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PlantaDOI 10.1007/s00425-014-2035-0

OrIgInal artIcle

Arabidopsis nudix hydrolase 7 plays a role in seed germination

Xin Zeng · Yong‑Fang Li · Ramamurthy Mahalingam

received: 24 October 2013 / accepted: 23 January 2014 © Springer-Verlag Berlin Heidelberg 2014

Keywords Arabidopsis · atnudt7 · Dormancy · nudix hydrolase · Pyridine nucleotides · reactive oxygen species

Introduction

Seed dormancy is usually defined as failure of a viable seed to complete germination in conditions favorable for germination to proceed (Bewley 1997). Seed dormancy is an important developmental checkpoint, allowing plants to regulate when and where they grow and plants have evolved different mechanisms for inducing dormancy (Bentsink et al. 2006; Finch-Savage and leubner-Metzger 2006).

In nature, environmental signals such as changes in tem-perature, light, soil hydration that allow plants to restrict the timing of their establishment to certain seasons can also modulate dormancy levels. Other than environmental factors, hormone abscisic acid (aBa) and genetic factors such as the delay of germination (DOg1) are known to promote seed dormancy (Penfield and King 2009; Bent-sink et al. 2006; nakabayashi et al. 2012). In the laboratory conditions, numerous chemical and physical treatments have been described that can reduce seed dormancy. For example, a period of dry after-ripening (several weeks to months), moist-chilling or cold stratification (for few days in dark) or application of gibberellic acid (ga), nitrate and nitric oxide could reduce seed dormancy level (Debeaujon and Koornneef 2000).

Seed after-ripening is an extraordinary process since it occurs in dry seeds with very low moisture levels that are not particularly favorable for metabolic processes. non-enzymatic processes involving reactive oxygen species were shown to alleviate dormancy in sunflower (Bazin et al. 2011; Oracz et al. 2007). In arabidopsis, seed

Abstract Arabidopsis nudix hydrolase 7 (Atnudt7) mutants exhibit reduced seed germination phenotype fol-lowing after-ripening. the role of atnUDt7 in seeds and during early stages of imbibition was examined. Seeds of Atnudt7-1 and col-0 following 3 days of imbibition were used to profile changes in naDH- and aDP-ribose pyroph-osphohydrolase enzyme activities, expression of nudix family genes closely related to AtNudt7, and atnUDt7 protein levels. changes in pyridine nucleotides, phytohor-mones, reactive oxygen species and poly(aDP-ribose) lev-els in after-ripened seeds and 1 day after imbibition were also analyzed. changes in AtNUDT7 gene expression, pro-tein levels and enzyme activities in Wt seeds and during early stages of imbibition were correlated. Atnudt7-1 seeds lacked naDH pyrophosphohydrolase activity that led to very high catabolic redox charge. abscisic acid (aBa) lev-els were higher in Atnudt7-1 mutant while salicylic acid, gibberellic acid, and reactive oxygen species (rOS) levels were higher in Wt seeds. In Atnudt7-1, there was excess rOS accumulation 1 day after imbibition. Par levels were significantly higher in Atnudt7-1 mutant when compared to Wt during imbibition. Based on these observations, we conclude naDH pyrophosphohydrolase activity conferred by atnUDt7 is important for naD:naDH homeostasis in seeds. Perturbations to this key redox couple alter aBa and rOS levels in the seeds that in turn lowers germination.

Electronic supplementary material the online version of this article (doi:10.1007/s00425-014-2035-0) contains supplementary material, which is available to authorized users.

X. Zeng · Y.-F. li · r. Mahalingam (*) Department of Biochemistry and Molecular Biology, 246 noble research center, Oklahoma State University, Stillwater, OK 74078, USae-mail: ramamurthy.mahalingam@okstate.edu

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after-ripening was shown to be a discrete developmental pathway associated with specific gene networks (carrera et al. 2008). Perhaps both non-enzymatic processes involv-ing reactive oxygen species (rOS) and regulated gene transcription and translation occurring in discrete moisture pockets within seeds are involved in seed after-ripening and/or in breaking dormancy.

