1. Arabidopsis WRKY45 Interacts with the DELLA Protein RGL1 to Positively Regulate Age-Triggered Leaf Senescence 
Ligang Chen, Shengyuan Xiang, Yanli Chen, Daibo Li, Diqiu Yu 
Molecular Plant 
Volume 10, Issue 9, Pages (September 2017)
DOI: /j.molp
Copyright © 2017 The Author Terms and Conditions

2. Figure 1 Loss of WRKY45 Function Delays Age-Triggered Leaf Senescence.
(A) Real-time qPCR analysis of WRKY45 transcript levels in wild-type leaves at different developmental stages. RNA was isolated from leaves as indicated in Supplemental Figure 2A from more than 20 plants. Transcript levels of WRKY45 in non-senescent (NS) leaves were arbitrarily set to 1. ACTIN2 was used as an internal control. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with NS leaves.
(B) Real-time qPCR analysis of WRKY45 transcript levels in different parts of a senescing wild-type leaf. RNA was isolated from leaves as indicated in Supplemental Figure 2B from more than 20 plants. Transcript levels of WRKY45 in NS leaves were arbitrarily set to 1. ACTIN2 was used as an internal control. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with NS leaves.
(C) The senescence phenotypes of 7-week-old Col-0 and wrky45 mutant plants. Plants were grown under long days (16 h light/8 h dark) at 22°C. Three independent experiments were performed with each replica containing more than 30 plants for each line. Representative plants were photographed.
(D) Relative chlorophyll content of Col-0 and wrky45 mutant plants at the indicated leaf age. Leaves three to four from 12 to 15 plants of each genotype (approximately 26 leaves) were harvested and pooled. Values are means ± SD of three independent biological replicates. *P < 0.05, **P < 0.01, Student's t-test compared with Col-0. Asterisks indicate the differences between WRKY45 RNAi-9 or wrky45 and wild-type at the same time.
(E) Membrane ion leakage of Col-0 and wrky45 mutant plants at the indicated leaf age. Leaves three to four from 12 to 15 plants of each genotype (approximately 26 leaves) were harvested and pooled. Values are means ± SD of three independent biological replicates. *P < 0.05, **P < 0.01, Student's t-test compared with Col-0. Asterisks indicate the differences between WRKY45 RNAi-9 or wrky45 and wild-type.
(F–L) Real-time qPCR analysis of transcript levels of senescence marker genes in age-triggered senescing leaves of Col-0 and wrky45 mutant plants. RNA was isolated from leaves three to four of each genotype in (C). Transcript levels of senescence marker genes in Col-0 plants were arbitrarily set to 1. ACTIN2 was used as an internal control. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with Col-0.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

3. Figure 2
Overexpression of WRKY45 Accelerates Age-Triggered Leaf Senescence.
(A) The senescence phenotypes of 6-week-old Col-0 and WRKY45-overexpressing plants. Plants were grown under long days (16 h light/8 h dark) at 22°C. Three independent experiments were performed with each replica containing more than 30 plants for each line. Representative plants were photographed.
(B) Relative chlorophyll content of Col-0 and WRKY45-overexpressing plants at the indicated leaf age. Leaves three to four from 12 to 15 plants of each genotype (approximately 26 leaves) were harvested and pooled. Values are means ± SD of three independent biological replicates. *P < 0.05, **P < 0.01, Student's t-test compared with Col-0.
(C) Membrane ion leakage of Col-0- and WRKY45-overexpressing plants at the indicated leaf age. Leaves three to four from 12 to 15 plants of each genotype (approximately 26 leaves) were harvested and pooled. Values are means ± SD of three independent biological replicates. *P < 0.05, **P < 0.01, Student's t-test compared with Col-0.
(D–J) Real-time qPCR analysis of transcript levels of senescence marker genes in age-triggered senescing leaves of Col-0- and WRKY45-overexpressing plants. RNA was isolated from leaves three to four of each genotype in (A). Transcript levels of the senescence marker genes in Col-0 plants were arbitrarily set to 1. ACTIN2 was used as an internal control. Values are means ± SD of three independent biological replicates. *P < 0.05, **P < 0.01, Student's t-test compared with Col-0.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

