Arabidopsis CIA2 and CIL have distinct and overlapping functions in regulating chloroplast and flower development.

Arabidopsis CHLOROPLAST IMPORT APPARATUS 2 (CIA2) and its paralogous protein CIA2-LIKE (CIL) are nuclear transcription factors containing a C-terminal CCT motif. CIA2 promotes the expression of nuclear genes encoding chloroplast-localized translocons and ribosomal proteins, thereby increasing the efficiency of protein import and synthesis in chloroplasts. We have previously reported that CIA2 and CIL form a homodimer or heterodimer through their C-terminal sequences and interact with other nuclear proteins, such as CONSTANS (CO), via their N-terminal sequences, but the function of CIL had remained unclear. In this study, we verified through transgenic cia2 mutant plants expressing the CIL coding sequence that CIL is partially functionally redundant to CIA2 during vegetative growth. We also compared phenotypes and gene expression profiles of wildtype Col-0, cia2, cil, and cia2/cil mutants. Our results indicate that CIA2 and CIL coordinate chloroplast biogenesis and function mainly by upregulating the expression of the nuclear factor GOLDEN2-LIKE 1 (GLK1) and chloroplast transcription-, translation-, protein import-, and photosynthesis-related genes, with CIA2 playing a more crucial role. Furthermore, we compared flowering phenotypes in single, double, and triple mutant plants of co, cia2, and cil. We found that CIA2 and CIL participate in modulating long-day floral development. Notably, CIA2 increases flower number and height of the inflorescence main axis, whereas CIL promotes flowering.

INTRODUCTION

The main function of chloroplasts is photosynthesis, using solar energy to convert carbon dioxide into organic compounds. They also carry out a number of other functions, including synthesis of amino acids, pigments, lipids, and phytohormones, as well as storage of starch/oil compounds and stress and immune responses during plant growth and development. Chloroplasts are believed to have evolved through endocytosis of cyanobacteria. Most cyanobacterial genes were subsequently amalgamated into the host nuclear genome (Martin et al., 2002). Chloroplasts harbor about 3,000 proteins in higher plants, of which ~100 are encoded by the chloroplast genome and the remainder by the nuclear genome (Paila, Richardson, & Schnell, 2015). Therefore, proteins involved in chloroplast development and other vital functions require cooperation between the nuclear and chloroplast genomes.

Nucleus‐encoded chloroplast proteins cross the plastid double‐membrane posttranslationally. Apart from a few outer‐envelope membrane proteins, most of the precursor proteins contain a transit peptide (TP) at their N‐terminus. The chloroplast translocon complex, comprised of Translocon at Outer/Inner envelope membrane of Chloroplast (Toc/Tic), recognizes TPs and facilitates transport of these precursor proteins into the chloroplast stroma. The TPs are removed in the stroma by peptidase, enabling proper folding of mature proteins with the assistance of chaperone proteins (Paila, Richardson, & Schnell, 2015).

Screening for Arabidopsis point‐mutation lines defective in chloroplast protein import identified the chloroplast import apparatus 2 (cia2) mutant. This cia2 mutant is denoted cia2‐1 hereafter because several such allelic mutants are now available from the Arabidopsis Biological Resource Center (ABRC). Compared with wildtype Col‐0, cia2‐1 exhibits a pale‐green phenotype and a ~ 50% reduction in total chlorophyll and carotenoid contents. Nevertheless, leaf size and shape are similar to or slightly smaller than Col‐0 at the same developmental stage. In addition, cia2‐1 displays an ~50% reduction in chloroplast protein import efficiency relative to Col‐0 (Sun, Chen, Lin, & Li, 2001). Moreover, gene transcript levels of 2 translocons (i.e., TOC33 and TOC75), 1 chaperonin (i.e., CPN10), and 17 chloroplast ribosomal proteins (cpRP) are at least 1.5‐fold lower in cia2‐1 than wild‐type, as revealed by microarray and reverse transcription‐polymerase chain reaction (RT‐PCR) analyses (Sun, Huang, & Chang, 2009). Thus, CIA2 is thought to upregulate protein import and synthesis efficiency in chloroplasts.

Arabidopsis transcription factors CIA2 and CIA2‐LIKE (CIL) comprise 435 amino acids (aa) and 394 aa, respectively (Sun, Chen, Lin, & Li, 2001). Both host a 43‐aa CCT motif [CONSTANS (CO), CO‐LIKE (COL), and TIMING OF CAB EXPRESSION 1 (TOC1)] (Putterill, Robson, Lee, Simon, & Coupland, 1995) at their C terminus (Sun, Chen, Lin, & Li, 2001). CCT motifs function as a nuclear localization signal (NLS) and as an interaction target for CO, COL and TOC1 (Robson et al., 2001; Strayer et al., 2000). However, the CCT motif of CIA2 and CIL has been characterized as a protein–protein interaction region but not as an NLS (Yang & Sun, 2020).

The CCT family of proteins all possesses a CCT motif at their C terminus, and they can be classified into three subgroups based on their N‐terminal structure: (1) COL, having one or two zinc‐finger B‐box domains; (2) pseudo‐response regulators (PRR), having a PRR domain; and (3) the CCT‐motif family (CMF), hosting unknown domains (Cockram et al., 2012). COL proteins are mainly involved in circadian clock and photoperiodic flowering regulation (Putterill, Robson, Lee, Simon, & Coupland, 1995; Shim, Kubota, & Imaizumi, 2017). PRR proteins modulate circadian rhythms and light‐signaling transduction (Liu, Newton, Liu, Shiu, & Farré, 2016; Nakamichi et al., 2010). The functions of CMF proteins seem diverse and have yet to be fully ascertained. For example, five Arabidopsis CMF proteins have been characterized. CIA2 and CIL are involved in regulating chloroplast development (Gawroński et al., 2021; Li et al., 2021; Sun, Chen, Lin, & Li, 2001; Sun, Huang, & Chang, 2009) and possibly flower development (Yang & Sun, 2020). ACTIVATOR OF SPORAMIN::LUC 2 (ASML2) differentially regulates the expression of sugar‐inducible genes (Masaki et al., 2005). ORBITALLY MANIFESTED GENE 1 (OMG1) regulates pollen tube formation and reactive oxygen species (ROS) homeostasis (Sng, Kolaczkowski, Ferl, & Paul, 2018). FITNESS also regulates ROS homeostasis and enhances expression of three antioxidant and defense genes (Osella et al., 2018). Similarly, rice (Oryza sativa) OsCMF8 (or GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7, Ghd7), and OsCMF1 have been reported to modulate flowering time, plant height, and grain number (Xue et al., 2008). In barley (Hordeum vulgare), HvCMF7 (or ALBOSTRIANS, HvAST) and HvCMF3 (or HvAST‐LIKE, HvASL) participate in modulating chloroplast translation (Li et al., 2019).

Unlike known CCT proteins, the NLSs of CIA2 and CIL are not localized within the CCT motif. CIA2 has two functional NLSs localized at residues 62–65 and 291–308. Truncated CIA2 containing a single NLS is primarily localized in the cytoplasm, but the presence of both NLSs ensures CIA2 entry into the nucleus. In contrast, CIL only possesses one NLS located at residues 47–55 (Yang & Sun, 2020). Twelve CIA2‐interacting proteins have been identified by screening a yeast two‐hybrid (Y2H) Arabidopsis cDNA library, including itself, CIL, ABSCISIC ACID INSENSITIVE 3 (ABI3), ARABIDOPSIS RESPONSE REGULATOR 3 (ARR3), CO, and three subdomains of nuclear factor Y subunit (NF‐Y; Yang & Sun, 2020). CO and the NF‐Y protein complex bind to the promoter of FLOWERING LOCUS T (FT) to increase FT expression and induce flowering (Tiwari et al., 2010; Wenkel et al., 2006). ABI3 and ARR3 are partially involved in regulating flowering time (Hong, Lee, Lee, Kim, & Ryu, 2019; Kurup, Jones, & Holdsworth, 2000), as well as signaling cascades in response to rhythmic expression of oscillator genes (Kakimoto, 2003; Salomé, To, Kieber, & McClung, 2006). Thus, CIA2 and CIL evidentially interact with proteins involved in controlling flowering, strongly implying a role for CIA2 and CIL in flowering development and warranting validation.

In this study, we stably overexpressed the coding sequence (CDS) of Arabidopsis CIL in the cia2‐1 mutant, driven by endogenous CIL or cauliflower mosaic virus 35S (35S) promoters to determine if CIL is functionally redundant to CIA2. Previous Y2H analysis has demonstrated CIA2 and CIL interaction (Yang & Sun, 2020). Herein, we further confirm CIA2‐CIL interaction in the nuclei of mesophyll cells by means of biomolecular fluorescence complementation (BiFC) assays. We have also identified additional cia2 and cil mutant alleles for which we have characterized phenotypes, pigment contents, plant weight, chloroplast size, and structure, as well as steady‐state transcript and protein levels of specific nuclear genes encoding chloroplast‐localized proteins in single and double mutants relative to Col‐0. Furthermore, we used microarray analyses to explore downstream targets of CIA2 and CIL. Since CIA2 and CIL both interact with flowering regulator CO (Yang & Sun, 2020), we assessed the flowering time and flower phenotypes of Col‐0, double and triple cia2, cil, and co mutants. Overall, we present a thorough functional study of CIA2 and CIL in Arabidopsis. Based on our findings, we propose that both CIA2 and CIL not only regulate chloroplast function but also contribute to flower development in distinct ways. Specifically, CIA2 is a primary regulator of flower number and the height of the inflorescence main axis, whereas CIL is a principal flowering‐time controller.

