Plant Physiology Preview. Published on September 23, 2015, as DOI:10.1104/pp.15.00858
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Running head: AtGASA6 regulates seed germination
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Authors for correspondence:
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Xiaojing Wang Ph.D.
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School of Life Sciences,
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South China Normal University,
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Guangzhou 510631, China
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Tel/Fax: 86-20-85216417
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E-mail:
[email protected]
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Shengchun Zhang Ph.D.
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School of Life Sciences,
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South China Normal University,
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Guangzhou 510631, China
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Tel/Fax: 86-20-85216417
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E-mail:
[email protected]
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Reasearch Area: Signaling and Responses; Genes, Development and Evolutions
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Copyright 2015 by the American Society of Plant Biologists
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AtGASA6 Serves as an Integrator of Gibberellin-, Abscisic Acid- and
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Glucose-Signaling during Seed Germination in Arabidopsis
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Chunmei Zhong1, Hao Xu1, Siting Ye, Shiyi Wang, Lingfei Li, Shengchun Zhang*,
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Xiaojing Wang*
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Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of
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Life Sciences, South China Normal University, Guangzhou 510631, China
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*Authors for correspondence 1
These authors contributed equally to this work
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Summary: A cell wall-localized protein stimulates embryonic axis elongation and
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seed germination, and modulates cross-talk between gibberellins, abscisic acid and
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glucose signaling pathways.
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Footnotes
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This work is supported by the National Natural Science Foundation of China
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(90917011 for XJ Wang, 31370350 for SC Zhang), Guangzhou Pearl River new star
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project for science and technology (2012J2200033 for SC Zhang).
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*Authors for correspondence:
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Xiaojing Wang,
[email protected]
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Shengchun Zhang,
[email protected]
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3 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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Abstract
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The DELLA protein RGL2 plays an important role in seed germination under
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different conditions through a number of transcription factors. However, the functions
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of the structural genes associated with RGL2-regulated germination are less defined.
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Here, we report the role of an Arabidopsis cell wall-localized protein, AtGASA6, in
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functionally linking RGL2 and a cell wall loosening expansin protein (AtEXPA1),
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resulting in the control of embryonic axis elongation and seed germination. AtGASA6
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over-expressing seeds showed precocious germination, while T-DNA and RNAi
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mutant seeds displayed delayed seed germination under abscisic acid, paclobutrazol
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and glucose stress conditions. The differences in germination rates resulted from
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corresponding variation in cell elongation in the hypocotyl-radicle transition region of
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the embryonic axis. AtGASA6 was down-regulated by RGL2, GIN2 and ABI5 genes,
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and loss of AtGASA6 expression in the gasa6 mutant reversed the insensitivity shown
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by the rgl2 mutant, to paclobutrazol, and of the gin2 mutant to glucose-induced stress,
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suggesting that it is involved in regulating both the GA and glucose signaling
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pathways. Furthermore, it was found that the promotion of seed germination, and
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length of embryonic axis by AtGASA6, resulted from a promotion of cell elongation at
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the embryonic axis mediated by AtEXPA1. Taken together, the data indicate that
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AtGASA6 links RGL2 and AtEXPA1 functions, and plays a role as an integrator of
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GA-, ABA- and glucose- signaling, resulting in the regulation of seed germination via
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a promotion of cell elongation.
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Introduction
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The transition from the dormant embryonic stage to seed germination is pivotal
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in the plant life cycle (Bewley, 1997), and is influenced by both environmental and
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intrinsic signals (Koornneef et al., 2002), including the two phytohormones, abscisic
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acid (ABA) and gibberellin (GA) (Gubler et al., 2005). The roles of ABA in
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modulating seed development, dormancy, germination and plant adaptation to abiotic
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environmental stresses, have been well studied (Gubler et al., 2005; Finkelstein et al.,
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2008), and it has been found that ABA specifically inhibits endosperm rupture, rather
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than testa rupture, in the seed germination process (Muller et al., 2006; Lee et al.,
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2010). ABA signaling components include ABSCISIC ACID-INSENSITIVE 3 (ABI3),
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which encodes a B3 domain transcription factor, and ABSCISIC ACID-INSENSITIVE
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5 (ABI5), encoding a basic leucine-zipper (bZIP) transcription factor, both of which
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are necessary for growth arrest when germinating seeds encounter unfavorable
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conditions (Giraudat et al., 1992; Finkelstein and Lynch, 2000). ABI5 functions
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downstream of ABI3 and is essential for executing ABA-dependent growth arrest,
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which occurs after the breakage of seed dormancy, but prior to autotrophic growth
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(Lopez-Molina et al., 2002). Such growth arrest occurs through the recruitment of de
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novo late embryogenesis programs, and confers osmotic tolerance to harsh
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environmental conditions (Lopez-Molina et al., 2002).
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GA promotes seed germination and growth, and its biosynthesis and responses
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are highly coordinated during seed germination (Ogawa et al., 2003). Complex
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regulatory events in the GA signaling pathway include crosstalk with other hormones
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and the regulation of genes involved in promoting cell elongation and division
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(Ogawa et al., 2003). Accumulation of GA during seed germination is accompanied
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by a reduction in ABA levels, suggesting that GA and ABA play antagonistic roles in
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this process (Olszewski et al., 2002). GA signaling is known to be regulated by a
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group of repressors collectively called DELLA proteins, including REPRESSOR OF
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ga1-3 (RGA), GA-INSENSITIVE (GAI), and RGA-LIKE1/2/3 (RGL1/2/3) (Tyler et
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al., 2004), of which RGL2 appears to be the major DELLA factor involved in 5 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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repressing seed germination (Lee et al., 2002; Tyler et al., 2004; Cao et al., 2005).
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Recent studies have shown that RGL2 stimulates ABA biosynthesis by inducing the
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expression of XERICO and ABI5, while ABA enhances RGL2 expression (Ko et al.,
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2006; Zentella et al., 2007; Piskurewicz et al., 2008), indicating that RGL2 mediates
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the interaction of GA and ABA during seed germination.
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During seed germination, sugar signaling also plays an important role, by
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interacting with phytohormones, especially ABA (Leon and Sheen, 2003).
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Arabidopsis HEXOKINASE 1 (HXK1) has been characterized as a glucose sensor
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(Jang et al., 1997; Moore et al., 2003). HXK1 dependent and independent glucose
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signaling leads to increased ABA levels by inducing the expression of ABA synthesis
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genes (ABA1/2/3), and enhancing signaling through ABI3 and ABI5 to control seed
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germination (Ramon et al., 2008). Sugar signaling has also been reported to modulate
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the effects of other phytohormones, including cytokinin, ethylene and auxin (Ramon
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et al., 2008). However, the interaction between GA and sugar signaling in the control
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of seed germination is not clear and factors mediating the GA and sugar signaling
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interaction remain to be identified.
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To date, in a wide range of plant species, the Gibberellic Acid-Stimulated
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Arabidopsis (GASA) peptide family, which comprises cysteine-rich peptides, has
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been identified as GA-responsive (Roxrud et al., 2007), and the tomato GA-stimulated
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transcript 1 (GAST1) has been identified as the first GASA gene member (Shi et al.,
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1992). GASA proteins contain a putative signal peptide at the N-terminus for
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targeting to the secretory pathway, and a conserved C-terminal region of
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approximately 60 amino acids that contains 12 cysteine residues at conserved sites.
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Fourteen GASA genes have been identified in A. thaliana (Roxrud et al., 2007). Most
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of them are regulated by phytohormones, such as GA and ABA, and participate in
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hormone signaling pathways by mediating hormonal levels and responses (Zhang and
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Wang, 2008; Zhang et al., 2009; Rubinovich and Weiss, 2010; Sun et al., 2013). Thus
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far, GASA proteins have been demonstrated to play important roles in various
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developmental programs, including seed germination (Rubinovich and Weiss, 2010),
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root formation (Taylor and Scheuring, 1994; Zimmermann et al., 2010), the 6 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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establishment of seed size (Roxrud et al., 2007), stem growth and flowering time
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(Ben-Nissan et al., 2004; Zhang et al., 2009), fruit development and ripening (de la
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Fuente et al., 2006; Moyano-Canete et al., 2013), fiber development (Liu et al., 2013),
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and leaf expansion (Sun et al., 2013). Some GASAs have been reported to be
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involved in cellular processes such as the promotion or inhibition of cell elongation
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and cell division; however, the detailed cellular regulation mechanisms through which
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GASAs operate have not been established.
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In this study we report the characterization of the A. thaliana GASA protein
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AtGASA6 and describe both its involvement in hormonal cross-talk during seed
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germination, and the mechanism by which it affects cell elongation in germinating
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seeds.
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Results
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AtGASA6 and AtEXPA1 are up-regulated in imbibed della mutant seeds
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Although GA and DELLA proteins are considered to play central regulatory
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roles in seed germination, the downstream structural components of this pathway,
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especially the putative genes affecting cell wall properties during cell elongation, have
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not been well clarified. Previous transcriptome data suggest that several GASA and
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EXPANSIN family genes, which encode putative cell wall localized proteins, likely
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function in GA-mediated seed germination (Gonzali et al., 2006; Stamm et al., 2012).
