6 research outputs found
EIN3 and ORE1 Accelerate Degreening during Ethylene-Mediated Leaf Senescence by Directly Activating Chlorophyll Catabolic Genes in <i>Arabidopsis</i>
<div><p>Degreening, caused by chlorophyll degradation, is the most obvious symptom of senescing leaves. Chlorophyll degradation can be triggered by endogenous and environmental cues, and ethylene is one of the major inducers. ETHYLENE INSENSITIVE3 (EIN3) is a key transcription factor in the ethylene signaling pathway. It was previously reported that EIN3, <i>miR164</i>, and a NAC (NAM, ATAF, and CUC) transcription factor ORE1/NAC2 constitute a regulatory network mediating leaf senescence. However, how this network regulates chlorophyll degradation at molecular level is not yet elucidated. Here we report a feed-forward regulation of chlorophyll degradation that involves <i>EIN3</i>, <i>ORE1</i>, and chlorophyll catabolic genes (<i>CCGs</i>). Gene expression analysis showed that the induction of three major <i>CCGs</i>, <i>NYE1</i>, <i>NYC1</i> and <i>PAO</i>, by ethylene was largely repressed in <i>ein3 eil1</i> double mutant. Dual-luciferase assay revealed that EIN3 significantly enhanced the promoter activity of <i>NYE1</i>, <i>NYC1</i> and <i>PAO</i> in <i>Arabidopsis</i> protoplasts. Furthermore, Electrophoretic mobility shift assay (EMSA) indicated that EIN3 could directly bind to <i>NYE1</i>, <i>NYC1</i> and <i>PAO</i> promoters. These results reveal that EIN3 functions as a positive regulator of <i>CCG</i> expression during ethylene-mediated chlorophyll degradation. Interestingly, ORE1, a senescence regulator which is a downstream target of EIN3, could also activate the expression of <i>NYE1</i>, <i>NYC1</i> and <i>PAO</i> by directly binding to their promoters in EMSA and chromatin immunoprecipitation (ChIP) assays. In addition, EIN3 and ORE1 promoted <i>NYE1</i> and <i>NYC1</i> transcriptions in an additive manner. These results suggest that ORE1 is also involved in the direct regulation of <i>CCG</i> transcription. Moreover, ORE1 activated the expression of <i>ACS2</i>, a major ethylene biosynthesis gene, and subsequently promoted ethylene production. Collectively, our work reveals that EIN3, ORE1 and CCGs constitute a coherent feed-forward loop involving in the robust regulation of ethylene-mediated chlorophyll degradation during leaf senescence in <i>Arabidopsis</i>.</p></div
EIN3 directly associates with and transactivates the promoters of <i>NYE1</i>, <i>NYC1</i>, and <i>PAO</i>.
<p>(A) Left panel: Schematic diagrams of EIN3 binding site (EBS) in the promoter or 5’-UTR regions of <i>NYE1</i>, <i>NYC1</i>, and <i>PAO</i>. Right panel: EIN3 physically interacts with the promoters of <i>NYE1</i>, <i>NYC1</i>, and <i>PAO</i> in EMSA. About 30-bp DNA fragments containing the EBS in the promoters or 5’-UTR of <i>NYE1</i>, <i>NYC1</i>, and <i>PAO</i> were used as probes for EMSA. Mutated version of competitor DNA (m) was added in 800-fold molar excess. “‒” and “+” represent absence or presence, respectively. Triangle indicates the DNA-protein complex. (B) Kinetic analysis of <i>NYE1</i>, <i>NYC1</i>, and <i>PAO</i> expression in leaves of WT and <i>ein3 eil1</i> in response to ethylene. Experiments were performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g001" target="_blank">Fig 1A</a>. The expression of each corresponding gene in the WT at 0 hr was set to 1. Data are mean ± SEM of 3 biological replicates with technical duplicates for each. (C) Left panel: Schematic diagrams of effector and reporter constructs used in the transient dual-luciferase assays. CaMV 35S promoter driving <i>EIN3</i> (<i>35S</i>:<i>EIN3</i>) was used as effector, and the empty vector was used as a control. The dual-luciferase reporter constructs consist of <i>35S</i> driving <i>Renilla</i> luciferase (REN) reporter gene for internal normalization, and the promoters of <i>NYE1</i> (2012 bp), <i>NYC1</i> (493 bp), <i>PAO</i> (365 bp) driving firefly luciferase (LUC) reporter gene. Right panel: transient dual-luciferase assay of EIN3 transactivating the promoters of <i>NYE1</i>, <i>NYC1</i>, and <i>PAO</i> in <i>Arabidopsis</i> protoplasts. The procedure was as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g001" target="_blank">Fig 1B</a>. Data are mean ± SEM of at least 3 biological replicates. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 (<i>t</i>-test).</p
ORE1 directly activates the expression of <i>NYE1</i>, <i>NYC1</i>, <i>NOL</i> and <i>PAO</i>.
