Mol. Cells 2018; 41(12): 1033-1044
Published online November 14, 2018
https://doi.org/10.14348/molcells.2018.0363
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: jlim@konkuk.ac.kr
As sessile organisms, plants have evolved to adjust their growth and development to environmental changes. It has been well documented that the crosstalk between different plant hormones plays important roles in the coordination of growth and development of the plant. Here, we describe a novel recessive mutant,
Keywords Arabidopsis, auxin biosynthesis, ethylene, PDX3, PLP
Plants, which are sessile in nature, need to adapt their growth and development to a changing environment for survival and reproduction. Plant hormones play important roles in ensuring flexible growth and development by coordinating the interactions between environmental signals and genetic programs (Wolters and Jürgens, 2009). Furthermore, accumulating evidence indicates that the crosstalk between different hormone pathways is crucial for the plasticity of plant growth and development under various environmental conditions (reviewed in Depuydt and Hardtke, 2011; Gazzarrini and McCourt, 2003; Vanstraelen and Benková, 2012). Substantial data have been published on the interactions between auxin and ethylene in the regulation of Arabidopsis (
ACSs, TAA1 and TARs all belong to the pyridoxal-5′-phosphate (PLP)-dependent class of enzymes (He et al., 2011; Huai et al., 2001; Stepanova et al., 2008; Tao et al., 2008). PLP, a B6 vitamer, acts as a cofactor in numerous enzymatic reactions, including ethylene and auxin biosynthesis. Vitamin B6 collectively refers to PLP and its vitamers, which comprise pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5′ phosphorylated forms: pyridoxine 5′-phosphate (PNP), pyridoxamine 5′-phosphate (PMP), and PLP (Colinas et al., 2016; Fitzpatrick et al., 2007; Rueschhoff et al., 2013). PLP can be produced by both
To identify additional molecular components involved in ethylene-auxin crosstalk, in this study, we performed a genetic screen for root-specific growth responses to ethylene. By utilizing the GAL4/UAS activation-tagging system previously reported (Waki et al., 2013), we identified a novel Arabidopsis mutant with insensitivity to root growth inhibition in the presence of ACC. Through genetic, physiological, and molecular analyses, we revealed that the PNP/PMP oxidase (PDX3) in the salvage pathway plays a role in root-growth response to ethylene via ethylene-induced auxin biosynthesis.
For identification of
Digital images of seedlings at given times were taken with SP-560UZ digital camera (Olympus, Japan). Lengths of hypocotyls and roots from the digital images were measured using ImageJ software (
Total RNA was extracted from roots of 4-d-old WT and mutant seedlings using the RNeasy Plant Mini Kit (Qiagen, Germany), as previously described (Lee et al., 2012). Approximately 0.5 μg of the isolated RNA samples were used for synthesis of cDNA using TOP script™ RT DRY MIX (d18/dN6 Plus) (Enzynomics, Korea) according to the manufacturer’s instructions. The RT-qPCR assays were performed with an Mx3000P QPCR machine (Agilent Technologies, USA) using RbTaq™ qPCR 2X PreMIX (Enzynomics, Korea). The
To generate transgenic plants with a
The histochemical detection of GUS activity was carried out as previously described with minor modifications (Lee et al., 2012; Yoon et al., 2016). Dark-grown seedlings were incubated in GUS staining solution [0.4 mM 5-bromo-4-chloro-3-indoxyl-b-D-glucuronic acid, 2 mM K3Fe(CN)6, 2 mM K4Fe(CN)6, 0.1 M sodium phosphate, 10 mM EDTA, and 0.1% Triton X-100] for 2 h. After GUS staining, samples were rinsed with 70% EtOH for 30 min. Subsequently, samples were observed with differential interference contrast (DIC) optics using an Axio Imager.A1 microscope (Carl Zeiss, Germany). Digital images of the samples were obtained with an AxioCam MRc5 digital camera (Carl Zeiss, Germany). For GFP images, samples were stained with 10 μM propidium iodide (Sigma-Aldrich, USA) and observed with an Olympus FV-1000 confocal laser scanning microscope (Olympus, Japan).