Majority of the dormancy- or germination-related mutants identified to date are associated with hormone bio-synthesis or hormone signaling (Koornneef et al. 2002). these include the well-characterized aBa mutants (Kuc-era et al. 2005), ga mutants (russell et al. 2000; Steber et al. 1998) and ethylene pathway mutants (Beaudoin et al. 2000; ghassemian et al. 2000). another class of dormancy- or germination-associated mutants is structural mutants wherein the seed coat or testa was shown to have an impor-tant role in regulating germination (Debeaujon et al. 2000). Mutations in chromatin remodeling factors such as histone monoubiquitination (hub1, hub2) (liu et al. 2007), histone deacetylases (HD2) (colville et al. 2011) and PIcKle (Henderson et al. 2004) were shown to affect seed germi-nation processes. recently, mutants associated with redox metabolism have been shown to play a key role in seed dormancy and include mutants of the naDPH oxidase (AtrbohB) (Muller et al. 2009), and nicotinamidase (nic2) (Hunt et al. 2007).

One of the early biochemical events following seed imbibition is the changes in pyridine nucleotide (Pn) lev-els (gallais et al. 1998). In the embryos of non-dormant caryposes of Avena sativa, significant increases in naDH levels were observed within 6 h of imbibition, while in the dormant caryopses, levels of naDH remained unchanged (gallais et al. 1998). In the highly dormant cape Verdes Island (cvi) ecotype of arabidopsis, levels of naD were high and naDP levels were low when compared with Ws ecotype that has intermediate dormancy or col-0 ecotype with least or no dormancy (Hunt and gray 2009). Measure-ment of Pns in fresh and after-ripened seeds of cvi showed that breaking of dormancy was associated with a reduction in naD levels (Hunt and gray 2009). In Arabidopsis seeds that have reduced nicotinamidase activity (nic2-1), levels of naD were high, and this was associated with increase in seed dormancy (Hunt et al. 2007). these studies show that Pns homeostasis plays an important role in seed dormancy.

nudix (nucleoside diphosphates linked to moiety X) hydrolases are pyrophosphohydrolases that work on a wide range of metabolites including Pns, aDP-ribose, coenzyme a, dinucleoside polyphosphates, ribo and deoxyribo-nucleoside triphosphates (Dunn et al. 1999). It has been speculated increases in the activity of nudix enzymes may be important for converting naD to nMn during after-ripening (Hunt and gray 2009). In a proteom-ics-based analysis of seed dormancy, a nudix hydrolase,

atnUDt3 was shown to vary between dormant and non-dormant seeds (chibani et al. 2006). We and several other researchers have reported that Arabidopsis nudix hydrolase 7 (atnUDt7) is naDH pyrophosphohydrolase and also an aDP-ribose pyrophosphohydrolase (ge and Xia 2008; Ishikawa et al. 2009; Jambunathan and Mahalingam 2006; Ogawa et al. 2005; Olejnik and Kraszewska 2005; ge et al. 2007). loss-of-function of atnUDt7 leads to stunted plant growth, increased levels of rOS and naDH, and constitu-tive activation of stress response genes (Jambunathan and Mahalingam 2006). In this study, we report reduced ger-mination phenotype in the seeds of Atnudt7 mutant. We show that naDH pyrophosphohydrolase activity in seeds is important for realizing maximal germination rates in arabi-dopsis. We propose a model showing the interconnections between atnUDt7, Pns, rOS, and phytohormones in dry seeds and during early stages of imbibition in Arabidopsis.

Materials and methods

Plant materials

Seeds of Arabidopsis thaliana col-0, Atnudt7-1 (SalK_046441), Atnudt7-2 (SalK_104293), and atnt-nudt7t complementation lines in the Atnudt7-1 background were used in this study. col-0 and Atnudt7 mutants were grown at the same time under long day conditions (16-h light and 8-h dark period and temperature of 20–22 °c) and seeds from 8 to 10 plants of each genotype were bulked together to obtain sufficient quantities of seeds for the vari-ous experiments described in this study. Seeds were consid-ered fresh for up to 2 weeks after harvest and after-ripened after 1 month of dry storage at room temperature.

Atnudt7-1 complementation lines

to generate AtNUDT7t complementation transgenic lines, primers with Kpn I and Hin dIII restriction sites (atnUD t7compl-F: 5′-ggtaccgacttatcgaatcatctactt aatta-3′ and atnUDt7compl-r: 5′-aagcttgaag tcgatggcaagttttacag-3′) were designed to amplify about 2,400 base pairs of genomic Dna that included the full length atnudt7 gene sequence along with promoter region. Pcr product was ligated into pgeM-t easy vector. restriction enzymes Kpn I and Hind III were used to digest the plasmid and release the insert from pgeM-t easy vector. released insert was fused into pZP121 binary vector, transformed into DH5 alpha cells and purified plasmid was obtained. this plasmid was transfected into Agrobacterium and then Atnudt7-1 plants were trans-formed using the floral dip method (clough and Bent 1998). Plants were allowed to set seeds and t0 seeds were screened

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on MS agar plates containing gentamycin (100 μg/ml). gentamycin screening was repeated for two more cycles to obtain t3 generation seeds. Several individual transformants were subjected to germination assays, followed by western analysis with atnudt7 polyclonal antibodies.

germination assays

Arabidopsis seeds (100 seeds/plate) were placed on top of wet double layer whatman paper in petri-dish plates, sealed with parafilm and placed under light for 10 h at 22 °c in a plant growth chamber. the numbers of seeds that ger-minated were recorded every day for a period of 7 days. germination assays were conducted four times using three plates of 100 seeds for each replicate experiment.

