrease
or remain unchanged (https://www.genevestigator.com/
gv/; Zimmermann et al., 2004; Supplemental Fig. S4). This
could lend support for a specific role of APX6 in seeds. An
Electronic Fluorescent Pictograph browser developmental
heat map in developing seeds further shows that APX6 is
uniformly abundant in all the seed’s tissues (Supplemental
Fig. S5; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi;
Winter et al., 2007). The Electronic Fluorescent Pictograph browser profile of germinating seeds has revealed
that the level of APX6 decreased sharply already within
3 h after imbibition (HAI; Winter et al., 2007). To learn
more about the expression of APX6 and the division of
labor among the three cAPXs, we determined their wildtype steady-state transcriptional levels in late maturing
siliques (stages 16–20; i.e. in mature desiccating seeds) as
well as in germinating seeds (0–72 HAI; Fig. 5). Real-time
PCR analysis uncovered reciprocal trends in the expression of APX1 and APX6; while the abundance of APX1
declined as seeds matured and desiccated, the APX6
mRNA level slowly accumulated (Fig. 5A), and the opposite occurred during germination (Fig. 5B). The maximal abundance of APX6 was detected in mature dry
seeds, which was about 30-fold higher than the level of
APX1 (Fig. 5B). APX6 transcript levels decreased sharply,
almost 20-fold, within 12 HAI, while the APX1 level slowly
increased, reaching a comparable but relatively low level
(Fig. 5B). The low levels of the three cAPXs persisted until
germination was completed with the emergence of the
radicle at 48 HAI. Following germination, the level of APX1
increased sharply, becoming once again the dominant
cAPX. The APX2 level also increased during seed maturation; however, it still remained much lower compared
with APX1 and APX6 and was undetectable throughout
germination (Fig. 5). Taken together, the gradual replacement of APX1 with APX6 in desiccating seeds and contrariwise in germinating seeds indicates specialization and
tight control over the expression of cAPXs.
ABA Response and Metabolic Profiling in Seeds
The reversal of germination of apx6 seeds by stratification treatment (Fig. 2B) strongly pointed to the involvement of ABA. Cross talk between ABA and ROS
was previously shown in several independent studies
(Sarath et al., 2007; Liu et al., 2010; Bahin et al., 2011;
Ishibashi et al., 2012; Ye et al., 2012; Lariguet et al.,
2013). To further investigate the possible relationship
between APX6 activity in seeds and ABA, freshly harvested seeds of the wild type, apx6, and abi4 as a positive
control were germinated on MS medium containing
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Chen et al.
Figure 4. ROS levels in dry seeds and germination response to oxidative stress. A, Relative levels of H2O2, normalized to the
wild-type level, were measured using the Amplex Red fluorescence assay. B, Relative levels of superoxide radicals (O2∙2) were
measured as formazan accumulation in nitroblue tetrazolium staining. C, Protein oxidation assay, showing carbonylated
proteins (right) on a western blot of 10 mg of total seeds and Coomassie Blue staining (left) as a loading control. D, Germination
assay was conducted on 0.53 MS medium containing Paraquat (PQ). SD values for all samples represent averages of eight
replicates of 50 seeds. Asterisks indicate Student’s t test significance at *P , 0.05 and **P , 0.01. Col-0, Columbia-0. [See
online article for color version of this figure.]
0.5 mM ABA (Fig. 6, A and B). ABA strongly inhibited
the germination of apx6-1 and apx6-3 compared with the
germination rate of wild-type and abi4 seeds. Five DAI,
only 54% of apx6 seeds showed emerged radicles (Fig. 6A),
with only minor progression within the following
4 d (Fig. 6B). Stratification treatment partially alleviated
the inhibition of ABA on the germination of apx6, resulting in a uniform and complete germination within
7 d after stratification. Yet, apx6 germination and the
development of the seedlings were still retarded compared with the wild type (Supplemental Fig. S6). These
results suggest that APX6 is involved in the release of
Figure 5. Developmental expression pattern of
cAPXs in the wild type. Relative expression of
APX1, APX2, and APX6 was determined by realtime PCR analyses during the final stages of silique development (seed maturation; A) and seed
germination (B). The fold change values, presented above and below the bars, are normalized
to the APX6 level in stage 16 siliques (in A) or to
the APX6 level in mature dry seeds (0 HAI; in B).
SD values represent averages of three replicates
for each stage. ND, Not determined.
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Role of AP6 in Seed Physiology
the ABA inhibitory effect during germination and perhaps also during early stages of seedling development.
Next, we examined whether differences in the expression of ABA-related genes in dry and imbibed seeds
could account for the hypersensitivity to ABA (Fig. 6,
C and D). We examined the expression of the ABA
signal transduction genes ABI3, ABI4, and ABI5 and the
expression of the ABA response marker genes EARLY
METHIONINE-LABELLED6 (EM6) and RESPONSIVE
TO DESSICATION 29B (Rd29b) and dehydrin Late Embryogenesis Protein (LEA) (At2g21490). Apart from a
slightly reduced EM6 level in apx6-1 imbibed seeds, no
significant changes were detected in ABA response
marker genes between apx6-1 and the wild type (Fig. 6,
C and D). In contrast, the transcript levels of ABI3 and
ABI5 showed mild suppression in dry seeds (a decrease
of 22% and 35% relative to the wild type, respectively)
that was further increased during imbibition (2.7- and
2.4-fold decrease relative to the wild type, respectively).
