Mol. Cells 2014; 37(7): 532-539
Published online July 31, 2014
https://doi.org/10.14348/molcells.2014.0128
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: jjeon@khu.ac.kr
We isolated a rice (
Keywords metallothionein,
Senescence is a final developmental process in the life cycle of plants and a type of programmed cell death. Senescence is controlled by multiple developmental factors and also external biotic and abiotic stress signals (Buchanan-Wollaston et al., 2005; Lim et al., 2007; Munne-Bosch and Alegre, 2004). Comprehensive analyses of altered gene expression patterns in genetically controlled senescent leaves, as a study model for plant senescence, has identified important regulatory factors. To date, over 100 transcription factors, most notably belonging to the WRKY, NAC, C2H2 zinc finger, AP2/EREBP, and MYB families, have been identified to be highly upregulated in senescent leaves in
The WRKY transcription factors constitute one of the largest transcription factor families in plants. WRKY proteins have the highly conserved DNA-binding domain of one or two 60 amino acid regions harboring WRKYGQK and a zinc finger structure. WRKYs have been shown to play important roles in the senescence process. For instance, the
The production of reactive oxygen species (ROS) is one of the earliest components of the leaf senescence pathway (Jing et al., 2008; Mittler et al., 2004; Zentgraf and Hemleben, 2008). For instance, AtWRKY53 and its regulators are controlled by hydrogen peroxide (H2O2) (Miao et al., 2007; 2008). Cellular levels of ROS have been positively correlated with the severity of leaf senescence in
Few studies to date have focused on leaf senescence in rice, a vital commercial crop plant that feeds more than half of the world’s population. In particular, the molecular regulatory mechanism of transcription factors underlying leaf senescence remains largely unknown in rice. The OsWRKY family comprises over 100 members in the rice genome (Rice WRKY Working Group, 2012). To date, most of the functionally characterized OsWRKYs have been reported to play roles in defense responses to biotic pathogens and also abiotic stress responses to environmental stimuli and hormones (De Vleesschauwer et al., 2013; Jang et al., 2010; Ryu et al., 2006). In our present study, we describe the isolation and characterization of a leaf senescence-inducible
Rice [japonica cultivar (cv.) Dongjin] plants were grown in a greenhouse under a 14/10 h light and dark period, at 24?28°C temperature and 70?80% humidity.
Total RNA was prepared from various tissues of rice plants using Trizol reagent (Invitrogen, USA) with DNase treatment (TURBO DNA-free kit; Ambion-Life technologies, USA). The extracted RNA was reverse-transcribed using AMV reverse transcriptase XL (2620A; Takara, Japan) with RNase inhibitor (2312A; Takara) and an oligo-dT primer. The synthesized first strand cDNA was used in subsequent PCR reactions with gene-specific primers and control primers for
To examine the transcriptional activation ability of OsWRKY42, the effector vector was constructed by fusing the
To analyze transcriptional repression by
The
The
For the detection of H2O2, fully expanded second leaves from the top of six-week-old wild type and transgenic plants were placed in solution of 1 mg ml?1 3,3-diaminobenzidine (DAB) and vacuum infiltrated at 650 mmHg for 10 min. The leaves were then immersed in DAB solution for 3 h in darkness. Subsequently, the treated leaves were decolorized in 80% ethanol, placed onto glass slides and observed under a stereomicroscope (Olympus, USA). The H2O2 content was also measured colorimetrically in the same fully expanded second leaves from the tops of six-week-old wild type and transgenic plants using 50 mM phosphate buffer (pH 6.5) containing 1 mM hydroxylamine (Jana and Choudhuri, 1982). After centrifugation at 6,000 ×
Rice leaves were prepared from the fully expanded second leaves from the tops of six-week-old wild type and transgenic plants. The total chlorophyll contents were measured spectrophotometrically after extraction in 80% acetone using the method of Porra et al. (1989).