MATERIALS AND METHODS

Plant materials and growth conditions

Arabidopsis thaliana ecotype Columbia‐0 (Col‐0) was used as wildtype in this study. The cia2‐1 mutant was isolated from our previous screening (Sun, Chen, Lin, & Li, 2001). Col‐0‐background mutants cia2‐2 (SALK_004037), cil‐3 (SALK_068498), and cil‐4 (CS925818) were obtained from the ABRC (Alonso et al., 2003). The co‐1 (CS3387, Landsberg background) mutant was obtained from the ABRC and backcrossed to Col‐0 for more than five generations to generate a Col‐0‐background co‐1 mutant (referred to as co in this study). Double and triple mutants were generated by crossing. Transgenic plants P :CIA2 (cia2‐1 background) and P :CIA2 (cia2‐1 background) have been reported previously (Sun, Huang, & Chang, 2009). The P :CIL (cia2‐1 background) and P :CIL (cia2‐1 background) transgenic plants were generated in this study. Seeds were surface‐sterilized with 25% (v/v) bleach and soaked in water for 48 h at 4°C, then sown on 1X Murashige and Skoog agar medium with Gamborg's vitamins and 1% (w/v) sucrose, or grown on soil. Plants were grown at 22°C under long‐day conditions (16 h/8 h light/dark cycle) under fluorescent light of 80–100 μmol m−2 s−1 for various numbers of days.

Plasmid construction and plant transformation

Sequences of the CaMV 35S promoter and NOS terminator from vector pBI121 (Invitrogen) and a 2xHA epitope were ligated into the binary vector pPZP221 (Hajdukiewicz, Allison, & Maliga, 1997) to create plasmid pCS188. The CIL CDS was PCR‐amplified from Col‐0 cDNA using specific primers (CIL‐BamHI‐F and CIL‐SmaI‐R, supporting information Table S1), before being in‐frame fused after the 2xHA tag of pCS188 to create pCS190. The upstream sequence (1.5 kilobases) of CIL (promoter and 5′ untranslated region) was PCR‐amplified from Col‐0 genomic DNA using specific primers (CILpro‐HindIII‐F and CILpro‐SpeI‐R, supporting information Table S1) to replace the 35S promoter sequence in pCS190, creating pCY158. The pCS190 or pCY158 plasmids were transformed into cia2‐1 individually using an Agrobacterium GV3101‐mediated floral dipping method (Clough & Bent, 1998). Transformants were selected on agar plates containing 30 μg/ml G418 and verified by PCR using construct‐specific primers. Western blotting using antibodies against the HA tag (Santa Cruz Biotechnology) was performed to ensure specific interaction between HA antiserum and HA‐CIL fusion proteins in transgenic plants. These transgenic lines harboring pCS190 or pCY158 were named P :CIL(cia2‐1) and P :CIL(cia2‐1), respectively. All transgenic plants were selected for more than two generations, and only homozygous transgenic plants (T3) were used in our experiments.

Mesophyll protoplast transformation and BiFC assay

Full‐length CDSs of CIA2 and CIL were PCR‐amplified from Col‐0 cDNA template using specific primers (CIA2‐XhoI‐F and CIA2‐SmaI‐R for CIA2, CIL‐XhoI‐F and CIL‐SmaI‐R for CIL, supporting information Table S1) and then inserted into the pSCYNE(R) or pSCYCE(R) vectors (Waadt et al., 2008). The forward primer containing the NLS sequence of simian virus 40 large T‐antigen (Kalderon, Roberts, Richardson, & Smith, 1984) was used to amplify the GFP sequence from p326GFP‐nt plasmid, and it replaced the original GFP fragment between the Bam HI and Sma I sites to generate the NLS‐GFP construct, which we used as a nuclear marker. The resulting plasmids were transiently expressed in Arabidopsis mesophyll protoplasts isolated from 18‐day‐old mature leaves according to a polyethylene glycol (PEG) transformation protocol described previously (Yoo, Cho, & Sheen, 2007). After 16‐h incubation at 22°C under continuous low‐light conditions, signals of cyan fluorescent protein (CFP) and green fluorescent protein (GFP) were detected by Ziess LSM880 confocal microscopy. CFP fluorescence (406–470 nm) and GFP fluorescence (490–518 nm) were excited by Diode and Argon2 lasers (488 nm), respectively.

Measurement of chloroplast areas

Chloroplasts were isolated from the seventh leaf of 28‐day‐old plants according to the method of Grabsztunowicz and Jackowski (2013). Purified chloroplasts were visualized by Ziess LSM880 confocal microscopy. The areas of >500 chloroplasts from each plant were analyzed in ZEN software (blue edition, Carl Zeiss GmbH).

Quantification of pigment content

Leaves (21 days old) were weighed, immediately frozen in liquid N2, and then ground in 80% acetone. After centrifugation to remove debris, absorbance of the tissue supernatant was measured at 470, 645, and 663 nm using a Multiskan GO spectrophotometer (Thermo Fisher Scientific). Chlorophyll and carotenoid concentrations were calculated as reported previously (Lichtenthaler, 1987). All measurements were performed in triplicate using three independent leaf samples.

RNA isolation and quantification of transcript levels

Total RNAs were isolated from 10‐day‐old to 28‐day‐old plants using TRIzol solution (Invitrogen) as per Chomczynski and Sacchi (1987). First‐strand cDNAs were synthesized via Moloney murine leukemia virus RNase H2 reverse transcriptase (Promega) using 5 μg of total RNAs as template and oligo dT19‐N as primer. Transcript levels for various genes were analyzed by RT‐PCR or real‐time quantitative RT‐PCR (RT‐qPCR). Primers specific for each gene are described in the supporting information Table S1. For RT‐PCR assays, these gene‐specific primers were used to amplify each transcript by means of 25 cycles with first‐strand cDNA as template. PCR products were electrophoresed on 1.5% agarose gels, before visualization using ethidium bromide, and UV light. For RT‐qPCR analysis, cDNA and specific primers were employed with 2X SYBR FAST qPCR reagents (KAPA Biosystems) to detect each transcript in a StepOne Plus thermal cycler (Applied Biosystems) by means of programs recommend by the manufacturer (3 min at 95°C, 40 cycles of 95°C for 3 s, and 60°C for 30s). We adopted the comparative CT method described by Livak and Schmittgen (2001) to determine relative gene expression, with expression of UBQ10 acting as an internal control. Mean values from three independent experiments were determined.

Transmission electron microscopy

True leaves of 10‐day‐old Col‐0, cia2‐1, cil‐3, and cia2‐1/cil‐3 seedlings were sliced and fixed at room temperature for 4 h in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1‐M sodium phosphate buffer, pH 7. After rinses, the samples were postfixed in 1% OsO4 in the same buffer for 4 h at room temperature. After three rinses, samples were dehydrated in an acetone series, embedded in resin, and sectioned with a Leica EM UC6 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 Gatan Digital Micrograph acquisition software.

In vitro protein import into chloroplasts and protein analyses

The protocol for isolating intact chloroplasts from 21‐day‐old leaves was described previously (Sun, Chen, Lin, & Li, 2001). [35S]‐methionine‐labeled proteins were synthesized through in vitro transcription and in vitro translation of wheat germ extracts (TnT Quick Coupled Transcription/Translation System, Promega) according to the manufacturer's specifications. Labeled proteins were separated by 10% SDS‐PAGE and quantified with a PhosphorImager SP system (Molecular Dynamics). For immunoblot analysis, 25 μg of chloroplast proteins were fractionated by 4%–12% SDS‐PAGE, blotted onto polyvinylidene difluoride membrane, and hybridized with anti‐Toc33, anti‐RbcL, anti‐RbcS, or anti‐GS2 antiserum (Agrisera, Sweden) according to manufacturer's suggestion. Western blot signals were quantified using a LAS 4000 image analyzer (GE Healthcare) and calibrated based on the signals for Col‐0.

Microarray analysis

Total RNAs (0.2 μg) from 18‐day‐old plants were amplified using a Low Input Quick‐Amp Labeling kit (Agilent Technologies) and labeled with Cy3 (CyDye, Agilent Technologies). Cy3‐labled cRNA (1.65 μg) was fragmented at 60°C for 30 min. Individual fragmented labeled cRNA was hybridized to an Agilent Arabidopsis V4 4 × 44 K Microarray (Agilent Technologies) at 65°C for 17 h, conducted in duplicate. After washing, the microarray was scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm for Cy3. The array image was analyzed with default settings in Feature Extraction software version 10.7.1.1.

The microarray data were normalized by log conversion, and expression ratios were determined in Genespring GX 11.8 software (Agilent Technologies). The threshold for fluorescence intensity was set at 100. Genes displaying at least a 1.5‐fold and statistically significant (P < .05) change in both microarray slides are listed in supporting information Tables S2 and S3. Gene annotations were compiled from The Arabidopsis Information Resource (Rhee et al., 2003) and Gene Ontology Consortium (2001), and subcellular localizations were predicted based on SUBAcon of the SUBA3 database (Hooper et al., 2014; Tanz et al., 2013).

Histochemical staining to detect ROS

Plants were grown on agar plates for 18 days under normal growth conditions and then treated under fluorescent light (200 μmol m−2 s−1) for 1 h. For superoxide anion (O2 −) detection, detached rosette leaves were vacuum‐infiltrated with 3.5 mg/ml nitroblue tetrazolium (NBT) (N6876, Sigma‐Aldrich) staining solution in 10‐mM potassium phosphate buffer containing 10‐mM sodium azide. After vacuum infiltration, stained leaves were bleached in boiling 100% acetic acid‐glycerol‐ethanol (1/1/3) (v/v/v) solution for 5 min (Ramel, Sulmon, Bogard, Couée, & Gouesbet, 2009).

