Mol. Cells 2014; 37(11): 795-803
Published online November 5, 2014
https://doi.org/10.14348/molcells.2014.0127
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: shchoe@snu.ac.kr
To withstand ever-changing environmental stresses, plants are equipped with phytohormone-mediated stress resistance mechanisms. Salt stress triggers abscisic acid (ABA) signaling, which enhances stress tolerance at the expense of growth. ABA is thought to inhibit the action of growth-promoting hormones, including brassinosteroids (BRs). However, the regulatory mechanisms that coordinate ABA and BR activity remain to be discovered. We noticed that ABA-treated seedlings exhibited small, round leaves and short roots, a phenotype that is characteristic of the BR signaling mutant,
Keywords ABA, abiotic stress, BIN2, brassinosteroids, RD26, Root
Due to their sessile nature, plants have developed strategies to cope with abiotic challenges and biotic stresses (Chung et al., 2012; Kim et al., 2014; Maharjan and Choe, 2011). Plants exposed to abiotic stresses display severe growth retardation and reduced productivity. Growth is regulated by plant hormones, which modify endogenous programs in response to exogenous signals. However, the hormone-dependent mechanisms by which growth is inhibited under stress conditions are not fully understood. The molecular mechanisms that impart tolerance to water stress can be divided into abscisic acid (ABA)-dependent and ABA-independent pathways (Shinozaki and Yamaguchi-Shinozaki, 2007). ABA plays vital roles in adaptation to environmental changes, seed dormancy, and the regulation of stomatal closure (Grill and Himmelbach, 1998; Lee and Luan, 2012).
Under stressed conditions, plants rapidly produce ABA, which stimulates the resistance mechanism. In the ABA-dependent pathway, ABA binds to soluble receptors of the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCARs) family, which induces the
In addition to ABFs/AREBs, other ABA-induced transcription factors are known to participate in the ABA response and stress tolerance. For instance, the NAC (NAM, ATAF1/2 and CUC2) transcription factor family, which largely consists of NO APICAL MERISTEM (NAM) and
Brassinosteroids (BRs) are a class of plant steroidal hormones (Chung and Choe, 2013). Like mammalian steroid hormones such as estrogen, ecdysone, and progesterone, BRs play key roles in plant development, regulating processes such as cell elongation, vascular system differentiation, senescence, and stress responses (Choe, 2006; Clouse and Sasse, 1998). BRs and other phytohormones have numerous target genes in common, and complex crosstalk mechanisms exist among these hormone signal transduction pathways (Chung et al., 2011; Nemhauser et al., 2006). Brassinolide (BL), the most active form of BRs, binds to an island domain in the extracellular domain of
In contrast to the ABA pathway, the majority of signaling components have been elucidated in the BR signaling cascade (Vriet et al., 2013). Given the importance of ABA and BRs, much research has focused on identifying the mechanism of crosstalk between BRs and ABA. It has been demonstrated that ABA and BRs antagonistically regulate each other during seed germination and root growth inhibition (Steber and McCourt, 2001; Zhang et al., 2009)
In this study, we sought to understand the mechanisms by which BRs and ABA interact. First, we selected putative marker genes that are antagonistically regulated by BRs and ABA from a microarray database deposited in AtGenExpress (Nemhauser et al., 2006). Secondly, we found that chemical inhibition of BIN2 decreased the plant’s tolerance to salt stress, suggesting that BIN2 is involved in ABA-mediated salt tolerance processes. Based on our findings, we propose a model in which transcription factors that bind to common target genes and are specific to either BRs or ABA are antagonistically regulated by each other to bring about ABA-dependent stress responses.
Arabidopsis plants of Columbia ecotype were grown in 0.5×Murashige and Skoog (MS) medium containing 0.5% sucrose and 0.8% plant agar. Plants or seedlings were kept in a growth room at 22°C with a 16 h light/8 h dark cycle. To measure the survival rate in salt media, seeds were germinated and grown in MS agar medium and transferred to medium containing the indicated concentrations of hormone or chemical. After 1 day, seedlings were transferred again to medium containing NaCl.