Samples for rna, protein isolations and various bio-chemical assays were collected from col-0, Atnudt7-1 and Atnudt7-1t complementation lines before placing the seeds in petri plates (0 days), 1, 2, and 3 days after imbibition (DaI). the samples were frozen in liquid nitrogen and stored at −80 °c.

aDP-ribose and naDH pyrophosphohydrolase activity assays

arabidopsis seed samples (0.2 g) were homogenized with 0.5 ml of 100 mM tris–Hcl (pH 8.0) containing 20 % glycerol (Ishikawa et al. 2009). after centrifugation (20,000g) for 20 min at 4 °c, the supernatant was used for analysis of enzymatic activity. aDP-ribose and naDH pyrophosphohydrolase activities were assayed colorimetri-cally by coupling to alkaline phosphatase and measuring the amount of inorganic phosphate formed at 37 °c (ames and King 1966; ribeiro et al. 2001). the standard assay mixture contained, in a volume of 0.1 ml, 50 mM tris–Hcl (pH 8.0), 5 mM Mgcl2, 0.1 mM substrates, 0.7 units of alkaline phosphatase, 1 mg/ml bovine serum albumin, and crude seed extract (approximately 10 μg of protein). the solution was incubated at 37 °c for 15 min (Jambuna-than and Mahalingam 2006) and the reaction was stopped and color was developed by addition of 1 ml of standard inorganic phosphate reagent (6 volumes of 3.4 mM ammo-nium molybdate in 0.5 M H2SO4, 1 volume of 570 mM asa, and 1 volume of 130 mM SDS) (ribeiro et al. 2001). after 20 min incubation at 45 °c, absorbance at 820 nm was measured. Blanks without enzyme and/or substrate were run in parallel. enzyme activities were linear with time and amount of enzyme.

rt-Pcr and qPcr analysis

total rna was isolated from col-0 and Atnudt7-1 (0, 1, 2 and 3 DaI) using rneasy kit (Qiagen, Valencia, ca) and

was diluted to 200 ng/μl. two μg of this total rna was used for cDna synthesis following manufacturer’s instruc-tions (Invitrogen). Sequences of the primers used for this analysis are given in Online resource 4. reverse-transcrip-tion Pcr was conducted using the following thermal con-ditions: 94 °c for 5 min followed by 25 cycles of 94 °c for 30 s, 60 °c for 30 s and 72 °c for 1 min and a final extension at 72 °c for 10 min. Pcr products were visual-ized on 1.5 % agarose gel. real-time Pcr was conducted in triplicates using Maxima SYBr green qPcr master mix (Fermentas) in a 7500 real-time Pcr machine (applied Biosystems). Pcr was performed using the following ther-mal conditions: 94 °c for 10 min followed by 35 cycles of 94 °c for 15 s, 55 °c for 20 s and 72 °c for 40 s. amplifi-cations from actin2 gene were used for normalization.

Western analysis

total protein was extracted from seed samples with protein extraction buffer. Protein concentration was determined using Bradford assay (Bio-rad). about 10–15 μg of each sample was used for the western blot and hybridizations were conducted using atnUDt7 polyclonal antibodies as described earlier (Jambunathan et al. 2010).

Pn extraction and measurement

glycyl glycine method was used to measure the naD and naDH levels in seeds (Hayashi et al. 2005; Jambunathan et al. 2010). Seed tissues collected from after-ripened col-0 and Atnudt7-1 mutant (0, and 1 day after imbibition) were used. three technical replicates and three biological repli-cates of each sample were analyzed.

