阿拉伯芥核蛋白CIA2和CIL調控葉綠體發育機制研究

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(1)國立臺灣師範大學理學院生命科學系博士論文 Department of Life Science College of Science. National Taiwan Normal University Doctoral Dissertation. 阿拉伯芥核蛋白 CIA2 和 CIL 調控葉綠體發育機制研究 Arabidopsis Nuclear Proteins CIA2 and CIL Regulate Chloroplast Development. 楊俊彥 YANG, Chun-Yen 指導教授：孫智雯博士 Advisor: Chih-Wen SUN, Ph.D.. 中華民國 109 年 8 月 August 2020.

(2) Acknowledgments 本研究得以完成要感謝我的指導教授孫智雯老師，感謝她耐心的指導和提供學術研究相關等各方面的協助。特別感謝中研院植微所吳素幸老師實驗室和其中成員，除了提供設備及技術指導以完成本研究中許多成果外，在實驗方向及論文寫作也提供了很多的討論與指導。感謝中研院植微所微矩陣及次世代定序實驗室的研究技師周素珍學姊協助並指導分析結果以完成 Microarray analysis 及電子顯微鏡實驗室協助完成 TEM 的結果。在論文寫作及實驗方向上，感謝中研院分生所李秀敏老師、植微所涂世隆老師及台大細分所蔡皇龍老師悉心的指導與修正，並給予了很多寶貴的建議。 D207 實驗室相關工作的協助要感謝心嚴、彥民、昱龍、耿禎、瓊丹等成員；在課程上或生活上，聖凱、許庭、與祥、孟緯、廷漢跟其他與我幫助過我及支持我的朋友及同學。感謝學輔中心的諮商老師協助我渡過追求學位的後期，讓我能獲得這個學位。能獲的這個學位真的不容易，感謝曾經幫助過我的人；也謝謝家人這些年的關心、體諒與支持，讓我能盡心地進行研究，特別是爸媽，讓您們擔心了這麼多年。最後，謹以此論文獻給我的家人及曾經幫助過我的人。. I.

(3) 中文摘要 CHLOROPLAST IMPORT APPARATUS 2 (CIA2)與 CIA2-LIKE (CIL)隸屬於一羣植物特有、會參與開花時間或蓋日韻律調節的 CCT [CONSTANS (CO), CO-LIKE (COL), TIMING OF CAB EXPRESSION1 (TOC1)] 轉錄因子。有別於其他 CCT 蛋白，CIA2 會提高一些葉綠體蛋白合成基因的表現量，進而確保葉綠體的正常發育。 CIL 與 CIA2 的胺基酸序列具有 65％的相似性；CIL 在葉子和花苞中表現，其表現型態與 CIA2 相似，這表明 CIL 和 CIA2 在阿拉伯芥中是同源的。CIL 在 cia2 突變株中表現量增加，而 cia2 /cil 雙突變株的葉色比 cia2 突變株更為淺綠。對 cia2 /cil 雙突變株的微陣列分析 (Microarray analysis) 顯示，與葉綠體發育有關的細胞核表現基因，包括與光合作用和葉綠素生合成相關的基因，表現量明顯降低，顯示 CIA2 和 CIL 共同調節了 GOLDEN2-LIKE 1 和葉綠體發育相關基因的表現。微觀結構觀察(Microstructure observation) 顯示 10 天齡的 cia2 /cil 雙突變株具有特定的發育異常。 CIA2 是細胞核轉錄蛋白，包含位於氨基酸 62-65 和 291-308 的兩個細胞核導引訊息 (nuclear localization signal, NLS)。 CIL 也是細胞核轉錄蛋白，其 NLS 位於胺基酸 47-50。 CIA2 和 CIL 的 CCT 結構不具有核定位信號功能。酵母雙雜交（yeast two-hybrid, Y2H）篩選確認了與 CIA2 相互作用的蛋白。除了自身和 CIL 外，還確認了諸如 CO、NUCLEAR FACTOR Y B1 (NF-YB1)、NF-YC1、NF-YC9 和 ABSCISIC ACID-INSENSITIVE 3 等蛋白與葉綠體功能和開花時間的調節有關。Y2H 進一步確認 CIA2 和 CIL 的 N 和 C 端區域對於與其他蛋白的交互作用很重要。儘管 CIA2 和 CIL 的 CCT 結構是兩個蛋白質之間的主要相互作用片段，但 CIA2 和 CIL N 端的 CIA2 and CIL conserved 1 (CC1) 結構使上述 CIA2、CIL 和開花時間調節蛋白之間能夠交互作用。本研究中顯示阿拉伯芥 CIA2 和 CIL 與 CO 和 NF-Y 複合體(complex) 交互作用，並參與 CO 相關的開花機制調控。CIA2 和 CIL 的 N 端 CC1 結構與 CO 和 NF-Ys (B1、C1 和 C9) 相互作用形成更高階的複合體，並且 CC1 結構中的胺基酸序列與 NF-Ys 中 NF-YA1 結構的序列相似。其中，NF-YAs 蛋白是利用 NF-YA1 結構與 NF-YB / NF-YC 複合體交互作用。最後，本研究的結果顯示 CIA2 和 CIL 參與葉綠體發育和 CO 相關開花機制的調控。. II.

(4) 關鍵詞：阿拉伯芥、CHLOROPLAST IMPORT APPARATUS 2 (CIA2)、CIA2-LIKE (CIL)、CCT 結構、葉綠體發育、細胞核導引訊息、酵母菌雙雜交、植物開花調節. III.

(5) English Abstract Chloroplast import apparatus 2 (CIA2) and CIA2-like (CIL) are classified to plant-specific, CCT [CONSTANS (CO), CO-LIKE (COL), TIMING OF CAB EXPRESSION1 (TOC1)] motif-containing transcription factors involving in regulation of flowering time or circadian rhythm. In contrast to other CCT proteins, CIA2 is able to increase the expression yields of genes encoding chloroplast proteins, and therefore to ensure the proper development of chloroplast. CIL shares 65% similarity of amino acid sequence with CIA2; CIL is expressed in leaves and young flower buds, and its expression pattern is similar to that of CIA2, suggesting that CIL and CIA2 are homologous in Arabidopsis. CIL is overexpressed in cia2 plants, and the pale-green phenotype of cia2/cil is more severe than that of cia2. Microarray analysis of cia2/cil double mutants revealed evidently decreased expression of nuclear genes involved in chloroplast development, including genes associated with photosynthesis and chlorophyll biosynthesis, indicating that CIA2 and CIL co-regulate the expression of GLK1 and chloroplast development-related genes. Microstructure observations revealed a specific developmental abnormality of chloroplasts in the 10-day-old cia2/cil double mutants. CIA2 is a nuclear protein containing two nuclear localization signals (NLSs) located at amino acid (aa) positions 62-65 and 291-308. CIL is also a nuclear protein, with an NLS located at 47-50 aa. The CCT motifs of CIA2 and CIL do not function as an NLS. CIA2-interacting candidates were identified by using yeast two-hybrid (Y2H) screening. In addition to CIA2 and CIL, CIA2-interacting proteins identified from Y2H such as CO, NUCLEAR FACTOR Y B1 (NF-YB1), NF-YC1, NF-YC9, and ABSCISIC ACID-INSENSITIVE 3 were speculated to be involved in the regulation of chloroplast function and flowering time. Y2H experiments revealed that the N- and C-terminal regions of CIA2 and CIL are important for interactions with other candidate proteins. Although the CCT motifs of CIA2 and CIL are the major interacting fragments between the two protein, the N-terminal CIA2 and CIL conserved 1 (CC1) motif enables the interactions among CIA2, CIL, and the flowering time regulatory proteins mentioned above. This study proposes that the Arabidopsis CIA2 and CIL interact with the CO and NF-Y complex IV.

(6) and participate in CO-related flowering regulation. Moreover, the N-terminal CC1 motifs of CIA2 and CIL interact with CO and NF-Ys (B1, C1, and C9) to form a high-order complex, and the residues in the CC1 motif are similar to those in the NF-YA1 subdomain of NF-YAs, which interact with the NF-YB/NF-YC complexes. Finally, the results of this study suggest that CIA2 and CIL co-regulate the expression of genes involved in chloroplast development and CO-related flowering regulation.. Keywords: Arabidopsis, CHLOROPLAST IMPORT APPARATUS 2 (CIA2), CIA2-LIKE (CIL), CCT motif, chloroplast development, nuclear localization signal, yeast two-hybrid, flowering regulation. V.

(7) Table of Contents Acknowledgments………………………………………………………………………………...I Chinese Abstract……………………………………………………………………….………….II English Abstract………………...…………………………………………………………………IV Introduction………………………………………………………………………………………..1 Material and Methods……………………………………………………………………………...7 Results……………………………………………………………………………………………...14 Part I. Functional analysis of CIA2 and CIL……………………………………………………...14 Part II. Analysis of protein structure and interactions of CIA2 and CIL………………………….21 Part III. Analysis of the evolutionary relationships between CIA2 and CIL……………………...30 Discussion………………………………………………………………………………………….34 Conclusions………………………………………………………………………………………...44 References………………………………………………………………………………………….45 Tables……………………………………………………………………………………………....56 Figures……………………………………………………………………………………………...65 Supplemental Data……………………………………………………………………………...…..88. VI.

