Journal of Integrative Plant Biology 2012, 54 (9): 663673

Research Article

Proanthocyanidins Inhibit Seed Germination by Maintaining a High Level of Abscisic Acid in Arabidopsis thaliana
F

Liguo Jia1 , Qiuyu Wu1 , Nenghui Ye1 , Rui Liu1 , Lu Shi1 , Weifeng Xu1 , Hui Zhi1 , A. N. M. Rubaiyath Bin Rahman1 , Yiji Xia1 and Jianhua Zhang2
1 Department 2 School

of Biology, Hong Kong Baptist University, Hong Kong, China of Life Sciences and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China Corresponding author Tel: +852 3943 6288; Fax: +852 2603 6382; E-mail: jhzhang@cuhk.edu.hk F Articles can be viewed online without a subscription. Available online on 5 July 2012 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipb doi: 10.1111/j.1744-7909.2012.01142.x

Abstract
Proanthocyanidins (PAs) are the main products of the avonoid biosynthetic pathway in seeds, but their biological function during seed germination is still unclear. We observed that seed germination is delayed with the increase of exogenous PA concentration in Arabidopsis. A similar inhibitory effect occurred in peeled Brassica napus seeds, which was observed by measuring radicle elongation. Using abscisic acid (ABA), a biosynthetic and metabolic inhibitor, and gene expression analysis by real-time polymerase chain reaction, we found that the inhibitory effect of PAs on seed germination is due to their promotion of ABA via de novo biogenesis, rather than by any inhibition of its degradation. Consistent with the relationship between PA content and ABA accumulation in seeds, PA-decient mutants maintain a lower level of ABA compared with wild-types during germination. Our data suggest that PA distribution in the seed coat can act as a doorkeeper to seed germination. PA regulation of seed germination is mediated by the ABA signaling pathway. Keywords:
Abscisic acid; Arabidopsis; Brassica napus; proanthocyanidins; seed germination.

Jia L, Wu Q, Ye N, Liu R, Shi L, Xu W, Zhi H, Rahman ANMRB, Xia Y, Zhang J (2012) Proanthocyanidins inhibit seed germination by maintaining a high level of abscisic acid in Arabidopsis thaliana. J. Integr. Plant Biol. 54(9), 663673.

Introduction
Seed germination is a complex process and many signals are involved, such as abscisic acid (ABA), gibberellins (GA), ethylene, nitric oxide (NO), and hydrogen peroxide (H 2 O 2 ) (Bewley 1997; Zhou et al. 1998; Ghassemian et al. 2000; Bethke and Jones 2001; Bethke et al. 2006). Following the uptake of water by the quiescent dry seed, germination commences by remobilizing a series of essential biological processes including DNA repair, transcription, and translation (Barroco et al. 2005). Besides water, other environmental factors are needed to help the seed break dormancy, including appropriate levels of light, temperature, oxygen and more.

However, because these environmental factors interact with each other to affect the seed, no single one of them needs to be present at an optimal level for germination to commence (Cone and Spruit 1983). Many chemicals, both natural and synthetic, have been shown to promote or inhibit seed germination, and work by regulating hormone levels. For example, nordihydroguaiaretic acid (NDGA) and diniconazole were used to regulate ABA as inhibitors of 9-cis-epoxycarotenoid dioxygenase and ABA 8 -hydroxylase, respectively (Han et al. 2004; Kitahata et al. 2005). The hormones ABA and GA both play a major role in regulating seed germination. According to their inuences on dormancy and control of germination, physiological seed 2012 Institute of Botany, Chinese Academy of Sciences

