Arrested Differentiation of Proplastids Into Chloroplasts in Variegated Leaves Characterized by Plastid Ultrastructure and Nucle

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Arrested Differentiation of Proplastids into Chloroplasts in Variegated Leaves Characterized by Plastid Ultrastructure Special Issue – Regular Paper and Nucleoid Morphology Wataru Sakamoto 1 ,∗ , Yasuyuki Uno 1 , 3, Quan Zhang 2, Eiko Miura 1, Yusuke Kato 1 and Sodmergen 2 1 Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, 710-0046 Japan 2Key Laboratory of Cell Proliferation and Differentiation (Ministry of Education), College of Life Sciences, Peking University, Beijing 100871, PR China

Leaf variegation is seen in many ornamental plants and is Keywords: Arabidopsis thaliana • Chloroplast development • often caused by a cell-lineage type formation of white Electron microscopy • Leaf variegation • Plastid nucleoid • Downloaded from sectors lacking functional chloroplasts. A mutant showing Thylakoid membrane . such leaf variegation is viable and is therefore suitable Abbreviations : Col , Columbia ; cpDNA , chloroplast DNA; for studying chloroplast development. In this study, the DAPI , 4 ′ ,6-diamidino-2-phenylindolyle ; FTL , fi rst true leaf; formation of white sectors was temporally investigated in MRL , mature rosette leaf; PEND , plastid envelope DNA the Arabidopsis leaf-variegated mutant var2 . Green binding; ptDNA , plastid DNA; PLB , prolamellar body; PVB , http://pcp.oxfordjournals.org/ sectors were found to emerge from white sectors after the plastidic vacuolated body; SiR , sulfi te reductase; SYBG , SYBR formation of the fi rst true leaf. Transmission electron Green I; TEM , transmission electron microscopy; var1 , yellow microscopic examination of plastid ultrastructures variegated 1; var2 , yellow variegated 2. confi rmed that the peripheral zone in the var2 shoot meristem contained proplastids but lacked developing chloroplasts that were normally detected in wild type. Introduction These data suggest that chloroplast development proceeds

very slowly in var2 variegated leaves. A notable feature in Leaf variegation is defi ned as a patch of different colors in by guest on April 29, 2016 var2 is that the plastids in white sectors contain remarkable the same leaf and is often seen in a variety of ornamental globular vacuolated membranes and prolamellar body- plants. When the variegation pattern is identical in each leaf, like structures. Although defective plastids were hardly it is likely formed by leaf tissues differentiating into specifi c observed in shoot meristems, they began to accumulate cell types. This type of variegation is termed ‘fi gurative during early leaf development. Consistent with these (or structural) variegation’ ( Kirk and Tilney-Bassett 1978 ). observations, large plastid nucleoids detected in white On the other hand, non-identical variegation is termed ‘true sectors by DNA-specifi c fl uorescent dyes were characteristic variegation’ and is frequently caused by genetic mutations of those found in proplastids and were clearly distinguished or by environmental stresses such as nutrient defi ciency and from those in chloroplasts. These results strongly imply pathogen infection. True variegation caused by genetic muta- that in white sectors, differentiation of plastids into tions sometimes follows cell lineage and is characterized by chloroplasts is arrested at the early stage of thylakoid splashes of green/white sectors ( Kirk and Tilney-Bassett development. Interestingly, large plastid nucleoids were 1978 , Rodermel 2002 ). Other non-cell-lineage types show detected in variegated sectors from species other than unequal variegation patterns such as irregularly shaped mar- Arabidopsis. Thus, plastids in variegated leaves appear ginal sectors. Mutations that result in leaf variegation or to share a common feature and represent a novel stripes have been known to occur in many plant species plastid type. for a long time. Such mutants are invaluable resources to

3 Present address: Nanto Seeds Co., Kashihara, Nara, 634-0077 Japan. ∗ Corresponding author: E-mail, [email protected] ; Fax, + 81-86-434-1206 . Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127, available online at www.pcp.oxfordjournals.org © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. 2069 W. Sakamoto et al.

functionally study chloroplast development (Sakamoto The results in var1 and var2 appear to imply that a thresh- 2003 , Aluru et al. 2006 , Yu et al. 2007 ). Unlike albino mutants, old of FtsH levels (or levels of other molecules regulated by which are completely defective in chloroplast development FtsH) is an important functional constituent for proper and thus do not grow into maturity, the formation of green/ chloroplast development. Meanwhile, studies on FtsH in white sectors allows us to simultaneously dissect both Arabidopsis and Synechocystis demonstrated that FtsH is sectors. Chloroplast biogenesis is a complex process and involved in avoiding photoinhibition by contributing to the various plastid types are known (Wise 2006, López-Juez 2007, light-dependent degradation of the Photosystem II (PSII) Sakamoto et al. 2008). Plastids in white sectors may repre- reaction center protein D1 (Nixon et al. 2005, Komenda et al. sent a novel plastid type. 2007 , Kato and Sakamoto 2009 ). Thus, FtsH is currently In the last decade, many variegation/stripe mutants have proposed to play a dual role in chloroplasts; an unrelated been characterized at the molecular level in model plant role to variegation in the PSII repair cycle and a variegation- species including Arabidopsis ( Carol and Kuntz 2001 , related role in thylakoid formation. In addition, several trans - Rodermel 2002, Sakamoto 2003, Aluru et al. 2006, Yu et al. acting recessive mutations that suppressed leaf variegation 2007), maize (Han et al. 1992) and tomato (Keddie et al. in var2 have been identiﬁ ed. A majority of the suppressors 1996 , Carol and Kuntz 2001 ). With the exception of muta- appear to act on chloroplast protein synthesis ( Miura et al.

tions caused by transposons and organelle genomes (leading 2007, Yu et al. 2008). Based upon these ﬁ ndings, the balance Downloaded from to a genetic mosaic), most of the mutants are recessive and between protein synthesis and degradation was proposed to are nuclear encoded. The recessive nature of variegation mitigate the variegation phenotype. The accumulating indicates that two plastid types separated in green/white amount of genetic evidence implied more complexity in the sectors are formed in the same genetic background. formation of variegation sectors.