RNA extraction and cDNA synthesis were conducted according to a previous report (Chung et al., 2012). Quantitative RT-PCR analysis was performed using SYBR-mix (KAPA Biosystems).
Five-day-old
MEME (
We observed that treatment of Arabidopsis seedlings with ABA often resulted in phenotypes that resembled those of BR-deficient dwarf mutants. The small, curled leaves of ABA-treated Col wild-type plants were similar to those of mock-treated
Because ABA treatment mimicked the BR mutant phenotype, we reasoned that
To identify the genes that are oppositely regulated by ABA and BRs, we analyzed a set of publicly available microarray data that were reported by Nemhauser et al. (2006). They reported that 383 genes upregulated by BRs, 268 were downregulated by BRs, 1440 were upregulated by ABA, and 1476 were downregulated by ABA (Nemhauser et al., 2006). Because BRs and ABA have a tendency to reverse the effects on Arabidopsis growth (Fig. 1), we focused on genes that respond oppositely to BRs and ABA. We determined the union (62) of genes downregulated by BRs and upregulated by ABA, and the union (50) upregulated by BRs and downregulated by ABA (Fig. 2A). These genes were chosen for further investigation.
Because BZR1 can function either as an inducer or repressor of BR-responsive genes (He et al., 2005), we determined the number of BR-response elements (BRREs, CGTG[TC][GA], (He et al., 2005)) in the promoter DNA sequences of these genes (Fig. 2B). Whereas 25% of genes in the whole genome contained the BRRE motif, 48% of genes upregulated by ABA and downregulated by BR contained this motif (Fig. 2B). Since the BRRE was not enriched in the group of genes downregulated by ABA and upregulated by BR, we propose that genes that are upregulated by ABA and downregulated by BR are likely targeted by BZR1 through direct binding to BRRE in the promoter sequences.
We next determined the frequency of the BRRE in genes reported to be regulated by other hormones or a biosynthetic precursor of hormone, such as 1-aminocyclopropane-1-carboxylic acid (ACC), indole-3-acetic acid (IAA), methyl jasmonate (MeJA), gibberellins (GA), and cytokinins (CK). Whereas the percentage of genes possessing the BRRE is enriched in the ABA-upregulated group (36.4%) and BR-downregulated group (35.2%) compared to the whole genome control (25%), the percentage was similar for other hormone-response genes (Table 1), except for those upregulated by MeJA, suggesting that BRs negatively regulate JA signaling, as previously noted (Kim et al., 2013).
Of the genes upregulated by ABA and downregulated by BRs, seven encoded
Similar to BR mutants,
Interestingly, previous reports showed that overexpression of other transcription factors involved in the response to abiotic stresses, including
It was reported that BR mutants are hypersensitive to ABA. Moreover, BR-overproducing and constitutive BR signaling mutants tend to be less tolerant to stresses (Chung et al., 2012; Kim et al., 2013), suggesting that BR negatively regulates stress responses. Since
Previously, it was reported that
To quantify the expression of
We have demonstrated that BRs repress the expression of the ABA-responsive genes to ameliorate ABA-dependent stress tolerance. To test which component in the BR signaling pathway is the focal point of ABA and BR crosstalk in the salt stress tolerance mechanism, we first examined the salt stress tolerance of wild-type seedlings treated with the BIN2 inhibitors, Lithium (Klein and Melton, 1996; Stambolic et al., 1996) and bikinin (De Rybel et al., 2009). Lithium inhibits BIN2 activity by competing with Mg2+ (Klein and Melton, 1996; Stambolic et al., 1996). In presence of Lithium (LiCl), the effects of NaCl was greatly enhanced such that seedlings turned yellow and died (Fig. 4A). More quantitatively, the survival rate of seedlings treated with 3 mM LiCl was 42% that of control seedlings treated with 3 mM KCl (Figs. 4A and 4B). We obtained the similar results when repeating this experiment using bikinin. Induction of GUS activity in the
To further understand how the genes are co-regulated by BRs and ABA, we compared the promoter sequences of the gene identified and displayed in Fig. 2. Firstly, we found that the motif sequence conserved among the ABA-upregulated and BR-downregulated genes was clearly an ABRE (motif 1, Fig. 5A). Motif 1 is similar to the BRRE; therefore, it is tempting to suggest that BZR1 and ABF/AREB transcription factors may competitively regulate their target genes by binding to the same response elements.
