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Arabidopsis thaliana Trihelix Transcription factor AST1 mediates
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abiotic stress tolerance by binding to a novel AGAG-box and some
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GT motifs
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Hongyun Xu, Lin He, Yong Guo, Xinxin Shi, Dandan Zang, Hongyan Li, Wenhui
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Zhang, Yucheng Wang *
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State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University),
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26 Hexing Road, Harbin 150040, China
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* Corresponding author:
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Name: Yucheng Wang
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State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University),
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26 Hexing Road, Harbin 150040, China
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Tel.: +86 451 82190607x11;
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Fax: +86 451 82190607x11.
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E-mail: [email protected]
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Hongyun Xu: [email protected]; Lin He: [email protected];
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Yong Guo: [email protected]; Xinxin Shi: [email protected]
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Dandan Zang: [email protected]; Hongyan Li: [email protected]
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Wenhui Zhang: [email protected]
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The date of submission: 2017/03/27
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The number of tables and figures: 9 figures
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The word count: 6689 words
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The number of supplementary figures: 9 figures
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The number of supplementary figures: 5 tables
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Arabidopsis thaliana Trihelix Transcription factor AST1 mediates
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abiotic stress tolerance by binding to a novel AGAG-box and some
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GT motifs
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：
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Highlight AST1 could bind a novel AGAG-box and some GT motifs to regulated
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stress-related genes to cause physiological changes, and then improve abiotic
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stress tolerance .
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Abstract
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Trihelix transcription factors are characterized by containing a conserved trihelix
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(helix-loop-helix-loop-helix) domain that bind to GT elements required for light
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response, play roles in light stress, and also in abiotic stress responses. However, only
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few of them have been functionally characterised. In the present study, we
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characterized the function of AST1 (Arabidopsis SIP1 clade Trihelix1) in response to
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abiotic stress. AST1 shows transcriptional activation activity, and its expression is
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induced by osmotic and salt stress. The genes regulated by AST1 were identified
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using qRT-PCR and transcriptome assays. A conserved sequence highly present in the
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promoters of genes regulated by AST1 was identified, which is bound by AST1, and
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termed AGAG-box with the sequence [A/G][G/A][A/T]GAGAG. Additionally, AST1
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also binds to some GT motifs including GGTAATT, TACAGT, GGTAAAT and
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GGTAAA, but failed in binding to GTTAC and GGTTAA. Chromatin
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immunoprecipitation combined with qRT-PCR analysis suggested that AST1 binds to
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AGAG-box and/or some GT motifs to regulate the expression of stress tolerance
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genes, resulting in reduced reactive oxygen species, Na+ accumulation, stomatal
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apertures, lipid peroxidation, cell death and water loss rate, and increased proline
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content and reactive oxygen species scavenging capability. These physiological
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changes mediated by AST1 finally improve abiotic stress tolerance.
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Key words: abiotic stress, Arabidopsis thaliana, Trihelix AST1, AGAG-box, GT
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motifs, transcriptional regulation.
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Introduction
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Trihelix transcription factors are characterized by a conserved trihelix
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(helix-loop-helix-loop-helix) domain that binds specifically to GT elements required
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for the light response, and are also termed GT factors (Zhou, 1999; Nagano et al.,
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2001). Compared with other transcription factor families, the trihelix family is
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relatively small, having 30 members in A. thaliana and 31 members in rice. According
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to the structure of the trihelical domain, the trihelix family is classified into five
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groups, GT-1, GT-2, SH4, GTγ and SIP1 (Kaplan-Levy et al., 2012; Qin et al., 2014).
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The trihelix family binds to light-responsive GT elements in target promoters.
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These GT elements have different sequences, including GGTTAA, GGTAATT,
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GGTAAAT, GTTAC, TACAGT and GGTAAA, and are found in the promoters of
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light regulated genes, and are mainly involved in the light response (Green et al.,
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1987; Kay et al., 1989; O'Grady et al., 2001; Gao et al., 2009; Yoo et al., 2010).
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Moreover, GT elements are involved in biotic or abiotic stress responses, being found
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in many promoters of genes associated with drought, salt stress and pathogen
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infection (Buchel et al., 1996; Park et al., 2004; Yoo et al., 2010).
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Trihelix transcription factors mainly respond to light stress and regulate the
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expression of light-responsive genes (Kaplan-Levy et al., 2012). Trihelix proteins are
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also involved in various developmental processes, including chloroplasts, embryonic
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development, seed germination and dormancy, stomatal aperture and the
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developments of trichomes and flowers (Willmann et al., 2011; Kaplan-Levy et al.,
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2014; O'Brien et al., 2015; Wan et al., 2015). Additionally, trihelix proteins are have
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roles in abiotic stresses, such as cold, oxygen, drought and salt stresses. For example,
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Arabidopsis GT-2 LIKE 1 (GTL1) is involved in plant water stress responses and
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drought tolerance, and gtl1 mutations regulate stomatal density by reducing leaf
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transpiration to improve water use efficiency (Yoo et al., 2010). Poplar GTL1 has
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functions in water use efficiency and drought tolerance; when exposed to
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environmental stresses, PtaGTL1 induces Ca2+ signatures to modulate stomatal
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development and regulate plant water use efficiency (Weng et al., 2012). Arabidopsis
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GT-4 Trihelix can improve plant salt stress tolerance by regulating the expression of
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Cor15A to protect plants from the damage to the chloroplast membrane and enzymes
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caused by salt stress (Wang et al., 2014). A. thaliana AtGT2L and rice OsGTγ-1 were
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both induced by salt, drought, cold stress and abscisic acid (ABA) treatment (Fang et
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al., 2010; Xi et al., 2012). Although trihelix have roles in plants’ adaptation to various
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environmental stresses, their mechanisms of action in abiotic stress tolerance are
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largely unknown. For example, besides binding to GT motifs, whether they bind other
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cis-acting elements to regulate gene expression in response to abiotic stress, the
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identities of the target genes regulated by Trihelix and the physiological response
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mediated by Trihelix when exposed to abiotic stress remain to be revealed.
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The function of Arabidopsis SIP1 clade Trihelix1 (AST1, At3g24860), which
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belongs to the trihelix subfamily of SIP1, has not been characterized. In this study, we
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characterized the function of AST1 in response to abiotic stress. Our study showed
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that AST1 plays an important role in plant salt and osmotic stresses, and we revealed
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the physiological responses mediated by AST1. Additionally, we identified a novel
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cis-acting element (the AGAG-Box) that is recognized by AST1. AST1 regulates
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stress-related genes by binding to the AGAG-Box and/or GT motifs to mediate salt
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and osmotic stress tolerance.
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Materials and methods
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Plant materials
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A. thaliana ecotype Columbia was used in this study. The AST1 (AT3G24860)
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T-DNA insertion mutants, SALK_038594C, were obtained from the Arabidopsis
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Biological Resource Centre (ABRC). Three-week-old A. thaliana plants were watered
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with 150 mM NaCl or 200 mM mannitol for 3, 6, 12, 24 and 48 h, respectively. Roots
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and leaves were harvested for expression analysis, and plants treated with fresh water
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were also harvested at the corresponding time points as controls.
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Beta-glucuronidase (GUS) Staining and GUS Activity Quantification
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The 1500 bp promoter of AST1 together with the full 5′ UTR of AST1 replaced the
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CaMV 35S promoter in vector PBI121 to drive GUS gene expression
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(ProAST1:GUS), and transformed into A. thaliana using the floral dip method
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(Clough et al., 1998). The T3 homozygous transgenic plants at different
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developmental stages were used for GUS staining and activity assays according to the
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methods described by Cheng et al. (2013) and Lu et al. (2007).
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Subcellular Localization assay
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The coding sequence (CDS) of AST1 was fused to the N-terminus of the green
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fluorescent protein (GFP) gene, under the control of CaMV 35S promoter
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(35S:AST1-GFP) and GFP under the control of 35S promoter was also generated
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(35S:GFP), and were transformed into A. thaliana plants. The root tips of 5-day-old
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transgenic seedlings were visualized using a fluorescence microscope Imager
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(Zeiss,Germany). The construct of 35S:AST1-GFP and 35S:GFP were also
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transformed separately into onion epidermal cells using the particle bombardment
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method and visualized using a confocal laser scanning microscopy LSM410 (Zeiss,
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Jena, Germany).
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Overexpression and knockout of AST1 in A. thaliana
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The CDS of AST1 was inserted into pROK2 (Hilder et al., 1987) under the control of
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35S promoter (35S:AST1), and were transformed into A. thaliana. Empty pROK2
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was also transformed as the control. The expression of AST1 in T3 homozygous
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transgenic lines or SALK_038594C plants was monitored by quantitative real-time
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reverse transcription PCR (qRT-PCR).
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Stress tolerance analysis
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A. thaliana seeds were placed on 1/2 MS solid medium supplied with 150, 185 mM
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mannitol or 100, 125 mM NaCl for 10 days, and the proportion of seedlings survival
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rate was calculated. The 4-d-old seedlings grown on 1/2 MS solid medium were
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transferred to 1/2 MS medium supplied with 100 and 125 mM NaCl or 200 and 300
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mM mannitol for 12 days, and the root lengths and fresh weights were measured.