Molecular characterization of corresponding genes in these In contrast to the intensive genetic studies, little is known http://pcp.oxfordjournals.org/ mutants revealed that variegation is not caused by identical regarding how these abnormal plastids are formed along genes but rather by various redundant mechanisms associ- with leaf development. Variegation in var2 is only observed ated with chloroplast function. While these studies demon- in true leaves but not in cotyledons. It is more severe in the strate that a threshold of chloroplast factors exists and ﬁ rst emerging leaves than in laterally developing leaves determines plastid differentiation into chloroplast in a cell- ( Zaltsman et al. 2005a , Kato et al. 2007 ). In this study, plastid autonomous manner, the precise mechanisms leading to ultrastructures and nucleoids in var2 were deeply examined the formation of variegated sectors still remain unclear. during the early stage of true leaf development. Our results

The permanent appearance of variegation sectors in these demonstrate that white sectors contain a novel type of by guest on April 29, 2016 mutants also suggests that plastid differentiation into chlo- plastid that is likely formed as a result of the arrest at the roplast is determined at a particular stage of leaf develop- early stage of chloroplast development. ment and is not an irreversible event. In Arabidopsis, the yellow variegated2 ( var2) mutant shows Results a variegation pattern typical of cell-lineage types and is thus an ideal model for studying chloroplast differentiation. It Characteristic features of plastids in var2 white was originally reported along with var1 by Martínez-Zapater sectors in mature leaves (1992) and mapped on chromosome 2. The gene responsible We fi rst prepared ultrathin sections of mature rosette for variegation encodes FtsH2, one of the isoforms in the leaves (MRLs) at 30 d after sowing, and examined plastids in chloroplast FtsH, which is localized in the thylakoid wild type (ecotype Columbia, Col) and var2 variegated membrane ( Chen et al. 2000 , Takechi et al. 2000 ). FtsH is an sectors by transmission electron microscopy (TEM). Green ATP-dependent metalloprotease that belongs to the AAA + sectors in var2 MRLs contain chloroplasts that were protein family (Neuwald et al. 1999, Ito and Akiyama 2005). almost indistinguishable from those observed in wild type It is one of the major prokaryotic proteases in chloroplasts ( Fig. 1A, B ). In contrast, plastids in white sectors clearly and performs processive degradation to maintain functional lacked developed thylakoid membranes and granal stacks, chloroplasts ( Adam et al. 2006 , Sakamoto 2006 ). Plastidic but instead contained globular vacuolated membrane struc- FtsH forms a hetero-complex with two types of isoform that tures (termed hereafter as ‘plastidic vacuolated body’, PVB). are functionally distinguishable: Type A includes FtsH1 and They appeared to vary in size but were mostly globular and FtsH5, and Type B includes FtsH2 and FtsH8 (Yu et al. 2005, constituted of a single membrane (Fig. 1C, D , Supplemen- Zaltsman et al. 2005b). FtsH levels are coordinately regulated tary Fig. S1). In addition to PVBs, we observed prolamellar between the two types (Sakamoto et al. 2003, Yu et al. 2004), body (PLB)-like structures in MRL plastids in white sectors and we confi rmed that leaf variegation in var1 (similar ( Fig. 1D, E ). Subsequently to TEM analysis, we examined but not as intense as var2 ) is caused by the lack of FtsH5 plastid nucleoids, plastid DNA (ptDNA)–protein complexes ( Sakamoto et al. 2002 ). that are detectable with DNA fl uorescence dyes such as

2070 Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors Downloaded from http://pcp.oxfordjournals.org/

Fig. 1 Characteristic ultrastructures of plastids and their nucleoids observed in var2 mature leaves (30 d old). Lens-shaped chloroplasts detected in Col (A) and var2 green sectors (B), and a plastid detected in a var2 white sector (C–E). P, PVB. PLB, PLB-like structure. Plastid nucleoids detected by SYBG stain in Col (F) and var2 (G). In (G), the image was taken at the border between the green and white sectors. Areas corresponding

to single chloroplasts or a plastid are enclosed by red or white circles, respectively. Signals corresponding to nuclei are indicated by yellow by guest on April 29, 2016 arrowheads. Bars, 1 µm in (A)–(E); 10 µm in (F) and (G).

4 ′ ,6-diamidino-2-phenylindolyle (DAPI) and SYBR Green I completely white. Photographs taken every day after the (SYBG) (Kuroiwa 1991) (Fig. 1F, G ). The plastid nucleoids in emergence of the FTL demonstrated that green sectors white sectors were large and resembled those in proplastids began to form at 7 d after sowing under our growth condi- ( Miyamura et al. 1986, Miyamura et al. 1990, Fujie et al. tions. The sectors formed at this stage became clear as the 1994), suggesting that plastid development into chloroplasts leaf matured (at 9 d) and it appeared permanent in mature is arrested in white sectors. The remarkable inner-membrane leaves (after 13 d). These fi ndings indicate that at least in structures and nucleoid structures thus appeared to be FTL, the differentiation of proplastids into chloroplasts characteristic of var2 white sectors. To examine these two occurs at approximately day 7. features during an early stage of true leaf development, we focused on the fi rst true leaf (FTL) and characterized sector Plastid ultrastructure during variegation formation formation in detail. in the FTL in var2 To examine when abnormal plastids are detectable, ultra- Formation of green sectors in FTLs in var2 thin sections were prepared from 6-day-old seedlings (except Previous reports by Chen et al. (1999) showed that the varie- for cotyledons and roots) and 8-day-old FTLs in Col and gation pattern in var2 remained unchanged, when mature var2, and were examined by TEM ( Figs. 3, 4). No green sec- leaves were examined. Consistent with these reports, the tors were visible at this stage ( Fig. 2 ), although cells that variegation patterns that we observed in MRL remained became green sectors could be included in the preparation. unchanged at least for 10 d (Supplementary Fig. S2). When To follow chloroplast development, we fi rst examined we observed expanding FTL in var2 , however, we found that plastids at the peripheral zones of the shoot meristem. Cells some green sectors emerged from the white sector ( Fig. 2). in Col contained mostly globular chloroplasts of ∼2 µm with FTL was highly variegated in var2 and often looked a limited number of thylakoids and no starch granules ( Fig. 3A ).

