Running Head - Plant Physiology...2014/07/21 · ROS than dormant seeds during imbibition (Leymarie...

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Running Head

Role of APX6 in seed physiology

Corresponding author:

Gad Miller,

The Mina & Everard Goodman Faculty of Life Sciences,

Bar-Ilan University, Ramat-Gan 5290002, Israel

Tel/fax: 972-3-7384553

Email: [email protected]

Research Area

Signaling and Response

Plant Physiology Preview. Published on July 21, 2014, as DOI:10.1104/pp.114.245324

Copyright 2014 by the American Society of Plant Biologists

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Ascorbate peroxidase 6 protects Arabidopsis thaliana desiccating and

germinating seeds from stress and mediates crosstalk between ROS,

ABA and auxin

Changming Chen1, Ilya Letnik1, Yael Hacham2,3, Petre Dobrev3, Bat-Hen Ben-

Daniel1, Radomíra Vanková4, Rachel Amir2,3 and Gad Miller1*

1 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University,

Ramat-Gan 5290002, Israel 2 Laboratory of Plant Science, Migal Galilee Technology Center, P.O. Box 831,

Kiryat Shmona 12100, Israel 3 Tel Hai College, Upper Galilee, Israel 4 Institute of Experimental Botany AS CR, Rozvojová 263, 16502 Prague 6, Czech

Republic

One sentence summary

An ascrobate peroxidase functions to protect maturing and early germinating seeds

from stress and modulate the seed’s cellular metabolism and signaling.



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Financial sources: 1 This work was supported by the Marie Curie Actions-International Career

Integration Grant (grant no. 293999), the Israel Science Foundation (grant no.

938/11), and the Czech Science Foundation, project no. 206/09/2062.

* Corresponding author; e-mail [email protected]



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Abstract

The seed ability to properly germinate largely depends on its oxidative poise.

However the regulation of this state is not well understood. The level of reactive

oxygen species (ROS) in Arabidopsis thaliana is controlled by a large gene network,

which includes the gene coding for the H2O2-scaveging enzyme, cytosolic ascorbate

peroxidase 6 (APX6), yet its specific function 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

that is 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 increase 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 (TCA) cycle,

changes in amino acid levels, and elevated metabolism of ABA and auxin in drying

apx6 mutant seeds. Further germination assays showed impaired response of the apx6

mutants to ABA and to IAA. Relative suppression of ABI3 and ABI5 expression, two

of the major ABA-signaling downstream components controlling dormancy,

suggested that an alternative signaling route inhibiting germination was activated. Our

study, thus uncovered a new role for APX6, in protecting mature desiccating and

germinating seeds from excessive oxidative damage and suggest that APX6 modulate

the ROS signal crosstalking with hormone signals to properly execute the germination

program in Arabidopsis.



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Introduction

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., 2011). Over-accumulation 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 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, grasses and Arabidopsis in which suppression of ROS production inhibited

germination, demonstrates 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 non-dormant 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; Finch-

Savage and Leubner-Metzger, 2006; Finkelstein et al., 2008; Holdsworth et al., 2008;

Rajjou et al., 2011). 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 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 release of dormancy, which is controlled by

abscisic acid (ABA) and the activation of gibberelic acid (GA) that control



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germination-promoting signals (Finch-Savage and Leubner-Metzger, 2006;

Finkelstein et al., 2008).

Recent studies in Arabidopsis and barley have shown that 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

specific oxidation of embryonic proteins, of fatty acids and of mRNA molecules (Job

et al., 2005; Oracz et al., 2007; Oracz et al., 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 and Arabidopsis seeds (Job et al.,

2005; Oracz et al., 2007; Oracz et al., 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 evidences 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 hydrogen peroxides (H2O2), superoxide ions (O2.-) and

others, to their lesser reactive forms (Mittler et al., 2004; Miller et al., 2008; Miller et

al., 2010). Ascorbate peroxidases (APXs) comprise a small family of nine enzymes in

Arabidopsis that uses ascorbic acid as a substrate to reduce H2O2 to water (Mittler

2004; TAIR10). Of the three cytosolic (c) APXs, the function of APX1 and APX2, is

relatively well established (Panchuk et al., 2002; Davletova et al., 2005; Suzuki et al.,

2013). APX1 is the most abundant APX, that function 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., 2013). In contrast, 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., 2013). However,



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very little is known about the expression 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

in protecting mature desiccating seeds as well as germinating seeds from excessive

oxidative stress and also function 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 T-DNA insertion lines

were obtained from ABRC (Figure 1A). Phenotypic evaluation of the identified

homozygous lines indicated relative slower and poorer germination rates on soil as

compared to the wild type (WT). The germination phenotype of apx6-1 and apx6-3

was consistent and these lines were later shown in RT-PCR to be true knockout lines

(Figure 1B). In contrast, the 3’UTR insertion line apx6-2, showed inconsistent

germination phenotype and showed APX6 expression similar to the WT (Figure 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 to WT plants, exhibiting similar growth rates and

mature plant sizes (Figure S1).