Accession numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this study are as follows: AGL6 (AT2G45650), AP1 (AT1G69120), AP3 (AT3G54340), CAL (AT1G26310), CIA2 (AT5G57180), CIL (AT4G25990), CPN10‐II (AT2G44650), CPN10‐III (AT3G60210), CO (AT5G15840), FLC (AT5G10140), FT (AT1G65480), GLK1 (AT2G20570), GLK2 (AT5G44190), GNC (AT5G56860), KNATM (AT1G14760), PHYA (AT1G09570), PHYB (AT2G18790), PHYD (AT4G16250), PIF4 (AT2G43010), PKS4 (AT5G04190), RPS6 (AT4G31700), RPL11 (AT1G32990), RPL15 (AT3G25920), RPL28 (AT2G33450), RUP2 (AT5G23730), SEP2 (AT3G02310), SPA2 (AT4G11110), TOC33 (AT1G02280), TOC34 (AT5G05000), UBQ10 (AT4G05320), VRN2 (AT4G16845), and WOX1 (AT3G18010).

RESULTS

Partial functional redundancy between CIA2 and CIL

That CIA2 and CIL that are functionally redundant in Arabidopsis are supported by three lines of evidence presented previously (Sun, Chen, Lin, & Li, 2001): (1) CIA2 and CIL share high protein sequence identity; (2) both CIA2 and CIL display similar expression patterns, that is, mostly in green tissues and barely detectable in roots; and (3) CIL transcript levels are enhanced in the cia2‐1 mutant, perhaps to compensate for CIA2 loss. To verify if they exert similar functions, we expressed CIA2 or CIL in cia2‐1. Transgenic plants expressing the CIA2 CDS under control of the endogenous 1.5‐kb CIA2 upstream region or 35S promoter in the cia2‐1 background were named P :CIA2(cia2‐1) and P :CIA2(cia2‐1), respectively (Sun, Huang, & Chang, 2009). Both types of transgenic plant rescued the pale‐green phenotype of cia2‐1 (Figure 1a). Furthermore, though chlorophyll and carotenoid levels in cia2‐1 were reduced to half those of Col‐0, they were fully restored in these transgenic lines (Figure 1b). Finally, reductions in mature plant weight and chloroplast area of cia2‐1 were recovered in the transgenic plants (Figure 1c, d). These results confirm that cia2‐1 defects can be rescued by expressing functional CIA2 in either P :CIA2(cia2‐1) or P :CIA2(cia2‐1).

FIGURE 1

The functions of CHLOROPLAST IMPORT APPARATUS 2 (CIA2) and CIA2‐LIKE (CIL) are partially redundant. (a) Phenotype of 28‐day‐old Col‐0, cia2‐1, and P :CIA2(cia2‐1), P :CIA2(cia2‐1), P :CIL(cia2‐1), and P :CIL(cia2‐1) transgenic plants. (b) Quantification of chlorophyll a, chlorophyll b, carotenoids, and ratios of chlorophyll a/b in 21‐day‐old plants. Data represent means ± SD of three independent experiments. (c) Fresh weight of 28‐day‐old plants. Values represent means ± SE (N > 10). (d) Chloroplast area of the 7th leaf of 28‐day‐old plants. Data represent means ± SE (N > 500). Statistical analysis was conducted by one‐way ANOVA with a Tukey post hoc test (p <  .05)

We employed a similar strategy to reveal if CIL functions as a CIA2 backup. Transgenic plants expressing the CIL CDS in the cia2‐1 background under the control of the endogenous 1.5‐kb CIL upstream region or the 35S promoter were named P :CIL(cia2‐1) and P :CIL(cia2‐1), respectively. We acquired eight homozygous T3 transformants representing P :CIL(cia2‐1) or P :CIL(cia2‐1). The phenotypes of the four P :CIL(cia2‐1) lines (named P :CIL(cia2‐1) #1–4) were all similar to each other, so we selected P :CIL(cia2‐1) #1 as representative of P :CIL(cia2‐1) plants, and it was subjected to further assessments (supporting information Figure S1A). Likewise, P :CIL (cia2‐1) #1 is representative of P :CIL(cia2‐1) lines. Expression of transgenes in two complemented plants was verified by RT‐qPCR (supporting information Figure S1B).

P :CIL(cia2‐1) appeared greener than cia2‐1 but still paler than Col‐0 (Figure 1a). Chlorophyll contents and plant weight of P :CIL(cia2‐1) were higher than those of cia2‐1 but still much lower than for Col‐0 plants (Figure 1b,c). P :CIL(cia2‐1), in which the CIL CDS is expressed via a strong promoter (supporting information Figure S1B), was greener than cia2‐1 and P :CIL(cia2‐1) (Figure 1a). However, fresh weight of P :CIL(cia2‐1) was similar to that of cia2‐1, but lower than that of Col‐0 and P :CIL(cia2‐1) (Figure 1c). Higher levels of chlorophyll a and b in P :CIL(cia2‐1) relative to cia2‐1 and P :CIL(cia2‐1) indicated that both CIA2 and CIL participate in regulating chlorophyll accumulation (Figure 1b). The chlorophyll a/b ratio was similar across plant types. Carotenoid levels and chloroplast area in P :CIA2(cia2‐1) and P :CIA2(cia2‐1) plants were similar to those of Col‐0 (Figure 1b,d). In contrast, carotenoid levels for P :CIL(cia2‐1) and P :CIL(cia2‐1) were similar to or higher than in cia2‐1 but still less than for Col‐0 (Figure 1b), indicating that CIA2 affects carotenoid accumulation much more than CIL does. Chloroplast areas of P :CIL(cia2‐1) and P :CIL(cia2‐1) were similar to those in cia2‐1 yet still smaller than for wildtype (Figure 1d). Taken together, CIA2 and CIL appear to modulate pigment levels and chloroplast size, but CIA2 seems more important than CIL.

CIA2‐CIL interaction in the nucleus of Arabidopsis mesophyll cells

Transient expression of CIA2 and CIL in onion epidermal cells previously revealed that they both localize in the nucleus. Moreover, Y2H assays had also shown that CIA2 interacts with itself and CIL via their CCT motifs (Yang & Sun, 2020). To further validate the CIA2‐CIL interaction, we conducted BiFC assays on mesophyll cells. The full‐length CDS of CIA2 or CIL was ligated into either pSCYNE(R) or pSCYCE(R) vectors. The former vector encompasses the N‐terminal 1‐173 aa of cyan fluorescent protein (nCFP), whereas the latter vector contains the C‐terminal 156‐239 aa of CFP (cCFP). Various combinations of nCFP and cCFP plasmids were co‐transformed into protoplasts isolated from 18‐day‐old mesophyll cells. An NLS‐GFP construct harboring the NLS of simian virus 40 large T‐antigen and the CDS of GFP was used as a nuclear marker (Figure 2). Individual constructs and original vector were also transformed as controls and did not generate fluorescence signal (Figure 2b–e). CFP was detected in the nucleus of mesophyll cells, suggesting that CIA2 and CIL interact in the nucleus as homodimers and heterodimers (Figure 2f–i).

FIGURE 2

Biomolecular fluorescence complementation (BiFC) reveals that CHLOROPLAST IMPORT APPARATUS 2 (CIA2) and CIA2‐LIKE (CIL) interact in the nucleus of Arabidosis mesophyll cells. In addition to the NLS‐GFP construct, plasmids expressing different combinations of nCFP‐CIA2, cCFP‐CIA2, nCFP‐CIL, and cCFP‐CIL were co‐transformed into mesophyll protoplasts isolated from 18‐day‐old Col‐0 plants. NLS‐GFP acted as a nuclear marker. Plasmids N‐terminal 1‐173 aa of cyan fluorescent protein (nCFP) and C‐terminal 156‐239 aa of CFP (cCFP) (cCFP) were controls. Left to right columns represent CFP signal (blue), combined CFP and GFP (green) signals, and superimposition of CFP + GFP + chlorophyll fluorescence (magenta), respectively. Scale bar is 5 μm

Additive phenotypic defects of cia2‐1/cil‐3, and cil2–2/cil‐3 double mutants

To explore the functions of CIA2 and CIL further, we obtained seeds of additional cia2 and cil mutant alleles from the ABRC. We illustrate the gene structures of CIA2 and CIL in Figure 3a, highlighting the positions of mutations. The cia2‐1 mutant possesses a point mutation at nucleotide 770 of the CIA2 CDS that converts a tryptophan residue into a stop codon at residue 257, resulting in a truncated 256‐aa CIA2 protein (Sun, Chen, Lin, & Li, 2001). The cia2‐2 mutant (SALK_004037) hosts a T‐DNA insertion in the first exon of CIA2. No full‐length CIA2 transcript is present in cia2‐2 (Gawroński et al., 2021). The protein structures of CIA2 and CIL are presented in Figure 3b. Both proteins harbor a CCT motif in their C termini (residues 383–425 in CIA2 and 341–383 in CIL; Sun, Chen, Lin, & Li, 2001). In addition to the CCT motif, CIA2 and CIL share five conserved motifs, denoted CC 1–5 (representing conserved regions of CIA2 and CIL 1 to 5). The CC1 motif (residues 45–86 in CIA2 and 30–71 in CIL) contains an NLS and is essential for protein–protein interaction (Yang & Sun, 2020). CIA2 has an additional NLS at residues 291–308. Most truncated CIA2 protein from cia2‐1 remains in the cytosol of epidermal cells because it lacks this second NLS (Yang & Sun, 2020).