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Fig. 2 Appearance of the green sectors in TFL of var2 mutants. Photographs of identical plant at 7 d (A, E), 9 d (B, F), 11 d (C, G) and 13 d (D, H)

after sowing. (E)–(H) are close-up images corresponding to (A)–(D), respectively. Bars, 2 mm. by guest on April 29, 2016

In contrast, cells at the peripheral zone in var2 contained are dominated by white sectors, most of the cells in FTL smaller size (∼1 µm) proplastid-like plastids and only few contain abnormal plastids ( Fig. 4E). We rarely observed detectable thylakoids ( Fig. 4A ). These results suggest that chloroplast-like plastids that retained thylakoids. These the formation of chloroplasts, represented by thylakoid chloroplast-like plastids were found to coexist with abnor- development, proceeds slowly in var2 . mal plastids (not shown). In general, a series of developing chloroplasts can be found by observing cells from the base to the tip of a single Formation of abnormal plastids in white sectors developing leaf ( Leese and Leach 1976 ). In fact, the FTL in Col Ultrastructures of plastids in Col and var2 during FTL devel- showed that chloroplasts became enlarged (from ∼2 to 8 µm) opment are summarized in Fig. 5 . In Col, the formation of and lens shaped, contained an increased number of granal leaf primordia around the meristem is followed by initial stacks and ﬁ nally accumulated starch grains ( Fig. 3B–E ). thylakoid development. During this stage of development, When he FTL in var2 was examined, chloroplast develop- chloroplasts are globular and thylakoids begin to develop. ment was found to be remarkably different. In var2, proplas- Subsequently, chloroplasts became oval and appeared to be tids in shoot meristematic cells appeared to develop distributed in proximity to plasma membranes. Afterwards, thylakoids along with leaf maturation ( Fig. 4A, B). As afore- thylakoid membranes constituted granal networks and mentioned, however, this process was slow in comparison mature chloroplasts accumulated large starch bodies. In with that in Col. We also found that the middle to the tip parts contrast to the normal chloroplast development as described of 6-day-old FTLs accumulated abnormal plastids that were above, var2 contained many proplastids within the periph- characterized by irregularly shaped envelopes ( Fig. 4C, D). eral zones of the shoot meristem. Overall, we observed dis- No apparent PVBs and PLB-like structures were detectable tinct plastid development in var2 as schematically presented in 6- and 8-day-old FTLs we observed. Because FTLs in var2 in Fig. 6 . Three features characteristic of var2 were detected.

2072 Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors Downloaded from http://pcp.oxfordjournals.org/ Fig. 3 TEM analysis of chloroplasts in Col. Chloroplasts in 6-day-old plants at the shoot meristem (A), the basal part of FTL (B), the middle part of FTL (C) and the tip part of FTL (D). Chloroplasts in 8-day-old plants at the tip part of FTL (E). Bar, 2 µm. by guest on April 29, 2016

Fig. 4 TEM analysis of plastids in var2 . Plastids (indicated by red arrowheads) in 6-day-old plants at the shoot meristem (A), the basal part of FTL (B), the middle part of FTL (C) and the tip part of FTL (D). Plastids in 8-day-old plants at the tip part of FTL (E). Bar, 2 µm.

Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. 2073 W. Sakamoto et al.

6 days 8 days

Tip Meristem Base Middle Col Downloaded from http://pcp.oxfordjournals.org/ var2 by guest on April 29, 2016

1µm

Fig. 5 Summarized plastid ultrastructures during leaf development in Col and var2 leaves. Representative images of plastids in 6-day-old (meristem, base, middle, tip parts of FTL) and 8-day-old (tip part of FTL) plants are shown. In var2 , two types of plastid representing abnormal plastids and chloroplasts that likely form green sectors afterwards (after 6 d, middle part) are indicated as separate rows.

First, we found that abnormal plastids started to accumulate leaf development and concomitant formation of abnormal from the tip to the middle part of the FTL at 6 d. These data plastids (feature 3, Fig. 6). suggested that this is the stage where cells are destined to undergo normal chloroplast differentiation or a possible Formation of plastid nucleoids during leaf arrest of thylakoid development (feature 1, Fig. 6). Secondly, development cells in green sectors form normal chloroplasts, but this pro- The morphologies of plastid nucleoids, as detected by DAPI cess occurs at a much slower rate than the normal chloro- staining of thin sections, are known to change along with plast development in Col (feature 2, Fig. 6). However, chloroplast development ( Lawrence and Possingham 1986 , chloroplast development in var2 appears to proceed into Miyamura et al. 1990 , Rowan et al. 2004 ). These changes are maturity, since green sectors in mature leaves contain fully also well correlated with thylakoid development and granal developed chloroplasts. Finally, abnormal plastids accumu- stacking. Because the observation of plastid nucleoids in late PVBs and PLB-like structures instead of thylakoids, var2 MRL indicated that white sectors contained nucleoids but these bodies were not formed during an early step of that were larger and less abundant than those observed in

