Mol. Cells 2017; 40(8): 577-586
Published online July 31, 2017
https://doi.org/10.14348/molcells.2017.0075
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: colim@gnu.ac.kr
Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors that are implicated in protein turnover and stress responses. Here, we characterized a PhyCYS from
Keywords abscisic acid,
Plant cystatins, or phytocystatins (PhyCYSs), are proteinaceous inhibitors of the papain-like (C1A) and legumain (C13) families of plant cysteine proteases (CPs) (MEROPS peptidase database;
The establishment of seed germination and early seedling growth is strongly influenced by various unfavorable environmental conditions, which induce stress responses, therefore negatively affecting these processes. The joint action of papain-like CPs and legumains plays a key role in the degradation of reserve proteins (Zakharov et al., 2004), and their activity is inhibited by PhyCYSs under unfavorable conditions (Julián et al., 2013), including drought (Rodriguez et al., 2010), heat (Je et al., 2014), high alkalinity (Sun et al., 2014), high salinity (Tan et al. 2016), and low temperature stress (Zhang et al., 2008), as well as osmotic imbalance (Bae et al., 2010), but their complex interactions make it difficult to establish clear connections between a particular stress and the corresponding response at the level of
The transcriptional regulation of
The rate of seed germination is also influenced by the contents of abscisic acid (ABA), which increase in response to various stresses and negatively affect germination and early seedling growth (Verma et al., 2016). ABA-responsive genes, including
In the present study, we investigated the activity of the
The
A T-DNA insertional mutant containing a single T-DNA insertion in
To analyze the expression of
To monitor the activity of the
GUS activity in transgenic
Quantitative measurement of GUS activity in protein extracts was performed using the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (4-MUG; Sigma-Aldrich, USA). Protein extracts were isolated by grinding the tissues in extraction buffer (50 mM sodium phosphate [pH 7.0], 10 mM EDTA, 0.1% SDS, 0.1% Triton X-100, 10 mM β-mercaptoethanol, and 25 μg/ml phenylmethylsulfonyl fluoride), followed by centrifugation (12,000 rpm for 10 min). The supernatants were combined with extraction buffer containing 0.8 mM 4-MUG, and GUS activity at various time points was determined in triplicate and calculated as pmoles 4-methylumbelliferone (4-MU)/min/mg protein as described by Chen et al. (2010).
To investigate the effect of
To examine the inhibitory activity of AtCYS5 in
To measure seedling growth, stratified seeds treated with HS at 50°C for 115 min or normal temperature conditions (22°C) were germinated for 7 days prior to fresh weight and primary root length measurements.
To compare the effects of ABA on germination and seedling growth in various plants, seeds from
Transcriptional activators of the
EMSA was performed as described by Je et al. (2014), using double-stranded (ds)-oligonucleotide probes corresponding to a fragment of the
To determine whether AtCYS5 might also function in HS responses, we isolated total RNA from germinating
If AtCYS5 is required for germination,
To determine whether increased
To confirm the function of AtCYS5 in seed germination and seedling growth
We imbibed seeds from the two independent
To investigate the effect of ABA, an inhibitor of seed germination, on
Since seeds of the
HS increases ABA levels in plant cells (Toh et al., 2008). Therefore, the increased germination in
ABA- and stress-responsive
ABREs represent a subset of the C- and G-box sequences [CACGT(C/G)] present in the promoter regions of many light-regulated genes (Giuliano et al., 1988). The
Since in the presence of ABA, the expression of
In the case of ABF3, the addition of unlabeled
In conclusion, group A bZIPs ABF1 and ABF3 regulate
(A) Imbibed wild-type Col-0
Time course of germination (in hours after imbibition) for freshly harvested seeds of untransformed wild-type (WT),
Phenotypes are shown for 7-day-old untransformed wild-type (WT),
(A) Seeds of untransformed wild-type (WT), two independent
(A) Schematic representation of the effector and reporter constructs used for the transient promoter activation assays. The effector constructs used to express the bZIP factor tagged with green fluorescent protein (GFP) contained the
(A) Schematic diagram of the
(A) 32P-labeled
Mol. Cells 2017; 40(8): 577-586
Published online August 31, 2017 https://doi.org/10.14348/molcells.2017.0075
Copyright © The Korean Society for Molecular and Cellular Biology.