(8) Introduction Chloroplast is a plant-specific organelle, which differentiates from the proplastid and is responsible for photosynthesis during plant growth and development. According to the endosymbiont theory, chloroplasts were introduced into plant cells and most genes were transferred into the nuclei of plant cells through endocytosis of cyanobacteria (Martin et al., 2002; Imamura et al., 2009; Pfalz and Pfannschmidt, 2013). Chloroplasts possess 3,100 proteins (Leister, 2003; Paila et al., 2015), although only a few of these are translated from their own genomes, whereas over 90% are translated by the nucleus and transported to the chloroplast (Martin et al., 2002; Pfalz and Pfannschmidt, 2013). Therefore, proteins involved in chloroplast development and regulatory functions must be produced by the cooperation of genes in the nucleus and chloroplast. Nucleus-encoded chloroplast proteins are transferred to the chloroplast via post-translational translocation. Expect some outer membrane proteins, most nuclear-encoded precursor proteins harbor a transit peptide sequence at the N-terminal, which is recognized by the chloroplast translocon complex. The proteins are then imported into the stroma, after which the transit peptides are removed by stromal processing peptidase to turn the precursor proteins into mature proteins through association with chaperone proteins (Paila et al., 2015). In a previous study, Arabidopsis thaliana was used a model plant to screen for mutants with loss of chloroplast protein transport mechanism using ethyl methanesulfonate as a mutagen and antibiotic resistance screening methods. In that study, a nuclear transcription factor associated with the chloroplast protein input—CHLOROPLAST IMPORT APPARATUS 2 (CIA2)—was isolated. In cia2 mutants, translation is pre-terminated owing to point mutations in CIA2, resulting in the formation of an incomplete CIA2 protein (Sun et al., 2001). The cia2 mutant exhibits a pale green phenotype, and the total chlorophyll a and b content of the mutant plants is reduced to ~50% of the content of the wildtype plants. However, the mutant plants show similar leaf shape and size to wildtype plants at the same developmental stage (Sun et al., 2001; Sun et al., 2009). In addition, the efficiency of chloroplast protein import is reduced in cia2 mutants (Sun et al., 2001; Sun et al., 2009). Furthermore, microarray analysis showed that CIA2 enhances the expression of 1.

(9) TRANSLOCON AT OUTER ENVEIOPE MEMBRANE OF CHLOROPLASTS75 (Toc75), Toc33, CHAPERONIN10 (CPN10), and CHLOROPLAST RIBOSOMAL PROTEINs (cpRPs), which in turn regulate chloroplast functions (Sun et al., 2009). Alignment of the full-length (FL) 435 amino acid (aa) sequence of CIA2 has revealed a 43 aa C-terminal CCT [CONSTANS (CO), CO-LIKE (COL), TIMING OF CAB EXPRESSION1 (TOC1)] motif (Putterill et al., 1995; Robson et al., 2001; Strayer et al., 2000; Gangappa and Botto, 2014). In Arabidopsis, CIA2 has a homologous gene, CIA2-LIKE (CIL). CIL encodes a 394 aa protein with 65% similarity to CIA2 and harbors a C-terminal CCT motif (Sun et al., 2001). In cia2 mutants, CIL expression is up-regulated, indicating that its function may be redundant with that of CIA2; however, the precise function of CIL remains unclear. The CCT motif is a sequence in plant-specific CCT class transcription factors that functions as a nuclear localization signal (NLS) or protein-interacting region (Strayer et al., 2000; Kurup et al., 2000; Robson et al., 2001); however, whether the CCT motifs of CIA2 and CIL function as NLSs or protein-interacting regions remains unknown. CCT motif proteins, which harbor a C-terminal CCT motif, are classified into three types according to their N-terminal structure: COL proteins with an N-terminal B-box domain, proteins with an N-terminal pseudo-receiver (PRR) domain, and the CCT motif family (CMF) proteins without any specific structure or a known N-terminal domain (Cockram et al., 2012). COL proteins are involved in the regulation of photoperiodism and flowering (Putterill et al., 1995; Yano et al., 2000; Turner et al., 2005); PRR proteins are involved in the regulation of circadian rhythm and light signal transduction (Salome et al., 2006; Nakamichi et al., 2010; Nakamichi et al., 2012); and little is known about the functions of CMF proteins, although CMF3 has been reported to be involved in the regulation of glycan synthesis-related gene expression (Masaki et al., 2005). Although CIA2 and CIL are classified as CMF14 and CMF9 (Cockram et al., 2012), their roles in the regulation of physiological processes mentioned above remain unknown. Arabidopsis GOLDEN2-LIKE (GLK) proteins, belonging the plant GARP (GOLDEN2, ARR-B class, and PSR1) superfamily of proteins, are the other nuclear transcription factors involved in the 2.

(10) regulation of chloroplast development. GARP superfamily proteins are groups of plant-specific transcription factors containing similar DNA-binding domains (Riechmann et al., 2000), including GOLDEN2 in maize (Hall et al., 1998), ARABIDOPSIS RESPONSE REGULATOR-B (ARR-B) class proteins and GLK proteins (Imamura et al., 1999; Fitter et al., 2002) in Arabidopsis, and PHOSPHATE STARVATION RESPONSE 1 (PSR1) protein in Chlamydomonas (Wykoff et al., 1999). Arabidopsis GLKs, including GLK1 and GLK2, are nuclear transcription factors and increase the expression of photosynthesis-related genes including photosystem components, such as LIGHT-HARVESTING COMPLEX 2.2 (Lhcb2.2), Lhcb3, and Lhcb4.2, as well as chlorophyll biosynthetic enzymes, such as CHLOROPHYLL a OXYGENASE (CAO) and PROTOCHLOROPHYLLIDE B (PORB), to regulate chloroplast development (Waters al., 2009). The phenotype of glk1 or glk2 single mutants is similar to that of wildtype; however, the leaf size of glk1/glk2 double mutant is smaller than that of wildtype, and its chlorophyll a and b content is reduced by 35% of the wildtype. Introduction of GLK1 or GLK2 in glk1/glk2 double mutants (Fitter et al., 2002; Waters et al., 2008) restored the phenotype to wildtype, demonstrating the functional redundancy of GLK1 and GLK2. The phenotype of cia2 mutant is similar to that of glk1/glk2 double mutant, and its leaves are pale green; however, the leaf size does not differ between cia2 and wildtype. In cia2 mutants, CIL expression is up-regulated, indicating the functional redundancy of CIA2 and CIL; therefore, the physiological roles of CIA2 and CIL may be elucidated under simultaneous functional defects. In angiosperms, flowers are important organs for reproduction. Floral transition is regulated by various factors, such as photoperiod, ambient temperature, plant hormones, vernalization, and nutrients, which control the vegetative to reproductive development of plants under optimal conditions (Mouradov et al., 2002; Boss et al., 2004; Song et al. 2015). Among these pathways, photoperiodic flowering mainly relies upon the regulation of three groups of proteins. The first group of proteins include phytochromes and cryptochromes, which receive red and blue light and transmits the light signals to a second group of central oscillator transcription factors, such as CIRCADIAN CLOCK ASSOCIATED1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), 3.

(11) EARLY FLOWERING4 (ELF4), and TOC1, associated with circadian rhythm (Schaffer et al., 1998; Green and Tobin, 1999; Doyle et al., 2002; Makino et al., 2002). Subsequently, the central metronome regulates the third group of flowering pathway-associated proteins CO and FLOWERING LOCUS T (FT), which in turn promote flowering (Mouradov et al., 2002; Boss et al., 2004). The central oscillator genes are expressed rhythmically in the diurnal cycle and are regulated by the circadian clock. CCA1 and LHY transcript and protein expression peaks in the early morning, and TOC1 mRNA expression peaks in the evening; these gene expression profiles produce an auto-regulatory transcriptional and translational negative feedback loop (Alabadí et al., 2001; Boss et al., 2004). During the late evening, the TOC1 protein degradation activates CCA1 and LHY, resulting in peak expression at the start of the day. This in turn increases CCA1 and LHY expression during the day, which suppresses TOC1 expression. Consequently, CCA1 and LHY mRNA expression drops at the end of the day, resulting in the abundant of TOC1 proteins. Reduced CCA1 and LHY expression abrogates the suppression of TOC1, and the cycle resumes, forming an oscillatory loop (Makino et al., 2002; Matsushika et al., 2002a, 2002b; Shim et al., 2017). Studies of changes in photoperiodic flowering time in mutant plants have shown CO to be the main output of circadian oscillations (Suárez-López et al., 2001; Valverde et al. , 2004; Shim et al., 2017), which regulated by the circadian clock and light. The co mutants exhibit delayed floral transition on long days, but they flower early on short days. CO overexpression induces early flowering under either photoperiod (Putterill et al., 1995; Suárez-López et al., 2001). Light-stabilized CO induces FT transcription, which controls shoot apical meristem floral transition (Abe et al., 2005; Corbesier et al., 2007; Jaeger et al., 2007). In Arabidopsis, CO form a complex with the nuclear protein NUCLEAR FACTOR Y (NF-Y) through the C-terminal CCT motif, and the CO–NF-Y complex binds to the CO response element (CORE) and CCAAT box of the FT promoter to increase FT expression and promote flowering (Wenkel et al., 2006; Tiwari et al., 2010). The NF-Y protein, also known as CCAAT-BOX FACTOR (CBF) or HEME ACTIVATOR PROTEIN (HAP), has been independently identified in yeast, mammals, and plants, and it is a 4.