2074 Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors

Col

(3)

var2 (1)

(2)

Fig. 6 A schematic representation of the green/white sector formation in var2 . Temporal chloroplast development in Col FTL is indicated from

left to right. Green bars represent the degree of grana formation. Three features characteristic of var2 are indicated by numbers. Feature 1 is the Downloaded from point when the abnormal plastids start to accumulate. This point corresponds to 6 day after sowing. Feature 2 is the fact that chloroplast development, as represented by granal stacking, proceeds very slowly in var2 compared with Col. Feature 3 is the formation of PVBs in plastids. PVB is predominantly seen in MRL, but not at the early step of leaf development in FTL. http://pcp.oxfordjournals.org/ by guest on April 29, 2016

Fig. 7 Morphological change in plastid nucleoids during leaf development as observed by SYBG-stained thin sections of FTL in Col (A–D) and var2 (E–H). Cells in 8-day-old plants at the basal part of FTL (A, E), the middle part of FTL (B, F) and the tip part of FTL (C, G). Cells in 10-day-old plants at the tip part of FTL (D, H). Note that ﬂ uorescent signals in each image were incorporated by a digital camera at the identical time setting. Signals corresponding to nuclei are indicated by yellow arrowheads. Areas corresponding to single chloroplasts or plastids are enclosed by red circles. Bar, 10 µm. green sectors ( Fig. 1G ), we characterized plastid nucleoids In Col, developing chloroplasts at the tip and the middle during leaf development in Col and var2 FTLs in this study. parts of the FTL contained a few large nucleoids that were We ﬁ xed Col and var2 seedlings at 6 or 8 d from which frequently observed as surrounding the envelope ( Fig. 7A, B ). roots and cotyledons were removed (Supplementary As chloroplasts matured and contained granal stacks (tip Fig. S3A, B ). Fixed tissues were embedded into Technovit part of 8-day-old leaf), nucleoids became smaller in size and resin and a cross-section of FTL was stained with SYBG to more abundant (Fig. 7C, D). The nucleoids appeared dense visualize plastid nucleoids (Supplementary Fig. S3C). (images in Fig. 7 were recoded by the same time length) and

Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. 2075 W. Sakamoto et al.

were dispersed in the stroma. In some instances, nucleoids not accompany an overall decrease in ptDNA amounts. were observed in proximity to the exterior of thylakoid In fact, our previous semi-quantitative PCR analysis did not membranes. In var2 , however, it was evident that abnormal reveal signifi cant changes in ptDNA level between green and plastids contained nucleoids whose structures differed from white sectors of var2 (Kato et al. 2007). To examine ptDNA those in Col. Throughout leaf development, plastids retained levels carefully, we separately isolated single green and white nucleoids that appeared at the initial stage. These nucleoids protoplasts from both sectors and performed quantitative did not become smaller in size and did not disperse within PCR ( Fig. 8A ). Protoplasts from white and green cells were plastids and rather remained as large aggregates ( Fig. 7E–H). clearly distinguishable from each other due to the presence In some leaf tips at 6 or 8 d, prominent large signals were of chlorophyll auto-fl uorescence ( Fig. 8B–E ). The number of occasionally detected. These results are consistent with our chloroplasts per protoplast was not signifi cantly different TEM observation, which revealed that thylakoid formation between Col and var2 green sectors (Supplementary is strongly impaired in the plastids of white sectors, suggest- Fig. S4 ). Single protoplasts from Col were also subjected to ing that abnormal plastids resulted from an arrest of proper our PCR analysis. The results using two primer sets, detect- chloroplast development. ing ptDNAs corresponding to psbA or ndhA , showed that relative ptDNA levels were not signifi cantly different between

ptDNA levels in the white sectors Col, and green and white sectors in var2 ( Fig. 8F ). We also Downloaded from Since our cytological observations were not quantitative, we measured the area size of protoplasts and found that cells assumed that the structural change in plastid nucleoids did in white sectors are smaller than those in green sectors and http://pcp.oxfordjournals.org/ F psbA

1 by guest on April 29, 2016

ndhA

2 Relative cpDNA amount

Col var2 var2 green white

Fig. 8 Estimation of cpDNA levels in protoplasts derived from green and white cells. (A) Capture of protoplasts with a glass capillary (denoted by red arrowhead, bar = 200 µm). Protoplasts from green sectors (B, C) and from white sectors (D, E) are shown. Images captured by bright fi eld (B, D) and by G fi lter set (C, E, detecting chlorophyll autofl uorescence) are shown (bar, 10 µm). (F) CpDNA levels estimated by real-time PCR analysis using a single protoplast from Col, var2 green and white sectors (n = 10). Results from two gene-specifi c primers (psbA and ndhA ) are shown.

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Ipomoea nil Oryza sativa

Artemisia princeps Pampan. Felicia amelloides Downloaded from http://pcp.oxfordjournals.org/

Habenaria radiata Ficus benzyamina by guest on April 29, 2016

Alternanthera ficoidea Arabis glabra

Fig. 9 Plastid nucleoids detected by DAPI-stained thin sections from variegated leaves. Images of eight species were shown along with the photograph of each corresponding variegated leaf. For each species, representative images from green sectors (left) and white sectors (right) are shown (bars, 10 µm). Note that ﬂ uorescent signals in each image were incorporated by a digital camera at the identical time setting. Signals corresponding to nuclei are indicated by yellow arrowheads. Areas corresponding to single chloroplasts or plastids are enclosed by red and white circles, respectively.

