Visualization of plastid nucleoids in situ using the PEND-GFP fusion protein

Plastid DNA is a circular molecule of 120-150 kbp, which is organized into a protein-DNA complex called a nucleoid. Although various plastids other than chloroplasts exist, such as etioplasts, amyloplasts and chromoplasts, it is not easy to observe plastid nucleoids within the cells of many non-green tissues. The PEND (plastid envelope DNA-binding) protein is a DNA-binding protein in the inner envelope membrane of developing chloroplasts, and a DNA-binding domain called cbZIP is present at its N-terminus. We made various PEND-green fluorescent protein (GFP) fusion proteins using the cbZIP domains from various plants, and found that they were localized in the chloroplast nucleoids in transient expression in leaf protoplasts. In stable transformants of Arabidopsis thaliana, PEND-GFP fusion proteins were also localized in the nucleoids of various plastids. We have succeeded in visualizing plastid nucleoids in various intact tissues using this stable transformant. This technique is useful in root, flower and pollen, in which it had been difficult to observe plastid nucleoids. The relative arrangement of nucleoids within a chloroplast was kept unchanged when the chloroplast moved within a cell. During the division of plastid, nucleoids formed a network structure, which made possible equal partition of nucleoids.     

Characterization of the activity of a plastid-targeted green fluorescent protein in Arabidopsis.

In Arabidopsis thaliana the PALE CRESS (PAC) gene product is required for both chloroplast and cell differentiation. Transgenic Arabidopsis plants expressing a translational fusion of the N-terminal part of the PAC protein harboring the complete plastid-targeting sequence and the green fluorescent protein (GFP) exhibit high GFP fluorescence. Detailed analyses based on confocal imaging of various tissues and cell types revealed that the PAC-GFP fusion protein accumulates in chloroplasts of mature stomatal guard cells. The GFP fluorescence within the guard cell chloroplasts is not evenly distributed and appears to be concentrated in suborganellar regions. GFP localization studies demonstrate that thin tubular projections emanating from chloroplasts and etioplasts often connect the organelles with each other. Furthermore, imaging of non-green and etiolated tissue further revealed that GFP fluorescence is present in proplastids, etioplasts, chromoplasts, and amyloplasts. Even photobleaching of carotenoid-free plastids does not affect PAC-GFP accumulation in the organelles of the guard cells indicating that the protein translocation machinery is functional in all types of plastids. The specific accumulation of GFP in guard cell chloroplasts, their tubular connections, the translocation of the precursor polypeptide into the different types of organelles, as well as the use of a plastid-targeted GFP protein as a versatile marker is discussed in the context of previously described observations.     

FtsZ ring formation at the chloroplast division site in plants

Among the events that accompanied the evolution of chloroplasts from their endosymbiotic ancestors was the host cell recruitment of the prokaryotic cell division protein FtsZ to function in chloroplast division. FtsZ, a structural homologue of tubulin, mediates cell division in bacteria by assembling into a ring at the midcell division site. In higher plants, two nuclear-encoded forms of FtsZ, FtsZ1 and FtsZ2, play essential and functionally distinct roles in chloroplast division, but whether this involves ring formation at the division site has not been determined previously. Using immunofluorescence microscopy and expression of green fluorescent protein fusion proteins in Arabidopsis thaliana, we demonstrate here that FtsZ1 and FtsZ2 localize to coaligned rings at the chloroplast midpoint. Antibodies specific for recognition of FtsZ1 or FtsZ2 proteins in Arabidopsis also recognize related polypeptides and detect midplastid rings in pea and tobacco, suggesting that midplastid ring formation by FtsZ1 and FtsZ2 is universal among flowering plants. Perturbation in the level of either protein in transgenic plants is accompanied by plastid division defects and assembly of FtsZ1 and FtsZ2 into filaments and filament networks not observed in wild-type, suggesting that previously described FtsZ-containing cytoskeletal-like networks in chloroplasts may be artifacts of FtsZ overexpression.     