<p>(A) Relative expression of <i>CCG</i>s in leaves of WT and <i>ein3 eil1</i>with ethylene treatment for 24 hr. Gene expression was relative to that in the WT at 0 hr. Data are mean ± SEM of 3 biological replicates with technical duplicates for each. * <i>p</i> < 0.05 (<i>t</i>-test). (B) Schematic diagrams of ORE1 binding site (OBS) in the promoter or 5’-UTR regions of <i>NYE1</i>, <i>NYC1</i>, <i>NOL</i> and <i>PAO</i>. (C) ORE1 physically interacts with the promoters of <i>NYE1</i>, <i>NYC1</i>, <i>NOL</i>, and <i>PAO</i> in EMSA. About 30-bp DNA fragments containing the OBS in the promoter or 5’-UTR regions of <i>NYE1</i>, <i>NYC1</i>, <i>NOL</i>, and <i>PAO</i> were used as probes for EMSA, with purified MBP or MBP-ORE1 protein expressed in <i>E</i>. <i>coli</i>. “‒” and “+” represent in absence or presence, respectively. “m” represents mutated competitor. Triangle indicates the DNA-protein complex. (D) ORE1 associated with the promoters of <i>NYE1</i>, <i>NYC1</i>, <i>NOL</i>, and <i>PAO</i> in ChIP-qPCR assay. Chromatins isolated from <i>35S</i>:<i>ORE1-GFP</i> transgenic line and WT control were immunoprecipitated with anti-GFP antibody followed by qPCR to amplify regions covering the putative ORE1 binding sites. Input sample was used to normalize the qPCR results in each ChIP sample. <i>BFN1</i>, reported as a direct target of ORE1, was used as a positive control. A retrotransposon (At4g03770) located within the heterochromatic region associated with di-methylated H3-K9 was used as a negative control. Fold enrichment was presented as a ratio of normalized results from <i>35S</i>:<i>ORE1-GFP</i> plants and WT. Data are mean ± SEM of at least 3 technical replicates. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 (<i>t</i>-test). The experiment was repeated twice with similar results. (E) Left panel: Schematic diagrams of effector and reporter constructs used in the transient dual-luciferase assays. CaMV 35S promoter driving <i>ORE1</i> (<i>35S</i>:<i>ORE1</i>) was used as effector, and empty vector as a negative control. A 309-bp fragment upstream from ATG of <i>NOL</i> was used to make the <i>pNOL</i>:<i>LUC</i> reporter construct and all other reporters were as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g002" target="_blank">Fig 2C</a>. Right panel: Transient dual-luciferase assay of ORE1 transactivates the promoters of <i>NYE1</i>, <i>NYC1</i>, <i>NOL</i>, and <i>PAO</i> in <i>Arabidopsis</i> protoplasts. The procedure was as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g002" target="_blank">Fig 2C</a>. Data are mean ± SEM of at least 3 biological replicates. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 (<i>t</i>-test). (F) EIN3 and ORE1 transactivate the promoters of <i>NYE1</i> and <i>NYC1</i> in <i>Arabidopsis</i> protoplasts in an additive manner. The transient expression procedure, and the constructs used for the assay were as in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g002" target="_blank">2C</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g003" target="_blank">3E</a>. The amount of each effector was half that used in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g002" target="_blank">2C</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g003" target="_blank">3E</a>. The same amount of corresponding empty vector was used if one effector was absent in a transformation so that the total amount of plasmids was the same among all assays. Data are mean ± SEM of at least 3 biological replicates. * <i>p</i> < 0.05 (<i>t</i>-test).</p
<i>ORE1</i> is directly activated by EIN3 and repressed by <i>miR164</i>.