FIGURE 3

The cia2‐1/cil‐3 double mutant displays more severe phenotypic dysfunction. (a) Gene structures of CHLOROPLAST IMPORT APPARATUS 2 (CIA2) and CIA2‐LIKE (CIL). Arrow (point mutation) and solid triangles (T‐DNA insertion) represent the locations of mutations in CIA2 and CIL. Ecotype in parentheses indicates the genetic background of the corresponding mutant line. Black box, exon. White box, 5′‐ or 3′‐UTR. Line, intron. (b) Protein structures of CIA2 and CIL. CIA2 and CIL comprise 435 aa and 394 aa, respectively. CC1, conserved region 1 of CIA2 and CIL (gray box). NLS, nuclear localization signal (white box). CCT, CCT domain (hatched box). (c) Phenotype of 28‐day‐old Col‐0, cia2‐1, cia2‐2, cil‐3, cil‐4, cia2‐1/cil‐3, and cia2‐2/cil‐3 plants. (d) Fresh weight of 28‐day‐old plants. Values represent means ± SE (N > 10). (e) Detailed gene structure of cil‐3. Arrows indicate the positions of the three primers (CIL‐F, CIL‐R, and NTPII‐F; supporting information Table S1) used in (f). ATG and TAA represent start and stop codons, respectively. (f) Homozygosity of the cil‐3 and cia2‐1/cil‐3 mutants was confirmed by PCR. Genomic DNA (gDNA) acted as a loading control. (g) Transcript levels of CIL in 18‐day‐old Col‐0, cia2‐1, cia2‐2, cil‐3, and cia2‐1/cil‐3 plants, as determined by RT‐PCR. Expression of UBQ10 acted as an internal control. (h) Quantification of chlorophyll a, chlorophyll b, carotenoids, and chlorophyll a/b ratio in 21‐day‐old plants. Data from three independent experiments, with results representing means ± SE (N = 50 ~ 70). Statistical analysis was conducted by one‐way ANOVA with a Tukey post hoc test (p <  .05)

Allelic mutants cil‐1 to cil‐4 have T‐DNA insertions in the exons or first intron of CIL. Given that the cil‐1 (Col‐3 background) and cil‐2 (Col‐4 background) mutants are not in the Col‐0 background, we focused on the cil‐3 (SALK_068498) and cil‐4 (CS925818) mutants for this study. The phenotypes and fresh weights of 28‐day‐old cia2‐1, cia2‐2, cil‐3, and cil‐4 are compared in Figure 3c,d. Relative to cia2‐1, cia2‐2 is greener, larger, and has more fresh weight, but it is still not as green or has as much fresh weight as Col‐0. Therefore, cia2‐2 exhibits a more benign phenotype than cia2‐1. Phenotype and fresh weight of Col‐0, cil‐3, and cil‐4 are indistinguishable during vegetative growth. Gawroński et al. (2021) reported that cil‐1 and cil‐2 displayed phenotypes similar to their corresponding wildtype plants. Moreover, a cil knockout mutant (AtCIL_P4_2 _ 18) generated by Cas9 endonuclease site‐directed mutagenesis was also observed to have a wildtype phenotype (Li et al., 2021). Since cia2‐1 displayed greater impairment than cia2‐2 and the T‐DNA insertion of cil‐3 is located closer to the CIL N terminus, we have concentrated on cia2‐1 and cil‐3 as representative of cia2 and cil mutants, respectively, for most of our subsequent experiments. Double mutants (dm) were generated by crossing the cia2‐1 or cia2‐2 lines with cil‐3 and then selecting for several generations to obtain homozygous lines.

In cil‐3, an ~5‐kb T‐DNA sequence was inserted between nucleotides 328–725 of the first exon of the CIL CDS (Figure 3e). Homozygosity of the cil‐3 and cia2‐1/cil‐3 mutants was validated by PCR using CIL‐ and T‐DNA‐specific primers (supporting information Table S1), with cil‐3 and the dm amplifying the CIL‐F+NPTII‐F fragment but not the endogenous CIA‐F+CIL‐R fragment. No full‐length CIL transcript was observed for cil‐3 or the dm by RT‐PCR (Figure 3g). CIL transcripts were slightly increased in cia2‐1, perhaps to compensate for the CIA2 deficit. Phenotypes and fresh weight of homozygous dm plants are summarized in Figure 3c,d. Both cia2‐1/cil‐3 and cia2‐2/cil‐3 are paler and smaller than the cia2‐1 or cia2‐2 single mutants, especially with respect to young leaves. However, defective phenotypes are more prounounced for the cia2‐1/cil‐3 than cia2‐2/cil‐3. Chlorophyll a, b and carotenoid levels were significantly reduced in cia2‐1/cil‐3 (Figure 3h), indicating that CIA2 and CIL contribute to chlorophyll and carotenoid accumulation. The chlorophyll a/b ratio in both dm was also increased relative to wildtype (Figure 3d), implying that both CIA2 and CIL act redundantly to regulate the chlorophyll ratio. However, CIA2 might play a more important role in regulating plant size and pigment accumulation.

Regulation of chloroplast development by CIA2 and CIL

CIA2 acts as a transcription factor to upregulate expression of genes encoding chloroplast ribosomal proteins, chaperonin protein (CPN10, AT2G44650, referred to as CPN10‐II hereafter) and components of the translocon machinery required for protein import into chloroplast, so it enhances the efficiency of protein synthesis and protein import in chloroplasts (Sun, Huang, & Chang, 2009). Even though cil mutants display no significant phenotypic defects during vegetative growth, we used RT‐qPCR to establish if CIL also regulates the expression of the aforementioned genes. TOC34 and CPN10‐III (AT3G60210) are paralogs of TOC33 and CPN10‐II, respectively, and they were used as transcript controls because their expression is not regulated by CIA2 (Sun, Huang, & Chang, 2009). Transcript levels of all target genes were indistinguishable in Col‐0 and cil‐3, except for higher expression of RPL15 in cil‐3 relative to wildtype (Figure 4a). Again, mRNA levels of all target genes were significantly decreased in cia2‐1. Furthermore, expression of TOC33, CPN10‐II, RPS6, and RPL28 in the dm was lower than in cia2‐1. Together, these results indicate that (1) similar to CIA2, CIL does not regulate the expression of TOC34 or CPN10‐III and (2) CIA2 and CIL act, respectively, as major and minor expression modulators of certain chloroplast ribosomal and translocon genes in the nucleus.

FIGURE 4

Expression levels of genes related to chloroplast development and function in Col‐0, cia2‐1, cil‐3, and cia2‐1/cil‐3 plants. (a) Total RNAs were isolated from leaves of 18‐day‐old plants. Relative transcript levels for various genes were measured by real‐time quantitative RT‐PCR (RT‐qPCR) using gene‐specific primers (supporting information Table S1). Expression of UBQ10 acted as an internal control and gene expression levels is presented as means ± SD of three independent experiments. (b) Steady‐state levels of chloroplast proteins were analyzed by Western blot analysis. Intact chloroplasts were isolated from leaves of 21‐day‐old plants. Chloroplast proteins (25 μg) were analyzed by SDS‐PAGE and immunoblotting using anti‐Toc33, anti‐RbcL, anti‐RbcS or anti‐GS2 antiserum. Closed and opened circles indicate the Toc33 and Toc34 proteins, respectively. (c) Western blot signals in (b) were quantified using an image analyzer and calibrated based on the signals for Col‐0. Data represent means ± SD of three independent experiments. Statistical analysis was conducted by ANOVA with a Tukey test (p < .05)

Next, we assessed the steady‐state levels of chloroplast translocon and stromal proteins by immunoblot analyses. Proteins isolated from intact chloroplasts of 21‐day‐old leaves were separated by SDS‐PAGE followed by Western blot analysis (Figure 4b), which were quantified and calibrated against the respective signals for Col‐0 (Figure 4c). Toc33 and Toc34 are receptors on the chloroplast outer membrane responsible for recognizing and importing photosynthetic and housekeeping proteins into chloroplasts, respectively (Kessler & Schnell, 2009), whereas ribulose‐1,5‐bisphosphate carboxylase‐oxygenase (RuBisCo) large and small subunits (RbcL and RbcS), as well as GLUTAMINE SYNTHASE 2 (GS2), are stromal proteins. We observed no significant differences in protein levels between Col‐0 and cil‐3. Mirroring transcript levels, protein levels of Toc33 were reduced in cia2‐1 and more so in cia2‐1/cil‐3, relative to wildtype. Protein levels of RbcL, RbcS, and GS2 were also reduced in cia2‐1 and again even more so in the dm, likely due to defective protein synthesis and import. RbcL is a chloroplast genome‐encoded protein, and translation efficiency of RbcL in chloroplasts was previously tested by pulse‐labeling experiments, revealing a reduction in cia2‐1 compared with Col‐0 (Sun, Huang, & Chang, 2009). Thus, the reduced RbcL protein levels are likely due to plastid translation deficiencies of cia2‐1 and dm. RbcS and GS2 are nuclear genome‐encoded proteins. Since the transcript and protein levels of TOC33 decreased in both cia2‐1 and cia2‐1/cil‐3 (Figure 4a,b), RbcS and GS2 levels might be diminished because of Toc33 paucity in those mutants. Thus, amounts of Toc33, RbcL, RbcS, and GS2 are all reduced in the cia2 and cia2/cil mutants.

RuBisCo and GS2 are involved in carbon assimilation and ammonium reassimilation generated by photorespiration, respectively. Consequently, chloroplast structure and function might be altered due to the protein import and metabolic defects arising from CIA2 and CIL deficiencies. Accordingly, we examined chloroplast ultrastructure of 10‐day‐old Col‐0, cia2‐1, cil‐3, and cia2‐1/cil‐3 via transmission electron microscopy (Figure 5a–l). Chloroplast size in the dm was significantly smaller than in Col‐0, whereas chloroplast size of cil‐3 lay between those of Col‐0 and cia2‐1 chloroplasts. To confirm this chloroplast size difference and to avoid size bias arising from the sectioning angle of the specimens, we purified intact chloroplasts from the 7th leaf of 28‐day‐old plants and assessed chloroplast area. As observed for chloroplast size, the area of isolated chloroplasts followed the hierarchy Col‐0 > cil‐3 > cia2‐2 > cia2‐1 > cia2‐2/cil‐3 > cia2‐1/cil‐3 (Figure 5m). Next, we assessed RbcS import efficiency using equal numbers of chloroplasts isolated from 21‐day‐old plants. In Figure 5n, we show that Col‐0 and cil‐3 exhibited similar import efficiencies, whereas that of cia2‐1 was lower and that of cia2‐1/cil‐3 was the lowest. Based on our chloroplast area and protein import results, we assert that CIA2 and CIL both modulate chloroplast development and function but that CIA2 is the major regulator.