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In order to verify this, we evaluated their expressions in imbibed della mutant seeds
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and found that only AtGASA6 (At1g74670) and AtEXPA1 (At1g69530), are
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up-regulated in rga-24, gai-t6, rgl2-1 and gai-t6/rga-24 imbibed seeds (Fig. 1A-B). In
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addition, the GeneMANIA program predicted that the expression patterns of
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AtGASA6 and AtEXPA1 are similar to those of RGL2 and that these three genes are
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co-expressed (Fig. S1) (Zuberi et al., 2013), implying that both AtGASA6 and
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AtEXPA1 may function with RGL2.
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RT-PCR was performed to determine the temporal and spatial expression
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patterns of AtGASA6, which is expressed in various organs, including roots, 7 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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inflorescence stems, rosette and cauline leaves, flowers, as well as mature siliques
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(Fig. S2A). To confirm these results, pGASA6::GUS plants (Col) were generated and
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examined. During the seedling stage, GUS activity was observed in petioles (Fig.
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S2B-a), the vascular tissue of rosette leaves (Fig. S2B-b) and cotyledons (Fig. S2B-c),
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the root meristem (Fig. S2B-d), and emerging lateral roots (Fig. S2B-e). During the
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reproductive stage, strong GUS activity was observed in ovules and anthers (Fig.
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S2B-f). Interestingly, GUS activity was observed in developing and immature seeds at
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stage 12 (Fig. S2B-g), 13 (Fig. S2B-h), 15 (Fig. S2b-i) and 16 (Fig. S2B-j), but not in
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mature seeds at stage 17 (Fig. S2B-k). GUS staining was apparently specific in the
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hypocotyl-radicle transition zone of the embryonic axis in germinated seeds (Fig.
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S2B-l), although no staining was observed in the endosperm layer and testa (Fig.
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S2B-m). Thus, the GUS expression patterns were in close agreement with the
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RT-PCR analyses (Fig. S2A). Importantly, AtGASA6 was found to be highly
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expressed in developing seeds and the embryonic axis of imbibed seeds, suggesting
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that it may play roles in seed germination.
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AtGASA6 is a positive regulator of seed germination under stress conditions
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In order to elucidate the function of the AtGASA6 gene, a T-DNA insertion line
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in the third exon (Fig. 2A), gasa6-1, was identified from ABRC. RT-PCR showed
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that gasa6-1 is an AtGASA6 knock-down mutant (Fig. 2B), and phenotypic analysis
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revealed that germination was slightly delayed in the gasa6-1 mutant under normal
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conditions, but significantly delayed as a consequence of exposure to high
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concentration of glucose, ABA, or the GA antagonist paclobutrazol (PAC; Fig. 2D).
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The difference in germination status between gasa6-1 mutant and wild-type seeds
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under various conditions was also documented (Fig. 2E).
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In order to determine the function of the AtGASA6 gene during seed germination,
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AtGASA6 over-expressing and RNAi suppressed transgenic A. thaliana lines were
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generated for further analysis. RT-PCR analysis confirmed that AtGASA6 expression
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levels were increased or decreased according to the particular transgenic line (Fig.
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2C). And the expression levels of the closest homologous genes of AtGASA6, which 8 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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are classified to the same sub-family (Zhang and Wang, 2008), in AtGASA6–RNAi
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lines were not affected significantly by RNAi (Fig. S3). Finally, the AtGASA6
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over-expressing lines OE#2-1, OE#5-5 and OE#6-1, and AtGASA6-RNAi lines R#3-5,
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R#4-3 and R#6-7, were chosen for subsequent experiments. Under normal conditions,
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although the seed germination rate of the AtGASA6 over-expressing lines appeared
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somewhat faster than that of the wild-type, and germination rate of the
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AtGASA6-RNAi seeds was slightly slower, the differences were not statistically
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significant (Fig. 2E). However, the seed germination rates were clearly different when
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the various AtGASA6 genotypes were challenged using specific stress conditions.
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Compared to wild-type seeds, AtGASA6 over-expressing seeds germinated faster, and
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the RNAi seeds germinated significantly more slowly under 6% glucose, 0.5 μM PAC
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or 1 μM ABA conditions (Fig. 2E). In order to assess whether the germination
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difference was caused by osmotic stress, seed germination rates were tested using 6%
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mannitol, and it was found that the OE and RNAi lines did not show significant
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difference compared to the wild-type (Fig. 2E). The AtGASA6-RNAi seeds began to
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germinate after 46 hrs on 6% glucose, 36 hrs on 0.5 μM PAC or 1 μM ABA plates,
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while the percentage of germinated AtGASA6 over-expressing seeds had reached 20%
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at these time points (Fig. 2E). As a result of these treatments, seed germination rate
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differences between the three AtGASA6 genotypes remained significant until the end
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of the germination process, indicating that AtGASA6 is associated with glucose, GA
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and ABA signaling pathway-mediated seed germination.
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AtGASA6 expression is regulated by glucose, ABA and GA
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It is well documented that GA and ABA act antagonistically in the regulation of
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seed germination, and ABA and GA levels change substantially during seed
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imbibition (Karssen et al., 1983; Yamauchi et al., 2004; Okamoto et al., 2006). Since
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the seed germination rates of AtGASA6 over-expressing and RNAi lines were affected
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by glucose, ABA or PAC, we hypothesized that AtGASA6 might be up-regulated by
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GA, and down-regulated by ABA and glucose, during seed germination. To test this,
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wild-type (Col-0) seeds were planted on minimal medium (MS medium without 9 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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sucrose) with 10 μM ABA, 10 μM PAC, 6% glucose or 10 μM GA3 after 10 h of
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exposure to light. Gene expression analyses showed that AtGASA6 transcripts were
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up-regulated almost five-fold after 6 h of GA treatment, while very low level of
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transcript was detected after ABA, PAC or glucose treatment for 6 h, compared to the
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mock treatment (Fig. 3A), indicating that AtGASA6 is indeed down-regulated by ABA
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and glucose, and up-regulated by GA in germinating seeds. To determine if the effects
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of glucose, ABA and GA on AtGASA6 expression were developmentally regulated,
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expression was measured in 14-day old wild-type seedlings treated with 6% glucose,
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10 μM ABA, or 10 μM GA3 for 3 h, and the effects of all treatments were observed to
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be the same as during seed germination (Fig. S4).
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To elucidate the effects of GA and ABA concentrations on AtGASA6 expression,
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germinating seeds were treated with 1 μM or 10 μM PAC, and 10 μM or 100 μM
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ABA. As expected, the effect of PAC on the expression of AtGASA6 was the opposite
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of that of GA, and a significant inhibition effect was positively correlated with
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concentration. After 1 μM or 10 μM PAC treatments for 6 h, AtGASA6 transcript
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levels declined by approximately 70% or 90%, respectively (Fig. 3B). Similarly, the
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down-regulation effect of high ABA concentrations was more severe than that of the
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lower concentration, and AtGASA6 transcripts were barely detectable after treatment
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with 100 μM ABA (Fig. 3B). Although GA induced AtGASA6 expression, there was
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no significant difference between the 1 μM and 10 μM GA3 treatments (Fig. 3C).
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Glucose has been reported to inhibit seed germination in a similar manner to
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ABA, and this was also apparent in our studies (Fig. 2E). As GA antagonizes ABA
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function in seed germination, we speculated that the down-regulating effect of glucose
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on AtGASA6 expression might be counteracted by the inducing effect of GA. As
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expected, we found that a 6% glucose treatment inhibited AtGASA6 transcript levels,
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but that this effect was abolished by 1 μM or 10 μM GA3 applications (Fig. 3C).
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Basing on the above data that AtGASA6 is down-regulated by glucose, ABA and
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up-regulated by GA, we speculated that the lower seed germination rate of AtGASA6
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RNAi and gasa6-1 mutants under ABA or 6% glucose treatment also can be rescued
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by exogenous GA treatment. As expected, the significant germination rate differences 10 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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caused by ABA and glucose among the AtGASA6 mutants, could all be countered by
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addition of exogenous GA (Fig. 3D-E). However, exogenous GA treatment could not
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eliminate the germination rate differences among the different AtGASA6 genotype
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seeds absolutely under normal condition (Fig. S5). Taken together these data confirm
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the regulation of AtGASA6 by glucose, ABA and GA.
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AtGASA6 is an integrator of glucose, GA and ABA signaling crosstalk
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Since AtGASA6 is regulated by glucose, ABA and GA, its role in each of the
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three signaling pathways were further investigated, by evaluating AtGASA6
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expression in the seeds of mutant genotypes, corresponding to important steps in the
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different pathways. ABI3, ABI4, and ABI5 are critical regulators of seed development
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and seed germination in the ABA signaling pathway (Lopez-Molina et al., 2001;
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Lopez-Molina et al., 2002; Piskurewicz et al., 2008), the DELLA protein RGL2 is an
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important regulator of seed germination in the GA signaling pathway (Lee et al.,
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2002; Tyler et al., 2004), and GIN1 and GIN2 both play important roles in glucose
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signaling and seed germination (Moore et al., 2003; Lin et al., 2007). After exposure
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to light for 10 h, germinating seeds were harvested for analysis. In the della mutants
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rag-24, rgl2-1 and gai-t6/rga-24, the AtGASA6 transcript levels were significantly
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higher (Fig. 1A), and the same was true for the ABA signaling mutants, abi3, abi4,
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and abi5, and glucose signaling mutants gin1 and gin2 (Fig. 4A), indicating that
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AtGASA6 mRNA levels are regulated by these components in the GA, ABA and
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glucose signaling pathways.
