Table_2.PDF

<p>Leaf senescence is an integral part of plant development, and the timing and progressing rate of senescence could substantially affect the yield and quality of crops. It has been known that a circadian rhythm synchronized with external environmental cues is critical for the optimal coordination of various physiological and metabolic processes. However, the reciprocal interactions between the circadian clock and leaf senescence in plants remain unknown. Here, through measuring the physiological and molecular senescence related markers of several circadian components mutants, we found that CIRCADIAN CLOCK-ASSOCIATED 1 inhibits leaf senescence. Further molecular and genetic studies revealed that CCA1 directly activates GLK2 and suppresses ORE1 expression to counteract leaf senescence. As plants age, the expression and periodic amplitude of CCA1 declines and thus weakens the inhibition of senescence. Our findings reveal an age-dependent circadian clock component of the process of leaf senescence.</p

Image_4.TIF

<p>Leaf senescence is an integral part of plant development, and the timing and progressing rate of senescence could substantially affect the yield and quality of crops. It has been known that a circadian rhythm synchronized with external environmental cues is critical for the optimal coordination of various physiological and metabolic processes. However, the reciprocal interactions between the circadian clock and leaf senescence in plants remain unknown. Here, through measuring the physiological and molecular senescence related markers of several circadian components mutants, we found that CIRCADIAN CLOCK-ASSOCIATED 1 inhibits leaf senescence. Further molecular and genetic studies revealed that CCA1 directly activates GLK2 and suppresses ORE1 expression to counteract leaf senescence. As plants age, the expression and periodic amplitude of CCA1 declines and thus weakens the inhibition of senescence. Our findings reveal an age-dependent circadian clock component of the process of leaf senescence.</p

Image_2.TIF

<p>Leaf senescence is an integral part of plant development, and the timing and progressing rate of senescence could substantially affect the yield and quality of crops. It has been known that a circadian rhythm synchronized with external environmental cues is critical for the optimal coordination of various physiological and metabolic processes. However, the reciprocal interactions between the circadian clock and leaf senescence in plants remain unknown. Here, through measuring the physiological and molecular senescence related markers of several circadian components mutants, we found that CIRCADIAN CLOCK-ASSOCIATED 1 inhibits leaf senescence. Further molecular and genetic studies revealed that CCA1 directly activates GLK2 and suppresses ORE1 expression to counteract leaf senescence. As plants age, the expression and periodic amplitude of CCA1 declines and thus weakens the inhibition of senescence. Our findings reveal an age-dependent circadian clock component of the process of leaf senescence.</p

Image_1.TIF

<p>Leaf senescence is an integral part of plant development, and the timing and progressing rate of senescence could substantially affect the yield and quality of crops. It has been known that a circadian rhythm synchronized with external environmental cues is critical for the optimal coordination of various physiological and metabolic processes. However, the reciprocal interactions between the circadian clock and leaf senescence in plants remain unknown. Here, through measuring the physiological and molecular senescence related markers of several circadian components mutants, we found that CIRCADIAN CLOCK-ASSOCIATED 1 inhibits leaf senescence. Further molecular and genetic studies revealed that CCA1 directly activates GLK2 and suppresses ORE1 expression to counteract leaf senescence. As plants age, the expression and periodic amplitude of CCA1 declines and thus weakens the inhibition of senescence. Our findings reveal an age-dependent circadian clock component of the process of leaf senescence.</p

Image_5.TIF

<p>Leaf senescence is an integral part of plant development, and the timing and progressing rate of senescence could substantially affect the yield and quality of crops. It has been known that a circadian rhythm synchronized with external environmental cues is critical for the optimal coordination of various physiological and metabolic processes. However, the reciprocal interactions between the circadian clock and leaf senescence in plants remain unknown. Here, through measuring the physiological and molecular senescence related markers of several circadian components mutants, we found that CIRCADIAN CLOCK-ASSOCIATED 1 inhibits leaf senescence. Further molecular and genetic studies revealed that CCA1 directly activates GLK2 and suppresses ORE1 expression to counteract leaf senescence. As plants age, the expression and periodic amplitude of CCA1 declines and thus weakens the inhibition of senescence. Our findings reveal an age-dependent circadian clock component of the process of leaf senescence.</p

Table_1.XLSX

<p>Leaf senescence is an integral part of plant development, and the timing and progressing rate of senescence could substantially affect the yield and quality of crops. It has been known that a circadian rhythm synchronized with external environmental cues is critical for the optimal coordination of various physiological and metabolic processes. However, the reciprocal interactions between the circadian clock and leaf senescence in plants remain unknown. Here, through measuring the physiological and molecular senescence related markers of several circadian components mutants, we found that CIRCADIAN CLOCK-ASSOCIATED 1 inhibits leaf senescence. Further molecular and genetic studies revealed that CCA1 directly activates GLK2 and suppresses ORE1 expression to counteract leaf senescence. As plants age, the expression and periodic amplitude of CCA1 declines and thus weakens the inhibition of senescence. Our findings reveal an age-dependent circadian clock component of the process of leaf senescence.</p

Image_6.TIF

<p>Leaf senescence is an integral part of plant development, and the timing and progressing rate of senescence could substantially affect the yield and quality of crops. It has been known that a circadian rhythm synchronized with external environmental cues is critical for the optimal coordination of various physiological and metabolic processes. However, the reciprocal interactions between the circadian clock and leaf senescence in plants remain unknown. Here, through measuring the physiological and molecular senescence related markers of several circadian components mutants, we found that CIRCADIAN CLOCK-ASSOCIATED 1 inhibits leaf senescence. Further molecular and genetic studies revealed that CCA1 directly activates GLK2 and suppresses ORE1 expression to counteract leaf senescence. As plants age, the expression and periodic amplitude of CCA1 declines and thus weakens the inhibition of senescence. Our findings reveal an age-dependent circadian clock component of the process of leaf senescence.</p

Image_3.TIF

<p>Leaf senescence is an integral part of plant development, and the timing and progressing rate of senescence could substantially affect the yield and quality of crops. It has been known that a circadian rhythm synchronized with external environmental cues is critical for the optimal coordination of various physiological and metabolic processes. However, the reciprocal interactions between the circadian clock and leaf senescence in plants remain unknown. Here, through measuring the physiological and molecular senescence related markers of several circadian components mutants, we found that CIRCADIAN CLOCK-ASSOCIATED 1 inhibits leaf senescence. Further molecular and genetic studies revealed that CCA1 directly activates GLK2 and suppresses ORE1 expression to counteract leaf senescence. As plants age, the expression and periodic amplitude of CCA1 declines and thus weakens the inhibition of senescence. Our findings reveal an age-dependent circadian clock component of the process of leaf senescence.</p

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

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