FIGURE 5

Ultrastructure and protein import efficiency of Col‐0, cia2‐1, cil‐3, and cia2‐1/cil‐3 chloroplasts. (a–d) Images of chloroplasts in 10‐day‐old true leaves, as observed by transmission electron microscopy. Panels (e–h) and (i–l) are magnified images of the regions encompassed by red and green boxes in (a) to (d), respectively. (m) Chloroplast area of the 7th leaf of 28‐day‐old plants. Results represent means ± SE (N > 500). (N) Radioactive RuBisCo small subunit (RbcS) was imported into chloroplasts isolated from 21‐day‐old plants. Proteins (25 μg) from intact chloroplasts after import were analyzed by SDS‐PAGE. Relative import efficiency was quantified using an image analyzer and calibrated based on the signals for Col‐0. Opened and closed circles indicate the precursor and mature RbcS, respectively. Data represent means ± SD of five independent experiments. Statistical analysis was conducted by ANOVA with a Tukey test (p < .05)

Identification of genes downregulated or upregulated in cia2‐1, cil‐3, and cia2‐1/cil‐3 mutants

To identify additional CIA2‐ and CIL‐regulated genes, we adopted Agilent oligonucleotide microarrays (Agilent Arabidopsis V4 4 × 44 K Microarray) to compare via two independent experiments the transcription profiles of Col‐0 and the cia2‐1, cil‐3, and cia2‐1/cil‐3 mutants. Genes displaying >1.5 fold‐change (FC ≥ 1.5, P ≤ .05) in expression in both experiments are summarized in Figure 6a,b and in supporting information Tables S2 and S3. Overall, we identified 294 or 297 genes downregulated or upregulated, respectively, in the cia2‐1, cil‐3, and cia2‐1/cil‐3 lines.

FIGURE 6

Differentially expressed genes in Col‐0, cia2‐1, cil‐3, and cia2‐1/cil‐3 plants. (a) Venn diagram showing the number of genes displaying >1.5‐fold downregulated expression in the cia2‐1, cil‐3, and cia2‐1/cil‐3 mutants. Changes in expression levels were calculated by dividing the normalized mutant transcript level by that of the wildtype Col‐0. (b) Venn diagram showing the number of genes displaying >1.5‐fold upregulated expression in the cia2‐1, cil‐3, and cia2‐1/cil‐3 plants. (c) Subcellular localization of proteins encoded by the 279 downregulated genes in cia2‐1/cil‐3. (d) Subcellular localization of proteins encoded by the 277 upregulated genes in cia2‐1/cil‐3

Downregulated genes observed in the cia2‐1/cil‐3 mutant reveal that they are co‐upregulated by CIA2 and CIL (Figure 6a). Based on Gene Ontology functional annotation (Gene Ontology Consortium, 2001), downregulated gene products could be classified into six subcellular localizations (Figure 6c). Given that most downregulated gene products in the dm are chloroplast proteins, this outcome provides further evidence supporting that CIA2 and CIL both regulate chloroplast function and development. Detailed analysis of the 155 downregulated chloroplast proteins revealed ribosomal proteins to be the most abundant (Table 1 and supporting information Table S2). Since most of those ribosomal proteins were previously reported as displaying reduced transcript levels in cia2‐1 (Sun, Huang, & Chang, 2009), CIL appears to function supplementary to CIA2 in inducing expression of various cpRP genes. Forty‐three photosynthesis‐related proteins can be categorized into three functional groups, that is, photosystem, chlorophyll‐biosynthetic, and electron transport proteins (Table 1). CPO1, HEMC, ABC4, and PDE327 have all been characterized previously as downstream target genes of CIA2 (Sun, Huang, & Chang, 2009). Transcript levels of the remaining 39 proteins were reduced only in the dm, indicating that CIA2 and CIL may coordinate the expression of these genes. However, CIA2 remains the major protein responsible for regulating photosynthesis‐related genes.

TABLE 1

Representative chloroplast biogenesis‐related proteins displaying altered gene expression in cia2‐1/cil‐3 mutants

AGI code a	Description	Slide1			Slide2			PSL b
		cia2‐1	cil‐3	cia2‐1/cil‐3	cia2‐1	cil‐3	cia2‐1/cil‐3
FC c ≥ 1.5 in cia2‐1/cil‐3
Chloroplast transcription‐related proteins (12) d
AT1G06190	Rho termination factor	0.61	0.90	0.58	0.57	0.99	0.57	CP
AT2G36990	RNA polymerase sigma‐subunit F (SIGF, SIG6)	0.76	0.87	0.63	0.72	1.05	0.65	CP
AT3G09210	Plastid transcriptionally active 13 (pTAC13)	0.80	0.97	0.66	0.75	1.09	0.66	CP
AT4G20130	Plastid transcriptionally active 14 (pTAC14)	0.57	1.01	0.35	0.51	1.06	0.35	CP
AT5G24314	Plastid transcriptionally active 7 (pTAC7)	0.76	1.03	0.57	0.68	1.22	0.58	CP
Chloroplast ribosomal proteins (35)
AT1G32990	50S ribosomal protein L11 (cpRPL11)	0.66	1.01	0.51	0.55	1.12	0.52	CP
AT2G33450	50S ribosomal protein L28 (cpRPL28)	0.72	1.02	0.55	0.66	0.94	0.52	CP
AT3G17170	30S ribosomal protein S6 (cpRPS6)	0.66	0.96	0.63	0.53	1.05	0.59	CP
AT5G47190	50S ribosomal protein L19‐2 (cpRPL19‐2)	0.66	0.99	0.47	0.58	1.03	0.42	CP
Chloroplast protein‐folding factors (5)
AT1G55490	Chaperonin‐60 beta1 (CPN60b1) A_84_P812113	0.50	0.84	0.44	0.55	1.29	0.59	CP
AT2G44650	Chloroplast chaperonin 10 (CPN10‐II)	0.66	0.94	0.59	0.65	0.88	0.56	CP
Chloroplast protein targeting factors (5)
AT1G02280	Chloroplast outer membrane translocon 33 (Toc33)	0.58	1.04	0.56	0.55	1.09	0.45	CP
AT1G63900	SUPPRESSOR OF PPI1 LOCUS 1 (SP1)	0.81	0.89	0.66	0.94	0.94	0.58	CP
AT2G18710	Preprotein translocase subunit SecY1	0.73	1.02	0.64	0.70	1.08	0.51	CP
AT5G03940	Signal recognition particle 54 kDa subunit (CpSRP54)	0.64	0.95	0.51	0.76	1.21	0.46	CP
Chlorophyll‐biosynthetic proteins (14)
AT1G03475	Coproporphyrinogen III oxidase (CPO1, HEMF1)	0.80	0.97	0.66	0.64	0.94	0.61	CP
AT1G44446	Chlorophyllide a oxygenase (CAO, CH1)	0.71	0.97	0.47	0.72	1.04	0.59	CP
AT3G48730	Glutamate‐1‐semialdehyde aminomutase 2 (GSA2)	0.69	0.95	0.52	0.68	1.12	0.57	CP
AT3G59400	GENOMES UNCOUPLED 4 (GUN4)	0.93	1.14	0.64	1.02	0.92	0.62	CP
AT4G27440	Protochlorophyllide reductase B (PORB)	0.94	1.00	0.53	0.93	0.88	0.62	CP
AT5G08280	Porphobilinogen deaminase (HEMC)	0.91	1.09	0.66	0.89	1.07	0.57	CP
AT5G54190	Protochlorophyllide reductase A (PORA)	1.20	0.87	0.34	1.25	0.85	0.55	CP
Photosystem proteins (22)
AT1G03130	Photosystem I subunit D‐2 (PsaD‐2)	0.98	1.29	0.52	0.99	0.98	0.48	CP
AT1G49975	Photosystem I reaction center subunit N (PsaN)	0.81	0.91	0.63	0.98	0.93	0.63	CP
AT2G05070	LHC II chlorophyll a/b binding protein 2.2 (LHCB2.2)	0.92	1.10	0.45	0.99	0.92	0.60	CP
AT2G34430	LHC II chlorophyll a/b binding protein 1.4 (LHCB1.4)	0.92	1.12	0.55	0.92	0.95	0.40	CP
AT3G08940	LHC II chlorophyll a/b binding protein 4.2 (LHCB4.2)	0.96	1.23	0.37	1.07	0.95	0.59	CP
AT3G47470	LHC II chlorophyll a/b binding protein 2.4 (LHCB2.4)	0.92	1.06	0.55	0.94	0.96	0.66	CP
AT5G27390	Photosystem II reaction center PsbP family protein	0.78	0.89	0.55	0.88	1.01	0.59	CP
Electron transport proteins (7)
AT1G60600	Aberrant chloroplast development 4 (ABC4)	0.62	0.91	0.36	0.45	1.07	0.32	CP
AT2G01918	Oxygen evolving enhancer 3 (PsbQ)‐like 3 (PQL3)	0.89	1.05	0.53	0.60	1.16	0.34	CP
AT4G30720	FAD/NAD(P)‐binding oxidoreductase (PDE327)	0.53	0.86	0.43	0.54	0.89	0.47	CP
AT5G45040	Cytochrome C6A (CytC6A)	0.82	1.03	0.63	0.73	1.07	0.45	CP
Oxidative stress proteins (6)
AT3G06730	Thioredoxin‐like protein (TrxZ)	0.66	0.97	0.56	0.72	1.17	0.66	CP
AT5G23310	Fe superoxide dismutase 3 (FSD3)	0.63	0.89	0.57	0.59	1.07	0.62	CP

AGI, arabidopsis genome initiative.