Phytohormone analysis

Seeds of col-0 and Atnudt7-1 mutant were set up on petri plates as described above and samples were collected 24 h after transfer to plates. about 100 mg seed tissue from each sample was collected and the hormone extraction, lc–MS/MS analysis was conducted at Donald Danforth Plant Sci-ence center Proteomics and Mass Spectrometry Facility using previously described methods (chen et al. 2009). Samples were analyzed for aBa, Indole-3-acetic acid (Iaa), jasmonoyl-isoleucine (Ja-Ile), and salicylic acid (Sa). Data were normalized based on internal standards: D6aBa, D5Iaa, H2Ja and D4Sa. For the analysis of ga, the method described in (Zhang et al. 2011) was followed.

rOS measurements

Seeds of col-0 and Atnudt7-1 mutant were set up on petri plates as described above and samples were collected 0

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and 1 day after imbibition. rOS measurement was done using the H2-DcFDa (Sigma) as described previously (Joo et al. 2005). the average fluorescence value obtained from three measurements was divided by the protein content and expressed as relative fluorescence units per milligram of protein. the analysis was repeated with three biological replicates.

Poly(aDP-ribose) immunoassay

levels of Par in col-0 and Atnudt7-1 after-ripened seeds (0, and 1 day after imbibition) were measured by dot blot-ting three different concentrations of total protein extract (1, 1.5 and 2 μg) and detected using polyclonal antibody to Par (BD BioSciences). autoradiograms were scanned using ImageMaster Pro software to measure intensity and volume of signals. the experiment was replicated twice.

Statistical analysis

Microsoft excel was used for conducting student’s t test for determining the statistical significance of results comparing Wt and Atnudt7-1 mutant for differences in germination rates, enzyme activities, Pns, rOS, Par and phytohor-mone levels.

Results

loss of atnUDt7 lowers germination of arabidopsis seeds

germination of after-ripened seeds of Wt and Atnudt7-1 was examined on petri plates for a period of 7 days. ger-mination of Atnudt7-1 seeds was significantly lower compared with Wt seeds (p < 0.0001) (Fig. 1a). We also examined the germination potential in the seeds of a differ-ent t-Dna insertion line, Atnudt7-2 (Bartsch et al. 2006). Similar to Atnudt7-1, after-ripened seeds of Atnudt7-2 showed significantly lower germination potential com-pared with Wt seeds (p < 0.001) (Fig. 1a). atnudt7-1t23 and atnudt7-1t37, two different complementation lines in Atnudt7-1 background (Online resource 1), carrying the Wt version of the AtNUDT7 gene (including the promoter region) showed close to 100 % germination similar to Wt seeds (Fig. 1a) providing conclusive genetic evidence that a functional atnUDt7 is important for high germination of arabidopsis seeds.

germination of freshly harvested Atnudt7-1 and Atnudt7-2 seeds (less than 2 weeks old) was significantly lower than Wt seeds (Fig. 1b). germination of Atnudt7 mutant seeds over a 3-month time period was around 65 % (Fig. 1b). to examine if asynchronous germination was a

factor leading to lower germination of Atnudt7 mutants, seeds were cold stratified at 4 °c for 3 days and used for germination assays. the cold-stratification treatment did not improve the germination of Atnudt7 mutant seeds

Fig. 1 arabidopsis t-Dna insertion knock-out lines of AtNUDT7 exhibit lower germination phenotype. a germination kinetics of after-ripened seeds (3 months) of col-0, Atnudt7-1, Atnudt7-2, Atnudt7-1t23 and Atnudt7-1t37 complementation lines over a 7-day time period. b germination potential of col-0, Atnudt7-, Atnudt7-2, and Atnudt7-1t23 seeds tested at different time points after harvest. ger-mination percentages were calculated following 7 days of imbibition. c germination potential of after-ripened seeds of col-0, Atnudt7-1, Atnudt7-2, Atnudt7-1t23 and Atnudt7-1t37 complementation lines subjected to cold stratification at 4 °c for 3 days. Percentage germi-nation was assessed by radicle emergence under a dissection micro-scope. three independent experiments with approximately 100 seeds for each experiment were used. Bars represent standard error (p < 0.0001)

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(Fig. 1c). the germination rates did not change when the observations were extended to a 14-day time period (data not shown).

naDH pyrophosphohydrolase enzyme activity in seeds and during early stages of seed imbibition are conferred by atnUDt7

Previous studies from our group and several others have established that atnUDt7 is a naDH and aDP-ribose pyrophosphohydrolase enzyme (ge and Xia 2008; Ishi-kawa et al. 2009; Jambunathan and Mahalingam 2006; Ogawa et al. 2005; Olejnik and Kraszewska 2005). Hence, enzyme assays were conducted to measure the naDH pyrophosphohydrolase and aDP-ribose pyroph-osphohydrolase activities in after-ripened seeds and 1–3 DaI in Col-0 and Atnudt7-1 seeds. High naDH pyroph-osphohydrolase activity was observed in col-0 seeds and none was detectable in Atnudt7-1. One day follow-ing imbibition, naDH pyrophosphohydrolase activity in col-0 was reduced to about 50 %, while their levels in Atnudt7-1 were barely detectable (p < 0.001) (Fig. 2a).

