Phosphorylation of WHIRLY1 by CIPK14 Shifts Its Localization and Dual Functions in Arabidopsis  Yujun Ren, Yanyun Li, Youqiao Jiang, Binghua Wu, Ying Miao  Molecular Plant  Volume 10, Issue 5, Pages 749-763 (May 2017) DOI: 10.1016/j.molp.2017.03.011 Copyright © 2017 The Author Terms and Conditions

Figure 1 WHY1 Protein Immunodetection in Different Compartments of Arabidopsis Cells from the Pwhy1-WHY1-HA Line and at Different Development Stages. (A) WHY1 protein isolated from plastid and the nucleus fractions of 6-week-old rosette leaves. (B) Nuclear WHY1 and nuclear WHY1 treated with phosphatase from 6-week-old rosette leaves. Asterisk indicates the phosphorylated form of WHY1. (C) 12 μg of plastid WHY1 and nuclear WHY1 or nuclear WHY1 treated with phosphatase isolated from 3- to 8-week-old Arabidopsis rosette leaves. An antibody directed against the HA tag was used for detection of the WHY1 fusion proteins, The purity of the subcellular protein fractions was shown by immunological detection of the plastid protein cytochrome b559 (α-Cytb559) and the nuclear protein histone H2B (α-Histone H2B). Asterisk indicates the phosphorylated form of WHY1. Molecular Plant 2017 10, 749-763DOI: (10.1016/j.molp.2017.03.011) Copyright © 2017 The Author Terms and Conditions

Figure 2 Localization of CIPK14 Protein and Interaction of CIPK14 and WHY1 by Bimolecular Fluorescence Complementation Assay and CoIP. (A) Onion epidermal cells or Arabidopsis wild-type protoplasts were transformed with 35S:CIPK14-GFP. Green fluorescence can be detected in the cytoplasm and the nucleus (a, f). Onion epidermal cells were transformed with 35S:WHY1-GFP and 35S:nWHY1-HA-GFPc155 plus 35S:nWHY1-cmyc-GFPn173 as positive controls (d, e) (Grabowski et al., 2008). Onion epidermal cells and Arabidopsis protoplasts were transformed with 35S:WHY1-HA-GFPc155 plus 35S:CIPK14-cmyc-GFPn173 and 35S:nWHY1-HA-GFPc155 plus 35S:CIPK14-cmyc-GFPn173. Green fluorescence can be detected (left) in the cytoplasm and the nucleus according to the time course (2, 4, 10, and 16 h) and bright field (right) (b, c, g, and i). WHY2 protein instead of WHY1 was used as a negative control (h). The bar represents 100 μm. (B) Western blot detection of CIPK14-GFP and WHY1-HA proteins isolated from the transformed protoplast according to the time course (2, 4, 10, and 16 h) using antibodies against GFP peptide and HA tag. (C) The interaction detection of CIPK14 and WHY1 in the nucleus by co-immunoprecipitation. Protein extracts (plastid protein, P; nuclear protein, N; and nuclear plus cytoplasm protein, N + C) of overexpressing CIPK14-GFP in a PWHY1-HA background line as input. CoIP with antibody against an HA tag; the antibody against GFP was used to immunodetect the pull-down protein. Molecular Plant 2017 10, 749-763DOI: (10.1016/j.molp.2017.03.011) Copyright © 2017 The Author Terms and Conditions

