Role of CIA2 and CIL in the regulation of chloroplast photomorphogenesis in Arabidopsis

Role of CIA2 and CIL in the regulation of chloroplast photomorphogenesis in Arabidopsis | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Role of CIA2 and CIL in the regulation of chloroplast photomorphogenesis in Arabidopsis Roshanak Zarrin Ghalami , Pawel Burdiak , Muhammad Kamran , Maria Duszyn , Anna Rusaczonek , Ewa Muszyńska-Sadłowska , Stanisław Karpiński doi: https://doi.org/10.1101/2025.11.24.690140 Roshanak Zarrin Ghalami 1 Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Warsaw University of Life Sciences , Nowoursynowska Street 159, 02-776 Warsaw, Poland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pawel Burdiak 1 Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Warsaw University of Life Sciences , Nowoursynowska Street 159, 02-776 Warsaw, Poland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Muhammad Kamran 1 Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Warsaw University of Life Sciences , Nowoursynowska Street 159, 02-776 Warsaw, Poland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maria Duszyn 1 Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Warsaw University of Life Sciences , Nowoursynowska Street 159, 02-776 Warsaw, Poland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anna Rusaczonek 2 Department of Botany, Institute of Biology, Warsaw University of Life Sciences , Nowoursynowska 159, 02-776 Warsaw, Poland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ewa Muszyńska-Sadłowska 2 Department of Botany, Institute of Biology, Warsaw University of Life Sciences , Nowoursynowska 159, 02-776 Warsaw, Poland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stanisław Karpiński 1 Department of Plant Genetics, Breeding and Biotechnology, Institute of Biology, Warsaw University of Life Sciences , Nowoursynowska Street 159, 02-776 Warsaw, Poland Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: stanislaw_karpinski{at}sggw.pl Abstract Full Text Info/History Metrics Supplementary material Preview PDF 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 assembly and related gene expression. Although their precise molecular roles remain unclear, our findings support their involvement in chloroplast development. This is indicated by deregulation of foliar chlorophyll content, chlorophyll a fluorescence parameters, chloroplast size, and gene expression of PSII molecular markers in 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 transcription of genes such as LHCB2.1 and psbA . According to the immunoblotting and microscopy imaging results, there is an impaired assembly 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. 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 some biochemical, physiological and morphological changes such as stem shortening, leaf expansion, chlorophyll synthesis, and functional chloroplast development [ 1 – 3 ]. De-etiolation involves optimizing a plant’s ability to receive and convert absorbed light into biochemistry and 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 – 6 ]. Chloroplast development is a process that requires the interaction of nuclear and plastid gene expression to provide the efficient assembly and optimal function of the photosynthetic apparatus [ 7 , 8 ]. Moreover, this process is also involved in transporting nuclear-encoded proteins across the double chloroplast membrane, mediated by specialized translocon complexes, to facilitate proper chloroplast biogenesis and function [ 9 ]. The translocons at the outer membrane of chloroplasts (TOC) and the inner membrane of chloroplasts (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 – 16 ]. Several TFs are involved in the chloroplast biogenesis process. HY5 is a transcription regulator of light-mediated genes, which is responsible for chlorophyll and photosynthetic proteins synthesis and thylakoid membrane formation [ 17 – 19 ]. Phytochrome-interacting transcription factors (PIFs) integrates 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 – 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 ]. Other proteins like Early light-induced protein 1 (ELIP1), Photosystem II protein D1 (psbA), and LIGHT-HARVESTING CHLOROPHYLL A/B-BINDING (LHCB2) are also involved in protecting chloroplasts against photo-oxidative damage through efficient energy dissipation, and energy transfer within PSII for optimal photosynthetic activity [ 25 – 28 ]. 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 – 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 – 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 assembly and chloroplast ultrastructure during de-etiolation. Results 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 de-etiolation. 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 hours ( Figure 1B , S2). The cia2cil double mutant maintained a pale green phenotype in all analyzed time points compared to Col-0 (WT), whereas its phenotype was reverted in complementation lines. To determine the changes in CIA2 and CIL promoter activity during de-etiolation, a luciferase assay was performed using the CIA2 and CIL promoters. The analysis revealed that both these transcription factors are upregulated during the first hours of de-etiolation (T0-T8) ( Figure 1C ). This supports the involvement of CIA2 and CIL in the early stages of the photomorphogenic transition. To check the difference between complementation lines, we quantified CIA2 transcript levels at T0-T96 time points in Col-0, 3 5Sp::CIA2 cia2cil, and CIA2p::CIA2 cia2cil . CIA2 expression in 35Sp::CIA2 cia2cil was highest across all time points except T0. While CIA2p::CIA2 cia2cil showed lower expression overall, with a transient peak at 0 hours after light exposure (Figure S1). Download figure Open in new tab Figure 1: Pigment accumulation and CIA2/CIL promoter activity during de-etiolation in Arabidopsis seedlings. (A) Arabidopsis thaliana seedlings of the following genotypes: Col-0, cia2, cil, cia2cil , CIA2p::CIA2 cia2cil , 35Sp::CIA2 cia2cil during the first 96 hours of de-etiolation (T0, T4, T8, T12, T24, T48, T72 and T96). (B) Analysis of chlorophyll content. (C) Luciferase reporter assay showing CIA2 and CIL promoter activity during the first 96 hours of de-etiolation. This study involved two genetic constructs carrying luciferase genes driven by CIA2 or CIL promoters. Mean values were derived from 9 measurements (n = 9), and statistical significance (ANOVA and Tukey HSD test) is shown relative to Col-0 (P < 0.05 (*), P < 0.005 (**), or P < 0.001 (***)). To investigate chloroplast development during de-etiolation in cia2 , cil, and cia2cil mutants, confocal and transmission electron microscopy (TEM) were used. In confocal microscopy observations, chloroplasts in cia2cil exhibited abnormal structures and were noticeably smaller than those in the wild type (Col-0) and single mutants ( Figure 2A-D , 2G ). Complementation lines significantly reduced these structural defects, with CIA2p::CIA2 cia2cil restoring chloroplast morphology and size to nearly wild-type levels ( Figure 2A-G ). Download figure Open in new tab Figure 2. Ultrastructural analysis of chloroplast morphology during de-etiolation. (A-F) Confocal laser scanning microscopy (CLSM) images of chloroplasts (scale bar = 10 μm) in the (A) Col-0, (B) cia2, (C) cil, (D) cia2cil mutants and (E) CIA2p::CIA2 cia2cil and (F) 35Sp::CIA2 cia2cil complementation lines after 48h of de-etiolation. The red fluorescence corresponds to the chlorophyll fluorescence in the chloroplasts of mesophyll cells. (G) Quantitative analysis of chloroplast diameter (in μm), derived from confocal images using Fiji (ImageJ) (n=25). (H-O) Transmission Electron Microscopy (TEM) images of chloroplasts (scale bar = 1μm) in the (H,I) Col-0 and the (J,K) cia2, (L,M) cil, (N,O) cia2cil mutants after 48h. (P) The number of granum and quantitative analysis of granum (Q) length and (R) width (in μm), derived from TEM images using Fiji (ImageJ) (n = 40), and statistical significance (ANOVA and Tukey HSD test) is shown relative to Col-0 (P < 0.05 (*), P < 0.005 (**), or P < 0.001 (***)). A detailed analysis of the thylakoid structure by TEM revealed that the length and width of the grana were significantly decreased in cia2cil compared to all other analyzed genotypes. However, the double mutant granum number (per chloroplast) remained unchanged, and interestingly, cil single mutants exhibited an increase in both grana width and length, while the cia2 single mutant displayed no significant differences compared to wild-type ( Figure 2 P-R ). Moreover, TEM analysis of chloroplasts in the cia2cil showed defects in thylakoid membranes and less well-organized grana structures ( Figure 2 N-Q, P-R ). Role of CIA2 and CIL in maintaining PSII function during de-etiolation To further confirm if CIA2 and CIL are required for 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 cia2cil double mutant plants after 72 h ( Figure 3A ). In complementation lines, Fv/Fm 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 24h 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. Download figure Open in new tab Figure 3. Photosystem II performance during de-etiolation monitored by chlorophyll a fluorescence parameters. (A) Maximum quantum efficiency of photosystem II ( F v / F m ) and (B) non-photochemical quenching (NPQ) in Col-0, cia2 , cil , cia2cil , CIA2p::CIA2 cia2ci l, and 35Sp::CIA2 cia2cil seedlings during 96h of de-etiolation. Mean values were derived from 12 measurements (n = 12), and statistical significance (ANOVA and Tukey HSD test) is shown relative to the Col-0 (P < 0.05 (*), P < 0.005 (**), or P < 0.001 (***)). CIA2 and CIL might regulate the expression of photosynthesis-related genes during de-etiolation CIA2 and CIL have been demonstrated to function as transcription factors that regulate the expression of genes encoding chloroplast-targeted proteins, including key components of the chloroplast translational machinery. They also exhibited dual subcellular localization to the nucleus and chloroplasts, suggesting that CIA2 and CIL might play a role in coordinating gene expression between the nucleus and chloroplasts, which is critical for chloroplast biogenesis and development [ 30 , 32 , 33 ]. RT-qPCRs were performed to investigate whether these proteins also regulate gene expression in response to de-etiolation. The study revealed severe abnormalities in gene expression levels, particularly in the double mutant plants ( Figure 4 , S3). During early time points (T0-T24), the cia2cil double mutant displayed significantly increased expression of ELIP1 , which encodes early light-inducible protein that is involved in protecting PSII against photooxidative stress. On the other hand, transcriptomic analysis in cia2cil revealed a significant downregulation of genes, such as GLK1 and GLK2, which are involved in chloroplast development , as well as LHCB2 and psbA , which are necessary for the functional PSII ( Figure 4 , S3). Deregulated expression patterns observed in double mutant plants correlated with delayed greening and reduced chlorophyll accumulation. Two other genes, TIC110 and TOC159 , which encode the chloroplast protein import machinery system, were also downregulated in the double mutant, indicating a disorder in the import of nuclear-encoded proteins required for chloroplast development. Light signaling regulators, HY5 and PIFs, showed the opposite expression pattern. In wild-type plants, HY5 expression, which promotes photomorphogenesis and chloroplast biogenesis, was induced between T8 and T24 of de-etiolation, while PIF1 and PIF4 , both negative regulators of photomorphogenesis , were downregulated at these periods. This pattern was opposite in the cia2cil double mutant, indicating a failure to switch from skotomorphogenic (dark-adapted) to photomorphogenic (light-adapted) development. Furthermore, down-regulation of ribosomal genes such as RPL11 , RPL18 , RPL28 , and RPS6 in cia2cil plants directly correlates with the delayed chloroplast development and PSII assembly observed in the cia2cil mutant ( Figure 4 , S3). Notably, the expression of genes involved in chloroplast development, photosynthesis, and ribosome biogenesis was partially or fully restored in both complementation lines ( CIA2p::CIA2 cia2cil and 35S::CIA2 cia2cil ), suggesting functional rescue by CIA2. In contrast, the expression of PIF1 and PIF4 remained downregulated in both lines, further supporting their negative regulatory relationship with CIA2 activity during de-etiolation ( Figure 4 , S3). Download figure Open in new tab Figure 4: Gene expression heatmaps of chloroplast biogenesis-related pathways during de-etiolation. Heat-map of the expression of genes involved in (A) chloroplast development and transcriptional regulation, (B) protein translation and import, and (C) photosystem components and light harvesting in cia2 , cil , cia2cil , CIA2p::CIA2 cia2cil , 35Sp::CIA2 cia2cil in 0-96 hours. During de-etiolation, CGA1 expression showed strong suppression in the cia2 , cil , and especially cia2cil mutants at 12 hours, a key time point for light-induced chloroplast gene activation. The complementation line CIA2p::CIA2 cia2cil restored CGA1 expression to wild-type levels, while 35S::CIA2 cia2cil showed variable expression, particularly at later time points. MYBS1 expression was elevated in cia2 seedlings at the first