Conversely, ABI4 levels increased 45% and 75% relative
to the wild type in dry and imbibed seeds, respectively
(Fig. 6, C and D).
Next, we performed liquid chromatography-mass
spectrometry analyses to measure the levels of ABA, its
metabolic intermediates, as well as other phytohormones
that may be involved in regulation of the germination
phenotype of apx6 (Table I). The ABA level in apx6-1 was
40% higher than in the wild type. Furthermore, the levels
of inactivated forms of ABA were higher in the mutant,
particularly dihydrophaseic acid, which was more than
5-fold higher (P , 0.0001, Student’s t test). These findings
suggest that apx6 seeds have a higher level of ABA metabolism during maturation compared with the wild type.
Furthermore, the level of the active GA4 was 79%
higher (P , 0.005) in the mutant, while the level of GA19,
the precursor of GA1, was similar in both seed types.
Some effects on cytokinin metabolism were detected, as
differences were found in active and storage forms of
cytokinins and several inactive forms. However, no clear
trend was observed. Interestingly, the most striking
Figure 6. Response of apx6 mutants to ABA. A and B, Germination assay on 0.53 MS medium containing 0.5 mM ABA (A) and a
representative image showing germination at 9 DAI (B). SD values represent averages of eight replicates. C and D, Relative
transcript levels of ABA-responsive and signaling genes in dry seeds (C) and at 12 HAI (D). SD values represent averages of three
replicates. Asterisks indicate Student’s t test significance at *P , 0.05 and **P , 0.01. Col-0, Columbia-0. [See online article for
color version of this figure.]
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Chen et al.
Table I. Levels of phytohormones in dry seeds
Values are means 6 SD (pmol g21 fresh weight) and ratios as indicated.
significance at *P , 0.05 and **P , 0.01.
Group
ABAs
Auxins
GAs
Cytokinins
SD
values represents five replicates. Asterisks indicate Student’s t test
Compound
Feature
Wild Type
apx6-1
Ratio, apx6-1:Wild Type
ABA
PA
Dihydrophaseic acid
9OH-ABA
IAA
IPA
IAM
IAN
IAA-Asp
IAA-Glu
OxIAA
OxIAA-GE
GA4
GA19
tZ
tZR
DZR
iP
iPR
cZ
cZR
tZ7G
tZ9G
DZ9G
iP7G
iP9G
cZ7G
tZRMP
iPRMP
tZOG
tZROG
DZROG
cZOG
cZROG
Active
Deactivated
Deactivated
Deactivated
Active
Precursor
Precursor
Precursor
Degradation
Degradation
Degradation
Degradation
Active
Precursor
Active
Active
Active
Active
Active
Active
Active
Deactivated
Deactivated
Deactivated
Deactivated
Deactivated
Deactivated
Precursor
Precursor
Storage
Storage
Storage
Storage
Storage
335.62 6 83.82
2.38 6 1.03
4.50 6 0.87
0.37 6 0.29
330.80 6 78.02
55.28 6 11.07
1.6 6 1.40
1,919.9 6 1,122.84
557.29 6 248.39
2.96 6 1.69
8,193 6 2,090
257 6 94
55.50 6 17
29.36 6 20
1.53 6 0.62
4.13 6 1.06
0.56 6 0.20
0.22 6 0.16
0.97 6 0.32
0.75 6 0.30
5.07 6 1.76
2.26 6 0.43
0.43 6 0.14
0.06 6 0.02
49.36 6 12.25
0.46 6 0.24
6.46 6 0.98
1.43 6 0.39
0.99 6 0.27
0.51 6 0.10
2.19 6 0.87
0.25 6 0.11
0.56 6 0.34
0.42 6 0.22
483.59 6 136.17
3.65 6 1.16
23.10 6 4.98**
0.86 6 0.64
636.68 6 174.61*
67.67 6 19.78
8.22 6 6.20
1,028.79 6 184.31
1,233.18 6 692.24
33.58 6 23.79*
12,100.48 6 2,777.42
436.12 6 179.87
99.14 6 27.73*
27.79 6 22.34
2.23 6 0.36
4.54 6 1.10
0.40 6 0.08
0.08 6 0.01
0.90 6 0.25
1.73 6 0.81
6.80 6 1.41
2.52 6 0.75
1.00 6 0.32*
0.05 6 0.03
64.96 6 9.94
1.06 6 0.38*
11.17 6 3.11*
1.24 6 0.29
1.28 6 0.11
0.36 6 0.10
2.08 6 0.86
0.30 6 0.14
0.66 6 0.31
1.57 6 1.11
1.44
1.53
5.14
2.32
1.92
1.22
5.64
0.54
2.21
11.34
1.48
1.70
1.79
0.95
1.46
1.10
0.71
0.37
0.92
2.31
1.34
1.11
2.32
0.87
1.32
2.29
1.74
0.87
1.29
0.72
0.95
1.20
1.19
3.79
finding was the changes observed in auxin homeostasis. Indole-3-acetic acid (IAA) level in apx6 seeds
was almost twice that found in the wild type (P ,
0.05). Furthermore, the level of IAA-Glu, the auxin
degradation by-product, was 11 times higher in the
mutant (P , 0.005). Taken together, these metabolic
changes suggest that APX6 may play an important
role in regulating major hormone metabolic pathways
in desiccating seeds.