On the other hand, the aDP-ribose pyrophosphohydro-lase activity (Fig. 2b) was not different between col-0 and Atnudt7-1 mutant in 0 and 1-day sample. this sug-gested that naDH was the preferred substrate for atnUDt7 in the dry seeds and during the early stages following imbibition. In addition, the relatively high aDP-ribose pyrophosphohydrolase and naDH pyroph-osphohydrolase activities at 2 and 3 DaI in both col-0 and Atnudt7-1 (Fig. 2a, b) indicated there may be some other enzymes, may be other members of the nudix fam-ily that could play a role.

expression of arabidopsis nudix family naDH and aDP-ribose pyrophosphohydrolase genes in seeds and during germination

Previous study has shown that atnUDt2, atnUDt6 and atnUDt10 also utilize naDH and aDP-ribose as primary substrates (Ogawa et al. 2005). this prompted us to examine the gene expression level of AtNUDT 2, 6, and 10 along with AtNUDT7 in dry seeds and 1, 2, and 3 days following imbibition in col-0, as well as Atnudt7-1 mutant. expression of AtNUDT7 was higher in col-0 dry seeds and 1-day sample than in the 2- and 3-day samples (Fig. 3) and was not detectable in the mutant (Fig. 3; table 1). expression levels of AtNUDT10 were higher in col-0 when compared to Atnudt7-1 mutant, especially in later stages of germination (2- and 3-day samples) (Fig. 3; table 1). AtNUDT6, the clos-est homolog of AtNUDT7, did not show significant dif-ferences in expression in the seeds and 1 DaI (table 1). However, it was not detectable in the mutant at 2 DaI. Interestingly, 3 DaI, AtNUDT6 levels showed more than

Fig. 2 naDH and aDP-ribose pyrophosphohydrolase enzyme activ-ity during arabidopsis seed germination. a naDH pyrophospho-hydrolase activity; b aDP-ribose pyrophosphohydrolase activity in col-0 and Atnudt7-1 after-ripened seeds and 1, 2 and 3 days after imbibition. Asterisk represents statistically significant differences in the enzyme activities in col-0 samples when compared with corre-sponding Atnudt7-1 samples

Fig. 3 expression profiles of arabidopsis nudix hydrolase genes during germination. rt-Pcr was conducted using rna from col-0 and Atnudt7-1 after-ripened seeds and 1, 2 and 3 days after imbibi-tion. gene-specific primers to amplify approximately 400 bp were designed for atnUDt6, atnUDt7, and atnUDt10. actin gene was used as a control for assessing the quality of the cDnas. See table S1 for primer sequences used in this experiment

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twofold higher expression in Wt compared to Atnudt7-1 mutant (table 1). Higher expression of AtNUDT6 and AtNUDT10 could confer higher aDP-ribose pyroph-osphohydrolase activity observed in both col-0 and Atnudt7-1 mutant. expression of AtNUDT2 was very low and did not show any significant changes in expression between the Wt and Atnudt7-1 mutant.

rapid changes in atnUDt7 protein levels during imbibition

atnUDt7 protein levels during imbibition were analyzed using polyclonal antibodies. Interestingly, atnUDt7 pro-tein levels were reduced to nearly 50 % within 24 h after imbibition when compared with their levels in dry seeds, (Fig. 4, Online resource 2). atnUDt7 protein levels at the 2-day time point were extremely low and at the 3-day time point were not detectable. a finer time-course experi-ment was conducted wherein samples were taken at 6–12 h intervals. Western analysis indicated that reduction in atnUDt7 levels as early as 6 h after imbibition and con-tinued to decline steadily up to 36 h and was not detectable by 48 h (Fig. 4, Online resource 2).

the well-correlated rt-Pcr, western analysis and enzyme activity assays confirmed the naDH pyrophosphohydrolase

activity in dry seeds and during early hours following imbibi-tion was conferred by atnUDt7.

rapid changes in naD and naDH levels during imbibition

naDH levels in Atnudt7-1 mutant seeds were nearly fourfold higher than the Wt seeds (0-day samples) (p < 0.002) (Fig. 5a). at 24 h after imbibition, levels of