The sequences at approximately 1.5-kb upstream from the coding regions of
For chromatin immunoprecipitation (ChIP) analysis, the
Through a systematic screen of the digital expression profile of rice
To next examine the subcellular localization of OsWRKY42, we generated an
The transcriptional activator ability of OsWRKY42 was examined using the maize protoplast transient expression system (Cho et al., 2009). The effector vectors used for this experiment contain either BD alone, or
To further investigate the role of
To examine whether
A disruption of the critical and tightly regulated balance between the production and scavenging of ROS has been well established as a promoter of leaf senescence (Jing et al., 2008; Mittler et al., 2004; Zentgraf and Hemleben, 2008). The early leaf senescence phenotype we observed in our current analysis prompted us examine whether the ROS level was increased in
To examine whether any ROS producing and scavenging genes were disrupted in
Analysis of about 1.5 kb upstream regulatory sequences of
The
The processes underlying senescence influence many important agricultural traits including the number and quality of seeds and timing of seed setting. However, our knowledge concerning the regulatory mechanisms that control senescence is still limited in rice, one of the world’s most agronomically important crops. It has been evident that ROS trigger leaf senescence (Zentgraf and Hemleben, 2008). Plants have developed a fine-tuned network of enzymatic and low-molecular-weight antioxidative components to regulate their ROS status. Oxidative stress and senescence occur when this critical balance is disrupted because of a depletion of antioxidants or excess accumulation of ROS.
The association between ROS and the WRKY transcription factors was revealed by the finding that the
ROS participate in a diverse range of plant processes including pollen development (Hiscock and Allen, 2008; McInnis et al., 2006ba; 2006b). In this regard, it is noteworthy that the
Histochemical GUS analysis of a serial deletion of the rice
The
In summary, we have here isolated and characterized a rice leaf senescence-inducible factor,
Mol. Cells 2014; 37(7): 532-539
Published online July 31, 2014 https://doi.org/10.14348/molcells.2014.0128
Copyright © The Korean Society for Molecular and Cellular Biology.
Muho Han1,2, Chi-Yeol Kim1,2, Junok Lee1, Sang-Kyu Lee1, and Jong-Seong Jeon1,*
1Graduate School of Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin 446-701, Korea
Correspondence to:*Correspondence: jjeon@khu.ac.kr
We isolated a rice (
Keywords: metallothionein,
Senescence is a final developmental process in the life cycle of plants and a type of programmed cell death. Senescence is controlled by multiple developmental factors and also external biotic and abiotic stress signals (Buchanan-Wollaston et al., 2005; Lim et al., 2007; Munne-Bosch and Alegre, 2004). Comprehensive analyses of altered gene expression patterns in genetically controlled senescent leaves, as a study model for plant senescence, has identified important regulatory factors. To date, over 100 transcription factors, most notably belonging to the WRKY, NAC, C2H2 zinc finger, AP2/EREBP, and MYB families, have been identified to be highly upregulated in senescent leaves in
The WRKY transcription factors constitute one of the largest transcription factor families in plants. WRKY proteins have the highly conserved DNA-binding domain of one or two 60 amino acid regions harboring WRKYGQK and a zinc finger structure. WRKYs have been shown to play important roles in the senescence process. For instance, the
The production of reactive oxygen species (ROS) is one of the earliest components of the leaf senescence pathway (Jing et al., 2008; Mittler et al., 2004; Zentgraf and Hemleben, 2008). For instance, AtWRKY53 and its regulators are controlled by hydrogen peroxide (H2O2) (Miao et al., 2007; 2008). Cellular levels of ROS have been positively correlated with the severity of leaf senescence in
Few studies to date have focused on leaf senescence in rice, a vital commercial crop plant that feeds more than half of the world’s population. In particular, the molecular regulatory mechanism of transcription factors underlying leaf senescence remains largely unknown in rice. The OsWRKY family comprises over 100 members in the rice genome (Rice WRKY Working Group, 2012). To date, most of the functionally characterized OsWRKYs have been reported to play roles in defense responses to biotic pathogens and also abiotic stress responses to environmental stimuli and hormones (De Vleesschauwer et al., 2013; Jang et al., 2010; Ryu et al., 2006). In our present study, we describe the isolation and characterization of a leaf senescence-inducible
Rice [japonica cultivar (cv.) Dongjin] plants were grown in a greenhouse under a 14/10 h light and dark period, at 24?28°C temperature and 70?80% humidity.