(12) functionally conserved transcription factor and histone-fold protein across species (Mantovani, 1999). NF-Y consists of three subunits: NF-YA (CBF-B; HAP2), NF-YB (CBF-A; HAP3), and NF-YC (CBF-C; HAP5). Each of the NF-Y subunits is encoded by a single gene in yeast and mammals (Mantovani, 1999). The Arabidopsis genome encodes 10 homologs each of NF-YA, NF-YB, and NF-YC (Petroni et al., 2012). The NF-Y proteins form heterodimeric complexes and interact with other proteins to form various complexes that serve pivotal functions in many plant life processes, such as seed development, flowering, primary root elongation, abscisic acid signaling, and drought resistance, among others (Mantovani, 1999; Petroni et al., 2012; Zhao et al., 2017). Previous experiments have revealed that NF-YB1 and NF-YC1 can interact with CO (Wenkel et al., 2006). In plants, nf-yb1 T-DNA-insertion mutants produce flowers at the same time as wildtype plants. In addition, NF-YB1 and CO co-overexpression promotes early flowering, whereas NF-YB1 overexpression delays flowering (Wenkel et al., 2006). Although the flowering time of nf-yc1 mutants has not been observed, nf-yc9 T-DNA-insertion single mutants show no difference in flowering time from wildtype plants, but nf-yc3/nf-yc4/nf-yc9 triple mutants show delayed flowering (Kumimoto et al., 2010). These results suggest the functional redundancy of NF-Y subunits and their involvement in CO-mediated flowering time regulation. ABI3 also interacts with CO (Kurup et al., 2000). ABI3, whose orthologue in maize is named VIVIPAROUS 1 (VP1, Finkelstein et al., 2002), is a B3 domain transcription factor and plays essential roles in abscisic acid-mediated regulation of seed maturation, desiccation sensitivity, and precocious germination (Ooms et al., 1993; Nambara et al., 1994; Parcy et al., 1994, 1997). However, the late-flowering phenotype of ectopic ABI3 expression and the early-flowering phenotype of abi3-4 mutant suggest its other roles in the regulation of flowering time (Kurup et al., 2000; Hong et al., 2019). In this study, CIL transgenic plants were used to demonstrate the functional redundancy between CIA2 and CIL. Microarray analysis and microstructure observation showed that CIA2 and CIL co-regulate chloroplast biogenesis and maintenance. Moreover, chloroplast dysfunction leads to the accumulation of reactive oxygen species (ROS) and up-regulation of nuclear-encoded 5.

(13) mitochondrial genes in cia2/cil mutants. Additionally, the reporter gene -glucuronidase (GUS) was used to confirm that the CIA2 homologous protein CIL is a plant nuclear protein, and NLSs in CIA2 and CIL were identified. The yeast two-hybrid (Y2H) system was used to screen interacting candidates and confirm the interaction between CIA2 and CIL. In addition, the interaction between CIA2 and CIL was further confirmed using biomolecular fluorescence complementation (BiFC) in plant cells. Finally, CIA2 and CIL were found to interact with flowering time regulation-related proteins, such as CO, NF-YB1, NF-YC1, and NF-YC9. Therefore, CIA2 and CIL proteins are speculated to be involved in the regulation of flowering time.. 6.

(14) Material and Methods Plant Materials and Growth Condition Arabidopsis thaliana ecotype Columbia (Col-0) was used as wildtype in this study. The cia2 mutant was isolated from our previously study (Sun et al., 2001). The cil mutants (Col ecotype, SALK_068498; Alonso et al., 2003) were obtained from the Arabidopsis Biological Resource Center (ABRC). Seeds were disinfected with 25% (v/v) commercial bleach and soaked in water for 48 hours (h) at 4oC, then grown on 1X Murashige and Skoog (1962) agar medium with 1% (w/v) sucrose or grown in soil after soaked without disinfection. Plants were grown at 22oC under a 16-h-light/8-h-dark cycle at an illumination of 80 to 100 mol m-2s-1 for various numbers of days.. Plasmid Construction and Plant Transformation The sequences of the cauliflower mosaic virus (CaMV) 35S promoter (35Spro) and NOS terminator from vector pBI121 (Invitrogen) and the 2xHA epitope coding sequence were ligated into the binary vector pPZP221 (Hajdukiewicz et al., 1997) to create plasmid pCS188. The coding sequence (CDS) of CIL with 3’ untranslated region amplified by polymerase chain reaction (PCR) from Col cDNA was in-frame fused to 2xHA sequence and inserted into pCS188 to create pCS190. The cia2 mutant was transformed with pCS190 using the floral dipping method (Clough and Bent, 1998), mediated by Agrobacterium tumefaciens strain GV3101. Transgenic plants were selected on agar plates containing 30 g/mL G418 and verified by PCR using construct-specific primers (Supplemental Table S1). This transgenic strain was named 35Spro:HA-CIL/cia2. The promoter sequence of CIL (-1504 ~ -1) was PCR-amplified from Col genomic DNA replaced the 35S promoter of pCS190 to create pCY190 plasmid. The same strategies of above used to generate transgenic strain CILpro:HA-CIL/cia2. Two homozygous transgenic plants (T3) were used for this study.. Quantification of Pigment Content The content of chlorophyll and carotenoid in 18-day-old leaves was measured as described by 7.

(15) Lichtenthaler (1987). Leaf tissues (20 mg) were frozen in liquid N2, grounded in an Eppendorf tube, then suspended in 500 l 80% acetone. After the removal of debris by centrifugation, the OD of the supernatant was measured at 470, 645 and 663 nm. All measurements were performed in triplicate using three independent leaf samples.. RNA Isolation and Quantification of Transcript Level Total RNAs isolated from 18-day-old plants by TRIzol solution (Invitrogen; Chomczynski and Sacchi, 1987) and treated with RQ1 DNase I (Promega) prior to cDNA synthesis. First-stranded cDNAs were synthesized using Moloney murine leukemia virus RNase H2 reverse transcriptase (Promega) and an oligo dT19-N primer with 5 g of total RNAs. Amounts of transcripts for various genes were analyzed by reverse transcription-PCR (RT-PCR) or real-time quantitative RT-PCR (qRT-PCR). Primers specific for each gene were described in Supplemental Table S1. In RT-PCR assay, these gene-specific primers were used to amplify each transcript with 25 PCR cycles using the first-stranded cDNA as templates. PCR products were electrophoresis on 1.5% agarose gel, visualized by ethidium bromide and UV light. In qRT-PCR, the cDNA and specific primers with 2X SYBR FAST qPCR reagents (KAPA Biosystems) were used to detect each transcript by StepOne Plus thermal cycler (Applied Biosystems) with programs recommend by manufacturer (1 cycle at 95 oC for 3 min, 40 cycles of 95 oC for 3 sec and 60 oC for 30 sec). The comparative CT method which described by Livak and Schmittgen (2001) was used to determine the relative amount of gene expression and the expression of UBQ10 used as internal control. The mean values of three independent experiments were calculated.. Microarray Analysis 0.2 μg of total RNA from 18-day-old plants was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies) and labeled with Cy3 (CyDye, Agilent Technologies) during the in vitro transcription process. 1.65 μg of Cy3-labled cRNA was fragmented at 60°C for 30 min. Correspondingly fragmented labeled cRNA is then pooled and hybridized to Agilent Arabidopsis 8.

(16) V4 4×44K Microarray (Agilent Technologies) at 65°C for 17 h. The microarrays were scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm for Cy3. The array image was analyzed by the Feature Extraction software version 10.7.1.1 using the default setting. The microarray data were normalized by the log conversion, and the expression ratios were analyzed using Genespring GX 11.8 software (Agilent Technologies). The threshold of fluorescence intensities were cutoff below 100. The genes had at least a 1.5-times fold change in natural logarithm statistically of both slides, which the gene expression in wildtype is 1 and the expression change is ≥1.5 and ≤ 0.68 are listed in Supplemental Table S2 and S3. Gene annotations were compiled from The Arabidopsis Information Resource (TAIR; www.arabidopsis.org) and the predicting subcellular location bases on SUBcon of SUBA3 database (Tanz et al., 2013; Hooper et al., 2014).. Transmission Electron Microscopy The true leaves of 10-day-old seedling of Col, cia2, cil, and cia2/cil were cut and fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.0, at room temperature for 4 h. After rinses, the samples were post-fixed in 1% osmium tetroxide in the same buffer for 4 h at room temperature. After rinsing three times, samples were dehydrated in sequentially increased concentration of acetone, embedded in resin, and sectioned with Leica EMUC6 ultramicrotome (Leica Microsystems GmbH). The ultrathin sections (75–90 nm) were stained with uranyl acetate and lead citrate. Images were captured using a Tecnai G2 Spirit TWIN electron microscope (FEI) and Digital Micrograph acquisition software (Gatan).. Histochemical Staining of Reactive Oxygen Species Detection The amount of leaf ROS was measured from 18-day-old plants grown on the agar plate under 200 -. mol m-2s-1 light treatment for 1 h. The detection of H2O2 or O2 was strained by 3,3’-diaminobenzidine (DAB) or nitroblue tetrazolium (NBT), respectively (Lv et al., 2011). Leaves were vacuum infiltrated in freshly prepared 0.1% (w/v) DAB-HCl (pH 3.8) solution, and then 9.