Secondly, in the case of the BR-up and ABA-downregulated genes, a GAGA motif was enriched (Fig. 5B). Previously, it was reported that BASIC PENTACYSTEINE (BPC) proteins bind to this GAGA motif (Meister et al., 2004; Monfared et al., 2011; Sing et al., 2009). It is likely that BPC may function in the antagonistic regulation of ABA and BR. However, we cannot rule out the possibility that conserved sequences can be bound by currently unidentified transcription factors (Rozhon et al., 2010; Yan et al., 2009).
BR-deficient mutants are known to be hypersensitive to ABA (Steber and McCourt, 2001; Zhang et al., 2009), suggesting that ABA can efficiently induce ABA responses when growth is minimized. Therefore, we identified the genes antagonistically regulated by ABA and BRs, including
Since it is not fully understood how ABA inhibits growth, we studied the mechanisms controlled by ABA and the growth-promoting BRs. Plants grown in medium supplemented with ABA or NaCl displayed a growth retardation phenotype similar to that of BR-deficient mutants (Fig. 1). One explanation for this is that BR downregulates the genes that may not be directly required for stress-tolerance processes. In support of this, the expression of
Salt-treated roots tend to break easily and this effect is severe when BR is added. In contrast, when Pcz is added, roots remain thick and relatively intact compared to those subjected to salt treatment alone (Supplementary Fig. S2). In Arabidopsis, cell wall loosening is temporarily required for BR-mediated cell expansion, and this is catalyzed by
It was interesting to observe that
Seedlings in which BIN2 activity was inhibited by either LiCl or bikinin displayed a decreased survival rate when subjected to a salt stress of 200 mM NaCl (Fig. 4). GUS activity was absent in
We predict that salt stress controls a step below BIN2 in the BR signaling pathway, because NaCl treatment in combination with BIN2 inhibition altered the expression of
In conclusion, we sought to determine why stressed plants display stunted growth. We analyzed the relationship between BRs, which promote growth, and ABA, which is involved in the response to environmental stress. Based on the phenotypic similarities between ABA-treated seedlings and
. Proportion of genes containing the BRRE
ABA | BRs | ACC | IAA | GA | MeJa | CK | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Up or Down | U | D | U | D | U | D | U | D | U | D | U | D | U | D |
No. genes | 1440 | 1476 | 268 | 383 | 167 | 365 | 430 | 355 | 40 | 82 | 806 | 701 | 332 | 163 |
BRRE-containing gene | 524 | 356 | 59 | 135 | 45 | 87 | 123 | 78 | 11 | 26 | 272 | 181 | 81 | 46 |
BRRE/Total | 36.4 | 24.1 | 22.0 | 35.2 | 26.9 | 23.8 | 28.6 | 22.0 | 27.5 | 31.7 | 33.7 | 25.8 | 24.4 | 28.2 |
Mol. Cells 2014; 37(11): 795-803
Published online November 30, 2014 https://doi.org/10.14348/molcells.2014.0127
Copyright © The Korean Society for Molecular and Cellular Biology.