time points (0-12 h), but was generally reduced in the cil mutant and unstable in cia2cil , indicating disrupted regulation. The CIA2p::CIA2 cia2cil line maintained expression close to wild-type across the time course, while 35S::CIA2 cia2cil exhibited inconsistent and often lower expression. Immunoblot analysis ( Figure 5A ) revealed a time-dependent pattern of ELIP1 protein accumulation, with high levels during the early hours of de-etiolation, followed by a gradual decline in all genotypes. The cia2cil double mutant consistently exhibited higher ELIP1 levels than the other genotypes throughout the time course. The accumulation of LHCB2 and psbA proteins in the double mutant was delayed, and protein buildup could be seen only after 72 hours of light exposure. Single mutants have a level of LHCB2 and psbA protein that is nearly identical to that of Col-0 throughout the de-etiolation process. In complementation lines, LHCB2 and psbA began to accumulate within 12 hours of light exposure and continued to increase with time, similar to Col-0. Download figure Open in new tab Figure 5. Immunoblot analysis of chloroplast protein accumulation during de-etiolation. Immunoblot analysis using total protein isolated from seedlings of (A) Col-0, cia2 , cil , cia2cil during 96h of de-etiolation and (B) complementation lines (Col-0, cia2cil , CIA2p::CIA2 cia2cil , 35Sp::CIA2c ia2cil ) in T0-T48 time points. The level of ELIP1, LHCB2 and the D1 protein of PSII reaction center are shown. The level of the 50 kDa unknown protein, stained with Coomassie blue, confirmed equal gel loading. Discussion The study reveals the potential roles of CIA2 and CIL in chloroplast biogenesis. Our data show that CIA2 and CIL function synergistically in chlorophyll synthesis, chloroplast development, and PSII activity. The cia2cil double mutant displays a pale-green phenotype, reduced pigment levels, disrupted chloroplast structure, and decreased PSII efficiency. It’s consistently lower chlorophyll a/b ratio compared to other genotypes suggests impaired PSII core assembly and an imbalance between PSII and its associated light-harvesting complexes. Complementation of the double mutant restored chloroplast structure, pigment content, and PSII performance to near wild-type levels. These findings suggest that CIA2 and CIL may play roles in PSII biogenesis and chloroplast maturation, either directly or indirectly ( Figure 1 - 3 ). Furthermore, luciferase reporter assays revealed that the CIA2 and CIL promoters are strongly induced shortly after the transition from dark to light ( Figure 1C ). These findings support a role for CIA2 and CIL in early light-driven chloroplast development during de-etiolation. These findings offer new insights into the roles of CIA2 and CIL in photomorphogenesis, extending previous research that highlighted their importance in chloroplast biogenesis only in mature plants [ 29 , 29 , 30 , 33 ]. Numerous regulators have been described to play roles in chloroplast biogenesis. Among these, GLKs synergistically influence the balance of chlorophyll biosynthesis, chloroplast development, and the level of PhANGs (Photosynthesis Associated Nuclear Genes) [ 23 ]. Furthermore, it has been suggested that GLKs play a more direct role in coordinating and regulating the expression of genes crucial for chloroplast development, chlorophyll biosynthesis, light harvesting, and carbon fixation ( GUN4, CAO, LHCB1-6 ) [ 22 ]. Yang et al. (2022) suggested that CIA2 and CIL might regulate chloroplast development directly or indirectly via GLK1. Furthermore, their results implied that CIA2 plays a more crucial role than CIL in upregulating GLK1 expression [ 22 , 33 ]. Disturbed chlorophyll synthesis, along with lower transcript and protein levels associated with light-harvesting complex (LHCB2) and photosystem assembly (D1) in cia2cil ( Figure 4 , 5 ), led to delayed and diminished PSII efficiency during de-etiolation ( Figure 3 ), indicating a disturbed capacity of energy utilization and light capture [ 34 , 35 ]. A failure to assemble fully functioning PSII complexes in mutant plants results in elevated non-photochemical quenching (NPQ) after 24 h of exposure, indicating a dependence on protective energy dissipation processes. These results are consistent with those of Wang et al. (2022), which also demonstrated PSII inefficiency in mutants with chlorophyll deficiencies and highlighted the dependence of PSII functionality on pigment and protein biosynthesis pathways [ 36 ]. Lutein plays a role in stabilizing the LHCII and in enabling thermal energy dissipation in protecting photosystem II under high-light conditions [ 37 – 39 ]. Lower NPQ in cia2cil plants was accompanied by lower lutein contents ( Figure 3B , S2), and lower LHCB2 level ( Figure 5A ), when compared to Col-0, suggesting impaired photoprotective mechanisms. Therefore, a lower level of LHCB2, which is required for the proper organization of antenna complexes, presumably resulted in impaired PSII assembly, as evidenced by the visible stacked thylakoid grana [ 34 ]. This association implies that the cia2cil photoprotection is compromised due to deficiencies in both carotenoid production and LHCII integrity. Additionally, decreased Fv/Fm, an indicator of PSII dysfunction, is commonly correlated with lower D1 protein levels [ 40 – 42 ]. In our studies, the cia2cil showed delayed development of PSII efficiency, along with a gradual increase of D1 accumulation from T0 to T96 ( Figure 5A ), which suggests that during this process, the CIA2 and CIL play a positive and putative regulatory role in D1 synthesis. On the other hand, the incomplete rescue of NPQ with complementation lines suggests that CIA2 alone is insufficient to fully restore the photoprotective capacity of the double mutant. NPQ involves rapid energy dissipation mechanisms that may rely more on CIL. According to immunoblot analysis ( Figure 5 ), the delayed accumulation of LHCB2 and psbA in the cia2cil double mutant suggests a deficiency in the nuclear and plastid protein translation during chloroplast development. Single mutants did not exhibit this delay, indicating that CIA2 and CIL have overlapping activities. Complementation with CIA2 partially improved early protein accumulation, especially after 12 hours of de-etiolation, which suggests that CIA2 might has a function in regulating photosynthetic protein synthesis since it has been shown that the translation of chloroplast mRNA and the maturation and accumulation of 23S rRNA are influenced by CIA2 and CIL [ 30 ]. Overall, CIA2 is critical for maintaining a balance between photoprotective and photosynthetic protein synthesis throughout the transition from etiolation to