(17) incubated in darkness at room temperature for 10 h (Orozco-Cardenas and Ryan, 1999). Plant leaves were vacuum infiltrated with 0.1% (w/v) NBT in 10 mM potassium phosphate buffer (pH 7.8) containing 10 mM NaN3, and then incubated in darkness at room temperature for 1 h (Kawai-Yamada et al., 2004). After incubation, both stained plant leaves were placed in acetic acid: glycerol: ethanol (1: 1: 3, v/v/v) solution at 95oC for 10 min and stored in 95% ethanol until images captured.. Nuclear Localization Assay The process of nuclear localization assay was described previously (Sun et al., 2001). PCR-amplified CIA2 or CIL fragments were digested with Bam HI and Xba I and cloned into the Bgl II- and Xba I-digested plasmid pRG/NIa1-76 (Carrington et al., 1991), replacing the NIa1-76 fragment. CIA262-65 and CIA2291-308 fragments were replaced by Mun I (Mfe I) and Kpn I sites, respectively. CIL47-50 and CIL245-262 fragments were substituted by Hpa I and Alf II sites. All constructs were further sequence confirmed. These constructs were transiently expressed in onion epidermal cells by microparticle bombardment using the Biolistic PDS-1000/He Particle Delivery System (Bio-RAD) as described (Varagona et al., 1992). SYTOX-stained nuclei were observed using Leica TCS SP2 confocal microscope in the fluoresce channel, and localization of 5-bromo-4-chloro-3-indolyl--glucuronic acid (X-gluc) staining was observed using the same microscope in the transmission channel. The SYTOX Green Stain (Molecular Probes) is the green-fluorescent nuclear dye.. Yeast Two-Hybrid Screening A normalized library made from 11 Arabidopsis tissues was used for Y2H screening (Mate and Plate Library - Universal Arabidopsis, Clontech). The cDNA library was transformed into yeast strain Y187 and the screening process followed The Matchmaker Gold Yeast Two-Hybrid System protocol (Clontech). The CIA2 full-length CDS as bait was cloned into the GAL4 binding domain (BD) containing pGBKT7 vector via Nco I and BamH I sites then transformed into the AH109 yeast 10.

(18) strain (Clontech). Mating of the two sexually different strains was carried out at 30 oC for 24 h and the resulting zygotes were plated directly on SD/–Leu–Trp–His growth medium supplemented with 5 mM 3-Amino-1,2,4-triazole (3-AT). After 6 days of incubation in a 30 oC condition, colonies with a growth surface diameter of more than 1 mm were transferred on SD/-Leu-Trp-His containing 10 mM 3-AT. The β-galactosidase activity assay was carried out after 6 days incubation. Plasmids from blue yeast colonies that survived this stringent selection was extracted and sequenced using the GAL4 activing domain (AD) 5'AD or T7P primers. The cDNA sequence of library which amino acid sequence was in-frame with the GAL4-AD would be selected and further confirmed.. Yeast Two-Hybrid Assay The Y2H experimental process is referring to the steps of The Matchmaker Yeast Two-Hybrid System 2 (Clontech). The restriction sites of pAS2-1 and pACT2 were used to inset the CDS respectively such as ABI3 (Bam HI and Sal I), ARR3 (Nco I and BamH I), CO fragments (Sma I and BamH I), CIA2 (Nco I and Sma I), CIL (Nco I and Sma I), NF-YB1 (Nco I and EcoR I), NF-YC1 (Nco I and EcoR I), NF-YC9 (BamH I and Sal I), which were amplified by PCR from plasmids of cDNA library of candidates or cDNA synthesized from 18 or 28 day-old wildtype plants and sequencing for confirmation. These plasmids were introduced to AH109 yeast strain and verified the protein interaction by SD/–Leu–Trp–His growth medium supplemented with various concentrations of 3-AT. After 6~9 days of incubation in a 30 oC condition, the expression of reporter gene -galactosidase (lacZ) of yeast colonies were further confirmed by colony-lift filter assay. O-nitrophenyl-D-galactopyranoside (ONPG) was used as substrate to quantify LacZ activity, following an experimental procedure as described previously (Miller et al. 1972).. Bimolecular Fluorescence Complementation Assays Full-length CDS of CIA2 and CIL were PCR-amplified and inserted into the vectors pVYNE(R) and pVYCE(R) (Waadt et al. 2008). The BiFC constructs were sequence-verified and then transient expressed in onion epidermal cells by microparticle bombardment using the Biolistic PDS-1000/He 11.

(19) Particle Delivery System (Bio-RAD) as described previously (Varagona et al., 1992). For BiFC involving two plasmid constructs, 1.25 g of each plasmid which was 1:1 molar ratio of the two plasmids and give rise to 2.5 g total plasmid DNA should be mixed well for the cartridges preparation. Signals of 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei and enhanced yellow fluorescent protein (eYFP) were detected using a Leica TCS SP2 confocal microscope. Excitation of the UV laser was used to observe DAPI fluorescence (455-465 nm), and the Ar/KrAr laser (488 nm) was used to visualize eYFP fluorescence (525-545 nm).The DAPI (Molecular Probes) is the blue-fluorescent nuclear dye. PCR-amplified CIA2 FL and CIL FL fragments were also inserted into the vectors pSCYNE(R) and pSCYCE(R) (Waadt et al. 2008) and transiently expressed in Arabidopsis mesophyll protoplasts by polyethylene glycol (PEG) transformation (Yoo et al. 2007). Signals of cyan fluorescent protein (CFP) and green fluorescent protein (GFP) were detected by confocal microscope Ziess LSM880 after continuous low-light condition incubation at 22°C for 16 h. Excitation of the Diode laser was used to observe CFP fluorescence (406-470 nm) and the Argon2 laser (488 nm) was used to visualize GFP fluorescence (490-518 nm). The forward primer containing NLS sequence of simian virus 40 large T-antigen (Kalderon et al., 1984) was used to amplify the GFP sequence from p326GFP-nt plasmid and replaced the original GFP fragment by the Bam HI and Sma I sites to generate the NLS-GFP construct. The NLS-GFP construct was sequence-verified and used as a nuclear marker.. Phylogenetic analyses The 43-aa CCT motif of CIA2 was subjected to BLAST analysis (Altschul et al., 1997) for full-length homologous sequences in the TAIR database. MUSCLE software (Edgar RC, 2004a and 2004b) with default settings was used to compare the full length sequences. MAGA-X software (Kumar et al., 2018) was used to analyze and draw a phylogenetic tree based on a Neighbor-Joining and bootstrap methods.. 12.

(20) The N-terminal 200-aa sequences of Arabidopsis CIA2 and CIL were subjected to BLAST analysis (Altschul et al., 1997) for homologous sequences against the Gramene (www.gramene.org) and NCBI (National Center for Biotechnology Information; www.ncbi.nlm.nih.gov) databases. The phylogenetic tree of CIA2 and CIL homologous proteins was generated follow the process described above. MEME software (Bailey et al., 2009) was used to identify conserved motifs in full-length sequences of homologous proteins.. Reporter Gene Activity Analysis The 1.5kb promoter of CIA2 and CIL were ligated to Bam HI and Nco I sites of pJD301 plasmid containing Luciferase 2 (LUC2) gene (Wang et al., 2011). The 35Spro:GUS-NOS fragment digested from pBI121 (Invitrogen) was inserted into Hind III and EcoR I sites of pUC119 to generate pCS126 plasmid as internal control. Leaves from 18-day-old Col plants were used to this reporter assay. Plasmid DNA (2 g) was precipitated on gold particles (diameter, 1.0 m; 160 g) and then bombarded to 18-day-old Col leaves using the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad) with a pressure of 1,100 pound-force per square inch. Tissues were incubation at 22oC under 4 h light- 8 h dark- 4 h light condition. The protocol of GUS and LUC activity assay was described as Jefferson (1987). The intensity of LUC and GUS activity was detected by SpectraMax L and Gemini XPS spectrophotomer (Molecular Devices), respectively.. 13.

(21) Results Part I. Functional analysis of CIA2 and CIL. CIL and CIA2 show functional redundancy The cia2 mutants showed pale green phenotype and reduced chlorophyll a and b content; however, the leaf shape and size of the mutant plants were similar to those of wildtype plants (Col) (Figure 1). A previously study has shown that CIA2, driven by the 35S and CIA2 promoters, increased chlorophyll content of cia2 mutants to recover the phenotype to those of wildtype, indicating that CIA2 expression could rescue the cia2 mutant phenotype (Sun et al., 2009). Therefore, we used similar strategies to confirm the functional redundancy of CIL and CIA2. Vectors containing FL CIL CDS driven by the 35S or CIL promoter (-1504 ~ -1) were introduced into cia2 mutants via floral dipping, and four independent strains each of CILpro:HA-CIL/cia2 and 35Spro:HA-CIL/cia2 homozygous transgenic plants were obtained. The phenotype and chlorophyll content of the four CILpro:HA-CIL/cia2 strains were similar (Supplemental Figure S1); therefore, the transgenic strain CILpro:HA-CIL/cia2#1 was used as a representative. Similarly, the transgenic strain 35Spro:HA-CIL/cia2#1 was used as a representative (Figure 1). The phenotype and chlorophyll content of plants of the CILpro:HA-CIL/cia2#1 strain were similar to those of cia2 mutants; however, the phenotype and chlorophyll content of plants of the 35Spro:HA-CIL/cia2#1 strain were similar to those of Col plants, suggesting that adequate CIL expression could restore the cia2 phenotype to wildtype.. CIL and CIA2 show similar expression patterns CIA2 is expressed in leaves and young flower buds (Sun et al., 2001), but the expression pattern of CIL remains unknown. To understand the expression pattern of CIL in various tissues and at different developmental stages, we used RT-PCR and qRT-PCR. CIL was not expressed in roots, and its expression was restricted to aboveground tissues, including leaves and young flower buds, indicating that the expression patterns of CIL and CIA2 are similar (Figure 2). Therefore, based on 14.