Germination of freshly harvested non-stratified apx6-1 and apx6-3 seeds on soil

pellets resulted in a dramatic delay and reduced level of germination, measured as

appearance of cotyledons (Figure 2A). Four days after imbibition (DAI), only ~5% of

the mutants were germinated compared to 50% of the WT. The germination rate of

apx6 mutants continued steadily and slowly, reaching less than 45% germination 8

DAI (Figure 2A). Stratification treatment (48h at 4ºC) dramatically improved

germination rate, although it did not completely reverse it (Figure 2B). At 5 DAI and

6 DAI, germination was 63% and 85% relative to WT, respectively (P values <5E-6

and < 0.01, respectively). In contrast, germination on plant growth media (0.5 MS,

0.8% agar) did not result in apparent differences in the rate of germination (radicle



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emergence) between WT and apx6 mutants (Figure 2C). This delayed germination

phenotype on soil and the lack of it on MS media was consistent.

Soil is relatively hyperosmotic compared to MS plates, which has 100% humidity

and a relative hypoosmotic environment. This 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 WT and apx6 mutants on MS media

supplemented with sorbitol (100, 200 mM) and NaCl (50, 100 mM) (Figure 3). Both

apx6 lines showed severe inhibition of germination in both treatments compared to

WT. The 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 WT (Figure 4).

The levels of H2O2 and superoxide radicals measured in dry seeds extracts were 28-

35% and 20-25% higher, respectively, in both apx6 lines compared with the WT

(Figure 4A and 4B). In addition, the higher levels of ROS in the mutants were

correlated with oxidative damage demonstrated by increased accumulation of

carbonylated proteins (Figure 4C). A general peroxidase activity assay shows reduced

activity in dry seeds of apx6-1 (Figure S2), which is in agreement with the elevated

level of H2O2 and increased oxidative damage. We further measured ascorbic acid

(AA) and total glutathione in dry seeds of WT and apx6-1 (Figure S3). The level of

total glutathione accumulated in WT seeds was ~25% higher in average compared to

apx6-1. The level of reduced AA in apx6-1 was 5% lower and the oxidation of AA

(dehydroascorbate) was 25% higher in average compared to the WT, although these

differences were not significant. However, the ratio of reduced to oxidized AA was

significantly higher in the WT (P < 0.05, student 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 (PQ). The apx6 lines

showed increased sensitivity, as demonstrated by the dose-response delayed

germination of the mutant relative to the WT (Figure 4D), indicating that the apx6

lines are more sensitive to oxidative stress. Taken together the results presented in

figure 4 and figure S3 suggested that there is higher oxidative level in dry apx6 than

in the WT.



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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 explains

why inhibition of germination in both WT and apx6 mutants was not acute. A

Genevestigator developmental expression graph of all 9 Arabidopsis APXs reveal that

in seeds only APX6 show increase while the level of all others decrease or remains

unchanged (www.genevestigator.com; (Zimmermann et al., 2004); Figure S4). This

could lend support for a specific role of APX6 in seeds. An EF browser

developmental heat map in developing seeds further shows that APX6 is uniformly

abundant in all the seed’s tissues (Winter et al., 2007). EF browser profile of

germinating seeds has reveals that the level of APX6 sharply decrease already within

3 HAI (Winter et al., 2007). To learn more about the expression of APX6 and the

division of labor among the three cAPXs, we determined the WT 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 hours after imbibition, Figure 5). Real-

time PCR analysis uncovered reciprocal trends in the expression of APX1 and APX6;

while the abundance of APX1 declines as the seeds mature and desiccate, APX6

mRNA level slowly accumulates (Figure 5A) and the opposite occurrs during

germination (Figure 5B). The maximal abundance of APX6 was detected in mature

dry seeds, which was about 30 fold higher than the level of APX1 (Figure 5B). APX6

transcript levels sharply decreased almost 20 fold within 12 HAI, while APX1 level

slowly increased, reaching a comparable but relatively low level (Figure 5B). The low

levels of the 3 cAPXs persisted until germination was completed with the immergence

of the radicle at 48 HAI. Following germination, the level of APX1 sharply increases,

becoming once again the dominant cAPX. APX2 level also increased during seed

maturation however it still remained much lower compared to APX1 and APX6 and

was undetectable throughout germination (Figure 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 (Figure 2B)

strongly pointed to the involvement of ABA. Crosstalk 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



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further investigate the possible relationship between APX6 activity in seeds and ABA,

freshly harvested seeds of WT, apx6 and abi4 as a positive control were germinated

on MS media containing 0.5 µM ABA (Figure 6A and 6B). ABA strongly inhibited

germination of apx6-1 and apx6-3 compared with the germination rate of the WT and

abi4 seeds. Five days after imbibition, only 54% of apx6 seeds showed an emerged

radicle (Figure 6A), with only minor progression within the following four days

(Figure 6B). Stratification treatment partially alleviated the inhibition of ABA on

germination of apx6, resulting in a uniform and complete germination within 7 days

after stratification. Yet apx6 germination and development of the seedlings were still

retarded compared to the WT (Figure S6). These results suggest that APX6 is

involved in the release of 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 (Figure 6C and