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To determine whether AtGASA6 transcript levels in GA, ABA or glucose
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signaling mutants, are affected by other signaling factors, a series of treatments were
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performed. In the wild-type, AtGASA6 expressions were significantly inhibited by
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more than 50% by PAC, ABA or glucose, while in abi5 mutant seeds, the inhibitions
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of AtGASA6 expression by glucose, PAC or ABA were alleviated, indicating that the
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glucose-, GA-, and ABA-mediated AtGASA6 expression are partially dependent on
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ABI5 (Fig. 4B). AtGASA6 expression was not inhibited by glucose treatment, but
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showed a moderate decrease following ABA or PAC treatment in the gin2 mutant, 11 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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indicating that glucose-induced AtGASA6 inhibition is mediated by GIN2 (Fig. 4B).
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Interestingly, under glucose, ABA or PAC treatments, the significant down-regulation
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of AtGASA6 expression levels in the wild type plant were alleviated greatly in the rgl2
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mutant, suggesting that RGL2, at least, partially involved in the glucose, ABA and
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GA signaling pathways regulated AtGASA6 expression (Fig. 4B). These results
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indicating that the AtGASA6 expression is regulated by the crosstalk of glucose, ABA
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and GA signaling.
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Considering the data above, we speculated that AtGASA6 might function
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downstream of RGL2. To test this, the gasa6-1 knock down mutant was crossed with
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the rgl2-13 (Col) mutant. Under normal and 0.5 μM PAC treatment conditions, the
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rgl2-13 mutant seeds germinated faster, and the rgl2-13/gasa6-1 double mutant seeds
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germinated slightly slower than wild-type seeds and similar to that of gasa6-1 seeds
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(Fig. 4C-D), indicating AtGASA6 functions downstream of RGL2 in GA pathway. To
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further demonstrate that AtGASA6 operates downstream of GIN2 in the
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glucose-signaling pathway, AtGASA6-RNAi plants in the gin2 and wild-type (Ler)
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backgrounds, were generated and expression levels were detected by quantitative
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real-time PCR (Fig. S6). The responses of gin2/AtGASA6-RNAi, gin2, and
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AtGASA6-RNAi (Ler) seeds, to 6% glucose, were tested. The gin2/AtGASA6-RNAi
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seeds showed a significantly lower seed germination response to glucose treatment
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compared to those of gin2, indicating that down-regulation of AtGASA6 increased the
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sensitivity of gin2 to glucose (Fig. 4E-F). These results suggested that AtGASA6
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indeed functions downstream of GIN2 and RGL2, and plays an integral role in glucose,
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ABA and GA signaling pathways.
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AtGASA6 is located downstream of RGL2 to integrate glucose and GA signaling
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Since RGL2 plays an essential role in integrating GA and ABA signaling during
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seed germination (Piskurewicz et al., 2008; Lee et al., 2010), and our results suggest
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that AtGASA6 plays an integral role in glucose, ABA and GA signaling pathways, and
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functions downstream of RGL2 in GA pathway, we hypothesized that the response of
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AtGASA6 to glucose and GA is also regulated by RGL2. To verify this, mutant seeds 12 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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of rgl2-13, rgl2-13/gasa6-1 and gasa6-1, were challenged with 6% glucose. As
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expected, rgl2-13 seeds were insensitive to the treatment; however, importantly,
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rgl2-13/gasa6-1 seeds showed the same hypersensitivity to 6% glucose as the gasa6-1
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seeds (Fig. 5A). Since the inhibition of AtGASA6 expression by glucose could be
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reversed by addition of exogenous GA, the germination difference to 6% glucose
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between rgl2-13 and gasa6-1 was predicted to be reduced, but not dispelled by
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addition of exogenous GA. Although the germination rate of rgl2-13 was not changed,
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all other mutant germination rates were significantly increased by treating with 6%
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glucose plus 1μM GA3, compared with 6% glucose only. The germination rates of
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gasa6-1 and rgl2-13/gasa6-1 were significantly lower than those of rgl2-13, although
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they were more similar at late germination stages (Fig. 5B). Taken together, these data
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suggest that AtGASA6 integrates glucose and GA signaling, and functions
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downstream of the RGL2-mediated seed germination pathway.
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AtGASA6 regulates seed germination by promoting embryonic axis elongation
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The GUS staining of AtGASA6 specifically expressed in the hypocotyl-radicle
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transition zone of the embryonic axis in germinated seeds (Fig. S2B-l), indicates
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AtGASA6 might function in embryonic axis development during seed germination. To
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confirm this hypothesis, pAtGASA6::GUS seeds were germinated under different
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conditions that are known to change the germination process. GUS activity was
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observed at different time intervals around the threshold before or after endosperm
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rupture. Under normal germination conditions, AtGASA6 was first expressed at the
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embryonic axis prior to endosperm rupture. The expression level at the embryonic
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axis was 45% after exposure to light for 18 h, when the embryonic axis began to
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rupture the endosperm. This rate rose to 100%, after exposure to light for 25 h and
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once all the seeds were germinated (Fig. 6), indicating that expression of AtGASA6 in
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hypocotyls is a marker for the completion of seed germination. Importantly, seed
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germination and GUS activity were delayed significantly and synchronously by 6%
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glucose, ABA or PAC treatments, but enhanced by GA treatment. Under the GA
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condition, AtGASA6 expression time was advanced to 14 h, and the expression rate 13 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
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reached 100% by 18 h after stratification, while glucose, ABA and PAC treatments
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delayed the time point significantly, when both AtGASA6 expression and endosperm
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rupture rates all reached 100% (Fig. 6). These results suggest that AtGASA6 is
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expressed in the embryo hypocotyl to promote endosperm rupture and, together with
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data showing that AtGASA6 over-expression promotes seed germination in the
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presence of ABA, 6% glucose or PAC (Fig. 2D-E), indicate that AtGASA6 may
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promote seed germination by influencing embryo hypocotyl growth. To test this
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hypothesis, the hypocotyl growth of wild-type, AtGASA6 over-expressing, and RNAi
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seeds treated with glucose, ABA, PAC, or mannitol, were observed and their lengths
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were measured at the time of endosperm rupture. The hypocotyls of AtGASA6
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over-expressing lines were longer, but AtGASA6 RNAi lines were shorter than those
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of the wild-type. Under normal conditions and mannitol treatment, the hypocotyl
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lengths of all genotypes were similar (Fig. 7A-B).
374
It is well documented that completion of germination is the result of cell
375
expansion, and not cell division within the hypocotyl-radicle transition region
376
(Sliwinska et al., 2009). Thus, cell length and width of hypocotyl-radicle transition
377
region of embryo axis, were measured to determine whether the effects of AtGASA6
378
on embryo axis growth were caused by cell expansion. Cell length was observed to be
379
greater in the AtGASA6 over-expressing seeds, and smaller in the RNAi seeds, under
380
glucose, ABA and PAC treatments. Moreover, the cell width was also greater in
381
AtGASA6 over-expressing seeds (Fig. 7C-D), indicating that AtGASA6 accelerates
382
seed germination by promoting cell expansion within the hypocotyl-radicle transition
383
region of the embryonic axis under glucose, ABA and PAC treatments.
384 385
AtGASA6 regulates seed germination and cell elongation through the action of
386
AtEXPA1
387
Bioinformatical analysis revealed that AtGASA6 is co-expressed with the
388
expansin
1
(AtEXPA1)
and
expansin
8
(AtEXPA8)
genes
(Fig.
S7,
389
http://atted.jp/cgi-bin/locus.cgi?loc=At1g74670), which encode cell wall loosening
390
expansin proteins. AtGASA6 encodes a small polypeptide (101 amino acids) that 14 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
391
shares common structural features with other GASA family proteins: an N-terminal
392
putative signal peptide sequence, a variable hydrophilic intermediate region, and a
393
highly conserved 60 amino acid C-terminal domain containing 12 conserved cysteine
394
residues, named the GASA domain (Fig. S8). The subcellular localization of the
395
AtGASA6 protein was determined by analysis of the AtGASA6-EGFP fusion protein,
396
transiently and stably expressed in onion epidermal, and A. thaliana root cells, under
397
the regulation of the cauliflower mosaic virus (CaMV) 35S promoter, respectively.
398
GFP fluorescence in the cell periphery was observed in both cell types, while a
399
control soluble GFP protein was uniformly localized to the nucleus and cytoplasm
400
(Fig. S2C), indicating that AtGASA6 is mainly localized in the cell wall as the eFP
401
Browser suggested (Fig. S8D) (Winter et al., 2007). In addition, our results suggested
402
that AtGASA6 accelerates seed germination by promoting hypocotyl cell expansion.
403
Thus, we speculated that the AtGASA6 protein somehow functions with AtEXPA1,
404
and/or AtEXPA8, in cell expansion. To investigate their relationship, we first
405
analyzed whether the two expansin genes share the same responses to exogenous
406
stimulus with AtGASA6. The expression of AtEXPA1 was higher in the AtGASA6 OE
407
lines, and lower in the AtGASA6 RNAi lines (Fig. 8A). In addition, we observed that
408
the expression of AtGASA6 was not changed in the AtEXPA1 mutants (Fig. 8B).