In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana

Among the protein translocation pathways of the thylakoid membrane in chloroplasts, the DeltapH/TAT pathway is unique in several aspects. In vitro transport assays with isolated chloroplasts or thylakoids have defined the trans-thylakoidal proton gradient as the sole requirement for effecting transport. From these studies, evidence has also accumulated indicating that, in contrast to the remaining protein transport pathways present in the thylakoid membrane, the DeltapH/TAT pathway is able to mediate the transport of folded proteins. The present work has established a novel approach to demonstrate the transport of folded proteins by this pathway in vivo. For this purpose, Arabidopsis thaliana plants were stably transformed with gene constructs expressing enhanced green fluorescent protein (EGFP) alone or fused to the transit peptides of different chloroplast proteins under the control of the 35S CAMV promoter. The intracellular and intraorganellar distribution of EGFP in the resulting transformants showed that while all the chloroplast transit peptides efficiently mediated the transport of EGFP into plastids, only those specific for the DeltapH/TAT pathway were able to direct the protein into the thylakoid lumen as well. This could be demonstrated both by fluorescence and immunoelectron microscopy. Analysis of isolated and fractionated chloroplasts using western blot and spectrofluorometric assays confirmed the presence of folded EGFP solely within the thylakoid lumen of these lines. These results strongly suggest that the protein adopts a folded state in the chloroplast stroma and thus, can only be translocated further into the chloroplast lumen by the DeltapH/TAT pathway.     

金黄滴虫叶绿体拟核的荧光镜检及电镜观察

金黄滴虫细胞在用DNA特异的荧光染料DAPI处理后,在荧光显微镜下细胞核和叶绿体拟核均散发蓝色荧光,穗晰可见。每一叶绿体有一拟核,拟核沿叶绿体的周缘排列,形状相当于叶绿体的轮廓,成不规则的两叶形环。环的全长约在20—30υm之间。 拟核环大多是单线的,有些拟核环出现或短或长的双线部分,有时甚至几乎整个拟核环都可变为双线。这表明拟核环通过“纵裂”而形成双环,在叶绿体分裂时,分别进入两个子叶绿体。这一情况在电镜照片上得到了证实。 叶绿体分裂和细胞分裂之间似乎不存在严格的相关性,这是导致细胞中叶绿体数目多于1个的原因。     

Chloroplast targeting of chloroplast division FtsZ2 proteins in Arabidopsis

Plant nuclear genomes encode chloroplast division proteins homologous to the eubacterial cell division protein FtsZ. In higher plants, FtsZ genes constitute a small gene family that consists of two subgroups, FtsZ1 and FtsZ2. It was previously hypothesized that members of one family (FtsZ1) targeted chloroplasts, while members of the other family (FtsZ2) localized in the cytoplasm. We determined the full-length cDNA sequences of two FtsZ2 genes from Arabidopsis thaliana (AtFtsZ2-1 and AtFtsZ2-2) and found that the genes encode polypeptides of 478 and 473 amino acids, respectively, and both contain N-terminal extensions beyond what have previously been predicted. The N-terminal regions of both AtFtsZ2-1 and AtFtsZ2-2 were expressed as green fluorescent protein (GFP) fusions under the cauliflower mosaic virus 35S promoter in bombarded tobacco cells. Confocal laser scanning microscopy revealed both fusions exclusively localized to chloroplasts, demonstrating that the N-terminal regions function as chloroplast-targeting signals in vivo. Thus, FtsZ2 proteins function within chloroplasts.     

Arabidopsis ACCELERATED CELL DEATH2 modulates programmed cell death

The Arabidopsis thaliana chloroplast protein ACCELERATED CELL DEATH2 (ACD2) modulates the amount of programmed cell death (PCD) triggered by Pseudomonas syringae and protoporphyrin IX (PPIX) treatment. In vitro, ACD2 can reduce red chlorophyll catabolite, a chlorophyll derivative. We find that ACD2 shields root protoplasts that lack chlorophyll from light- and PPIX-induced PCD. Thus, chlorophyll catabolism is not obligatory for ACD2 anti-PCD function. Upon P. syringae infection, ACD2 levels and localization change in cells undergoing PCD and in their close neighbors. Thus, ACD2 shifts from being largely in chloroplasts to partitioning to chloroplasts, mitochondria, and, to a small extent, cytosol. ACD2 protects cells from PCD that requires the early mitochondrial oxidative burst. Later, the chloroplasts of dying cells generate NO, which only slightly affects cell viability. Finally, the mitochondria in dying cells have dramatically altered movements and cellular distribution. Overproduction of both ACD2 (localized to mitochondria and chloroplasts) and ascorbate peroxidase (localized to chloroplasts) greatly reduces P. syringae-induced PCD, suggesting a pro-PCD role for mitochondrial and chloroplast events. During infection, ACD2 may bind to and/or reduce PCD-inducing porphyrin-related molecules in mitochondria and possibly chloroplasts that generate reactive oxygen species, cause altered organelle behavior, and activate a cascade of PCD-inducing events.     