Yuhee Chung1,4, Soon Il Kwon2, and Sunghwa Choe1,2,3,*
1School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea, 2Convergence Research Center for Functional Plant Products, Advanced Institutes of Convergence Technology, Suwon 443-270, Korea, 3Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-921, Korea, 4Present address: Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, USA
Correspondence to:*Correspondence: shchoe@snu.ac.kr
To withstand ever-changing environmental stresses, plants are equipped with phytohormone-mediated stress resistance mechanisms. Salt stress triggers abscisic acid (ABA) signaling, which enhances stress tolerance at the expense of growth. ABA is thought to inhibit the action of growth-promoting hormones, including brassinosteroids (BRs). However, the regulatory mechanisms that coordinate ABA and BR activity remain to be discovered. We noticed that ABA-treated seedlings exhibited small, round leaves and short roots, a phenotype that is characteristic of the BR signaling mutant,
Keywords: ABA, abiotic stress, BIN2, brassinosteroids, RD26, Root
Due to their sessile nature, plants have developed strategies to cope with abiotic challenges and biotic stresses (Chung et al., 2012; Kim et al., 2014; Maharjan and Choe, 2011). Plants exposed to abiotic stresses display severe growth retardation and reduced productivity. Growth is regulated by plant hormones, which modify endogenous programs in response to exogenous signals. However, the hormone-dependent mechanisms by which growth is inhibited under stress conditions are not fully understood. The molecular mechanisms that impart tolerance to water stress can be divided into abscisic acid (ABA)-dependent and ABA-independent pathways (Shinozaki and Yamaguchi-Shinozaki, 2007). ABA plays vital roles in adaptation to environmental changes, seed dormancy, and the regulation of stomatal closure (Grill and Himmelbach, 1998; Lee and Luan, 2012).
Under stressed conditions, plants rapidly produce ABA, which stimulates the resistance mechanism. In the ABA-dependent pathway, ABA binds to soluble receptors of the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCARs) family, which induces the
In addition to ABFs/AREBs, other ABA-induced transcription factors are known to participate in the ABA response and stress tolerance. For instance, the NAC (NAM, ATAF1/2 and CUC2) transcription factor family, which largely consists of NO APICAL MERISTEM (NAM) and
Brassinosteroids (BRs) are a class of plant steroidal hormones (Chung and Choe, 2013). Like mammalian steroid hormones such as estrogen, ecdysone, and progesterone, BRs play key roles in plant development, regulating processes such as cell elongation, vascular system differentiation, senescence, and stress responses (Choe, 2006; Clouse and Sasse, 1998). BRs and other phytohormones have numerous target genes in common, and complex crosstalk mechanisms exist among these hormone signal transduction pathways (Chung et al., 2011; Nemhauser et al., 2006). Brassinolide (BL), the most active form of BRs, binds to an island domain in the extracellular domain of
In contrast to the ABA pathway, the majority of signaling components have been elucidated in the BR signaling cascade (Vriet et al., 2013). Given the importance of ABA and BRs, much research has focused on identifying the mechanism of crosstalk between BRs and ABA. It has been demonstrated that ABA and BRs antagonistically regulate each other during seed germination and root growth inhibition (Steber and McCourt, 2001; Zhang et al., 2009)
In this study, we sought to understand the mechanisms by which BRs and ABA interact. First, we selected putative marker genes that are antagonistically regulated by BRs and ABA from a microarray database deposited in AtGenExpress (Nemhauser et al., 2006). Secondly, we found that chemical inhibition of BIN2 decreased the plant’s tolerance to salt stress, suggesting that BIN2 is involved in ABA-mediated salt tolerance processes. Based on our findings, we propose a model in which transcription factors that bind to common target genes and are specific to either BRs or ABA are antagonistically regulated by each other to bring about ABA-dependent stress responses.
Arabidopsis plants of Columbia ecotype were grown in 0.5×Murashige and Skoog (MS) medium containing 0.5% sucrose and 0.8% plant agar. Plants or seedlings were kept in a growth room at 22°C with a 16 h light/8 h dark cycle. To measure the survival rate in salt media, seeds were germinated and grown in MS agar medium and transferred to medium containing the indicated concentrations of hormone or chemical. After 1 day, seedlings were transferred again to medium containing NaCl.
