Role of CIA2 and CIL in the Regulation of Chloroplast Development During Photomorphogenesis in Arabidopsis

Highlights

What are the main findings?

• CIA2 and CIL participate and optimize chloroplast biogenesis and development, affecting chloroplast ultrastructure, pigment accumulation, PSII function, and expression of key photosynthetic and protein-import genes.
• Complementation of the double cia2cil mutant with functional CIA2 restores early greening, chloroplast morphology, PSII performance, and gene expression dynamics to near wild-type levels, revealing CIA2 as the dominant factor in this regulatory module.

What is the implication of the main finding?

• The results suggest that CIA2 and CIL may contribute not only in the regulation of responses to UV-AB, high light, heat shock and chloroplast translation regulation as it was indicated before, but also play a role in establishment of functional chloroplasts during early development, providing a basis for future studies for amelioration of crops chloroplast performance and photosynthetic capacity and in general plant light stress responses.

Abstract

Chloroplast development plays a crucial role in plant de-etiolation, a process in which plants switch from growth in darkness to light-driven development, known as photomorphogenesis. This study provides evidence that CIA2 (Chloroplast Import Apparatus 2) and CIL (CIA2-Like) contribute to chloroplast biogenesis, likely by affecting and regulating PSII activity and related gene expression. Although their precise molecular roles remain unclear, our findings support their possible involvement in chloroplast development. This is indicated by downregulation of foliar chlorophyll content, chlorophyll a fluorescence parameters, chloroplast size, and gene expression of PSII molecular markers in the cia2cil double mutant during de-etiolation. Chlorophyll a fluorescence and quantitative gene expression analysis during de-etiolation revealed a significant reduction in PSII maximal efficiency and non-photochemical quenching, as well as deregulated expression of genes such as LHCB2.1 and psbA. According to the immunoblotting and microscopy imaging results, there is an impaired function of PSII and a compromised ultrastructure of the chloroplast membranes in cia2cil plants. However, in CIA2p::CIA2_cia2cil and 35Sp::CIA2_cia2cil complementation lines, reversion of this phenotype was observed. These results suggest a supporting role for CIA2 and CIL in the plant de-etiolation process, expanding our understanding of chloroplast biogenesis regulation.

1. Introduction

Plants have evolved several ways of coping with different and highly fluctuating environmental conditions. The transition from a dark-grown, etiolated state to a light-adapted, photosynthetically active state, known as de-etiolation, is considered one of the most critical steps in plant development, enabling the plant to achieve its capability for photosynthesis. In this process, plants undergo various biochemical, physiological, and morphological changes, including stem shortening, leaf expansion, chlorophyll synthesis, and functional chloroplast development [1,2,3]. De-etiolation involves optimizing a plant’s ability to receive and convert absorbed light into biochemical energy, thereby promoting plant development. It is controlled by the complex cross-talk of several signaling pathways, including plastid and chloroplast retrograde signaling, as well as phytochromes and cryptochromes [1,2]. Regulatory mechanisms of chloroplast biogenesis are highly complex and include the interactions of numerous transcription factors (TFs) that regulate gene expression in response to light signals [2,4,5,6].

Chloroplast development is a process that requires the interaction of nuclear and plastid gene expression to provide the efficient and optimal function of the photosynthetic apparatus [7,8]. Moreover, this process is required for transporting nuclear-encoded proteins across the double-chloroplast envelope, mediated by specialized translocon complexes, to facilitate proper chloroplast biogenesis and function [9]. The translocons at the outer envelope membrane (TOC) and the inner envelope membrane (TIC) collaborate to import precursor proteins required for chloroplast formation and photosynthesis [10]. The TOC complex functions as a selective gateway, recognizing and transporting preproteins in a GTP-dependent manner, whereas the TIC complex controls their migration into the stroma [11]. Light triggers TOC159 activity to enhance the transport of photosynthetic proteins, and hormonal signals like gibberellins and cytokinins regulate translocon function to balance chloroplast differentiation and development under changing environmental conditions [12,13]. The ultimate end of chloroplast development is regulated by the cell death mechanism that depends on cell death regulators (LSD1, EDS1, PAD4), non-photochemical quenching (NPQ), and PsbS [14,15,16].