4. Figure 3 SAG12, SAG13, SAG113, and SEN4 Are Direct Targets of WRKY45.
(A) Upper panel shows schematic representation of SAG12, SAG13, SAG113, and SEN4 promoter regions containing W box clusters. Only perfect W boxes (T/CTGACC/T, black bar) are depicted. The number and relative position of W boxes in respective promoters relative to the ATG start codon are indicated. The prefix “p” indicates the promoter. Pink lines indicate the sequences detected by ChIP assays. ChIP assays were performed with chromatin prepared from HA-WRKY45 plants, using an anti-HA antibody (IP) or preimmune serum (negative control, no Ab). ChIP results are presented as a percentage of input DNA. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with controls.
(B) Schematic of the SAG12pro:NLS-GFP reporter and WRKY45 and GUS effectors.
(C) Transient expression assays showed that WRKY45 activates the expression of SAG12. GFP fluorescence was detected 48 h after co-infiltration with the indicated constructs. The experiment was repeated three times with similar results and representative photos are shown.
(D) Real-time qPCR analysis of the accumulation of GFP transcripts. Total RNA was extracted from the leaves of N. benthamiana co-infiltrated with combinations of various constructs in (B). N. benthamiana ACTIN was used as an internal control. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with controls.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

5. Figure 4 Physical Interaction between RGL1 and WRKY45.
(A) Yeast two-hybrid assay. Protein-protein interaction was indicated by the ability of cells to grow on synthetic dropout medium lacking Leu, Trp, His, and Ade. The GAL4 activation domain expressed by pGADT7 (shown as AD) was used as a negative control. The experiment was repeated three times with similar results and representative images are displayed.
(B) Co-IP analysis. Myc-fused RGL1 was immunoprecipitated using an anti-Myc antibody, and co-immunoprecipitated HA-WRKY45 was then detected using an anti-HA antibody. Protein input for Myc-RGL1 in immunoprecipitated complexes was also detected. The experiment was repeated three times with similar results and representative images are displayed.
(C) BiFC analysis. Fluorescence was observed in the nuclear compartments of N. benthamiana leaf epidermal cells; the fluorescence resulted from complementation of the C-terminal portion of YFP fused to RGL1 (RGL1-cYFP) with the N-terminal portion of YFP fused to WRKY45 (WRKY45-nYFP). No signal was observed from negative controls. DAPI, 4′,6-diamidino-2-phenylindole. The experiment was repeated three times with similar results and representative images are displayed.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

6. Figure 5 Overexpression of RGL1 Delays Age-Triggered Leaf Senescence.
(A) The senescence phenotypes of 7-week-old Col-0 and RGL1-overexpressing plants. Plants were grown under long days (16 h light/8 h dark) at 22°C. Three independent experiments were performed with each replica containing more than 30 plants for each line. Representative plants were photographed.
(B) Relative chlorophyll content of Col-0 and RGL1-overexpressing plants at the indicated leaf age. Leaves three to four from 12 to 15 plants of each genotype (approximately 26 leaves) were harvested and pooled. Values are means ± SD of three independent biological replicates. *P < 0.05, **P < 0.01, Student's t-test compared with Col-0.
(C) Membrane ion leakage of Col-0- and RGL1-overexpressing plants at the indicated leaf age. Leaves three to four from 12 to 15 plants of each genotype (approximately 26 leaves) were harvested and pooled. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with Col-0.
(D–J) Real-time qPCR analysis of transcript levels of senescence marker genes in age-triggered senescing leaves of Col-0- and RGL1-overexpressing plants. RNA was isolated from leaves three to four of each genotype in (A). Transcript levels of the senescence marker genes in Col-0 plants were arbitrarily set to 1. ACTIN2 was used as an internal control. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with Col-0.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

7. Figure 6 RGL1 Represses Transcriptional Activation Function of WRKY45.
(A) Schematic of the SAG12pro:NLS-GFP reporter and WRKY45, RGL1, and GUS effectors.
(B) Transient expression assays showed that RGL1 represses the transcriptional activation of WRKY45. GFP fluorescence was detected 48 h after co-infiltration with the indicated constructs. The experiment was repeated three times with similar results and representative images are displayed.
(C) Real-time qPCR analysis of the accumulation of GFP transcripts. Total RNA was extracted from the leaves of N. benthamiana co-infiltrated with combinations of various constructs in (A). N. benthamiana ACTIN was used as an internal control. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with controls.
(D) RGL1 interferes with the binding of WRKY45 to its target genes (shown in Figure 4A). 35Spro:HA-WRKY45 was crossed with 35Spro:RGL1-GFP to obtain HA-WRKY45/RGL1-GFP plants. ChIP assays were performed with chromatin prepared from GA3 or PAC pre-treated HA-WRKY45/RGL1-GFP plants, using an anti-HA antibody (IP) or preimmune serum (negative control, no Ab). ChIP results are presented as a percentage of input DNA. Values are means ± SD of three independent biological replicates. **P < 0.01, *P < 0.05, Student's t-test compared with controls.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