To gain a better understanding of the effect of APX6 on
metabolic processes in seeds, we performed a comparative nontargeted gas chromatography-mass spectrometry
(GC-MS) analysis for several primary metabolites (Table II),
including analysis for amino acids (Table III). Principal
component analysis comparison of all metabolites included in Tables II and III showed divergent distribution
for apx6-1 and wild-type samples, demonstrating the
dissimilarities between them (Supplemental Fig. S7).
The most notable changes in the primary metabolites
appeared in the tricarboxylic acid cycle intermediates,
succinate, citrate, fumarate, and malate, which were all
significantly elevated in apx6-1 (Table II). These results
suggest that desiccating apx6 seeds had an altered
tricarboxylic acid cycle activity during development and
that an increased respiration rate might exist. In contrast,
very little or no significant changes in the levels of sugars
and polyols were observed.
The levels of most amino acids were significantly
elevated in the mutant. Among the highly accumulated
amino acids (more than 2-fold) were Asn, Asp, and Pro
(Table III). Asp is produced from Glu and oxaloacetate,
which is also the tricarboxylic acid cycle intermediate
linking malate and citrate. This finding further corresponds with the accumulation in tricarboxylic acid cycle
metabolites (Table II) and suggests that the oxaloacetate
production rate is elevated in apx6 seeds. Pro functions
as a compatible solute that is well known to accumulate
during drought and salt stress to counteract the reduction in osmotic potential (Lehmann et al., 2010). The
levels of Leu, Ile, and Val, collectively known as coordinately regulated branched-chain amino acids (BCAAs),
similarly increased by 60% to 70% in apx6 seeds. Like
Pro, the three BCAAs are known to be involved in protection during abiotic stresses, particularly osmotic stress
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Role of AP6 in Seed Physiology
Table II. Levels of primary metabolites in dry seeds
Values are means 6 SD (peak area per 1,000) and ratios as indicated.
nificance at *P , 0.05, **P , 0.01, and ****P , 5 3 1024.
Group
Sugars
Polyols
Organic acids
Miscellaneous organic acid
Inorganic acids
SD
values represent five replicates. Asterisks indicate Student’s t test sig-
Compound
Wild Type
Fru monophosphate
Gluconic acid
Glc biphosphate
Glc monophosphate
Suc
Erythritol
Glycerol
Myoinositol
Sorbitol
Xylitol
Citrate
Fumarate
Malate
Succinate
Benzoate
Nicotinic acid
Palmitate
Pyro-Glu
Ethanolamine
Phosphoric acid
Hydroxylamine
2,725 6 871
872 6 168
3,629
25,475 6 9,644
111,163 6 13,640
494 6 129
4,114 6 402
11,692 6 691
1,789 6 748
161 6 16
4,727 6 839
1,568 6 322
2,294 6 321
337 6 54
3,245 6 346
1,687 6 148
2,765 6 1,005
31,141 6 8,245
10,923 6 4,439
1,066 6 203
5,345 6 528
(Joshi et al., 2010). In contrast, Trp is the only amino acid
depleted in apx6 seeds, showing a 2.7-fold decrease
compared with the wild type (Table I). This reduction is
inversely correlated with the accumulation of auxins,
which is logical, since Trp is the precursor for auxin
biosynthesis.
Auxin was most recently shown to promote seed
dormancy through the stimulation of ABA signaling (Liu et al., 2013). Our results in the apx6 mutant
apx6-1
Ratio, apx6-1:Wild Type
3,633 6 1,679
1,362 6 489*
5,050 6 1,155
34,960 6 6,984
144,169 6 33,243
634 6 82*
4,847 6 622
11,003 6 2,911
2,259 6 1,386
150 6 39
10,510 6 2,214****
8,963 6 2,432****
6,071 6 1,625****
471 6 83**
4,801 6 489****
3,177 6 327****
3,895 6 2,220
55,645 6 16,678**
9,078 6 2,216
2203 6 830**
7,277 6 1,739*
1.3
1.6
1.4
1.4
1.3
1.3
1.2
0.9
1.3
0.9
2.2
5.7
2.6
1.4
1.5
1.9
1.4
1.8
0.8
2.1
1.4
point to a possible link between IAA and ABA through
H2O2. To test the possibility that IAA is involved in the
reduced-germination phenotype of apx6 mutants, seeds
were germinated on plant medium containing IAA.
Both mutant lines showed clear inhibition at 50 mM and
an almost complete suppression at 100 mM (P , 0.001;
Fig. 7). This result suggests that there is interplay between IAA and H2O2 in the ABA-mediated dormancy
that is regulated by APX6 in seeds.
Table III. Levels of amino acids in dry seeds
Values are means 6 SD (nmol g21 fresh weight) and ratios as indicated. SD values represent five replicates. Asterisks indicate
Student’s t test significance at *P , 0.05, **P , 0.01, ***P , 5 3 1023, and ****P , 5 3 1024.