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dormancy can be described as embryo dormancy, testa dormancy, and endosperm dormancy (Finch-Savage and Leubner-Metzger 2006). Both the seed coat and the embryo control seed dormancy and germination in Arabidopsis thaliana (Debeaujon and Koornneef 2000; Debeaujon et al. 2000; Muller et al. 2006). There is much evidence in support of the positive role of ABA in the induction of dormancy and in the maintenance of the dormant state in imbibed seeds (Hilhorst 1995; Kucera et al. 2005). Many ABA biogenesis, metabolism and signaling mutants have been found to exhibit abnormal seed dormancy and germination (Finkelstein et al. 2002; Himmelbach et al. 2003). ABA-insensitive mutants such as abi1, abi2 and abi3 have decreased primary dormancy, and ABA-decient mutants such as aba1, aba2, and aao3 show an absence of primary dormancy in mature Arabidopsis seeds (Leon-Kloosterziel et al. 1996; Raz et al. 2001; Finkelstein et al. 2002; Himmelbach et al. 2003; Nambara and Marion-Poll 2005; Holdsworth et al. 2008). Besides those genes responding to ABA signaling, many important genes for ABA biogenesis and catabolism have been documented. 9-cis-epoxycarotenoid dioxygenases (NCEDs) are responsible for the cleavage of 9-cis-epoxycarotenoids to xanthoxin, which is considered to be the key regulatory step of ABA biosynthesis. Altogether, nine members were found of which NCED6 and NCED9 are required for ABA biosynthesis and are involved in seed dormancy and germination in Arabidopsis (Lefebvre et al. 2006). Among four ABA degradation genes, CYP707A2 plays a major role in ABA catabolism during imbibition, and regulates seed dormancy in Arabidopsis (Kushiro et al. 2004; Okamoto et al. 2006; Liu et al. 2009). Some changes, including seed structure or/and accumulated metabolic products, can affect seed germination and dormancy, which might be due to natural variation or articial mutagenesis (Koornneef et al. 2002; Koornneef et al. 2004). Most investigated groups of mutants are transparent testa (tt ) and transparent testa glabra (ttg) which show reduced seed dormancy, among which TT1, TT2 and TT8 belong to regulatory genes. TT2 and TT8 together with TTG1 participate in forming the MBW complex to regulate BAN expression (Nesi et al. 2001; Baudry et al. 2004). TT1 encodes a WIP-type Zn-nger transcriptional factor and regulates proanthocyanidin (PA) biogenesis, but its detailed regulatory mechanism is still not clear (Sagasser et al. 2002). TT18 encodes a leucocyanidin dioxygenase, which is located downstream of the avonoid pathway and is essential for PA biosynthesis (Abrahams et al. 2003). These mutants exhibit a yellow or pale brown seed coat color because their avonoid pigmentation and accumulation are greatly reduced (Debeaujon et al. 2000; Lepiniec et al. 2006). Flavonoids are a large group of secondary metabolites that are present in most plant seeds and grains in no fewer than 6000 different varieties (Shirley 1998; Harborne and Williams 2000; Lepiniec et al. 2006). The main types of avonoids in seeds are avonols, anthocyanins, phlobaphenes,

isoavones, and PAs (also called condensed tannins), based on the oxidation level of the C-ring. Stereochemistry, combination and degree of polymerization, position and nature of substitutions, and linkages between basic units contribute to avonoid complexity and diversity (Lepiniec et al. 2006). Only avonols and PAs are accumulated and account for 50% each of measurable avonoids in Arabidopsis seeds (Routaboul et al. 2006). Flavonols are mainly located in both the testa and the embryo, while PAs are biosynthesized and accumulated in the seed coat (Marles et al. 2003; Routaboul et al. 2006). Most PAs found in seeds are soluble procyanidin polymers of two stereoisomers: epicatechin (EC, 23cis) and catechin (C, 23trans), and their degree of polymerization can be up to 9 (Harborne and Williams 2000; Routaboul et al. 2006). However, only EC, with a mean degree of polymerization between 5 and 8, is found in Arabidopsis seeds (Abrahams et al. 2002; Routaboul et al. 2006). As the end-product of the avonoid biosynthetic pathway, PAs are not only benecial to the human diet but also inuence the quality of foods such as wines, fruit juices, and teas (Shirley 1998; Dixon et al. 2005). Their biological functions in plants have been extensively investigated, including UV protection (Li et al. 1993; Landry et al. 1995), protection against microbial pathogens, insect pests, and larger herbivores (Dixon et al. 2005), and binding metals, especially iron and zinc (House 1999). Some mutants of PA biogenesis have been identied based on seed color, because PAs become oxidized and confer a brownish color to the testa in Arabidopsis (Koornneef 1981; Koornneef 1990; Shirley et al. 1995; Devic et al. 1999; Nesi et al. 2000; Nesi et al. 2001; Baudry et al. 2006). Using these mutants, Debeaujon et al. (2000) investigated the inuence of the testa on seed dormancy, germination, and longevity, and found that most mutants exhibit reduced dormancy. The authors proposed that this reduced dormancy may be attributed to a change in the testa itself. However, it is unknown whether the change in the mutant testa affects water absorption, mechanical resistance, or both (Debeaujon et al. 2000). Further analysis demonstrated that the differential dormancy between wild-types and mutants is associated with a difference in gibberellin requirement (Debeaujon and Koornneef 2000). ABA plays a major role in regulating seed dormancy/germination by inhibiting radical protrusion and rupture of the endosperm (Muller et al. 2006; Graeber et al. 2010; Weitbrecht et al. 2011). We therefore asked whether PAs also have an effect on regulating ABA levels in seeds.