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Three-week-old plants grown in the soil were treated separately with 150 mM NaCl
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or 200 mM mannitol for 10 days, and their fresh weights and chlorophyll contents
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were calculated; total chlorophyll contents were measured following the method of
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Gitelson et al. (2003).
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Detection of reactive oxygen species (ROS) and cell death
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Three-week-old A. thaliana under normal conditions were watered with 150 mM
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NaCl or 200 mM mannitol for 24 h. To detect H2O2 and O2.- content, leaves were
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infiltrated with nitroblue tetrazolium (NBT) or 3, 30-diaminobenzidine (DAB)
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solutions, as described by Zhang et al. (2011). For cell death determination, the
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detached leaves were incubated in Evans blue solution and stained according to Kim
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et al. (2003). For propidium iodide (PI) staining, 7-d-old seedlings in plates were
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treated with 150 mM NaCl or 200 mM mannitol for 24 h, and were used for PI
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staining according to Jones et al. (2016).
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Physiological analysis
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Three-week-old A. thaliana plants under normal conditions were watered with 150
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mM NaCl or 200 mM mannitol for 5 days, and were used for the following
7
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physiological measurements. Electrolyte leakage rate analysis was performed
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following the procedures described by Fan et al. (1997), and malonic dialdehyde
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(MDA) was determined according to the method of Madhava et al. (2000). Peroxidase
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(POD) and Superoxide Dismutase (SOD) were assayed as described previously (Han
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et al., 2008). The water loss rate was determined according to Hsieh et al. (2013). The
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proline content was determined as described by Han et al. (2014).
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Stomatal Aperture Analysis
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Lower epidermal peels of 3-week-old plants leaves were stripped to float in a solution
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of 10 mM MES-KOH, pH 6.15, with 30 mM KCl, and were incubated under light for
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2.5 h at 22°C to open the stomata. The leaves were then transferred to MES-KCl
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buffer, including 150 mM NaCl or 200 mM mannitol, for 3 h. Stomatal apertures were
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viewed using a light microscope (Olympus BX43, Japan) and measured by the
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software IMAGEJ 1.36b (http://brokensymmetry.com) (Watkins et al., 2014).
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Quantitative real-time reverse transcription PCR (qRT-PCR)
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Total RNA was isolated using TRIzol reagent (Invitrogen). RNA (1 μg) was reverse
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transcribed into cDNA using oligo(dT) as primers, and diluted to 100 μl. For
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qRT-PCR, the reaction system (20 μl) included 10 μl of SYBR Green Realtime PCR
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Master Mix, 10 μM of forward or reverse primer and 2 μl cDNA dilution products.
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ACT7 (AT5G09810) and TUB2 (AT5G62690) were used as internal controls. All
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primers for qRT-PCR were shown in Table S1. The PCR was performed with an
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Opticon 2 System (Bio-Rad, Hercules, CA, USA) with following conditions: 94°C for
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2 min; 45 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 40 s; and 79 °C for 1 s for
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plate reading. The relative expression levels were calculated using delta-delta Ct
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method (Livak and Schmittgen et al., 2001).
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Visualization and Measurement of Na+ and K+ contents
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One week-old A. thaliana seedlings grown under normal conditions were treated with
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150 mM NaCl for 24 h, and seedlings grown under normal conditions were used as
8
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controls. The plants were stained with 10 μM CoraNa-Green (Sigma, USA) for 2 h in
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the dark, then the root tips was visualized under an LSM710 microscope (Zeiss, Jena,
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Germany). After 150 mM NaCl or water treatment for 5 days, the roots and leaves
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were harvested for Na+ and K+ content analysis, which were performed as described
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preciously (Han et al., 2014).
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RNA-seq
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Three-week-old AST1 over-expressing plants and SALK plants were treated with 200
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mM Mannitol for 24 h, and then the leaves were harvested for RNA-Seq. Statistical
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selection of differentially expressed genes between overexpression line 3 (OE3) and
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knockout line 2 (KO1.2) was based on a minimal 2.5 log2 fold change, together with
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a P-value ≤ 0.05 for the t-test, for three biological repetitions.
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MEME analysis
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The promoter sequences (from -1 to -1000 bp) of the genes that are upregulated by
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AST1 were analyzed using the MEME program (http://meme-suite.org/tools/meme),
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with the same parameters used by Bailey et al. (2006).
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Yeast One-Hybrid (Y1H) Assays
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The CDS of AST1 was inserted into vector pGADT7-rec2 (Clontech) as the prey and
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one copy of each conserved sequence predicted by MEME was cloned into pHIS2 as
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baits (the primers were listed in Table S2). The positive clones were screened on
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SD/-Leu/-Trp (DDO) or SD/-His/-Leu/-Trp (TDO) medium supplied with 3-AT
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(3-Amino-1, 2, 4-triazole).
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Transient Expression Assay
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The sequences that were confirmed to interact with AST1 by Y1H were cloned
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separately into a reformed pCAMBIA1301 vector (where 35S:hygromycion had been
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deleted, and a 46 bp minimal promoter was inserted between the BglII site and ATG
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of GUS) as reporter constructs (the primers were listed in Table S3). The 35S:AST1
9
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
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was used as effector vector. The reporters and effector vector were co-transformed
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into tobacco by the transient transformation method (Zang et al., 2015), and 35S:LUC
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was cotransformed to normalize transformation efficiency. The GUS and LUC
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activities were determined as described previously (Lu et al., 2007).
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Electromobility shift assay (EMSA)
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The CDS of AST1 was cloned into the pMAL-c5X vector between the BamHI and
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EcoRI enzyme digest sites and were induced to express by IPTG into Escherichia coli
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strain ER2523. Then the AST1 protein was extracted and purified following the
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Instruction Manual (NEB, pMAL™ Protein Fusion & Purification System). The
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probes were labeled with biotin using EMSA Probe Biotin Labeling Kit according to
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the manuals (Beyotime, China), and the unlabeled probe was used for the competitor.
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The EMSA was performed using Chemiluminescent EMSA kit (Beyotime, China).
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The primers used for EMSA were listed in Table S4
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ChIP Assays
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Three-week-old A. thaliana expressing the AST1-GFP fusion gene were used for
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ChIP analysis. The plants were treated with 150 mM NaCl or 200 mM Mannitol for
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24 h, and then harvested for the ChIP assays. ChIP experiments were performed as
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described by Haring et al. (2007). The cross-linked chromatin was sonicated and
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incubated with an anti-GFP antibody (Beyotime, China) (ChIP+), and the chromatin
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incubated with a rabbit anti-haemagglutinin (HA) antibody was used as the negative
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control (ChIP-). The DNA was detected by qPCR with the CDS of Actin2
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(At3G18780) as an internal control. The primers used for ChIP were listed in Table
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S5.
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Accession Numbers
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Sequence data from this article can be found in The Arabidopsis Information
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Resource (http://www.arabidopsis.org/) under the following accession numbers:
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SOD2 AT2G28190
（
），FSD1（AT4G25100）, SOD(AT5G11000), SOD(AT3G10920),
10
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
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PER4(AT1G14540), POD(AT1G24110), POD(AT1G71695), PRX37(AT4G08770),
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PRX72(AT5G66390), POD(AT5G58400), ATMYB61
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(AT2G39880),
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(AT5G62520), HKT1 (AT4G10310), NHX2(AT3G05030), NHX3(AT5G55470),
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NHX6(AT1G79610), SOS2(AT5G35410), SOS3 (AT5G24270), LEA3(AT1G02820),
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LEA7(AT1G52690),
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ATCOR47(AT1G20440),
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(AT3G49580), SAUR16 (AT4G38860) .
P5CS2(AT3G55610),
（ AT1G09540 ） ,
P5CS1
(AT4G34590),
P5CDH
PRODH
COR15(AT2G42520),
ERD10(AT1G20450),
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307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
11
LEA14(AT1G01470),
ABR
(AT3G02480),
LSU1
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Results
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Spatial and temporal expression profiles of AST1
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GUS staining was performed on the transgenic A. thaliana plant expressing
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ProAST1:GUS to determine the expression profile of AST1. AST1 was expressed at
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each studied developmental stage and in different tissues. The expression of AST1
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increased from 5-d- to 20-d-old seedlings, but reduced in plants older than 20 d
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(Figure 1A, 1-6), displaying a temporal expression pattern. AST1 was highly
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expressed in leaves, stems and anthers compared with roots and siliques (Figure 1A,
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7-11). Consistently, qRT-PCR showed that AST1 was highly expressed in stems,
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leaves and flowers, but had relative lower expression levels in roots and siliques
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(Figure 1B). Interestingly, although AST1 was expressed in leaves, it had relative
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higher expression in guard cells, root and leaf vascular systems (Figure 1A8, 12-13).