RNA extraction and cDNA synthesis were conducted according to a previous report (Chung et al., 2012). Quantitative RT-PCR analysis was performed using SYBR-mix (KAPA Biosystems).
Five-day-old
MEME (
We observed that treatment of Arabidopsis seedlings with ABA often resulted in phenotypes that resembled those of BR-deficient dwarf mutants. The small, curled leaves of ABA-treated Col wild-type plants were similar to those of mock-treated
Because ABA treatment mimicked the BR mutant phenotype, we reasoned that
To identify the genes that are oppositely regulated by ABA and BRs, we analyzed a set of publicly available microarray data that were reported by Nemhauser et al. (2006). They reported that 383 genes upregulated by BRs, 268 were downregulated by BRs, 1440 were upregulated by ABA, and 1476 were downregulated by ABA (Nemhauser et al., 2006). Because BRs and ABA have a tendency to reverse the effects on Arabidopsis growth (Fig. 1), we focused on genes that respond oppositely to BRs and ABA. We determined the union (62) of genes downregulated by BRs and upregulated by ABA, and the union (50) upregulated by BRs and downregulated by ABA (Fig. 2A). These genes were chosen for further investigation.
Because BZR1 can function either as an inducer or repressor of BR-responsive genes (He et al., 2005), we determined the number of BR-response elements (BRREs, CGTG[TC][GA], (He et al., 2005)) in the promoter DNA sequences of these genes (Fig. 2B). Whereas 25% of genes in the whole genome contained the BRRE motif, 48% of genes upregulated by ABA and downregulated by BR contained this motif (Fig. 2B). Since the BRRE was not enriched in the group of genes downregulated by ABA and upregulated by BR, we propose that genes that are upregulated by ABA and downregulated by BR are likely targeted by BZR1 through direct binding to BRRE in the promoter sequences.
We next determined the frequency of the BRRE in genes reported to be regulated by other hormones or a biosynthetic precursor of hormone, such as 1-aminocyclopropane-1-carboxylic acid (ACC), indole-3-acetic acid (IAA), methyl jasmonate (MeJA), gibberellins (GA), and cytokinins (CK). Whereas the percentage of genes possessing the BRRE is enriched in the ABA-upregulated group (36.4%) and BR-downregulated group (35.2%) compared to the whole genome control (25%), the percentage was similar for other hormone-response genes (Table 1), except for those upregulated by MeJA, suggesting that BRs negatively regulate JA signaling, as previously noted (Kim et al., 2013).
Of the genes upregulated by ABA and downregulated by BRs, seven encoded
Similar to BR mutants,
Interestingly, previous reports showed that overexpression of other transcription factors involved in the response to abiotic stresses, including
It was reported that BR mutants are hypersensitive to ABA. Moreover, BR-overproducing and constitutive BR signaling mutants tend to be less tolerant to stresses (Chung et al., 2012; Kim et al., 2013), suggesting that BR negatively regulates stress responses. Since
Previously, it was reported that
To quantify the expression of
We have demonstrated that BRs repress the expression of the ABA-responsive genes to ameliorate ABA-dependent stress tolerance. To test which component in the BR signaling pathway is the focal point of ABA and BR crosstalk in the salt stress tolerance mechanism, we first examined the salt stress tolerance of wild-type seedlings treated with the BIN2 inhibitors, Lithium (Klein and Melton, 1996; Stambolic et al., 1996) and bikinin (De Rybel et al., 2009). Lithium inhibits BIN2 activity by competing with Mg2+ (Klein and Melton, 1996; Stambolic et al., 1996). In presence of Lithium (LiCl), the effects of NaCl was greatly enhanced such that seedlings turned yellow and died (Fig. 4A). More quantitatively, the survival rate of seedlings treated with 3 mM LiCl was 42% that of control seedlings treated with 3 mM KCl (Figs. 4A and 4B). We obtained the similar results when repeating this experiment using bikinin. Induction of GUS activity in the
To further understand how the genes are co-regulated by BRs and ABA, we compared the promoter sequences of the gene identified and displayed in Fig. 2. Firstly, we found that the motif sequence conserved among the ABA-upregulated and BR-downregulated genes was clearly an ABRE (motif 1, Fig. 5A). Motif 1 is similar to the BRRE; therefore, it is tempting to suggest that BZR1 and ABF/AREB transcription factors may competitively regulate their target genes by binding to the same response elements.