D). We examined the expression of the ABA signal transduction genes, ABI3, ABI4,

ABI5 and the expression of the ABA response marker genes, EM6, Rd29b and

dehydrin LEA protein (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 WT (Figure 6C, 6D). In contrast, the transcript levels of

ABI3 and ABI5 showed mild suppression in dry seeds (a decrease of 22% and 35%

relative to the WT, respectively) that was further increased during imbibition (2.7 and

2.4 fold decrease relative to the WT, respectively). Conversely, ABI4 levels increased

45% and 75% relative to the WT in dry and imbibed seeds, respectively (Figure 6D,

6D).

Next, we performed LC-MS 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 1). The ABA level in apx6-1 was by 40%

higher than in the WT. Furthermore, the levels of inactivated forms of ABA were

higher in the mutant, particularly dihydrophaseic acid (DPA), which was more than 5

fold higher (P <0.0001, Student T-test). These findings suggest that apx6 seeds have a

higher level of ABA metabolism during maturation compared with the WT.

Furthermore, the level of the active gibberellin GA4 was by 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



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in active and storage forms of cytokinins and several inactive forms. However, no

clear trend was observed. Interestingly, the most striking finding was the changes

observed in auxins homeostasis. IAA level in apx6 seeds was almost twice as that

found in WT (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 non-targeted GCMS analysis for several primary

metabolites (Table 2) including analysis for amino acids (Table 3). Principal

Component Analysis (PCA) comparison of all metabolites included in tables 2 and 3

show divergent distribution for apx6-1 and WT samples, depicting the dissimilarities

between them (Figure S7). The most notable changes in the primary metabolites

appeared in the tricarboxylic acid (TCA) cycle intermediates, succinate, citrate,

fumerate and malate, which were all significantly elevated in apx6-1 (Table 2). These

results suggest that desiccating apx6 seeds had an altered TCA cycle activity during

development and that an increased respiration rate might exist. In contrast, very little

or no significant changes in 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 (> 2 fold) were asparagine, aspartate and

proline (Table 3). Aspartate is produced from glutamate and oxaloacetae, which is

also the TCA cycle intermediate linking malate and citrate. This finding further

corresponds with the accumulation in TCA cycle metabolites (Table 2), and suggests

that the oxaloacetate production rate is elevated in apx6 seeds. Proline functions as a

compatible solute that is well known to accumulate during drought and salt stress to

counteract reduction in osmotic potential (Lehmann et al., 2010). The levels of

leucine, isoleucine and valine, collectively known as coordinately regulated branched-

chain amino acid (BCAAs), similarly increased by 60-70% in apx6 seeds. Like

proline, the three BCAAs are known to be involved in protection during abiotic

stresses, particularly osmotic stress (Joshi et al., 2010). In contrast, tryptophan is the

only amino acid depleted in apx6 seeds, showing 2.7 fold decrease compared to the

WT (Table 1). This reduction is inversely correlated with the accumulation of auxins,

which is logical since tryptophan is the precursor for auxin biosynthesis.



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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 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 media containing IAA. Both mutant lines showed clear inhibition at 50µM and

an almost complete suppression at 100µM (P value <0.001) (Figure 7). This result

suggests that there is interplay between IAA and H2O2 in the ABA mediated

dormancy, which is regulated by APX6 in seeds.

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 days

old flowering plants bearing siliques (stages 17-18) to HS. Plants were subjected to a

daily regime of climbing temperatures starting at 24°C and peaking at 39°C or 42°C

midday. The dry seeds were collected after 5 and 6 days.

The impact of the 42°C HS on the seeds’ vigor was tested by germination assays of

freshly harvested seeds on either soil pellets or MS agar (Figure 8A and 8B). In both

assays, apx6 seeds showed dramatic retardation in germination compared to WT seeds

(Figure 8A-B). Western blot analysis on dry seeds collected from 39°C or 42°C

stressed plants revealed higher accumulation of the HS-responsive proteins MBF1c

and Hsps in apx6 seeds (Figure 8C). In addition, APX1 and APX2 transcripts

increased 2 and 2.5-fold, respectively in apx6 seeds collected at 42 °C (Figure 8D).

The higher accumulation of both HS-responsive APX1 and APX2 transcripts (Suzuki

et al., 2013) indicate the activation of a compensatory mechanism to protect apx6

seeds from HS-associated oxidative stress that 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 WT and apx6-1 collected under control

conditions (i.e. 24°C) were exposed to 42°C for 6, 12, 24 or 42 consecutive hours.