Results
Effect of exogenous PAs on seed germination
Dormancy-broken wild-type (Col0) seeds were selected to investigate seed germination under gradient PA treatments.

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Approximately 1.5 mg/g (dry weight) soluble PAs in mature wild-type (Col0) seeds were detected in our pilot experiment. Based on this concentration of PAs in natural Arabidopsis seeds, gradient concentrations of 0, 0.025%, 0.05%, 0.1% and 0.2% PAs were supplemented into a Murashige and Skoog (MS) medium for a seed germination assay (Figure 1A). Seed germination percentages were 88.4%, 39.2%, 30.1%, 19.2% and 5.6% under the 0, 0.025%, 0.05%, 0.1% and 0.2% PA treatments, respectively, after 3 days of imbibition (Figure 1B). The maximum germination percentage was only 79.6% under 0.2% PA treatment after 7 days (Figure 1A, B). In order to determine if and how much PAs were absorbed by seeds cultured in the PA medium, PA content was detected during 12 h imbibition on the medium with the gradient PAs. Compared with the control PAs, absorption increased with increasing concentration of PAs in the medium (Figure 1C). To further explore which process of seed germination (testa rupture or radicle elongation) is affected by PAs, it is necessary to use seeds with their seed coats removed. The Arabidopsis seed is small, making it too difcult to separate the embryo from the seed coat. Brassica napus and Arabidopsis belong to the Brassicaceae family, but the B. napus seed size is far larger than that of Arabidopsis (Lysak et al. 2009). The black-seeded line of B. napus has been shown to accumulate PAs, and some important genes for its biogenesis have been cloned and analyzed (Wei et al. 2007; Auger et al. 2009; Chai et al. 2009). We thus examined the effect of PAs on radical elongation of coatless B. napus seeds. Consistent with our hypothesis, the radicle elongation of peeled B. napus seeds decreased with the increase of PA concentration supplemented in the medium (Figure 2A, B). These results suggest that PAs inhibitory effect on seed germination is at least partly due to their restraining radicle elongation.

Effect of PAs on ABA accumulation

To further explore the mechanism of PAs inhibitory effect on seed germination, the changes in ABA content were investigated during the early seed germination stage. We found that ABA accumulation decreased gradually with the extension of imbibition time, which is consistent with previous studies. We also detected a low level of ABA in wild-type seeds, and this low level of ABA was maintained at later germination stages (Ali-Rachedi et al. 2004; Chiwocha et al. 2005; Weitbrecht et al. 2011). Unlike the tendency for ABA to change when germinating seeds are imbibed with water, ABA content increased rapidly in the initial 3 h and then stayed at a high level when seeds were imbibed with 0.1% PAs (Figure 3A). The evidence provided here illustrates that PA inhibition of seed germination is mediated by the ABA signaling pathway. Abscisic acid is synthesized in both the endosperm and the embryo, but the testa does not participate in hormonal

Figure 1. Effects of proanthocyanidins (PAs) on seed germination in Arabidopsis. (A) Pictures of 7 d wild-type (Col0) seedlings on MS medium supplemented with and without 0.025%, 0.05%, 0.1% and 0.2% PAs, respectively. (B) Effect of PAs on seed germination behavior in seven consecutive days. (C) PA content of wild-type seeds after 12 h imbibition on MS medium with and without 0.025%, 0.05%, 0.1% and 0.2% PAs. Values are means with SE (n = 3 for b and c). Means denoted by the same letter did not signicantly differ at P < 0.05 according to Duncans multiple range test.

