Continuous expression in tobacco leaves of a Brassica napus PEND homologue blocks differentiation of plastids and development of palisade cells

Abstract

SummaryBrassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope.

Development-Dependent Changes in the Amount and Structural Organization of Plastid DNA

“…Furthermore, this study identi fi ed approximately 35 other proteins possibly associated with nucleoids including a family of DNA gyrases, as well as proteins involved in DNA repair, and plastid transcription and translation (Phinney and Thelen 2005 ) . A plastid envelope DNA-binding protein (PEND) is thought to attach the nucleoids to the envelope membrane early in chloroplast development in order to provide an anchor for plastome replication and segregation during organelle division (Sato et al , 1998Terasawa and Sato 2005 ;Wycliffe et al 2005 ) . However, the PEND protein, was not detected in maize nucleoids which did also not contain the bisul fi te reductase (Majeran et al 2012 ) .…”

Inducible Knockdown of MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE1 Reveals Roles of Galactolipids in Organelle Differentiation in Arabidopsis Cotyledons

“…Considering that cell proliferation for cotyledon formation is nearly completed during embryogenesis and the cell expansion mainly contributes to the postgerminative cotyledon growth in the light (Tsukaya et al, 1994;StoynovaBakalova et al, 2004), the morphological disorder and the dwarf phenotype in the white L4 cotyledons may be due to distorted mesophyll cell expansion during postgerminative growth. The distorted organization of mesophyll cells has been observed in several mutants with defective chloroplast development (Wang et al, 2000;Wycliffe et al, 2005;Garcion et al, 2006;Sulmon et al, 2006), including the mgd1-2 mutant (Kobayashi et al, 2007), thus showing a tight developmental link between chloroplasts and host mesophyll cells. Furthermore, application of norflurazon, which inhibits carotenoid biosynthesis and impairs chloroplast development, to Arabidopsis seedlings was recently found to affect the transition of leaf cells from the proliferation stage to the expansion stage, thus resulting in decreased cell size in young true leaves (Andriankaja et al, 2012).…”

New insights into plastid nucleoid structure and functionality

“…The cbZIP domain of PEND was shown to bind selectively to AT-rich regions of plastidic DNA containing the canonical sequence TAAGAAGT (Sato and Ohta 2001). Interestingly, both the PEND protein from Brassica napus and its rapeseed homolog, GSBF1, were found to repress the expression of the nuclear-encoded rbcS gene (Waldmüller et al 1996;Wycliffe et al 2005). That PEND indeed fulfills a direct role in the nucleus was supported by the recent observation that a PEND:GFP fusion protein is targeted to the nucleus when the N-terminal presequence is deleted (Terasawa and Sato 2009).…”