(22) the similarity in expression patterns between CIL and CIA2 and the fact that adequate CIL expression restored the cia2 mutant phenotype, CIL is the homologous protein of CIA2 in Arabidopsis and CIL and CIA2 are complementary genes.. CIA2 and CIL co-regulate chloroplast development CIA2 has previously been reported to regulate the expression of chloroplast development-related genes (Sun et al., 2001; 2009). Owing to the similar expression patterns of CIA2 and CIL and their complementary functions (Figure 2), CIL may also be involved in the regulation of chloroplast developmental. To determine the function of CIL, cil mutants were used in this study. The phenotype of cil mutants did not differ from that of Col plants (Figure 3A). PCR was performed to confirm T-DNA insertion and verify the homozygous knockout of CIL in cil mutants. T-DNA (~5 kilobases, kb) was inserted into CIL between 328 and 725 basepair (bp) positions after the start codon to form an incomplete CIL transcript (Figure 3B and C). Furthermore, although the FL CIL CDS could not be amplified using PCR from the genomic DNA of cil mutants, the intersection fragment of CIL and T-DNA could be amplified, indicating that the generated cil mutants were homozygous (Figure 3D). Furthermore, we crossed the cia2 and cil mutants to obtain homozygous cia2/cil double mutants. The leaf size of cia2/cil double mutant was smaller than that of cia2 single mutant, and cia2/cil mutant showed a more severe pale-green phenotype than that of cia2 mutant (Figure 3A). Therefore, the functional defects caused by cil mutant could be observed once CIA2 is absent. CIA2 enhances the expression of several nuclear genes, including Toc75, Toc33, CPN10-II, and cpRPs (RPL11, RPL15, RPL18, RPL28, RPL29, and RPS6), to regulate chloroplast development (Sun et al., 2009). CIL expression pattern is similar to CIA2 expression pattern; therefore, qRT-PCR was performed to assess whether the genes described above are regulated by CIL as well. Toc75 and Toc33 expression showed no significant difference between cil mutants and Col plants; Toc75 expression was decreased to similar levels in cia2 and cia2/cil mutants; and the decrease extant of Toc33 expression was in a greater extent in cia2/cil double mutants than that in cia2 single mutants 15.

(23) (Figure 4A). The expression levels of other Toc genes showed no significant difference between Col and mutant plants, with the exception of Toc34 expression, which was slightly increased in cia2/cil mutants (Figure 4A), suggesting that the Toc34 up-regulation somewhat compensated Toc33 down-regulation. CPN10-II expression was decreased to a greater extent in cia2/cil double mutants than in cia2 single mutants, but the expression level was similar between cil mutants and Col plants. In contrast, CPN10-III expression showed no significant difference across the three mutants and wildtype plants (Figure 4B). Regarding the cpRPs, there was no difference in RPS6, RPL11, and RPL29 expression between cil mutants and Col plants. In cia2 and cia2/cil mutants, RPL15 expression was slightly increased but RPS6, RPL11, RPL15, and RLP29 expression was decreased (Figure 4C). These results indicate that CIL might regulate the expression of Toc75and cpRPs through CIA2 function and the expression of Toc33 and CPN10-II synergistically with CIA2; however, CIA2 and CIL are not involved in the regulation of CPN10-III. Therefore, CIL functions similar to CIA2 in the regulation of chloroplast development. To further confirm that CIA2 and CIL co-regulate chloroplast functions, chloroplast microstructures were observed in leaves of 10-day-old Col plants as well as cia2, cil, and cia2/cil mutants. The results indicated that the chloroplasts of cia2/cil double mutants showed fewer granal thylakoids and greater development abnormalities (Figure 5J-L). Thus, CIA2 and CIL co-regulate chloroplast development.. CIA2 and CIL co-regulate downstream genes The cia2/cil double mutants with smaller leaf size and paler green phenotype than the cia2 single mutants revealed functional redundancy of CIA2 and CIL (Figure 3A). To identify the genes downstream that are co-regulated by CIA2 and CIL, microarray analysis of two independent experiments was performed and genes that showed significant foldchange in expression of ≥ 1.5-fold compared to Col (P ≤ 0.05) were determined. A total of 103, 26, and 279 genes were down-regulated and 70, 20, and 277 genes were up-regulated in cia2, cil, and cia2/cil mutants, respectively (Figure 6A and B; Supplemental Tables S2 and S3). These results suggest that only 16.

(24) cia2/cil double mutants affected the genes co-regulated by CIA2 and CIL, further confirming their functional redundancy. In cia2/cil double mutants, the chloroplast development-related and nuclear transcription-related genes constituted 55.6% (155/279) and 30.0% (84/279) of the downregulated genes, respectively (Figure 6C; Supplemental Table S2). Plastid translation-related genes, such as cpRPs, photosystem genes, and chlorophyll biosynthetic genes constituted 23.2% (36/155), 16.1% (25/155), and 9% (12/155) of the chloroplast development-related genes (Figure 6D; Supplemental Table S2). The expression of these genes was much lower in cia2/cil double mutants than in cia2 single mutants and showed no obvious change in cil single mutants, indicating that these genes are primarily regulated by CIA2 and supplemented by CIL. During chloroplast development, the plastid genome of land plants expresses photosynthesis-related genes. This plastid gene expression involves two distinct types of RNA polymerases, namely NUCLEUS-ENCODED RNA POLYMERASE (NEP) and PLASTID-ENCODED RNA POLYMERASE (PEP) (Hajdukiewicz et al., 1997; Yu et al., 2014). PEPs form a complex involving interaction with at least 12 PEP-ASSOCIATED PROTEINs (PAPs) (Steiner et al., 2011; Pfalz and Pfannschmidt, 2013) and PLASTID RNA POLYMERASE SIGMA-SUBUNITs (SIGs) that are involved in PEP regulation (Schweer et al., 2010). All PAPs in the proteomes of a transcriptionally active chromosome of the chloroplast nucleoid have recently been identified (Majeran et al., 2012; Melonek et al., 2016). SIGF expression in cia2/cil double mutants is much lower than that in cia2 single mutants but slightly lower than that in cil single mutants (Table 1; Supplemental Table S2). In addition, the expression of PLASTID-TRANSCRIPTIONALLY ACTIVE 7 (pTAC7/PAP12), pTAC13, pTAC14/PAP7, and PLASTIDIAL THIOREDOXIN Z (TrxZ/PAP10) is much lower in cia2/cil double mutants than in cia2 single mutants but slightly down-regulation of that in cil single mutants. These results indicate loss of CIA2 and CIL functions affect plastid transcriptional genes, and these genes are primarily regulated by CIA2 and supplemented by CIL. Conversely, mitochondrial-related, nuclear transcription-related, and chloroplast development-related genes constituted 48.4% (134/277), 26.0% (72), and 17.3% (48/277) of the 17.

(25) up-regulated genes in cia2/cil double mutants, respectively (Figure 6E and F; Supplemental Table S3). In cia2/cil double mutants, the mitochondrial respiration- and oxidative stress-related genes constituted 14.9% (20/134) and 10.4% (14/134) of the mitochondria-related genes, respectively (Figure 6F; Table 2 and 3; Supplemental Table S3). The products of mitochondria-related genes, including TRANSLOCASE AT MITOCHONDRIALE INNER MEMBRANE (Tim) genes (e.g., Tim8, Tim13, and Tim23-3), mitochondrial respiratory chain complex I-related genes [e.g., PROHIBITIN 1 (PHB1), PHB2, and PHB4], and mitochondrial respiratory chain complex II-related genes (e.g., SUCCINATE DEHYDROGENASE 2), are encoded by the nucleus and transported to the mitochondria. Therefore, in the presence of chloroplast developmental defect and increased cellular oxidative stress, the nuclear-encoded mitochondria-related genes may be up-regulated to increase mitochondrial function for maintaining cell redox hemostasis and generating energy. On the other hand, functional chlorophyll biosynthesis involves the tetrapyrrole and methylerythritol 4-phosphate (MEP) pathways (Lange and Ghassemian, 2003). GLKs, including GLK1 and GLK2, are nuclear transcription factors that up-regulate the expression of photosynthesis-related and chlorophyll biosynthetic genes to promote chloroplast development (Waters et al., 2009). The chlorophyll biosynthetic genes regulated by GLKs are mainly involved in the tetrapyrrole pathway (Lange and Ghassemian, 2003; Waters al., 2009). In cia2/cil double mutants, the expression of GLK1 and its downstream regulated genes, such as GENOMES UNCOUPLED 4 (GUN4), PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA), and PORB, was significantly decreased, but it remained unchanged in cia2 or cil single mutants (Figure 7; Supplemental Tables S2 and S4). These results indicate that CIA2 and CIL co-regulate the expression of GLK1 and its regulated chlorophyll biosynthetic genes. In addition, CIA2 and CIL regulate other chlorophyll biosynthetic genes, including GLUTAMATE-1-SEMIALDEHYDE AMINOTRANSFERASE 2 (GSA2), COPROPORPHYRINOGEN-III OXIDASE HEMB1 (HEMB1), HEMF1, FLOURESCENT IN BLUE LIGHT (FLU), and 1-DEOXY-D-XYLULOSE 5-PHOSPHATE SYNTHASE (DXS) (Supplemental Tables S2 and S4). GSA2, HEMB1, and HEMF1 are tetrapyrrole pathway enzyme genes, and DXS is an MEP pathway enzyme gene (Lange and Ghassemian, 2003); 18.