PSL, predicted subcellular location, based on SUBAcon of the SUBA3 database (Hooper et al., 2014; Tanz et al., 2013). CP, chloroplast.

FC, fold‐change of differential gene expression comparing mutants to Col‐0. FC ≥ 1.5 is underlined.

Numbers in parentheses represent the number of genes with FC ≥ 1.5 in the particular functional category.

Transcription of plastid genes requires a multisubunit plastid‐encoded RNA polymerase (PEP) complex. However, the activity and specificity of the core PEP enzyme in Arabidopsis are regulated by nuclear‐encoded transcription factors and oxidative stress‐related proteins such as sigma factors (SIG), plastid transcriptionally active chromosome (pTAC), thioredoxins type Z (TrxZ), and Fe‐type superoxide dismutase (FSD) (Börner, Aleynikova, Zubo, & Kusnetsov, 2015; Yu, Huang, & Yang, 2014). Various ptac, trxZ, and fsd3 mutants have been reported as exhibiting suppressed expression of photosynthetic genes and defective chloroplast development and de‐etiolation (Arsova et al., 2010; Gao et al., 2011; Myouga et al., 2008; Pfalz, Liere, Kandlbinder, Dietz, & Oelmüller, 2006; Steiner, Schröter, Pfalz, & Pfannschmidt, 2011).

Transcripts of six oxidative stress‐related genes were reduced in cia2‐1/cil‐3. Moreover, chlorophyll and carotenoid levels in the dm were also reduced by 30%–50% relative to Col‐0 (Figure 3h). Thus, due to decreased pigments, photosynthetic, and oxidative stress‐related proteins, more ROS might be retained by the cia2‐1/cil‐3 mutant. Using a NBT assay to evaluate ROS accumulation, we found that cia2‐1/cil‐3 indeed harbored more superoxide anions (O2 −) than wildtype and single mutants (supporting information Figure S2).

In terms of upregulated genes among the three mutant lines, we identified 277 in cia2‐1/cil‐3, indicating that these genes are also co‐downregulated by CIA2 and CIL (Figure 6b). Given that almost half of the upregulated gene products in dm are mitochondrial proteins (Figure 6d), CIA2 and CIL might also regulate mitochondrial function, such as transcription, translation, electron transport, oxidative stress, protein targeting, and folding (supporting information Table S3).

Notably, 88 and 73 genes encoding nuclear protein were downregulated or upregulated, respectively, among mutants (supporting information Tables S2 and S3). The transcript level of representative genes listed in Table 2 was confirmed by RT‐qPCR (Figure 7). Expression of GOLDEN2‐LIKE 1 (GLK1) was lower than wildtype in both cia2‐1 and dm, suggesting that CIA2 is more essential than CIL in upregulating GLK1 expression (Table 2 and Figure 7). In contrast, expression of its homologous gene GLK2 did not appear to be modulated by CIA2 or CIL. Both GLK1 and GLK2 are transcription factors that promote photosynthesis‐related gene expression and integrate chloroplast retrograde signaling (Tokumaru et al., 2017; Waters et al., 2009). GLK1 overexpression in a glk1/glk2 double mutant enhanced transcript levels of chlorophyll‐biosynthetic genes (e.g., CAO, GUN4, and PORB), as well as light‐harvest complex II chlorophyll a/b‐binding protein genes (e.g., LHCB2.2, LHCB2.4, and LHCB4.2), with chromatin immunoprecipitaion (ChIP) confirming that GLK1 binds to the promoter sequences of those genes (Waters et al., 2009). Thus, CIA2 and CIL might regulate chloroplast development directly or indirectly via GLK1. Moreover, expression of GATA NITRATE‐INDUCIBLE CARBON‐METABOLISM‐INVOLVED (GNC) was higher in cia2‐1 and the dm compared with Col‐0, indicating that CIA2 is more important than CIL in downregulating GNC expression (Table 2 and Figure 7). GNC promotes chloroplast biogenesis by repressing the expression of photomorphorgenesis‐related repressor genes, such as PHYTOCHROME‐INTERACTING FACTOR (PIFs; Zubo et al., 2018). Thus, CIA2 and CIL benefit chloroplast development by restraining GNC expression.

TABLE 2

Representative photomorphogenesis or flowering‐related transcription factors displaying altered gene expression in the cia2‐1, cil‐3, and cia2‐1/cil‐3 mutants based on microarray data

AGI a code	Description	Slide1			Slide2			PSL b	RT‐qPCR c
		cia2‐1 cil‐3 cia2‐1/cil‐3 cia2‐1 cil‐3 cia2‐1/cil‐3
FC d ≥ 1.5 in cia2‐1, cil‐3 and cia2‐1/cil‐3
AT1G69120	APETALA 1 (AP1)	3.55	0.64	4.55	3.76	0.46	4.86	N	+
AT2G45650	AGAMOUS‐like 6 (AGL6)	2.68	0.78	3.46	1.80	0.55	2.16	N	+
FC ≥ 1.5 in cil‐3 and/or cia2‐1/cil‐3
AT4G11110	Suppressor of Phy A‐105 related 2 (SPA2)	1.09	0.49	0.98	1.13	0.50	1.40	N	+
AT4G16250	Phytochrome D (PhyD)	0.80	0.43	0.83	0.85	0.55	0.84	N	+
AT4G16845	Vernalization 2 (VRN2)	1.28	0.04	1.24	1.30	0.03	1.41	N	+
AT4G25990	CCT motif family protein (CIL)	1.18	0.54	0.43	1.23	0.49	0.34	N	+
AT5G10140	FLOWERING LOCUS C (FLC)	0.97	1.95	0.61	0.92	1.61	0.68	N	+
FC ≥ 1.5 in cia2‐1 and cia2‐1/cil‐3
AT1G14760	KNOX MEINOX (KNATM)	0.42	0.93	0.42	0.63	1.09	0.34	N	+
AT2G20570	GOLDEN2‐like 1 (GLK1)	0.66	1.01	0.39	0.66	0.87	0.23	N	+
AT3G18010	WUSCHEL‐related homeobox 1 (WOX1)	0.30	0.74	0.33	0.51	0.88	0.42	N	+
AT1G26310	CAULIFLOWER (CAL)	9.17	1.18	33.94	4.84	1.22	7.07	N	+
AT3G02310	SEPALLATA 2 (SEP2)	4.09	1.26	4.03	2.83	1.00	4.25	N	+
AT3G54340	APETALA 3 (AP3)	2.86	0.94	4.10	3.71	0.83	7.62	N	+
AT5G23730	Repressor of UVB photomorphogenesis 2 (RUP2)	1.80	1.17	2.16	1.89	1.05	2.24	N	+
AT5G56860	GATA transcription factor 21 (GNC)	1.56	0.97	2.33	1.36	1.05	1.70	N	+
FC ≥ 1.5 in cia2‐1/cil‐3
AT1G09570	Phytochrome A (PhyA)	0.70	1.07	0.57	0.91	0.94	0.59	N
AT2G43010	Phytochrome interacting factor 4 (PIF4)	1.06	1.41	0.65	1.01	1.07	0.66	N	+
AT4G34530	Cryptochrome‐interacting bHLH 1 (CIB1)	0.67	1.02	0.50	0.67	1.00	0.48	N	+
AT5G04190	Phytochrome kinase substrate 4 (PKS4)	0.70	0.94	0.52	0.67	0.92	0.30	N	+

AGI, Arabidopsis Genome Initiative.

PSL, Predicted Subcellular Location, based on SUBAcon of the SUBA3 database (Hooper et al., 2014; Tanz et al., 2013). N, nucleus.

+, confirmed FC ≥ 1.5 by RT‐qPCR.

FC, fold‐change of differential gene expression comparing mutants to Col‐0. FC ≥ 1.5 is underlined.

FIGURE 7

Confirmation of altered gene expression from microarray analysis by real‐time quantitative RT‐PCR (RT‐qPCR). Total RNAs were isolated from leaves of 16‐day‐old plants. Relative transcript levels for various genes were measured by RT‐qPCR using gene‐specific primers (supporting information Table S1). Expression of UBQ10 acted as an internal control and gene expression levels are presented as means ± SD of three independent experiments. Asterisks indicate statistically significant differences compared with Col‐0, as determined by Student's t‐test (* p < .05, ** p < .01, *** p < .005)

Apart from transcription factor genes involved in regulating chloroplast development, many participate in modulating flower development (see Table 2). In particular, AGL6 and AP1 displayed a fold‐change in transcript levels ≥1.5 in mutants. AGL6 is a negative regulator of FLC and a positive regulator of FT in Arabidopsis (Yoo, Wu, Lee, & Ahn, 2011). FLC is a coordinator that mediates flowering time via the vernalization and autonomous pathways (Dennis & Peacock, 2007). FT is a key flowering‐time integrator gene and AP1 is a floral meristem identity gene (Gregis, Sessa, Dorca‐Fornell, & Kater, 2009). Similar to cia2‐1, AGL6 and AP1 in the dm displayed 2.16‐ to 6.25‐fold greater expression relative to Col‐0 based on microarray and RT‐qPCR analyses (Table 2 and Figure 7). In contrast, expression of both genes was 1.48‐ to 2.17‐fold lower in the cil‐3 mutant compared with Col‐0. Together, these results signify that CIA2 and CIL exert opposing effects on AGL6 and AP1, with CIA2 and CIL acting as their negative and positive regulators, respectively.