409
Notably, the expression of AtEXPA1 in response to the addition of glucose, GA, and
410
ABA, is similar to that of AtGASA6 (Fig. 8C). These results suggest that AtEXPA1
411
functions downstream of AtGASA6 action.
412
We further investigated the potential function of AtEXPA1 in seed germination
413
through the use of transgenic plants with reduced or elevated AtEXPA1 expression
414
(Fig. S9). Our results indicate AtEXPA1 indeed functions in the seed germination
415
process in a similar fashion to AtGASA6, as it promotes seed germination under
416
glucose, PAC, or ABA treatment. Specifically, the germination rate of the AtEXPA1
417
over-expressing (OE) seeds was greater than that of wild-type seeds following 6%
418
glucose, PAC, or ABA treatments, while the germination rate of seeds from the expa1
419
knock-out line was significantly less (Fig. 9). Additionally, the embryonic axis and
420
cell lengths of AtEXPA1-OE lines were greater than for wild-type seeds, but less than 15 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
421
for the expa1 seeds, following glucose, ABA, and PAC treatments (Fig. 10).
422
Given the similar expression patterns and function of AtEXPA1 and AtGASA6 in
423
the process of seed germination, as well as the fact that AtEXPA1 expression was
424
affected by AtGASA6, we crossed the expa1 mutant with AtGASA6 over-expressing
425
plants to elucidate the genetic relationship between these genes. As expected, the
426
faster germination rate and greater cell length of the AtGASA6 over-expressing plants
427
was abolished by the AtEXPA1 knock-out mutation (Fig. 9, 10), suggesting that the
428
AtGASA6 mediated seed germination under 6% glucose, ABA, or PAC treatments, are
429
dependent on the action of the AtEXPA1 gene.
430 431
Discussion
432
Numerous studies show that a typical GA response is the promotion of seed
433
germination. The DELLA protein RGL2 is involved in integrating phytohormones
434
and environmental factors controlling seed germination (Lee et al., 2002; Ariizumi
435
and Steber, 2007; Piskurewicz et al., 2008; Ariizumi et al., 2013). However,
436
downstream events of RGL2 are not well understood. The GAST1-like family,
437
members of which encode small proteins with a conserved cysteine-rich domain, is
438
known as one of the few identified GA-induced gene families (Lin et al., 2011;
439
Roxrud et al., 2007; Shi et al., 1992; Zhang and Wang, 2008). Although many studies
440
have attempted to elucidate their roles in plant development, GA responses, in
441
particular, is still unclear. Here, we provide evidence supporting the role of AtGASA6
442
protein in mediating, not only GA responses, but also ABA and glucose responses, in
443
regulating the timing of seed germination, and suggest that GASA family members
444
function on a multiple phytohormones network.
445
AtGASA6 integrates the GA, ABA and glucose signaling pathways
446
Seed germination is known to be governed by two major counteracting
447
phytohormones, ABA and GA, in response to various environmental factors
448
(Finkelstein et al., 2008). More signaling components of these two phytohormones
449
pathways, however, need to be found in regulating seed germination. AtGASA6 16 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
450
expression was induced by GA, and repressed by PAC and ABA, in both germinating
451
seeds and seedlings (Fig. 3, S4). Expression of several other AtGASA6 homologous
452
genes are also induced by GA and inhibited by ABA (Zhang and Wang, 2008; Lin et
453
al., 2011), indicating GASA family members are some important factors involve in
454
GA and ABA signaling network. However, gasa6-1 seeds did not show significant
455
germination phenotype under the normal condition probably due to functional
456
redundancy, this problem is also reported on AtGASA4, the homologous gene of
457
AtGASA6 (Rubinovich and Weiss, 2010). Over-expression of AtGASA6 can
458
effectively overcome the inhibiting effects of PAC and ABA (Fig. 2), while the
459
difference in seed germination rates under ABA treatment, between AtGASA6
460
over-expressing and RNAi seeds, was eliminated by exogenous GA (Fig. 3D),
461
indicating that AtGASA6 operates in both GA and ABA signaling pathways during
462
seed germination. To identify the specific position of AtGASA6, in the GA and ABA
463
signaling network, we examined AtGASA6 expression in DELLA mutants, rga24,
464
gai-t6 and rgl2-1, and ABI mutants, abi3, abi4 and abi5. Results showed that all of
465
the tested DELLA proteins down-regulate AtGASA6 expression, and that RGL2 is the
466
main protein modulating AtGASA6 expression in germinating seeds (Fig. 1). This
467
expression pattern is consistent with the finding that AtGASA6 expression is repressed
468
in the RGL2-mediated seed germination transcriptome (Stamm et al., 2012). Our
469
results show that under condition with added PAC, the highest seed germination rates
470
of rgl2-13 were reduced by the loss of AtGASA6 function (Fig. 4C-D), suggesting that
471
AtGASA6 is an important factor for RGL2-mediated seed germination under PAC
472
treatment. Interestingly, AtGASA4 may also function downstream of RGL2 proteins in
473
seed germination (Ariizumi et al., 2013). In addition, several GASA family genes have
474
been reported to function downstream of the DELLA proteins (Sun et al., 2013;
475
Zhang and Wang, 2008). These findings show us that GA mediates GASA proteins
476
function in different plant developmental processes, through the regulation of DELLA
477
proteins. In the absence of ABI5, which acts downstream of ABI3 and represses seed
478
germination in response to ABA and GA (Lopez-Molina et al., 2002; Piskurewicz et
479
al., 2008), up-regulation of AtGASA6 occurs and the repression of AtGASA6 by PAC 17 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
480
is diminished (Fig. 4B). AtGASA6 expression was also up-regulated in abi3 and abi4
481
mutants (Fig. 4A). These results suggest that AtGASA6 acts downstream of ABI5 to
482
mediate the interaction between GA and ABA. When GA levels are low, RGL2
483
promotes ABA biosynthesis by stimulating the expression of XERICO, which encodes
484
a RING-H2 zinc finger factor (Ko et al., 2006; Zentella et al., 2007; Piskurewicz et
485
al., 2008). In turn, elevation of endogenous ABA levels promotes expression of RGL2
486
and ABI5, and a subsequent increase in protein levels (Piskurewicz et al., 2008).
487
Presumably, RGL2 allows AtGASA6 to respond to both the GA and ABA pathways,
488
by generating a single signal output that does not permit germination under
489
unfavorable environmental conditions.
490
To date, at least three types of glucose signal transduction mechanisms have
491
been found in plants, of which, the hexokinase (HXK)-dependent pathway may play a
492
leading role (Xiao et al., 2000). Glucose-induced ABA accumulation has been
493
suggested to be essential for HXK-mediated glucose responses (Arenas-Huertero et
494
al., 2000), and inhibition of seed germination by glucose has been shown to be caused
495
by a slowing of the decline in endogenous ABA levels through modulating the
496
transcription of ABA biosynthesis and perception genes (Price et al., 2003); however,
497
the role of GA in sugar signaling, and the issue of whether the HXK-dependent
498
sugar-signaling pathway is affected by GA, is not well understood. In the present
499
work, AtGASA6 expression was substantially down-regulated by 6% glucose, in both
500
germinating seeds and seedlings (Fig. 3, S4), and AtGASA6 over-expressing lines
501
displayed enhanced tolerance of glucose-induced inhibition seed germination (Fig. 2)
502
and cotyledon greening (Fig. S10). Consistent with these results, AtGASA6 has also
503
been characterized as an in vivo sugar marker gene (Gonzali et al., 2006), and is
504
severely repressed by AtTZF1, which serves as a positive regulator of ABA/sugar, and
505
a negative regulator of GA responses (Lin et al., 2011). These findings indicate that
506
AtGASA6 plays important roles in the sugar signaling pathway, and may participate in
507
the crosstalk of sugar/GA and sugar/ABA signaling. The expressions of AtGASA6
508
were clearly higher in the gin2 and gin1 mutants (Fig. 4A), and the glucose-inhibited
509
AtGASA6 expression was absent in gin2 (Fig. 4B). Additionally, the reduction of 18 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
510
AtGASA6 expression in the gin2 mutant, resulted in decreased gin2 seed tolerance to
511
6% glucose (Fig. 4F), indicating that AtGASA6 functions downstream of GIN2.
512
Notably, the inhibition of AtGASA6 expression by the 6% glucose treatment, was
513
abolished by GA application (Fig. 3C), as was the glucose-induced inhibition of
514
AtGASA6 RNAi seed germination (Fig. 3E). These results indicate that GA signaling
515
affects the glucose signaling pathway, and that AtGASA6 may integrate their crosstalk.
516
Among the GA signaling components, only the RGL2 gene is found to be
517
up-regulated by glucose, and the inhibitory effect of glucose on seed germination has
518
been suggested to be accomplished via the inactivation of the GA signaling pathway
519
through RGL2 (Yuan and Wysocka-Diller, 2006). Interestingly, the expression of
520
GIN2 was lower in germinating rgl2-1 mutant seeds (Fig. S11), indicating that
521
HXK-dependent glucose-signaling may be regulated by RGL2, although the
522
regulatory mechanisms of RGL2 in glucose signaling are unclear. Genetic analysis
523
revealed that AtGASA6 also plays an important role downstream of RGL2, in glucose
524
signal-inhibited seed germination, since reduction of AtGASA6 altered the tolerance of
525
rgl2 seeds to the glucose signal (Fig. 5).