Col (Supplementary Fig. S5). As a result, our quantitative differentiation is made at a very early stage of leaf develop- PCR with single protoplasts supported our previous obser- ment. The FTL in var2 is dominated by white sectors. In the vation that larger plastid nucleoids in white sectors do not shoot meristem, Col contained many small globular chloro- refl ect an altered amount of ptDNA per cell. plasts with thylakoids and a few granal stacks, whereas the var2 mutant contained proplastids and a limited number of Enlarged plastid nucleoids are commonly found thylakoids with no stacking. Thus, one notable feature of in variegated leaves var2 that was revealed in this study was a clear delay in We assumed that enlarged plastid nucleoids are characteris- chloroplast development. Given the observation that the tic of undifferentiated plastids lacking thylakoid membranes var2 shoot apical meristem predominantly contained pro- and that white sectors in variegated leaves in species other plastids, we conclude that proplastids are normal in shoot than Arabidopsis may contain similar plastid nucleoids. To meristems and have a potential to become functional chlo- test this, mature variegated leaves from eight species were roplasts. It should be noted that prior to the appearance of selected to examine plastid nucleoids with DAPI staining. green sectors, the FTL appeared yellow at 7 d, an observation The pattern and degree of variegation in these species that perhaps indicates that proplastids can initiate chloro- were variable. Specifi cally, Ipomoea , Oryza , Artemisia and plast differentiation at the very initial step ( Fig. 2E). The Alternanthera species showed cell-lineage type variegation green sectors became more evident as the yellow color Downloaded from and other species showed non-cell-lineage type variegation. somewhat disappeared ( Fig. 2F ). Collectively, all of these Except for the Oryza stripe mutant, it was unclear whether data support our hypothesis that chloroplast development leaf variegation is caused by a mutation. Nevertheless, is initiated but arrested at the initial step. variegated sectors were permanently observed in each The delayed emergence of green sectors that we observed species under our growth conditions in a greenhouse ( Fig. 9 , in Fig. 2 has not been previously reported in var2 , because http://pcp.oxfordjournals.org/ pictures of leaves). previous studies focused on expanded leaves. In this study, Leaf tissues containing both green and white sectors were we showed that expanding FTL contains a patch of the cells fi xed and embedded in Technovit resin and thin sections that escape from the arrest and undergo chloroplast devel- were stained with DAPI to visualize plastid nucleoids. We set opment ( Fig. 2F–H ). Namely, the gradual appearance of the time period unchanged when recording images with green patches in white sectors indicates that chloroplast fl uorescence microscopy and the images from both sectors development proceeds very slowly in var2. Variegation is were compared ( Fig. 9 ). In all species, green sectors were more severe in the FTL than in the second and subsequent found to contain small/dense DAPI signals that were dis- leaves, partly because other FtsHs than FtsH2 are upregu- by guest on April 29, 2016 persed within stroma ( Fig. 9 , red circles in left panels). In lated in the second and subsequent leaves in var2 (Zaltsman strong contrast however, white sectors contained enlarged et al. 2005a ). Increased FtsH levels in the second and subse- DAPI signals that were brighter and resembled those quent leaves may be one of the reasons that FTL only showed observed in var2 white sectors ( Fig. 9 , white circles in right a marked emergence of green sectors. The delay of chloro- panels). Similar to those in var2 , the enlarged nucleoids plast development in var2 can be connected to the suppres- detected in cell-lineage type species (e.g. Artemisia and sor mutations in which leaf variegation was rescued ( Park Ipomoea) were located along with the envelope. Given that and Rodermel 2004 , Miura et al. 2007 , Yu et al. 2008 , Zhang enlarged nucleoids represent plastids with undifferentiated et al. 2009). One possibility is that a suppressor mutation can thylakoids, these results suggest that abnormal plastids in delay overall chloroplast development in the var2 back- white sectors of variegated leaves generally contain plastids ground. As a consequence, the threshold level of FtsH (or with arrested thylakoid formation. other factors regulated by FtsH) was mitigated, thereby allowing most of the mesophyll cells to contain chloroplasts Discussion of normal appearance. In this scenario, it is possible that any mutations delaying chloroplast differentiation may also act Formation of green and white sectors in var2 as a suppressor. It also implies that the balance between the Although plastids in white sectors of variegated leaves leaf maturation process and chloroplast development, rather have been previously observed in many species (Kirk and than the balance between protein synthesis and degrada- Tilney-Bassett 1978 ), there has not been a systematic analy- tion in chloroplasts, is important for proper chloroplast sis to examine at what stage abnormal plastids arise. In this formation. study, we focused on FTLs of Arabidopsis Col and var2, and The other important fi nding from our TEM analysis per- characterized sector formation during leaf development by tains to PVBs and PLB-like structures that were found in observing plastid ultrastructure and nucleoid morphology. abnormal plastids. Because the PVBs were predominantly Detailed observations of white sectors demonstrated that detected in MRL white sectors, we predicted that PVBs arise the decision for a leaf cell to undergo normal chloroplast during leaf development. Unexpectedly, their precursor-like

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structures were hardly detectable in the early stage of FTL study indicated that expression of plastid genes is not signifi - development, suggesting that PVB formation is very slow or cantly altered between green and white sectors in var2 (Kato predominantly proceeds in mature leaves. An alternative et al. 2007 ). It thus appears that the altered nucleoid mor- possibility is that apparent PVBs are not formed in FTL. This phologies in var2 white sectors do not affect plastid gene possibility seems unlikely but not completely ruled out, since expression at the transcript level. Some chloroplast proteins we did not examine FTL at 30 d. Although the origin of PVB were biochemically identifi ed to interact with ptDNAs and remains unclear, the co-existence of PLB-like structures constitute plastid nucleoids ( Sato et al. 2003 , Sakai et al. raises a possibility that PVBs are derived from PLB-like struc- 2004, Sakamoto et al. 2008). Those include, for example, tures. The PLB is a particular type of membrane structure in plastid envelope DNA-binding protein (PEND; Sato et al. etioplasts and is thought to represent a novel type of thyla- 1998) and sulfi te reductase (SiR; Sato et al. 2001), MFP1 koid under dark conditions ( Kirk and Tilney-Bassett 1978 , ( Jeong et al. 2003 ) and CND41 ( Murakami et al. 2000 ) whose Wise 2006). It is reasonable to assume that the abnormal precise roles in nucleoid formation and gene expression plastids in FTL are unable to stay arrested in development remain unclear. PEND is located in the inner envelope and and show characteristics similar to etioplasts by forming was shown to anchor ptDNAs to the plastid envelope. PLBs ( Park et al. 2002 , Philippar et al. 2007 ). Supporting this A study using truncated PEND–green fl uorescent protein observation is the previous study by Chen et al. (2000) that showed that nucleoids change their morphology within dif- Downloaded from demonstrated that etioplast formation in cotyledons is ferent tissues ( Terasawa and Sato 2005 ). Large plastid nucle- comparable between Col and var2 . It is possible that the oids detected in white sectors may be associated with PEND white plastids in MRL perhaps have a potential to develop since they are located along with the envelope. DAPI-stained but they fail to form a functional thylakoid network from plastid nucleoids were also studied previously by Fujie et al.

PLB-like structures due to the lack of photosynthetic pig- (1994). Plastid nucleoids detected in 6- and 8-day-old http://pcp.oxfordjournals.org/ ments and other protein components. As a result, these Col FTL in this study were similar to those reported in the defective plastids form PVBs instead. Thus, our observations previous studies. Plastid nucleoids detected in white tissues demonstrate that plastids in the white sectors of variegated resemble those from non-photosynthetic tissues such as leaves represent a novel plastid type. Membrane structures roots and petals (Fujie et al. 1993). Once again, these data like PVBs in a mutant defective in chloroplast development indicate that nucleoid morphologies are well correlated with have been previously reported in other studies ( Motohashi the development of thylakoid membranes. et al. 2001 , Abdelkader et al. 2007 , Okegawa et al. 2007 ). On Although our focus was on nucleoid morphology between the other hand, we did not detect invaginated membrane the green and white sectors in var2, it appeared to accom- by guest on April 29, 2016 structures along with the inner envelope, which were pany quantitative differences in DNA signals. To clarify this reported in several mutants defective in thylakoid formation question, we carefully estimated ptDNA levels using single (Kroll et al. 2001, Kobayashi et al. 2007). We do not assume protoplasts. However, no signifi cant differences were that PVBs directly derive from inner envelope. detected between Col, white and green sectors. This result was consistent with our previous semi-quantitative PCR Nucleoid structures in variegated leaves analysis ( Kato et al. 2007 ) and confi rms that alteration of Similar to our TEM analysis, our initial characterization of nucleoid morphologies do not represent ptDNA amounts. plastid nucleoids was carried out only in MRL. Therefore we Regarding chloroplast DNA (cpDNA) amounts during leaf decided to observe plastid nucleoids during FTL develop- development, contradictory results were reported from dif- ment in this study. As previously reported, a series of sec- ferent laboratories (Rowan and Bendich 2009). Chloroplasts tions in 6- and 8-day-old FTLs demonstrated that plastid in mature leaves from Arabidopsis were shown to contain a nucleoids follow the typical morphological alteration substantially reduced level of ptDNAs based on fl uorescence (Kuroiwa 1991, Fujie et al. 1994, Sakamoto et al. 2008). Imma- detection and quantitative RT-PCR (Rowan et al. 2004). On ture chloroplasts tend to contain nucleoids that are large, the other hand, cpDNA levels were reported to remain less packed and dispersed along with the inner envelope. unchanged based on Southern blot analysis ( Li et al. 2006 , As the chloroplasts mature with many granal stacks, the Zoschke et al. 2007 ). Given that the var2 white sectors con- nucleoids appeared to move in stroma and they become tain plastids whose development into chloroplasts is arrested small and packed ( Fig. 7A–D ). It is evident that in var2 , the at the initial step, our data indirectly indicate that ptDNA prominent large SYBG signals remain in abnormal plastids of amounts do not signifi cantly change throughout chloroplast white sectors ( Fig. 7E–H ). These results are consistent with development. It is possible, however, that ptDNAs were our previous observation and support our hypothesis that unchanged between the two sectors because the number of thylakoid development is arrested in white sectors. plastids decreased in white sectors. These data also suggest DNA compaction in nucleoids was suggested to affect that each plastid in the white sectors indeed contained gene expression (Sekine et al. 2002). In contrast, our previous a larger amount of DNA than chloroplasts in green sectors.

We were unable to exclude this possibility since the plastid cacodylate buffer (pH 7.4) for at least 24 h at room tempera- number in white cells was not determined. ture. After four separate rinses with cacodylate buffer, the Besides the quantitative change of ptDNA amounts, we samples were treated with 2% (w/v) osmium tetraoxide for raised a possibility in this study that many variegated sec- 4–6 h at room temperature. After four separate rinses with tors, other than var2 in Arabidopsis, display large plastid cacodylate buffer, the samples were stained by 2% (w/v) nucleoids due to the impairment of normal thylakoid devel- uranyl acetate in 50 % (v/v) ethanol for 1 h at room tempera- opment. The examination of variegated leaves from differ- ture. Samples were further dehydrated with a graded etha- ent species, including both cell-lineage and non-cell-lineage nol series [50% , 70% , 85% , 95% , 100% (v/v)]. Ethanol was types, demonstrated that white sectors contain large nucle- subsequently replaced by a series of Spurr’s resin dilutions oids as expected. It is unclear whether variegated leaves [25 %, 50% , 75% , 100% (v/v)] and the resin was hardened examined in this study were due to a genetic deﬁ ciency for 16 h at 65 ° C. Ultrathin sections (60–70 nm) were made (except for rice) or from physiological effects. In addition, with an Ultracut N (Reichert-Nissei). Thin sections were cell area size, number of chloroplasts per cell and the size of stained with 0.25% (w/v) lead citrate and examined with chloroplasts varied among these species. Nevertheless, white a JEM-1010 JEOL transmission electron microscope (at the sectors always tended to be abundant with large nucleoids College of Life Sciences, Peking University.

