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...cloned into pENTR-1A and recombined into pHGWFS7. All binary vector constructs were introduced into Agrobacterium strain GV3101 and transformed into Arabidopsis Columbia-0 using the floral dip method =-=[47]-=-. All the transgenic lines were first selected based on their antibiotics resistance and further confirmed by expression level of the EXP2 or histochemical GUS assays. RNA Extraction and Real-time RT-...

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... downstream of GA and ABA signaling. We then performed GA and ABA treatments and found AtEXP2 expression was GAinducible, but not affected by ABA treatment, which was consistent with previous studies =-=[1,17,60,64]-=-. GA is well known to be a pivotal phytohormone in breaking seed dormancy and promoting seed germination [1–2,53]. Previous studies showed that active GAs are synthesized mainly in the radicle and mic...

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...hat AtEXP2 is required for seed germination in Arabidopsis. The Participation of AtEXP2 in Seed Germination is Regulated by GA Since seed germination is largely controlled by phytohormones GA and ABA =-=[2,53]-=-, we next determined whether the expression of AtEXP2 was regulated by GA and ABA. As shown in Figure 4, the expression of AtEXP2 was significantly higher in GA3-treated wild type seeds than in mock-t...

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...sing exp2 mutant seeds, which showed that exp2 seeds germinate later than wild type. Further through analyzing promoter sequence of AtEXP2 using PLACE tools (Plant cis-acting regulatory DNA elements) =-=[63]-=- we found there were putative GA-responsive elements (GARE) and ABA-responsive elements (ABRE) in the promoter region of AtEXP2, indicated that AtEXP2 would function downstream of GA and ABA signaling...

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... involved in cell wall extensibility modification [7–8]. Expansin was firstly identified from young cucumber seedlings for its ability to mediate the acidinduced extension of cucumber hypocotyl walls =-=[7,9]-=-. Expansins induce cell wall extension by disrupting non-covalent linkages between cellulose microfibrils and the cross-linking matrix glycans in cell wall [8–10]. Expansins are encoded by a multigene...

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...er genes. Primers used in real-time RT-PCR as follows : EXP2-F: 59CATAAACTCCGACGACAACG-39, EXP2-R: 59-TACCCACAAGCACCACCCAT-39; TUB2-F: 59-ATCCGTGAAGAGTACCCAGAT-39, TUB2-R: 59-AAGAACCATGCACTCATCAGC-39 =-=[52]-=-. The PCR program was as follows: 30 s at 95uC, followed by 40 cycles of 5 s at 95uC, 30 s at 60uC. GUS Activity Analysis Tissues were prefixed in 90% acetone on ice for 20 min and incubated in GUS st...

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...s [10–12]. Expansins can be divided into four divergent subfamilies based on genomic and phylogenetic analyses, denoted as a-expansin (EXPA), bexpansin (EXPB), EXP-like A (EXLA) and EXP-like B (EXLB) =-=[10,13]-=-, and there are thirty-six expansins encoding by 26 EXPAs, 6 EXPBs, 3 EXLAs and 1 EXLBs in Arabidopsis (http://www. personal.psu.edu/fsl/ExpCentral). The extensive occurrence of the expansins superfam...

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...hat AtEXP2 is required for seed germination in Arabidopsis. The Participation of AtEXP2 in Seed Germination is Regulated by GA Since seed germination is largely controlled by phytohormones GA and ABA =-=[2,53]-=-, we next determined whether the expression of AtEXP2 was regulated by GA and ABA. As shown in Figure 4, the expression of AtEXP2 was significantly higher in GA3-treated wild type seeds than in mock-t...

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...on by phytohormones, expansin genes are also differentially regulated by various environmental cues [54], such as abiotic stresses including salt stress, heat stress, drought stress, and water stress =-=[30,37,43,70]-=-. Here we tested the germination phenotype of exp2 and the overexpression line in response to salt stress and osmotic stress, and we found that exp2 mutant exhibited hypersensitivity to salt stress an...

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... involved in cell wall extensibility modification [7–8]. Expansin was firstly identified from young cucumber seedlings for its ability to mediate the acidinduced extension of cucumber hypocotyl walls =-=[7,9]-=-. Expansins induce cell wall extension by disrupting non-covalent linkages between cellulose microfibrils and the cross-linking matrix glycans in cell wall [8–10]. Expansins are encoded by a multigene...

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...may inhibit seed germination by repressing AtEXP2-mediated cell wall expansion. In contrast to our observation, some other expansin coding genes in other species are subject to upregulation by stress =-=[37,43,71]-=-. This variable gene-specific expression pattern of expansin genes under abiotic stress suggest that individual expansin genes are subjected to different regulation by stress conditions and that the m...