526 527
AtGASA6 regulates cell elongation at the embryonic axis through AtEXPA1
528
The completion of seed germination is marked by the appearance of the radicle
529
through the surrounding endosperm and testa (Bewley, 1997; Bentsink and Koornneef,
530
2008; Weitbrecht et al., 2011). Physiologically, rupture of the endosperm and testa is
531
associated with cell division and enlargement, which require cell wall loosening
532
(Cosgrove, 2000a). Some cell wall-localized proteins are suggested to be involved in
533
cell expansion/elongation in seed germination, e.g. expansins (Chen and Bradford,
534
2000; Chen et al., 2001; Yan et al., 2014), and several cell wall-localized GASA
535
proteins from different plant species are involved in cell division and elongation (de la
536
Fuente et al., 2006; Zhang et al., 2009), indicating that GASAs may play a role in cell
537
wall extensibility modification. AtGASA6 is localized mainly in the cell wall (Fig.
538
S2C), and AtGASA6 was specifically expressed in the hypocotyl-radicle transition
539
zone of embryonic axis in germinating seeds (Fig. 6), suggesting that AtGASA6 19 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
540
regulates seed germination, possibly by mediating embryonic axis elongation.
541
Interestingly, further results demonstrated that AtGASA6 accelerates cell expansion
542
within the hypocotyl-radicle region during seed germination under glucose, ABA and
543
PAC treatment (Fig. 7). However, AtGASA6 has not play roles in the mannitol
544
-mediated seed germination inhibition, Under mannitol treatment, all the seeds
545
showed reduced seed germination rate, and shorter embryonic axis length, but slightly
546
larger cell expansion compared to the normal condition (Fig. 2E, Fig. 7B, and Fig.
547
7D), it is imply that the relationship between cell expansion and embryonic axis
548
length is complex under mannitol treatment, and the cell division is needed to be
549
taken into consideration to elucidate the relationship among cell expansion,
550
embryonic axis length and seed germination under mannitol treatment in the future
551
work. Combined with the observation that AtGASA6 co-expresses with several cell
552
wall loosing genes (Fig. S7), it is reasoned that the function of AtGASA6 protein may
553
contribute to cell wall loosening by association with expansin action. Expansins are
554
thought to induce cell wall extension by disrupting non-covalent load-bearing
555
linkages between cellulose microfibrils and the cross-linking matrix glycans
556
(Cosgrove, 2005). Different expansins perform diverse tissue- or organ-specific roles
557
(Cosgrove, 2000b), including endosperm rupture and seed germination (Chen and
558
Bradford, 2000; Chen et al., 2001; Yan et al., 2014), and leaf development (Pien et al.,
559
2001; Goh et al., 2012). LeEXP4 is expressed specifically in the micropylar
560
endosperm cap region and is associated with endosperm cap weakening (Chen and
561
Bradford, 2000). In contrast, LeEXP8 mRNA is localized to the radicle cortex of the
562
embryo, whereas LeEXP10 mRNA is expressed throughout the embryo during seed
563
germination (Chen et al., 2001). Our data show that the expression of AtEXPA1 was
564
markedly higher in AtGASA6 over-expressing seeds, and lower in RNAi seeds, while
565
AtGASA6 expression did not change in AtEXPA1 mutants (Fig. 8). In addition,
566
AtEXPA1 can also promote seed germination by regulating cell elongation at the
567
embryonic axis, similar to AtGASA6 (Fig. 9; Fig. 10). The faster germination rates,
568
and greater cell length phenotypes of AtGASA6 over-expressing seeds, were abolished
569
by expa1 (Fig. 9; Fig. 10). Taken together, these results suggested that the function of 20 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
570
AtGASA6 on cell expansion depends on the actions of AtEXPA1. Therefore, we
571
hypothesized the existence of a direct physical interaction between AtGASA6 and
572
AtEXPA1, but this was not supported by bimolecular fluorescence complementation
573
and yeast two-hybrid analyses (data not shown). In the RGL2-regulated seed
574
germination transcriptome, several EXPANSIN genes, including AtEXPA1, AtEXPA3,
575
AtEXPA8 and AtEXPA9, are repressed by RGL2 (Stamm et al., 2012). AtEXPA2 is
576
also involved in seed germination and abiotic stress responses, and its expression
577
levels in germinating seeds is repressed by expression of DELLA genes, including
578
RGL1, RGL2, RGA and GAI (Yan et al., 2014). Whether the DELLA genes repress
579
expansin genes, directly or indirectly, to regulate seed germination, is not known;
580
however, our results indicate that AtGASA6 influences seed germination by mediating
581
a signal between DELLA proteins and expansins.
582 583
Conclusions
584
In conclusion, our results suggest a role for AtGASA6 as a positive regulator of
585
seed germination. We propose a model (Fig. 11), where GA promotes, and glucose
586
and/or ABA inhibits, AtGASA6 expression, the cumulative effects of which govern
587
GA- and ABA-mediated seed germination. In addition, AtGASA6 promotes seed
588
germination via AtEXPA1 action by promoting cell elongation, and consequently
589
embryonic hypocotyls length. However, the functional mechanism of AtGASA6 in
590
regulating seed germination and cell elongation, and the working relationship between
591
AtGASA6 and AtEXPA1, and even the other expansins, are needed more evidence to
592
elucidate in the future.
593 594
Materials and Methods
595
Plant materials
596
Arabidopsis thaliana ecotype Columbia (Col-0) was used in the generation of all
597
transgenic plants, and as the experimental control in this study, unless specifically
598
indicated. The mutant lines gin2-1, abi3-1, gai-t6, rgl2-1, rga24 and gai-t6/rga24, are
599
in the Landsberg gerecta (Ler) background, and were generously provided by 21 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
600
Professor Xiangdong Fu (Institute of Genetics and Developmental Biology of the
601
Chinese Academy of Sciences); rgl2-13 is in the Col background (Provided by
602
Professor Ling Li in South China Normal University); gasa6-1 (SALK_072904),
603
expa1 (SALK_010506C), AtEXPA1-OE (Provided by Professor Xuechen Wang in
604
China Agriculture University) and abi4-102, is in the Col background; gin1 and abi5
605
are in the Wassilewskija (Ws) background. All mutant seeds were bought from the
606
ABRC stock center of Ohio State University, unless otherwise indicated. PCR was
607
utilized on genomic DNA using gene-specific and T-DNA border-specific primers to
608
obtain plants homozygous for the T-DNA insertion. The double mutant lines utilized
609
in the present study were created by cross-pollination between the relevant mutants,
610
and the genotypes were verified via resistance and PCR.
611 612
Plant growth conditions and seed germination assay
613
Plants were grown at 22°C under long days (16 h light/ 8 h Dark), light was supplied
614
by cool and warm white fluorescent bulbs, reaching an intensity of approximately 100
615
μmol m-2s-1 on the shelf surface. Seeds were surface sterilized with 70% (v/v) ethanol
616
for 10 s, then 1%（v/v）sodium hypochlorite for 10min, and rinsed four to five times
617
with sterile distilled water. Sterilized seeds were subsequently sown on Murashige and
618
Skoog Basal Medium (Signa-Aldrich) containing 0.8% (w/v) agar (MBCHEM) and
619
1.5% sucrose at pH 5.7. Plates were kept at 4°C in darkness for 3 d for stratification,
620
and then transferred to a plant culture room set at 22°C with a 16 h light/ 8 h dark
621
photoperiod. For the germination assay, at least 80 seeds for each genotype were
622
sterilized and sown on MS basal medium supplemented with or without 6% glucose,
623
6% mannitol, 1.5% sucrose, 1 μM GA3, 0.5 μM PAC or 1 μM ABA, MS basal
624
medium as control, 6% mannitol as osmotic control. The radical tip emergence was
625
defined as the first sign of seed germination. Seeds were scored daily until seed
626
germination rate reached over 98%. The germination results were calculated based on
627
at least three independent experiments. For seedling genotype assay, at least 80 seeds
628
for each genotype were sterilized and sown on MS basal medium with 6% glucose
629
and 6% mannitol, and at least 60 growing similar seedlings, for each genotype, were 22 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
630
measured based on at least three independent experiments, MS medium was used as
631
control. For Real-time PCR assays of different treatments, seeds were collected at 4
632
hours after stratification and sown on MS basal medium supplemented with or
633
without 6% glucose, 10 μM GA3, 0.5 μM PAC or 10 μM ABA, for 6 hours. For the
634
control, seeds were collected at 10 hours after seeds were transferred into light at 22°C
635
from dark at 4°C. The GA3, PAC and ABA stock solutions were made with 100%
636
alcohol and stored frozen at -20°C in dark, then used depending upon the experiment.
637 638
Plasmid construction and plant transformation
639
The GASA6 gene, its promoter (1200 bp before the 5’ untranslated region), and the
640
Coding sequence (CDS), were PCR amplified using high-fidelity DNA polymerase
641
(Pfu Ultra DNA polymerase) from Arabidopsis genomic DNA and cDNA,
642
respectively, and cloned into a T-Vector (Takara Bio., Japan). The sequence of the
643
cloned AtGASA6 gene, promoter and CDS, were verified by DNA sequencing and
644
were sub-cloned into pBI101 and modified pBin19 binary vectors, respectively. For
645
overexpression experiments, the cauliflower mosaic virus (CaMV) 35S promoter was
646
used to drive the expression of the GASA6 CDS. For promoter analysis, the promoter
647
was sub-cloned into pBI101 to drive expression of the β-glucuronidase (GUS) gene
648
(pGASA6::GUS). For RNAi experiments, the hydrophobic region fragment was
649
subcloned into pRNAi-35S vector (Provided by Professor Yaoguang Liu in South
650
China Agriculture University). The fusion of GASA6 and enhanced green fluorescent
651
protein (EGFP), was made by cloning GASA6 (with the stop codon removed) in
652
frame to the N terminus of the GFP protein of the modified pBin19 binary vectors.