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Under NaCl stress conditions, in leaves, the expression of AST1 was highly induced
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at 6 to 12 h, but continually decreased after 6 h of stress (Figure 1C). In roots, AST1
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was highly induced by stress for 3, 12 and 48 h, downregulated at 6 h, and recovered
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at 24 h under NaCl stress conditions. Under mannitol stress conditions, in leaves, the
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expression of AST1 was downregulated at 3 h, but increased continually from 6 to 12
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h, reaching its expression peak at 12 h, after which it decreased continually (Figure
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1C). In roots, AST1 was slightly induced by stress for 3 to 12 h, highly induced at 24
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and 48 h, and reached its expression peak at mannitol stress for 48 h (Figure 1C).
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Consistently, determination of GUS activity in Arabidopsis plant expressing
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ProAST1:GUS also confirmed that the expression of AST1 was significantly induced
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in leaves and roots after exposed to mannitol or NaCl for 12 h (Figure 1D). These
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results suggested that the expression of AST1 responded to salt and mannitol stress,
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and might play a role in salt and osmotic stress tolerance.
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Subcellular localization of AST1
The results showed that the GFP signal was detected in the whole cells of root tips
12
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or root elongation zone in A. thaliana plants expressing 35S:GFP (Figure S1A).
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However, the GFP signal was only detected in the nucleus of the root tips to the root
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hair zone in A. thaliana expressing AST1-GFP (Figure S1A). Additionally, transient
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transformation of onion epidermal cells also indicated that AST1 was localized in the
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nucleus (Figure S1B). Taken together, these results indicated that AST1 was target to
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the nucleus.
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Generation of overexpression or knockout plants for AST1
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The T3 generation of A. thaliana plants overexpressing AST1 (OE) and the AST1
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mutant plants (SALK_038594C) (KO plants) were generated, and the T-DNA
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sequence was inserted at the position that was at the 388 bp down-stream of the ATG.
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The qRT-PCR results showed that the expression of AST1 was significantly increased
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in the OE plants and highly decreased in the KO plants (Figure S2), indicating that
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AST1 had been successfully overexpressed and knocked-out, respectively, and that
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these plants were suitable for gain and loss-of-function analysis. Three
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AST1-overexpressing lines (OE1, OE2 and OE3) that had relative high AST1
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expression and three homozygous mutant plants (KO1.1, KO1.2 and KO1.3) that had
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the lowest AST1 expression were selected for further study. Wild-type plants (WT)
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and WT plants transformed with the empty pROK2 vector (35S) were used as the
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controls.
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AST1 improves Drought and Salt tolerance
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Under normal conditions, there was no difference in seedling survival rates among
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all the studied plants (Figure 2A). Under salt or osmotic stress conditions, compared
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with the WT, all OE plants showed significantly higher seedling survival rates, all KO
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plants showed significant lower seedling survival rates, and 35S plants showed similar
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seedling survival rates (Figure 2A). Root length and fresh weight were analyzed to
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determine stress tolerance. There was no difference in growth phenotype, root
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elongation and fresh weights among all the studied lines under normal conditions
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(Figure S3). Under stress conditions, compared with WT plants, the root elongation
13
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and fresh weights of KO plants were significantly reduced, but all the OE plants
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showed significantly increased root elongation and fresh weights; the 35S plants were
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similar to the WT (Figure S3). Stress tolerance was further studied in seedlings grown
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in soil. There was no difference in growth phenotype, fresh weights, and chlorophyll
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content among the studied plants under normal conditions (Figure 2B). Under salt or
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osmotic stress conditions, compared with the WT and 35S plants, all the OE plants
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showed increased chlorophyll content and fresh weights, and the KO lines displayed
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decreased fresh weights and chlorophyll contents. These results suggested that
395
overexpression or knockout of AST1 didn’t affect the growth and phenotype of plants.
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However, AST1 could regulate salt and osmotic stress tolerance positively.
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Stomatal aperture and water loss rate analysis
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As AST1 is highly expressed in guard cells (Figure 1A), we studied whether it
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played a role in controlling stomatal apertures. Under normal conditions, all the lines
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had similar stomatal apertures and width/length ratios (Figure 3A). When exposed to
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salt and osmotic stress, the WT and 35S plants had similar stomatal apertures and
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width/length ratios. Compared with the WT plants, the OE lines displayed decreased
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stomatal apertures and lower width/length ratios, and the KO plants showed increased
405
stomatal apertures and higher width/length ratios (Figure 3A). Protein AtMYB61 was
406
found to control the stomatal aperture ( Liang et al., 2005); therefore, we further
407
studied whether AST1 could regulate AtMYB61 expression. The expression of
408
AtMYB61 was significantly increased in the OE plants compared with the WT and
409
35S plants, and was significantly decreased in the KO plants (Figure 3B). The
410
stomatal aperture is closely related with the water loss rate; therefore, we further
411
studied the water loss rates under dehydration conditions. WT and 35S lines had
412
similar water loss rates; however, the KO plants exhibited increased water loss rates,
413
and the OE plants displayed decreased water loss rates compared the WT plants
414
(Figure 3C). These results together indicated that AST1 regulates AtMYB61
415
expression positively to control stomatal aperture, resulting in a reduced water loss
416
rate.
14
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417
418
Determination of Na+ and K+ contents
419
The accumulation of Na+ in root tips was visualized by CoroNa-Green, a
420
sodium-specific fluorophore. Under normal conditions, there was no substantial
421
difference in Na+ accumulation among the studied plants. However, under salt stress
422
conditions, KO plants displayed substantially stronger fluorescence than the WT and
423
35S plants, and the OE plants showed the weakest fluorescence (Figure 4A),
424
indicating that Na+ was highly accumulated in the KO plants, but was accumulated
425
lowly in the OE plants compared with in WT plants. Na+ and K+ contents were further
426
determined using a Flame spectrophotometer. All the studied lines had generally
427
similar Na+ and K+ contents under normal conditions. Under NaCl stress condition,
428
Na+ was increased and K+ was decreased in all plants. However, in both the leaves
429
and roots, the Na+ content was highly accumulated in KO plants, followed by the WT
430
and 35S plants; the OE plants had lowest Na+ level (Figure 4B), which was consistent
431
with CoroNa-Green staining. Meanwhile, the OE plants had higher K+ levels, and KO
432
plants had lower K+ level compared with those in the WT and 35S plants in both
433
leaves and roots (Figure 4B). The K+/Na+ ratios were similar in the leaves and roots of
434
all plants under normal conditions. Under salt stress conditions, the OE plants had the
435
highest K+/Na+ ratio, followed by the WT and 35S lines, and the KO plants had the
436
lowest K+/Na+ ratio (Figure 4B). We further examined the expression of genes related
437
to Na+ or K+ transport, including those encoding 1 sodium transporter (HKT1), and
438
three Na+ (K+)/H+ transport proteins (NHX2, NHX3, NHX6), two salt overly sensitive
439
(SOS) family proteins (SOS2 and SOS3), which control plant K+ and Na+ nutrition.
440
The results showed that HKT1 had its highest expression in KO plants, followed by
441
that in the WT and 35S plants, and was lowest in the OE plants (Figure 4C).
442
Conversely, NHX2, NHX3, NHX6 and SOS2 showed their highest expression levels in
443
the OE plants, followed by the WT and 35S plants, and showed their lowest
444
expression levels in the KO plants. The expression of SOS3 was not significantly
445
different among the studied lines (Figure 4C).
446
15
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447
Analysis of proline metabolism
448
Proline is an important osmotic adjustment substance and also plays a role in ROS
449
scavenging; therefore we measured the proline contents in the plant lines. The results
450
showed that all the lines had similar proline contents under normal conditions.
451
However, when exposed to salt or osmotic stress, the OE plants had highest proline
452
level, followed by the WT and 35S plants, and the KO plants had the lowest proline
453
contents (Figure 5A). We further investigated the genes involved in proline
454
metabolism, including two proline biosynthesis genes, D(1)-pyrroline-5-carboxylate
455
synthetase (P5CS) gene, p5CS1 and p5CS2; two proline degradation genes,
456
D(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) and proline dehydrogenase
457
(PRODH). When exposed to salt or osmotic stress conditions, the expression of both
458
p5CS1 and p5CS2 were increased in the OE lines and decreased in the KO lines,
459
compared with the WT and 35S lines. Conversely, P5CDH and PRODH showed the
460
highest expression levels in the KO plants, followed by the WT and 35S lines, and the
461
lowest level in the OE plants (Figure 5B). These results indicated that AST1 could
462
increase proline content by affecting the expression of proline metabolism genes.
463
464
Cell death and MDA content analysis
465
Evans blue and PI fluorescence staining were used to detect cell death in leaves and
466
roots, respectively. There was no difference in Evans blue and PI staining among all
467
the plants under normal conditions. Under NaCl and mannitol conditions, compared
468
with the WT and 35S plants (they have similar cell death rates according to the
469
staining), both Evans blue and PI staining showed that cell death was substantially
470
decreased in OE plants. By contrast, KO plants displayed increased cell death (Figure
471
S4A, B). To measure cell death quantitatively, the electrolytic leakage rates were
472
determined. All the studied lines shared similar electrolytic leakage rates under
473
normal conditions. Under salt or osmotic stress, the WT and 35S plants shared similar
474
electrolytic leakage rates; however, compared with the WT and 35S plants, all KO and
475
OE plants showed increased and decreased electrolytic leakage rates, respectively
476
(Figure S4C), which was consistent with the results from Evans blue and PI staining.