Fig. 4 Protein profile of atnUDt7 during arabidopsis seed germi-nation. Western blot analysis was conducted using the atnUDt7 pol-yclonal antibodies. Fifteen micrograms of total protein extracts from col-0 loaded in each lane; in the top panel, proteins were extracted from after-ripened dry seeds (0), 1, 2 and 3 days after imbibition. For the bottom panel, proteins were extracted from after-ripened dry seeds (0), 6, 12, 24, 36 and 48 h after imbibition

Fig. 5 Pyridine nucleotide analysis during arabidopsis seed germi-nation. col-0 and Atnudt7-1 mutant 0 (after-ripened seeds), and 1 day after imbibition were analyzed. a naDH; b naD+; c catabolic redox charge which is defined as ratio of naDH to naD++naDH. Values shown are the means of three independent experiments. Bars repre-sent standard errors. Asterisk represents statistically significant differ-ences between col-0 and corresponding Atnudt7-1 samples

Table 1 real-time Pcr analysis of arabidopsis nudix hydrolase genes during seed germination

DaI refers to days after imbibition. Up refers to an increase in the expression in the Wt samples while there was no detectable expres-sion in the Atnudt7-1 mutant. Mean and standard deviations from three replicate experiments are reported

Wt:Atnudt7-1 Seeds 1 DaI 2 DaI 3 DaI

AtNUDT2 1.2 ± 0.1 0.9 ± 0.4 1.1 ± 0.1 1.2 ± 0.3

AtNUDT6 1 ± 0.6 1 ± 0.3 Up 2.3 ± 0.4

AtNUDT7 Up Up Up Up

AtNUDT10 2.5 ± 0.2 3.7 ± 0.7 8.6 ± 1.1 6.8 ± 0.8

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naDH were lowered by more than 60 % in Atnudt7-1 while the levels of this metabolite increased in the Wt (p < 0.001) (Fig. 5a). On the contrary, naD+ levels remained stable in Wt while their levels in Atnudt7-1 seeds were significantly lower than in Wt, 24 h after imbibition (Fig. 5b). changes in naD+ and naDH levels lead to significant alterations in the seed redox levels dur-ing the early stages of imbibition (Fig. 5c). crc defined as naDH/naD++naDH showed opposite patterns in col-0 dry seeds (low) when compared to 24 h after imbi-bition (Fig. 5c). On the contrary, crc values were very high in Atnudt7-1 seeds and it showed a decline by 24 h after imbibition. changes in redox brought about during the imbibition phase will impact the biochemical milieu of the seeds facilitating transition from quiescence state to germination state and naDH pyrophosphohydro-lase activity of atnUDt7 may play a crucial role in this transition.

rOS profiles of atnudt7-1 and Wt are different in seeds and during imbibition

Several studies have highlighted the importance of rOS in breaking dormancy and during seed germination (el-Maarouf-Boteau and Bailly 2008; Sarath et al. 2007). rOS levels in the after-ripened seeds of Atnudt7-1 were sixfold to eightfold lower, compared with Wt seeds (p < 0.02) (Fig. 6a). Interestingly, within 24 h following imbibition,

levels of rOS increased more than 20-fold in Atnudt7-1 mutant compared to Wt (p < 0.02) (Fig. 6a).

Par levels are altered during seed imbibition

We observed that Par levels in after-ripened Atnudt7-1 seeds were increased by fourfold (p < 0.005) by 24 h after imbibition compared with their levels in col-0 (Fig. 6b). Significant increases in the Par levels in Atnudt7-1 mutant following imbibition are consistent with the observed increase in rOS, since the latter can lead to oxidative dam-age of proteins and Dna, which in turn triggers the ParP enzyme (Ishikawa et al. 2009; Mahalingam et al. 2007).

aBa and ga levels are significantly altered in atnudt7-1 mutant

We observed more than twofold higher levels of aBa in after-ripened seeds of Atnudt7-1 mutant than col-0 seeds (p < 0.02). aBa levels were barely detectable in both Wt and Atnudt7-1 mutant 1 day after imbibition (Fig. 7a). ga4 is one of the major active gas that stimulate seed germina-tion (Ogawa et al. 2003). ga4 was undetectable in dry seeds of Wt while the mutant contained low levels of this bioac-tive ga (Fig. 7b). However, within 24 h after imbibition, ga4 levels surged by nearly 1,000-fold in Wt, while in the mutant the increase was a modest 2.5-fold (p < 0.05). lev-els of ga1 were higher in Wt seeds compared to Atnudt7-1