653
The 35S promoter was used to drive expression of the fusion protein. Plants were
654
transformed with Agrobacterium tumefaciens by the floral dipping method (Clough
655
and Bent, 1998). Transformants were selected by 50 μg/ml kanamycin, and verified
656
by PCR using specific primers. RNAi seeds were selected by using 25 μg/ml
657
Hygromycin B. All transformed plants were selected for two more generations, and
658
homozygous transgenic plants (T3) were used for further characterization.
659 23 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
660
GUS staining and GFP localization
661
T3 transgenic lines carrying the pGASA6::GUS construct were assayed for GUS
662
staining. The samples were immersed into the GUS staining solution (50 mM sodium
663
phosphate buffer, pH 7.0, 1 mM EDTA, 0.1% Triton X-100, 1 mg/ml of X-Gluc, 2
664
mM ferricyanide, and 2 mM ferrocyanide), and incubated at 37°C for 12 h or
665
overnight. Samples were washed in 70% ethanol for several times until the material
666
was bleached. Finally, the samples were observed and photographed using an
667
Olympus BX Microscope (Olympus Corporation, Japan). For GASA6-EGFP
668
localization analysis, the roots of 4-days-old young seedlings were observed using a
669
confocal laser-scanning microscopy (CLSM) combination system (LSM510/
670
ConfoCor2; Zeiss, Jena, Germany). The recombinant sequences were incubated with
671
gold particle, and the constructed gold-coated DNA particles were delivered into
672
onion epidermal cells using the PDS-1000/He particle bombardment system (Bio-Rad,
673
USA). The samples were observed using a confocal laser-scanning microscopy
674
(CLSM) combination system, or a fluorescence microscope (Nikon TS-100, Japan)
675
and photographed using a Nikon 5400 camera (Nikon, Japan).
676 677
Genomic DNA extraction and T-DNA insertion mutant screening
678
Homozygous T-DNA insertion mutants were identified following the protocol
679
described
680
(http://signal.salk.edu/tabout.html) by PCR. 2 ~ 3 leaves of 4-weeks-old seedlings,
681
growing in soil, were collected from individual plants. Genomic DNA was extracted
682
using a quick CTAB method (Rogers and Bendich, 1985), and used for PCR reactions
683
with the primers recommended in the SALK protocols.
at
the
SALK
Insertion
Sequence
Database
684 685
Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)
686
Total RNAs from tissues were isolated using the total RNA isolation kit, with
687
genomic DNA removed, according to the manufacturer’s instructions (Takara Bio,
688
Japan). The cDNAs were synthesized from 1 μg total RNA using the Prime ScriptTM
689
first strand cDNA synthesis Kit (Takara Bio, Inc.), and 1 μl cDNA was used for 24 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
690
subsequent PCR. All PCR reactions were performed in a total volume of 20 μl using
691
22 ~ 30 cycles under the following conditions: denaturation, 94°C, 30 s; annealing,
692
58°C, 30 s; extension, 72°C, 30 s. PCR products were subject to 1% agarose gel
693
electrophoresis, photographed using the Bio Imagine system (Gene Company Ltd.),
694
and analyzed with Gene Tools software (Gene Company Ltd.). Gene expressions were
695
normalized to the expression of Actin1. Primer sequences were used as previously
696
reported (Zhang et al., 2009).
697 698
Real-Time PCR
699
Total RNA from germinating seeds or 2-weeks-old seedlings were extracted using the
700
total RNA isolation kit with genomic DNA removed, according to the manufacture’s
701
instructions, and reverse transcribed using the PrimeScriptTM RT reagent kit with
702
gDNA Eraser (TAKARA, Japan). Quantitative real-time PCR was performed using
703
the Promega Real-time PCR Kit (Promega, America). Gene expression was
704
normalized to the expression of ubiquitin 1 (UBQ1). Primer sequences used were as
705
previously reported (Jiang et al., 2012).
706 707
Hypocotyl length assay
708
For hypocotyl length assay, seeds were germinated on MS basal medium with 0.8%
709
(w/v) agar, with or without GA3, PAC, ABA, glucose or mannitol, stratified for 3d in
710
the dark at 4 °C, and then grown at 22 °C under light condition. Hypocotyl length was
711
quantified with Image J software (http://rsbweb.nih.gov/ij/) after photography of
712
seeds collected at the corresponding time points.
713 714
Observation of embryonic axis and cotyledon epidermal cells
715
For embryonic axis epidermal cells observation, seeds were collected in the
716
corresponding time points under stress conditions, and stained with 100 μM propidum
717
iodide (PI) solution for 10 ~ 30 s, rinsed with water for three times, and photographed.
718
For cotyledon epidermal cells observation, the cotyledons of 6-day-old young
719
seedlings were collected and immersed in 75% ethanol until the tissue was bleached. 25 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
720
The samples were stained with 100 μM PI for 30 min, and were subsequently rinsed
721
with water for three times and photographed. All the pictures were photographed by
722
using a commercial laser-scanning microscope (LSM510/ConfoCor2) combination
723
system (Zeiss).
724 725
Accession numbers
726
Sequence data from this article can be found in the Arabidopsis Genome Initiative or
727
GenBank/EMBL database under the following accession numbers: ABI3, At3g24650;
728
ABI4, At2g40220; ABI5, At2g36270; Actin1, At2g37620; AtEXP1, At1g69530; GAI,
729
At1g14920; AtGASA6, At1g74670; GIN1, At1g52340; GIN2, At4g29130; RGA,
730
At2g01570; RGL2, At3g03450; UBQ1, At3g52590. All the primers are listed in
731
Supplementary Table 1.
732 733
Acknowledgements
734
We thank Professor Xiangdong Fu from Institute of Genetics and Developmental
735
Biology of the Chinese Academy of Sciences for providing della mutants seeds,
736
Professor Xuechen Wang in China Agriculture University for providing AtEXP1-OE
737
seed line, Professor Ling Yuan (University of Kentucky, America) and Dr. Sitakanta
738
Pattanaik (University of Kentucky, America) for giving helpful comments on the
739
manuscript, and Dr. Kathy Shen (University of Kentucky, America) for proof reading
740
the manuscript.
741 742
Author Contributions
743
S.Z. and X.W. conceived this project and designed all research. C.Z., H.X., S.Y.,
744
S.W., L.L., and S.Z. performed the research. S.Z., C.Z., H.X., and X.W. analyzed data.
745
S.Z. and X.W. wrote the article.
746 747
Reference
748 749
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921
Figure Legends
922
Figure 1. DELLA proteins inhibit AtGASA6 and AtEXPA1 expression in germinating
923
seeds. Relative transcript levels of AtGASA6 (A) and AtEXPA1 (B) in germinating
924
wild-type (Col) and della mutant seeds (rga-24, gai-t6, rgl2-1, gai-t6/rga-24). All
925
seeds were collected at 10 h after seeds were transferred into light at 22°C from dark
926
at 4°C. The transcript levels were normalized to UBQ1 gene expression. Results are
927
averages ± SE (n ＝ 3). All experiments were repeated at least three times with
928
similar results.
929 930
Figure 2. AtGASA6 over-expressing seeds are hyposensitive, but RNAi and gasa6-1
931
mutant seeds are hypersensitive to glucose (Glc), ABA and PAC. (A) The diagram of
932
T-DNA insertion position in gasa6-1 mutant. (B) Relative transcript levels of
933
AtGASA6 in 2-week-old gasa6-1 mutant seedlings detected by RT-PCR, Actin1 was
934
used to standardize gene expression. (C) Relative transcript levels of AtGASA6 in
935
germinating seeds of AtGASA6 over-expressing and RNAi seeds. The transcript levels
936
all were normalized to Actin1 gene expression. (D) Photographs of germination
937
phenotypes of wild-type, gasa6-1, AtGASA6 over-expressing (OE#2-1, OE#5-5,
938
OE#6-1), and RNAi (R#3-5, R#4-3, R#6-7) seeds, treated with 0.5 μM PAC, 1 μM
939
ABA, 6% glucose, or 6% mannitol, minimal medium was used as control. Seeds were
940
photographed at 24 h, 64 h, 52 h, 64 h, or 48 h. At least 40 seeds were used. (E) Seed
941
germination rates of wild-type (Col), gasa6-1, AtGASA6 over-expressing (OE#2-1,
942
OE#5-5, OE#6-1), and RNAi (R#3-5, R#4-3, R#6-7) seeds, untreated or treated with
943
0.5 μM PAC, 1 μM ABA, 6% glucose, or 6% mannitol, minimal medium was used as
944
control. The asterisk indicates significant differences compared with control seeds
945
(One-way ANOVA was used to analyze the significant difference: * P ＜ 0.01).
946
Three technical replicates were performed for each of three biological replicates.