16
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
477
Malonic dialdehyde (MDA) contents were measured to evaluate the level of
478
membrane lipid peroxidation. Under normal conditions, all the plants had similar
479
MDA levels. Under salt or osmotic stress conditions, the KO plants had the highest
480
MDA content, followed by the WT and 35S plants (they shared similar MDA level),
481
and the OE plants showed the lowest MDA contents (Figure S4D). These results
482
indicated that expression of AST1 could reduce membrane lipid peroxidation under
483
abiotic stress conditions.
484
485
ROS scavenging assay
486
We first studied the contents of O2.- and H2O2 by nitroblue tetrazolium (NBT) and
487
3, 30-diaminobenzidine (DAB) in situ staining, respectively, and a deeper the blue
488
or brown color indicated the accumulation of O2.- and H2O2, respectively. There was
489
no observable difference in NBT and DAB staining among the WT, OE, 35S and KO
490
plants under the normal conditions (Figure S5A). When exposed to NaCl or mannitol,
491
the WT and 35S plants had similar O2. - and H2O2 levels; compared with them, OE
492
plants displayed substantially reduced O2.- and H2O2 accumulation, and all KO plants
493
showed increased O2.- and H2O2 accumulation.
494
The reactive oxygen species (ROS) levels were altered; therefore, we further
495
studied whether this was caused by altered ROS scavenging capability. Peroxidase
496
(POD) and Superoxide Dismutase (SOD) activities were measured. Under normal
497
conditions, there was no difference in SOD and POD activities among all the plants.
498
However, under NaCl or mannitol conditions, the activities of SOD and POD in the
499
OE plants were the highest, followed by the WT and 35S plants, and the KO plants
500
had the lowest SOD and POD activities (Figure S5B). The expression of the SOD and
501
POD genes were further studied, and the genes that have known SOD or POD activity
502
were selected for study. Under salt and mannitol conditions, the expression levels of
503
all the POD and SOD genes (except for ATSOD1) in OE plants were the highest,
504
followed by the WT and 35S, and the KO plants had the lowest expression levels
505
(Figure S5C). This result indicated that AST1 could induce the expression of SOD and
506
POD genes to elevate the SOD and POD activities when exposed to salt and osmotic
17
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
507
stress.
508
509
AST1 induced the expression of LEA family genes in response to salt and
510
drought stresses
511
Seven LEA (late embryogenesis abundant) family genes that had been reported to
512
be involved in abiotic stress tolerance were studied. Under normal conditions, there
513
was no difference in expression levels among the plants (Figure S6). When exposed to
514
NaCl or Mannitol, except for the ABA-RESPONSE PROTEIN (ABR) gene, all the
515
studied LEA family genes displayed their highest expression levels in the OE plants,
516
followed by the WT and 35S plants, and showed their lowest expression levels in the
517
KO plants (Figure S6). These results showed that AST1 could induce certain LEA
518
family genes to improve abiotic stress tolerance.
519
520
RNA-Seq analysis
521
A transcriptomic analysis was carried out to identify differentially expressed genes
522
(DEGs) between the OE3 and KO1.2 lines. In total, 144 DEGs (fold change ≥ 2 and
523
false discovery rate (FDR) < 0.05) were identified, among which 65 genes were
524
upregulated and 77 genes were downregulated. These DEGs were listed in Table S6
525
and the hierarchical clustering analysis was shown in Figure S7. Gene ontology (GO)
526
analysis showed that these DEGs were mainly involved in signaling, immune system
527
process, reproduction and cell killing in biological process (Figure S8A). Kyoto
528
encyclopedia of genes and genomes (KEGG) analysis showed that the DEGs were
529
mainly associated with plant hormone signal transduction and plant-pathogen
530
interaction pathways (Figure S8B). These results indicated that AST1 played a key
531
role in regulating these pathways.
532
533
A novel motif recognized by AST1
534
To study the motif mainly bound by AST1 in regulating gene expression when
535
exposed to abiotic stress, the MEME motif discovery tool (http://meme-suite.org) was
536
used. As shown in Figure 7, AST1 could bind to GT2, GT3, GT4 and GT5 to active
18
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
537
gene expression, suggesting that AST1 should play an expressional activation role.
538
Therefore, the promoters of 54 genes that were highly upregulated by AST1 according
539
to the qRT-PCR and RNA-Seq analyses were employed for further study. The MEME
540
results showed that there was a 12 base conserved sequence present in most of the
541
studied promoters (Figure 6A).
542
Y1H results showed that AST1 could bind to this 12 base conserved sequences
543
“AGAGAGAGAAAG” (Figure 6B). The 1st to 8th base of the 12 base conserved
544
sequence appeared with the highest frequency and might be the core sequence of this
545
motif, therefore, they were subjected for further study. The 1st to 8th base of 12 bp
546
conserved sequences were represented by 32 types of sequences, and were all
547
subjected to Y1H assays. The Y1H results showed that only some of the eight base
548
sequences were bound by AST1; however, when the 7th base was G or the 8th was A,
549
their binding to AST1 were lost (Figure 6B). Therefore, the eight base sequences that
550
were
551
[A/G][G/A][A/T]GAGAG, and was termed the AGAG-box. To further determine the
552
bindings of the AGAG-box by AST1, we produced GUS gene reporter constructs that
553
contained all the sequences of AGAG-box separately, the 12 base conserved sequence,
554
or the six sequences (AGTGAGAA, AGTGAGGG, AGTGAGGA, GAAGAGAA,
555
GAAGAGGG and GAAGAGGA) that could not be bound by AST1 according to
556
Y1H (Figure 6B). Each reporter was co-transformed with the effector (35S:AST1)
557
into tobacco plants. The GUS/LUC ratio showed that AST1 recognized all the
558
AGAG-box sequences and the 12-base conserved sequence, but failed to bind to the
559
other sequences (Figure 6C). This result was consistent with that of Y1H.
bound
by
AST1
were
represented
by
the
consensus
sequence
560
To further determine whether AGAG-box sequences could be bound by AST1,
561
five types of AGAG-box sequences that showed highly transactivation when
562
interacted with AST1 (Figure 6C) were labeled with biotin as the probes, and were
563
used for EMSA. The results showed that the DNA-protein complexes were observed,
564
and the complex binds were gradually decreased with increasing the unlabeled probes
565
(Figure 6D), showing that AST1 could bind to these AGAG-box sequences.
566
Meanwhile, the two sequences (AGTGAGGG and GAAGAGAA) that were not
19
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
567
bound by AST1 according to Y1H were also studied, and EMSA result confirmed that
568
they could not be bound by AST1 (Figure 6D).
569
To determine whether AST1 could bind to AGAG-box in A. thaliana plants, ChIP
570
analysis was performed. Three genes whose promoters contained only AGAG-boxes
571
and no GT motifs were used for ChIP analysis. The ABR gene (AT3G02480) whose
572
promoter region did not contain both AGAG-box and GT motifs, and only contained
573
an AST1 non-recognized sequence (GAAGAGAA) was used as the negative control.
574
When using ChIP+ ( immunoprecipitated with the anti-GFP antibody) as the template,
575
the promoter region containing the AGAG-box were PCR amplified; however, the
576
promoter region far way from AGAG-box all failed PCR (Figure 6E), indicating that
577
AST1 really bound to AGAG-box in A. thaliana. Additionally, the promoter region of
578
ABR (containing GAAGAGAA that was not bound by AST1 according this study)
579
also failed in PCR amplification when use ChIP+ as the template (Figure 6E).
580
Meanwhile, the promoter regions could all be amplified from the Input, and the ChIP-
581
( immunoprecipitated with the anti-HA antibody) failed PCR for all the promoter
582
regions, indicating that ChIP results were reliable (Figure 6E). These results suggested
583
that AST1 indeed bound to the AGAG-box to regulate the expression of genes in A.
584
thaliana.
585
586
AST1 binds to GT cis-acting elements
587
Previous studies showed that Trihelix proteins could bind to GT motifs, including
588
GGTTAA (GT1), GGTAATT (GT2), TACAGT (GT3), GGTAAAT (GT4), GGTAAA
589
(GT5) and GTTAC (GT6) (Green et al., 1987; Kay et al., 1989; O'Grady et al., 2001;
590
Gao et al., 2009; Yoo et al., 2010). We first investigated the binding of AST1 to these
591
GT motifs using Y1H. The results showed that AST1 bound to GGTAATT (GT2),
592
TACAGT (GT3), GGTAAAT (GT4), GGTAAA (GT5), but failed to binds to
593
GGTTAA (GT1) and GTTAC (GT6) (Figure 7A). The interaction between AST1 and
594
these GT motifs were further performed in Tobacco. Three copies of each GT motif
595
were fused with 35S minimal promoter to drive a GUS gene as a reporter, and were
596
transformed with 35S:AST1 into Tobacco plants. The results showed that AST1 could
20
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
597
bind to GT2, GT3, GT4 and GT5, but failed to bind to GT1 and GT6, which was
598
consistent with the Y1H results (Figure 7B).