(26) therefore, CIA2 and CIL likely co-regulate genes involved in all chlorophyll biosynthesis pathways and key regulators in the tetrapyrrole pathway (Figure 7; Supplemental Tables S2 and S4).. Chloroplast developmental defects in cia2/cil double mutants lead to ROS overproduction In plants, photosynthetic processes usually generate ROS; therefore, the reaction centers of photosystem I (PSI) and PSII in chloroplast thylakoids are the major ROS generation sites. In PSI, photoreduction of oxygen to superoxide radicals (O2-) and their subsequent disproportionation produces hydrogen peroxide (H2O2) and oxygen is catalyzed by SUPEROXIDE DISMUTASE (SOD). Thereafter, H2O2 is reduced to water, catalyzed by ASCORBATE PEROXIDASE (APX) (Mehler et al, 1951; Asada et al., 1974; Miyake and Asada, 1994; Asada, 2006). In PSII, singlet oxygen (1O2) generates in the reaction center of chlorophyll (Telfer et al., 1994; Hideg et al., 1998). ROS production levels in plant cells are low under normal conditions, but ROS production is enhanced in the presence of chloroplast dysfunction, and SOD expression increases (Myouga et al., 2008). Arabidopsis expresses three COPPER/ZINC-SODs (CSD1-3), three FE-SODs (FSD1-3), and one MANGANESE-SOD (MSD1). FSD1 is localized in the stroma of chloroplasts, plasma membrane, and mitochondrial membrane (Kliebenstein et al., 1998; Brugie`re et al., 2004; Marmagne et al., 2004; Peltier et al., 2006). FSD2, FSD3, and CSD2 are attached to the thylakoid membranes, whereas FSD2 and FSD3, also known receptively as PAP9 and PAP4, are evenly colocalized in chloroplast nucleoids (Ogawa et al., 1995; Myouga et al., 2008; Pfalz and Pfannschmidt, 2013). CSD1, CSD3, and MSD1 are respectively localized in the cytoplasm, peroxisomes, and mitochondria (Peck, 2005; Baginsky and Gruissem, 2006). FSD3/PAP4 was down-regulated by nearly 0.60-fold in cia2 and cia2/cil mutants, and CSD2 was up-regulated by 1.66-fold in cia2 mutants and by 2.2-fold in cia2/cil double mutants (Table 2; Supplemental Table S2). MSD1 was only up-regulated by approximately 1.74-fold in cia2/cil double mutants (Table 2; Supplemental Table S3). There was no significant difference in the expression levels of other SOD genes between 19.

(27) Col and mutant plants. Additionally, APXs showed no change in expression between Col and mutant plants. These results of microarray analysis suggest increased oxidative stress downregulates FSD3 and up-regulates CSD2, MSD1, and mitochondria-related genes. Histochemical staining was used to detect ROS levels in plants. cia2/cil double mutants, but not cia2 and cil single mutants, showed high O2- accumulation in leaves (Figure 8A). No H2O2 accumulation was detected in both Col and mutant plants (Figure 8B). Therefore, we hypothesize that chloroplast dysfunction increases cellular oxidative stress, resulting in the phenotype of cia2/cil double mutants, was proven.. CIA2 and CIL affect the flowering time of Arabidopsis The cil and cia2/cil mutants showed obvious delay in flowering under LD (Table 4). In addition, CIA2 and CIL interact with CO (Results in Part II), and CO is involved in the regulation of flowering time (Putterill et al., 1995). In this study, microarray analysis found changed in the expression of several genes related to flowering time regulation in cia2, cil, and cia2/cil mutants, including the positive regulators of flowering such as APETAL 1 (AP1), CAULIFLOWER (CAL), AGAMOUS-LIKE 6 (AGL6), and REPRESSOR OF UV-B PHOTOMORPHOGENESIS 2/EARILYFLOWERING BY OVEREXPRESSION, among others (Irish and Sussex, 1990; Ma et al., 1991; Kempin et al., 1995; Wang et al., 2011), as well as negative regulators of flowering such as BBX30, BBX31, and BBX32 (Graeff et al., 2016; Tripathi et al., 2017) (Table5; Supplemental Table S2 and S3). In addition, the expression of 13 genes that regulate flowering time was reduced in cia2, cil, or cia2/cil mutants, including positive regulators such as COL5 and LONG VEGETATIVE PHASE 1, among others (Hassidim et al., 2009; Xu et al., 2012), as well as negative regulators such as FLOWERING LOCUS C and MADS AFFECTING FLOWERING 4, among others (Rouse et al., 2002; Xu et al., 2013). Among these, the expression of AP1, CAL, and AGL6 was increased in cia2 and cia2/cil mutants but decreased in cil mutants. FLC expressing did not show significant change in cia2 single mutants, increased in cil single mutants, and decreased in cia2/cil double mutants, suggesting that CIA2 and CIL are involved in the regulation of flowering time; however, the 20.

(28) underlying mechanism remains unclear. Therefore, to confirm whether CIA2 and CIL affect flowering time, Col and mutant (cia2, cil, cia2/cil, and ppi1) strains were used. CIA2 transgenic strains (35Spro:CIA2/cia2 and CIA2pro:CIA2/cia2) were also used to observe flowering time under different daylight conditions. As CIA2 regulates Toc33 expression and affects chloroplast development, we explored whether chloroplast dysplasia affected flowering time using a ppi1 mutant strain. Under LD condition, the flowering time of cil and cia2/cil mutants was delayed by 2 days compared with that of other plants, and the average rosette leaf number of cil mutants was greater (by 3) than that of Col plants (Table 4). Under MD condition, cia2, cil, cia2/cil, and ppi1 mutants flowered 7 to 15 days later but 35Spro:CIA2/cia2 and CIA2pro:CIA2/cia2 mutants flowered 10 to 13 days earlier than Col plants (Table 4). Under SD condition, cia2, cil, cia2/cil, and ppi1 mutants flowered 4 to 44 days later but 35Spro:CIA2/cia2 and CIA2pro:CIA2/cia2 mutants flowered 7 to 13 days earlier than Col plants (Table 4). Based on these results, under MD and SD conditions, cia2, cil, and cia2/cil mutants showed delayed flowering, whereas 35Spro:CIA2/cia2 and CIA2pro: CIA2/cia2 mutants showed early flowering. Although the flowering time of 35Spro:CIL/cil plants were not determined, CIA2 and CIL might be involved in the regulation of flowering time given that these two genes are complementary. This would be further verified by expressing the CIL protein in transgenic plants. In addition, the Y2H experiments showed that CIA2 and CO proteins interaction with each other; therefore, CO interaction likely regulates the effects of CIA2 and CIL on flowering time. This would be further verified by crosses of cia2 and cil mutants with co mutants to establish double and triple mutants.. Part II. Analysis of protein structure and interactions of CIA2 and CIL. NLSs of CIA2 are located at 62-65 and 291-308 aa A previous study has shown that CIA2 is a nuclear protein (Sun et al., 2001). To identify the NLS of CIA2, the CIA2 amino acid sequence was resolved by P-SORT (psort.nibb.ac.jp; Nakai and 21.

(29) Kanehisa, 1992). In addition to the CCT motif (383-426 aa) as a possible NLS, CIA2 was predicted to contain three other NLSs: 56-59 aa (RKPR), 62-65 aa (RKRP), and a bipartite NLS at 291-308 aa (KKVVISGEKSNKKKKKKK). To verify the predicted NLSs of CIA2, the C terminus of GUS fusion constructs with CIA2 FL protein or different deletion fragments of CIA2 driven by the CaMV 35S promoter was transiently expressed in onion epidermal cells through microparticle bombardment. Cells transformed with GUS alone showed uniform staining throughout, and cells transformed with GUS fused to a potyvirus nuclear inclusion protein (Figure 9A and B) (Carrington et al., 1991; Varagona et al., 1992) or CIA2 FL (Figure 9C) showed nuclear localization. These data indicate that CIA2 is a nuclear protein, which corroborates the findings of the previously study (Sun et al., 2001). When cells expressed GUS fused to CIA2 with 1-61 aa fragment deletion, the nuclear localization of CIA2 fragment was not affected; however, when cells expressed GUS fused to CIA2 with 1-65 aa fragment deletion, CIA2 did not show nuclear localization and cells showed uniform staining throughout (Figure 9D and E). These results indicate that the second predicted NLS of CIA2 at 62-65 aa is required for nuclear localization. When cells expressed GUS fused to CIA2 with 291-435 aa fragment deletion, CIA2 did not show nuclear localization (Figure 9F); however, deletion of the 309-435 aa fragment of CIA2 did not affect its nuclear localization (Figure 9G). Therefore, the predicted bipartite NLS located at 291-308 aa is also essential for nuclear localization. In addition, cells transformed with GUS fused to CIA2 fragment with 309-435 aa fragment deletion, which removed the CCT motif, did not affect its nuclear localization, indicating the CCT motif of CIA2 does not function as an NLS (Figure 9G). Furthermore, cells transformed with GUS fused to CIA2 with 62-65 or 291-308 aa fragment deletion showed uniform staining throughout and no nuclear localization of CIA2 (Figure 9H and I). These results indicate that 62-65 and 291-308 aa fragments of CIA2 are essential for its nuclear localization and CIA2 must harbor both NLSs at same time to enter the nucleus. However, the CCT motif of CIA2 does not function as an NLS.. 22.