Interestingly, we observed extremely low VERNALIZATION 2 (VRN2) expression in cil‐3, yet similar levels in Col‐0, cia2‐1, and the dm (Table 2 and Figure 7). VRN2 is a repressor of FLC in vernalization pathways (Dennis & Peacock, 2007). Moreover, cil‐3 displayed higher FLC expression than wildtype, but cia2‐1 exhibited equivalent expression to wildtype and it was slightly lower for cia2‐1/cil‐3. Thus, CIL might be a direct or indirect repressor of FLC via VRN2 regulation, whereas CIA2 is less important in regulating FLC expression. Accordingly, CIL might play a more important role in regulating flowering time.

Flower meristem identity genes (e.g., CAL; Gregis, Sessa, Dorca‐Fornell, & Kater, 2009) and organ development genes (e.g., AP3 and SEP2; Pelaz, Ditta, Baumann, Wisman, & Yanofsky, 2000) were specifically upregulated in the cia2‐1 and cia2‐1/cil‐3 mutants, demonstrating that CIA2 represses these genes. Moreover, expression yield of WOX1, which promotes shoot length and flower number (Dolzblasz et al., 2016), was lower in cia2‐1 and dm, so CIA2 promotes WOX1 expression. Thus, CIA2 appears to be more significant than CIL in terms of regulating floral organ development.

Genes involved in light‐mediated photomorphogenesis, photoperiod, and light perception pathways of flowering were more likely to be downregulated in the cia2‐1/cil‐3, indicating that both CIA2 and CIL participate in these developmental processes. For example, expression of CIB1, PIF4 and PKS4 was lower in the dm. However, some of those genes are specifically regulated either by CIA2 or CIL. For instance, CIA2 represses RUP2 expression, whereas CIL promotes expression of SPA2. Since PIF4, PKS4, RUP2, and SPA2 are all repressors of photomorphogenesis and flowering pathways (Arongaus et al., 2018; Balcerowicz et al., 2011; Castillon, Shen, & Huq, 2007; Jenkitkonchai et al., 2021; Liu et al., 2018; Schepens, Boccalandro, Kami, Casal, & Fankhauser, 2008), CIA2 and CIL might be equally involved in regulating light‐mediated photomorphogenesis and photoperiod pathways of flowering.

Regulation of flower development by CIA2 and CIL

Our microarray analyses provide evidence that CIA2 and CIL regulate the expression of genes responsible for flowering‐time control, flower meristem identity and organ development (Table 2). Accordingly, we examined flowering time and flower phenotype under long‐day conditions for the Col‐0, cia2‐1, cil‐3, and cia2‐1/cil‐3 mutants (Figure 8). Relative to Col‐0, cia2‐1 exhibited slightly early flowering, but a similar rosette leaf number (Figure 8a–c). The cil‐3 mutant presented delayed flowering by ~2.7 days and an increase in rosette leaves compared with wildtype. Even though flowering was delayed in the dm by ~3.6 days, they had fewer rosette leaves. Overall then, CIL seems to be the principal flowering‐time determinator because the late flowering phenotype of cil‐3 corresponds to having additional rosette leaves. Inflorescence main axis height, flower number on the main axis, and total flower number were clearly reduced for cia2‐1 compared with Col‐0 and cil‐3, and the respective values for the dm were even lower (Figure 8d–f). Thus, CIA2 is a major and CIL is a minor modulator by which flower number and main axis length can be increased.

FIGURE 8

Comparison of flowering time and phenotypes in Col‐0 and various mutants grown under a 16 h light/8 h dark photoperiod. (a) Phenotypes of 35‐day‐old plants plants (Col‐0, cia2‐1, cil‐3, and cia2‐1/cil‐3, left panel) and 46‐day‐old plants (co, cia2‐1/co, cil‐3/co, and cia2‐1/cil‐3/co, right panel). (b) Flowering time of plants after sowing. (c–f) Rosette leaf number, main axis height, flower number on main axis, and total flower number of 56‐day‐old plants (Col‐0, cia2‐1, cil‐3 and cia2‐1/cil‐3) and 87‐day‐old plants (co, cia2‐1/co, cil‐3/co, and cia2‐1/cil‐3/co). Scale bar in (a) is 1 cm. In (b) to (f), values represent means ± SE (N = 45 for plants on left panel, N = 14 for plants on right panel). Statistically significant differences compared with Col‐0 (left panel) or co (right panel), respectively, as determined by Student's t‐test (*p < .05, ** p < .01, *** p < .005)

Since CIL and CO promote flowering and CIA2 increases flower number under long‐day conditions, we anticipated that phenotypic comparison of their respective double and triple mutants would help establish if they act coordinately in the flowering process. We obtained a co‐1 mutant (CS3387, Landsberg background) from the ABRC and backcrossed it to Col‐0 to get a co mutant in the Col‐0 background. Double and triple mutants were generated by crossing and then sequential selection to isolate homozygous cia2‐1/co, cil‐3/co, and cia2‐1/cil‐3/co. The flowering time of co was delayed by ~20 days relative to Col‐0 (Figure 8b). Moreover, the co single mutant displayed 2.8‐fold more rosette leaves than the wildtype, together revealing that CO is indeed a key flowering‐time determinator in Arabidopsis. Flowering time and rosette leaf number in the cia2‐1/co double mutant were similar to co (Figure 8b,c). Nevertheless, cil‐3/co bolted 6 days later than co and also grew more rosette leaves. These phenotypic differences were more obvious in the cia2‐1/cil‐3/co triple mutant. Thus, CO and CIL promote flowering, whereas CIA2 only weakly delays it. Inflorescence main axis height, flower number on the main axis, and total flower number of the co, cia2‐1/co, and cil‐3/co mutants were indistinguishable when bolting day was extended to more than 50 days, but values for all of these parameters were dramatically decreased in the cia2‐1/cil‐3/co (Figure 8d–f). Accordingly, CO, CIA2, and CIL act in concert to increase flower number and main axis height under long‐day conditions when plants grow slowly.

DISCUSSION

CIA2 and CIL represent major and minor regulators of chlorophyll accumulation and the chlorophyll a/b ratio

Our observations of pale‐green, dark‐green, and light yellow‐green phenotypes for the rosette leaves of the cia2, cil and cia2‐1/cil‐3 mutants are mainly due to 25%–50%, almost 0%, and 50%–70% reductions, respectively, in chlorophyll relative to wildtype (Figure 3). Moreover, CIA2 or CIL overexpression in cia2‐1 could fully or partially compensate for its reduced chlorophyll levels (Figure 1). Furthermore, microarray analysis indicated that 14 chlorophyll‐biosynthetic genes were downregulated in the dm (Table 1). Together, these data confirm that CIA2 and CIL coordinately regulate chlorophyll levels in Arabidopsis leaves, with CIA2 especially being more important than CIL in this process. Notably, we observed that the chlorophyll a/b ratio was significantly increased in the dm (Figure 3h), perhaps due to decreased expression of CAO in cia2‐1/cil‐3 plants (Table 1). CAO converts chlorophyllide a to b or chlorophyll a to b, resulting in raised chlorophyll b levels (Wójtowicz, Jagielski, Mostowska, & Gieczewska, 2021), so reduced CAO expression in the dm diminishes chlorophyll b levels and thereby enhances the chlorophyll a/b ratio.

Carotenoid levels were obviously decreased in cia2 and dm (Figure 3h), but no carotenoid biosynthesis‐related genes exhibited significantly altered expression (FC ≥ 1.5) in these mutants (supporting information Tables S2 and S3). Since carotenoid production is coordinated with the production of chlorophyll and pigment‐bearing proteins in chloroplasts (Pribil, Labs, & Leister, 2014), the decreased carotenoid levels of cia2 and dm may be a side‐effect of significantly reduced chlorophyll levels. This assumption is supported by our findings in transgenic plants, with P :CIA2(cia2‐1) and P :CIA2(cia2‐1) in which the chlorophyll defect of cia2‐1 was fully restored also displaying carotenoid levels equivalent to wildtype, whereas P :CIL(cia2‐1) and P :CIL(cia2‐1) in which chlorophyll levels were only partially restored only displaying moderately rescued carotenoid levels (Figure 1).

CIA2 and CIL homodimers and heterodimers localize in the nucleus of mesophyll cells

Previous screening of a Y2H Arabidopsis cDNA library using CIA2 as bait revealed that CIA2 interacts with itself and CIL through their CCT motifs (Yang & Sun, 2020). In the current study, we demonstrate by PEG‐mediated protoplast transformation that CIA2 and CIL interact in the nucleus of mesophyll cells (Figure 2). Since CFP signal was visualized solely in the nucleus of mesophyll cells, homodimeric and heterodimeric CIA2 and CIL are restricted to nuclei.

CIA2 and CIL have at least 72 orthologues in various plants (Yang & Sun, 2020). A barley orthologue, HvCMF7, was observed to localize to plastids by biolistic bombardment assay and to partially complement the cia2‐1 defect in transgenic plants (Li et al., 2019, 2021). Thus, Gawroński et al. (2021) reported previously that CIA2 might be a chloroplast protein based on transient expression of p35S::CIA2:YFP and p35S::CIA21‐100:YFP transgenes in Arabidopsis seedlings and tobacco leaves. However, CIA2 and CIL do not enter chloroplasts, as revealed by protein import experiments (Li et al., 2021). In addition, a pCIA2::YFP:CIA2 transgene, in which YFP blocked the potential chloroplast TP of CIA2 and presented no chloroplast localization, complemented the cia2 mutant, indicating that a chloroplast localization for CIA2 is not critical (Gawroński et al., 2021).