Amino Acid
Wild Type
apx6-1
Ratio, apx6-1:Wild Type
Ala
Asn
Asp
Cys
Glu
Gln
Gly
His
Homoserine
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
158.26 6 18.59
134.57 6 58.47
127.64 6 26.59
3.39 6 1.34
776.44 6 136.10
35.31 6 8.01
233.84 6 129.56
2.55 6 0.93
12.90 6 3.60
110.16 6 17.65
86.06 6 14.30
5.80 6 1.00
46.33 6 14.27
158.19 6 45.92
83.76 6 21.09
175.02 6 15.83
96.65 6 9.51
480.54 6 77.42
54.50 6 7.30
245.46 6 38.87
217.08 6 2.59****
290.98 6 86.70**
403.31 6 126.01****
3.84 6 0.69
1,149.97 6 211.60***
68.33 6 18.73***
255.62 6 174.45
4.36 6 1.13*
15.90 6 2.87
174.54 6 24.64****
139.16 6 19.71****
10.47 6 3.41**
52.08 6 8.75
240.10 6 36.42**
237.77 6 17.94****
280.70 6 66.30***
146.62 6 37.41**
177.25 6 31.61****
65.75 6 14.40
411.542 6 65.52****
1.37
2.16
3.16
1.13
1.48
1.94
1.09
1.71
1.23
1.58
1.62
1.80
1.12
1.52
2.84
1.60
1.52
0.37
1.21
1.68
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Chen et al.
Response and Tolerance of Seeds to Heat
Stress Conditions
Desiccation (i.e. drought stress) and heat stress (HS)
are two conflicting stresses, which are more harmful
when combined (Rizhsky et al., 2004; Suzuki et al.,
2014). To test whether desiccating apx6 seeds are more
sensitive to HS, we exposed 45-d-old flowering plants
bearing siliques (stages 17 and 18) to HS. Plants were
subjected to a daily regime of climbing temperatures,
starting at 24°C and peaking at 39°C or 42°C at midday. The dry seeds were collected after 5 and 6 d.
The impact of the 42°C HS on seed vigor was tested
by germination assays of freshly harvested stressed
seeds on either soil pellets or MS agar under controlled
conditions (Fig. 8, A and B). In both assays, apx6 seeds
showed dramatic retardation in germination compared
with wild-type seeds (Fig. 8, A and B). Western-blot
analysis of dry seeds collected from 39°C- or 42°C-stressed
plants revealed higher accumulation of the HS-responsive
proteins multiprotein bridging factor 1C (MBF1c) and
heat shock proteins in apx6 seeds (Fig. 8C). In addition,
APX1 and APX2 transcripts increased 2- and 2.5-fold,
respectively, in apx6 seeds collected at 42°C (Fig. 8D).
The higher accumulation of both HS-responsive APX1
and APX2 indicates the activation of a compensatory
mechanism to protect apx6 seeds from HS-associated
oxidative stress, which most likely prevents greater oxidative damage.
We next tested whether mature apx6 seeds are also
more sensitive to HS during germination. For this purpose, seeds of the wild type and apx6-1 collected under
control conditions (i.e. 24°C) were exposed to 42°C for 6,
12, 24, or 42 h. Following the HS period, the seeds were
transferred to 24°C for recovery and germination completion. The results revealed that the longer the stress
period persisted, the greater was the impairment in the
germination of apx6 compared with the wild type
(Supplemental Fig. S8). Since APX6 expression declines sharply within a few hours after imbibition
(Fig. 5B; Supplemental Fig. S5B), these findings suggest
that APX6 activity is required for seed thermotolerance
during the initial stage of germination and further
suggest that this stage is most vulnerable to stress. Taken
together, these results further support the function of
APX6 as an important antioxidative mechanism that
protects seeds from abiotic pressures during maturation
and germination.
DISCUSSION
In this study, new varied roles were identified for
cytosolic APX6 in Arabidopsis seeds during maturation and germination. We have shown that APX6 is a
major component of the antioxidative mechanism that
is important for seed vigor under favorable conditions
and even more so during stress.
The expression pattern of APX6 (Fig. 5; Supplemental
Fig. S5), together with the germination phenotypes of
the mutants, highlight the specialized function of APX6
Figure 7. Response of apx6 mutants to auxin. Freshly harvested seeds
were germinated on 0.53 MS medium with IAA. Sample images of
seed germination (A) and histograms of germination at 5 DAI (B) are
shown. SD values represent averages of eight replicates of 50 seeds.
****Student’s t test significance at P , 5 3 1024. Col-0, Columbia-0.
[See online article for color version of this figure.]
during seed desiccation and during the early stage of
germination. The function of APX6 seems to be critical
during the maturation-drying phase, in which the metabolism of the seed shifts from a general decrease in
unbound metabolites to the accumulation of a set of
specific metabolites (Angelovici et al., 2010). In addition,
the stored reservoir of APX6 in the dry seed would serve
in protecting the embryo from excessive oxidative pressure that accompanies the increased respiratory metabolism during imbibition. Furthermore, APX6 is required
to protect seeds against osmotic stress (Fig. 3), and in its
absence increased accumulation of Pro and BCAAs,
which function in osmotolerance (Joshi et al., 2010;
Lehmann et al., 2010), may provide partial compensation.