photomorphogenesis. Confocal microscopy revealed abnormally formed chloroplasts in the cia2cil mutant, with decreased development and overall size ( Figure 2 ). A smaller granum size only in cia2cil double mutant but not in single mutants implies that CIA2 and CIL regulatory proteins might have complementary functions in regulating the stacking and organization of thylakoid membranes and granum structure; however, they are not necessary for granum initiation, as shown by the unchanged granum number ( Figure 2P ). The single mutants, cia2 and cil , exhibit fewer structural defects and show better photosynthetic function, suggesting that both CIA2 and CIL contribute to chloroplast development, and their combined loss exacerbates the phenotype. Ultrastructural observation using TEM revealed significant abnormalities, including undeveloped thylakoid membranes and diminished stacking at T48 ( Figure 2P-R ), consistent with another study that suggested similar defects in fully developed plants [ 31 ]. These findings are in accordance with reduced PSII assembly ( Figure 3A ) and emphasize the possible regulatory impact of CIA2 and CIL on chloroplast structure and function. A high level of ELIP1 in the double mutant ( Figures 4 and 5A ) suggests that plants attempt to reduce photooxidative damage, which is consistent with the findings of Kleine et al. (2007), who found comparable responses in chloroplast-stressed mutants. In both complementation lines, ELIP1 expression is partially or fully rescued, indicating that CIA2 alone can restore ELIP1 expression and likely its downstream chloroplast-related functions, even in the absence of CIL, thereby reinforcing the idea that CIA2 is a possible key regulator. Beyond defects in chlorophyll synthesis and photosystem assembly, our data suggest broader disruptions in the chloroplast biogenesis machinery. In particular, the observed downregulation of key components of the protein import apparatus, such as TIC110 and TOC159 in all analyzed mutants ( Figure 4 , S3) is consistent with Sun et al. (2009) study, which showed CIA2 crucial role in protein import and translation processes. Also, the lack of expression of ribosomal genes such as RPL11, RPL18, RPL28 , and RPS6 reveals another reason for chloroplast development deficiency and delay in the cia2cil mutant, which is associated with inefficient chloroplast-encoded protein synthesis and assembly. These findings are consistent with Gawronski et al. (2021) study, which found that ribosomal deficits in cia2cil lead to a delay in chloroplast-targeted protein synthesis and assembly. CPN also showed decreased expression in cia2cil which is associated with the failure of post-import protein folding and maturation, a side effect of deficient protein import machinery [ 43 ]. The combined disruptions in protein import, folding, and assembly suggest that chloroplast development in cia2cil may be impaired not only locally within the organelle but also through defects in nuclear regulatory programs. The phenotypic similarities between cia2cil and glks mutants also support the hypothesis that CIA2 and CIL are involved in chloroplast biogenesis. Both double mutant lines exhibit pale green phenotypes and deficiencies in the formation of the photosynthetic apparatus [ 22 , 32 ]. Furthermore, their putative localization to chloroplasts implies possible functions as environmental sensors, directing adaptive responses via retrograde signaling [ 44 ]. Fitter et al. (2017) showed that these GLKs are required for chloroplast formation and the activation of photosynthetic machinery genes, while Yang et al. (2022) found that CIA2 and CIL coordinate chloroplast biogenesis and function primarily by enhancing the expression of the nuclear factor GLK1 and genes associated with chloroplast transcription, translation, protein import, and photosynthesis. Downregulation of CGA1 in cia2cil mutants emphasises the involvement of CIA2 and CIL in activating early light-responsive genes during de-etiolation, supporting their function in transcriptional coordination [ 45 ]. MYBS1 expression is upregulated in the cia2 single mutant but is downregulated in the cia2cil double mutant, indicating a compensatory response to the loss of CIA2 alone, and a loss of regulatory control when both CIA2 and CIL are absent, which is consistent with MYBS1 functioning along with GLKs in Arabidopsis [ 24 ]. The reduced expression of GLKs in the double mutant reinforces the upstream role of CIA2 and CIL, while CGA1 , MYBS1 , GLK1 and GLK 2 expression were restored in the CIA2p::CIA2 cia2cil complementation line but not in 35S::CIA2 cia2cil , emphasizing the importance of native promoter-driven expression for proper timing and coordination of chloroplast biogenesis. The differences between CIA2p::CIA2 cia2cil and 35S::CIA2 cia2cil lines likely reflect that constitutive overexpression of CIA2 may disrupt regulatory balance, either by overriding normal developmental controls or by triggering negative feedback that suppresses downstream gene activation. Together, these results reveal that CIA2 and CIL may redundantly regulate key transcription factors (CGA1, MYBS1, GLK1 and GLK2), and that their absence disrupts chloroplast development by impairing the transcriptional network required for de-etiolation. Both complementation lines restored HY5 activation and photomorphogenic development, but the 35Sp::CIA2 cia2cil line showed stronger upregulation of PIF4 , likely reflecting excessive CIA2 expression. This suggests that while proper HY5 activation is sufficient for recovery, native levels of CIA2 provide a more balanced regulation of the transcriptional network. Beyond GLK-mediated regulation, the transcriptional balance between HY5 and PIFs is another critical component of light-dependent development. HY5 promotes light responses, whereas PIFs drive skotomorphogenesis; their opposite expression patterns in cia2cil , with HY5 downregulated and PIFs upregulated in the double mutant, indicate impaired photomorphogenic development and an important role of CIA2 and CIL in balancing these opposing signaling pathways. These transcriptional patterns indicate a coordinated regulatory network in which CIA2 and CIL might play a role. The downregulation of TIC110, TOC159, ribosome-encoded genes, and CPN , which leads to impaired protein synthesis and import, along with GLK1, GLK2 , and HY5 low expression and the upregulation of PIFs in the double mutant, indicates a widespread failure in chloroplast biogenesis. While in complementation lines, partial restoration was observed and highlights the importance of CIA2 in chloroplast protein import (e.g., TOC33 and TOC75 ), plastid translation, protein folding, and photosynthesis