599
To further determine the bindings of AST1 to GT motifs, EMSA was performed.
600
When the GT-1 and GT-6 probe was added, only the free DNA probe was observed,
601
further indicating that GT1 and GT-6 were not bound by AST1. When GT2, 3, 4, and
602
5 sequences were respectively added with AST1 protein, the DNA-protein complexes
603
could be observed (Figure 7C), confirming that GT2-5 sequences all could be bound
604
by AST1.
605
To determine whether AST1 could bind to the GT motifs in A. thaliana, ChIP
606
analysis was performed. Six genes whose promoters contained only GT1, GT2, GT3,
607
GT4, GT5 or GT6 motifs, and no other known Trihelix binding motif (including the
608
AGAG-box), were used for the ChIP analysis. When ChIP+ was used as the PCR
609
template, the promoter fragments containing GT2, GT3, GT4, or GT5 motifs were
610
amplified, and the promoter regions containing distant GT2, GT3, GT4, or GT5
611
motifs failed in PCR amplification. In addition, the promoter regions containing
612
proximal or distal GT1 or GT6 motifs all failed in PCR amplification using ChIP+
613
(Figure 7D). At the same time, all the chosen promoter region could be amplified
614
from the Input sample, and ChIP- failed to PCR amplify any of the promoter regions
615
(Figure 7D), indicating that the ChIP-PCR results are reliable. These results together
616
indicated that AST1 could bind to GT2, GT3, GT4 and GT5 (GT2-5), but not to GT1
617
and GT6 in A. thaliana.
618
619
ChIP analysis of the genes directly regulated by AST1
620
To further determine the genes regulated directly by AST1, ChIP analysis was
621
performed. The stress tolerance genes whose expressions were affected by AST1
622
according to qRT-PCR or RNA-seq were studied for ChIP analysis. The schematic
623
diagram of the promoter fragments from different AST1-upregulated genes used for
624
qChIP-PCR was shown as Figure S9. The results showed that besides ABR, SOS3 and
625
ATSOD1, the chosen promoter regions contained AGAG-box or GT2-5 motifs of all
626
the studied genes were significantly enriched, suggesting that they were regulated
21
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
627
directly by AST1 (Figure 8). Importantly, SOS3, ABR and ATSOD1 did not contain an
628
AGAG-box, GT2, GT3, GT4, or GT5 in their promoters, and their promoters could
629
not be bound by AST1 (Figure 8). Furthermore, the expressions of SOS3, ABR and
630
ATSOD1 were not affected by AST1 according to the qRT-PCR results (Figure 4,
631
Figure S6 and Figure S5b). These results further confirmed that AST1 could bind to
632
the AGAG-box and the GT2, GT3, GT4, or GT5 (GT2-5) motifs to regulate the
633
expression of genes. In addition, according to the ChIP results (Figure 8), the genes
634
involving in water loss rate, ion homeostasis, and proline contents and ROS
635
scavenging capability were mainly directly regulated by AST1.
636
637
Discussion
638
AST1 is a GT transcription factor, whose function involved in abioic stress had not
639
been characterized previously. In the present study, we identified the motifs bound by
640
AST1 and further revealed the stress tolerance related genes regulated by AST1 and
641
the physiological changes mediated by AST1 in response to abiotic stress.
642
AST1 binds to a novel motif AGAG-box to regulate the expression of genes
643
Previous studies showed that some Trihelix proteins could bind to different types of
644
GT-motifs (Kaplan-Levy et al., 2012). However, our results showed that AST1 could
645
only bind to GGTAATT (GT2), TACAGT (GT3), GGTAAAT (GT4) and GGTAAA
646
(GT5), but not to GGTTAA (GT1) and GTTAC (GT6) (Figure 7). Additionally, AST1
647
also binds to a novel motif, the AGAG-box, which contains eight types of sequences.
648
Among these sequences, when the sequences “AAAGAGAG”, “AGAGAGAG”,
649
“GGAGAGAG” and “GATGAGAG” were present, AST1 showed relatively higher
650
activation of gene expression (Figure 6C). By contrast, when the other four
651
sequences, “GAAGAGAG”, “GGTGAGAG”, “AATGAGAG” and “AGTGAGAG”,
652
were present, AST1 showed relatively lower gene expression activation (Figure 6C).
653
These results suggested that AST1 might show higher binding affinities to
654
“AAAGAGAG”, “AGAGAGAG”, “GGAGAGAG” and “GATGAGAG” compared
655
with
656
“AGTGAGAG”. These two groups of sequences only had differences in the first to
those
to
“GAAGAGAG”,
“GGTGAGAG”,
22
“AATGAGAG”
and
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
657
the third nucleic acids, indicating that these three nucleic acids might be relatively
658
important for AST1 binding.
659
We screened the frequency of the occurrence of the AGAG-box and GT2-5 motifs
660
in the promoters of genes regulated by AST1, including the 24 genes identified by
661
qRT-PCR, and the 62 genes that were upregulated by AST1 according to RNA-Seq.
662
Among these promoters, 58% (50 genes) contained AGAG-box motifs, and 65% (56
663
genes) contained different GT2-5 motifs. The occurrence frequencies of AGAG-box
664
and GT2-5 motifs were similar, suggesting that like the GT motifs, the AGAG-box
665
also played a very important role in AST1-mediated gene expression.
666
AST1 binds to AGAG box and GT motifs serving as a transcriptional activator
667
We studied the binding of AST1 to different GT motifs or AGAG box in Tobacco
668
plants. The results showed that AST1 could bind to AGAG box and GT2-5 to activate
669
the expression of GUS gene (Figure 6C; Figure 7B), suggesting that AST1 should
670
serve as a gene expression activator when binding to these motifs.
671
The physiological response mediated by AST1
672
Plant guard cells form stomatal pores that played important roles in CO2 uptake for
673
photosynthesis and in transpirational water loss. Transpiration accounts for most of
674
the water loss in plants. Plants reduce transpirational water loss by inducing stomatal
675
closure in response to drought stress (Munemasa et al., 2015). In the present study, we
676
found that AST1 was highly expressed in guard cells (Figure 1A), and induces
677
stomatal closure to reduce water loss (Figure 3). Previous studies showed that
678
AtMYB61 directly controls the stomatal aperture (Liang et al., 2005). Our study
679
showed that AST1 could upregulate the expression of AtMYB61 directly (Figure 3B
680
and Figure 8). These results indicated that AST1 controlled stomatal closure and
681
opening by regulating AtMYB61 expression directly, thereby aiding water stress
682
tolerance.
683
Maintenance of K+/Na+ homeostasis was quite important for plant salt tolerance
684
(Sergey et al., 2007). Our study showed that AST1 reduced Na+ accumulation and
685
decreases K+ loss (Figure 4). Meanwhile, AST1 also regulated genes involved in Na+
686
and K+ homeostasis, including HKT1, NHX2, NHX3, NHX6 and SOS2 (Figure 8).
23
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
687
These results indicated that AST1 could reduce Na+ accumulation and decrease K+
688
loss by regulating the expressions of Na+ and K+ transporter genes, which will
689
contribute to alleviating salt stress.
690
Proline is the main solute used in osmotic potential adjustment. In A. thaliana,
691
P5CS is the key enzyme in proline biosynthesis, and the degradation of proline is
692
catalyzed by two enzymes, PRODH and P5CDH (Silva-Ortega et al., 2008; Szabados
693
et al., 2010). Our results indicated that AST1 controlled the proline content and the
694
expression of P5CS genes positively, and downregulates PRODH and P5CDH (Figure
695
5). These results suggested that AST1 induced the expression of P5CS to increase
696
proline biosynthesis; simultaneously, it decreased the expression of PRODH and
697
P5CDH to inhibit proline degradation, resulting proline accumulation to enhance
698
osmotic potential, thereby improving salt and osmotic stress tolerance.
699
ROS scavenging is important for abiotic stress tolerance in plants. Excess ROS
700
generated by abiotic stress attack all macromolecules, leading to serious damage to
701
DNA, including lesions and mutations, cellular components, metabolic dysfunction
702
and cell death (Karuppanapandian et al., 2011). Proline not only acts as osmotic
703
adjuster but also serves as ROS scavenger. The proline content had been found to be
704
highly induced by AST1 (Figure 5A). Additionally, SOD and POD are the two most
705
important antioxidant enzymes in ROS scavenging. AST1 induced the expression of
706
both SOD and POD genes to increase SOD and POD activities (Figure S5), which
707
enhanced ROS scavenging capability and reduced ROS accumulation (Figure S5) to
708
improve abiotic stress tolerance.
709
AST1 regulates the expression of LEA genes to improve stress tolerance
710
The plant LEA family proteins, which are important for abiotic stress tolerance,
711
stabilize the cell membrane, and serve as molecular chaperones or shield to prevent
712
irreversible protein aggregation caused by abiotic stress, thus protecting the plant
713
from damage (Serrano et al., 2003). Some LEA family proteins that had been
714
confirmed to play a role in stress tolerance were studied here, and AST1 was found to
715
induce the expression of most of the studied LEA genes (Figure S6 and Figure 8).
