1 Comparative proteomics analysis of coregulation of 2 CIPK 14 and WHIRLY 1 / 3 mediated leaf pale 3 yellowing in Arabidopsis 4

Leaf variegation pale yellowing is observed in the Calcineurin B-Like-Interacting Protein 11 Kinase14 (CIPK14) overexpression line (oeCIPK14) and double knockout WHIRLY1/WHIRLY3 12 (why1/3) lines of Arabidopsis, the distribution of WHIRLY1 (WHY1) protein between plastids and 13 the nucleus are affected by the phosphorylation of WHY1 by CIPK14. To elucidate the coregulation 14 of CIPK14 and WHIRLY1/WHIRLY3 mediated leaf pale yellowing, a differential proteomic analysis 15 is conducted between the oeCIPK14 variegated (oeCIPK14-var) line, why1/3 variegated (why1/3-var) 16 line and wild type (WT). More than 800 protein spots are distinguished on each gel, 67 differential 17 abundance proteins (DAPs) are identified by matrix-assisted laser desorption ionization-time of 18 flight/time of flight mass spectrometry (MALDI-TOF/TOF-MS), of which, 34 DAPs are in the 19 oeCIPK14-var, 33 DAPs are in the why1/3-var compared to WT. Five overlapping proteins differentially 20 change both in the oeCIPK14-var and in the why1/3-var. They are ATP-dependent Clp protease 21 proteolytic subunit-related protein 3 (ClpR3), Ribulose bisphosphate carboxylase large chain (RBL), 22 Beta-amylase 3 (BAM3), Ribosome-recycling factor (RRF), Ribulose bisphosphate carboxylase small 23 chain (RBS). Bioinformatics analysis show that most of DAPs are involved in photosynthesis, 24 defense and antioxidation pathway, protein metabolism, amino acid metabolism, energy 25 metabolism, malate biosynthesis, lipid metabolism and transcription. Thus, the photosystem 26 parameters are measured that the content of chlorophyll, the photochemical efficiency of PSII 27 (Fv/Fm), and electron transport rates (ETR) decrease in the why1/3-var and oeCIPK14-var, but the 28 non-photochemical quenching (NPQ) increases. Both mutants show high sensitivity to strong light. 29 Based on the annotation of DAPs from both why1/3-var and oeCIPK14-var lines, we conclude that 30 CIPK14 phosphorylation mediated WHY1 deficiency in plastids is related to impairment of protein 31 metabolism leading to chloroplast dysfunction. 32

intriguing, the variegated phenotype can be recovered partially by overexpression of plastid-located 97 WHY1 [28].

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This study focuses on comparable analysis of the phenotype and proteomic alteration between 99 why1/3 variegated (why1/3-var) lines and oeCIPK14 variegated (oeCIPK14-var) lines for evaluating the 100 relationship of CIPK14 and WHY1/WHY3 producing pale yellow leaf. The total proteins of wild type 101 (WT), why1/3-var and oeCIPK14-var rosette leaves are separated by two-dimensional gel 102 electrophoresis analysis (2-DE) and the differential expression proteins are identified by matrix-   Table 1.

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In order to evaluate the quality of 2-DE differentially display proteins, two light-dependent 127 reaction complex related proteins are selected to do immunodetection by using consumable 128 antibodies against the ATP synthase subunit alpha (atp A) and the photosystem Ⅱ (PS Ⅱ) complex   Table 1. The majority of the proteins are photosynthesis-151 associated proteins, followed by defense and antioxidation proteins, protein metabolism and amino 152 acid metabolism, energy metabolism, malate biosynthesis, lipid metabolism and transcription related 153 proteins (Table 1 and Figure 2). Comparative analysis of differentially expression proteins between 154 why1/3-var/WT and oeCIPK14-var/WT are shown in Figure 2A and 2B, respectively. The five 155 overlapping proteins between why1/3-var/WT and oeCIPK14-var/WT are ATP-dependent Clp 156 protease proteolytic subunit-related protein 3 (ClpR3), Ribulose bisphosphate carboxylase large chain  Figure            mutants are measured to determine the photosynthetic performance. Consistent with the pale-green 258 phenotype, the chlorophyll content is lower in the oeCIPK14-var plants than wild-type, whereas it 259 remains unaltered in the why1/3-var ( Figure 6A). Interestingly, the chlorophyll content slightly 260 increases in the oeWHY1 and cipk14 plants ( Figure 6A). The maximum photochemical efficiency of 261 photosystem II (Fv/Fm) significantly decreases in the why1/3-var and oeCIPK14-var plants ( Figure 6B).

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The electron transport rates (

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The fluorescence images of the whole plant of different genotype are taken by using Image-PAM

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(Pulse-Amplitude Modulation) measuring system, as showed in Figure 6E.   in abundance of cyt f may accelerate the ETR that are lower in the why1/3-var and oeCIPK14-var plants.

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The HCF101 has been shown to serve as a chloroplast scaffold protein for the assembly and

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Chlorophyll fluorescence is measured and chlorophyll fluorescence image is taken using an 432 Imaging-PAM-Maxi (Walz, Germany) as described by Shao [60].

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The protein pellets are dissolved in sample lysis buffer (

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We concluded that in the why1/3-var or oeCIPK14-var lines most of proteins are involved in 501 photosynthesis, amino acids and protein metabolism, or defense and antioxidation (Figure 7). They 502 are related to photosynthesis and insufficiencies in energy supply. The co-regulation of CIPK14 503 phosphorylation mediated WHIRLY1/WHIRLY3 deficiency in plastids are speculated that they 504 might be controlled by mechanism of amino acids and plastid protein metabolism at the 505 posttranscriptional level. The detail mechanism will be addressed in future.