functionality and defective ribosome assembly [ 30 – 32 ]. The observed differences in gene expression between the CIA2p::CIA2 cia2cil and 35Sp::CIA2 cia2cil complementation lines likely reflect variation in promoter strength and positional effects. The greening phenotype observed in both lines indicates that a threshold level of CIA2 expression is sufficient to support chloroplast development. However, the elevated and constitutive expression driven by the 35S promoter may alter the transcriptional fine-tuning required during de-etiolation, thereby contributing to the observed transcriptional differences. These interpretations are supported by the CIA2 expression levels shown in Figure S1. The gene expression patterns of complementation lines highlight the role of CIA2 as an important positive and putative regulator of chloroplast biogenesis and photomorphogenesis. In both CIA2p::CIA2 cia2cil and 35S::CIA2 cia2cil lines, CIA2 complementation led to increased expression of genes related to photosynthesis ( GLK1 , GLK2 , LHCB2 , psbA ), ribosome biogenesis ( RPS6 , RPL11 , RPL18, RPL28 ), and protein import into the chloroplast machinery ( TIC110 , TOC159 ). Luciferase activity results show that CIA2 is much more upregulated than CIL during the de-etiolation process ( Figure 1C ). This corresponds with the CIA2 putative role in the activation of genes required for chloroplast protein import and ribosomal protein production, and CIL has more supplementary role in this process [ 29 , 31 – 33 ]. In conclusion, our results show that CIA2 and CIL either directly or indirectly affect several aspects of chloroplast gene expression and PSII assembly ( Figure 6 ). These TFs may have multiple roles, but their precise molecular functions and regulatory mechanisms require further investigation. Nonetheless, our study highlights the role of CIA2 and CIL in chloroplast biogenesis through the coordinated regulation of key genes. By integrating phenotypic, biochemical, molecular, and structural data, we provide new insights into the mechanisms of chloroplast formation. These findings also open up potential avenues for improving photosynthetic efficiency and stress tolerance in plants through genetic and biotechnological approaches. Download figure Open in new tab Figure 6. Proposed model of CIA2 and CIL regulatory roles in chloroplast biogenesis during de-etiolation in Arabidopsis thaliana. Light perception activates photoreceptors, leading to induction of HY5, a key transcription factor that promotes the expression of CIA2 and CIL. In turn, CIA2 and CIL reinforce HY5 expression and inhibit PIFs, negative regulators of photomorphogenesis, thereby promoting a successful transition from skotomorphogenesis to photomorphogenesis. CIA2 and CIL also activate nuclear genes required for chloroplast development, including GLK1/2, CGA1, MYBS1 (transcriptional regulators), TOC159/TIC110 (protein import machinery), RPLs/RPSs (ribosomal proteins), and LHCB2/psbA (PSII components). These regulatory cascades coordinate chloroplast biogenesis and etioplast-to-chloroplast transition. Bidirectional communication between the nucleus and chloroplast via anterograde and retrograde signaling ensures tight coordination of gene expression and organelle development. Material and Methods Plant material and growth conditions Arabidopsis thaliana seeds (Columbia-0 (Col-0) , cia2 (SALK_004037), cil (SAIL_228_C01) , cia2cil, CIA2p::CIA2 cia2cil , and 35Sp::CIA2 cia2cil were surface-sterilized with chlorine gas generated by the reaction of sodium hypochlorite and concentrated hydrochloric acid (HCl). Seeds were sown in spots containing 50 seeds (to facilitate rapid harvest) on agar plates containing 0.5 Murashige and Skoog salt mixture (Duchefa Biochemie, Haarlem, Netherlands) without sucrose. Following stratification in the dark for 3 days at 4°C, plates with seeds were kept for 2 hours at the light intensity of 40 µmol photons m -2 s -1 at 21°C and then transferred to the dark for 3 days at 21°C. After 3 days of darkness, plates were transferred to long photoperiod (16/8 h) growing conditions with the light intensity of 80 µmol photons m -2 s -1 . Seedlings were collected at each time point (T0, T4, T8, T12, T24, T48, T72, T96) in three replicates and transferred into a 1.5 ml tube, flash-frozen in liquid nitrogen, and stored at -80°C until further analysis [ 3 , 46 ]. 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 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 Table S1 and S2. PCR products were purified and inserted into the entry clone using the pENTR/D-TOPO cloning kit (Invitrogen). 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). [ 47 – 49 ]. Luciferase Activity Assay To analyze luciferase activity, the Promega Luciferase Assay System kit 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, Lumat LB9507) [ 50 ]. Chlorophyll a fluorescence Chlorophyll a fluorescence parameters were measured on seedlings using a pulse amplitude-modulated FluorCam 800 MF and the associated software (Photon Systems Instruments, Drasov, Czech Republic). Before measurements, the plates were kept in the dark for 30 min to determine F 0 and F m . Chlorophyll fluorescence terminology has been previously described [ 51 , 52 ]. Pigments analysis 20 –50 mg of frozen tissue was homogenized in a Mixer Mill MM 400 (Retsch, Düsseldorf, Germany) (5 min, 4 °C, 30 Hz) with 1 ml of cold acetone (−20 °C). The homogenate was evaporated using Savant DNA120 SpeedVac (Thermo Scientific, Waltham, MA, USA), dissolved in cold solvent A (acetonitrile: methanol; 90:10; v/v), and re-homogenized for 1 min. The extract was filtered through a 0.2 μm nylon filter (Whatman) into an auto-sampler vial, capped, and stored in the dark at −80 °C for HPLC analysis (Shimadzu, Kyoto, Japan). The pigments were separated on a Synergi TM 4 μm MAX-RP 80 Å LC Column 250 × 4.6 mm (Phenomenex, Torrance, CA, USA) at 30 °C. Solvent A was used for 10 min to elute all xanthophylls, followed by solvent B (methanol: ethyl acetate; 68:32; v/v) for 10 min at a flow rate of 1 ml/min. The results are given as the peak area per μg of fresh weight, according to the protocol previously used [ 53 , 54 ]. Immunoblot analysis Proteins were extracted from whole seedlings in four volumes (w/v) of SDS-PAGE sample buffer (0.2M Tris/HCL pH 6.8, 0.4 M dithiothreitol, 8% (w/v) SDS, 0.4% (w/v) Bromophenol blue, and 40% (v/v) glycerol). Proteins were denatured for 15 min at 95°C, and cell debris was removed by centrifugation for 5 min at 16,000 g. Proteins (40 µg) were separated on SDS-PAGE (10–15% (w/v) polyacrylamide concentrations depending on the molecular weight of the protein of interest and transferred onto an Immobilon-P PVDF membrane (Merck) by semi-dry transfer. Immunodetections were performed using specific antibodies and dilutions: 1:500 LHCB2 (AS01 003, Agrisera), 1:10000 D1(psbA) (AS05 084, Agrisera) and 1:1000 ELIP1 (PHY0842A, PhytoAB). After incubation with primary antibodies overnight at 4°C, membranes were incubated for 1 h at RT with horseradish peroxidase–conjugated secondary antibodies (1:3000 (v/v) anti-rabbit secondary antibodies, Agrisera). Protein bands were immunodetected using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) according to the manufacturer’s recommendations, visualized with the ChemiDoc XRS+ System (Bio-Rad) and analyzed with ImageLab Software 5.2.1 (Bio-Rad)[ 55 , 56 ]. Quantitative Real-Time PCR RNA extraction was performed using Plant RNA Reagent (Life Technologies) according to the manufacturer’s instructions. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies). All PCR amplifications were run on a 7500 Fast Real-Time PCR System (Applied Biosystems) using a Power SYBR Green PCR master mix (Life Technologies) UPL7 and PP2AA3 were used as reference genes to calculate relative expression [ 57 ]. All primers used in this study are listed in Table S1. Transmission electron microscopy Sample preparation for TEM analysis involved fixation of leaf fragments (seedlings (T48)) in 2 % (v/v) glutaraldehyde and 2 % (v/v) paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) for 3 h and washing off four times in the same cacodylate buffer [ 58 ]. Next, the plant samples were secondary fixed in 2 % (v/v) osmium tetroxide for 2 h at low temperature, dehydrated in an ethanol gradient, and replaced with propylene oxide. Finally, samples were embedded in EPON epoxy resin. Its polymerization was performed at 60 ° C overnight. Ultra-thin sections of about 80 nm thickness were prepared using a UCT ultramicrotome (Leica Microsystems), stained with uranyl acetate dissolved in a saturated ethanol solution, followed by incubation in lead citrate. An FEI 268D ‘Morgagni’ transmission electron microscope (FEI Company, Hillsboro, OR, USA) equipped with an Olympus-SIS ‘Morada’ digital camera (Olympus) was applied for the ultrastructural examination [ 58 ]. Quantitative analysis of thylakoids was performed using Fiji (ImageJ) [ 59 ]. Confocal microscopy Seedlings (T48) were analysed for chloroplast structure. Confocal microscopy observations were performed using a Zeiss LSM700 microscope equipped with 20x and 40x EC Plan-Neofluar objectives. Chlorophyll fluorescence was excited using a 488 nm laser. Signals were detected using 652-682BP (chlorophyll) filters with a beam splitter set at 601 nm. Quantitative analysis of chloroplast diameter (in μm), derived from confocal images, was performed using Fiji (ImageJ) [ 59 , 60 ]. Statistical analysis All statistical analyses were carried out using GraphPad Prism version 8. ANOVA and Tukey’s HSD test were used to examine differences across several experimental groups, followed by 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 (***)). Supplementary data Table S1 and S2: Primer sequence used for quantitative real-time PCR (qRT-PCR) and constructs. Fig. S1. CIA2 expression in complementation lines. Fig. S2. Pigments analysis, the content of carotenoids and lutein. Fig. S3. Transcriptome Analysis. qRT-PCR analysis of gene expression in graphs. Author contributions RZ, MK, MD, PB, AR, and EMS: investigation; RZ, PB, and MD: formal analysis and visualization; RZ: writing the original draft; PB, MD, and SK: writing - review & editing; SK: supervision and funding acquisition. Conflict of interest The authors declare no conflict of interest. Funding This work was supported by the Polish National Science Center (Narodowe Centrum Nauki; OPUS20, UMO-2020/39/B/NZ3/02103) given to S.K. Funder Information Declared Narodowe Centrum Nauki , OPUS20, UMO-2020/39/B/NZ3/02103 References 1. ↵ Banaś , A.K. ; Leja , K. ; Zgłobicki , P. ; Jedynak , P. ; Kowalska , E. ; Strzałka , W. ; Grzyb , J. ; Myśliwa-Kurdziel , B . De-Etiolation Is Almost Color Blind: The Study of Photosynthesis Awakening under Blue and Red Light . Plant and Cell Physiology 2024 , 65 , 1993 – 2017 . OpenUrl PubMed 2. ↵ Armarego-Marriott , T. ; Sandoval-Ibañez , O. ; Kowalewska , Ł . Beyond the Darkness: Recent Lessons from Etiolation and de-Etiolation Studies . Journal of experimental botany 2020 , 71 , 1215 – 1225 . OpenUrl CrossRef PubMed 3. ↵ Pipitone , R. ; Eicke , S. ; Pfister , B. ; Glauser , G. ; Falconet , D. ; Uwizeye , C. ; Pralon , T. ; Zeeman , S.C. ; Kessler , F. ; Demarsy , E . A Multifaceted Analysis Reveals Two Distinct Phases of Chloroplast Biogenesis during De-Etiolation in Arabidopsis . Elife 2021 , 10 , e62709 . OpenUrl CrossRef PubMed 4. ↵ Wu , S.-H . Gene Expression Regulation in Photomorphogenesis from the Perspective of the Central Dogma . Annual review of plant biology 2014 , 65 , 311 – 333 . OpenUrl CrossRef PubMed 5. Ma , L. ; Li , J. ; Qu , L. ; Hager , J. ; Chen , Z. ; Zhao , H. ; Deng , X.W . Light Control of Arabidopsis Development Entails Coordinated Regulation of Genome Expression and Cellular Pathways . The Plant Cell 2001 , 13 , 2589 – 2607 . OpenUrl Abstract / FREE Full Text 6. ↵ Armarego-Marriott , T. ; Kowalewska , Ł. ; Burgos , A. ; Fischer , A. ; Thiele , W. ; Erban , A. ; Strand , D. ; Kahlau , S. ; Hertle , A. ; Kopka , J . Highly Resolved Systems Biology to Dissect the Etioplast-to-Chloroplast Transition in Tobacco Leaves . Plant physiology 2019 , 180 , 654 – 681 . OpenUrl Abstract / FREE Full Text 7. ↵ Pogson , B.J. ; Woo , N.S. ; Förster , B. ; Small , I.D . Plastid Signalling to the Nucleus and Beyond . Trends in plant science 2008 , 13 , 602 – 609 . OpenUrl CrossRef PubMed Web of Science 8. ↵ Pogson , B.J. ; Ganguly , D. ; Albrecht-Borth , V . Insights into Chloroplast Biogenesis and Development . Biochimica et Biophysica Acta (BBA)-Bioenergetics 2015 , 1847 , 1017 – 1024 . OpenUrl 9. ↵ Richardson , L.G. ; Paila , Y.D. ; Siman , S.R. ; Chen , Y. ; Smith , M.D. ; Schnell , D.J . Targeting and Assembly of Components of the TOC Protein Import Complex at the Chloroplast Outer Envelope Membrane . Frontiers in plant science 2014 , 5 , 269 . 10. ↵ Thomson , S.M. ; Pulido , P. ; Jarvis , R.P . Protein Import into Chloroplasts and Its Regulation by the Ubiquitin-Proteasome System . Biochemical Society Transactions 2020 , 48 , 71 – 82 . OpenUrl CrossRef PubMed 11. ↵ Schleiff , E. ; Jelic , M. ; Soll , J . A GTP-Driven Motor Moves Proteins across the Outer Envelope of Chloroplasts . Proceedings of the National Academy of Sciences 2003 , 100 , 4604 – 4609 . OpenUrl Abstract / FREE Full Text 12. ↵ Demarsy , E. ; Lakshmanan , A.M. ; Kessler , F . Border Control: Selectivity of Chloroplast Protein Import and Regulation at the TOC-Complex . Frontiers in plant science 2014 , 5 , 483 . 