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PAs enhance ABA biogenesis rather than suppress its catabolism

Abscisic acid content is controlled by the balance between its biogenesis and its catabolism. To understand the cause of ABA accumulation by PA treatments, we investigated the expression of selected genes involved in ABA biogenesis and catabolism. Our data show that two biosynthetic genes, NCED6 and NCED9, had higher expression under PA imbibition than under water imbibition (Figure 3C, D). However, we did not detect a signicant difference in the expression of the ABA catabolic gene CYP707A2 in treatments of water compared to treatments of PAs (Figure 3E). This suggests that the accumulation of ABA in the PA-treated germinating seeds is due to the enhancement of ABA biogenesis rather than the suppression of ABA catabolism. While ABA content remained high at 12 h under PA treatment, the transcript levels of ABA synthesis genes decreased dramatically. Interestingly, the ABA catabolism gene CYP707A2 showed a great decrease in transcript levels as well. Thus, the high ABA level observed at 12 h under PA treatment is probably due to a decrease in the catabolism of ABA. A pharmacological approach was used to further investigate the relationship between PA and ABA levels. NDGA, an ABA biogenesis inhibitor, or Diniconazole, an ABA catabolism inhibitor (Han et al. 2004; Kitahata et al. 2005), were supplemented into MS medium with and without 0.1% PAs to test seed germination. NDGA was found to negate the inhibitory effect of PAs on seed germination (Figure 4). However, more severe inhibition of seed germination was observed when Diniconazole and PAs were used together.

Changes in ABA content in PA-decient mutants

Figure 2. Effect of proanthocyanidins (PAs) on radicle elongation of Brassica napus seeds. (A) Photos of 3 d peeled B. napus seedlings on MS medium supplemented with and without 0.025%, 0.05%, 0.1% and 0.2% PAs, respectively (bar was set as 1 cm). (B) Radicle length of B. napus seeds 3 d culture on MS medium with and without 0.025%, 0.05%, 0.1% and 0.2% PAs. Values are means with SE (n = 3 for b). Means denoted by the same letter did not signicantly differ at P < 0.05 according to Duncans multiple range test.

To determine whether PAs also have an effect on ABA content during seed maturation, we quantied ABA in wild-type and PA-decient mutant seeds. We identied and conrmed ve mutant lines, tt16, tt25, tt84, tt85, and tt183, that were obtained from the Arabidopsis Biological Research Center (ABRC) (see supplemental data). Our data show that wild-type seeds accumulated more ABA compared to PA-decient seeds (Figure 5A). Furthermore, lower ABA levels were maintained during the germination process when compared to wild-type seeds (Figure 5B).

Discussion
balance to control seed germination behavior (Lefebvre et al. 2006). We found more ABA accumulation in PA-imbibed seeds compared to those imbibed with water on a per-grain basis at two time points we selected (Figure 3B). These data suggest that exogenous PA treatment may have triggered de novo ABA synthesis in the seeds. Flavonoids are secondary metabolites that exist extensively throughout the plant kingdom. To this point, at least 6000 different types have been found, mainly including avonols, anthocyanins, phlobaphenes, isoavones, and PAs (Harborne and Williams 2000; Lepiniec et al. 2006). These important

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Figure 3. Effects of exogenous proanthocyanidins (PAs) on abscisic acid (ABA) content, and the transcript level of genes involved in ABA synthesis and catabolism during the imbibition of Arabidopsis and Brassica napus seeds. ABA content in seeds treated with water or 0.1% PAs in (A) Arabidopsis and (B) in peeled B. napus seeds. Transcript levels of ABA biosynthetic gene (C) NCED6 and (D) NCED9, and ABA catabolic gene (E) CYP707A2 when seeds were treated with water or 0.1% PAs during imbibitions. Values are means with SE (n = 3 for a, b, c, d and e). Means denoted by the same letter did not signicantly differ at P < 0.05 according to Duncans multiple range test.