EdU staining was performed as described previously, but with minor modifications (Choe et al., 2017; Hong et al., 2015). Four-day-old seedlings were transferred to 0.5X MS media with 0.8% agar containing 5 μM EdU (Invitrogen, USA) for 30 min. The seedlings were fixed in fixative solution (3.7% formaldehyde and 1% Triton x-100 in 1X PBS) for 10 min with vacuum infiltration. After fixation, samples were incubated for 50 min at room temperature. Subsequently, samples were washed twice with 3% bovine serum albumin (BSA) in 1X PBS. Finally, the seedlings were incubated with 250 μL of freshly prepared Click-iT® reaction cocktail for 2 h in the dark at room temperature, according to the manufacturer’s instructions (Invitrogen, USA). The EdU-stained seedlings were washed once with 3% BSA in 1X PBS and stored in 1X PBS in the dark. Stained seedlings were observed with an Olympus FV-1000 confocal laser scanning microscope (Olympus, Japan).
Sequence data from this article can be found in the Arabidopsis Genome Initiative under the following accession numbers:
Genetic screening for mutants with root-specific ethylene insensitivity has previously been used to successfully identify molecular components involved in the ethylene-auxin crosstalk (Alonso et al., 2003; Stepanova et al., 2005; 2008). To identify additional root-specific modulators of the ethyleneauxin crosstalk, we utilized the GAL4/UAS activation-tagging system. In particular, we used transgenic plants harboring a driver of the QC-specific
Taken together, our genetic and physiological results suggest that the mild insensitive phenotype is likely due to an insertional mutation, which results in a defect in the root-specific ethylene response. Therefore, we named the newly isolated loss-of-function mutant as
Previous studies have reported that under IAA treatment,
Given that the root growth phenotypes of dark-grown
To further evaluate the role of
Collectively, our results strongly support the hypothesis that
In a root growth assay, besides its insensitive root growth to ethylene under dark-grown conditions, we found that
When measured as described previously (Achard et al., 2009; Dello Ioio et al., 2007; Heo et al., 2011; Lee et al., 2012; Ubeda-Tomás et al., 2009), the root meristem size of
To identify the locus responsible for the
To further verify whether mutations in the
Previous studies have shown that by adding Trp or IAA,
We had initially attempted to tissue-specifically overexpress T-DNA with tandem UAS sequences (pBIB-UAS) by using the QC-specific driver (
Ethylene is known to inhibit root growth primarily by affecting cell elongation (Alarcón et al., 2014; Bleecker and Kende, 2000; Le et al., 2001; Růžička et al., 2007; Swarup et al., 2007). We also found that cell elongation in the
Vitamin B6 plays crucial roles in plant development and hormone homeostasis (Boycheva et al., 2015; Chen and Xiong, 2005; Percudani and Peracchi, 2003; Titiz et al., 2006). Therefore, a complete loss of
Our genetic, physiological, and molecular analyses provide important information on the role of MINE/PDX3 in ethylene-induced auxin biosynthesis and plant growth, with a focus on the root. Subsequent molecular cloning led us to the conclusion that the insensitivity of
In summary, this study provides lines of evidence that MINE/PDX3-mediated production of PLP in the salvage pathway participates in TAA1/TAR-dependent auxin biosynthesis induced by ethylene, which in turn influences the crosstalk between ethylene and auxin in the Arabidopsis root.
Mol. Cells 2018; 41(12): 1033-1044
Published online December 31, 2018 https://doi.org/10.14348/molcells.2018.0363
Copyright © The Korean Society for Molecular and Cellular Biology.