13. ↵ Rochaix , J . Chloroplast Protein Import Machinery and Quality Control . The FEBS journal 2022 , 289 , 6908 – 6918 . OpenUrl CrossRef PubMed 14. ↵ Czarnocka , W. ; Karpiński , S . Friend or Foe? Reactive Oxygen Species Production, Scavenging and Signaling in Plant Response to Environmental Stresses . Free Radical Biology and Medicine 2018 , 122 , 4 – 20 . OpenUrl PubMed 15. Czarnocka , W. ; Van Der Kelen , K. ; Willems , P. ; Szechyńska-Hebda , M. ; Shahnejat-Bushehri , S. ; Balazadeh , S. ; Rusaczonek , A. ; Mueller-Roeber , B. ; Van Breusegem , F. ; Karpiński , S . The Dual Role of LESION SIMULATING DISEASE 1 as a Condition-dependent Scaffold Protein and Transcription Regulator . Plant, Cell & Environment 2017 , 40 , 2644 – 2662 . OpenUrl 16. ↵ Kamran , M. ; Burdiak , P. ; Karpiński , S . Crosstalk Between Abiotic and Biotic Stresses Responses and the Role of Chloroplast Retrograde Signaling in the Cross-Tolerance Phenomena in Plants . Cells 2025 , 14 , 176 . 17. ↵ Xiao , Y. ; Chu , L. ; Zhang , Y. ; Bian , Y. ; Xiao , J. ; Xu , D . HY5: A Pivotal Regulator of Light-Dependent Development in Higher Plants . Frontiers in plant science 2022 , 12 , 800989 . 18. Chattopadhyay , S. ; Ang , L.-H. ; Puente , P. ; Deng , X.-W. ; Wei , N . Arabidopsis bZIP Protein HY5 Directly Interacts with Light-Responsive Promoters in Mediating Light Control of Gene Expression . The Plant Cell 1998 , 10 , 673 – 683 . OpenUrl Abstract / FREE Full Text 19. ↵ Gangappa , S.N. ; Botto , J.F . The Multifaceted Roles of HY5 in Plant Growth and Development . Molecular plant 2016 , 9 , 1353 – 1365 . OpenUrl CrossRef PubMed 20. ↵ Leivar , P. ; Monte , E . PIFs: Systems Integrators in Plant Development . The Plant Cell 2014 , 26 , 56 – 78 . OpenUrl Abstract / FREE Full Text 21. ↵ Fitter , D.W. ; Martin , D.J. ; Copley , M.J. ; Scotland , R.W. ; Langdale , J.A . GLK Gene Pairs Regulate Chloroplast Development in Diverse Plant Species . The Plant Journal 2002 , 31 , 713 – 727 . OpenUrl CrossRef PubMed Web of Science 22. ↵ Waters , M.T. ; Moylan , E.C. ; Langdale , J.A . GLK Transcription Factors Regulate Chloroplast Development in a Cell-autonomous Manner . The Plant Journal 2008 , 56 , 432 – 444 . OpenUrl CrossRef PubMed Web of Science 23. ↵ Zubo , Y.O. ; Blakley , I.C. ; Franco-Zorrilla , J.M. ; Yamburenko , M.V. ; Solano , R. ; Kieber , J.J. ; Loraine , A.E. ; Schaller , G.E . Coordination of Chloroplast Development through the Action of the GNC and GLK Transcription Factor Families . Plant physiology 2018 , 178 , 130 – 147 . OpenUrl Abstract / FREE Full Text 24. ↵ Frangedakis , E. ; Yelina , N.E. ; Billakurthi , K. ; Hua , L. ; Schreier , T. ; Dickinson , P.J. ; Tomaselli , M. ; Haseloff , J. ; Hibberd , J.M . MYB-Related Transcription Factors Control Chloroplast Biogenesis . Cell 2024 , 187 , 4859 – 4876 .e22 , doi: 10.1016/j.cell.2024.06.039 . OpenUrl CrossRef 25. ↵ Górecka , M. ; Lewandowska , M. ; Dąbrowska-Bronk , J. ; Białasek , M. ; Barczak-Brzyżek , A. ; Kulasek , M. ; Mielecki , J. ; Kozłowska-Makulska , A. ; Gawroński , P. ; Karpiński , S . Photosystem II 22kDa Protein Level-a Prerequisite for Excess Light-inducible Memory, Cross-tolerance to UV-C and Regulation of Electrical Signalling. Plant , Cell & Environment 2020 , 43 , 649 – 661 . OpenUrl 26. Hutin , C. ; Nussaume , L. ; Moise , N. ; Moya , I. ; Kloppstech , K. ; Havaux , M . Early Light-Induced Proteins Protect Arabidopsis from Photooxidative Stress . Proceedings of the National Academy of Sciences 2003 , 100 , 4921 – 4926 . OpenUrl Abstract / FREE Full Text 27. Luciński , R. ; Jackowski , G . The Structure, Functions and Degradation of Pigment-Binding Proteins of Photosystem II . Acta biochimica polonica 2006 , 53 , 693 – 708 . OpenUrl PubMed Web of Science 28. ↵ Zarter , C.R. ; Adams , W.W. ; Ebbert , V. ; Adamska , I. ; Jansson , S. ; Demmig-Adams , B. Winter Acclimation of PsbS and Related Proteins in the Evergreen Arctostaphylos Uva-ursi as Influenced by Altitude and Light Environment . Plant, Cell & Environment 2006 , 29 , 869 – 878 . OpenUrl CrossRef PubMed Web of Science 29. ↵ Yang , C.-Y. ; Sun , C.-W . Sequence Analysis and Protein Interactions of Arabidopsis CIA2 and CIL Proteins . Botanical Studies 2020 , 61 , 1 – 13 . OpenUrl PubMed 30. ↵ Gawroński , P. ; Burdiak , P. ; Scharff , L.B. ; Mielecki , J. ; Górecka , M. ; Zaborowska , M. ; Leister , D. ; Waszczak , C. ; Karpiński , S . CIA2 and CIA2-LIKE Are Required for Optimal Photosynthesis and Stress Responses in Arabidopsis Thaliana . The Plant Journal 2021 , 105 , 619 – 638 . OpenUrl CrossRef PubMed 31. ↵ Li , M. ; Ruwe , H. ; Melzer , M. ; Junker , A. ; Hensel , G. ; Tschiersch , H. ; Schwenkert , S. ; Chamas , S. ; Schmitz-Linneweber , C. ; Börner , T . The Arabidopsis AAC Proteins CIL and CIA2 Are Sub-Functionalized Paralogs Involved in Chloroplast Development . Frontiers in Plant Science 2021 , 12 , 681375 . 32. ↵ Yang , C. ; Yan , W. ; Chang , H. ; Sun , C . Arabidopsis CIA2 and CIL Have Distinct and Overlapping Functions in Regulating Chloroplast and Flower Development . Plant Direct 2022 , 6 , e380 . OpenUrl 33. ↵ Sun , C.-W. ; Huang , Y.-C. ; Chang , H.-Y . CIA2 Coordinately Up-Regulates Protein Import and Synthesis in Leaf Chloroplasts . Plant Physiology 2009 , 150 , 879 – 888 . OpenUrl Abstract / FREE Full Text 34. ↵ Tikkanen , M. ; Aro , E.-M . Integrative Regulatory Network of Plant Thylakoid Energy Transduction . Trends in plant science 2014 , 19 , 10 – 17 . OpenUrl CrossRef PubMed Web of Science 35. ↵ Huesgen , P.F. ; Schuhmann , H. ; Adamska , I . Photodamaged D1 Protein Is Degraded in Arabidopsis Mutants Lacking the Deg2 Protease . FEBS letters 2006 , 580 , 6929 – 6932 . OpenUrl CrossRef PubMed Web of Science 36. ↵ Wang , G. ; Zeng , F. ; Song , P. ; Sun , B. ; Wang , Q. ; Wang , J . Effects of Reduced Chlorophyll Content on Photosystem Functions and Photosynthetic Electron Transport Rate in Rice Leaves . Journal of plant physiology 2022 , 272 , 153669 . 37. ↵ Ruban , A.V. ; Johnson , M.P . Dynamics of Higher Plant Photosystem Cross-Section Associated with State Transitions . Photosynthesis Research 2009 , 99 , 173 – 183 . OpenUrl CrossRef PubMed Web of Science 38. Ilioaia , C. ; Johnson , M.P. ; Liao , P.-N. ; Pascal , A.A. ; van Grondelle , R. ; Walla , P.J. ; Ruban , A.V. ; Robert , B . Photoprotection in Plants Involves a Change in Lutein 1 Binding Domain in the Major Light-Harvesting Complex of Photosystem II . Journal of Biological Chemistry 2011 , 286 , 27247 – 27254 . OpenUrl Abstract / FREE Full Text 39. ↵ Ilioaia , C. ; Johnson , M.P. ; Duffy , C.D. ; Pascal , A.A. ; van Grondelle , R. ; Robert , B. ; Ruban , A.V . Origin of Absorption Changes Associated with Photoprotective Energy Dissipation in the Absence of Zeaxanthin . Journal of Biological Chemistry 2011 , 286 , 91 – 98 . OpenUrl Abstract / FREE Full Text 40. ↵ Wang , Z. ; Li , G. ; Sun , H. ; Ma , L. ; Guo , Y. ; Zhao , Z. ; Gao , H. ; Mei , L . Effects of Drought Stress on Photosynthesis and Photosynthetic Electron Transport Chain in Young Apple Tree Leaves . Biology open 2018 , 7 , bio035279. 41. Chaumont , M. ; Morot-Gaudry , J. ; Foyer , C . Effects of Photoinhibitory Treatment on CO2 Assimilation, the Quantum Yield of CO2 Assimilation, D1 Protein, Ascorbate, Glutathione and Xanthophyll Contents and the Electron Transport Rate in Vine Leaves . Plant, Cell & Environment 1995 , 18 , 1358 – 1366 . OpenUrl 42. ↵ Bethmann , S. ; Melzer , M. ; Schwarz , N. ; Jahns , P. The Zeaxanthin Epoxidase Is Degraded along with the D1 Protein during Photoinhibition of Photosystem II . Plant Direct 2019 , 3 , e00185. 43. ↵ Sun , C.-W. ; Chen , L.-J. ; Lin , L.-C. ; Li , H . Leaf-Specific Upregulation of Chloroplast Translocon Genes by a CCT Motif–Containing Protein, CIA 2 . The Plant Cell 2001 , 13 , 2053 – 2061 . OpenUrl Abstract / FREE Full Text 44. ↵ Estavillo , G.M. ; Crisp , P.A. ; Pornsiriwong , W. ; Wirtz , M. ; Collinge , D. ; Carrie , C. ; Giraud , E. ; Whelan , J. ; David , P. ; Javot , H . Evidence for a SAL1-PAP Chloroplast Retrograde Pathway That Functions in Drought and High Light Signaling in Arabidopsis . The Plant Cell 2011 , 23 , 3992 – 4012 . OpenUrl Abstract / FREE Full Text 45. ↵ Chiang , Y.-H. ; Zubo , Y.O. ; Tapken , W. ; Kim , H.J. ; Lavanway , A.M. ; Howard , L. ; Pilon , M. ; Kieber , J.J. ; Schaller , G.E . Functional Characterization of the GATA Transcription Factors GNC and CGA1 Reveals Their Key Role in Chloroplast Development, Growth, and Division in Arabidopsis . Plant Physiology 2012 , 160 , 332 – 348 , doi: 10.1104/pp.112.198705 . OpenUrl Abstract / FREE Full Text 46. ↵ Lindsey III , B.E. ; Rivero , L. ; Calhoun , C.S. ; Grotewold , E. ; Brkljacic , J . Standardized Method for High-Throughput Sterilization of Arabidopsis Seeds . Journal of visualized experiments: JoVE 2017 , 56587 . 47. ↵ Nakagawa , T. ; Kurose , T. ; Hino , T. ; Tanaka , K. ; Kawamukai , M. ; Niwa , Y. ; Toyooka , K. ; Matsuoka , K. ; Jinbo , T. ; Kimura , T . Development of Series of Gateway Binary Vectors, pGWBs, for Realizing Efficient Construction of Fusion Genes for Plant Transformation . Journal of bioscience and bioengineering 2007 , 104 , 34 – 41 . OpenUrl CrossRef PubMed Web of Science 48. Emami , S. ; Yee , M. ; Dinneny , J.R . A Robust Family of Golden Gate Agrobacterium Vectors for Plant Synthetic Biology . Frontiers in plant science 2013 , 4 , 339 . 49. ↵ Engler , C. ; Kandzia , R. ; Marillonnet , S. A One Pot, One Step, Precision Cloning Method with High Throughput Capability . PloS one 2008 , 3 , e3647 . OpenUrl CrossRef PubMed 50. ↵ Manzoor , H. ; Kelloniemi , J. ; Chiltz , A. ; Wendehenne , D. ; Pugin , A. ; Poinssot , B. ; Garcia-Brugger , A . Involvement of the Glutamate Receptor A t GLR 3.3 in Plant Defense Signaling and Resistance to H Yaloperonospora Arabidopsidis . The Plant Journal 2013 , 76 , 466 – 480 . OpenUrl CrossRef PubMed Web of Science 51. ↵ Kramer , D.M. ; Johnson , G. ; Kiirats , O. ; Edwards , G.E . New Fluorescence Parameters for the Determination of QA Redox State and Excitation Energy Fluxes . Photosynthesis research 2004 , 79 , 209 – 218 . OpenUrl CrossRef PubMed Web of Science 52. ↵ Baker , N.R . Chlorophyll Fluorescence: A Probe of Photosynthesis in Vivo . Annu. Rev. Plant Biol . 2008 , 59 , 89 – 113 . OpenUrl CrossRef PubMed Web of Science 53. ↵ Rusaczonek , A. ; Czarnocka , W. ; Kacprzak , S. ; Witoń , D. ; Ślesak , I .; Szechyńska-Hebda , M. ; Gawroński , P. ; Karpiński , S . Role of Phytochromes A and B in the Regulation of Cell Death and Acclimatory Responses to UV Stress in Arabidopsis Thaliana . Journal of experimental botany 2015 , 66 , 6679 – 6695 . OpenUrl CrossRef PubMed 54. ↵ Rusaczonek , A. ; Czarnocka , W. ; Willems , P. ; Sujkowska-Rybkowska , M. ; Van Breusegem , F. ; Karpiński , S . Phototropin 1 and 2 Influence Photosynthesis, UV-C Induced Photooxidative Stress Responses, and Cell Death . Cells 2021 , 10 , 200 . 55. ↵ Laemmli , U.K . Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. nature 1970 , 227 , 680 – 685 . 56. ↵ Burnette , W.N . “Western Blotting”: Electrophoretic Transfer of Proteins from Sodium Dodecyl Sulfate-Polyacrylamide Gels to Unmodified Nitrocellulose and Radiographic Detection with Antibody and Radioiodinated Protein A . Analytical biochemistry 1981 , 112 , 195 – 203 . OpenUrl CrossRef PubMed Web of Science 57. ↵ Gawroński , P. ; Witoń , D. ; Vashutina , K. ; Bederska , M. ; Betliński , B. ; Rusaczonek , A. ; Karpiński , S . Mitogen-Activated Protein Kinase 4 Is a Salicylic Acid-Independent Regulator of Growth but Not of Photosynthesis in Arabidopsis . Molecular Plant 2014 , 7 , 1151 – 1166 . OpenUrl CrossRef PubMed 58. ↵ Morris , J.K . A Formaldehyde Glutaraldehyde Fixative of High Osmolality for Use in Electron Microscopy . J. cell Biol 1965 , 27 , 1A – 149A . OpenUrl CrossRef 59. ↵ Schindelin , J. ; Arganda-Carreras , I. ; Frise , E. ; Kaynig , V. ; Longair , M. ; Pietzsch , T. ; Preibisch , S. ; Rueden , C. ; Saalfeld , S. ; Schmid , B . Fiji: An Open-Source Platform for Biological-Image Analysis . Nature methods 2012 , 9 , 676 – 682 . OpenUrl PubMed 60. ↵ Pawley , J. Handbook of Biological Confocal Microscopy ; Springer Science & Business Media , 2006 ; Vol. 236 ; ISBN 0-387-25921-X . View the discussion thread. 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Share Role of CIA2 and CIL in the regulation of chloroplast photomorphogenesis in Arabidopsis Roshanak Zarrin Ghalami , Pawel Burdiak , Muhammad Kamran , Maria Duszyn , Anna Rusaczonek , Ewa Muszyńska-Sadłowska , Stanisław Karpiński bioRxiv 2025.11.24.690140; doi: https://doi.org/10.1101/2025.11.24.690140 Share This Article: Copy Citation Tools Role of CIA2 and CIL in the regulation of chloroplast photomorphogenesis in Arabidopsis Roshanak Zarrin Ghalami , Pawel Burdiak , Muhammad Kamran , Maria Duszyn , Anna Rusaczonek , Ewa Muszyńska-Sadłowska , Stanisław Karpiński bioRxiv 2025.11.24.690140; doi: https://doi.org/10.1101/2025.11.24.690140 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41910) Biophysics (21436) Cancer Biology (18576) Cell Biology (25480) Clinical Trials (138) Developmental Biology (13368) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15598) Genomics (22482) Immunology (17726) Microbiology (40360) Molecular Biology (17163) Neuroscience (88534) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)