(30) CIL is a nuclear protein with an NLS located at 47-50 aa CIL is a homologous protein of CIA2 in Arabidopsis with a highly conserved amino acid sequence (Sun et al., 2001). The CIL protein was also predicted to be nuclear protein using P-SORT analysis, with three predicted NLSs: 41-44 aa (RRPR), 47-50 aa (RKRP), and 255-262 aa containing a lysine-rich domain. Using a similar strategy as CIA2, cells transformed with GUS fused to CIL FL showed nuclear localization, indicating that CIL is a nuclear protein (Figure 10C). When cells were transformed with GUS fused to CIL with 1-46 aa fragment deletion, nuclear localization of CIL was not affected (Figure 10D); however, when cells were transformed with GUS fused to CIL with 1-50 aa fragment deletion, they showed uniform staining throughout (Figure 10E). These results indicate that the second predicted NLS of CIL at 47-50 aa is essential for its nuclear localization, and the sequence of this NLS is the same as that of CIA2 NLS at 62-65 aa. When cells expressed GUS fused to CIL with 254-394-aa fragment deletion, which removed the CCT motif, CIL showed nuclear localization (Figure 10F). Therefore, removal of the CCT motif did not affect nuclear localization of CIL, which is similar to the result obtained for CIA2. Therefore, the 255-262 aa fragment containing a lysine-rich domain and the CCT motif of CIL do not function as NLSs (Figure 10F and H). The 47-50 aa fragment of CIL is a unique NLS, as confirmed by staining results of cells expressing GUS fused to CIL with 47-50 aa fragment deletion (Figure 10G). Based on the above results, the unique NLS of CIL is located at 47-50 aa (RKRP), and its sequence is identical to that of CIA2 NLS at 62-65 aa as well as to that of GLK1 and GLK2 NLSs (Fitter et al., 2002). GLKs are other transcription factors in Arabidopsis that increase the expression of genes involved in photosynthesis and regulation of chloroplast development (Waters et al., 2009). However, CIA2 contains two NLSs at 62-65 aa and 291-308 aa, which must both exist at same time to enter the nucleus, although the CCT motifs of CIA2 and CIL do not functions as an NLS.. CIA2 interacts with transcription factors regulating flowering CO has been shown to interact with ABI3 by CCT motif and regulate Arabidopsis flowering time 23.

(31) (Kurup et al., 2000) and the CCT motif of CO also shown to function as interaction region with NF-YB/NF-YC subunits (Wenkel et al., 2006; Tiwari et al., 2010); therefore, CIA2 may form complexes with different nuclear proteins by CCT motif to regulate cellular physiological mechanisms. To verify this, candidate nuclear proteins interacting with CIA2 were identified by Y2H screening. The C-terminal GAL4-binding domain (BD) was fused to the CIA2 protein as a bait, and the Arabidopsis cDNA library was used to screen interacting candidates. Because CIA2 is a nuclear protein, a total of 14 nuclear proteins were screened out (Table 6), which included both CIA2 and CIL, suggesting that CIA2 can form a protein dimer with itself and also with CIL to regulate the expression of chloroplast development-related genes. These candidates included six proteins, namely ABI3, ARR3, CO, NF-YB1, NF-YC1, and NF-YC9, involved in the regulation of flowering time, as described above, suggesting that CIA2 functions as a flowering time regulator. In addition to the flowering time regulators mentioned above, ARABIDOPSIS RESPONSE REGULATOR 3 (ARR3) and ARR4 have been reported to regulate the rhythmic expression of oscillator genes. The expression periods of oscillator genes such as CCA1, LHY, and TOC1 are prolonged in arr3/arr4 double mutants (Salomé et al., 2006). ARR3 and ARR4 are functionally redundant and belong to the response regulator family of proteins involved in cytokinin response signaling cascades, which includes 23 members: 10 type-A regulators, 11 type-B regulators, and 2 others (Kakimoto, 2003; Salomé et al., 2006). Plants with mutations in CCA1, LHY, and TOC1 show shortened the expression periods and confer the early-flowering phenotype (Schaffer et al., 1998; Green and Tobin, 1999; Makino et al., 2002). Prolonged expression periods of these genes in arr3/arr4 mutants suggest the roles of ARR3 and ARR4 in flowering regulation.. CIA2 and CIL interact with flowering regulators through their N-terminals To further confirm the interactions among CIA2, CIL, and the six flowering regulators and to identify the fragment of CIA2 that interacts with CIL and flowering regulators, CIA2 fragments of different length were constructed with pAS2-1 fused with GAL4-BD and FL CDSs of candidate 24.

(32) proteins were constructed with pACT2 containing the GAL4 activation domain (AD) for verification using Y2H screening. BD-fused CIA2 FL and CIA2 1-189 aa interacted with AD-linked ABI3, ARR3, CIA2, CIL, CO, NF-YB1, NF-YC1, and NF-YC9, whereas BD-CIA2 190-379 aa and BD-CIA2 383-435 aa fragments did not interact with these candidates (Figure 11A). BD-CIA2 330-435 aa fragment could interact with CIA2 and CIL but not with any other candidate. Moreover, CIA2 interacted with CIL, all flowering regulators, and itself through an N-terminal 1-189 aa fragment. In addition, CIA2 interacted with CIL and itself through a C-terminal 330-435 aa fragment. However, the 383-435 aa CCT motif of CIA2 alone was not sufficient to complete the interaction with any interacting candidate. As CIL is the CIA2 homologous protein in Arabidopsis, the conserved amino acid sequences of the two proteins are similar, making CIL a CIA2-interacting candidate; thus, a similar strategy as that for CIA2 was used to detect whether CIL interacts with CIA2-interacting proteins and similar results were obtained regarding the interactions. CIL, BD-fused CIL FL, and CIL 1-158 aa also interacted with CIA2-interacting proteins, except with CIA2 and itself. As opposed to CIA2, BD-CIL 290-394 aa interacted only with CIA2 and itself (Figure 12A). BD-CIL 154-337 aa and BD-CIL 337-394 aa could not interact with any candidate protein (Figure 12A). The N-terminal 1-158 aa fragment of CIL interacted with these CIA2-interacting proteins, except with CIA2 and itself. CIL interacted with CIA2 and itself through its C-terminal 290-394 aa fragment. However, the 337-394 aa CCT motif of CIL alone was not enough to complete interactions with any candidate (Figure 12A). LacZ activity was used to analyze the intensity of interaction; the interaction between CIA2 and CIL was higher than that among CIL and other interacting proteins as well as the interaction of CIL with itself to form a homodimer (Figure 11B and 12B). Moreover, the interaction between BD-CIA2 and AD-CIL is realized through the binding of CIL C-terminal with CIA2 N-terminal (Figure 11B and 12B). However, CIA2 also binds to the N- or C-terminal of itself to form a homodimer. To determine the effects of GAL4-BD and AD on experimental results, a domain swap 25.

(33) analysis was performed by fusing GAL4-BD with GAL4-AD at the N-terminals of CIA2 and CIL fragments, respectively. CIA2 interacts with itself at two positions—the N-terminal 1-189 aa fragment and the C-terminal 330-435 aa fragment containing the CCT motif (Figure 13A). CIA2 interacts with CIL through the conserved C-terminal 330-435 aa fragment, and CIL interacts with itself through the CIL 290-394 aa fragment (Figure 13A and 14A). Further confirmation of these interactions in swap experiments revealed that BD-CIA2 1-189 aa and BD-CIA2 330-435 aa fragments interacted with AD-CIA2 1-189 aa, AD-CIA2 330-435 aa, and AD-CIL 290-394 aa fragments (Figure 13A). BD-CIL 290-394 aa fragment interacted with AD-CIA2 330-435 aa and AD-CIL 290-394 aa fragments but not with AD-CIA2 1-189 aa fragment (Figure 14A). CIA2 383-435 aa and CIL 337-394 aa fragments containing the CCT motif alone did not interact with other fragments or with themselves after fusion with GAL4-BD or AD (Figure 13A and 14A). GUS activity quantitative analysis showed that the interaction between BD-CIA2 1-189 aa and AD-CIL 290-39 aa fragment was the strongest (1.4-fold) (Figure 13B). The interaction between BD-CIA2 1-189 aa and AD-CIA2 330-435 aa fragment was similar to that between positive controls (PCs) (Figure 13B). However, the interaction between BD-CIA2 1-189 aa and AD-CIA2 1-189 aa fragment or between BD-CIL 290-394 aa and AD-CIA2 330-435 aa fragment was weaker (0.4-fold) (Figure 13B and 14B). Thus, the N-terminal CIA2 1-189 aa fragment and the C-terminal conserved sequence of the CCT motif at CIA2 330-435 aa were the sites through which CIA2 interacted with itself; the C-terminal CIA2 330-435 aa fragment was the site through which CIA2 interacted with CIL; and CIL 290-394 aa was the site through which CIL interacted with itself. Based on these results, CIL could interact with CIA2, suggesting that CIA2 and CIL participated in the same regulatory mechanisms. CIA2 regulated the expression of genes involved in chloroplast development; thus, CIL may also participate in chloroplast development.. CIA2 interacts with CIL and itself in plant cells, as confirmed by bimolecular fluorescence complementation analysis Y2H screening proved that CIA2 interacts with CIL and itself, but it is necessary to know 26.