Following the HS period, the plants were transferred to 24°C for recovery and

germination completion. Results revealed that the longer the stress period persisted,

the greater the impairment was in the germination of apx6 compared to the WT

(Figure S8). Since APX6 transcript levels sharply decline within a few hours after

imbibition (Figures 5B and S5B), these findings suggest that APX6 activity is



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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 anti-oxidative mechanism that

protect seeds from abiotic pressures during maturation and during 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 (Figures 5 and S5) together with the germination

phenotypes of the mutants, highlight the specialized function of APX6 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 for protecting seeds against osmotic stress (Figure 3),

and in its absence increased accumulation of proline and BCAAs, which function in

osmo-tolerance (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 sensors activation (Baena-

Gonzalez 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 increase respiration rate, as indicated by the elevated expression of the AOX1

gene (Figure 8D). Indeed, increased activation of the TCA cycle, as indicated by the

accumulation of its intermediates (Table 2), 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 protein degradation of oxidized proteins (Figure 4D), since

carbonylated proteins are destined for degradation (El-Maarouf-Bouteau et al., 2013).



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Positive feedback for TCA cycle activity was recently shown due to contribution of

catabolism of the Asp-family pathway amino acids, Lys, Met, Thr, Ile and Gly, to the

TCA cycle (Galili, 2011). Activation of such positive feedback in apx6 seeds might

explain the fact that the level of Asp-family amino acids showed only little or no

increase compared with the WT, while Asp level was 3 fold higher (Table III).

Additionally, an increased flow through the TCA cycle could be achieved by the

activation of the �-aminobutirate (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 (Bouche et al., 2003). Our results showing that increase in

cytosolic H2O2 in desiccating seeds triggers metabolic reprogramming, indicates 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, reveals a strong crosstalk

with ABA signaling. This was initially suggested by stratification treatment, which

almost completely alleviated the germination phenotype on soil (Figure 2), and was

further confirmed by increased sensitivity to ABA (Figures 6 and S6).

Correspondingly, germination of apx6 mutants is also hypersensitive to NaCl and

sorbitol (Figure 3). Contrary, ABA insensitive mutants, such as abi4, abi5, sag and

ABA deficient mutants, such as aba2, precariously germinate in the presence of NaCl

or manitol compared to WT seeds which are protected by dormancy governed by

ABA (Quesada et al., 2000; Gonzalez-Guzman 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 1). 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 to the WT. However, ABA and GA are antagonistic

and 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 WT and apx6 in the expression of the ABA-responsive marker-genes Em6,

the dehydrin-LEA protein and RD29b (Figure 6C, 6D). Interestingly, the transcripts



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level 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 ABA

inhibition of seed germination (Bentsink and Koornneef, 2008). ABI5 functions

downstream of ABI3 and in concert they promote dormancy and regulate 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 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 ascorbic acid

(Arrigoni and De Tullio, 2000; Foyer et al., 2012; Ye and Zhang, 2012; Ye et al.,

2012). The activity of the NCED 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

ascorbic acid deficient mutant vtc1, the level of ABA was 60% higher than in the WT

due to an increase in ABA synthesis transcripts including that of NCED (Pastori et al.,

2003). In apx6 seeds there is slightly less reduced AA and more DHA than in the WT,

yet the total AA (reduced + oxidized) is comparable in both (Figure S3). Potentially,

the changes in the ratio between reduced 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 1, Figure 7) were likely to affect the

germination phenotype of apx6. Since trypthophan is the principal precursor for IAA

biosynthesis routes (Korasick et al., 2013), its depletion in dry seeds of apx6 implies

that the accumulation in mutant was due to de novo synthesis. The possibility that

oxidative conditions in apx6 seeds enhance auxin biosynthesis was yet examined and

will be tested in the near future.



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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 crosstalk 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 crosstalk between auxin homeostasis and ROS in

seed germination and primary root growth (He et al., 2012; Jiao et al., 2013). Whether

ABI4 participates in 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 feedback on ROS generation. The involvement of IAA

crosstalk 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 decrease in auxin

availability and 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 addition of IAA inhibited germination (Table I, Figure 7). Therefore, we suggest

that our findings point to a different node of this crosstalk that is activated by increase

in cytosolic H2O2 and that is involved in dormancy, germination control and stress

responsiveness of seeds. This further suggests that under favorable conditions the

activation of this crosstalk is suppressed by APX6 activity.

Our study adds to recent reports portraying a complex relationship between ROS,

ABA and other hormones in seeds’ physiology. The crosstalk unraveled in this study

involving ROS, ABA and IAA, does not activate ABI3-mediated dormancy program

and therefore may constitute a novel interplay that is associated with oxidative stress

in desiccating seeds. Consideration should be taken for the involvement of other

signals in the oxidative stress response in seeds, since the levels of GA and several



17

cytokinins were also altered in the mutant (Table I). APX6 emerge 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 WT

seeds (Figure 5), raises 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.

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 ABRC

(https://abrc.osu.edu/). Homozygous lines were PCR-identified according to the

SIGnAL Laboratory recommendations (http://signal.salk.edu/tdnaprimers.2.html).

The abi4-1 mutant was used for germination control in the presence of ABA.