Multiple types of stress cause fluctuations in energy
that ultimately converge and generate energy deficiency
signals, resulting in energy sensor activation (BaenaGonzález and Sheen, 2008). This could be the case for
apx6 seeds undergoing maturation drying under favorable conditions and even more so during HS, which
further increases respiration rate, as indicated by the
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Role of AP6 in Seed Physiology
Figure 8. Effect of heat stress on mature drying seeds. A and B, Germination
assay on soil pellets (A) and on 0.53 MS medium (B) of seeds harvested from
plants grown under favorable conditions at 24˚C and seeds harvested from
plants exposed to HS (42˚C) for the last 5 to 6 d of their maturation (see
“Materials and Methods”). SD values represent averages of eight replicates of
50 seeds. C, Western blots showing HS-responsive proteins, MBF1c and
Hsp17.6, in dry seeds that experienced HS (39˚C and 42˚C) prior to harvesting
(during maturation on the mother plant). D, Relative transcript abundance of
the three cAPXs and other stress-responsive genes in the 42˚C-stressed dry
seeds. SD values represent averages of three replicates. Col-0, Columbia-0.
elevated expression of the ALTERNATIVE OXIDASE 1a
AOX1a) gene (Fig. 8D). Indeed, increased activation of
the tricarboxylic acid cycle, as indicated by the accumulation of its intermediates (Table II), could increase
energy generation (Angelovici et al., 2011; Galili, 2011)
to compensate for energetic depletion in the absence
of APX6.
The relative elevation of most amino acids could
potentially be attributed, at least in part, to an increase
in the degradation of oxidized proteins (Fig. 4D), since
carbonylated proteins are destined for degradation
(El-Maarouf-Bouteau et al., 2013). Positive feedback for
tricarboxylic acid cycle activity was recently shown due
to the contribution of catabolism of the Asp family
pathway amino acids, Lys, Met, Thr, Ile, and Gly (Galili,
2011). Activation of such positive feedback in apx6 seeds
might explain the fact that the level of Asp family
amino acids showed little or no increase compared with
the wild type, while the Asp level was 3-fold higher
(Table III). Additionally, an increased flow through the
tricarboxylic acid cycle could be achieved by the activation of the g-aminobutyrate (GABA) shunt, generating succinate from Glu via GABA. Activation of the
GABA shunt is associated with stress conditions and
seed desiccation (Angelovici et al., 2011; Fait et al., 2011)
and has been shown to prevent ROS accumulation
(Bouché et al., 2003). Our results showing that increase
in cytosolic H2O2 in desiccating seeds triggers metabolic
reprogramming indicate that APX6 is a major modulator of ROS signals in desiccating seeds. The new ROS
signal, seen in apx6 seeds and resulting in reduced
germination, revealed a strong cross talk with ABA
signaling. This was initially suggested by stratification
treatment, which almost completely alleviated the germination phenotype on soil (Fig. 2), and was further
confirmed by increased sensitivity to ABA (Fig. 6;
Supplemental Fig. S6). Correspondingly, germination of
apx6 mutants was also hypersensitive to NaCl and
sorbitol (Fig. 3). In contrast, ABA-insensitive mutants,
such as abi4 and abi5, and ABA-deficient mutants, such
as aba2, precariously germinate in the presence of NaCl
or mannitol compared with wild-type seeds, which are
protected by dormancy governed by ABA (Quesada
et al., 2000; González-Guzmán et al., 2002; Tezuka
et al., 2013).
Despite a 40% increase in the ABA content of dry
apx6 seeds, the higher and more significant accumulation was observed for ABA breakdown products
(Table I). This finding is in agreement with a previous
report showing that H2O2 mediates ABA catabolism
(Liu et al., 2010). In addition, H2O2 was shown to
promote GA biosynthesis (Liu et al., 2010; Bahin et al.,
2011), and indeed, the level of GA4 was 80% higher in
apx6 compared with the wild type. However, ABA and
GA are antagonistic; therefore, increased ABA homeostasis cannot solely account for the germination
phenotype of apx6.
In contrast with ABA metabolic changes, only minor
differences were observed between the wild type and
apx6 in the expression of the ABA-responsive marker
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Chen et al.
genes EM6 and RD29b and the dehydrin LEA protein
(Fig. 6, C and D). Interestingly, the transcript levels of
ABI3 and ABI5 were relatively lower in dry and imbibed
apx6 seeds, yet these seeds display reduced germination
and enhanced ABA sensitivity.
ABI3 has long been recognized as a major regulator
of seed dormancy and the ABA inhibition of seed germination (Bentsink and Koornneef, 2008). ABI5 functions
downstream of ABI3, and in concert they promote
dormancy and regulate the ABA-inducible expression
of LEA proteins such as EM1 and EM6 (Gampala et al.,
2002; Lopez-Molina et al., 2002). Taken together, our
results suggest that the germination phenotype in apx6
mutants is not mediated by ABI3 but rather through
another ABA-responsive signaling route.
In contrast, the level of ABI4 was elevated in dry and
imbibed apx6 seeds. ABI4 was recently shown to positively regulate dormancy by promoting ABA synthesis (Shu et al., 2013). However, it is still debatable
whether ABI4 actually affects seed dormancy (Liu et al.,
2013). Interestingly, recent evidence has shown that
ABI4 is involved in redox regulation and oxidative
challenges in Arabidopsis leaves (Giraud et al., 2009;
Foyer et al., 2012).