that were clearly distinguishable from those in the green Downloaded from sectors. Our results indicate that despite various patterns Technovit thin sections and staining with SYBG found among different variegated leaves, white sectors have and DAPI common features that represent an arrest in thylakoid For preparation of Arabidopsis sections, cotyledons and differentiation. roots were removed from 6- and 8-day-old seedlings under

a dissection microscope (as illustrated in Supplementary http://pcp.oxfordjournals.org/ Materials and Methods Fig. S3). The leaf tissues were ﬁ xed in FAA solution [5 % (v/v) Formalin, 5% (v/v), acetic acid, 45% (v/v) ethanol] for at Plant materials and growth conditions least 3 h or stored at 4 °C. For other plant species, a 1 × 2 mm Arabidopsis thaliana ecotype Columbia (Col) was used as region of leaves containing green and white sectors was a wild-type plant in this study. For observing leaf variegation excised prior to ﬁ xation. Fixed tissues were pre-stained by in var2, the var2-1 allele was used (with the exception that 2.5 % (w/v) safranin and washed in 0.1 M phosphate-buffered photos shown in Supplementary Fig. S2 used var2-6 ). saline (PBS pH 7.4). The tissues were dehydrated by a graded

This var2-1 allele possesses a nonsense mutation at Gln597 series of ethanol dilutions (50% , 70% , 80% , 90% , 95% , by guest on April 29, 2016 ( Sakamoto et al. 2004 ). Sterilized seeds were germinated 99.5 %, 100% (v/v). Ethanol was replaced by a series of and grown on 0.7% (w/v) agar plates containing Murashige Technovit7100 resin dilutions [25% , 50% , 74% , 100% (v/v)]. and Skoog medium supplemented with Gamborg’s vitamins The samples were kept overnight at room temperature and (Sigma-Aldrich) and 1.5% (w/v) sucrose. After stratifi cation the resin was fi nally hardened overnight at 4° C. Thin sec- for 2 or 3 d at 4 ° C under darkness, plates were maintained tions (0.9 µm) were stained with 1 µg/ml DAPI (Invitrogen) under 12 h light (approximately 100 µ mol/m2 /s at a con- or 1 µg/ml SYBRG (Invitrogen) and examined by an Olympus stant temperature at 22° C. For further analysis, plants were AX-84-FLBD fl uorescent microscope (U-MWU2 fi lter set for transferred to soil after 4–5 weeks. Additional sources for DAPI and U-MW13 for SYBG). Images were recorded with variegated plants used in Fig. 9 (with the exception of the a DP71 digital camera (Olympus). Images from green and rice stripe mutant) were purchased from a local nursery and white sectors were recorded with identical time settings. subsequently grown in a greenhouse at the Research Insti- tute for Bioresources, Okayama University. The rice stripe Protoplast preparation and manipulation mutant (st-5 , mutation mapped on chromosome 4) was Mesophyll protoplasts were prepared as previously generously provided by Dr Masahiko Maekawa (Okayama described in Kato et al. (2007) . Leaves from Col and var2-1 University). plants were cut into small pieces and were gently suspended in enzyme solution [0.1% (w/v) cellulose Onozuka R10 Transmission electron microscopy (Yakult), 0.05% (w/v) pectolyase Y23 (Kyowa Chemical Leaf samples for TEM were prepared as follows. For 6-day-old Products), 400 mM mannitol, 10 mM CaCl2 , 20 mM KCl, FTLs, cotyledons and roots were removed from plate-grown 5 mM EGTA, 20 mM MES, pH 5.7]. After incubation at room seedlings and the whole remaining leaf tissues were fi xed. For temperature for 1 h, protoplasts were collected by centrifu- 8-day-old FTLs, only middle and tip regions from the FTLs gation at 60 × g for 1 min. The isolated protoplasts were then were excised and fi xed for microscopic observation. For resuspended in wash buffer (154 mM NaCl, 125 mM CaCl2 , 30-day-old MRLs, a 1 × 1 mm region of leaves was excised and 5 mM KCl, 2 mM MES, pH 5.7). For PCR analysis, protoplasts fi xed. Leaf samples were fi xed in 4% (v/v) glutaraldehyde in were placed on a slide glass and were detected via inverse

2080 Plant Cell Physiol. 50(12): 2069–2083 (2009) doi:10.1093/pcp/pcp127 © The Author 2009. Plastids in variegated leaf sectors