metabolites have been investigated in the model plant Arabidopsis, particularly in its biosynthetic pathway at the molecular level. PAs, as important end-products, have been a primary focus not only because of their biosynthetic process but also because of their important biological functions (Dixon et al. 2005). The cloning and functional analysis of BANYULS (BAN) was a breakthrough in the understanding of PA biogenesis, identifying the gene in the last step of PA production (Xie et al. 2003). Specic cell and tissue localization of PAs and BAN gene expression imply the role of PAs in seed development, dormancy, and germination behavior (Debeaujon et al. 2003). As early as 2000, the Koornneef group investigated the seed characteristics of transparent testa (PA-decient) mutants on dormancy, germination, and longevity, and found most mutants

show reduced seed dormancy (Debeaujon and Koornneef 2000; Debeaujon et al. 2000). The authors suggested that the reduced seed dormancy in the mutants might be due to a lack of pigments, but the detailed mechanism is still unclear. Reduced PA synthesis and thus fewer PA polymers in the testa of mutant seeds might enhance water absorption during germination. However, the evidence for this is not solid. First, the micropylar is the major entry point of water, but there is little PA accumulation in that area (Debeaujon et al. 2003; Manz et al. 2005; Wojtyla et al. 2006). Second, water uptake during seed germination is an active absorption, and aquaporins play a role in this process. No one has reported on a different expression of aquaporin genes between wild-type and PAdecient mutants (Schuurmans et al. 2003; Weitbrecht et al.

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Figure 4. The interaction of proanthocyanidins (PAs) and abscisic acid (ABA) during seed germination. Germination of the third day wild-type seeds on MS+PAs medium supplied with and without 100 M ABA biogenesis inhibitor, nordihydroguaiaretic acid (NDGA) and 100 M ABA catabolism inhibitor, diniconazole. Values are means with SE (n = 3). Means denoted by the same letter did not signicantly differ at P < 0.05 according to Duncans multiple range test.

2011). Moreover, PAs can cross-link cell wall components and increase testa thickness, which increases physical resistance and suppresses seed germination (Debeaujon et al. 2000). Another possibility is that PAs themselves as biochemical molecules could directly affect seed germination. Consistent with our speculation, exogenous PAs delay non-dormant seed germination, and this inhibition is stronger with the increase of PA concentration. Our data also show that radicle elongation decreases with the increase of PA concentration (Figure 2) in coatless B. napus seeds. Thus, PAs likely reduce seed germination in part by inhibiting radical elongation. ABA also inhibits seed germination by delaying radicle protrusion (Muller et al. 2006; Graeber et al. 2010; Weitbrecht et al. 2011). Thus, it is very possible that PA inhibition of seed germination is mediated by ABA signaling. Although the precise cause of PAinduced ABA production is unclear, we cannot discount that the release of PAs around seeds may contribute to a stressful micro-environment and promote ABA production. The endogenous ABA of non-dormant seeds rapidly declines upon imbibition during the early phase of germination, and then stays at a low level (Ali-Rachedi et al. 2004; Chiwocha et al. 2005; Weitbrecht et al. 2011). Consistent with our results, ABA content decreases in the initial hours during imbibition of water. In contrast, if imbibed in PA solution, ABA content increases rapidly at the beginning of imbibition and then stays at high levels in Arabidopsis (Figure 3A). The same changes were observed in B. napus embryos, in which more ABA was accumulated under PA imbibition (Figure 3B). This suggests that ABA is involved in the inhibition of seed germination

Figure 5. Effect of abscisic acid (ABA) content on germination for imbibition of Arabidopsis seeds. (A) Comparison of ABA content among the seeds of wild-types (col0), tt16, tt25, tt84, tt85 and tt183 at 60 d after harvesting. (B) Comparison of ABA content between wild-types and three selected mutants (tt16, tt84 and tt183) during the imbibitions of Arabidopsis seeds. Values are means with SE (n = 3 for A and B). Means denoted by the same letter did not signicantly differ at P < 0.05 according to Duncans multiple range test.

by PAs. The accumulation of ABA under PA treatments is probably due to the enhancement of ABA biogenesis based on gene expression analysis (Figure 3CE). This conclusion is further consolidated by a pharmacological experiment. NDGA, an ABA biogenesis inhibitor (Han et al. 2004), negated the inhibitory effects of PAs on seed germination. More severe inhibition of seed germination was observed when both Diniconazole and PAs were supplemented at the same time. This can be explained by the additive effect of two compounds: PAs stimulating ABA synthesis, and Diniconazole suppressing catabolism of ABA (Kitahata et al. 2005). This could result in an even higher level of ABA accumulation.

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Several PA-decient mutants were used to further test our hypothesis. At least 12 structural genes and six regulatory genes have been identied to regulate PA biogenesis (Lepiniec et al. 2006). Five randomly selected mutants belonging to four loci, tt16, tt25, tt84, tt85, tt183, were used for the investigation. Semi-quantitative PCR and histochemical analysis showed no PA accumulation in any of the selected mutants: tt16, tt2 5, tt84, tt85, tt183 (Figure S2; Figure S3a). Although we detected insoluble PAs in the mutants, the concentrations were far lower than those in wild-type seeds (Figure S3b). Our analysis showed that PA-decient mutants accumulated less ABA in dry seeds, and also had lower levels of ABA in germinating seeds. These results are consistent with other experiments in this study, suggesting that high PA levels are required to stimulate ABA synthesis during both seed maturation and germination. In summary, our data support the notion that PAs as biochemical molecules inhibit seed germination through promoting ABA synthesis and thus signaling. However, it is unknown how PAs that accumulate in the inner integument and pigment strand can affect ABA metabolism in the endorsperm/embryo. We speculate that certain soluble forms of PAs are released during the early stages of imbibition. These PAs will trigger ABA accumulation in the seed, which inhibits endosperm weakening and radical elongation, resulting in delayed germination in the Arabidopsis seed.

ined from the Arabidopsis Biological Research Center (ABRC). To maintain higher germination rates, the sterilized seeds were planted on agar plates for one week and then transferred to soil. Seeds were harvested separately from individual plants. Subsequently, PCR was performed to conrm the homozygous mutant lines with the genomic DNA of tt1 6, tt25, tt84, tt85 and tt183 mutants using gene-specic primers: tt16 (LP, 5 -AGCGATTGAATGTCTTGAAGC-3 ; RP, 5 -AACCCTGCAATGACAAAGTTG-3 ); tt25 (LP, 5 -GCGGG TCAGAATCTAGTTTCC-3 ; RP, 5 -AAGCAGATGGTCGTT GATAGC-3 ); tt84 (LP, 5 -CGAGGAAGACAACTCAACCAG3 ; RP, 5 -TACCACGTTTTCGTATCTCCG-3 ); tt85 (LP, 5 CGAGGAAGACAACTCAACCAG-3 ; RP, 5 -AGTTCCACAAC ACCATCAAGC-3 ); tt183 (LP, 5 -TTCAACCATTTTGATAC TTGCG-3 ; RP, 5 -GTGTCTTCGTTTGCTAGCGAC-3 ) and LBb1.3 (5 -ATTTTGCCGATTTCGGAAC-3 ).

Germination assay
Two-month-stored seed samples after harvesting were washed with 70% (v/v) ethanol for 30 s, sterilized with 2% hypochlorite for 10 min, and rinsed four times with sterile water. Roughly one hundred seeds were selected for each treatment, and were spread in Murashige and Skoog (MS) medium (SigmaAldrich, St. Louis, MO, USA) containing 0.8% (w/v) Bacto Agar (Difco/BD) supplemented with 1% sucrose. Plates were kept at 4 C in darkness for 3 d for stratication, and were then transferred to a growth chamber set at 22 C with a 16 h light/8 h dark photoperiod. Germination was dened as the rst sign of radicle tip emergence and scored daily until the 7th day of the incubation, and the germination results were calculated based on three independent experiments (Xi et al. 2010). For radicle elongation analysis of B. napus, uniformly round seeds with a black coat were picked out and immersed in water for 15 min. The seed coat was peeled carefully with forceps to make sure the embryo was not injured. Twenty peeled seeds were transferred to an MS medium with and without 0.025%, 0.05%, 0.1% and 0.2% PAs. Seedling photos were taken after 3 d of culture, and radicle elongation was calculated using ImageJ software.

Materials and Methods

Plant materials
Arabidopsis (Arabidopsis thaliana L.) (Col0 and some T-DNA insertion mutants with Col0 background) plants were grown in a growth chamber at 22 2 C with a 16 h photoperiod at a photon ux density of approximately 200 mol/m2 per s at daytime and a relative humidity of 80%. Seeds of wild-type and mutants for the experiment were grown and harvested at the same time. The seed samples after harvest were stored in a dehumidier cabinet for two months before being used for the experiments. Oilseed rape (Brassica napus L.) Yuhuang No.4 seeds were obtained from Chongqing Linong Agricultural Technology Co., Ltd. Seeds with a black coat and uniformly round sizes were selected for further radicle elongation analysis and ABA determination.

Chemical treatments
The exogenous PAs (Baoji Hongyuan Bio-technology Co., Ltd., Baoji, China) used in our experiment come from black soybean hull extracts and are water-soluble at room temperature. The extract contains at least 13.5% catechin and no less than 50.0% OPCs (oligomeric proanthocyanidins). For PA treatments, specic amounts were added into the MS medium before autoclaving according to the set concentration gradients. Nordihydroguaiaretic acid (NDGA) and Diniconazole (Sigma) were used as inhibitors of 9-cis-epoxycarotenoid dioxygenase

T-DNA insertion lines

The T-DNA insertion mutants tt16 (SALK_107737C), tt25 (SALK_005260), tt84 (SALK_030966), tt85 (SALK_048673) and tt183 (SALK_028793) generated by the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu/), were obta-

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and ABA 8 -hydroxylase, respectively (Han et al. 2004; Kitahata et al. 2005).

Total RNA extraction, RT-PCR and quantitative PCR analysis

Total RNAs from seeds were extracted using the Plant RNA Isolation Mini Kit (Agilent) according to the manufacturers instructions, and were reverse transcribed using the SuperScript reverse transcription-polymerase chain reaction (RTPCR) system (Invitrogen, Madison, WI, USA). The cDNA was then diluted 10 times and 3 L cDNA was used to perform the quantitative (q)RT-PCR. IQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and iCycle (Bio-Rad) were used in our experiment. The primers used for screening transparent testa mutants by semi-quantitative RT-PCR are as follows: TT1 (Forward, 5 -CACCACATTCAATGGAGTCAC-3 ; Reverse, 5 CCCACATGTGCATCTGAAGA-3 ); TT2 (Forward, 5 -AACCA AGCTGGTCTCAAGAGGTGT-3 ; Reverse, 5 -CGTTTCCAC AGTCCAAACCA-3 ); TT8 (Forward, 5 -GGTTTGCATTC CAATGCTTG-3 ; Reverse, 5 -TTGAGCACCCATTGTGACG3 ); TT18 (Forward, 5 -CATAGAAGCAACGAGTGAGTACG3 ; Reverse, 5 -AAGTCCGTGGAGGAAACTTAGC-3 ); ACT2 (Forward, 5 -GTGAAGGCTGGATTTGCAGGA-3 ; Reverse, 5 AACCTCCGATCCAGACACTGT-3 ). For qRT-PCR analysis, UBQ5 was used as an internal standard to normalize the data, and a second reference gene ACT8 was used to re-conrm the results (Chiang et al. 2011; Graeber et al. 2011). The primers used for gene expression analysis by qRT-PCR are as follows: NCED6 (Forward, 5 TGAGAGACGAAGAGAAAGAC-3 ; Reverse, 5 -GTTCCTTCA ACTGATTCTCG-3 ); NCED9 (Forward, 5 -GGAAAACGCCA TGATCTCACA-3 ; Reverse, 5 -AGGATCCGCCGTTTTAGGA T-3 ); CYP707A2 (Forward, 5 -AAATGGAGTGCACTCA TGTC-3 ; Reverse, 5 -CCTTCTTCATCTCCAATCAC-3 ); UBQ5 (Forward, 5 -GCATGCAAGCTTGGCGTAA-3 ; Reverse, 5 -TGAGCGGATAACAATTTCACACA-3 ); ACT8 (Forward, 5 -CTCAGGTATTGCAGACCGTATGAG-3 ; Reverse, 5CTGGACCTGCTTCATCATACTCTG-3 ).

PA extraction and analysis

Thirty milligrams of dry seeds were weighed for PA extraction and analysis. After treatments, the samples were ground in mortar with 1 mL acetonitrile/water (75:25; v/v) for 5 min on ice and were sonicated for 20 min. Following centrifugation, the pellet was extracted further with 1 mL acetonitrile/water (75:25; v/v) overnight at 4 C. The two extracts were pooled and concentrated under a ow of nitrogen, and the dry extract was dissolved in 200 L acetonitrile/water (75:25; v/v). Fifty microliter aliquots of the nal extracts alone and on all the remaining pellets were used for soluble and insoluble PA analysis, respectively. After adding 3 mL of butanolHCl reagent (butanol-concentrated HCl, 95:5, v/v) and 0.1 mL of ferric reagent (2% ferric ammonium sulfate in 2N HCl), the tubes were put in a boiling water bath adjusted to 98 C for 60 min. After cooling the tube on ice, PA-related absorbance was recorded at 550 nm (Porter et al. 1985; Routaboul et al. 2006). PA content was calculated according to the standard curve made by commercial PAs (Baoji Hongyuan Biotechnology Co., Ltd.). All values of PA content were equivalents of this standard product.