Secondly, in the case of the BR-up and ABA-downregulated genes, a GAGA motif was enriched (Fig. 5B). Previously, it was reported that BASIC PENTACYSTEINE (BPC) proteins bind to this GAGA motif (Meister et al., 2004; Monfared et al., 2011; Sing et al., 2009). It is likely that BPC may function in the antagonistic regulation of ABA and BR. However, we cannot rule out the possibility that conserved sequences can be bound by currently unidentified transcription factors (Rozhon et al., 2010; Yan et al., 2009).
BR-deficient mutants are known to be hypersensitive to ABA (Steber and McCourt, 2001; Zhang et al., 2009), suggesting that ABA can efficiently induce ABA responses when growth is minimized. Therefore, we identified the genes antagonistically regulated by ABA and BRs, including
Since it is not fully understood how ABA inhibits growth, we studied the mechanisms controlled by ABA and the growth-promoting BRs. Plants grown in medium supplemented with ABA or NaCl displayed a growth retardation phenotype similar to that of BR-deficient mutants (Fig. 1). One explanation for this is that BR downregulates the genes that may not be directly required for stress-tolerance processes. In support of this, the expression of
Salt-treated roots tend to break easily and this effect is severe when BR is added. In contrast, when Pcz is added, roots remain thick and relatively intact compared to those subjected to salt treatment alone (Supplementary Fig. S2). In Arabidopsis, cell wall loosening is temporarily required for BR-mediated cell expansion, and this is catalyzed by
It was interesting to observe that
Seedlings in which BIN2 activity was inhibited by either LiCl or bikinin displayed a decreased survival rate when subjected to a salt stress of 200 mM NaCl (Fig. 4). GUS activity was absent in
We predict that salt stress controls a step below BIN2 in the BR signaling pathway, because NaCl treatment in combination with BIN2 inhibition altered the expression of
In conclusion, we sought to determine why stressed plants display stunted growth. We analyzed the relationship between BRs, which promote growth, and ABA, which is involved in the response to environmental stress. Based on the phenotypic similarities between ABA-treated seedlings and
. Proportion of genes containing the BRRE.
ABA | BRs | ACC | IAA | GA | MeJa | CK | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Up or Down | U | D | U | D | U | D | U | D | U | D | U | D | U | D |
No. genes | 1440 | 1476 | 268 | 383 | 167 | 365 | 430 | 355 | 40 | 82 | 806 | 701 | 332 | 163 |
BRRE-containing gene | 524 | 356 | 59 | 135 | 45 | 87 | 123 | 78 | 11 | 26 | 272 | 181 | 81 | 46 |
BRRE/Total | 36.4 | 24.1 | 22.0 | 35.2 | 26.9 | 23.8 | 28.6 | 22.0 | 27.5 | 31.7 | 33.7 | 25.8 | 24.4 | 28.2 |
Dongwon Baek, Hyun Jin Chun, Songhwa Kang, Gilok Shin, Su Jung Park, Hyewon Hong, Chanmin Kim, Doh Hoon Kim, Sang Yeol Lee, Min Chul Kim, and Dae-Jin Yun
Mol. Cells 2016; 39(2): 111-118 https://doi.org/10.14348/molcells.2016.2188Chian Kwon, Jae-Hoon Lee, and Hye Sup Yun
Mol. Cells 2020; 43(6): 501-508 https://doi.org/10.14348/molcells.2020.0007Seo-wha Choi, Seul-bee Lee, Yeon-ju Na, Sun-geum Jeung, and Soo Young Kim
Mol. Cells 2017; 40(3): 230-242 https://doi.org/10.14348/molcells.2017.0002