716
Therefore, these LEAs highly expression would contribute to improve abiotic stress
24
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
717
tolerance. Therefore, one of pathways that AST1 improved salt and drought tolerance
718
was to induce the expression of LEAs involving abiotic stress tolerance.
719
In conclusion, our data suggested a working model for the function of AST1 in the
720
abiotic stress response. Abiotic stresses, such as salt or osmotic stress, induce the
721
expression of AST1. The induced AST1 protein binds to AGAG-boxes and/or GT2–5
722
motifs to regulate the expressions of genes involved in abiotic stress tolerance, such as
723
stomatal aperture, K+/Na+ homeostasis, proline biosynthesis, ROS scavenging, and
724
LEAs. The altered expressions of these genes lead to physiological changes, including
725
reduced water loss and Na+ accumulation, prevention of K+ loss, elevated proline
726
level, reduced ROS accumulation, and high expression of LEAs, which might play a
727
role in stabilizing the cell membrane and serving as molecular chaperones to prevent
728
protein aggregation caused by stress. These physiological changes ultimately
729
improved abiotic stress tolerance (Figure 9).
730
731
Acknowledgements
732
This work was supported by National Natural Science Foundation of China (No.
733
31270703), and the Fundamental Research Funds for the Central Universities
734
(2572014AA25).
735
736
Conflict of interest statement: We declare that we have no conflict of interest.
737
738
Supplementary data
739
The following supplemental materials are available
740
Supplemental Figure S1. Subcellular localization of AST1.
741
Supplemental Figure S2. Relative expression of AST1 in the OE and KO plants.
742
Supplemental Figure S3. Stress tolerance analysis on seedlings grown on 1/2 MS
743
medium.
744
Supplemental Figure S4. Detection of cell death.
745
Supplemental Figure S5. Analysis of ROS levels and ROS scavenging capability.
746
Supplemental Figure S6. The expression of LEA family genes
25
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
747
748
Supplemental Figure S7. Hierarchical clustering analysis of the differentially
regulated genes
749
Supplemental Figure S8. Go and KEGG analysis of differentially expressed genes.
750
Supplemental Figure S9. A schematic diagram of the promoter fragments used for
751
qChIP-PCR.
752
Supplemental Table S1. Primers used in qRT_PCR
753
Supplemental Table S2. Primers used in Y1H
754
Supplemental Table S3. Primers used in tobacco Transient Expression Assay
755
Supplemental Table S4. Primers used in EMSA assay
756
Supplemental Table S5. Primers used in ChIP assay
757
Supplemental Table S6. Regualted genes by AST1 in RNA-seq
758
759
760
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761
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897
and GT-factors. Trends Plant Science 4, 210–214.
898
899
Figure legends
900
Figure 1. Expression profiles of AST1.
901
(A) GUS staining analysis of ProAST1:GUS transgenic plants. (1–6) 2-, 5-, 10-, 15-,
902
20- and 30-d-old-seedling, (7) Root,
903
(11) Flower, (12) Rosette leave, (13) Leaf vascular tissue, (14) Guard cells. Bars: (1–3,
904
9, 10, 12,) 1 mm, (4–6) 1 cm, (7 and 8) 25 μm, (11) 250 μm, (13) 50 μm, (14) 10 μm.
905
(B) The expression of AST1 in different tissues of WT A. thaliana using qRT-PCR.
906
Tissues from four-week-old plants were used for analysis. The expression level in
907
roots was set as 1 to normalize the expression in other tissues. Asterisk (*) indicates
908
significant difference compared with the roots (P < 0.05).
909
(C) The expression of AST1 in response to abiotic stresses. The expression level in the
910
samples treated with fresh water harvested at each time point were as the controls, and
911
was set as 1 to normalize the expression at the corresponding time point. Three
912
biological replications were conducted. The error bars represent the standard deviation
913
(S.D.). Asterisk (*) indicates significant difference between treatments and controls (P
914
< 0.05).
915
(D) GUS staining of ProAST1:GUS transgenic plants under abiotic stress conditions.
916
A. thaliana plants containing ProAST1:GUS grown in 1/2 MS medium were treated
917
with NaCl or Mannitol for 12 h. At least 10 seedlings were included in each
918
experiment, and three biological replications were performed. The GUS activity in
919
control sample (no stress) was set as 1 to normalize the activity under stress
920
conditions. Bars: 1cm. Three biological replications were performed. Asterisk (*)
921
indicates a significant difference between treatments and controls (P < 0.05).
（8）root vascular tissue, (9) Stem, (10) Silique,
922
923
Figure 2. Abiotic Stress tolerance analysis of AST1
924
(A) Analysis of seed germination phenotypes under salt and osmotic conditions. (1)
925
Germination phenotype. A. thaliana grown in 1/2 MS medium were treated with NaCl
31
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
926
or Mannitol for 10 d. 35S: A. thaliana transformed with empty pROK2 (35S), OE:
927
transgenic plants overexpressing AST1; WT: Wild Type; KO: A. thaliana mutant
928
plants with knockout of AST1. The photographs showed representative seedlings. (2)
929
Seed germination assay. The survival rates under NaCl (100 or 125 mM) or Mannitol
930
(150 or 185mM) were calculated. A. thaliana plants grown in 1/2 MS medium were
931
used as the control. Data are means ± SD from three independent experiments.
932
Asterisk (*) indicates significant (t test, P < 0.05) difference compared with WT.
933
(B) Stress tolerance analysis on seedlings grown in soil. (1) Three-week-old A.
934
thaliana plants grown in soil were watered with 150 mM NaCl or 200 mM Mannitol
935
for 10 d, well watered plants were used as the control. (1) The growth of A. thaliana
936
plants under salt or osmotic stress for 10 d. (2, 3) Measurement of fresh weight and
937
chlorophyll content. Bars indicate the mean
938
three independent experiments (n=30). (*P < 0.05). Significant difference compared
939
with WT.
±standard deviation (SD) for each set of
940
941
Figure 3. Comparison of Stomatal closure and water loss rates.
942
(A) Stomatal closure assay. (1) The stomatal aperture under normal, salt and osmotic
943
stress conditions. Stomata were pre-opened under light and then incubated in the
944
solution of 150 mM NaCl or 200 mM Mannitol for 2.5 h under light. Water-mediated
945
stomatal closure was used as a control. (2) Measurement of stomatal aperture. Values
946
are mean ratios of width to length. Error bars represent standard errors of three
947
independent experiments (n=30–50). Bars: 10 um. Asterisk (*) indicates a significant
948
difference at P<0.05 compared with the WT.
949
(B) Analysis of the expression of stomatal aperture-related gene AtMYB61
950
(AT1G09540). The plants were treated with water (Control), 150 mM NaCl or 200
951
mM Mannitol, and the expression level of AtMYB61 in WT plants under normal
952
conditions was used to normalize all other expressions. Data are means ±SD from
953
three independent experiments. Asterisk (*) indicates a significant difference at
954
P<0.05 compared with the WT.
32
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
955
(C) Analysis of water loss rates. Leaves from three-week-old plants were harvested
956
for transpiration at room temperature. Values are means of the percentage of leaf
957
water loss ± SD (n=30). Three independent experiments were performed.
958
959
Figure 4. Analysis of Na+ and K+ contents.
960
(A) Image of Na+ distribution in root tips. Five-d-old plants were treated with water
961
(control) and 150 mM NaCl, respectively, for 24 h for staining with CoroNa-Green.
962
The roots of thirty seedlings were used for each type of plant, and some roots were
963
randomly selected to be photographed.
964
(B) Measurement of Na+ and K+ contents in leaves and roots. Na+ and K+ were
965
measured from 3-week -old plants of 150 mM NaCl treatment, and then K+/ Na+ ratio
966
were respectively calculated. Results are presented as means and standard errors from
967
three independent biological replicates.
968
(C) The relative expression of genes involved in Na+ or K+ transporting. The
969
expression of each gene in WT plants under normal conditions was set as 1 to
970
normalize its expression in different lines under different conditions. Data are means
971
± SD from three independent biological replicates. Asterisk (*) indicates a significant
972
difference at P<0.05 compared with the WT.
973
974
Figure 5. The regulation of proline metabolism genes by AST1.
975
(A) Proline content assay. Values represent the average of three biological replicates.
976
Significant differences from WT are indicated.
977
(B) The transcripts level of proline metabolism genes. The expression of each gene in
978
WT plants under normal condition was set as 1 to normalize its expression in different
979
lines under different conditions. Asterisk (*) indicates a significant difference at
980
P<0.05 compared with the WT.
981
982
Figure 6. Identification of AGAG-box recognized by AST1.
983
(A) MEME analysis of the conserved sequence present in the promoters of genes
984
regulated by AST1.
33
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
985
(B) Y1H assay of the interaction of AST1 with the full or the core conserved
986
sequences. The 12 bp conserved sequence or the 1st to 8th base of conserved
987
sequences (32 types in total) were tested for their interaction with AST1 using Y1H.
988
(C) Determination of the interaction between AST1 and AGAG-box in tobacco plants.