Fig. 6 analysis of total reactive oxygen species and poly-aDP-ribose levels during Arabidopsis seed germination. a rOS measure-ment in col-0 and Atnudt7-1 mutant 0 (after-ripened seeds), and 1 day after imbibition. Data represent average of three independ-ent experiments with standard error. On the Y-axis, rFU/mg repre-sents relative fluorescence units per mg of protein extract. b relative poly(aDP-ribose) (Par) levels during Arabidopsis seed germination. Protein extracts (three concentrations) from col-0 and Atnudt7-1

mutant collected from 0 (dry seeds), and 1 day after imbibition were used for making dot-blots. Poly(aDP) ribosylation levels were assayed with anti-Par antibodies. Hybridization intensities were extracted using the Imagene software. Data from two independent experiments were averaged and bars represent standard error. Aster-isk represents statistically significant differences between col-0 and corresponding Atnudt7-1 samples

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(Fig. 7b). levels of ga1 decreased by about fourfold within 24 h after imbibition in Wt (p < 0.01) and was not detect-able in Atnudt7-1. Furthermore, levels of ga29, the inac-tive form of ga was about twofold higher in the Atnudt7-1 seeds compared with Wt (p < 0.02) (Fig. 7b). though the levels of this inactive form of ga dropped by nearly 60 % following imbibition in Atnudt7-1, it was still nearly two-fold to threefold higher compared with Wt (p < 0.03) (Fig. 7b). these results suggest that the higher levels of aBa and inactive forms of ga in Atnudt7-1 could be sig-nificant factors for lowering germination. the levels of Iaa, Ja-Ile and Sa did not show significant differences between the Wt and Atnudt7-1 samples (data not shown).

Discussion

Previous studies have shown that changes in Pns are asso-ciated with seed dormancy (gallais et al. 1998; Hunt and

gray 2009). Based on these reports, we speculated that in the arabidopsis nudix hydrolase 7 mutant, Atnudt7, with increased naDH levels (ge and Xia 2008; Jambunathan and Mahalingam 2006), seed germination processes may be affected. the reduced germination in after-ripened seeds, fresh seeds and cold-stratified seeds (Fig. 1) con-firmed that a functional atnUDt7 is an important factor for normal germination of arabidopsis seeds.

It has been suggested that germination potential is pro-grammed during the seed maturation phase (Holdsworth et al. 2008) and stored proteins and translation of pre-existing mrnas are key players that determine potential of germination in arabidopsis (rajjou et al. 2004). Based on the information in the Arabidopsis eFP browser (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi?primarygene=at4g12720&modeInput=absolute) (Winter et al. 2007), atnudt7 gene is expressed during the early stages of embryo development, from the globular to the walking-stick stage (Online resource 3). Our studies demonstrate atnUDt7 is the major naDH pyrophosphohydrolase in dry after-ripened seeds and during early stages of seed imbibition based on enzyme activity, gene expression pat-tern and protein levels (Figs. 2, 3, 4; table 1).

Similar to the report in the non-dormant caryposes of avena (gallais et al. 1998), a rapid increase in crc within 24 h after imbibition in Wt arabidopsis seeds (Fig. 5) is caused by a surge in naDH levels. naDH pyrophosphohy-drolase activity of atnUDt7 is important for reducing the levels of naDH, which otherwise can lead to rOS buildup (see below). this begs the question what processes could lead to naDH buildup during seed imbibition? Upon imbi-bition, the quiescent dry seed rapidly absorbs water but the resumption of metabolic activity is more gradual. One of the earliest changes is the resumption of energy metabo-lism, especially respiration that can be detected within minutes (Bewley 1997; Botha et al. 1992; ehrenshaft and Brambl 1990; nonogaki et al. 2010; Powell et al. 1984). glycolytic and oxidative pentose phosphate pathway resume during the imbibition phase (aldasoro and nico-las 1979; Salon et al. 1988) and these processes can lead to naDH accumulation. Secondly, following imbibition, many seeds experience temporary anaerobic conditions leading to ethanol production (Kennedy et al. 1992). this ethanol is converted to acetaldehyde by alcohol dehydroge-nase. the highly reactive acetaldehyde is further converted to acetate by aldehyde dehydrogenase. each of these reac-tions also leads to production of naDH. the low oxygen availability in imbibed seeds also could restrict mitochon-drial atP production and favor the glycolytic pathway that in turn leads to an increase in naDH levels.