CIA2 is a principal modulator of chloroplast biogenesis during vegetative development

Coordinated expression of genes encoded in both the nuclear and plastid genomes ensures chloroplast biogenesis. Especially during early chloroplast developmental stages, nuclear‐encoded proteins responsible for the chloroplast‐localized import apparatus, plastid gene expression, and photosynthesis that should be massively expressed and imported into chloroplasts to fulfill their high functional and structural demands. Reduced chloroplast protein accumulation would result in decreased chloroplast area and plant size, as observed in our dm (Figures 3 and 5). More severe effects in young tissues are evidenced by obviously paler young leaves relative to mature leaves in both the cia2‐1/cil‐3 and cia2‐2/cil‐3 mutants (Figure 3c).

Previous results from ChIP, RT‐PCR, protein import, and synthesis experiments had shown that CIA2 directly binds and promotes transcript levels of its downstream genes, thereby increasing import and synthetic efficiency of photosynthetic proteins in chloroplasts (Sun, Huang, & Chang, 2009). Compared with Col‐0 and cil‐3, mRNA levels of TOC33, CPN10‐II, and cpRPs were significantly reduced in cia2‐1 and even further limited in cia2‐1/cil‐3 (Figure 4a). Moreover, accumulations of photosynthetic proteins and Toc33 (Figure 4c), as well as the import efficiency of RbcS (Figure 5n), were diminished in both cia2‐1 and the dm. Together, these outcomes indicate that relative to CIL, CIA2 plays a more important role in regulating photosynthetic protein import, synthesis, and folding.

Microarray data revealed additional downstream target genes of CIA2 and CIL that encode chloroplast proteins. Most of them participate in translation and photosynthesis (Table 1). CIA2 and CIL might modulate expression of these genes directly or indirectly via other nuclear factors, such as GLK1. Thus, CIA2 and CIL coordinate the upregulation of chloroplast biogenesis‐related gene expression during vegetative development (Figure 9). Most importantly, CIA2 plays a major role in modulating the expression of genes encoding proteins that participate in import, translation, folding, regulating other nuclear modulators (GLK1 or GNC), plastid gene transcription, chlorophyll biosynthesis, photosynthesis, and oxidative regulation, thereby ensuring normal chloroplast development.

FIGURE 9

Schematics of the hypothetical regulatory mechanisms exerted by CHLOROPLAST IMPORT APPARATUS 2 (CIA2) and CIA2‐LIKE (CIL) for vegetative and reproductive development in Arabidopsis under long‐day conditions. Preferential paths are indicated by thicker lines. Oval and rectangular boxes represent proteins and regulated genes, respectively

CIA2 and CIL deficiency increases ROS levels in chloroplasts and induces expression of genes encoding mitochondrion‐localized proteins

Photosynthesis in chloroplasts and respiration in mitochondria represent almost opposite catalytic reactions. However, they display sophisticated crosstalk that depends on metabolite homeostasis and anterograde regulation from the nucleus to organelles (Leister, 2005). ROS‐ and heme‐triggered retrograde signaling from plastids to nuclei has been demonstrated previously (Dietz, Turkan, & Krieger‐Liszkay, 2016; Terry & Smith, 2013). Nuclear factors (e.g., GLKs; Waters et al., 2009; Tokumaru et al., 2017) integrate such retrograde signals and reprogram the expression of nuclear genes encoding chloroplast‐ or mitochondrial‐localized proteins (Leister, 2005). Liao, Hsieh, Tseng, and Hsieh (2016) used lincomycin to block plastid ribosomes, thereby inhibiting protein synthesis and increasing ROS levels in plastids. Notably, not only were mitochondrial genes upregulated in lincomycin‐treated plants but so too were nuclear genes encoding mitochondrial proteins. In the current study, we identified genes encoding mitochondrial proteins that were upregulated in the dm (Figure 6d). Given reduced expression of 155 chloroplast‐related genes and increased ROS levels in the dm (Figure 6c and supporting information Figure S2), we surmise that these upregulated genes might be secondary targets of CIA2 and CIL. Chloroplast dysfunction may coordinately impact nuclear and mitochondrial genes to optimize mitochondrial function and reduce oxidative stress in the cells. However, whether CIA2 and CIL directly or indirectly regulate the expression of upregulated genes warrants further study.

CIL controls flowering time

VRN2 expression is extremely low in cil‐3 and slightly upregulated in cia2‐1 and the dm, implicating CIL as a primary participant in the vernalization pathway of flower development (Table 2 and Figure 7). Moreover, FLC expression is higher in cil‐3 relative to wildtype but normal in cia2‐1 and slightly lower in the dm. In addition, AGL6 expression is higher than wildtype for cia2‐1 and the dm but lower for cil‐3. Taken together, these findings indicate that CIL severely represses FLC expression by directly or indirectly regulating VRN2, but moderately enhances AGL6 expression. Since VRN2 and AGL6 both repress FLC to enhance FT expression (Dennis & Peacock, 2007; Yoo, Wu, Lee, & Ahn, 2011), overall the cil‐3 mutant displays transcript levels of FT ~50% those of Col‐0. In contrast, CIA2 moderately inhibits AGL6 expression and has a minimal effect on VRN2 and FLC, so CIL is the major controller of flowering relative to CIA2 (Figure 9).

These results help explain the differences in flowering time displayed by the double and triple mutants (Figure 8). In the presence of CO and under long‐day conditions, CIL transcript levels were slightly increased in cia2‐1, promoting earlier flowering relative to Col‐0. Due to cil‐3 lacking functional CIL and displaying higher CIA2 expression than wildtype (Figure 7), increased FLC and reduced AGL6 expressions downregulate FT, thereby delaying the flowering time. Lacking both functional CIA2 and CIL, cia2‐1/cil‐3 displayed the latest flowering phenotype and a much lower leaf number at bolting, implying the dm plants are highly stressed, so they develop flowers despite having fewer leaves (Kazan & Lyons, 2016). Mysteriously, Li et al. (2021) reported that cil (AtCIL_P4_2 _ 18) was early flowering, with that outcome probably due to the lower growth temperature and stronger light intensity (20°C day/17°C night under fluorescent light of 180 μmol m−2 s−1) used in their experiment.

In the absence of CO and under long‐day conditions, all co‐related mutant plants exhibited a flowering delay of at least 20 days compared with wildtype, implying that CO is a critical flowering‐time executor in Arabidopsis. Relative to co, we observed no early flowering or altered leaf number phenotype for cia2‐1/co. Nevertheless, cil‐3/co and cia2‐1/cil‐3/co exhibited 6‐day and even 15‐day delayed flowering relative to co. Leaf numbers were also significantly increased in cil‐3/co and cia2‐1/cil‐3/co, indicative of mutual interactions among CIA2, CIL, and CO being essential to regulate flowering time under long‐day conditions. However, CO and CIL still appear to play more crucial roles in modulating flowering time.

CIA2 modulates flower number and main axis height

Our microarray and RT‐qPCR analyses (Table 2 and Figure 7) show that CIA2 specifically represses the expression of two meristem identity genes (AP1 and CAL), two organ development genes (AP3 and SEP2), and one flower number‐inducing gene (WOX1). In contrast, apart from inducing AP1 expression, CIL does not affect the expression of these genes. These data explain why inflorescence main axis height, flower numbers on the main axis and total flower numbers are altered in our double and triple mutants (Figure 8). In the presence of CO, flower height and flower numbers are indistinguishable in wildtype and cil‐3, whereas they are clearly reduced in the cia2‐1 and cia2‐1/cil‐3. In the absence of CO, the co, cia2‐1/co, and cil‐3/co mutants did not present any difference in these parameters due to slow plant growth, but there was a dramatic reduction for the cia2‐1/cil‐3/co, implying that CO, CIA2, and CIL act in concert to increase flower number and main axis height under long‐day conditions. Nevertheless, CIA2 more prominently regulates flower number and main axis height relative to CIL by promoting WOX1 and repressing the expression of flower meristem identity and organ development genes (Figure 9). However, CIA2 activity in this regard might be altered by its interacting proteins, such as CO and NF‐Y subunits (Yang & Sun, 2020).

ONE‐SENTENCE SUMMARY

Loss‐of‐function mutants and complemented plants reveal that CIA2 is the main regulator of chloroplast and floral organ development, whereas CIL is the predominant flowering‐time controller.

FUNDING INFORMATION

This work was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 102‐2311‐B‐003‐001 and 104‐2311‐B‐003‐002‐MY3) to CWS.

CONFLICT OF INTEREST

The Authors did not report any conflict of interest.

AUTHOR CONTRIBUTIONS

CYY and CWS conceived the project and designed the experiments. CYY performed the experiments of Figures 1a, 2, 3e–g, 4a, and 6 and supporting information Figures S1 and S2. WYY performed the experiments of Figures 1c,d, 3c,d, 5m, 7, and 8. HYC performed part of experiments of Figures 1b, 3h, 5a–l, and 8. CWS performed the experiments of Figures 4b,c and 5n, and 9. CYY and CWS analyzed the data and wrote the manuscript. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors is: Chih‐Wen Sun (cwsun@ntnu.edu.tw).

Figure S1. P :CIL(cia2‐1) and P :CIL(cia2‐1) transgenic plants.

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Figure S2. Histochemical detection of ROS accumulation in Col‐0, cia2‐1, cil‐3, and cia2‐1/cil‐3 plants.

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Table S1. List of primers used in genotyping, cloning, RT‐PCR and RT‐qPCR.

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Table S2. Genes downregulated in cia2‐1, cil‐3, and cia2‐1/cil‐3.

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Table S3. Genes upregulated in cia2‐1, cil‐3, and cia2‐1/cil‐3.

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Dataset S1. Agilent microarray raw data.

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