8. Figure 7
Overexpression of RGL1 Partially Rescues the Early Senescence Phenotype of WRKY45-Overexpressing Plants.
(A) The senescence phenotypes of 6-week-old Col-0 and 35S:WRKY45-15, 35S:WRKY45-15/35S:RGL1, and 35S:RGL1 plants. Plants were grown under long days (16 h light/8 h dark) at 22°C. Three independent experiments were performed with each replica containing more than 30 plants for each line. Representative plants were photographed.
(B) Relative chlorophyll content of Col-0 and 35S:WRKY45-15, 35S:WRKY45-15/35S:RGL1, and 35S:RGL1 plants at the indicated leaf age. Leaves three to four from 12 to 15 plants of each genotype (approximately 26 leaves) were harvested and pooled. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with Col-0.
(C–I) Real-time qPCR analysis of transcript levels of senescence marker genes in age-triggered senescing leaves of Col-0 and 35S:WRKY45-15, 35S:WRKY45-15/35S:RGL1, and 35S:RGL1 plants. RNA was isolated from leaves three to four of each genotype in (A). Transcript levels of the senescence marker genes in Col-0 plants were arbitrarily set to 1. ACTIN2 was used as an internal control. Values are means ± SD of three independent biological replicates. **P < 0.01, *P < 0.05, Student's t-test compared with Col-0.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

9. Figure 8
Altered Response of wrky45 and WRKY45-Overexpressing Plants in GA-Mediated Leaf Senescence.
(A) Real-time qPCR analysis of WRKY45 transcript levels following GA treatment. RNA was isolated from leaves treated with GA3 or Mock. Transcript levels of WRKY45 in untreated Col-0 leaves were arbitrarily set to 1. ACTIN2 was used as an internal control. Values are means ± SD of three independent biological replicates. *P < 0.05, **P < 0.01, Student's t-test compared with mock.
(B) Real-time qPCR analysis of WRKY45 transcript levels in ga1, gid1agid1bgid1c, and della mutant plants. RNA was isolated from leaves three to four of the indicated genotype (6 weeks old). For ga1 and gid1agid1bgid1c mutants, transcript levels of WRKY45 in Col-0 were arbitrarily set to 1; for della mutants, transcript levels of WRKY45 in Ler plants were arbitrarily set to 1. ACTIN2 was used as an internal control. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with Col-0 or Ler.
(C). GA response of wild-type, WRKY45 mutants, and overexpressing plants. Plants were grown under long days (16 h light/8 h dark) at 22°C, and 8-day-old plants were sprayed with 500 μM GA3 or mock (ethanol 1%, Tween 0.1%) every other day. Three independent experiments were performed with each replica containing more than 30 plants for each line. The representative leaf six of each genotype was photographed.
(D) Relative chlorophyll content of 28-day-old wild-type, WRKY45 mutants, and overexpressing plants as treated in (C). Leaves three to four from 12 to 15 plants of each genotype (approximately 26 leaves) were harvested and pooled. Values are means ± SD of three independent biological replicates. *P < 0.05, Student's t-test compared with Col-0.
(E) Relative expression of SAG12 in the leaves of 28-day-old wild-type, WRKY45 mutants, and overexpressing plants as treated in (C). RNA was isolated from leaves three to four of the indicated genotype. Transcript levels of SAG12 in Col-0 plants were arbitrarily set to 1. ACTIN2 was used as an internal control. Values are means ± SD of three independent biological replicates. **P < 0.01, Student's t-test compared with Col-0.
(F) A working model illustrating WRKY45 functions in GA and age-triggered leaf senescence. WRKY45 is a senescence-associated gene that is upregulated during leaf aging and induced by GA. The GA pathway generally promotes leaf senescence in Arabidopsis. The DELLA protein RGL1 interacts with WRKY45 to inhibit its transcriptional activation function. WRKY45 directly binds the promoters of SAGs such as SAG12, SAG13, SAG113, and SEN4 to activate their expression during leaf senescence.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions