An unrecognized and crucial role of chloroplast division in leaf variegation in Arabidopsis thaliana

An unrecognized and crucial role of chloroplast division in leaf variegation in Arabidopsis thaliana | 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 An unrecognized and crucial role of chloroplast division in leaf variegation in Arabidopsis thaliana View ORCID Profile Wenjuan Wu , Wei Guo , Haojie Zhu , Di Li , View ORCID Profile Zhiyi Zhang , Danni Lin , Meiying Qu , Zhenjia Yu , View ORCID Profile Jirong Huang doi: https://doi.org/10.1101/2025.04.06.647415 Wenjuan Wu Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University , Shanghai 200432, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Wenjuan Wu Wei Guo Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University , Shanghai 200432, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Haojie Zhu Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University , Shanghai 200432, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Di Li Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University , Shanghai 200432, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhiyi Zhang Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University , Shanghai 200432, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zhiyi Zhang Danni Lin Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University , Shanghai 200432, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Meiying Qu Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University , Shanghai 200432, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhenjia Yu Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University , Shanghai 200432, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jirong Huang Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University , Shanghai 200432, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jirong Huang For correspondence: huangjr{at}shnu.edu.cn Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Leaf variegation provides a valuable model for elucidating the molecular mechanisms that underlie chloroplast biogenesis, including both chloroplast development and division. While there has been notable advancement in comprehending the role of the Arabidopsis Yellow Variegated2 ( VAR2 ), which encodes the AtFtsH2 subunit of the thylakoid FtsH metalloprotease complex, the exact mechanism governing leaf variegation formation remains elusive. In this study, time-course microscopy analyses of chloroplast number per cell from the first leaf pair revealed that most var2 cells lack chloroplasts. Interestingly, chloroplast biogenesis in the green sector of var2 leaves is significantly delayed but prolonged compared to the wild type (WT), leading to increased heterogeneity in chloroplast number and size among cells. These results suggest that chloroplast biogenesis in the green sector can partially compensate for the defect in the white sector. Additionally, approximately 15% of cells in var2 white sectors are devoid of plastids, and chloroplast number in guard cells is significantly lower in var2 than in WT, indicating that VAR2 mutations impair chloroplast division. Consistently, the var2 phenotype is exacerbated by knocking out key plastid division genes, including Paralog of Accumulation and Replication of Chloroplasts6 ( PARC6 ) and Plastid Division1 ( PDV1 ), but is fully rescued by overexpressing PDV1 or PDV2 . Furthermore, VAR2 mutations inhibit etioplast development, while accelerating chloroplast biogenesis by knocking out Constitutively Photomorphogenic1 ( COP1 ) rescues the var2 phenotype. Together, our findings reveal a novel role for VAR2 in plastid division and its essential function in chloroplast biogenesis, providing new insights into the mechanism underlying variegated leaf formation. Introduction Chloroplast biogenesis in higher plants is a fundamental process that reconstructs photosynthesis and provides sufficient energy and materials for autotrophic growth. In general, chloroplast biogenesis is regulated by the two basic events, namely development and division of chloroplasts ( Sakamoto et al ., 2008 ). Chloroplast development is a light-triggered dynamic event facilitating any kind of non-photosynthetic plastids, such as small, undifferentiated proplastids in meristematic cells and differentiated plastids (etioplasts, chromoplasts and leucoplasts), into chloroplasts. It is well documented that chloroplast development is finely regulated by a series of important processes, such as coordinated expression of nuclear and plastidic genes, protein import into chloroplasts and subsequent targeting to the sub-organelle compartments, biogenesis of thylakoid membranes and photosynthetic complexes, etc ( Jarvis and López-Juez, 2013 ). On the other hand, the number of semiautonomous chloroplasts in a cell is determined by its division through binary fission, which keeps pace with cell differentiation, proliferation and expansion. These two events of chloroplast biogenesis are closely linked but also independent, leading to dramatic variation in chloroplast number in different cell types ( Fang et al ., 2022 ; Pyke, 1997 ). For example, some arc (accumulation and replication of chloroplast) mutants exhibit an increased number of small chloroplasts, while some have only one or two large chloroplasts ( Chen et al ., 2018 ). This indicates that chloroplast number and size, which reflect the outcomes of chloroplast division and development, respectively, can mutually compensated for each other. The variegated leaf is an idea phenotype to elucidate molecular mechanisms underlying chloroplast biogenesis, since green sectors with normal chloroplasts and yellow or white sectors with non-photosynthetic plastids coexist in the same genetic background, and severity of leaf variegation is significantly affected by growth conditions and developmental stages ( Liu et al ., 2010 ). To date, many variegated-leaf mutants, such as immutans ( im ), chloroplast mutator ( chm ), thylakoid formation 1 ( thf1 ), variegated1 ( var1 ) and var2 , have been identified in the model plant Arabidopsis thaliana ( Liu et al ., 2010 ; Yu et al ., 2007 ). Among them, VAR1 and VAR2 , which encode FtsH5 and FtsH2 subunits of the thylakoid FtsH metalloprotease complex, respectively, are the most extensively studied. VAR1/FtsH5 and FtsH1 are functionally redundant and are collectively referred to as type A, while VAR2/FtsH2 and FtsH8 are interchangeable and classified as type B. The two major subunits, FtsH2/VAR2 and FtsH5/VAR1, assemble with the minor subunits FtsH1 and FtsH8 to form the hetero-hexameric FtsH complex, which plays an important role in the degradation of the D1 protein at the reaction center of photosystem II (PSII) as well as in the removal of misfolded polypeptides and protein aggregates ( Kato et al ., 2018 ; Kato et al ., 2009 ). Several hypotheses have been proposed to explain form the formation of leaf variegation, one of which is a threshold model. This model suggests that a certain level of FtsH activity is required for chloroplast development and green sector formation ( Aluru et al ., 2006 ). Genetic screening for second-site mutations that suppress the var2 phenotype has provided substantial support for this hypothesis. It has been concluded that reduced plastidic gene expression can restore the var2 phenotype. A rational explanation is that these mutations extend the duration of chloroplast development, thereby lowering the threshold level of FtsH activity and suppressing leaf variegation ( Yu et al ., 2004 ). However, it has also been reported that decreased plastidic gene expression in the suppressor lines can indirectly enhance the level of FtsH activity through an unknown mechanism ( Wu et al ., 2016 ; Yu et al ., 2004 ). Therefore, further investigations are necessary to elucidate the molecular mechanisms underlying the formation of variegated leaves. Only a few publications have reported that mutants with defective chloroplast division exhibit a leaf variegation phenotype. Kadirjan-Kalbach et al . (2012) reported that reduced expression of negative chloroplast division regulator ARC1 encoded by FtsHi1 led to leaf variegation. In addition, Arabidopsis homologs of the bacterial mechanosensitive (MS) channels of small conductance MscS-Like 2 (MSL2) and MSL3 act as components of the chloroplast division machinery to regulate filamentous temperature-sensitive Z (FtsZ) ring formation ( Wilson et al ., 2011 ); the msl2 msl3 double mutant displays a weak leaf variegation phenotype ( Haswell and Meyerowitz, 2006 ). These results indicate that leaf variegation is related to defective chloroplast division. However, the specific role of chloroplast division in variegated-leaf formation has yet to be investigated. We propose that if chloroplast division occurs more slowly than cell division due to genetic mutations or environmental stresses, the number of chloroplasts in daughter cells may diminish, potentially reaching zero. This reduction in chloroplasts could ultimately lead to the development of leaf variegation. Plastid division is driven by a ring-shaped machinery, which contains the plastid-dividing (PD) ring on the cytosolic side of the outer membrane, the FtsZ ring at the stromal face of the inner membrane, and dynamin-related protein 5B ring (DRP5B ring, also known as ARC5 ring) located at the site of chloroplast division ( Osteryoung and Pyke, 2014 ). The FtsZ ring is first formed by two homologues (FtsZ1 and FtsZ2) of the bacterial division protein FtsZ, which is a self-assembling, microtubulin-like GTPase. The correct placement of the FtsZ ring at the middle of a chloroplast is determined by the Min system composed of MinD1 (minicell D1), MinE1, MCD1 (multiple chloroplast division site1) and ARC3 ( Chen et al ., 2018 ). The assembly of the FtsZ ring is promoted by ARC6 (Accumulation and Replication of Chloroplasts6), which is localized to the chloroplast inner membrane division site and anchors the FtsZ ring to the site via interaction with FtsZ2, but is inhibited by PARC6 through interaction with ARC3. In addition, the intermembrane space domain of PARC6 and ARC6 recruits the transmembrane proteins PDV1 and PDV2, respectively ( Glynn et al ., 2008 ; Wang et al ., 2017 ; Zhang et al ., 2016 ). Finally, DRP5B is recruited by PDV1 and PDV2 and assembled into the DRP5B ring on the cytoplasmic surface of the outer envelope, and constriction initiates ( Gao et al ., 2003 ; Sun et al ., 2020 ). In eukaryotic unicellular algae that own one chloroplast, chloroplast division occurs in the synthesis phase of the cell cycle, and the two duplicated chloroplasts are evenly partitioned into the daughter cells at the mitosis phase ( Sumiya et al ., 2016 ). In contrast, the relationship between chloroplast division and cell division in higher plants is complex and probably varies according to cell types, developmental stages and environmental conditions ( Miyagishima and Kabeya, 2010 ; Pedroza-Garcia et al ., 2016 ). For instance, chloroplasts continuously divide to maintain its stable density during expansion of mesophyll cells ( He et al ., 2021 ; Miyagishima, 2011 ), while chloroplasts are partitioned into each daughter cell unbiasedly or stochastically with a tendency toward equality in leaf dividing cells (Birky, 1983; Sheahan et al ., 2004 ). Although it is not essential that chloroplasts have to divide before a cell starts division due to several chloroplasts existing in each dividing cell, chloroplast numbers must maintain at a sufficient level during leaf development. However, current knowledge of the regulation of chloroplast biogenesis during leaf development is quite limited. In this study, we investigated the mechanism underlying leaf variegation formation from the perspective of chloroplast biogenesis. Our results showed that VAR2 mutations significantly affect both the development and division of chloroplasts during the early stages of leaf development. We found that accelerating either chloroplast division or chloroplast development can suppress leaf variegation. Analyses of chloroplast biogenesis of another leaf variegation mutant im , and the chloroplast development mutant clpR4 , which exhibits a leaf virescent phenotype, further support the notion that chloroplast division plays an important role in variegated leaf formation. Overall, our findings reveal a coordinated yet independent relationship between chloroplast development and division during leaf growth, intricately linking these processes to the phenotype of leaf variegation, virescence, and pale green leaves. Results VAR2 mutations result in the majority of cells lacking chloroplasts in the first pair of leaves To evaluate the effect of VAR2 mutations on chloroplast biogenesis, we examined the number of chloroplasts during the development of the first pair of leaves. To do this, we made protoplasts from the leaves of 9-, 13- and 17-day-old seedlings grown on half-strength Murashige and Skoog (MS) media containing 1% sucrose under the long day (16 hr light/8 hr dark) condition ( Figure 1A ). Developmental status of chloroplasts were observed using confocal microscopy via chlorophyll autofluorescence. Our results showed that var2 protoplasts had more variations in autofluorescence than the wild type (WT) ( Figure 1B ). To better quantify differential chloroplast development, we classified leaf cells into three categories, type a, b and c, which represent cells with well-developed, poorly developed, and no chloroplasts, respectively, based on chlorophyll autofluorescence ( Figure 1C ). We found that over 90% of the cells in 9-, 13- or 17-d-old WT plants were classified as type a, whereas only 5%, 12% and 20% of the cells, respectively, were type a in the corresponding var2 plants ( Figure 1C ). In addition, the percentage of type c cells in var2 reached as high as 92% at day 9, whereas no type c cells were detected in WT ( Figure 1C ). Taken together, our results indicate that VAR2 mutations severely impair chloroplast biogenesis during leaf development. Download figure Open in new tab Figure 1. VAR2 mutations severely inhibit chloroplast biogenesis during leaf development. (A) Phenotypes of 9-, 13-, and 17-day-old WT and var2 seedlings. Bars, 0.5 cm. Red arrows indicate the first pair of true leaves. (B) Confocal microscopy images of protoplasts isolated from the leaves shown in (A). Red indicates chlorophyll autofluorescence. Bar, 5 μm. (C) Representative protoplasts of type a, b and c, and their corresponding percentage in the total protoplasts. More than 300 protoplasts were randomly selected. Bars, 10 μm. (D) Correlation analysis between chloroplast number and cell size. More than 100 protoplasts with red autofluorescence were analyzed for each genotype. Equations are computed via linear regression. The r 2 (correlation coefficient) value of the best-fit line is shown in each panel. (E) Percentage of cells with the different number of chloroplasts. More than 100 protoplasts were analyzed for each replicate. Chloroplast biogenesis is significantly delayed yet prolonged in var2 To further investigate the difference in chloroplast biogenesis between WT and var2 , we examined the correlation between cell size and chloroplast number in type a and b cells. As shown in Figure 1D , the distribution of the chloroplast number per cell over cell size was more dispersed in var2 than in WT across all three developmental stages, indicating that VAR2 mutations disturb the uniformity of chloroplasts. Correlation coefficients ( r 2 ) between cell size and chloroplast number per cell were 0.65 and 0.47 in 9-day-old WT and var2 seedlings, respectively. However these values became closer in 13-day-old (0.66 VS 0.52) or 17-day-old (0.67 VS 0.60) seedlings. In addition, 12.6% of chloroplast-containing cells in var2 , compared to only 1.7% in WT, were larger than 1500 μm 2 in the 17-day-old seedlings. These results demonstrate that chloroplast biogenesis is much slower and more heterogeneous in var2 leaves. We also analyzed the effect of VAR2 mutations on the number of chloroplasts per cell over time. In 9-day-old seedlings, 25.3% of the cells contained fewer than 5 chloroplasts in var2 , compared to only 1.6% in WT ( Figure 1E ). Conversely, 9.8% of var2 cells had more than 20 chloroplasts, which was 7% higher than in WT (2.8%) ( Figure 1E ). A similar trend was observed in 13-day-old seedlings. Interestingly, by 17 days, var2 cells with an extremely low number of chloroplasts were no longer detected, and the percentage of cells containing more than 50 chloroplasts was significantly higher in var2 (22.2%) than in WT (10.6%) ( Figure 1E ). These data indicate that VAR2 mutations reduce the frequency of plastid division while prolonging the duration of chloroplast biogenesis. Defective plastid division leads to the generation of plastid-free cells in var2 The above results promoted us to investigate whether plastid division is impaired in var2 . To address this, we labelled plastids by expressing plastid ribosomal protein L11 (PRPL11)-GFP fusion protein in var2 ( PRPL11 - GFP / var2 ), which exhibits similar phenotypes of leaf variegation and comparable percentages of three-type cells as var2 (Figure S1). We analyzed plastid numbers in individual protoplasts isolated from the green and yellow sectors of PRPL11 - GFP / var2 mature variegated leaves. In the green sector, 86.83% of the cells contained chloroplasts, showing both chlorophyll autofluorescence and GFP fluorescence, while 10.48% of the cells displayed only GFP signals, indicating the presence of plastids without chlorophyll, such as leucoplasts. In contrast, only 21.05% of the cells in the yellow sector owned chloroplasts, while 62.87% contained only GFP signals ( Figure 2A , 2B ). Surprisingly, neither GFP nor red chlorophyll autofluorescence were observed in some cells, suggesting the presence of plastid-free cells. The percentages of plastid-free cells were 14.04% in the yellow sector and 0.90% in the green sector ( Figure 2B ). Download figure Open in new tab Figure 2. VAR2 mutations impair chloroplast division and result in the formation of plastid-free cells. (A) Confocal microscopy analysis of protoplasts isolated from green and yellow sectors of mature leaves of PRPL11 - GFP / var2 plants grown in soil. White circles indicate plastid-free cells that lack both red autofluorescence and GFP signals. GFP, green fluorescence protein; Chl, chlorophyll autofluorescence; DIC, differential interference contrast microscopy. Bars, 20 μm. (B) Percentage of the three types of cells with chloroplasts, leucoplasts or without plastids shown in (A). More than 300 protoplasts were analyzed. (C) Correlation analysis between chloroplast number and cell size in green and yellow sectors of var2 . More than 150 protoplasts were analyzed per replicate. The r 2 values of the best-fit lines are 0.63 (green sectors) and 0.36 (yellow sectors). (D) Distribution of cells containing different chloroplast number in green and yellow sectors of var2 . More than 150 protoplasts were analyzed per replicate. (E) Confocal microscopy images of guard cells in RPL11-GFP/ WT and RPL11-GFP/var2 leaves. Bars, 4 μm. (F) Average of chloroplast number per guard cell. Significant differences between genotypes were marked with different letters (One-Way ANOVA, P < 0.05). (G) Percentage of cells with or without chlorophyll autofluorescence. More than 150 guard cells were analyzed in (F) and (G). We further quantified the number of chloroplasts and size per protoplast isolated from green and yellow sectors of var2 mature leaves. The average cell size in the yellow sector was 893.5 μm 2 , significantly smaller than 1846.1 μm 2 observed in the green sector. Consistently, the chloroplast number per cell was markedly lower in the yellow sector. Notably, 75.7% of the cells in the yellow sector possessed 1 to 20 chloroplasts, compared to only 11.6% in the green sector ( Figure 2C , 2D ). Overall, our findings reveal that VAR2 mutations impair plastid division, ultimately leading to the formation of plastid-free cells. Chloroplast division is also inhibited in var2 guard cells Since chloroplast numbers can be easily calculated in guard cells, we investigated whether VAR2 mutations have an impact on chloroplast division in guard cells. To do this, we introduced PRPL11 - GFP into the WT genetic background by crossing with PRPL11 - GFP / var2 . Our data showed that introduction of PRPL11-GFP into WT had no significant effect on chloroplast biogenesis (Figure S1). Chloroplast numbers in guard cells were analyzed in mature leaves of 20-day-old PRPL11 - GFP /WT and PRPL11 - GFP / var2 plants grown in soil. The average number of chloroplasts per guard cell were 3.35 in var2 green sectors, which were significantly less than in WT (3.83), and only 0.75 in the yellow sector ( Figure 2E , 2F ). In addition, all guard cells in the green sector contained chloroplasts, while only 63.4% of guard cells in the yellow sector had chloroplasts ( Figure 2G ). Interestingly, plastid-free guard cells were not detected in var2 , unlike in leaf cells,. Taken together, our data demonstrate that VAR2 mutations impair chloroplast division in guard cells as well. Cell division is not affected in var2 Although our data demonstrated that some var2 cells are devoid of plastids due to defective chloroplast biogenesis during leaf development, we cannot exclude the possibility that VAR2 mutations also affect cell division. To investigate this, we measured cell numbers in the first pair of true leaves and root meristems. As shown in Figure 3A and 3B , no significant differences in cell number were observed between the leaves of 17-day-old WT and var2 plants. Similarly, no obvious differences in root meristem size or meristem cell number were detected between 7-day-old WT and var2 seedlings ( Figure 3C - 3E ). These results indicate that cell division occurs normally in var2 . Download figure Open in new tab Figure 3. Cell division is not affected by VAR2 mutation. (A) Confocal microscopy images of WT and var2 leaf cells stained with FM4-64. Bar, 40 μm. (B) Mesophyll cell number in WT and var2 leaves. Data were means ± SD (n = 30). (C) Confocal microscopy images of WT and var2 root tips stained with propidium iodide. White and yellow arrows indicate quiescent centers and the ends of meristem zone, respectively. Bar, 20 μm. (D) and (E) are the size and cell number of root meristems. Data were means ± SD (n = 40). (F) Phenotypes of 20-day-old WT, amippd , var2 , and two independent lines of amippd var2 plants grown in soil. Bars, 1 cm. (G) Percentage of green sectors in var2 and amippd var2 plants. Data were means ± SD (n = 3). To further explore whether accelerated cell proliferation affects the leaf variegation phenotype of var2 , we knocked down the expression of Peapod ( PPD ), which suppresses the proliferation of dispersed meristematic cells in leaves, in the var2 background ( amippd / var2 ) using the artificial microRNA technique ( Schwab et al ., 2006 ). Surprisingly, amippd/var2 displayed a weaker leaf variegation phenotype and a higher percentage of green sectors, but no significant difference in the phenotype was observed compared to var2 ( Figure 3F , 3G ). Taken together, our data indicate that cell division is not affected by VAR2 mutations. Chloroplast division is involved in the formation of leaf variegation To investigate the role of chloroplast biogenesis in variegated leaf formation, we first examined whether plastid division contributes to leaf variegation of var2 . We analyzed the expression levels of key plastid-division genes, such as ARC6 , PARC6 , PDV1 and PDV2 , in var2 leaves during different stages of development. Quantitative PCR (qPCR) analysis showed that expression levels of these genes were not significantly different between WT and var2 at early stages of leaf development but appeared to decrease more rapidly in var2 than WT at later stages (Figure S2), indicating that VAR2 mutations have no substantial effect on mRNA levels of plastid division genes, particularly during early leaf development. To provide genetic evidence for the involvement of chloroplast division in leaf variegation, we made double mutants by crossing var2 with arc6 , parc6 , pdv1 or pdv2 mutants (Figure S3). Phenotypic analysis showed that the single mutants ( arc6 , parc6 , pdv1 and pdv2 ) displayed green leaves similar to WT ( Figure 4A , 4B ). Among the double mutants, parc6 var2 and pdv1 var2 displayed much more severe leaf variegation compared to var2 , whereas arc6 var2 and pdv2 var2 had similar variegation to var2 at both the seedling ( Figure 4A ) or juvenile stages ( Figure 4B ). Quantification analysis confirmed that the percentage of the green area in parc6 var2 and pdv1 var2 leaves was significantly reduced, compared to var2, while no difference was observed between var2 and arc6 var2 or pdv2 var2 had ( Figure 4C and 4D ). These result suggest that PARC6 and PDV1 play critical roles in the formation of leaf variegation in var2 . Download figure Open in new tab Figure 4. Chloroplast division is involved in the formation of leaf variegation (A) and (B) Phenotypes of 13-day-old (A) and 20-day-old (B) WT, var2 , arc6 , parc6 , pdv1 , pdv2 , arc6 var2 , parc6 var2 , pdv1 var2 , and pdv2 var2 plants. Bar in (A), 1 mm. Bar in (B), 1 cm. Red arrows in (A) indicate the first pair of true leaves. (C) and (D) Percentage of green sectors in the first pair of leaves shown in (A) and in plants shown in (B), respectively. Data were means ± SD (n = 3). (E) Percentage of type a, b and c cells isolated from the first pair of leaves shown in (A). Data were means ± SD (n = 6). (F) Chloroplast number per guard cell in green sectors of plants shown in (B). A total of 100 guard cells were analyzed. (G) Overexpression of PDV1 and PDV2 significantly rescues the var2 phenotype. PDV1 or PDV2 fused with Flag was overexpressed in WT and var2 genetic backgrounds. The shown were 20-day-old plants grown in soil. Bars, 1 cm. (H) Percentage of green sectors in plants shown in (G). Data were means ± SD (n = 3). Statistical analysis in all experiments was performed with One-Way ANOVA, and significant differences among genotypes were marked with different letters ( P < 0.05). To further evaluate the effects of mutations in PARC6 , ARC6, PDV1 and PDV2 on chloroplast biogenesis, we analyzed protoplasts isolated from the first pair of leaves. Confocal microscope analysis showed that the proportion of type a cells, which contain well-developed chloroplasts, was significantly lower in parc6 var2 and pdv1 var2 double mutants than in var2 , while it remained unchanged in arc6 var2 and pdv2 var2 ( Figure 4E ). In contrast, the percentage of type c cells, which lack chloroplasts, was markedly higher in parc6 var2 and pdv1 var2 than in var2 , whereas it was comparable to var2 in arc6 var2 and pdv2 var2 ( Figure 4E ). No significant difference in the proportion of type b cells was detected between var2 and any of the double mutants ( Figure 4E ). In addition, the number of chloroplasts in guard cells of all double mutants were significantly reduced, compared to var2 ( Figure 4F ). Taken together, these findings confirm that chloroplast division is impaired in var2 , and mutations in PARC6 and PDV1 exacerbate the variegated phenotype of var2 . Simultaneously, we tested whether accelerating chloroplast division could improve the leaf variegation phenotype of var2 . Since previous studies have shown that overexpression of PDV1 and PDV2 increases chloroplast numbers in mesophyll cells ( Miyagishima et al ., 2006 ), we generated transgenic plants overexpressing either PDV1 or PDV2 in the var2 background (Figure S4). As expected, transgenic plants overexpressing PDV1 ( PDV1 - Flag / var2 ) or PDV2 ( PDV2 - Flag / var2 ) significantly mitigated the variegated leaf phenotype of var2 ( Figure 4G ). The area of green sectors in PDV1 - Flag / var2 and PDV2 - Flag / var plants was approximately 90% and 95%, respectively, which was significantly greater than the 71.68% observed in var2 plants ( Figure 4H ). Notably, while PDV2 mutation did not affect leaf variegation in var2 , overexpression of PDV2 markedly improved the var2 phenotype, suggesting that PDV1 (a homolog of PDV2 ) may play a more critical role in plastid division in var2 . Collectively, our data indicate that chloroplast division is involved in leaf variegation of var2 . Early chloroplast biogenesis is critical for suppression of leaf variegation Genetic screening for suppressors of leaf variegation has identified many suppressor genes, such as Plastid Ribosomal Protein L11 ( PRPL11 ) and Suppressor of Thylakoid Formation1 ( SOT1 ), which are known to regulate chloroplast gene expression ( Wu et al ., 2016 ; Wu et al ., 2013 ). To inspect the role of the suppressor genes in chloroplast biogenesis, we analyzed the relationship between chloroplast number and cell size in the first pair of leaves from 13-day-old WT, var2 , prpl11 and prpl11 var2 seedlings ( Figure 5A ). Our results showed that type a cells with well-developed chloroplasts accounted 90.5% of cells in prpl11 , a proportion comparable to that of WT (92.6 %), whereas prpl11 var2 had a significantly higher proportion of type a cells (76.2%) than var2 (12.4 %). Notably, type c cells lacking chloroplasts were absent in prpl11 var2 ( Figure 5B ). These results indicate that PRPL11 mutations substanti ally improve chloroplast biogenesis in var2 . When chloroplast number was plotted against cell size, the correlation coefficient ( r 2 ) for prpl11 var2 (0.74) and prpl11 (0.77) were significantly higher than that of var2 (0.52) ( Figure 5C ). Interestingly, both prpl11 and prpl11 var2 exhibited a steeper slope in the regression equation than WT, indicating that cells of the same size in these genotypes contain more chloroplasts than WT. Similar trends were observed in another suppressor of leaf variegation, sot1 (Figure S5). Thus, our data underscore the importance of early chloroplast biogenesis in mitigating the variegation phenotype. Download figure Open in new tab Figure 5. Early chloroplast biogenesis is critical for suppression of leaf variegation (A) Phenotypes of 13-day-old WT, var2 , prpl11 and prpl11 var2 seedlings. Red arrows indicate the first pair of true leaves. Bars, 0.5 cm. Red arrows indicate the first pair of leaves. (B) Percentage of type a, b, or c cells isolated from the first pair of leaves. More than 400 protoplasts were analyzed in each sample. (C) Correlation analysis between chloroplast number and cell area in type a cells. More than 350 cells were analyzed for each sample. Correlation coefficients ( r 2 ) are shown in the panel. (D) Confocal microscopy images of the first pair of leaves from 2-day-old PRPL11-GFP /WT and PRPL11-GFP / var2 seedlings. Green signals indicate plastids; red signals indicate chlorophyll autofluorescence. Bar, 30 μm. (E) Schematic drawing of the SAM. Blue dots represent positions of tomographic pictures shown in (F). CZ, the central zone; BZ, the zone between the CZ and PZ; PZ, the peripheral zone. L1, L2 and L3 indicate the three layers of the SAM. (F) TEM analysis of the transition from proplastids into chloroplasts in the SAMs of 3-day-old WT and var2 seedlings cultured on half-strength MS media with 1% sucrose. Bars, 500 nm. To validate the role of VAR2 in early chloroplast biogenesis, we examined chloroplast numbers and development in the first pair of leaves using transgenic plants expressing PRPL11–GFP in WT and var2 . Our results showed that in var2 both chlorophyll autofluorescence intensity and GFP signal density were markedly reduced in whole leaves from 2-day-old seedling, compared to WT ( Figure 5D ). Moreover, a significant proportion of cells in var2 leaves contained only GFP signals ( Figure 5D ), indicating an increased prevalence of the non-photosynthetic plastids. Thus, these results confirm that chloroplast biogenesis at the early stage of leaf development is severely inhibited by VAR2 mutations. Proplastid-to-chloroplast transition is inhibited in the SAM of var2 It has been demonstrated that most proplastids in the shoot apical meristem (SAM), particularly in the L1 and L3 layers, are partially differentiated into chloroplasts ( Charuvi et al ., 2012 ). To determine whether VAR2 mutation affects the transition of proplastids to chloroplasts in the SAM, we analyzed plastid ultrastructure using transmission electron microscopy (TEM). In 3-day-old var2 seedlings grown on half-strength MS media with 1% sucrose in the 16 hr light/8 hr dark cycle, TEM analysis showed that plastids in var2 cells had fewer thylakoid networks compared to those in WT cells occupying equivalent SAM positions ( Figure 5E , 5F ). In the central zone (CZ), plastids in both L1 and L3 layers of WT cells exhibited distinct thylakoid membranes, whereas plastids in var2 cell displayed a near-complete absence of these structures ( Figure 5F ). In the peripheral zone (PZ), WT plastids from the L1 and L2 layers contained well-developed thylakoid membranes, whereas var2 plastids from the same layers were characterized by the presence of vesicles and tubules ( Figure 5F ). These results clearly indicate that VAR2 mutations significantly inhibit chloroplast development at a very early stage, affecting the transition from proplastids to chloroplasts within the SAM. Pchlide synthesis is defective in etiolated seedlings of var2 Chlorophyll synthesis is a crucial prerequisite for chloroplast development. It remains unknown if VAR2 mutations affect chlorophyll biosynthesis. To solve this question, we examined the greening process in etiolated var2 seedlings. Interestingly, 4-day-old var2 etiolated seedlings displayed a pale green coloration after exposure to light, in contrast to the greening cotyledon observed in WT seedlings ( Figure 6A ). Chlorophyll content in var2 seedlings was significantly lower than in WT after 30 min of de-etiolation ( Figure 6B ). Given the compromised chlorophyll synthesis, we postulated that the synthesis of protochlorophyllide (Pchlide), a precursor of chlorophyll, was blocked in var2 etiolated seedlings. As expected, Pchlide levels in 4-day-old var2 etiolated seedlings were approximately 50% of those in WT ( Figure 6C ). Furthermore, Pchlide content in var2 seedlings remained relatively stable following 30-min of light exposure, whereas it rapidly declined in WT seedlings ( Figure 6C ). These findings suggest that VAR2 is essential for Pchlide synthesis in the dark. Download figure Open in new tab Figure 6. COP1 mutations fully rescue leaf variegation in var2 (A) Phenotypes of 4-day-old WT and var2 etiolated and de-etiolated (for 30-min) seedlings. Bar, 1 mm. (B) Chlorophyll content of 4-day-old etiolated seedlings exposed to light for 30 min. The data were means ± SD (n = 3). Significant difference between WT and var2 was labelled with stars (student t- tes, P < 0.01). (C) Relative Pchlide content per seedling shown in (A). The data were means ± SD (n = 3). (D) Phenotypes of 4-day-old WT, var2 , cop1-4 and cop1-4 var2 etiolated seedlings. Bar, 1 mm. (E) Confocal microscopy images of the first pair of leaves (labelled with red arrows) from cop1-4 and cop1-4 var2 etiolated seedlings. Bar, 30 μm. (F) TEM images of plastids in the first pair of leaves shown in (E). Bar, 500 nm. (G) Phenotypes of 9- and 13-day-old WT, var2 , cop1-4 and cop1-4 var2 seedlings. Bars, 1 mm. Red arrows indicate the first pairs of leaves. (H) Percentage of type a, b and c cells from the first pair of leaves from 13-d-old seedlings shown in (G). The Data were means ± SD (n = 6). (I) Phenotypes of 20-day-old WT, var2 , cop1-4 , and cop1-4 var2 plants grown in soil in the 16 hr light/8 hr dark cycle, and the percentage of green sectors in var2 and cop1-4 var2 plants. Bar, 1 cm. The data were means ± SD (n = 3). Stars indicate significant difference between var2 and cop1-4 var2 (student t- tes, P < 0.01). (J) Chloroplast number per guard cell in plants shown in (I). A total of 130 guard cells were analyzed. Statistical analyses in (C), (H) and (J) were performed with One-Way ANOVA, and significant differences among genotypes were marked with different letters ( P < 0.05). COP1 mutations fully rescue leaf variegation of var2 COP1 is a central repressor of photomorphogenesis, and its mutations lead to photomorphogenic development, such as open and expanded cotyledons, short hypocotyls, and partially developed thylakoids in darkness ( Han et al ., 2020 ). To examine whether COP1 mutations affect chloroplast biogenesis in the var2 background, we generated the cop1-4 var2 double mutant through genetic crossing. Our data showed that 4-day-old etiolated seedlings of cop1-4 var2 displayed a constitutive photomorphogenic phenotype similar to those of cop1-4 , characterized by open cotyledons and shorter hypocotyls. In contrast, var2 and WT etiolated seedlings exhibited a typical skotomorphogenic phenotype with closed cotyledons and elongated hypocotyls ( Figure 6D ). In addition, the first pair of leaves emerged in 4-day-old cop1-4 and cop1-4 var2 , but not in var2 and WT etiolated seedlings ( Figure 6E ). TEM analysis showed partially developed thylakoid structures in leaves of both cop1-4 and cop1-4 var2 in darkness ( Figure 6F ). Under normal light conditions, cop1-4 var2 seedlings exhibited almost no leaf variegation at 9 and 13 days old when grown on half-strength MS media containing 1% sucrose ( Figure 6G ). Consistently, microscope analysis of protoplasts from the first pair of leaves of 13-day-old seedlings revealed significant changes in the percentages of type a and c cells in cop1-4 var2 . Type a cells with well-developed chloroplasts were significantly higher in cop1-4 var2 than in var2 , while type c cells were markedly reduced, compared to var2 ( Figure 6H ). Type B cells remained unaffected by the COP1 mutation in the var2 background ( Figure 6H ). When grown in soil, cop1-4 var2 plants exhibited almost no leaf variegation phenotype ( Figure 6I ). The area of green sectors in leaves was significantly higher in cop1-4 var2 than in var2 ( Figure 6I ). In addition, the number of chloroplasts in var2 guard cells was also significantly rescued by cop1-4 ( Figure 6J ). Finally, we investigated whether COP1 mutations could rescue the variegated leaf phenotype of var2 under various photoperiods. Across all tested light cycle conditions, cop1-4 almost fully suppressed the var2 phenotype (Figure S6). Taken together, our data suggest that COP1 mutations facilitate chloroplast biogenesis and rescue the leaf variegation phenotype of var2 under both dark and light conditions. Impaired chloroplast division as a common feature in variegation mutants but not in virescent mutants To determine whether defective plastid division is a common feature of other variegated mutants, we investigated chloroplast biogenesis in im , which encodes a chloroplast terminal oxidase. The im mutant exhibited more severe leaf variegation than var2 , as demonstrated by a significant reduction in the area of green sectors (Figure 8A, 8B). Like in var2 , the average number of chloroplasts per guard cell was significantly lower in both the green sector and yellow sector of im , compared to WT (Figure 8C). These results suggest that plastid division is impaired in im , as in var2. Mutants with defective chloroplast biogenesis can display a range of leaf coloration phenotypes, such as variegation, virescence, and pale green. However, it remains unclear whether virescent mutants are defective in plastid division. To address this, we examined chloroplast development and numbers in the virescent mutant clpr4 (Figure 8D-8G). Microscopy analysis of isolated protoplasts from the first pair of leaves showed that clpr4 exhibited a distribution of cell types similar to WT, predominantly consisting of type a cells. This pattern contrasts sharply with var2 , which showed a majority of type c cells devoid of chloroplasts (Figure 8E). To investigate whether ClpR4 mutations affect plastid division, we quantified the number of chloroplasts in guard cells. Interestingly, clpr4 had the significantly higher number of chloroplasts than WT and var2 (Figure 8G). These results suggest that enhanced chloroplast division in clpr4 compensates for its defects in chloroplast development. Discussion Over the past decades, studies on VAR2-mediated leaf variegation have primarily focused on elucidating the regulatory network of chloroplast development via genetic screening and functional analysis of suppressor and enhancer genes. However, the impact of VAR2 mutations on chloroplast division has remained largely overlooked. Here, we found dual roles of VAR2 in both chloroplast development and division, providing new insights into the mechanisms underlying variegated leaf formation in Arabidopsis thaliana . VAR2 is essential for both development and division of chloroplasts VAR2 mutations severely impair chloroplast biogenesis during early leaf development, leading to delayed chloroplast maturation and an increased proportion of cells with non-photosynthetic plastids. In addition, the biogenesis process is prolonged in var2 , resulting in greater variability in chloroplast number per cell and cell size. VAR2 also plays a pivotal role in the proplastid-to-chloroplast transition in the SAM, where its absence significantly disrupts thylakoid network formation. Thus, these findings underscore the fundamental role of VAR2 in establishing chloroplasts during early leaf development. Contrary to previous reports that indicated that the green sector of var2 are comparable to those in WT ( Sakamoto, 2003 ), we found that the green area in var2 harbor more and larger chloroplasts than that in WT, suggesting that green sectors may produce more photosynthate to meet the metabolic demands of the yellow sectors. In contrast, non-photosynthetic cells dominate the yellow sector due to defects in chloroplast development ( Kato et al ., 2007 ). Consistently, our quantitative analysis showed that about 80% of cells in the yellow sectors are non-autotrophic ( Figure 2 ). It is proposed that misfolded and photodamaged proteins will accumulate in the absence of VAR2 , and thylakoid membrane formation will be blocked at the early stage of chloroplast development, ultimately resulting in chloroplast dysfunction ( Dogra et al ., 2019 ). However, investigation has intensively focused on the role of VAR2 in maintaining homeostasis of PSII activity ( Kato et al ., 2018 ; Kato et al ., 2023 ). Therefore, it remains unclear how VAR2 mutations influence chloroplast development. In this study, we discovered that VAR2 mutations suppress de-etiolation of cotyledons due to less accumulation of Pchlide and its slower conversion in light, suggesting that VAR2 has distinct function in the dark from that in light. These findings provide a new approach to explore molecular mechanism by which VAR2 regulates chloroplast development. The L1 and L2 layers of the SAM give rise to the epidermis and outer mesophyll of a leaf, respectively, while the L3 layer contributes to the inner mesophyll and vasculature ( Tsukaya, 2002 ). The transition of proplastids into chloroplasts initiates across these layers, but thylakoid membranes are small, partially differentiated in the L1, L3 and PZ cells ( Charuvi et al ., 2012 ; Yadav et al ., 2019 ). These thylakoid networks keep till the young leaf primordium. Typical thylakoid ultrastructure with granal and stromal membranes appears in the old leaf primordium, where heterogeneity of chloroplast development is obvious due to different cell types ( Charuvi et al ., 2012 ). Our TEM analysis showed that thylakoid network formation was significantly inhibited in the SAM of var2 , leaving most plastids in a proplastids state ( Figure 5F ). This generally aligns with previous findings ( Sakamoto et al ., 2009 ) but challenges the assertion that differential thylakoid networks in WT and var2 PZs have no impact on chloroplast development in subsequent leaf development. Since the leaf primordium emerges from the PZ, we hypothesize that VAR2 mutations have an impact on chloroplast development in the young leaf primordia. This hypothesis is consistent with our genetic evidence showing that accelerating chloroplast biogenesis rescues the variegated phenotype of var2 ( Figure 4G , 6I ), highlighting the importance of timely chloroplast development in mitigating leaf variegation. Following the initiation of leaf primordia, the establishment of leaf polarities (dorsoventral, proximodistal and mediolateral) and subsequent development into the flattened leaf blade and petiole occur, accompanied by rapid cell division ( Tsukaya, 2013 ). Thus, it is important to keep chloroplast biogenesis in pace with cell division at early leaf development. Our results revealed that the yellow sector of var2 leaves predominantly consist of non-photosynthetic cells, which either lack chloroplasts or any kinds of plastids. Generally, the active degeneration of thylakoids is restricted to the maturation of leaf pavement cells and does not occur during the development of young leaf cells ( Charuvi et al ., 2012 ). A plausible explanation for the absence of chloroplasts in var2 cells is a self-protection mechanism aimed at mitigating photodamage caused by reactive oxygen species (ROS) generated from photosynthesis. This process likely leads to the formation of non-photosynthetic plastids and is in agreement with the observations that variegation severity is proportional to light intensity ( Rosso et al ., 2009 ). In addition, approximately 15% of cells in the yellow sector lack plastids entirely ( Figure 2B ). This phenomenon may stem from two potential mechanisms: (1) the rate of chloroplast division may lag behind that of cell division, resulting in plastid-free cells; or (2) damaged chloroplasts might be actively removed by autophagy pathways ( Niwa et al., 2004 ; Izumi et al., 2017 ). Together, these mechanisms provide a plausible explanation for the development of the yellow sector in var2 leaves. An important role of chloroplast division in variegated leaf formation One possible explanation for why most chloroplast division-related mutants do not exhibit variegation or virescent leaf phenotypes is that developmentally normal chloroplasts in these mutants can propagate through budding rather than binary fission (Forth and Pyke, 2006; Robertson et al ., 1995 ). In var2 , however, mutations disrupt both chloroplast development and division. Genetic interactions with division-related genes, such as PDV1 and PARC6 , highlight the importance of chloroplast division in the formation of variegated leaves in var2 . For instance, mutations in these genes exacerbated the variegation phenotype, while overexpression of PDV1 or PDV2 alleviated it. Interestingly, despite these defects, proplastid division in the SAM and RAM of var2 remains unaffected, suggesting that VAR2 does not participate in synchronizing proplastid division with the cell cycle. Large plastid nucleoids observed in the yellow sectors of various plant species ( Sakamoto et al ., 2009 ) might result from defective plastid division, similar to cellular DNA endoduplication. Kato et al . (2007) reported that all cells in the yellow sectors of var2 retain undifferentiated plastids, which contradicts our findings. We observed that approximately 15% of cells isolated from the yellow sectors of mature var2 leaves lacked any plastids ( Figure 2B ). This discrepancy may arise from differences in methods and materials. While Kato et al . (2007) examined fully expanded leaves from 7- or 8-week-old plants, we analyzed protoplasts from the yellow sectors of the first pair of leaves, which exhibit the most pronounced variegation. Our protoplast-based approach may have increased the likelihood of detecting abnormal cells in var2 . The presence of plastid-devoid cells has also been reported in crl mutants, which display pale green and variegated leaf phenotypes due to insufficient chloroplast division ( Asano et al ., 2004 ; Chen et al ., 2009 ). Furthermore, consistent with reports that aplastidic cells cannot complete the cell cycle ( Hudik et al ., 2014 ), we found that the proportion of plastid-lacking cells in the whole leaf was minor ( Figure 2B ). Chloroplast division defects are also evident in other variegated mutants, such as im . In the yellow sectors of im , guard cells contained significantly fewer chloroplasts compared to var2 ( Figure 7C ), suggesting that impaired chloroplast division is a common feature of variegated mutants. Conversely, the virescent mutant clpr4 , despite its defective chloroplast development, exhibits accelerated chloroplast division compared to WT ( Figure 7G ). This increased division rate ultimately restores leaf color to WT levels, indicating that enhanced chloroplast division can partially compensate for reduced chloroplast biogenesis caused by developmental defects. Collectively, our findings strongly suggest that defective chloroplast division plays a crucial role in the formation of variegated leaves. Download figure Open in new tab Figure 7. Chloroplast division is a key factor determining leaf variegation (A) Phenotypes of 20-day-old WT, var2 and im plants grown in soil. Bar, 1 cm. (B) The percentage of green sectors in leaves shown in (A). Data were means ± SD (n = 3). Stars indicate significant difference between var2 and im (student t -tes, P < 0.01). (C) Chloroplast number per guard cell in plants shown in (A). A total of 100 guard cells were analyzed. Significant differences among genotypes were marked with different letters (One-Way ANOVA, P < 0.05). (D) Phenotypes of 13-day-old WT, var2 and clpr4 seedlings grown on half-strength MS media with 1% sucrose. Bar, 1 mm. Red arrows indicate the first pairs of leaves. (E) Percentages of type a, b and c protoplasts isolated from the first pair of leaves. Data were means ± SD (n = 6). (F) Phenotypes of 20-day-old WT, var2 and clpr4 plants grown in soil. Bar, 1 cm. (G) Chloroplast number per guard cell in plants shown in (F). A total of 100 guard cells were analyzed. Statistical analyses were performed with One-Way ANOVA, and significant differences among genotypes were marked with different letters ( P < 0.05). (H) A general model illustrates the relationship between chloroplast biogenesis, including development and division, and various leaf color phenotypes observed in different mutants. Defects in either chloroplast development, division, or both can lead to distinct leaf phenotypes. Interdependence of chloroplast development and division Chloroplast development and division during leaf development have been suggested to operate independently while mutually compensating to maintain chloroplast biogenesis in response to intracellular and extracellular changes ( Pyke, 1999 ). Similar to the cell cycle, newly divided chloroplasts must grow to a specific size and replicate their DNA before undergoing another round of division ( Boffey and Leech, 1982 ; Ellis and Leech, 1985 ). Interestingly, impaired chloroplast division appears to have no significant impact on chloroplast development, as chloroplast ultrastructure remains normal in arc mutants ( Robertson et al., 1995 ). This interplay between chloroplast development and division is critical for sustaining chloroplast homeostasis during rapid cell proliferation. The presence of plastid-free cells in var2 leaves, along with normal cell division, suggests a disruption in the synchronization of chloroplast division with the cell cycle during leaf development. Delayed chloroplast development may hinder chloroplast division by restricting the growth of newly formed chloroplasts. Conversely, defective division exacerbates these developmental delays. This feedback loop likely contributes to the pronounced variegation observed in var2 leaves. Based on the available evidence on chloroplast biogenesis, we propose a general model to describe the relationship between chloroplast biogenesis and various leaf phenotypes, including variegation, virescence, and pale green or yellow leaves ( Figure 7H ). Defects in chloroplast development, as seen in mutants of chlorophyllide a oxygenase ( cao ) and divinyl protochlorophyllide 8-vinyl reductase ( pcb2 ), result in pale green or yellowish leaves. In contrast, defects in chloroplast division alone typically produce WT-like leaves. However, simultaneous defects in both chloroplast development and division, as in var2 and im , lead to leaf variegation. Accelerated chloroplast division in the context of defective development results in virescent leaves. We previously reported that virescent mutants with reduced plastid gene expression can rescue the var2 phenotype ( Liu et al., 2010 ; Hu et al., 2015 ; Ma et al., 2015 ). Consistent with this, our findings show that chloroplast division is accelerated in virescent mutants such as clpr4 and prpl11 ( Figures 5C , 7G ). This accelerated division may explain the suppression of leaf variegation in virescent mutants. These findings underscore the importance of the interaction between chloroplast development and division in shaping leaf coloration patterns. In summary, this study advances our understanding of the molecular mechanisms underlying leaf variegation and highlights the coordinated yet independent roles of chloroplast development and division in chloroplast biogenesis. Key conclusions include: (1) Leaf variegation arises from defects in both chloroplast development and division; (2) VAR2 plays a pivotal role in the proplastid-to-chloroplast transition and de-etiolation, likely by promoting chlorophyll biosynthesis; (3) Accelerating chloroplast biogenesis can suppress the variegation phenotype in var2 . Future research should focus on elucidating the molecular and biochemical mechanisms by which VAR2 regulates chloroplast biogenesis. Understanding these processes will further refine our knowledge of how chloroplast development and division contribute to leaf phenotypic diversity. Materials and methods Plant materials and growth conditions Arabidopsis seeds used in this study was the Columbia-0 ecotype, and mutants were var2-1 ( Martínez-Zapater, 1993 ), pdv1-1 ( Miyagishima et al ., 2006 ), pdv2-1 ( Miyagishima et al ., 2006 ), arc6-1 ( Pyke et al ., 1994 ), parc6-1 ( Glynn et al ., 2009 ), prpl11 ( Pesaresi et al ., 2001 ) and sot1 ( Wu et al ., 2016 ). Double mutants, pdv1 var2 , pdv2 var2 , arc6 var2 , parc6 var2 , prpl11 var2 and sot1 var2 were obtained by crossing and identified by sequencing PCR-based fragments. Seeds sterilized with 20% sodium hypochlorite solution were treated at 4 ℃ for 2-3 days, and then sown in half-strength MS plates supplemented with 1% (w/v) sucrose. Seedlings were grown under the long day condition with light intensity of 70-120 µmol m -2 s -1 at 20 ± 2℃. About 10-day-old seedlings were transplanted into soil for phenotypic observation. Plasmid construction and generation of transgenic plants To generate PRPL11 - GFP , PDV1 - FLAG , and PDV2 - FLAG constructs, PRPL11 , PDV1 and PDV2 CDS sequences were amplified by PCR with the primer pairs listed in Supplemental Table S1, and then cloned into the pENTER vector with an EasyGeno Assembly Cloning Kit (TIANGEN). The pENTER vectors containing target genes were recombined into the binary vector pGWB to get the final vectors. All constructs were confirmed by sequencing. Transgenic plants were screened on half-strength MS plates with antibiotics, and homozygous ones were applied in various assays. RNA extraction and quantitative RT-PCR analysis Total RNA was isolated from the first pair leaves of WT and var2 plants with an RNA Easy Fast Plant Tissue Kit (TIANGEN), and then reversely transcribed using an PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). The concentrations of cDNA templates were measured by a Nanodrop spectrophotometer before application to qRT-PCR. ACTIN2 was used as an internal control. Green sector size, chlorophyll, and Pchlide measurements Leaves from 13-d-old and 20-d-old plants were recorded by a stereomicroscope and a scanner. ImageJ software was used to measure the size of green sectors from the first pair of leaves or the whole plant. Percentage of green sector was calculated by the ratio of the green sector area to the whole leaf area. Chlorophyll was extracted from 0.1 g of rosette leaves with 4 ml of 80% acetone under the dark condition at 4°C on a shaker for 7-8 h until the leaves were completely white, and was measured at OD645 and OD663 using a Nanodrop (Thermo) spectrophotometer. Chlorophyll content was calculated according to the following equations: chlorophyll a (μg/ml) = 12.7 x OD663 -2.69 x OD645; chlorophyll b (μg/ml) = 22.9 x OD645 - 4.68 x OD663 ( Porra et al ., 1989 ). Pchlide was extracted from 4-d-old etiolated seedlings with 1.2 ml 80% acetone under the dark condition at 4°C on a shaker overnight, and was measured as previously described ( Czarnecki et al ., 2011 ). Protoplast isolation, confocal microscopy and TEM analysis Protoplasts were isolated by enzymatic hydrolysis as previously reported ( Tan et al ., 2019 ), and were observed by a laser confocal fluorescence microscope (Olympus, Tokyo, FV 3000). Plastid numbers were counted using ImageJ software. For FM4-64 dying, 10-day-old seedlings were fully immersed in 10 μM dye solution with a vacuum concentrator for 10 min. The leaf size was recorded by a stereomicroscope, and mesophyll cells were observed by confocal microscope. For propidium iodide (PI) dying, 7-day-old seedlings were stained with the solution (10 μg/ml) for 1.5 min. ImageJ software was used to measure root length and cells. Plastid and chloroplast numbers in guard cells of leaves from 20-day-old PRPL11-GFP /WT and PRPL11-GFP / var2 plants grown in soil were recorded by confocal microscope. Data were analyzed using GraphPad Prism 8.0 software. All experiments were repeated at least three times, and similar results were obtained. Statistical analyses were performed with One-Way ANOVA or t -tes, and significant differences among genotypes were marked with different letters or asterisks ( P < 0.05). For TEM observation, SAMs of 3-d-old light-grown seedlings and 4-d-old dark-grown seedlings were fixed and processed as previously described ( Zhou et al ., 2009 ). Ultrastructure of plastids was observed by a TEM (Tecnai Spirit G2 BioTWIN, FEI, The Kingdom of the Netherlands). Funding This work was supported by the National Natural Science Foundation of China (grant no. 32100191, 32370250); Shanghai Natural Science foundation (21ZR1447100) and Innovation Program of Shanghai Municipal Education Commission (2021-01-07-00-02-E00117). Author Contributions WW, WG and JH designed the study. HZ, WW and WG analyzed correlation between plastid number and cell size. WW, WG, DL, ZZ and MQ made transgenic plants and mutants. WW, WG, ZY and JH conducted confocal microscopy and TEM analyses. WW, WG and JH wrote the manuscript. All authors read and approved the manuscript. Declaration of interests The authors declare no competing interests. Supplementary information Table S1. Primers used in the experiments. Figure S1. Overexpression of RPL11-GFP has no obvious effect on leaf variegation of var2 . Figure S2. Quantitative PCR analysis of expression levels of chloroplast division-related genes. Figure S3. 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