Figure 3 The Phosphorylation of WHY1 by CIPK14 Affects the Binding Affinity of WHY1 at the WRKY53 Promoter by ChIP-qPCR and EMSA. (A and B) The phosphorylation of WHY1 by the recombinant purified CIPK14 kinase (A) and plant extracts (B) from oeCIPK141(oe5), kdCIPK14(kd1), and wild-type plants (wt) in an in vitro gel kinase assay. CIPK14, purified recombinant protein expressed in E. coli; WHY1, purified recombinant protein expressed in E. coli; CIPK14m, purified recombinant protein CIPK14 mutated 168T/D expressed in E. coli; CKII and casein used as controls. (C) The phosphorylation status of nuclear and plastid WHY1 protein isolated from 6-week-old rosette leaves of oeCIPK14 (oe5:oeCIPK14-5;oe6:oeCIPK14-6), kdCIPK14 (kd1:kdCIPK14-1;kd4:kdCIPK14-4) in a PWHY1-HA background and PWHY1-HA plants. Asterisk indicates the phosphorylated form of WHY1. An antibody against Pi-Thr168 (provided by the Biochemistry Department of Tuebingen University) was used to immunodetect the phosphorylated form WHY1. Coomassie staining shows the same amount of loading proteins. (D) The enrichment of WRKY53 and WRKY33 promoter (Pwrky53, Pwrky33) of WHY1-targeted genes detected by ChIP–qPCR (a) using 5-week-old rosette leaves of oeCIPK14 (oe5), kdCIPK14 (kd1) in the ID-nWHY1-HA background, and ID-nWHY1 plants in the wild-type background after 4 h induction with 20 μM estradiol. The induced nWHY1-HA protein was detected by antibody against an HA tag (b). Asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) show significant differences from the ID-nWHY1 line according to Student's t test. Actin was used as an inner control for ChIP–qPCR. (E) The binding affinity of WHY1 to the Pwrky53 element after incubating with plant extract proteins from oeCIPK14 (oe5,oe6), kdCIPK14 (kd1,kd4), and wild-type (wt) in a PWHY1-HA background. 25x, 50x, 25-fold and 50-fold specific competitor; Pwrky53 element, GTCAAAT…AAAAT (see Miao et al., 2013). The western blot with HA tag antibody after incubation of the plant extract proteins shows the same amount of WHY1 proteins. (F and G) The binding affinity of WHY1 to the Pwrky53 element after incubating with recombinant CIPK14 and CIPK14m protein from E. coli. With a time course (0,1, 2, 3, 4, and 5 h), the Pwrky53 element was incubated with purified CIPK14(F) and CIPK14m(G). 25x, 50x, 25-fold, and 50-fold specific competitor; Pwrky53 element, GTCAAAT…AAAAT (see Miao et al., 2013). The western blot with His tag antibody after incubation of the recombinant WHY1 proteins shows the same amount of WHY1 proteins. Molecular Plant 2017 10, 749-763DOI: (10.1016/j.molp.2017.03.011) Copyright © 2017 The Author Terms and Conditions

Figure 4 Phenotype Analyses of CIPK14 Mutants. (A) A T-DNA insertion line (a, koCIPK14 homozygous line; b, koCIPK14 heterozygous line; c, wild-type) of the CIPK14 gene shows a lethal phenotype. (B) 5% variegation pale-green phenotypes (oevar:oeCIPK14var) shown in oeCIPK14 lines; chlorophyll autofluorescence of the oevar line and the wild-type (wt) are observed under confocal microscopy. (C) The phenotypes of the leaves from plants grown for 10 weeks are observed by arrangement of leaves according to their age with number one being the oldest and a whole rosette upside down. An early senescence phenotype in two kdCIPK14 lines (kd1 and kd4) and a stay-green phenotype in two oeCIPK14 lines (oe5 and oe6) compared with wild-type are presented. (D) Senescent leaf proportions are characterized by 30 plants at week 10. The error bars indicate the standard error of 2 × 6 independent measurements. (E) Chlorophyll content of CIPK14 mutants at week 10; wt is set to 100%. The error bars indicate the standard error of five independent measurements. Molecular Plant 2017 10, 749-763DOI: (10.1016/j.molp.2017.03.011) Copyright © 2017 The Author Terms and Conditions