947 948
Figure 3. GA treatment abolishes the glucose- and ABA-induced differences in
949
germination of different AtGASA6 genotypes. (A) Relative transcript levels of 31 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
950
AtGASA6 in germinating seeds, in response to PAC, GA, Glucose and ABA. Seeds
951
were grown on minimal medium for 4 h at 22°C after seeds were transferred from
952
dark at 4°C, then were treated with 10 μM PAC, 10 μM GA, 10 μM ABA or 6%
953
glucose for 6 h, minimal medium was used as control. (B) Relative transcript levels of
954
AtGASA6 in response to different concentrations of PAC or ABA in germinating seeds.
955
Seeds germinated for 4 h at 22°C after seeds were transferred from dark at 4°C, then
956
were treated with 1 μM or 10 μM PAC, or 10 μM or 100 μM ABA for 6 h. (C)
957
Relative transcript levels of AtGASA6 in response to GA plus glucose in germinating
958
seeds. Seeds germinated for 4 h were treated with 6% glucose, 0.1 μM, 1 μM or 10
959
μM GA alone, or with 6% glucose for 6 h, minimal medium was used as control. (D)
960
Seed germination rate of wild-type (Col), AtGASA6 over-expressing (OE#2-1), RNAi
961
(R#6-7), and the gasa6-1 mutant treated with 1 μM ABA alone or plus 1 μM GA.
962
Error bars denote SD. Similar results were obtained in at least three independent
963
experiments, and at least 80 seeds were used. (E) Seed germination rate of wild-type
964
(Col), AtGASA6 over-expressing (OE#2-1), RNAi (R#6-7), and the gasa6-1 mutant
965
treated with 6% glucose alone or plus 1 μM GA. All transcript levels of gene
966
expression were normalized to UBQ1 gene expression. Results are averages ± SE (n
967
＝ 3). One-way ANOVA was used to analyze the significant difference: * P ＜ 0.01.
968
All experiments were repeated at least three times with similar results.
969 970
Figure 4. AtGASA6 functions downstream of RGL2, ABI5, GIN2, in the GA, ABA
971
and glucose signaling pathways. (A) Relative transcript levels of AtGASA6 in
972
germinating wild-type (Ler, Col-0, and Ws) and a set of abi and gin mutant seeds
973
(abi3-1, abi4-102, abi5-1, gin1-1, and gin2-1). All seeds were collected 10 h after
974
seeds were transferred into light at 22°C from dark at 4°C. (B) Relative transcript
975
levels of AtGASA6 in germinating wild-type (Ler, Ws), and rgl2-1, abi5-1, and gin2-1
976
mutant seeds, treated with 0.5 μM PAC, 10 μM ABA and 6% glucose for 6 h, minimal
977
medium was used as control. (C-D) Seed germination assays of Col, gasa6-1, rgl2-13,
978
and rgl2-13/gasa6-1, treated with 0.5 μM PAC. (E-F) Seed germination assays of Ler,
979
gin2, AtGASA6-RNAi (Ler/R2#3), and gin2/AtGASA6-RNAi (gin2/R2#1, gin2/R3#2, 32 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
980
gin2/R4#4) treated with 6% glucose. All transcript levels of gene expression were
981
normalized to UBQ1 gene expression. Results are averages ± SE (n ＝ 3). One-way
982
ANOVA was used to analyze the significant difference: * P ＜ 0.01. All experiments
983
were repeated at least three times with similar results.
984 985
Figure 5. An AtGASA6 mutation decreases rgl2-13 insensitivity to glucose, and GA
986
abolishes the glucose-induced germination differences between rgl2-13 and
987
rgl2-13/gasa6-1 mutants. Seed germination assays of Col, gasa6-1, rgl2-13,
988
rgl2-13/gasa6-1, treated with 6% glucose alone (A) or plus 1 μM GA3 (B). The
989
asterisk indicates significant differences compared with control plants (One-way
990
ANOVA was used to analyze the significant difference: * P ＜ 0.01). Three technical
991
replicates were performed for each of three biological replicates.
992 993
Figure 6. AtGASA6 is expressed at the embryonic axis to promote endosperm rupture
994
during seed germination. pGASA6::GUS seeds, untreated or treated with glucose,
995
ABA, PAC or GA, minimal medium was used as control. Numbers at left top corner
996
indicate time points after stratification, and values (%) at the bottom indicate the
997
percentage of the embryo containing GUS staining. Three technical replicates were
998
performed for each of three biological replicates.
999 1000
Figure 7. AtGASA6 accelerates embryonic axis elongation by promoting cell
1001
elongation and expansion. (A) Photographs of embryonic axisl elongation of
1002
AtGASA6 over-expressing, and RNAi seeds treated with 6% glucose, 0.5 μM PAC, 1
1003
μM ABA, or 6% mannitol, using minimal medium as the control and 6% mannitol as
1004
the osmotic control. Seeds under control, glucose, mannitol, PAC and ABA at 24 h, 64
1005
h, 64 h, 52 h and 48 h conditions were photographed. All pictures share the same scale
1006
bar in the bottom of the last picture, scale bar = 2 mm. (B) Embryonic axis length of
1007
AtGASA6 over-expressing, and RNAi seeds untreated or treated with 6% glucose, 6%
1008
mannitol, 0.5 μM PAC or 1 μM ABA. Seeds were harvested at the same time point as
1009
in (A). n > 50. (C) Embryonic axis cells of AtGASA6 over-expressing, and RNAi 33 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1010
seeds, untreated or treated with 6% glucose, 6% mannitol, 0.5 μM PAC or 1 μM ABA.
1011
Seeds were harvested at the same time point as (A). All the pictures share the same
1012
scale bar in the bottom of the last picture, scale bar = 20 μm. (D) Embryonic axis cell
1013
length and width in AtGASA6 over-expressing, and RNAi seeds, untreated or treated
1014
with 6% glucose, 6% mannitol, 0.5 μM PAC or 1 μM ABA. Seeds were harvested at
1015
the same time point as (A). The asterisk indicates significant differences compared
1016
with control seeds (One-way ANOVA was used to analyze the significant difference:
1017
* P ＜ 0.05, ** P ＜ 0.01). Three technical replicates were performed for each of
1018
three biological replicates.
1019 1020
Figure 8. AtGASA6 induces AtEXPA1 expression in germinating seeds. (A) Relative
1021
transcript
1022
over-expressing (OE#2-1, OE#6-1), RNAi (RNAi#4-3, RNAi#6-7), and gasa6-1 seeds.
1023
(B) Relative transcript levels of AtGASA6 in germinating wild-type (Col-0), expa1,
1024
and AtEXPA1 over-expressing (AtEXPA1-OE) seeds. (C) Relative transcript levels of
1025
AtEXPA1 in response to 6% glucose, 0.5 μM PAC or 1 μM ABA in germinating Col
1026
seeds. The transcript levels of gene expression were normalized to UBQ1 gene
1027
expression. Results are averages ± SE (n ＝ 3). All experiments were repeated at
1028
least three times with similar results.
levels
of
AtEXPA1
in
germinating
wild-type
(Col),
AtGASA6
1029 1030
Figure 9. AtEXPA1 is necessary for AtGASA6 function in response to GA, ABA and
1031
glucose during seed germination. (A) Photographs of germination phenotypes of
1032
wild-type
1033
AtGASA6-OE#2-1/expa1 seeds, untreated or treated with 6% glucose, 1 μM ABA or
1034
0.5 μM PAC. All pictures share the same scale bar in the bottom of the last picture,
1035
scale bar = 2 mm. (B) Seed germination assays of wild-type (Col), expa1,
1036
AtEXPA1-OE, GASA6-OE#2-1, and AtGASA6-OE#2-1/expa1 seeds, untreated or
1037
treated with 6% glucose, 1 μM ABA or 0.5 μM PAC. The transcript levels were
1038
normalized to UBQ1 gene expression. Results are averages ± SE (n ＝ 3). One-way
(Col),
expa1,
AtEXPA1-OE,
AtGASA6-OE#2-1,
and
34 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1039
ANOVA: * P ＜ 0.01. All experiments were repeated at least three times with similar
1040
results.
1041 1042 1043
Figure 10. AtGASA6 promoting cell elongation requires the participation of AtEXPA1.
1044
(A) Photographs of the embryonic axis of wild-type (Col), expa1, AtEXPA1-OE,
1045
GASA6-OE#2-1, and GASA6-OE#2-1/expa1 seeds, untreated or treated with 6%
1046
glucose, 1 μM ABA or 0.5 μM PAC, minimal medium was used as control. All the
1047
pictures share the same scale bar in the bottom of the last picture, scale bar = 2 mm.
1048
(B) Embryonic axis lengths of wild-type (Col), expa1, AtEXPA1-OE, GASA6-OE#2-1,
1049
and GASA6-OE#2-1/expa1 seeds, untreated or treated with 6% glucose, 1 μM ABA or
1050
0.5 μM PAC, minimal medium was used as control. Similar results were obtained in
1051
three independent experiments. (C) Photographs of cell size change of wild-type (Col),
1052
expa1, AtEXPA1-OE, GASA6-OE#2-1, and GASA6-OE#2-1/exp1 seeds, untreated or
1053
treated with 6% glucose, 1 μM ABA or 0.5 μM PAC, minimal medium was used as
1054
control. All pictures were share the same scale bar in the bottom of the last picture,
1055
scale bar = 2 μm. (D) Hypocotyl cell lengths of wild-type (Col), expa1, AtEXPA1-OE,
1056
GASA6-OE#2-1, and GASA6-OE#2-1/expa1 seeds, untreated or treated with 6%
1057
glucose, 1 μM ABA or 0.5 μM PAC. The transcript levels were normalized to UBQ1
1058
gene expression. Results are averages ± SE (n ＝ 3). One-way ANOVA: * P ＜
1059
0.05, ** P ＜ 0.01. All experiments were repeated at least three times with similar
1060
results.
1061 1062
Figure 11. Proposed model of AtGASA6 function integrating GA, ABA and glucose
1063
signals to regulate seed germination.
1064 1065 1066 1067 1068 35 Downloaded from www.plantphysiol.org on September 23, 2015 - Published by www.plant.org Copyright © 2015 American Society of Plant Biologists. All rights reserved.
1069
Supplemental data
1070
Supplementary Figure 1. Co-expression analysis of AtGASA6, AtEXPA1 and RGL2
1071
genes.
1072
Supplementary Figure 2. AtGASA6 gene expression pattern and AtGASA6 subcellular
1073
localization.
1074
Supplementary Figure 3. Transcript levels of AtGASA6 and its homologous genes in
1075
AtGASA6-RNAi plants.
1076
Supplementary Figure 4. AtGASA6 expression levels in response to GA, glucose and
1077
ABA in early seedling development.
1078
Supplementary Figure 5. Seed germination rates of different AtGASA6 genotype
1079
plants under GA treatment.
1080
Supplementary Figure 6. AtGASA6 expression levels in Ler/AtGASA6-RNAi and
1081
gin2/AtGASA6-RNAi plants.
1082
Supplementary Figure 7. Co-expression gene analysis of AtGASA6.
1083
Supplementary Figure 8. Bioinformatical analyses of AtGASA6 protein.
1084
Supplementary Figure 11. AtEXPA1 expression levels in the different EXPA1 genotype
1085
seeds.
1086
Supplementary Figure 10. AtGASA6 regulates cotyledon greening in response to 6%
1087
glucose in early seedling development.
1088
Supplementary Figure 11. GIN2 expression level in the rgl2-1 mutant.
1089
Supplementary Table 1. Gene-specific primer sequences.
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Zhong et al., Supplementary Figures
Supplementary Fig. 1. Co-expression analysis of AtGASA6, AtEXPA1 and RGL2 genes.
Co-expression
analysis
was
performed
using
the
GeneMANIA
(www.genemania.org). AtGASA6 (At1G74670), RGL2, and AtEXPA1 (named EXP1 in the picture) are highlighted in black.
Supplementary Fig. 2. AtGASA6 gene expression pattern and AtGASA6 subcellular localization. (A) RT-PCR analysis of AtGASA6 expression in various tissues. R, roots; S, inflorescence stem; RL, rosette leaves; CL, cauline leaves; F, flowers; P, mature siliques. Actin1 was used as the internal control gene. Similar results were obtained in two independent experiments. (B) Histochemical staining of GUS activity in pGASA6::GUS plants. a) 2-week-old seedling; b) rosette leaves of 2-weeks-old seedling; c) cotyledons of 2-week-old seedling; d) root of 2-week-old seedling; e) emerging lateral root of 2-week-old seedling; f) flower bud; g) the 13th stage of silique; h) the 14th stage of silique; i) the 15th stage of silique; j) the 16 ~ 17th stage of silique; k) the 18th stage of silique; l) embryo of 18hr-germinated seed; m) testa and endosperm of 18hr-germinated seed. (C) Subcellular localization of AtGASA6 protein. GASA6-EGFP localization in onion epidermal cells (upper panes), GASA6-EGFP in Arabidopsis root cells (middle panes), and EGFP in Arabidopsis root cells (lower
pane).
Supplementary Fig. 3. Transcript levels of AtGASA6 and its homologous genes in AtGASA6-RNAi plants. Relative transcript level of AtGASA6 expression in germinating seed. Similar results were obtained in two independent experiments.
Supplementary Fig. 4. AtGASA6 expression levels in response to GA, glucose and ABA in early seedling development. Relative transcript level of AtGASA6 expression in 2 -week-old seedling treated with 6% glucose, 10 µM ABA, or 10 µM GA for 3 h. Similar results were obtained in two independent experiments.
Supplementary Fig. 5. Seed germination rates of different AtGASA6 genotype plants under 1 µM GA treatment. Three technical replicates were performed for each of three biological replicates.
Supplementary Fig. 6. AtGASA6 expression levels in Ler/AtGASA6-RNAi (Ler/R#2-3) and gin2/AtGASA6-RNAi (gin2/R#2-6, gin2/R#4-4, gin2/R#5-1) plants. All transcript levels of gene expression were normalized to UBQ1 gene expression. Results are averages ± SE (n ＝ 3).
Supplementary Fig. 7. Co-expression gene analysis for AtGASA6. Co-expression analysis was performed using the ATTED-II Network Drawer. Octagonal blocks are DNA binding domain-containing proteins; AtGASA6 is highlighted in yellow; red asterisk indicates AtEXPA1.
Supplementary Fig. 8. Bioinformatical analyses of AtGASA6 protein. (A) Graphical representation for AtGASA6 protein structure. (B) The conserved cysteine-rich domain of AtGASA6 and other GAST-like genes. Alignment of whole sequences of AtGASA6 (accession number At1G74670) with that of Arabidopsis GASA4 (accession number At5g15230), GASA1 (accession number At1g75750) and GASA5 (accession number At3g02885), Lycopersicon esculentum GAST1 (accession number P27057), Gerbera hybrid GEG (accession number CAB45241), Petunia hybrid GIP1 (accession number AAG43509) and Fragaria×ananassa (accession number AAB97006). The conserved cysteines are marked with a bold asterisk. (C) A phylogenetic tree was constructed from the multiple-sequence alignment of AtGASA6 proteins and its homologs from different plant species, using the DNAMAN program. (D) Bioimformatical analysis of AtGASA6 protein subcellular localization according to
Arabidopsis
Cell
e-FP
Browser
at
http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi.The color scale at the right bottom corner indicates the absolute expression levels of GASA6 protein: red means higher while yellow indicates lower expression levels.
Supplementary Fig. 9. AtEXPA1 expression levels in the different EXPA1 genotype seeds. AtEXPA1 expression is reduced in the expa1 and AtGASA6-OE#2-1/expa1, and elevated in the EXPA1-OE germinating seeds. The seeds were germinated for 10 hrs under light. The transcripts were normalized to Actin1 gene expression level. Similar results were obtained in three independent experiments.
Supplementary Fig. 10. AtGASA6 regulates cotyledon greening in response to 6% glucose in early seedling development. Photographic representation for cotyledon greening of WT (Col), GASA6 over-expressing (OE#2, OE#5, OE#6), and RNAi (R#3, R#4, R#6) plants treated with 6% mannitol or 6% glucose, mannitol was used as an osmotic control. Seedlings were photographed in the 8th day. Scale bar = 2 mm. Similar results were obtained in three independent experiments.
Supplementary Fig. 11. GIN2 expression level in the rgl2-1 mutant. Relative transcript levels of GIN2 in the Ler and rgl2-1 mutant seedlings. Similar results were obtained in three independent experiments.
1 2
Table S1. Gene-specific primer sequences. Primer name
Sequences（5’-3’）
Actin1F-RT
GTATGTGGCTATTCAGGCTGT
Actin1R-RT
CTGGCGGTGCTTCTTCTCTG
AtGASA6F-RT
CGCTCTAGAGATGGCCAAACTCATAACTTC
AtGASA6R-RT
ATAGGATCCGATGGACATTTTGGTCCACC
AtGASA6F-QPCR
CAATGCACAAGGAGATGTAGCAAC
AtGASA6R-QPCR
AGGGACACAAAGGCATTTAGCA
AtGASA6-SALK-LP
CGCTCTAGAGATGGCCAAACTCATAACTTC
AtGASA6-SALK-RP
ATAGGATCCGATGGACATTTTGGTCCACC
LB
TGGTTCACGTAGTGGGCCATCG
RNAi-AtGASA6F
TATGGATCCCTTTCGTTTGTCTCACTATG
RNAi-AtGASA6F
ATAAGCTTTGGTCCACCTTGTTGAGTC
RNAi-AtGASA6R
TCATACGCGTCTTTCGTTTGTCTCACTATG
RNAi-AtGASA6R
ATCTGCAGTGGTCCACCTTGTTGAGTC
AtGASA6-EGFP-LP
CGCTCTAGAGATGGCCAAACTCATAACTTC
AtGASA6-EGFP-RP
ATAGGATCCGA TGGACATTTTGGTCCACC
pGASA6-GUS-F
CAGAAGCTTGAATTGTGAGTGTCGTAAC
pGASA6-GUS-R
ATAGGATCCAGTGAGACAAACGAAAGTG
AtGIN2F-QPCR
CGTCTCTCTGCTGCTGGAATCT
AtGIN2R-QPCR
GATTTCTGCACCTCCTCGTCTTT
AtEXPA1F-QPCR
ACTCTTACCTTAACGGACAA
AtEXPA1R-QPCR
TCTAACTGCTTCTACTGTGAA
UBQ1F-QPCR
GGCCAAGATCCAAGACAAAG
UQ1R-QPCR
GTTGACAGCTCTTGGGTGAA
3 4
1