ABA synthesis and changes in ROS or redox are
linked through AA (Arrigoni and De Tullio, 2000;
Foyer et al., 2012; Ye and Zhang, 2012; Ye et al., 2012).
The activity of the 9-cis-epoxycarotenoid dioxygenase
enzyme responsible for the oxidative cleavage of neoxanthin to xanthoxin in the ABA synthesis pathway
depends on ascorbate (Arrigoni and De Tullio, 2000;
Foyer et al., 2012). Nonetheless, in leaves of the AAdeficient mutant vitamin C defective1, the level of ABA
was 60% higher than in the wild type, due to an
increase in ABA synthesis transcripts, including that
of 9-cis-epoxycarotenoid dioxygenase (Pastori et al.,
2003). Seeds of apx6 mutants contained slightly less
AA and more DHA than in the wild type, yet their
total is comparable in both seed types (Supplemental
Fig. S3). Potentially, the changes in the ratio between
AA and DHA, acting as a redox couple signal, together with elevation in ABI4 expression, may increase the synthesis rate of ABA and its accumulation.
In addition to the changes in ABA metabolism and
signaling, changes in auxin metabolism and signal perception (Table I; Fig. 7) were likely to affect the germination phenotype of apx6. Since Trp is the principal
precursor for IAA biosynthesis routes (Korasick et al.,
2013), its depletion in dry seeds of apx6 implies that the
accumulation in the mutant was due to de novo synthesis. The possibility that oxidative conditions in apx6
seeds enhance auxin biosynthesis has yet to be examined
and will be tested in the near future.
It was recently shown that high levels of auxin and
the activation of IAA signaling enhance ABA-mediated
dormancy by supporting the persistence of ABI3 expression during imbibition (Liu et al., 2013). Our observations indicate that both auxin and ABA are involved
in the germination phenotype of the apx6 mutants.
However, since ABI3 and ABI5 expression were relatively
suppressed in apx6 seeds, it is likely that the cross talk
between IAA and ABA does not involve activation of
the ABI3 signaling route.
Interestingly, ABI4 was shown to regulate the development of Arabidopsis lateral roots by reducing
polar auxin transport (Shkolnik-Inbar and Bar-Zvi,
2010). In addition, two recent studies revealed cross
talk between auxin homeostasis and ROS in seed
germination and primary root growth (He et al.,
2012; Jiao et al., 2013). Whether ABI4 participates in
the regulation of auxin homeostasis in seeds remains
to be determined.
Altered levels of auxin may also influence the vigor
of apx6 seeds under stress conditions and further feed
back on ROS generation. The involvement of IAA
cross talk with H2O2 in plant stress tolerance was recently
reviewed; however, the mechanistic details are not
well understood (Krishnamurthy and Rathinasabapathi,
2013). Accumulation of mitochondrial ROS in an Arabidopsis ABA overly sensitive mutant, abo6, deficient
in a splicing regulator of mitochondrial complex I rendered it sensitive to germination on ABA. Furthermore,
abo6 showed a decrease in auxin availability, and the
addition of auxin released the inhibition of germination
(He et al., 2012). In contrast, the level of auxin homeostasis in APX6-deficient seeds increased, and the addition
of IAA inhibited germination (Table I; Fig. 7). Therefore,
we suggest that our findings point to a different node of
this cross talk that is activated by an increase in cytosolic
H2O2 and that is involved in dormancy, germination
control, and the stress responsiveness of seeds. This
further suggests that, under favorable conditions,
the activation of this cross talk is suppressed by APX6
activity.
Our study adds to recent reports portraying a
complex relationship between ROS, ABA, and other
hormones in seed physiology. The cross talk unraveled in this study involving ROS, ABA, and IAA
does not activate the ABI3-mediated dormancy program and, therefore, may constitute a novel interplay
that is associated with oxidative stress in desiccating
seeds. Consideration should be given to the involvement of other signals in the oxidative stress response
in seeds, since the levels of GA and several cytokinins
were also altered in the mutant (Table I). APX6 emerges
as the dominant ascorbate peroxidase in mature drying seeds, protecting against stress and serving as a
modulator of cellular signals. These newly discovered
specialized roles in seeds for APX6, together with the
replacement of APX1 with APX6 in mature desiccating
wild-type seeds (Fig. 5), raise questions regarding the
differences between these two enzymes. It might be
that APX6 is better suited to withstand severe desiccation, a matter that will require further examination. Understanding the degree of redundancy versus
specialization in family members such as APXs, peroxiredoxins, and NADPH oxidases will immensely
increase our understanding of the plant ROS network activity in developmental and physiological
challenges.
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Role of AP6 in Seed Physiology
MATERIALS AND METHODS
Plant Materials, Growth Conditions, Germination Assays,
and Stress Treatments
Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used in this study.
APX6 T-DNA insertion lines (apx6-1, WISCDSLOX321C09; apx6-2, WiscDsLox466F10; and apx6-3, WiscDsLox337H07) were obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu/). Homozygous lines
were PCR identified according to SIGnAL Laboratory recommendations
(http://signal.salk.edu/tdnaprimers.2.html).
The abi4-1 mutant was used as a germination control in the presence of
ABA.