<p>(A) Kinetic analysis of <i>ORE1</i> expression in leaves of the wild-type (WT) and <i>ein3 eil1</i> in response to ethylene treatment. Detached third and fourth rosette leaves from 4-week-old plants were treated with 100 μL/L ethylene for various periods of times. RT-qPCR was performed to quantify the <i>ORE1</i> mRNA levels. <i>ACT2</i> was used as an internal control to normalize different samples. The mRNA level of <i>ORE1</i> in the WT at 0 hr was arbitrarily set to 1. The x axis is shown in log2 scale. Data are mean ± SEM of 3 biological replicates with technical duplicates for each. (B) Transient dual-luciferase transactivation of the <i>ORE1</i> promoter by EIN3 in protoplasts from <i>Arabidopsis</i> leaves. Protoplasts were co-transformed with the <i>pORE1</i>:<i>Luc</i> reporter (1694 bp upstream from the translation start site of <i>ORE1</i>) and an effector overexpressing EIN3 (<i>35S</i>:<i>EIN3</i>). The <i>35S</i>:<i>REN</i> was serving as an internal control. Relative reporter activity was normalized by the ratio of LUC/REN. An empty vector was used as a negative effector control, with LUC/REN ratio arbitrarily set to 1. Data are mean ± SEM of 3 biological replicates. *** <i>p</i> < 0.001 (<i>t</i>-test). (C) Schematic diagram of putative EIN3 binding site (EBS) in the <i>ORE1</i> promoter. A 28-bp DNA fragment containing the EBS in <i>ORE1</i> promoter was used as the probe for EMSA. The putative EBS in the WT probe sequence is boxed. The consensus nucleotides of EBS in the competitor sequence (underlined) were mutated. (D) EIN3 proteins physically interact with <i>ORE1</i> promoter in EMSA. The N-terminus of EIN3 protein (aa 1–314 containing DNA binding domain) fused to maltose binding protein (MBP) was used to detect interaction (MBP-N-EIN3). MBP protein was used as a negative control. Biotin-labeled probes were added to each reaction mixture. WT competitor DNA was added in 80-fold and 800-fold molar excess. Mutated version of competitor DNA (m) was added in 800-fold molar excess. “‒” and “+” represent absence or presence, respectively. Triangle indicates the DNA-protein complex. (E) Representative leaves of WT, <i>ORE1ox</i>, <i>ein3</i>, and <i>ORE1ox ein3</i> plants subjected to ethylene treatment for 3 days. (F) qRT-PCR analysis of relative gene expression of <i>ORE1</i> and <i>miR164A</i> in leaves in (E). <i>ACT2</i> was used as reference gene. Expression of each gene in the WT was set to 1. Data are mean ± SEM of 2 biological replicates. ns: not significant.</p
A working model of the EIN3-ORE1-CCGs coherent feed-forward loop in regulation of ethylene-mediated chl degradation.
<p>According to our study and previous reports [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.ref027" target="_blank">27</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.ref031" target="_blank">31</a>], we propose a coherent feed-forward loop that involves EIN3 and ORE1 in regulating ethylene-mediated chl degradation. EIN3 directly represses the transcription of <i>miR164</i>, which negatively regulates <i>ORE1</i> at the post-transcriptional level. Meanwhile, EIN3 can directly bind to the <i>ORE1</i> promoter and induce <i>ORE1</i> transcription. Three <i>CCGs</i>, <i>NYE1</i>, <i>NYC1</i>, and <i>PAO</i>, are the direct targets of EIN3. As a transcription factor downstream of EIN3, ORE1 shares these 3 common direct targets with EIN3. However, ORE1 also has its own distinct target, <i>NOL</i>, during the regulation of chl degradation. The broad range of expression of <i>CCGs</i> leads to chl degradation, the early step of leaf senescence. In addition, ORE1 directly activates the expression of <i>ACS2</i>, which presumably triggers a positive feedback regulation of ethylene synthesis. Arrows and bars represent positive and negative regulations, respectively.</p
ORE1 is associated with <i>ACS2</i> promoter and transcriptionally activates its expression.
<p>(A) EMSA detection of binding of ORE1 to <i>ACS2</i> promoter <i>in vitro</i>. A 45-bp <i>ACS2</i> promoter fragment containing the putative ORE1 binding site was biotin-labeled and used as a probe. Purified MBP-ORE1 protein expressed in <i>E</i>. <i>coli</i> was used in EMSA. MBP was included as a negative control. “‒” and “+” represent absence or presence, respectively. “m” represents mutated competitor. Triangle indicates the DNA-protein complex. (B) ChIP-qPCR analysis of ORE1 binding to <i>ACS2</i> promoter <i>in vivo</i>. The <i>ACS2</i> promoter region containing a putative ORE1 binding site was amplified to detect the enrichment. The ChIP procedure and qPCR data processing were as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g003" target="_blank">Fig 3D</a>. Data are mean ± SEM of at least 3 technical replicates. ** <i>p</i> < 0.01 (<i>t</i>-test). The experiment was repeated twice with similar results. (C) Kinetic analysis of <i>ACS2</i> expression in WT and <i>nac2-1</i> with ethylene treatment for various periods of time (0, 1, 3, 24, 48, 72 hr). Expression in WT at 0 hr was set to 1. Data are mean ± SEM of 3 biological replicates with technical duplicates for each. (D) Transient dual-luciferase assay of transactivation of the <i>ACS2</i> promoter by ORE1 in <i>Arabidopsis</i> protoplasts. A 1019-bp <i>ACS2</i> promoter fragment covering the putative ORE1 binding site was used for making the <i>pACS2</i>:<i>LUC</i> reporter construct. The <i>35S</i>:<i>ORE1</i> effector construct was described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005399#pgen.1005399.g003" target="_blank">Fig 3E</a>. Data are mean ± SEM of 3 biological replicates. * <i>p</i> < 0.05 (<i>t</i>-test). (E) Ethylene production in the leaves of WT and <i>nac2-1</i> during senescence. Data are mean ± SEM (n = 9). ** <i>p</i> < 0.01 (<i>t</i>-test). The experiment was repeated twice with similar results.</p