One of the surprising findings from our study was the significantly lower level of rOS in the Atnudt7 mutant when compared to the Wt seeds (Fig. 6). Previously,

Fig. 7 Quantification of aBa and ga levels in arabidopsis after-ripened seeds and 1 day after imbibition. col-0 and Atnudt7-1 seeds, and 1 day after imbibition, were analyzed by lc–MS/MS. a aBa b ga1, ga4, and ga29. the data shown are means of three replicates for each sample. Bars represent standard error. Asterisk represents statistically significant differences between col-0 and corresponding Atnudt7-1 samples

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we have shown that rOS levels were threefold to four-fold higher in Atnudt7-1 leaves when compared with Wt (Jambunathan and Mahalingam 2006). the higher rOS in leaves may be due to higher levels of endogenous Sa (Bar-tsch et al. 2006), since Sa and rOS form a part of self-amplifying feed forward loop (Overmyer et al. 2003). It has been reported that seed Sa levels are negatively regulated by aBa (Kanno et al. 2010). lower levels of Sa observed in Atnudt7 mutant seeds could be due to antagonistic action of the elevated aBa levels (Fig. 7). lower Sa levels in turn could explain the observed lower levels of rOS in Atnudt7 seeds. Higher levels of aBa have been shown to inhibit rOS and enhance antioxidant enzyme activity (el-Maar-ouf-Boteau and Bailly 2008). rOS, especially H2O2 has been shown to break dormancy in several different plant

systems and this was attributed to a decrease in aBa levels (naredo et al. 1998; Wang et al. 1995). We speculate that higher levels of naDH in Atnudt7 seeds could play a role in the biosynthesis of aBa by xanthoxin dehydrogenase (gonzalez-guzman et al. 2002) and high levels of aBa in turn inhibit rOS that can lower germination rates (Muller et al. 2009). thus, atnUDt7 may play a role in regulating the seed aBa levels via regulating naD:naDH balance.

the significantly elevated levels of rOS 24 h after imbibition in Atnudt7-1 also suggests a functional atnUDt7 may be involved in maintaining rOS homeo-stasis during early phases of seed imbibition. the lack of naDH pyrophosphohydrolase activity of atnUDt7 may channel excess naDH for superoxide generation by the sequential actions of naDH kinase and naDPH

Fig. 8 Proposed model for atnUDt7 in dry seeds and early stages of imbibition. a atnUDt7 regulates naD+:naDH balance in after-ripened seeds. Byproduct of naDH pyrophosphohydrolase activity of atnUDt7, nicotinamide mononucleotide reduced form (nMnH), may be used for naD biosynthesis. Sequential action by naDH kinase and naDPH oxidase using naDH can lead to rOS. rOS and Sa levels increase due to the feed forward self-amplification process. Higher Sa levels can stimulate ga biosynthesis. b the extremely high catabolic redox charge caused by increase in naDH due to lack of atnUDt7 favors excess aBa biosynthesis that in turn suppresses rOS, lowers Sa and ga levels, that eventually promotes dormancy.

c Within 24 h after imbibition, the crc in col-0 is higher compared with dry seeds and may serve as phase transition marker to promote germination. aBa levels are negligible, hence relieves the repression on ga that in turn promotes germination. d lack of atnUDt7 per-turbs naD+:naDH homeostasis and crc is lower in imbibed seeds compared with dry seeds but is still higher than the values observed for col-0. negligible aBa levels and high crc lead to a de-repres-sion of rOS. this vast burst of rOS leads to oxidative stress as evi-denced by increased Par activity. this will impact the energy home-ostasis that eventually reduces germination potential of the imbibed Atnudt7-1 seeds

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oxidase (Hunt et al. 2004; Kwak et al. 2003; Murata et al. 2001; torres et al. 1998). this vast excess of rOS will cause an oxidative stress as evidenced by the significant increases in the Par levels (Fig. 7) and eventually lead to growth retardation phenotype observed in Atnudt7 mutant (Fig. 8). In fact, atnUDt7 was shown to have a signifi-cant impact on the Dna repair pathways via modulation of Par reaction under oxidative stress conditions (Ishi-kawa et al. 2009).

this study demonstrates that atnUDt7 enzyme activity is important in the dry seeds and in early stages of imbi-bition for regulating Pn homeostasis that in turn impacts hormonal and rOS metabolism (Fig. 8). Understanding the regulation of atnUDt7 gene will further aid in dissecting the complex interplay between redox homeostasis and phy-tohormones during early phases of seed imbibition and its impact on germination potential.

Acknowledgments this research was supported partially by national Science Foundation under grant no. ePS-081431. We thank Dr. Jane Parker (Max Planck Institute for Plant Breeding research) for providing us the Atnudt7-2 mutant seeds. We thank Dr. les-lie Hicks, Donald Danforth Plant Sciences center, Saint louis, MO for assistance with the phytohormone analysis. We thank Dr. Yixing Wang for technical assistance.

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