microscopy (Olympus CKX41N-31PH). Single protoplasts Acknowledgments were collected into a glass capillary (pore size 100 µm) by using a pressure-controlled PicoPipet device (Alatair Corp). We thank Dr Ryo Matsushima for microscopic analysis, Dr Protoplasts from green or white sectors in var2 were easily Masahiko Maekawa for providing rice stripe mutants, Dr distinguishable due to the presence or absence of chloro- Kazuhide Rikiishi for RT-PCR analyses. We also thank Rie plasts, respectively. Protoplasts from white sectors contained Hijiya for her technical assistance. abnormal plastids that were detectable under bright-fi eld microscopy, and were separated by protoplasts derived from References epidermal cells. Protoplasts were carefully transferred into a PCR tube, suspended in 9.2 µl of sterilized water, sonicated Abdelkader , A.F. , Aronsson , H. , Solymosi , K. , Boddi , B. and Sundqvist , C. in a water bath for 1 min (Type UR-20P; Tomy Seiko) and (2007 ) High salt stress induces swollen prothylakoids in dark-grown stored at 4° C until further use. For estimating the area of wheat and alters both prolamellar body transformation and protoplasts, they were suspended in wash buffer and reformation after irradiation. J. Exp. Bot. 58 : 2553 – 2564 . observed by fl uorescence microscopy. Images of protoplasts Adam , Z. , Rudella , A. and van Wijk, K.J. (2006 ) Recent advances in the study of Clp, FtsH and other proteases located in chloroplasts . from Col, green and white sectors in var2 were recorded by Curr. Opin. Plant Biol. 9 : 234 – 240 . digital camera and the area for each protoplast was calcu- Aluru , M.R. , Yu , F. , Fu , A. and Rodermel , S. (2006 ) Arabidopsis Downloaded from lated with Metamorph software (Molecular Devices). For variegation mutants: new insights into chloroplast biogenesis. counting chloroplast numbers, protoplasts were fi xed in J. Exp. Bot. 57 : 1871 – 1881 . 0.02 % (v/v) glutaraldehyde and immediately squashed on Carol , P. and Kuntz , M. (2001 ) A plastid terminal oxidase comes to a slide glass by gently pressing down on the cover glass. light: implications for carotenoid biosynthesis and chlororespiration .

Images of broken protoplasts with intact chloroplasts were Trends Plant Sci. 6 : 31 – 36 . http://pcp.oxfordjournals.org/ recorded by a digital camera and were then used to count Chen , M. , Choi , Y. , Voytas , D.F. and Rodermel , S. (2000 ) Mutations in chloroplasts. the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease . Plant J. 22 : 303 – 313 . Real-time PCR analysis Chen , M. , Jensen , M. and Rodermel , S. (1999 ) The yellow variegated mutant of Arabidopsis is plastid autonomous and delayed in The entire protoplast suspension in a PCR tube was used for chloroplast biogenesis. J. Hered. 90 : 207 – 214 . RT-PCR reactions (SYBR Premix ExTaq; Takara). RT-PCR was Fujie , M. , Kuroiwa , H. , Kawano , S. and Kuroiwa , T. (1993 ) Studies on the performed in 20 µl of a reaction mixture as recommended behavior of organelles and their nucleoids in the root apical by the supplier. PCR cycles were set to 50 under the follow meristem of Arabidopsis thaliana (L.) col. Planta 189 : 443 – 452 . by guest on April 29, 2016 conditions: 94° C for 13 s, 55 ° C for 15 s and 72 ° C for 20 s. Fujie , M. , Kuroiwa , H. , Kawano , S. , Mutoh , S. and Kuroiwa , T. (1994 ) LightCycler Software Version 4.0 (Roche Diagnostics) was Behavior of organelles and their nucleoids in the shoot apical used to quantify DNA levels. For detecting cpDNA, two sets meristem during leaf development in Arabidopsis thaliana L. Planta of primers, detecting the region corresponding to psbA 194 : 395 – 405 . and ndhA were used: psbA-F (5′ -TTGCGGTCAATAAGGT Han , C.D. , Coe , E.H. , Jr and Martienssen , R. A. (1992 ) Molecular cloning and characterization of iojap (ij ), a pattern striping gene of maize. AGGG-3 ′), psbA-R (5 ′-TAGAGAATTTGTGCGCTTGG-3 ′ ), ′ ′ ′ EMBO J. 11 : 4037 – 4046 . ndhA-F (5 -TGAGATCCGCTAAAACAAGG-3 ), ndhA-R (5 - Ito , K. and Akiyama , Y. (2005 ) Cellular functions, mechanism of action, ′ CTAGCCGATGGGACAAAA-3 ) and regulation of FtsH protease . Annu. Rev. Microbiol. 59 : 211 – 231 . Jeong , S.Y. , Rose , A. and Meier , I. (2003 ) MFP1 is a thylakoid-associated, Supplementary data nucleoid-binding protein with a coiled-coil structure. Nucleic Acids Res. 31 : 5175 – 5185 . Supplementary data are available at PCP online. Kato , Y. , Miura , E. , Matsushima , R. and Sakamoto , W. (2007 ) White leaf sectors in yellow variegated2 are formed by viable cells with undifferentiated plastids. Plant Physiol. 144 : 952 – 960 . Funding Kato , Y. and Sakamoto , W. (2009 ) Protein quality control in chloroplasts: The Ministry of Education, Culture, Sports, Science a current model of D1 protein degradation in the photosystem II and Technology Grant-in-Aid for Scientiﬁ c Research repair cycle. J Biochem. 146 : 463 – 469 . (No. 16085207 to W.S.), the Oohara Foundation (to W.S.), Keddie , J.S. , Carroll , B. , Jones , J.D. and Gruissem , W. (1996 ) The DCL gene of tomato is required for chloroplast development and palisade the Asahi Glass Foundation (to W.S.), the Novartis cell morphogenesis in leaves . EMBO J. 15 : 4208 – 4217 . Foundation (to W.S.) and the National Natural Science Kirk , J.T.O. and Tilney-Bassett , R.A.E. (1978 ) The Plastids: Their Foundation of China for a Creative Research Group Program Chemistry, Structure, Growth, and Inheritance . Elsevier/ (No. 30421004 to Sod.). Eiko Miura is a postdoctoral fellow North-Holland Biomedical Press, Amsterdam . supported by the Japan Society for the Promotion of Kobayashi , K. , Kondo , M. , Fukuda , H. , Nishimura , M. and Ohta , H. Science. ( 2007 ) Galactolipid synthesis in chloroplast inner envelope is

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