Total RNA was prepared from various tissues of rice plants using Trizol reagent (Invitrogen, USA) with DNase treatment (TURBO DNA-free kit; Ambion-Life technologies, USA). The extracted RNA was reverse-transcribed using AMV reverse transcriptase XL (2620A; Takara, Japan) with RNase inhibitor (2312A; Takara) and an oligo-dT primer. The synthesized first strand cDNA was used in subsequent PCR reactions with gene-specific primers and control primers for
To examine the transcriptional activation ability of OsWRKY42, the effector vector was constructed by fusing the
To analyze transcriptional repression by
The
The
For the detection of H2O2, fully expanded second leaves from the top of six-week-old wild type and transgenic plants were placed in solution of 1 mg ml?1 3,3-diaminobenzidine (DAB) and vacuum infiltrated at 650 mmHg for 10 min. The leaves were then immersed in DAB solution for 3 h in darkness. Subsequently, the treated leaves were decolorized in 80% ethanol, placed onto glass slides and observed under a stereomicroscope (Olympus, USA). The H2O2 content was also measured colorimetrically in the same fully expanded second leaves from the tops of six-week-old wild type and transgenic plants using 50 mM phosphate buffer (pH 6.5) containing 1 mM hydroxylamine (Jana and Choudhuri, 1982). After centrifugation at 6,000 ×
Rice leaves were prepared from the fully expanded second leaves from the tops of six-week-old wild type and transgenic plants. The total chlorophyll contents were measured spectrophotometrically after extraction in 80% acetone using the method of Porra et al. (1989).
The sequences at approximately 1.5-kb upstream from the coding regions of
For chromatin immunoprecipitation (ChIP) analysis, the
Through a systematic screen of the digital expression profile of rice
To next examine the subcellular localization of OsWRKY42, we generated an
The transcriptional activator ability of OsWRKY42 was examined using the maize protoplast transient expression system (Cho et al., 2009). The effector vectors used for this experiment contain either BD alone, or
To further investigate the role of
To examine whether
A disruption of the critical and tightly regulated balance between the production and scavenging of ROS has been well established as a promoter of leaf senescence (Jing et al., 2008; Mittler et al., 2004; Zentgraf and Hemleben, 2008). The early leaf senescence phenotype we observed in our current analysis prompted us examine whether the ROS level was increased in
To examine whether any ROS producing and scavenging genes were disrupted in
Analysis of about 1.5 kb upstream regulatory sequences of
The
The processes underlying senescence influence many important agricultural traits including the number and quality of seeds and timing of seed setting. However, our knowledge concerning the regulatory mechanisms that control senescence is still limited in rice, one of the world’s most agronomically important crops. It has been evident that ROS trigger leaf senescence (Zentgraf and Hemleben, 2008). Plants have developed a fine-tuned network of enzymatic and low-molecular-weight antioxidative components to regulate their ROS status. Oxidative stress and senescence occur when this critical balance is disrupted because of a depletion of antioxidants or excess accumulation of ROS.
The association between ROS and the WRKY transcription factors was revealed by the finding that the
ROS participate in a diverse range of plant processes including pollen development (Hiscock and Allen, 2008; McInnis et al., 2006ba; 2006b). In this regard, it is noteworthy that the
Histochemical GUS analysis of a serial deletion of the rice
The
In summary, we have here isolated and characterized a rice leaf senescence-inducible factor,
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