In all germination assays, the seeds were kept at 23°C under continuous low light

(50 μmol m–2 s–1) with 70% relative humidity. Freshly harvested seeds were surface

sterilized (4 min in 50% (v/v) ethanol, 3.4% (v/v) bleach mixture, rinsed with 100%

ethanol, and dried on filter paper) and sowed on 0.5 MS agar (0.8% w:v) or directly

placed on soil pellets (Jiffy 7000). All the germination experiments were conducted

within one week from harvesting, to preserve dormancy. Approximately 50 seeds in

6-8 replicates were placed in 0.5 MS plates per line per treatment. Plates were

supplemented with methyl viologen (paraquat), for oxidative stress; with NaCl or

sorbitol for osmotic stresses and ABA or IAA (Sigma Aldrich) for hormone

sensitivity determination. The germination rate on MS was scored as radicle

emergence under a dissecting microscope (Leica M80), and on soil as cotyledon



18

greening. Germination on soil pellets took place without cover at 24°C, at 70%

humidity. Heat stress on imbibed seeds was applied by incubating at 42°C for 6 to 48

hours, 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 light/dark cycle, 80

μmol m–2 s–1, and 70% relative humidity. The mutants and the wild type were always

grown side by side. Heat stress during seed maturation was done on 45-day-old plants

bearing green mature stage 17-18 siliques (yellow siliques were removed prior to

treatment), grown in chambers as described above. Gradual HS was applied according

to the following program: Light: 06:00–08:00, 28 °C; 08:00–10:00, 32 °C; 10:00–

12:00, 36°C; 12:00–16:00, 39°C or 42 °C; 16:00–18:00, 36 °C; 18:00–20:00, 32°C;

20:00–22:00, 28°C; Dark: 22:00–6:00, 23°C. Seeds were collected after 5 and 6 days.

Molecular and biochemical analyses

RNA extraction from dry seeds was carried out according to TRIzol-based method

previously described (Meng and Feldman, 2010). All other RNA extractions, PCR,

cDNA synthesis, real-time PCR were done as previously described (Miller et al.,

2009). First-strand complementary DNA was synthesized from 1 µg of total RNA

(treated with RNase-free DNase; New England Biolabs) at 42°C with Promega MV-

Reverse Transcriptase. Real-time PCR were performed on a 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 blots and protein oxidation analyses were done as previously described

(Miller et al., 2007).

Determination of relative H2O2 concentrations and relative total peroxidase

activity were performed as previously described (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, centrifuged for 10 min at 14,000g, 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 3 technical repeats and 6 biological



19

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 µM Amplex-Red in microplates and

fluorescence generation was measured as above described. The relative enzymatic

activity of peroxidases was normalized to total amount of protein.

Relative superoxide concentration was determined according to (Bournonville

and Diaz-Ricci, 2011) with minor modifications. About 5 mg of dry seeds were

crushed with 0.5 mg/ml NBT 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 DMSO 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.

Ascorbic acid level were determined calorimetrically as previously describes

(Gillespie and Ainsworth, 2007) with the following minor adaptation for seeds; 40 mg

of dry seeds were used for the extraction and were extracted to a final volume of 1 ml.

GC-MS and data analysis of primary metabolites including amino acids

Seeds from apx6-1 and WT 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 (SIM) of GC-MS, as previously described (Golan et al.,

2005). For GSH determination, Dry seeds (25 mg) were ground with a mortar and

pestle and then extracted and analyzed by HPLC, as previously described (Matityahu

et al., 2013). For primary metabolite analysis, samples were prepared as described for

the free amino acids and 7 μl of a retention-time-standard mixture (0.2 mg/ml n-

dodecane, n-pentadecane, n-nonadecane, n-docosane and n-octacosane, in pyridine).

In addition, 4.6 μl of a retention time standard mixture of norleucine and ribitol (2

mg/ml) were added prior to trimethylsilylation. Samples were run on GC-MS system

(Agilent 7890A series GC system coupled with Agilent 5975c Mass Selective

Detector), and a Gerstel ® multipurpose sampler (MPS2) was installed on this system

as previously described (Matityahu et al., 2013). The data collected were obtained



20

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 norleucine and ribitol. Substances were identified by comparison with

standards, and were also compared with the commercially available electron mass

spectrum libraries, NIST and WILEY.

Plant hormone analysis

Seed samples were purified and analyzed essentially as previously described (Dobrev

and Kaminek, 2002; Dobrev and Vankova, 2012). Dry seeds (30 mg) were

homogenized with ball mill (MM301, Retsch) and extracted in cold (-20oC) 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-transZ, 2H5-

transZR, 2H5-transZ7G, 2H5-transZ9G, 2H5-transZOG, 2H5-transZROG, 2H5-

transZRMP, 2H3-DHZ, 2H3-DHZR, 2H3-DHZ9G, 2H6-iP, 2H6-iPR, 2H6-iP7G, 2H6-

iP9G, 2H6-iPRMP (Olchemim). Extract was evaporated in vacuum concentrator

(Alpha RVC, Christ). Sample residue was dissolved into 1 ml 0.1 M formic acid and

applied to mixed mode reverse phase – cation exchange SPE column (Oasis-MCX,

Waters). Two hormone fractions were sequentially eluted: (1) fraction A eluted with

methanol – containing hormones of acidic and neutral character (auxins, ABA, GA),

and (2) fraction B eluted with 0.35 M NH4OH in 60% methanol – containing the

hormones of basic character (cytokinins). Fractions were evaporated to dryness in

vacuum concentrator and dissolved into 30 µl 10% methanol. An aliquot (10 µl) from

each fraction was separately analysed on HPLC (Ultimate 3000, Dionex) coupled to

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 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 (PCA) of GC-MS data was done by the MetaboAnalyst

