Recombinant Whirly1 translocates from transplastomic chloroplasts to the nucleus

Whirly1 was shown to be dually located in chloroplasts and nucleus of the same cell. To investigate whether the protein translocates from chloroplasts to the nucleus, we inserted a construct encoding an HA-tagged Whirly1 into the plastid genome of tobacco. Although the tagged protein was synthesized in plastids, it was detected in nuclei. Dual location of the protein was confirmed by immunocytological analyses. These results indicate that the plastidial Whirly1 is translocated from the plastid to the nucleus where it affects expression of target genes such as PR1. Our results support a role of Whirly1 in plastid-nucleus communication.     

A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage

All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer-tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent.     

MFP1 is a thylakoid-associated,nucleoid-binding protein with a coiled-coil structure

Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.     

杧果Whirly基因鉴定及其在病菌侵染过程中的表达分析

Whirly家族是一类植物特异的转录因子,无论在细胞核还是在细胞器内都有着广泛而复杂的生物学功能。该研究以杧果全基因组数据为基础,采用生物信息学的方法对杧果Whirly家族基因进行序列分析,并通过qRT-PCR技术分析杧果胶孢炭疽菌(Colletotrichum gloeosporioides,Cg)和细菌性黑斑病菌(Xanthomonas campestris pv.mangiferaeindicae,Xcm)侵染过程中杧果Whirly家族基因的相对表达量。结果表明:(1)从杧果基因组中鉴定了3个Whirly基因家族成员,分别命名为MiWHY1、MiWHY2和MiWHY3。(2)杧果Whirly蛋白均为不稳定亲水碱性蛋白;系统发育树显示,杧果Whirly基因与木薯、毛果杨、番茄Whirly基因亲缘关系最近,杧果与木薯、毛果杨、番茄Whirly家族共有1个高度保守的基序Motif1;杧果Whirly家族均含有Whirly超家族保守域,主要结构元件为无规则卷曲和α-螺旋;保守结构域的四聚体结构与马铃薯Whirly蛋白四聚体结构具有高度相似性。(3)qRT-PCR结果显示,Cg侵染过程中,杧果Whirly基因的相对表达量均显著上调;Xcm侵染12 h时,MiWHY1和MiWHY3相对表达量显著上调,初步确定杧果Whirly基因表达响应胶孢炭疽菌和细菌性黑斑病菌的侵染。研究认为,杧果中有3个Whirly基因家族成员,病菌侵染过程中可激发其表达活性,为后续研究杧果Whirly基因家族成员的功能和机制奠定基础。     

The chloroplast nucleoids of the bundle sheath and mesophyll cells of Zea mays

Dimorphic chloroplasts of Zea mays L. cv. GH5004 from bundle sheath and mesophyll cells contained similar amounts of DNA, while bundle sheath chloroplasts contained twice the number of nucleoids compared to mesophyll chloroplasts. On average bundle sheath nucleoids were half the size of mesophyll nucleoids and contained half as much DNA. Electron microscope autoradiography of the chloroplasts showed that the nucleoid DNA is associated with the thylakoids and in the case of mesophyll chloroplasts preferentially with the grana. These observations suggest that the differences in nucleoid distribution may be due to differences in membrane morphology, with the small nucleoids of agranal bundle sheath chloroplasts being widely dispersed.     

The 68 kDa DNA compacting nucleoid protein from soybean chloroplasts inhibits DNA synthesis in vitro

Nucleoids were purified from chloroplasts of dividing soybean cells and their polypeptide composition analyzed by SDS-polyacrylamide gel electrophoresis. Of the 15–20 nucleoid-associated polypeptides, several demonstrated DNA binding activity. Upon disruption of the nucleoids with high concentrations of NaCl, a subset of these proteins and the majority of chloroplast DNA were recovered in the supernatant after centrifugation. Removal of the salt by dialysis resulted in formation of nucleoprotein complexes resembling genuine nucleoids. Purification of these structures revealed three major proteins of 68, 35 and 18 kDa. After purification of the 68 kDa protein to homogeneity, this protein was able to compact purified chloroplast DNA into a nucleoid-like structure in a protein concentration-dependent fashion. Addition of the 68 kDa protein to an in vitro chloroplast DNA replication system resulted in complete inhibition of nucleotide incorporation at concentrations above 300 ng of 68 kDa protein per g of template DNA. These results led to in situ immunofluorescence studies of chloroplasts replicating DNA which suggested that newly synthesized DNA is not co-localized with nucleoids. Presumably, either the plastid replication machinery has means of removing nucleoid proteins prior to replication or the concentration of nucleoid proteins is tightly regulated and the proteins turned over in order to allow replication to proceed.     

Nucleotide sequences of the 4.5 S and 5 S ribosomal RNA genes from tobacco chloroplasts

Summary The DNA sequences of the 4.5 S and 5 S RNA genes from tobacco chloroplasts have been determined. The coding regions for the mature 4.5 S and 5 S RNAs were identified by sequencing these RNAs. The 4.5 S and 5 S RNA genes are composed of 103 and 121 base pairs respectively. These two genes are separated by the 256 base pair spacer. Several unique features in the spacer and in the region downstream from the 5 S coding region are discussed.