Gyuree Kim1,5, Sejeong Jang1,5, Eun Kyung Yoon1,3, Shin Ae Lee1,4, Souvik Dhar1, Jinkwon Kim1, Myeong Min Lee2, and Jun Lim1,*
1Department of Systems Biotechnology, Konkuk University, Seoul, Korea, 2Department of Systems Biology, Yonsei University, Seoul, Korea, 3Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, Singapore, 4Department of Agricultural Biology, National Institute of Agricultural Sciences, Wanju, Korea
Correspondence to:*Correspondence: jlim@konkuk.ac.kr
As sessile organisms, plants have evolved to adjust their growth and development to environmental changes. It has been well documented that the crosstalk between different plant hormones plays important roles in the coordination of growth and development of the plant. Here, we describe a novel recessive mutant,
Keywords: Arabidopsis, auxin biosynthesis, ethylene, PDX3, PLP
Plants, which are sessile in nature, need to adapt their growth and development to a changing environment for survival and reproduction. Plant hormones play important roles in ensuring flexible growth and development by coordinating the interactions between environmental signals and genetic programs (Wolters and Jürgens, 2009). Furthermore, accumulating evidence indicates that the crosstalk between different hormone pathways is crucial for the plasticity of plant growth and development under various environmental conditions (reviewed in Depuydt and Hardtke, 2011; Gazzarrini and McCourt, 2003; Vanstraelen and Benková, 2012). Substantial data have been published on the interactions between auxin and ethylene in the regulation of Arabidopsis (
ACSs, TAA1 and TARs all belong to the pyridoxal-5′-phosphate (PLP)-dependent class of enzymes (He et al., 2011; Huai et al., 2001; Stepanova et al., 2008; Tao et al., 2008). PLP, a B6 vitamer, acts as a cofactor in numerous enzymatic reactions, including ethylene and auxin biosynthesis. Vitamin B6 collectively refers to PLP and its vitamers, which comprise pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5′ phosphorylated forms: pyridoxine 5′-phosphate (PNP), pyridoxamine 5′-phosphate (PMP), and PLP (Colinas et al., 2016; Fitzpatrick et al., 2007; Rueschhoff et al., 2013). PLP can be produced by both
To identify additional molecular components involved in ethylene-auxin crosstalk, in this study, we performed a genetic screen for root-specific growth responses to ethylene. By utilizing the GAL4/UAS activation-tagging system previously reported (Waki et al., 2013), we identified a novel Arabidopsis mutant with insensitivity to root growth inhibition in the presence of ACC. Through genetic, physiological, and molecular analyses, we revealed that the PNP/PMP oxidase (PDX3) in the salvage pathway plays a role in root-growth response to ethylene via ethylene-induced auxin biosynthesis.
For identification of
Digital images of seedlings at given times were taken with SP-560UZ digital camera (Olympus, Japan). Lengths of hypocotyls and roots from the digital images were measured using ImageJ software (
Total RNA was extracted from roots of 4-d-old WT and mutant seedlings using the RNeasy Plant Mini Kit (Qiagen, Germany), as previously described (Lee et al., 2012). Approximately 0.5 μg of the isolated RNA samples were used for synthesis of cDNA using TOP script™ RT DRY MIX (d18/dN6 Plus) (Enzynomics, Korea) according to the manufacturer’s instructions. The RT-qPCR assays were performed with an Mx3000P QPCR machine (Agilent Technologies, USA) using RbTaq™ qPCR 2X PreMIX (Enzynomics, Korea). The
To generate transgenic plants with a
The histochemical detection of GUS activity was carried out as previously described with minor modifications (Lee et al., 2012; Yoon et al., 2016). Dark-grown seedlings were incubated in GUS staining solution [0.4 mM 5-bromo-4-chloro-3-indoxyl-b-D-glucuronic acid, 2 mM K3Fe(CN)6, 2 mM K4Fe(CN)6, 0.1 M sodium phosphate, 10 mM EDTA, and 0.1% Triton X-100] for 2 h. After GUS staining, samples were rinsed with 70% EtOH for 30 min. Subsequently, samples were observed with differential interference contrast (DIC) optics using an Axio Imager.A1 microscope (Carl Zeiss, Germany). Digital images of the samples were obtained with an AxioCam MRc5 digital camera (Carl Zeiss, Germany). For GFP images, samples were stained with 10 μM propidium iodide (Sigma-Aldrich, USA) and observed with an Olympus FV-1000 confocal laser scanning microscope (Olympus, Japan).