Several TFs are involved in the chloroplast biogenesis process. HY5 is a transcription regulator of light-mediated genes that is responsible for chlorophyll and photosynthetic proteins synthesis and thylakoid membrane formation [17,18,19]. Phytochrome-interacting transcription factors (PIFs) integrate light and hormonal signals, particularly gibberellin, to control chloroplast maturation and photomorphogenesis [20]. GLK1 and GLK2 (GOLDEN2-LIKE 1 and 2) enhance the expression of genes encoding chlorophyll biosynthesis enzymes and photosystem components [21,22,23]. CGA1 (cytokinin-responsive GATA factor 1) is a cytokinin-responsive transcription factor that upregulates photosynthesis-associated nuclear genes (PhANGs), along with MYB-related transcription factors (MYBS1), as regulators of chloroplast biogenesis, functioning in coordination with GLK transcription factors to ensure proper chloroplast biogenesis [24]. Proteins such as Early Light-Induced Protein 1 (ELIP1), Photosystem II protein D1 (psbA), and LIGHT-HARVESTING CHLOROPHYLL A/B-BINDING PROTEIN 2 (LHCB2) are structural or protective components of the photosynthetic machinery. psbA and LHCB2 encode core PSII proteins that contribute to light harvesting and energy transfer, whereas ELIP1 helps protect chloroplasts from photo-oxidative damage through efficient energy dissipation during periods of high light stress [25,26,27,28]. The transcription factors CIA2 and CIL, which are potentially dual-localized in the nucleus, have been suggested to regulate chloroplast protein import and biogenesis either directly or indirectly through GLK1 [29,30,31]. The phenotype of the cia2 mutant exhibits a pale-green appearance, likely due to deregulated protein import, which may be attributed to impaired chloroplast development [30,31,32]. In addition to their roles in chloroplast biogenesis, CIA2 and CIL also participate in other aspects of plant growth and development, including the heat shock response and flowering [30,32]. However, the precise mechanisms by which CIA2 and CIL might regulate chloroplast biogenesis during de-etiolation remain unclear and require further investigation. Therefore, this study aims to investigate the roles of CIA2 and CIL in chloroplast development, focusing on their contribution to PSII integrity and chloroplast ultrastructure during de-etiolation.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

Seeds of Arabidopsis thaliana (Col-0), the single mutants cia2 (SALK_004037) and cil (SAIL_228_C01), the double mutant cia2cil, and the complementation lines CIA2p::CIA2_cia2cil, and 35Sp::CIA2_cia2cil were sterilized by exposure to chlorine gas, produced by combining concentrated hydrochloric acid with sodium hypochlorite. Approximately 50 seeds were sown per spot on agar medium containing half-strength Murashige and Skoog salts (Duchefa Biochemie, Haarlem, The Netherlands) without added sucrose. After surface sterilization, seeds were stratified at 4 °C in darkness for three days. The plates were then illuminated for two hours at 40 µmol photons m⁻² s⁻¹ at 21 °C, followed by a three-day incubation in complete darkness at the same temperature. Subsequently, seedlings were transferred to a long-day photoperiod (16 h light/8 h dark) at 80 µmoL photons m⁻² s⁻¹. Samples were harvested at defined intervals during de-etiolation (T0, T4, T8, T12, T24, T48, T72, and T96). For each time point, seedlings were collected in triplicate, placed into 1.5 mL tubes, rapidly frozen in liquid nitrogen, and stored at −80 °C until further analyses [3,33].

2.2. Construct Generation and Transformation

To generate the CIA2p::CIA2_cia2cil and 35S::CIA2_cia2cil constructs, we employed the Golden Gate cloning method for precise assembly. Genomic DNA was extracted from Arabidopsis thaliana Col-0 using the CTAB method. We then amplified the full genomic sequence of CIA2 along with its native promoter region (1670 bp upstream of the start codon) by PCR For the CIA2p::CIA2_cia2cil construct, the native promoter was cloned upstream of the genomic CIA2 sequence, preserving its natural regulatory control, whereas for the 35S::CIA2_cia2cil construct, it was replaced with the constitutive CaMV 35S promoter. Both constructs were assembled in a single Golden Gate reaction using the Type IIS restriction enzyme BsaI and the binary vector pGoldenGate-SE9. Afterward, the constructs were introduced into the cia2cil double mutant background using Agrobacterium tumefaciens strain GV3101. Further experiments were conducted using the T3 generation of plants. For luciferase studies, the 2701 bp promoter region of CIA2 and the 2566 bp promoter region of CIL were amplified with primers listed in Tables S1 and S2. PCR products were purified and inserted into the entry clone using the pENTR/D-TOPO cloning kit (Invitrogen, Carlsbad, CA, USA). Next, CIA2 and CIL promoters were subcloned into the pGWB635 vector, with the luciferase reporter gene located upstream. The transgenic lines CIA2p::LUC and CILp::LUC were generated by floral dip transformation using Agrobacterium tumefaciens (GV3101) [34,35,36].

2.3. Luciferase Activity Assay

To analyze luciferase activity, the Luciferase Assay System kit (Promega, Madison, WI, USA) was used. Seedlings were collected at various time-points (T0, T4, T8, T12, T24, T48, T72, T96). 35 mg of seedlings were homogenized in liquid nitrogen, followed by the addition of 250 µL cell lysis buffer. The supernatant was separated after centrifugation and mixed with a luciferin substrate solution. Luminescence was quantified using a luminometer (Berthold Technologies, Bad Wildbad, Germany; Lumat LB9507) [37].

2.4. Chlorophyll a Fluorescence

Chlorophyll a fluorescence was assessed in seedlings using a pulse amplitude-modulated FluorCam 800 MF system operated with its dedicated control software (Photon Systems Instruments, Drasov, Czech Republic). Prior to recording fluorescence values, the plates were dark-adapted for 30 min to enable the determination of F₀ and F_m. The nomenclature and definitions of chlorophyll fluorescence parameters followed established descriptions in the literature [38,39].

2.5. Pigments Analysis

Approximately 20–50 mg of frozen plant material was ground at 4 °C in a Mixer Mill MM 400 (Retsch, Düsseldorf, Germany) at 30 Hz for 5 min in the presence of 1 mL of cold acetone (−20 °C). The resulting homogenate was concentrated using a Savant DNA120 SpeedVac system (Thermo Scientific, Waltham, MA, USA) to remove the solvent. The dried residue was re-suspended in chilled solvent A (acetonitrile: methanol, 90:10, v/v), briefly homogenized, and then passed through a 0.2 μm nylon membrane filter (Whatman, Maidstone, UK). The filtrate was transferred to autosampler vials, sealed, and kept at −80 °C in darkness until HPLC analysis (Shimadzu, Kyoto, Japan). Pigments were separated at 30 °C on a Synergi™ 4 μm MAX-RP 80 Å column (250 × 4.6 mm; Phenomenex, Torrance, CA, USA). The chromatographic run began with solvent A for 10 min to resolve xanthophylls, followed by solvent B (methanol:ethyl acetate, 68:32, v/v) for an additional 10 min, at a flow rate of 1 mL min⁻¹. Quantification was based on the peak area per microgram of fresh tissue, as per established procedures [40,41].

2.6. Immunoblot Analysis

Proteins were isolated from whole seedlings by extracting the tissue in four volumes (w/v) of SDS–PAGE sample buffer containing 0.2 M Tris/HCl (pH 6.8), 0.4 M dithiothreitol, 8% (w/v) SDS, 0.4% (w/v) bromophenol blue, and 40% (v/v) glycerol. The samples were heated at 95 °C for 15 min to denature the proteins, and insoluble material was removed by centrifugation at 16,000× g for 5 min. Aliquots containing 40 µg of total protein were loaded onto SDS-polyacrylamide gels (10–15% depending on the molecular weight of the target protein), separated electrophoretically, and transferred to Immobilon-P PVDF membranes (Merck, Darmstadt, Germany) using semi-dry blotting. Immunodetection was performed with specific primary antibodies at the following dilutions: LHCB2 (1:500, AS01 003, Agrisera, Vännäs, Sweden), D1/psbA (1:10,000, AS05 084, Agrisera), and ELIP1 (1:1000, PHY0842A, PhytoAB, San Jose, CA, USA). After overnight incubation at 4 °C, membranes were washed and incubated for 1 h at room temperature with horseradish peroxidase–conjugated anti-rabbit secondary antibodies (1:3000, Agrisera). Signal development was carried out using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Waltham, MA, USA), and immunoreactive bands were visualized with a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA) and quantified using ImageLab software (version 5.2.1, Bio-Rad) [42,43].

2.7. Quantitative Real-Time PCR

Total RNA was isolated from plant material using the Plant RNA Reagent (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol. First-strand cDNA synthesis was carried out with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative PCR was performed with a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the Power SYBR Green PCR Master Mix (Life Technologies). Relative transcript abundance was calculated using UPL7 and PP2AA3 as reference genes [44]. All primers used in this study are listed in Table S1.

2.8. Transmission Electron Microscopy

For transmission electron microscopy, small pieces of leaf tissue from seedlings (T48) were fixed for 3 h in 0.1 M cacodylate buffer (pH 7.2) containing 2% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde, followed by four washes in the same buffer. The material was then post-fixed in 2% (v/v) osmium tetroxide for 2 h at low temperature, dehydrated through a graded ethanol series, and transitioned to propylene oxide before being embedded in EPON epoxy resin. Polymerization of the resin was carried out overnight at 60 °C. Thin sections of approximately 80 nm were cut on a UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany). The sections were stained sequentially with uranyl acetate in saturated ethanol and then with lead citrate. Ultrastructural observations were made using an FEI 268D “Morgagni” transmission electron microscope (FEI Company, Hillsboro, OR, USA) equipped with an Olympus-SIS “Morada” digital camera (Olympus, Tokyo, Japan) [45]. Quantitative analysis of thylakoids was performed using Fiji (ImageJ) software (version 1.53) [46].

2.9. Confocal Microscopy

Chloroplast structure in T48 seedlings was assessed by confocal microscopy using a Zeiss LSM700 system (Zeiss, Oberkochen, Germany) equipped with EC Plan-Neofluar 20× and 40× objectives. Chlorophyll autofluorescence was excited at 488 nm, and emission was collected through a 652–682 nm bandpass filter with a 601 nm beam splitter. Chloroplast dimensions (in μm) were measured from the confocal images using the Fiji (ImageJ) software package [46,47].

2.10. Statistical Analysis

Statistical evaluations were performed in GraphPad Prism (version 8). Differences among experimental groups were assessed by ANOVA, and significant effects were further analyzed using Tukey’s post hoc test for pairwise comparisons. The data is provided as mean ± SEM, with statistical significance set at (p < 0.05 (*), p < 0.005 (**), or p < 0.001 (***)).

3. Results

3.1. CIA2 and CIL Promote Cotyledon Greening of Etiolated Seedlings During De-Etiolation and Are Essential for Chloroplast Biogenesis

Previous studies indicate that CIA2 and CIL impact chloroplast development and plastid rRNA maturation, processes that may indirectly influence translational capacity [30]. We further investigated whether these proteins are involved in regulating chloroplast development during the transition from dark to light. The phenotypic analysis highlights the developmental dynamics of all genotypes used in this study (Figure 1A). All genotypes, except the cia2cil double mutant, showed progressive greening and an increase in chlorophyll and carotenoid content over time, consistent with active chloroplast biogenesis in response to light. The chlorophyll a/b ratio also gradually increased in all lines during de-etiolation, reflecting gradual chloroplast development. In contrast, the double mutant consistently exhibited lower chlorophyll a/b ratios, particularly between 24 and 96 h following transition from darkness to light (Figure 1D). Nevertheless, the cia2cil maintained a pale green phenotype at all analyzed time points compared to Col-0 (WT), whereas its phenotype was reverted in the complementation lines. To determine the activity of CIA2 and CIL promoters during de-etiolation, a luciferase assay was performed using reporter constructs. Both promoters showed strong induction during early light exposure (T0–T8), with CIA2 promoter activity being notably higher than that of CIL, consistent with the more pronounced phenotypes observed in cia2 and cia2cil mutants (Figure 1C).

Subsequently, we used confocal and transmission electron microscopy (TEM) to investigate chloroplast development during de-etiolation in cia2, cil, and cia2cil mutants. It revealed altered chloroplast morphology in the cia2cil mutant compared to Col-0 and single mutants, while both CIA2p::CIA2_cia2cil and 35Sp::CIA2_cia2cil complementation lines restored chloroplast morphology (Figure S3). However, since chlorophyll autofluorescence does not define the chloroplast envelope, these observations were considered qualitative. Therefore, chloroplast size was quantitatively assessed using TEM, which allows direct visualization of chloroplast boundaries. TEM-based measurements demonstrated that chloroplast length was significantly reduced in the cia2cil double mutant and, to a lesser extent, in the cia2, whereas the cil mutant did not show any difference, compared to Col-0 (Figure 2I). In addition to reduced chloroplast size, TEM images revealed defects in thylakoid membrane organization in the cia2cil mutant, including less compact and irregular grana stacks compared to Col-0 and single mutants (Figure 2G,H). Quantitative analysis further showed that granum width and granum length were significantly reduced in the cia2cil mutant, whereas these parameters remained unchanged or showed slight increases in the cil plants (Figure 2J,K).

3.2. Role of CIA2 and CIL in Maintaining PSII Function During De-Etiolation

To further confirm whether CIA2 and CIL contribute to optimal PSII function during de-etiolation, we monitored chlorophyll a fluorescence to visualize the dynamic responses of photosystems after transition from dark to light conditions. PSII maximal photochemical efficiency (F_v/F_m) increased gradually in wild-type plants and achieved a maximal level 24 h after light exposure, while in cia2cil the process was delayed, reaching the same levels after 72 h (Figure 3A). In complementation lines, F_v/F_m values were fully restored; however, NPQ levels were only partially restored when compared to the wild type (Figure 3A,B). Additionally, the double mutant maintained increased non-photochemical quenching (NPQ) after 24 h of light exposure (Figure 3B), indicating a prolonged reliance on protective energy dissipation processes. On the other hand, complementation lines, particularly CIA2p::CIA2_cia2cil, exhibited a significant recovery in PSII efficiency, similar to that of wild-type plants.

3.3. CIA2 and CIL Might Regulate the Expression of Photosynthesis-Related Genes During De-Etiolation

CIA2 and CIL are dual-localized transcription factors implicated in regulating the expression of chloroplast-targeted genes, including those involved in protein translation and import, thylakoid assembly, and photoprotection [30,32,48]. To assess their impact on transcript abundance during chloroplast biogenesis, we profiled gene expression patterns across functional categories using pathway-based clustering (Figure 4 and Figure S4). Genes involved in chloroplast development and light-responsive gene expression, GLK1, GLK2, CGA1, MYBS1, and HY5, showed overall reduced transcript levels in the cia2cil double mutant compared to Col-0. Among these genes, GLK1 showed the most consistent reduction in transcript abundance, particularly at later stages, whereas changes in GLK2 and CGA1 were variable across time points and more modest in magnitude. Expression of MYBS1 was decreased in the double mutant, and HY5 also showed lower transcript levels at multiple time points. Light signaling regulators PIF1 and PIF4 exhibited distinct patterns. PIF4 transcript levels were higher in the cia2cil mutant during the early phase (T0–T12), while PIF1 showed a transient increase followed by levels comparable to Col-0. PIF1 was reduced in the single mutants across several time points.

Genes encoding components of the protein translation and import machinery, including RPL11, RPL18, RPL28, RPS6, TOC159, TIC110, and CPN, exhibited reduced expression in the cia2cil mutant. The decrease was consistent across most genes in this group, particularly during the early and mid-stages of de-etiolation (T12–T48).

Genes encoding photosystem components and light-harvesting proteins, including LHCB2 and psbA, exhibited more pronounced expression reductions in the cia2cil mutant compared to genes involved in chloroplast development, particularly during the mid-to-late stages of de-etiolation, coinciding with the observed delays in pigment accumulation and PSII. The expression of ELIP1 was induced in all genotypes during the early stages (T4–T8), then declined. Transcript levels in cia2cil were comparable to Col-0 across the time course. Immunoblot analysis showed that ELIP1 protein accumulated transiently during early de-etiolation, declined after approximately 12 h of light exposure, and thereafter remained at comparable levels across all genotypes (Figure 5). In contrast, LHCB2 and psbA proteins accumulated more slowly in cia2cil and were detected only after prolonged exposure to light. In both CIA2p::CIA2_cia2cil and 35Sp::CIA2_cia2cil complementation lines, LHCB2, psbA, and ELIP1 protein accumulation patterns were restored to wild-type levels, consistent with a functional rescue of chloroplast development.

4. Discussion

The study highlights the roles of CIA2 and CIL in supporting chloroplast biogenesis and photosynthetic development during de-etiolation. The cia2cil double mutant exhibited delayed greening, reduced chlorophyll content, and impaired PSII function, indicating that CIA2 and CIL contribute redundantly to these processes. This observation is consistent with previous findings showing that CIA2 and CIL are involved in chloroplast development and plastid rRNA maturation [30]. The modest reduction in chlorophyll a/b ratio in cia2cil mutants from T24 to T96 further suggests delayed photosynthetic maturation rather than complete loss of PSII functionality, supporting the notion of redundant but essential roles of CIA2 and CIL in early photomorphogenesis. Luciferase assays showed early promoter activation of both CIA2 and CIL shortly after light exposure (Figure 1C), consistent with their involvement in early stages of chloroplast development during de-etiolation. The stronger induction of the CIA2 promoter compared to CIL correlates with the more pronounced phenotypes observed in cia2 and cia2cil mutants, supporting previous findings that CIA2 plays a more dominant role [29,30,32,48]. This early light responsiveness aligns with their involvement in photomorphogenic regulation. In line with this, quantitative analysis of CIA2 transcript levels in the complementation lines (Figure S1) revealed that 35S::CIA2cia2cil exhibited the highest expression across all time points (except T0), while CIA2p::CIA2cia2cil showed a more transient peak early in de-etiolation. These differences suggest that constitutive 35S-driven expression may disrupt fine-tuning of gene expression required for chloroplast biogenesis, whereas native promoter-driven CIA2 expression provides a more physiologically regulated response. Together, these results support a model in which CIA2 and CIL act early in the light response pathway to contribute to coordinated chloroplast development, and highlight the importance of temporal regulation of CIA2 expression for balanced gene expression during early photomorphogenesis.

Our observations extend these studies by demonstrating that CIA2 and CIL are activated early during the dark-to-light transition and contribute to the initiation of light-driven chloroplast biogenesis. Pigment and PSII dynamics further reinforce the involvement of CIA2 and CIL in photosynthetic development. The cia2cil double mutant exhibited delayed recovery of PSII efficiency (Fv/Fm) and elevated NPQ levels during early de-etiolation (Figure 3), indicating a reliance on photoprotective mechanisms due to impaired PSII maturation. These observations are consistent with those of Wang et al. (2022), who demonstrated PSII inefficiency in mutants with chlorophyll deficiencies and highlighted the dependence of PSII functionality on pigment and protein biosynthesis pathways [49].

The lower lutein content in cia2cil (Figure 3B and Figure S2) and delayed accumulation of light-harvesting proteins (LHCB2) and D1 (psbA) support a compromised ability to protect PSII from photooxidative damage, consistent with the photoprotective role of lutein and LHCB2 in stabilizing LHCII and enhancing thermal energy dissipation under high-light conditions [50,51,52]. As shown by Tikkanen et al. (2014), reduced LHCB2 abundance can impair antenna organization and energy distribution, ultimately compromising PSII efficiency [53].

Immunoblot analysis revealed a delayed accumulation of LHCB2 and psbA proteins in the cia2cil mutant, consistent with reduced Fv/Fm and delayed D1 assembly. Gradual increases in D1 protein levels from T0 to T96 reflect slow PSII maturation rather than complete assembly failure, paralleling earlier reports correlating reduced D1 abundance with decreased PSII functionality [54,55,56,57]. These findings align with earlier work on mutants affecting chloroplast development, emphasizing the importance of temporal coordination between pigment synthesis and protein accumulation for proper photosystem assembly [30,31,48].

The chloroplast ultrastructure observed through confocal and TEM further supports the functional role of CIA2 and CIL in structural development. While confocal imaging showed smaller and less defined chloroplasts in cia2cil, TEM analysis confirmed significantly reduced chloroplast length and irregular grana formation at T48 (Figure 2). These defects mirror previous observations in mature cia2cil plants, where disrupted thylakoid organization and compromised plastid rRNA processing were reported [31]. The structural abnormalities correlate with impaired photosynthetic performance and emphasize that CIA2 and CIL are probably involved in both the regulation of gene expression patterns and the structural maturation of chloroplasts.

Analysis of gene expression patterns provided further insights into the molecular basis of the observed phenotypes. Gene expression analysis revealed that genes involved in chloroplast development, including GLK1, GLK2, HY5, CGA1, and MYBS1, exhibited moderate reductions in expression in the cia2cil double mutant (Figure 4 and Figure S4) [22,23]. Yang et al. (2022) also suggested that CIA2 and CIL may influence GLK1 expression, either directly or indirectly, a finding supported by our expression data [32]. Fitter et al. (2002) emphasized that GLKs are critical for chloroplast formation and activation of photosynthetic gene expression, supporting the functional relevance of this pathway [21]. Transcript levels of HY5 were reduced in cia2cil, while PIF4 and PIF1 showed variable increases early in de-etiolation. These patterns suggest altered light signaling dynamics, as HY5 promotes photomorphogenesis and represses PIF activity, whereas PIFs support skotomorphogenesis [17,18,19,58]. CGA1 transcript levels in the cia2cil mutant showed a transient increase at early stages of de-etiolation compared to Col-0, followed by a decline to levels that were lower than or comparable to those of Col-0 in later time points. Overall, these changes were modest and variable across the time course, suggesting that CGA1 expression is not strongly or persistently altered in the absence of CIA2 and CIL. Rather than indicating a direct regulatory role, this pattern is more consistent with subtle shifts in the expression dynamics of light-responsive genes during early photomorphogenesis [59]. In contrast, ELIP1 transcript levels remained largely unchanged between genotypes. At the protein level, ELIP1 accumulated transiently during the early phase of de-etiolation and declined thereafter, with comparable levels observed across genotypes at later time points. This behavior is consistent with previous reports indicating that ELIP1 protein abundance primarily reflects early light exposure and chloroplast stress–associated photoprotective responses rather than sustained, genotype-specific regulation [60].

Disruptions extended beyond photosynthetic genes to include core components of the protein import and translational machinery. Genes encoding translocon components (TIC110, TOC159), ribosomal proteins (RPL11, RPL18, RPL28, RPS6), and the chaperonin CPN were reduced in cia2cil (Figure 4). This is consistent with Sun et al. (2009), who reported that CIA2 contributes to chloroplast protein import and translation [48]. Gawroński et al. (2021) also demonstrated that ribosomal deficiencies in cia2cil mutants delay plastid protein synthesis and impair chloroplast development [30]. These findings suggest that CIA2 and CIL support chloroplast biogenesis by ensuring adequate translational and import capacity.

In summary, CIA2 and CIL function in a coordinated and partially redundant manner to support chloroplast biogenesis during de-etiolation. Their combined loss might disrupt pigment biosynthesis, impair PSII function, delay chloroplast structural maturation, and compromise expression of genes associated with light signaling, protein import, and translation. These findings reinforce and expand on prior studies, highlighting the multifaceted regulatory roles of CIA2 and CIL in establishing photosynthetic competence during the transition from dark to light growth.

5. Conclusions

In conclusion, our results demonstrate that CIA2 and CIL directly or indirectly support multiple aspects of nuclear gene expression encoding chloroplast proteins, thereby optimizing PSII efficiency and activity (Figure 6). Although these putative TFs are likely involved in distinct regulatory processes, their precise molecular functions and regulatory mechanisms remain to be elucidated. Nonetheless, our study highlights the contribution of CIA2 and CIL to chloroplast biogenesis through the coordinated regulation of key genes, integrating phenotypic, biochemical, and molecular analyses with TEM ultrastructural observations to provide insights into the mechanisms underlying chloroplast formation. While our work focused primarily on chloroplast development during de-etiolation, previous reports and our observations indicate that the cia2cil double mutant frequently exhibits growth retardation after greening. These growth defects are likely attributable to impaired chloroplast function, temperature responses, and reduced photosynthetic capacity. Future studies will address whether modulation of these putative TFs can be used to improve biomass accumulation, yield, or stress tolerance under diverse environmental conditions in crop species.