Chieun Song1, Taeyoon Kim1,2, Woo Sik Chung1,2, and Chae Oh Lim1,2,*
1Systems and Synthetic Agrobiotech Center and PMBBRC, Gyeongsang National University, Jinju 52828, Korea, 2Division of Life Science, Gyeongsang National University, Jinju 52828, Korea
Correspondence to:*Correspondence: colim@gnu.ac.kr
Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors that are implicated in protein turnover and stress responses. Here, we characterized a PhyCYS from
Keywords: abscisic acid,
Plant cystatins, or phytocystatins (PhyCYSs), are proteinaceous inhibitors of the papain-like (C1A) and legumain (C13) families of plant cysteine proteases (CPs) (MEROPS peptidase database;
The establishment of seed germination and early seedling growth is strongly influenced by various unfavorable environmental conditions, which induce stress responses, therefore negatively affecting these processes. The joint action of papain-like CPs and legumains plays a key role in the degradation of reserve proteins (Zakharov et al., 2004), and their activity is inhibited by PhyCYSs under unfavorable conditions (Julián et al., 2013), including drought (Rodriguez et al., 2010), heat (Je et al., 2014), high alkalinity (Sun et al., 2014), high salinity (Tan et al. 2016), and low temperature stress (Zhang et al., 2008), as well as osmotic imbalance (Bae et al., 2010), but their complex interactions make it difficult to establish clear connections between a particular stress and the corresponding response at the level of
The transcriptional regulation of
The rate of seed germination is also influenced by the contents of abscisic acid (ABA), which increase in response to various stresses and negatively affect germination and early seedling growth (Verma et al., 2016). ABA-responsive genes, including
In the present study, we investigated the activity of the
The
A T-DNA insertional mutant containing a single T-DNA insertion in
To analyze the expression of
To monitor the activity of the
GUS activity in transgenic
Quantitative measurement of GUS activity in protein extracts was performed using the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (4-MUG; Sigma-Aldrich, USA). Protein extracts were isolated by grinding the tissues in extraction buffer (50 mM sodium phosphate [pH 7.0], 10 mM EDTA, 0.1% SDS, 0.1% Triton X-100, 10 mM β-mercaptoethanol, and 25 μg/ml phenylmethylsulfonyl fluoride), followed by centrifugation (12,000 rpm for 10 min). The supernatants were combined with extraction buffer containing 0.8 mM 4-MUG, and GUS activity at various time points was determined in triplicate and calculated as pmoles 4-methylumbelliferone (4-MU)/min/mg protein as described by Chen et al. (2010).
To investigate the effect of
To examine the inhibitory activity of AtCYS5 in
To measure seedling growth, stratified seeds treated with HS at 50°C for 115 min or normal temperature conditions (22°C) were germinated for 7 days prior to fresh weight and primary root length measurements.
To compare the effects of ABA on germination and seedling growth in various plants, seeds from
Transcriptional activators of the
EMSA was performed as described by Je et al. (2014), using double-stranded (ds)-oligonucleotide probes corresponding to a fragment of the
To determine whether AtCYS5 might also function in HS responses, we isolated total RNA from germinating
If AtCYS5 is required for germination,
To determine whether increased
To confirm the function of AtCYS5 in seed germination and seedling growth
We imbibed seeds from the two independent
To investigate the effect of ABA, an inhibitor of seed germination, on
Since seeds of the
HS increases ABA levels in plant cells (Toh et al., 2008). Therefore, the increased germination in
ABA- and stress-responsive
ABREs represent a subset of the C- and G-box sequences [CACGT(C/G)] present in the promoter regions of many light-regulated genes (Giuliano et al., 1988). The
Since in the presence of ABA, the expression of
In the case of ABF3, the addition of unlabeled
In conclusion, group A bZIPs ABF1 and ABF3 regulate
(A) Imbibed wild-type Col-0
Time course of germination (in hours after imbibition) for freshly harvested seeds of untransformed wild-type (WT),
Phenotypes are shown for 7-day-old untransformed wild-type (WT),
(A) Seeds of untransformed wild-type (WT), two independent
(A) Schematic representation of the effector and reporter constructs used for the transient promoter activation assays. The effector constructs used to express the bZIP factor tagged with green fluorescent protein (GFP) contained the
(A) Schematic diagram of the
(A) 32P-labeled
Jung Eun Hwang, Chan Ju Lim, Huan Chen, Jihyun Je, Chieun Song, and Chae Oh Lim*
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(A) Imbibed wild-type Col-0
Time course of germination (in hours after imbibition) for freshly harvested seeds of untransformed wild-type (WT),
Phenotypes are shown for 7-day-old untransformed wild-type (WT),
(A) Seeds of untransformed wild-type (WT), two independent
(A) Schematic representation of the effector and reporter constructs used for the transient promoter activation assays. The effector constructs used to express the bZIP factor tagged with green fluorescent protein (GFP) contained the
(A) Schematic diagram of the
(A) 32P-labeled