EdU staining was performed as described previously, but with minor modifications (Choe et al., 2017; Hong et al., 2015). Four-day-old seedlings were transferred to 0.5X MS media with 0.8% agar containing 5 μM EdU (Invitrogen, USA) for 30 min. The seedlings were fixed in fixative solution (3.7% formaldehyde and 1% Triton x-100 in 1X PBS) for 10 min with vacuum infiltration. After fixation, samples were incubated for 50 min at room temperature. Subsequently, samples were washed twice with 3% bovine serum albumin (BSA) in 1X PBS. Finally, the seedlings were incubated with 250 μL of freshly prepared Click-iT® reaction cocktail for 2 h in the dark at room temperature, according to the manufacturer’s instructions (Invitrogen, USA). The EdU-stained seedlings were washed once with 3% BSA in 1X PBS and stored in 1X PBS in the dark. Stained seedlings were observed with an Olympus FV-1000 confocal laser scanning microscope (Olympus, Japan).
Sequence data from this article can be found in the Arabidopsis Genome Initiative under the following accession numbers:
Genetic screening for mutants with root-specific ethylene insensitivity has previously been used to successfully identify molecular components involved in the ethylene-auxin crosstalk (Alonso et al., 2003; Stepanova et al., 2005; 2008). To identify additional root-specific modulators of the ethyleneauxin crosstalk, we utilized the GAL4/UAS activation-tagging system. In particular, we used transgenic plants harboring a driver of the QC-specific
Taken together, our genetic and physiological results suggest that the mild insensitive phenotype is likely due to an insertional mutation, which results in a defect in the root-specific ethylene response. Therefore, we named the newly isolated loss-of-function mutant as
Previous studies have reported that under IAA treatment,
Given that the root growth phenotypes of dark-grown
To further evaluate the role of
Collectively, our results strongly support the hypothesis that
In a root growth assay, besides its insensitive root growth to ethylene under dark-grown conditions, we found that
When measured as described previously (Achard et al., 2009; Dello Ioio et al., 2007; Heo et al., 2011; Lee et al., 2012; Ubeda-Tomás et al., 2009), the root meristem size of
To identify the locus responsible for the
To further verify whether mutations in the
Previous studies have shown that by adding Trp or IAA,
We had initially attempted to tissue-specifically overexpress T-DNA with tandem UAS sequences (pBIB-UAS) by using the QC-specific driver (
Ethylene is known to inhibit root growth primarily by affecting cell elongation (Alarcón et al., 2014; Bleecker and Kende, 2000; Le et al., 2001; Růžička et al., 2007; Swarup et al., 2007). We also found that cell elongation in the
Vitamin B6 plays crucial roles in plant development and hormone homeostasis (Boycheva et al., 2015; Chen and Xiong, 2005; Percudani and Peracchi, 2003; Titiz et al., 2006). Therefore, a complete loss of
Our genetic, physiological, and molecular analyses provide important information on the role of MINE/PDX3 in ethylene-induced auxin biosynthesis and plant growth, with a focus on the root. Subsequent molecular cloning led us to the conclusion that the insensitivity of
In summary, this study provides lines of evidence that MINE/PDX3-mediated production of PLP in the salvage pathway participates in TAA1/TAR-dependent auxin biosynthesis induced by ethylene, which in turn influences the crosstalk between ethylene and auxin in the Arabidopsis root.
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