2.0, (http://metaboanalyst.ca; (Xia et al., 2009; Xia et al., 2012), on the log-

transformed (base 10) data sets and pareto scaling (mean-centered and divided by the

square-root of standard deviation of each variable) manipulation.



21

Gene identifier numbers

Arabidopsis Genome Initiative locus identifiers for genes mentioned in this article are

as follows: APX1 (AT1G07890), APX2 (AT3G09640), APX6 (AT4G32320),

Ubiquitin5 (AT3G62250), MBF1c (AT3G24500), ABI3 (AT3G24650), ABI4

(AT2G40220), ABI5 (AT2G36270), DREB1B (AT4G25490) DREB2B (AT3G11020),

Em6 (At2g40170), LEA (At2g21490), Rd29b (At5g52300), AOX1a (AT3G22370).

Supplemental Data

Supplental Table S1. Primers list and sequence.

Supplemental Figure S1. Mature WT and apx6 plants grown under control

conditions.

Supplemental Figure S2. Total peroxidase activity in dry seeds of WT and apx6.

Supplemental Figure S3. Ascorbic acid and glutathione levels in dry seeds.

Supplemental Figure S4. Genevestigator developmental expression plot of AtAPXs.

Supplemental Figure S5. EF browser developmental expression pattern of cAPXs in

WT

Supplemental Figure S6. Germination of stratified seeds on 0.5µM ABA.

Supplemental Figure S7. Principal component analysis of metabolites.

Supplemental Figure S8. Effect of HS during seed imbibition on germination.



22

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Figure legends

Figure 1. Gene structure and expression of APX6 in seeds. Gene map of the APX6

gene model (A). Exons are represented by boxes (UTRs in grey and cds in black) and

introns by a black line. The T-DNA insertions into the gene are shown as triangles.

Arrows below represent primers (P1-P4) used for absence verification of the APX6

transcript expression by semi-quantitative PCR (B) in dry seeds (DS) and in

germinating seeds two days after imbibition (DAI).

Figure 2. Germination phenotype of apx6 mutants under favorable conditions.

Germination rates of freshly harvested seeds on soil pellets (A) and following

stratification treatment at 4 °C for 48 h (B). Pictures above were taken 6 days after

imbibition (DAI) and 4 days after stratification (DAS), respectively. The germination

rate was scored as cotyledon immergence. Germination rate on 1/2MS agar (C),

germination was scored as radicle emergence. The pictures above were taken at 48

HAI. Standard deviations represent average of 8 replicates of 50 seeds.

Figure 3. Germination assays of freshly harvested seeds under hyperosmotic

conditions; ½ MS supplemented with NaCl or sorbitol. Standard deviations represent

an average of 8 replicates of 50 seeds.

Figure 4. ROS levels in dry seeds and germination response to oxidative stress.

Relative levels of H2O2, normalized to the WT level, were measured using the

amplex-red fluorescence assay (A). Relative levels of superoxide radicals were

measured as formazan accumulation in NBT staining (B). Protein oxidation assay (C),

showing carbonylated proteins (right) in western blot of 10µg total seeds and

coomasie stain (left) as loading control. The germination assay was conducted on

1/2MS containing paraquat (PQ). Standard deviations for all samples represent an

average of 8 replicates of 50 seeds. * and **Student’s t test significance at P < 0.05

and P < 0.01, respectively.

Figure 5. Developmental expression pattern of cAPXs in the WT. Relative expression

of APX1, APX2 and APX6 determined by real time PCR analyses during the final

stages of silique development (seed maturation) (A) and seed germination (B). The



28

fold change values presented above the bars, are normalized to APX6 level in stage

16 siliques (in A), or to APX6 level in mature dry seeds (0 HAI in B). Standard

deviations represent an average of 3 replicates for each stage.

Figure 6. Response of apx6 mutants to ABA. Germination assay on 1/2 MS

containing 0.5µM ABA (A) and representative picture showing germination at 9 DAI

(B). Standard deviations represent an average of 8 replicates. Relative transcript levels

of ABA-responsive and –signaling genes in dry seeds (C) and at 12 HAI (D).

Standard deviations represent an average of 3 replicates. * and **Student’s t test

significance at P < 0.05 and P < 0.01, respectively.