989
The studied sequences were fused separately with the 46-bp minimal promoter to
990
drive a GUS gene as reporters, and were then co-transformed with 35S:AST1 and
991
35S:LUC into tobacco plants. Diagrams of the reporter and effector vectors were
992
shown. Data are means ± SD from three independent biological replicates. Asterisk (*)
993
indicates a significant difference at P<0.05 compared with the sequence
994
“GAAGAGGA”.
995
(D) EMSA was carried out with AST1 protein and five type sequences of AGAG-box
996
as probes. Competition for the labeled sequences was tested by adding 10-, 30- and
997
100-fold excess of unlabeled probes. The free probes and DNA-AST1 complexes
998
were indicated with arrows.
999
(E) ChIP analysis of the binding of AST1 to the AGAG-box in A. thaliana. The gene
1000
promoters that contained only one AGAG-box and did not contain any GT motifs
1001
bound by AST1 were used. Schematic diagram showing the positions of the
1002
AGAG-box in the promoters.
1003
1004
Figure 7. Identification of the GT motifs recognized by AST1.
1005
(A) Y1H assay of the GT elements recognized by AST1. Six GT elements and their
1006
mutations were respectively cloned in pHIS2 vector, and their bindings to AST1 were
1007
studied using Y1H. The above motifs were mutated following this principle, i.e.
1008
“A/T” was mutated to “C” and “C/G” was mutated to “A”.
1009
(B) Determination of the interaction of AST1 with GT motifs in tobacco plants. GT
1010
motifs and their mutations were fused separately with the 46-bp minimal promoter to
1011
drive GUS as reporters; each reporter was co-transformed with 35S:AST1 and
1012
35S:LUC into tobacco. Diagrams of the reporter and effector vectors were shown.
1013
Data are means ± SD from three independent biological replicates. Asterisk (*)
1014
indicates a significant difference at P<0.05 compared with the mutations.
34
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1015
(C) EMSA was carried out with AST1 protein and GT-box sequences. Lane 1-6, GT1,
1016
2, 3, 4, 5 and 6 probes incubated with AST1 protein. The free probes and DNA-AST1
1017
complexes were marked.
1018
(D) ChIP analysis of the binding of AST1 to GT elements. The promoters that contain
1019
only one type of GT motifs and no other motif recognized by AST1 were used in this
1020
experiment. Schematic diagram showing the positions of GT elements in the
1021
promoters. Input (input sample), ChIP+ (immunoprecipitated with an anti-GFP
1022
antibody), ChIP– (immunoprecipitated with an anti-HA antibody).
1023
1024
Figure 8. qChIP-PCR analysis of the genes directly regulated by AST1.
1025
Three-week-old 35S:GFP and 35S:AST1-GFP transgenic plants treated with 150 mM
1026
NaCl or 200 mM Mannitol were used for ChIP analysis. The promoter fragments that
1027
contained AGAG-box or GT elements identified by qRT-PCR and transcriptome were
1028
studied. The expression values in 35S:GFP plants were set as 1 to normalize the
1029
expression in 35S:AST1-GFP plants. ABR, SOS3 and ATSOD1 that were not regulated
1030
by AST1 and did not containing ASTA1 binding motifs were used as negative
1031
controls. AT5G14410, AT1G27710, AT1G04770, AT3G24860, AT5G22460, LSU1,
1032
SAUR16 were the genes identified in RNA seq. The CDS of ACTIN2, which is not
1033
regulated by AST1, was used as internal control. Data are means ± SD from three
1034
independent biological replicates. Asterisk (*) indicates a significant difference at
1035
P<0.05 compared with the 35S:GFP.
1036
1037
Figure 9. Working model of AST1 in response to abiotic stress. Abiotic stresses
1038
including salt or drought stress triggers the expression of AST1. Activated AST1
1039
regulates the stress tolerance related genes by binding to the AGAG-box or GT2–5
1040
motifs, which results in reducing stomatal aperture, water loss rate, Na+ accumulation,
1041
K+ loss, and ROS accumulation, increased proline level. The induced stress tolerance
1042
LEA genes may also play a role in stabilizing cell membrane and preventing
1043
irreversible protein aggregation. These physiological changes finally improve salt and
1044
drought stress tolerance.
35
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1045
1046
36
Figure 1
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
（7）
A
(1)
B
(2)
(3)
(4)
(5)
AST1 in different tissues
(6)
Relative expression levels
4
（8）
(10)
(11)
(12)
(13)
(14)
*
C
AST1 in leaves under stresses
1
0
3.5
NaCl
*
10
8
6
*
4
*
2
0
3
6
12
24
6
12
24
Treatment time(h)
48
1.5
1
0.5
Relative expression levels
Relative expression levels
*
0
12
*
6
1
6
*
2
3
2
3
24
Mannitol
*
5
*
4
3
*
*
2
1
0
3
48
6
12
24
Treatment time(h)
Treatment time(h)
D
Control
150mM NaCl
200mM Mannitol
*
2
1
Relative GUS activity
*
3
Roots
2.5
Leaves
4
2
*
*
NaCl
M annitol
1.5
1
0.5
0
0
Control
NaCl
Silique
0
48
*
*
Flower
NaCl
2.5
Mannitol
3
Cauline
leaf
*
3
Treatment time(h)
4
Stem
AST1 in roots under stresses
Relative expression levels
Relative expression levels
12
*
*
2
Root
Relative GUS activity
(9)
*
3
M annitol
Control
48
Rosette
leaf
Figure 2
A
(1)
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
35S
35S
peer-reviewed)
is the author/funder. All rights 35S
reserved. No reuse allowed without permission.
OE1
KO1.1
OE2
OE1
KO1.2
OE3
KO1.1
OE2
KO1.3
OE1
KO1.2
OE3
OE2
KO1.3
WT
KO1.1
KO1.2
OE3
KO1.3
WT
1/2 MS
WT
125mM NaCl
185mM Mannitol
(2)
120
120
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
80
* *
*
60
**
40
*
*
* *
20
*
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
100
Seedling survival (%)
Seedling survival(%)
100
*
* *
*
80
60
*
40
*
* *
*
*
* * *
20
*
0
0
1/2MS
100
125
1/2MS
150
NaCl(mM)
185
Mannitol(mM)
B
(1)
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
Control
150 mM NaCl
For 10d
200 mM Mannitol
For 10d
(3)
OE1
KO1.1
35S
WT
270
240
210
180
150
120
90
60
30
0
*
**
*
*
Control
OE2
KO1.2
NaCl
**
*
OE3
KO1.3
*
***
Mannitol
Chlorophyll content（mg/g.FW）
Fresh weight (mg)
(2)
2
*
1.5
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
* **
**
*
* *
1
0.5
* *
*
0
Control
NaCl
Mannitol
Figure 3
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
A peer-reviewed)
(1)
is the author/funder. All rights reserved. No reuse allowed without permission.