In all germination assays, the seeds were kept at 23°C under continuous low
light (50 mmol m–2 s–1) with 70% relative humidity. Freshly harvested seeds
were surface sterilized (4 min in 50% [v/v] ethanol and 3.4% [v/v] bleach
mixture, rinsed with 100% [v/v] ethanol, and dried on filter paper) and sown
on 0.53 MS agar (0.8% [w/v]) or directly placed on soil pellets (Jiffy 7000). All
the germination experiments were conducted within 1 week from harvesting
to preserve dormancy. Approximately 50 seeds in six to eight replicates were
placed on 0.53 MS plates per line per treatment. Plates were supplemented
with methyl viologen (Paraquat) for oxidative stress; with NaCl or sorbitol for
osmotic stress; and with ABA or IAA (Sigma-Aldrich) for hormone sensitivity
determination. The germination rate on MS agar was scored as radicle
emergence observed with a dissecting microscope (Leica M80) and on soil as
cotyledon greening. Germination on soil pellets took place without cover at
24°C at 70% humidity. HS on imbibed seeds was applied by incubating at 42°C
for 6 to 48 h, following by transfer to 24°C for germination to progress.
Mature seeds were collected from plants grown in mixed soil (3:2:1, peat
moss:vermiculite:perlite, v/v/v) and grown under controlled conditions in
growth chambers (Percival E-30, AR-66; Percival Scientific): 24°C, 16/8-h light/
dark cycle, 80 mmol m–2 s–1, and 70% relative humidity. The mutants and the
wild type were always grown side by side. HS during seed maturation was
done on 45-d-old plants bearing green mature stage 17 and 18 siliques (yellow
siliques were removed prior to treatment) grown in chambers as described
above. Gradual HS was applied according to the following program: light, 6 to
8 AM, 28°C; 8 to 10 AM, 32°C; 10 AM to 12 noon, 36°C; 12 noon to 4 PM, 39°C or
42°C; 4 to 6 PM, 36°C; 6 to 8 PM, 32°C; 8 to 10 PM, 28°C; dark, 10 PM to 6 AM, 23°C.
Seeds were collected after 5 and 6 d.
Molecular and Biochemical Analyses
RNA extraction from dry seeds was carried out according to the TRIzolbased method described previously (Meng and Feldman, 2010). All other
RNA extractions, PCR, complementary DNA synthesis, and real-time PCR
were done as described previously (Miller et al., 2009). First-strand complementary DNA was synthesized from 1 mg of total RNA (treated with RNase-free
DNase; New England Biolabs) at 42°C with Promega MV-Reverse Transcriptase.
Real-time PCR was performed on the Bio-Rad CFX96 Touch Real-Time PCR
Detection System with 40 cycles and an annealing temperature of 60°C. Cycle
threshold values were determined by CFX Manager Software assuming 100%
primer efficiency.
Primer sequences are listed in Supplemental Table S1. Extraction of total
protein, western blotting, and protein oxidation analyses were done as described previously (Miller et al., 2007).
Determination of relative H2O2 concentrations and relative total peroxidase
activity was performed as described previously (Xiong et al., 2007) with minor
modifications. The dry seeds (2–3 mg) were crushed with 0.1 mL of 0.2 M
HClO4, incubated on ice for 5 min, and centrifuged for 10 min at 14,000g and
4°C. The supernatant was neutralized with 0.2 M KOH and was centrifuged
again at 12,000g for 1 min. Quantification of H2O2 in extracts was performed
using a reaction with the Amplex Red reagent (Molecular Probes, Invitrogen)
with three technical repeats and six biological replicates. Samples were measured with the Synergy 4 fluorescence plate reader (Bio-Tek) using 530/590-nm
excitation/emission.
Relative peroxidase activity in total protein extracts from dry seeds was
determined according to Liu et al. (2010) with modifications. H2O2 was added
to a final concentration of 2 mM to extracts containing 200 mM Amplex Red on
microplates, and fluorescence generation was measured as described above.
The relative enzymatic activity of peroxidases was normalized to the total
amount of protein.
Relative superoxide concentration was determined according to Bournonville
and Díaz-Ricci (2011) with minor modifications. About 5 mg of dry seeds was
crushed with 0.5 mg mL21 nitroblue tetrazolium prepared in 10 mM potassium
phosphate buffer, pH 7.8, for 1 h in the dark at room temperature. Fomazan was
extracted using 1 mL of 2 M potassium hydroxide:chloroform (1:1, v/v).
Chloroformic extracts were light protected and completely dried in vacuum at
room temperature. The solid residue was dissolved in 350 mL of dimethyl
sulfoxide and 300 mL of 2 M potassium hydroxide at room temperature and
immediately analyzed with a Synergy 4 spectrophotometer. Formazan quantification was performed at 630 nm.
AA levels were determined calorimetrically as described previously (Gillespie
and Ainsworth, 2007) with the following minor adaptation for seeds: 40 mg of dry
seeds was used for the extraction and was extracted to a final volume of 1 mL.
GC-MS and Data Analysis of Primary Metabolites,
Including Amino Acids
Seeds from apx6-1 and wild-type lines were pooled from at least 100 pods.