Figure 7. Response of apx6 mutants to auxin. Freshly harvested seeds were

geminated on 1/2MS with IAA. Sample pictures of seed germination (A) and

histograms (B) of germination at the 5th DAI. Standard deviations represent an

average of 8 replicates of 50 seeds. **** Student’s t test significance at P < 5*10-4.

Figure 8. Effect of heat stress on mature drying seeds. Germination assay on soil

pellets (A) and on 1/2 MS (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

5-6 days of their maturation (see materials and methods). Standard deviations

represent an average of 8 replicates of 50 seeds. Western blot 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) (C). Relative transcript

abundance of the three cAPXs and other stress-responsive genes in the 42°C-stressed

dry seeds (D). Standard deviations represent an average of 3 replicates.

Supplemental Figure S1. Mature (28 days old) WT and apx6 plants grown under

control conditions.

Supplemental Figure S2. Total peroxidase activity in dry seeds of WT and apx6.

Activity was measured in soluble protein extracts using amplex-red. Standard

deviations represent an average of 8 replicates.

Supplemental Figure S3. Reduced and oxidize ascorbic acid levels and total



29

glutathione level in dry seeds. Reduced acorbate (A) and dehydroascorbate (B) were

measured calorimetricaly (see material and methods). Total GSH was determined by

GCMS analysis. Standard deviations represent an average of 6 replicates.

* indicates t test significance at P < 0.05.

Supplemental Figure S4. Developmental gene expression graph for all nine APXs in Arabidopsis. The figure was generated by Genevestigator (www.genevestigator.com ; Zimmermann et al. 2004) APX1, 2, 6 are cytosolic; APX3 and 5 are microsomal; APX4, sAPX and tyl APX are plastidic; APX7 is currently considered as a pseudogene. Annotations are according to TAIR website (http://www.arabidopsis.org). Supplemental Figure S5. Expression of APX6 in germinating and developing seeds.

Plots were made by the EF browser website.

Supplemental Figure S6. Germination on 0.5µM ABA following stratification

treatment. Plates were kept for 48 h at 4°C and then transferred to 24°C. Control;

1/2MS. The picture was taken 7 days after stratification treatment.

Supplemental Figure S7. Principle component analysis (PCA) of different

metabolites that are shown in tables 2 and 3, which were detected by GCMS from dry

seeds. Six biological replicates of Col-0 (green) and apx6-1 mutant (red) are shown.

The variance explained by each component is given within parentheses. Six biological

replicates were taken.

Supplemental Figure S8. Effect of HS on imbibed seeds. Seeds germinated on

1/2MS were immediately exposed to 42°C for incremental time periods and then

transferred to 24°C to complete the germination process (radicle emergence).

Standard deviations represent an average of 8 replicates of 50 seeds. Standard

deviations represent an average of 6 replicates.



30

Table I. Level of Phytohormones in dry seeds

pmol/gFW

Col-0 apx6-1 apx6-1/WT

Group Compound Feature Mean (±SD) Mean (±SD) Ratio

Abscisic acids ABA Active 335.62 ± 83.82 483.59 ± 136.17 1.44

PA Deactivated 2.38 ± 1.03 3.65 ± 1.16 1.53

DPA Deactivated 4.50 ± 0.87 23.10 ± 4.98** 5.14

9OH-ABA Deactivated 0.37 ± 0.29 0.86 ± 0.64 2.32

Auxins IAA Active 330.80 ± 78.02 636.68 ± 174.61* 1.92

IPA Precursor 55.28 ± 11.07 67.67 ± 19.78 1.22

IAM Precursor 1.6 ± 1.40 8.22 ± 6.20 5.64

IAN Precursor 1919.9 ± 1122.84 1028.79 ± 184.31 0.54

IAA-Asp Degradation 557.29 ± 248.39 1233.18 ± 692.24 2.21

IAA-GLU Degradation 2.96 ± 1.69 33.58 ± 23.79* 11.34

OxIAA Degradation 8193 ± 2090 12100.48 ± 2777.42 1.48

OxIAA-GE Degradation 257 ± 94 436.12 ± 179.87 1.70

GA4 Active 55.50 ± 17 99.14 ± 27.73* 1.79

Gibbrrellins GA19 Precursor 29.36 ± 20 27.79 ± 22.34 0.95

Cytokinins tZ Active 1.53 ± 0.62 2.23 ± 0.36 1.46 tZR Active 4.13 ± 1.06 4.54 ± 1.10 1.10 DZR Active 0.56 ± 0.20 0.40 ± 0.08 0.71 iP Active 0.22 ± 0.16 0.08 ± 0.01 0.37 iPR Active 0.97 ± 0.32 0.90 ± 0.25 0.92

cZ Active 0.75 ± 0.30 1.73 ± 0.81 2.31 cZR Active 5.07 ± 1.76 6.80 ± 1.41 1.34 tZ7G Deactivated 2.26 ± 0.43 2.52 ± 0.75 1.11

tZ9G Deactivated 0.43 ± 0.14 1.00 ± 0.32* 2.32 DZ9G Deactivated 0.06 ± 0.02 0.05 ± 0.03 0.87 iP7G Deactivated 49.36 ± 12.25 64.96 ± 9.94 1.32 iP9G Deactivated 0.46 ± 0.24 1.06 ± 0.38* 2.29 cZ7G Deactivated 6.46 ± 0.98 11.17 ± 3.11* 1.74 tZRMP Precursor 1.43 ± 0.39 1.24 ± 0.29 0.87

iPRMP Precursor 0.99 ± 0.27 1.28 ± 0.11 1.29 tZOG Storage 0.51 ± 0.10 0.36 ± 0.10 0.72 tZROG Storage 2.19 ± 0.87 2.08 ± 0.86 0.95