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
Control
150 mM NaCl
200 mM Mannitol
Stomatal aperture
Ratio of width over length
(2)
1
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
0.8
*
0.6
0.4
*
0.2
*
*
*
* * *
* *
0
Control
B
NaCl
Mannitol
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
ATMYB61 AT1G09540
Relative expression levels
14
12
*
*
10
*
8
* *
6
4
* * *
2
*
* * *
0
Control
NaCl
Mannitol
C
35S
WT
70
OE1
KO1.1
OE3
KO1.3
OE2
KO1.2
Water loss rate（%）
60
50
40
30
20
10
0
0
0.5
1
1.5
2
Time (h)
3
4
5
*
Figure 4
A
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Control
150 mM NaCl
B
2.5
*
* *
2
1.5
*
*
1
250
250
*
200
*
150
*
100
* * *
50
150
100
*
* * *
0.5
0
0
Control
NaCl
Control
NaCl
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
*
*
20
*
15
* *
10
5
600
80
60
400
*
300
*
200
*
* * *
40
* *
20
100
0
0
Control
NaCl
500
K+/Na+
25
K+ content (mg/g DW)
Control
Na+ content (mg/g DW)
*
*
50
0
Roots
*
200
*
K+ /Na+
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
*
*
K+ content （mg/g DW)
Leaves
Na+ content (mg/g DW)
3
Control
NaCl
*
*
* *
0
NaCl
Control
NaCl
C
NHX2 AT3G05030
*
Relative expression levels
6
5
*
*
*
4
3
*
* *
*
2
* *
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
6
* *
* *
1
0
5
*
4
*
*
*
*
3
* *
2
* *
1
0
Control
NaCl
*
2
0
M annitol
*
*
3
1
Control
*
*
6
*
5
4
NHX3 AT5G55470
7
*
Relative expression levels
HKT1 AT4G10310
Relative expression levels
7
M annitol
Control
NaCl
Mannitol
NaCl
NHX6 AT1G79610
*
5
*
*
*
4
*
3
* * *
2
* *
Relative expression levels
Relative expression levels
6
SOS3 AT 5G24270
SOS2 AT5G35410
6
*
7
*
*
5
*
3
*
4
3
* *
2
*
*
1
1
0
0
Control
M annitol
NaCl
*
Relative expression levels
8
*
2.5
* *
2
1.5
1
0.5
0
Control
M annitol
NaCl
Control
Mannitol
NaCl
Figure 5
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
A peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Proline content (mg/g.FW)
1
35S
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
0.8
** *
0.6
**
*
* **
0.4
*
0.2
*
*
0
Control
NaCl
M annitol
B
P5CS2 AT3G55610
8
Relative expression levels
*
6
OE1
OE2
OE3
WT
KO1.1
KO1.2
KO1.3
*
*
5
*
4
3
* *
2
*
* *
7
35S
*
Relative expression levels
P5CS1 AT2G39800
*
7
* * *
6
*
5
*
4
*
*
3
2
**
1
1
0
0
Control
Mannitol
Control
NaCl
M annitol
NaCl
P5CDH AT5G62520
7
PRODH AT4G34590
*
8
6
*
4
*
*
6
*
*
*
2
*
Relative expression levels
Relative expression levels
10
*
*
*
5
4
* * *
3
*
* *
2
1
0
0
Control
Mannitol
NaCl
Control
M annitol
NaCl
Figure 6
A
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
B
Positive control
AGAGAGAGAAAG
AGAGAGAG
AGAGAGAA
AGAGAGGG
AGAGAGGA
AGTGAGAG
AGTGAGAA
AGTGAGGG
AGTGAGGA
Negative control
Positive control
AGAGAGAGAAAG
GGAGAGAG
GGAGAGAA
GGAGAGGG
GGAGAGGA
GGTGAGAG
GGTGAGAA
GGTGAGGG
GGTGAGGA
Negative control
DDO
Positive control
AGAGAGAGAAAG
AAAGAGAG
AAAGAGAA
AAAGAGGG
AAAGAGGA
AATGAGAG
AATGAGAA
AATGAGGG
AATGAGGA
Negative control
Positive control
AGAGAGAGAAAG
GAAGAGAG
GAAGAGAA
GAAGAGGG
GAAGAGGA
GATGAGAG
GATGAGAA
GATGAGGG
GATGAGGA
Negative control
TDO (70mM/3-AT)
C
DDO
TDO (70mM/3-AT)
Reporter
Effe ctor
Relative GUS/LUC activity
25
AS T1
CAMV 35S
CAMV 35S
（35S-AST1）
N OS-T
*
20
*
*
15
GU S
N OS-T
(35S-GUS)
GU S
NO S-T
(pCAMBI1301-elements)
cis 46bp-mini
*
*
*
10
*
*
*
*
5
0
GAAGAGGA
GAAGAGGG
GAAGAGAA
AGTGAGGA
AGTGAGGG
AGTGAGAA
-
GATGAGAG
-
+
GAAGAGAG
+
+
GGTGAGAG
+
+
GGAGAGAG
-
AATGAGAG
Protein
Probe
Competitor
AAAGAGAG
D
AGTGAGAG
Effector
AGAGAGAG
AGAGAGAGAAAG
35-GUS
Reporter
35S-AST1
+
+
+
+
-
+
+
+
+
- +
+
+
+
+
+
+
- -
+
+
+
+
+
+
+
- +
+
+
+
- +
+
+
+
- +
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
-
-
10X
-
-
10X
+
- -
- -
AAAGAGAG
GGAGAGAG
+
- -
+
DNA-protein
complexes
Free DNA probe
AGAGAGAGAAAG
AGAGAGAG
GATGAGAG
P5CS2
ABR
ATG
ATG
AT5G14410
ATCOR47
1（-874~ -675）
ABR
M
ChIP-
1
1
1
2
2
ATCOR47
P5CS2
ChIP+
2
M
Input
ChIP+
ChIP-
1
1
1
2
2
AGAG-box
2（-275~ -76）
1（-1000~ -801）
2（-229~ -30）
Input
GAAGAGAA
2（-280~ -81）
1（-952~ -753）
2（-210~ -11）
1（-701~ -502）
GAAGAGAA
ATG
ATG
E
AGTGAGGG
2
M
AT5G14410
Input
ChIP+
ChIP-
1
1
1
2
2
2
M
Input
ChIP+
ChIP-
1
1
1
2
2
2
Figure 7
A
Positive control
GT1: GGTTAA
pHIS2-GT1/pGADT7-AST1
GT1-M
: AACCCC
bioRxiv preprint first posted online Mar.
27, 2017;
doi: http://dx.doi.org/10.1101/121319pHIS2-GT1-M/pGADT7-AST1
. The copyright holder for this preprint (which was not
peer-reviewed) is
theGGTAATT
author/funder. All rights reserved. No reuse
allowed without permission.
GT2:
pHIS2-GT2/pGADT7-AST1
GT2-M: AACCCCC
pHIS2-GT2-M/pGADT7-AST1
GT3: TACAGT
pHIS2-GT3/pGADT7-AST1
pHIS2-GT3-M/pGADT7-AST1
GT3-M: CCACAC
GT4: GGTAAAT
pHIS2-GT4/pGADT7-AST1
GT4-M:AACCCCC
pHIS2-GT4-M/pGADT7-AST1
GT5:GGTAAA
pHIS2-GT5/pGADT7-AST1
GT5-M:AACCCC
pHIS2-GT5-M/pGADT7-AST1
GT6: GTTAC
pHIS2-GT6/pGADT7-AST1
pHIS2-GT6-M/pGADT7-AST1
GT6-M: ACCCA
Negative control
DDO
B
TDO (50mM/3-AT)
Reporter
Effe ctor
AS T1
CAMV 35S
CAMV 35S
（35S-AST1）
N OS-T
Relative GUS/LUC activity
30
GU S
N OS-T
GU S
NO S-T
(35S-GUS)
(pCAMBI1301-elements)
cis 46bp-mini
*
20
*
*
10
*
0
Reporter
35-GUS
GT1
GT1-M
GT2
GT2-M
GT3
Effector
GT3-M
GT4
GT4-M
GT5
GT5-M
GT6
GT6-M
35S-AST1
C
Protein
Probe
AST1
GT1
GT2 GT3 GT4 GT5
GT-6
DNA-protein complexes
Free DNA probe
D
ATG
GT1
1（-664~ -465）
ChIP+
ChIP-
1
1
2
M
2
1（-906~ -707）
AT5g58400
Input
ChIP+
ChIP-
1
1
2
2
2
M
Input
ChIP+
ChIP-
1
1
1
2
ChIP-
1
1
1
2
M
Input
ChIP+
ChIP-
1
1
1
2
2
2
2
ATSOD1
ERD10
ChIP+
2
2（-296~ -97）
1
AT4G34790
Input
2
ATSOD1
AT3G18773
SOS3
M
ATG
ERD10
1（-1000~ -801）
Input
2（-250~ -51）
GT6
GT5
2（-219~ -20）
2
1（-934~ -735）
ATG
AT4G34790
1
AT5G58400
2（-200~ -1）
1（-792~ -593）
GT4
M
GT3
AT3G18773
2（-200~ -1）
ATG
1（-862~ -663）
ATG
ATG
GT2
SOS3
2
M
Input
ChIP+
ChIP-
1
1
1
2
2
2
2（-325~ -126）
Figure 8
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
35S: GFP
35S: AST1-GFP
Mannitol
24
*
22
*
20
10
*
*
8
*
*
*
*
2
*
*
*
*
*
*
*
*
*
*
AT5G14410
*
*
ERD10
*
LEA7
4
*
*
*
6
AT5G58400
Fold enrichment (normalized to Input)
26
*
*
*
0
SAUR16
LSU1
AT5G22460
AT3G24860
AT1G04770
AT1G27710
PRX72
PER4
AT1G24110
COR15
ATCOR47
LEA14
LEA3
NHX2
NHX3
NHX6
P5CS2
P5CS1
AtMYB61
PRX37
AT1G71695
FSD1
SOD2
ATSOD1
SOS3
ABR
30
25
20
15
10
35S: GFP
35S: AST1-GFP
*
*
*
*
*
4
*
*
*
*
*
*
*
*
*
*
2
*
*
AT3G24860
*
AT1G04770
*
*
PRX72
*
*
*
*
*
*
AT5G58400
Fold enrichment (normalized to Input)
NaCl
*
0
SAUR16
LSU1
AT5G22460
AT1G27710
AT5G14410
ERD10
LEA7
PER4
AT1G24110
COR15
ATCOR47
LEA14
LEA3
NHX2
NHX3
NHX6
P5CS2
P5CS1
AtMYB61
PRX37
AT1G71695
FSD1
SOD2
ATSOD1
SOS3
ABR
bioRxiv preprint first posted online Mar. 27, 2017; doi: http://dx.doi.org/10.1101/121319. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Figure 9
Salt or drought stress
Induction of AST1
AST1
AST1 target genes
AGAG-box/GT2-5 motifs
NHX2,
AtMYB61
NHX3,
P5CS1,
POD and SOD
LEA family genes
P5CS2
family genes
involving
NHX6
stress
tolerance
Increased
Reduced
Reduced Na+
stomatal aperture accumulation and proline
level
and water loss rate
K+ from loss
Reduced
ROS
accumulation
Improved abiotic
stress tolerance
Stabilized cell membrane,
preventing irreversible
protein aggregation