Figure 5 WHY1 Protein and the Plastid Ribosomal RNA Analyses of oeCIPK14 (oe5:oeCIPK14-5; oe6:oeCIPK14-6; var:oeCIPK14var), kdCIPK14 (kd1:kdCIPK14-1; kd4:kdCIPK14-4) in a PWHY1-HA Background and PWHY1-HA Plants. (A and B) The distribution of plastid WHY1 protein and nuclear WHY1 protein from 5-week-old rosette leaves of oeCIPK14, kdCIPK14 in a PWHY1-HA background and PWHY1-HA plants (A). Antibodies against an HA tag and Pi-Thr168 were used for immunodetection; Coomassie staining was used as a loading control. The protein band signal was captured and calculated by the ImageJ program (http://www.di.uq.edu.au/sparqimagejblots) (B). The data are the average of three replicates. (C) Dot blots of total RNA (1 μg, 5 μg, 15 μg) isolated from 4-week-old rosette leaves of CIPK14 mutants performed with plastid rDNA (4.5S, 5S, 16S, 23S rDNA), ribosome protein with large and small subunits (RPL and RPS), ORF23 and ORF63 as probes, with pBluescript plasmid DNA as the control. (D) Northern blot analyses of chloroplast 4.5S, 5S, 16S, 23S, RPL, RPS, and cytoplasmic 18S rRNAs. Total RNA (3 μg) from 4-week-old leaves of CIPK14 mutants were used for the northern blot experiments. Equal loading controls for northern blot analyses are shown in 18S. Molecular Plant 2017 10, 749-763DOI: (10.1016/j.molp.2017.03.011) Copyright © 2017 The Author Terms and Conditions

Figure 6 Genetic Complementation of CIPK14 Mutants with Overexpression of Plastid WHY1 and Nuclear WHY1 Partially Recovers the CIPK14 Mutant Phenotype. (A) Phenotype analyses of a series of double mutants of CIPK14 and WHY1 (Supplemental Figure 3) Leaves from the transgenic plants are laid out in order of leaf emergence as indicated by the numbers. Number 1 is the oldest. Results from one of five biological replicates are shown. (B) The proportion of senescent leaves is characterized by 20 plants at week 7. The error bars indicate the standard error of 2 × 6 independent measurements. (C) 12 μg of plastid (P) and nuclear (N) protein from 4-week-old transgenic plants were immunodetected by antibody against an HA tag. WB, western blot. (D) The gene expression of WHY1 downstream senescence-related genes detected in a series of complementation lines of CIPK14 and WHY1 mutants by qRT–PCR. The relative expression level of the gene is normalized to Actin, with the wild-type as 1. The standard error bars present three time biological replicates and three technical replicates, n = 3 × 3 = 9; the values are shown as means ± SD. Asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) show significant differences compared to the kdCIPK14 line according to Student's t test. Molecular Plant 2017 10, 749-763DOI: (10.1016/j.molp.2017.03.011) Copyright © 2017 The Author Terms and Conditions

Figure 7 WHY1 Downstream Gene Expression by Quantitative RT–PCR in CIPK14 Mutants. qRT–PCR analyses of downstream related genes of WHY1 in CIPK14 mutants, including the DSB-related genes and plastid rRNA genes (A), the senescence-related and cell death-related genes (B). The relative gene expression level was normalized to Actin, with the wild-type as 1. The standard error bars present three biological replicates and three technical replicates, n = 3 × 3 = 9; the values are shown as means ± SD. Asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) show significant differences compared with Col-0 wild-type according to Student's t test. oe5:oeCIPK14-5; oe6:oeCIPK14-6; kd1:kdCIPK14-1; kd4:kdCIPK14-4. Molecular Plant 2017 10, 749-763DOI: (10.1016/j.molp.2017.03.011) Copyright © 2017 The Author Terms and Conditions

Figure 8 CIPK14 Level Control WHY1 Subcellular Localization and Its Prominent Functions in Plastids and the Nucleus, Providing a Model for Retrograde Signaling between Plastids and the Nucleus at the Early Leaf Developmental Stage in Arabidopsis. WHIRLY1 has different functions in plastid and the nucleus. The ratio of nWHY1/pWHY1 is determined by the CIPK14 expression level in the cell. The phosphorylation status of WHY1 by CIPK14 kinase affects the binding affinity of WHY1 protein with the promoter of the downstream gene to control gene expression and the phenotype. Therefore, CIPK14 plays a key role in switching the roles of WHY1 in the nucleus and plastids. Impairment of CIPK14 leads to seedling de-greening and plastid damage due to the distribution of WHY1 in plastids and the nucleus. The impairment of CIPK14 leads to a preference for senescence initiation due to the phosphorylation status of WHY1 in the nucleus. Molecular Plant 2017 10, 749-763DOI: (10.1016/j.molp.2017.03.011) Copyright © 2017 The Author Terms and Conditions