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...adicle needs to overcome the dual constraint of these structures to complete germination, therefore seed germination is defined as a two stage process with testa rupture followed by endosperm rupture =-=[1,3]-=-. Physiologically the rupture of endosperm and testa is associated with cell division and enlargement, which are accompanied by cell wall expansion and loosening [4–5]. The cell wall is an extracellul...

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...adicle needs to overcome the dual constraint of these structures to complete germination, therefore seed germination is defined as a two stage process with testa rupture followed by endosperm rupture =-=[1,3]-=-. Physiologically the rupture of endosperm and testa is associated with cell division and enlargement, which are accompanied by cell wall expansion and loosening [4–5]. The cell wall is an extracellul...

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...on by phytohormones, expansin genes are also differentially regulated by various environmental cues [54], such as abiotic stresses including salt stress, heat stress, drought stress, and water stress =-=[30,37,43,70]-=-. Here we tested the germination phenotype of exp2 and the overexpression line in response to salt stress and osmotic stress, and we found that exp2 mutant exhibited hypersensitivity to salt stress an...

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...ement needs to destroy this structural rigidity by modifying cell wall extensibility. Many enzymes are involved in modifying matrix polysaccharides including endo-b-mannanase and other endoglucanases =-=[6]-=-. In addition to those enzymes, expansins are involved in cell wall extensibility modification [7–8]. Expansin was firstly identified from young cucumber seedlings for its ability to mediate the acidi...

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...nfirmed by expression level of the EXP2 or histochemical GUS assays. RNA Extraction and Real-time RT-PCR Total RNAs were isolated from Arabidopsis seeds according to the protocol described previously =-=[48]-=-, and total RNAs from other tissues were extracted using TRIZOL reagent (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized using the M-MLV Reverse Transcriptase (Promega) fro...

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...gnaling pathway is mainly mediated by derepression of DELLA repressors [67–68], Stamm et al., (2012) have shown that RGL2 downregulates two expansin encoding genes EXPA3 and EXPA8 in seed germination =-=[69]-=-. In order to study whether AtEXP2 expression is subjected to the regulation of DELLA, we examined the AtEXP2 expression level in germinating seeds of various DELLA mutants and found that all four DEL...

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...lized to the radicle cortex of the embryo, and LeEXP10 mRNA is expressed throughout the embryo during seed germination, suggesting the specific roles of the two expansin genes during seed germination =-=[16]-=-. In the present study, we first used the public Arabidopsis microarray database to analyze the expression profile of all thirty-six expansin genes in Arabidopsis and found that only AtEXP2 was exclus...

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...lved in Response to Salt Stress and Osmotic Stress in Seed Germination The expression of expansin coding genes are not only regulated by developmental signals, but also affected by environmental cues =-=[17,54]-=-. In order to investigate whether AtEXP2 is involved in response to abiotic stress, we performed germination tests under salt stress and osmotic stress condition. As shown in Figure 8, when exposed to...

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...on by phytohormones, expansin genes are also differentially regulated by various environmental cues [54], such as abiotic stresses including salt stress, heat stress, drought stress, and water stress =-=[30,37,43,70]-=-. Here we tested the germination phenotype of exp2 and the overexpression line in response to salt stress and osmotic stress, and we found that exp2 mutant exhibited hypersensitivity to salt stress an...

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...lved in Response to Salt Stress and Osmotic Stress in Seed Germination The expression of expansin coding genes are not only regulated by developmental signals, but also affected by environmental cues =-=[17,54]-=-. In order to investigate whether AtEXP2 is involved in response to abiotic stress, we performed germination tests under salt stress and osmotic stress condition. As shown in Figure 8, when exposed to...

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...–15], and in the past several decades numerous studies have accumulated evidence about their involvement in diverse developmental processes, including seed germination [16–17], seedling morphogenesis =-=[18]-=-, root architecture [19–26], leaf development [27–30], fruit ripening [31–32], stoma opening [33–34], pollination [35], abscission, stress responses [36–43] and others. However, those characterized ex...

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...velopmental processes, including seed germination [16–17], seedling morphogenesis [18], root architecture [19–26], leaf development [27–30], fruit ripening [31–32], stoma opening [33–34], pollination =-=[35]-=-, abscission, stress responses [36–43] and others. However, those characterized expansins only represent a small proportion of the expansins superfamily in the plant kingdom, and the individual functi...

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...on by phytohormones, expansin genes are also differentially regulated by various environmental cues [54], such as abiotic stresses including salt stress, heat stress, drought stress, and water stress =-=[30,37,43,70]-=-. Here we tested the germination phenotype of exp2 and the overexpression line in response to salt stress and osmotic stress, and we found that exp2 mutant exhibited hypersensitivity to salt stress an...