Extraction and determination of ABA

Abscisic acid analysis was carried out using the radioimmunoassay (RIA) method modied according to Quarrie et al . (1988). Fifty milligrams of Arabidopsis seeds that absorbed distilled H 2 O or 0.1% PA solution were homogenized in 1 mL of distilled water and then shaken at 4 C overnight. The homogenates were centrifuged at 12 000 g for 10 min at 4 C, and the supernatant was directly used for the ABA assay. The 450 L reaction mixture contained 100 L of crude extract, 200 L of phosphate buffer (pH 6.0), 100 L of diluted antibody (Mac 252) solution (5 mg/mL bovine serum albumin (BSA) and 4 mg/mL soluble polyvinylpyrrolidone (PVP) in phosphate buffer <pH 6.0>), and 100 L of [3 H] ABA (8000 cpm) solution. The mixture was left standing for 45 min at 4 C, and precipitated with 50% saturated (NH 4 ) 2 SO 4 . After centrifuging at 9 000 rpm for 5 min and pouring off the supernatant, 200 L ddH 2 O and 1.2 mL scintillation uid were added to the pellet. The bound radioactivity in pellets was measured with a liquid scintillation counter (Quarrie et al. 1988). For B. napus, peeled seeds were transferred to an MS medium with and without 0.1% PAs. Samples were taken at set time points and 10 grains were selected for ABA determination for each treatment. The detailed procedure of extraction was the same as mentioned above in Arabidopsis.

Accession numbers
Sequence data described in this article can be found in the Arabidopsis Genome Initiative or The Institute for Genomic Research (TIGR) databases under the following accession numbers: NCED6 (AT3G24220); NCED9 (AT1G78390); TT1 (AT1G34790); TT2 (AT5G35550); TT8 (AT4G09820); TT18 (AT4G22880); ACT2 (AT3G18780); UBQ5 (AT3G62250); ACT8 (AT1G49240).

Acknowledgements
We are grateful for grant support from the Hong Kong Research Grants Council (CUHK 262809), the Hong Kong Baptist

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671

University Strategic Development Fund (SDF 090910P03), and the National Basic Research Program of China (973 project, 2012CB114300).

Chiwocha SD, Cutler AJ, Abrams SR, Ambrose SJ, Yang J, Ross AR, Kermode AR (2005) The etr12 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination. Plant J. 42, 35 48. Cone JW, Spruit CJP (1983) Imbibition conditions and seed dormancy

Received 16 Jan. 2012

Accepted 18 Jun. 2012

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Supporting Information
Additional Supporting Information may be found in the online version of this article: Figure S1. Detecting the reliability of the ABA determination method in solution containing PAs. (A) Comparison of the CPM (Counts per minute) values in an ABA-free solution with and without 0.1% PAs and 0.1% PAs + 4 mg/mL PVP. (B) Comparison of 60 ng/L ABA in water and in 0.1% PA solution. Values are means with SE (n = 3). Means denoted by the same letter did not signicantly differ at P < 0.05 according to Duncans multiple range test. Figure S2. Identication of PA-decient mutants of Arabidopsis. (A) T-DNA insertion sites of TT1, TT2, TT8 and TT18 genes. (B) Transcriptions of PA regulatory genes (TT1, TT2 and TT8) and structural gene (TT18) in WT, and respective mutants by semi-quantitative PCR. Figure S3. Phenotypic analysis and validation of selected PA-decient mutants. (A) Comparison of natural and stained seed coat color between wild-type and PA-decient mutants. Two kinds of histochemical methods, a Vanillin assay and a DMACA assay, were used. (B) Detection of insoluble PA concentration both in the seeds of wild-type and PA-decient mutants. (C) Soluble PA concentrations in the seeds of wild-type and PA-decient mutants; Values are means with SE (n = 3 for B and C). Means denoted by the same letter did not signicantly differ at P < 0.05 according to Duncans multiple range test. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. (Co-Editor: Lixin Zhang)