DZROG Storage 0.25 ± 0.11 0.30 ± 0.14 1.20 cZOG Storage 0.56 ± 0.34 0.66 ± 0.31 1.19 cZROG Storage 0.42 ± 0.22 1.57 ± 1.11 3.79

Standard errors represents 5 replicates. * and **Student’s t test significance at P <

0.05 and P < 0.01, respectively.



31

Table II. Level of primary metabolite in dry seeds

Peak area/1000

WT apx6-1 Ratio Group Compound Mean ± SD Mean ± SD apx6-1/WT Sugars Fructose MP 2,725 ± 871 3,633 ± 1,679 1.3 Gluconic acid 872 ± 168 1,362 ± 489* 1.6

Glucose BP 3,629 5,050 ± 1,155 1.4 Glucose MP 25,475 ± 9,644 34,960 ± 6,984 1.4 Sucrose 111,163 ± 13,640 144,169 ± 33,243 1.3

Polyols Erythritol 494 ± 129 634 ± 82* 1.3

Glycerol 4,114 ± 402 4,847 ± 622 1.2

Myo-inositol 11,692 ± 691 11,003 ± 2,911 0.9 Sorbitol 1,789 ± 748 2,259 ± 1,386 1.3 Xylitol 161 ± 16 150 ± 39 0.9

Organic acids Citrate 4,727 ± 839 10,510 ± 2,214**** 2.2

Fumarate 1,568 ± 322 8,963 ± 2,432**** 5.7

Malate 2,294 ± 321 6,071 ± 1,625**** 2.6 Succinate 337 ± 54 471 ± 83** 1.4 Benzoate 3,245 ± 346 4,801 ± 489**** 1.5

Nicotinic acid 1,687 ± 148 3,177 ± 327**** 1.9 Palmitate 2,765 ± 1,005 3,895 ± 2,220 1.4 Pyroglutamate 31,141 ± 8245 55,645 ± 16,678** 1.8

Misc organic Ethanolamine 10,923 ±4,439 9,078 ± 2,216 0.8

Inorrganic Phosphoric acid 1,066 ± 203 2203 ± 830** 2.1 Hydroxylamine 5,345 ± 528 7277. ± 1,739* 1.4

Standard errors represents 5 replicates. *,** and ****Student’s t test significance at P

< 0.05,P < 0.01and P < 5*10-4, respectively.



32

Table III. Level of amino acids in dry seeds

nmol/g FW

WT apx6-1 apx6-1/WT Amino acid Mean Mean Ratio Alanine 158.26 ± 18.59 217.08 ± 2.59**** 1.37 Asparagine 134.57 ± 58.47 290.98 ± 86.70** 2.16

Aspartate 127.64 ± 26.59 403.31 ± 126.01**** 3.16 Cysteine 3.39 ± 1.34 3.84 ± 0.69 1.13 Glutamate 776.44 ± 136.10 1,149.97 ± 211.60*** 1.48 Glutamine 35.31 ± 8.01 68.33 ± 18.73*** 1.94 Glycine 233.84 ± 129.56 255.62 ± 174.45 1.09 Histidine 2.55 ± 0.93 4.36 ± 1.13* 1.71

Homoserine 12.90 ± 3.60 15.90 ± 2.87 1.23 Isoleucine 110.16 ± 17.65 174.54 ± 24.64**** 1.58 Leucine 86.06 ± 14.30 139.16 ± 19.71**** 1.62

Lysine 5.80 ± 1.00 10.47 ± 3.41** 1.80 Methionine 46.33 ± 14.27 52.08 ± 8.75 1.12 Phenylalanine 158.19 ± 45.92 240.10 ± 36.42** 1.52 Proline 83.76 ± 21.09 237.77 ± 17.94**** 2.84 Serine 175.02 ± 15.83 280.70 ± 66.30*** 1.60 Threonine 96.65 ± 9.51 146.62 ± 37.41** 1.52

Tryptophan 480.54 ± 77.42 177.25 ± 31.61**** 0.37 Tyrosine 54.50 ± 7.30 65.75 ± 14.40 1.21 Valine 245.46 ± 38.87 411.542 ± 65.52**** 1.68

Standard errors represents 5 replicates. *,**, *** and ****Student’s t test significance

at P < 0.05,P < 0.01, P < 5*10-3 and P < 5*10-4, respectively.



















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