Free amino acids were extracted from 20 mg of dry seeds. The amino acids
were detected using the single-ion method of GC-MS, as described previously
(Amira et al., 2005). For reduced glutathione determination, dry seeds (25 mg)
were ground with a mortar and pestle and then extracted and analyzed by
HPLC, as described previously (Matityahu et al., 2013). For primary metabolite analysis, samples were prepared as described for the free amino acids
and 7 mL of a retention time standard mixture (0.2 mg mL21 n-dodecane,
n-pentadecane, n-nonadecane, n-docosane, and n-octacosane) in pyridine. In
addition, 4.6 mL of a retention time standard mixture of nor-Leu and ribitol
(2 mg mL21) was added prior to trimethylsilylation. Samples were run on a
GC-MS system (Agilent 7890A series gas chromatography system coupled
with Agilent 5975c Mass Selective Detector), and a Gerstel multipurpose
sampler (MPS2) was installed on this system as described previously (Matityahu
et al., 2013). The data collected were obtained using the Agilent GC/MSD
Productivity ChemStation software. All peaks above the baseline threshold
were quantified and grouped according to retention time, with areas normalized to nor-Leu and ribitol. Substances were identified by comparison with
standards and were also compared with the commercially available electron
mass spectrum libraries from the National Institute of Standards and Technology and the Wiley Registry of Mass Spectral Data.
Plant Hormone Analysis
Seed samples were purified and analyzed essentially as described previously (Dobrev and Kamínek, 2002; Dobrev and Vankova, 2012). Dry seeds (30
mg) were homogenized with a ball mill (MM301; Retsch) and extracted in cold
(220°C) extraction buffer consisting of methanol:water:formic acid (15:4:1,
v/v/v). To account for sample losses and for quantification by isotope dilution,
the following stable isotope-labeled internal standards (10 pmol per sample)
were added: [13C6]IAA (Cambridge Isotope Laboratories), [2H6]ABA, [2H2]GA4,
[2H2]GA8, [2H2]GA19, [2H5]trans-zeatin, [2H5]trans-zeatin riboside, [2H5]transzeatin-7-glucoside, [2H5]trans-zeatin-9-glucoside, [2H5]trans-zeatin-O-glucoside,
[2H5]trans-zeatin riboside-O-glucoside, [2H5]trans-zeatin riboside monophosphate,
[2 H3 ]dihydrozeatin, [ 2H 3]dihydrozeatin riboside, [2H 3 ]dihydrozeatin-9glucoside, [2H6]isopentenyl adenine, [2H6]isopentenyl adenosine, [2H6]isopentenyl
adenine-7-glucoside, [2H6]isopentenyl adenine-9-glucoside, and [2H6]isopentenyl
adenosine monophosphate (Olchemim). Extract was evaporated in a vacuum
concentrator (Alpha RVC; Christ). Sample residue was dissolved into 1 mL of
0.1 M formic acid and applied to a mixed-mode reverse-phase cation-exchange
solid-phase extraction column (Oasis-MCX; Waters). Two hormone fractions
were eluted sequentially: (1) fraction A, eluted with methanol, containing
hormones of acidic and neutral character (auxins, ABA, and GA); and (2)
fraction B, eluted with 0.35 M NH4OH in 60% (v/v) methanol, containing
hormones of basic character (cytokinins). Fractions were evaporated to
dryness in the vacuum concentrator and dissolved into 30 mL of 10% (v/v)
methanol. An aliquot (10 mL) from each fraction was separately analyzed by
HPLC (Ultimate 3000; Dionex) coupled to a hybrid triple quadrupole/linear
ion-trap mass spectrometer (3200 Q TRAP; Applied Biosystems) set in selected
reaction monitoring mode. Quantification of hormones was done using the
isotope dilution method with multilevel calibration curves (r2 . 0.99). Data
processing was carried out with Analyst 1.5 software (Applied Biosystems).
Statistical Analysis
Principal component analysis of GC-MS data was done by MetaboAnalyst
2.0 (http://metaboanalyst.ca; Xia et al., 2009, 2012) on the log-transformed
Plant Physiol. Vol. 166, 2014
381
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Chen et al.
(base 10) data sets and pareto scaling (mean centered and divided by the
square root of SD of each variable) manipulation.
Arabidopsis Genome Initiative locus identifiers for the genes mentioned in
this article are as follows: APX1 (At1g07890), APX2 (At3g09640), APX6
(At4g32320), UBIQUITIN5 (At3g62250), ACTIN2 (AT3G18780), MBF1c
(At3g24500), ABI3 (At3g24650), ABI4 (At2g40220), ABI5 (At2g36270), DRE
BINDING FACTOR 1B (DREB1B) (At4g25490) DREB2B (At3g11020), EM6
(At2g40170), LEA (At2g21490), Rd29b (At5g52300), and AOX1a (At3g22370).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Mature wild-type and apx6 plants grown under
control conditions.
Supplemental Figure S2. Total peroxidase activity in dry seeds of the wild
type and apx6.
Supplemental Figure S3. AA and glutathione levels in dry seeds.
Supplemental Figure S4. Genevestigator developmental expression plot of
AtAPXs.
Supplemental Figure S5. Electronic Fluorescent Pictograph browser developmental expression pattern of cAPXs in the wild type.
Supplemental Figure S6. Germination of stratified seeds on 0.5 mM ABA.
Supplemental Figure S7. Principal component analysis of metabolites.
Supplemental Figure S8. Effect of HS during seed imbibition on germination.
Supplemental Table S1. Primer list and sequences.
Received June 16, 2014; accepted July 20, 2014; published July 21, 2014.
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