(34) whether this interaction exists in plant cells. BiFC analysis can detect protein–protein interactions in plant cells (Waadt et al., 2008); therefore, this method was used to conform the interaction between CIA2 and CIL in plant cells. CIA2 FL and CIL FL fragments were separately fused to nEYFP and cEYFP at the N-terminal and transiently expressed in onion epidermal cells via particle bombardment. Laser was to excite the eYFP signal, which was compared with the signal displayed by the nucleic acid dye DAPI to confirming the position of interaction. nEYFP-CIA2 interacted with cEYFP-CIA2 and cEYFP-CIL in onion epidermal cells (Supplemental Figure S2A and B). In addition, nEYFP-CIL interacted with cEYFP-CIA2 and cEYFP-CIL in onion epidermal cells (Supplemental Figure S2D and E). Moreover, all interaction complexes were localized in the nucleus. Additionally, CIA2 FL and CIL FL fragments were separately fused to nCFP and cCFP at the N-terminal and transiently expressed in Arabidopsis protoplasts via PEG transformation (Yoo et al., 2007). Using laser to excite the CFP signal and nuclear label GFP (NLS-GFP) to reveal the position of interaction, the interaction of nCFP-CIA2 with cCFP-CIA2 and cCFP-CIL in the nucleus of Arabidopsis protoplast was confirmed (Figure 15F and G). Similar results were obtained for the interaction of nCFP-CIL with cCFP-CIA2 and cCFP-CIL in the nucleus of Arabidopsis protoplast (Figure 15H and I). Therefore, the interaction between CIA2 and CIL was confirmed in plant cells, and CIA2 and CIL were further proved to be nuclear proteins.. CIA2 and CIL interact with CO through the C-terminal CCT motif of CO Among the CIA2-interacting candidates, CO is another nuclear transcription factor with a CCT motif. Using the strategy similar to that for CIA2 and CIL, the interaction between CIA2 and CO was verified and interaction fragment was identified. Previous studies have shown that CO interacts with NF-YBs and NF-YCs through its C-terminal CCT motif and binds to the COREs and CCAAT box on the FT promoter to increase FT expression, thereby promoting plant flowering (Wenkel et al., 2006; Tiwari et al., 2010; Cao et al., 2014; Xu et al., 2016). Furthermore, CO generates a variant through alternative splicing and translates a CO FL protein (274 aa) without the C-terminal CCT 27.

(35) motif. This C-terminally truncated CO interacts with the original CO FL protein (373 aa) through the N-terminal to cause CO FL protein degradation, thus delaying flowering (Gil et al., 2017). To identify the position of interaction between CIA2 and CO and speculate the role of CIA2 in flowering regulation, CO fragments of different lengths were constructed and fused to BD or AD at the N-terminal for verifying the interactions of these fragments with CIA2. BD-linked CIA2 FL and CIA2 1-189 aa fragment interact with AD-linked CO FL, CO 181-373 aa, and CO 298-373 aa (comprising the CCT motif) fragments but not with AD-CO 1-274 aa fragment lacking the CCT motif (Figure 16A). Other CIA2 fragments such as BD-linked CIA2 190-379 aa, CIA2 330-435 aa, and CIA2 379-435 aa do not interact with other AD-fused CO fragments. As CO interacts with itself through its N-terminal B-box domain (Gil et al., 2017), the CO 1-274 aa fragment was replaced with a CO 1-115 aa fragment containing the B-box domain and domain swap analysis was used to further confirm the position of interaction between CIA2 and CO. BD-CO FL and BD-CO 181-373 aa interact with AD-CIA2 FL and AD-CIA2 1-189 aa, respectively, and, surprisingly, also with AD-CIA2 330-435 aa and AD-CIA2 379-435 aa, which harbor the CCT motif alone (Figure 17A); however, the BD-CO 298-373 aa fragment containing the CCT motif alone does not interact with any AD-fused CIA2 fragment. In addition, BD-CO 1-115 aa and BD-CO 298-373 aa do not interact with any AD-fused CIA2 fragment. In GUS activity analysis, the intensity of interaction of BD-CIA2 FL with AD-CO 181-373 aa, and AD-CO 298-373 aa fragments was respectively 1.23- and 1.22-fold higher than that of the PC group. The intensity of interaction between BD-CIA2 FL and AD-CO FL (0.63-fold) was lower than that of the PC group (Figure 16B). The intensity of interaction of BD-CIA2 1-189 aa with AD-CO 181-373 aa (0.95-fold) and AD-CO 298-373 aa (0.79-fold) was higher that of its interaction with AD-CO FL (0.27-fold) (Figure 16B). These results indicate that the N-terminal of BD-CIA2 interacts with the C-terminal CCT motif of AD-CO. In domain swap analysis, the intensity of interaction between BD-CO FL and AD-CIA2 330-435 aa (2.25-fold) was higher than that between BD-CO FL and AD-CIA2 1-189 aa (1.37-fold) as well as between BD-CO FL and AD-CIA2 361-435 aa (1.44-fold) (Figure 17B). Moreover, the intensity of interaction of BD-CO 181-373 aa 28.

(36) with AD-CIA2 330-435 aa (0.68-fold) and AD-CIA2 361-435 aa (0.32-fold) was higher than that of its interaction with AD-CIA2 1-189 aa (0.13-fold) (Figure 17B). The C-terminal CCT motif of BD-CO interacts with the N-terminal and C-terminal CCT motif of AD-CIA2. However, the intensity of interaction between the C-terminal fragment of BD-CO and the C-terminal fragment of AD-CIA2 was higher than that between the C-terminal of BD-CO and the N-terminal of AD-CIA2 (Figure 17B). Therefore, the C-terminal CCT motifs of CIA2 and CO are essential for protein– protein interaction. The results of interaction between CIL and CO were similar to those of interaction between CIA2 and CO. BD-CIL FL and BD-CIL 1-158 aa fragment interact with AD-CO FL, AD-CO 181-373 aa, and AD-CO 298-373 aa (comprising the CCT motif) fragments but not with AD-CO 1-274 aa fragment lacking the CCT motif (Figure 18A). Other fragments of CIL such as BD-CIL 155-289 aa, BD-CIL 290-394 aa, and BD-CIL 337-394 aa do not interact with other CO fragments (Figure 18A). In domain swap analysis, BD-CO FL and BD-CO 181-373 aa interact with AD-CIL FL and AD-CIL 1-158 aa as well as with AD-CIL 294-394 aa and AD-CIL 330-394 aa comprising the CCT motif; however, BD-CO 1-115 aa fragment containing the B-box domain and BD-CO 298-373 aa fragment containing the CCT motif do not interact with any AD-fused CIL fragment (Figure 19A). In GUS activity analysis, the intensity of interaction of BD-CIL FL or BD-CIL 1-158 aa with AD-CO FL or other AD-fused CO-truncated fragments ranged from 0.17- to 0.38-fold and was lower than the intensity of interaction of BD-CIA2 FL or BD-CIA2 1-189 aa with AD-CO FL or other AD-fused CO-truncated fragments (Figure 16B and 18B). However, the intensity of interaction between BD-CO FL and AD-CIL 1-158 aa (0.79-fold) was similar to that between BD-CO FL and AD-CIA2 379-435 aa (0.75 fold) but lower than that between BD-CO FL and AD-CIL 290-394 aa fragment (1.25-fold) (Figure 17B and 19B). These results indicate that the C-terminal CCT motifs of BD-CO and AD-CIL interact with each other, similar to the interaction between BD-CO and AD-CIA2. However, the intensity of interaction of the C-terminal of BD-CO with the N-terminals of AD-CIA2 and AD-CIL was similar to that of its interaction with the N-terminals of AD-CIA2 and AD-CIL (Figure 17B and 19B). 29.

(37) Based on the above interactions among CO, CIA2, and CIL, BD-linked CIA2 and CIL bind to the C-terminal CCT motif of CO through the N-terminal 1-189 aa fragment of CIA2 and 1-158 aa fragment of CIL but do not interact with the N-terminal B-box domain of CO. These results also suggest that CIA2 and CIL are involved in CO-mediated increase in FT expression to promote flowering. Furthermore, protein interactions targeting the CCT motif may be affected by combinatorial deconstruction and the length of binding fragments (Galletta and Rusan, 2015), as evidenced by the presence of interaction of BD-CIA2 FL and BD-CIA2 1-189 aa with AD-CO 298-373 aa but lack of interaction of BD-CO 298-373 aa with AD-CIA2 FL and AD-CIA2 1-189 aa in this study. Moreover, BD-CO FL interacts with AD-CIA2 330-435 aa fragment but not with AD-CIA2 379-435 aa fragment. Similar trends were also observed for the interaction between CIL and CO.. Part III. Analysis of the evolutionary relationships between CIA2 and CIL Arabidopsis CCT protein family Cockram et al. (2012) used biological information to classify proteins containing the CCT motif into three groups: COLs, PRRs, and CMFs. BLAST (Altschul et al., 1997) searches revealed CCT motif-containing proteins in Poaceae members, including rice (Oryza sativa), barley (Hordeum vulgare), and sorghum (Sorghum bicolour). Arabidopsis expresses 15 CMF proteins, including CIA2 and CIL, which are named CMF14 and CMF9, respectively. Previous studies have indicated that CIA2 affects chloroplast development by regulating the expression of Toc33, Toc75, CPN10, and cpRPs (Sun et al., 2001; 2009). ACTIVATOR OF SPOMIN::LUC2 (ASML2, also CMF8) regulates the expression of sugar synthesis genes (Masaki et al., 2005); however, little is known regarding the functions of other CMF proteins. For identifying the evolutionary relationships between CIA2, CIL, and other CMF proteins in Arabidopsis and further understanding the physiological mechanisms in which CIA2 and CIL are involved, bioinformatics was used to analyze the genetic relationships among Arabidopsis CCT proteins. The 43 aa sequence of the CIA2 CCT motif was blasted in the TAIR database, and the full-length amino 30.