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 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 2 Key Laboratory of Cell Proliferation and Differentiation (Ministry of Education), College of Life Sciences, Peking University, Beijing 100871, PR China – Regular Paper 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