Varying competence for protein import into chloroplasts during the cell cycle in Chlamydomonas.

By studying the import of radioactively labelled small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into chloroplasts of the green alga C. reinhardtii cw-15 protein delivery to chloroplasts was found to vary during the cell cycle. Chloroplasts were isolated from highly synchronous cultures at different time points during the cell cycle. When pSS was imported into 'young' chloroplasts isolated early in the light period about three times less pSS was processed to small subunit SS than in 'mature' chloroplasts from the middle of the light period. In 'young' chloroplasts also, less pSS was bound to the envelope surface. During the second half of the light period the import competence of isolated chloroplasts decreased again when based on chlorophyll content or cell volume, but did not change significantly when related to chloroplast number. Measurements of pSS binding to the surface of chloroplasts of different age indicated that the adaptation of protein import competence during the cell cycle is due to a variation of the number of binding sites per chloroplast surface area, rather than to modulation of the binding constant.     

An Arabidopsis gene encoding a chloroplast-targeted beta-amylase

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch. However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses beta-amylase activity. CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.     

The evolution of the regulatory mechanism of chloroplast division

Chloroplasts arose from a cyanobacterial endosymbiont and multiply by division, reminiscent of their free-living ancestor. However, chloroplasts can not divide by themselves, and the division is performed and controlled by proteins that are encoded by the host nucleus. The continuity of chloroplasts was originally established by synchronization of endosymbiotic cell division with host cell division, as seen in existent algae. In contrast, land plant cells contain multiple chloroplasts, the division of which is not synchronized, even in the same cell. Land plants have evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts (or other types of plastids) change along with their respective cellular function by changes in the division rate. We recently reported that PLASTID DIVISION (PDV) proteins, land-plant specific components of the chloroplast division apparatus, determined the rate of chloroplast division. The level of PDV protein is regulated by the cell differentiation program based on cytokinin, and the increase or decrease of the PDV level gives rise to an increase or decrease in the chloroplast division rate. Thus, the integration of PDV proteins into the chloroplast division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.Key words: chloroplast division, cell cycle, cell differentiation, cytokinin, endosymbiosis, evolution     

Characterization of chloroplast division using the Arabidopsis mutant arc5.

arc5 is a chloroplast division mutant of Arabidopsis thaliana. To identify the role of ARC5 in the chloroplast replication process we have followed the changes in arc5 chloroplasts during their perturbed division. ARC5 does not affect proplastid division but functions at a later stage in chloroplast development. Chloroplasts in developing mesophyll cells of arc5 leaves do not increase in number and all of the chloroplasts in mature leaf cells show a central constriction. Young arc5 chloroplasts are capable of initiating the division process but fail to complete daughter-plastid separation. Wild-type plastids increase in number to a mean of 121 after completing the division process, but in the mutant arc5 the approximately 13 plastids per cell are still centrally constricted but much enlarged. As the arc5 chloroplasts expand and elongate without dividing, the internal thylakoid membrane structure becomes flexed into an undulating ribbon. We conclude that the ARC5 gene is necessary for the completion of the last stage of chloroplast division when the narrow isthmus breaks, causing the separation of the daughter plastids.     

Optical manipulation reveals strong attracting forces at membrane contact sites between endoplasmic reticulum and chloroplasts

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.     

Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis

Chloroplasts move in a light-dependent manner that can modulate the photosynthetic potential of plant cells. Identification of genes required for light-induced chloroplast movement is beginning to define the molecular machinery that controls these movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that displays attenuated chloroplast movements under intermediate and high light intensities while maintaining a normal movement response under low light intensities. In wild-type plants, fluence rates below 20 micromol m(-2) s(-1) of blue light lead to chloroplast accumulation on the periclinal cell walls, whereas light intensities over 20 micromol m(-2) s(-1) caused chloroplasts to move toward the anticlinal cell walls (avoidance response). However, at light intensities below 75 micromol m(-2) s(-1), chloroplasts in pmi2 leaves move to the periclinal walls; 100 micromol m(-2) s(-1) of blue light is required for chloroplasts in pmi2 to move to the anticlinal cell walls, indicating a shift in the light threshold for the avoidance response in the mutant. The pmi2 mutation has been mapped to a gene that encodes a protein of unknown function with a large coiled-coil domain in the N terminus and a putative P loop. PMI2 shares sequence and structural similarity with PMI15, another unknown protein in Arabidopsis that, when mutated, causes a defect in chloroplast avoidance under high-light intensities.     

Molecular design of photosynthesis-elevated chloroplasts for mass accumulation of a foreign protein

In order to increase production of a useful protein by the chloroplast transformation technique, it seems to be necessary to determine the upper limit for the accumulation of a biologically active foreign protein in chloroplasts and then improve photosynthetic capacity and plant productivity. Here we show that the stromal fractions of tobacco chloroplasts could accommodate an additional 200-260 mg ml(-1) of green fluorescent protein in the stroma without any inhibition of gas exchange under various light intensity and growth conditions. The minimum amount of fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) limiting photosynthesis was then calculated. Analyses of the photosynthetic parameters and the metabolites of transformants into which FBP/SBPase was introduced with various types of promoter (PpsbA, Prrn, Prps2 and Prps12) indicated that a 2- to 3-fold increase in levels of FBPase and SBPase activity is sufficient to increase the final amount of dry matter by up to 1.8-fold relative to the wild-type plants. Their increases were equivalent to an increase of <1 mg ml(-1) of the FBP/SBPase protein in chloroplasts and were calculated to represent <1% of the protein accumulated via chloroplast transformation. Consequently, >99% of the additional 200-260 mg ml(-1) of protein expressed in the chloroplasts could be used for the production of useful proteins in the photosynthesis-elevated transplastomic plants having FBP/SBPase.     

Deg proteases and their role in protein quality control and processing in different subcellular compartments of the plant cell

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent serine endopeptidases found in almost every organism. Database searches revealed that 16 Deg paralogues are encoded by the genome of Arabidopsis thaliana, six of which were experimentally shown to be located in chloroplasts, one in peroxisomes, one in mitochondria and one in the nucleus. Two more Deg proteases are predicted to reside in chloroplasts, five in mitochondria (one of them with a dual chloroplastidial/mitochondrial localization) and the subcellular location of one protein is uncertain. This review summarizes the current knowledge on the role of Deg proteases in maintaining protein homeostasis and protein processing in various subcompartments of the plant cell. The chloroplast Deg proteases are the best examined so far, especially with respect to their role in the degradation of photodamaged photosynthetic proteins and in biogenesis of photosystem II (PSII). A combined action of thylakoid lumen and stroma Deg proteases in the primary cleavage of photodamaged D1 protein from PSII reaction centre is discussed on the basis of a recently resolved crystal structure of plant Deg1. The peroxisomal Deg protease is a processing enzyme responsible for the cleavage of N-terminal peroxisomal targeting signals (PTSs). A. thaliana mutants lacking this enzyme show reduced peroxisomal β-oxidation, indicating for the first time the impact of protein processing on peroxisomal functions in plants. Much less data is available for mitochondrial and nuclear Deg proteases. Based on the available expression data we hypothesize a role in general protein quality control and during acquired heat resistance.     

A plastid envelope location of Arabidopsis ent-kaurene oxidase links the plastid and endoplasmic reticulum steps of the gibberellin biosynthesis pathway

We have used fusions of gibberellin biosynthesis enzymes to green fluorescent protein (GFP) to determine the subcellular localization of the early steps of the pathway. Gibberellin biosynthesis from geranylgeranyl diphosphate is catalysed by enzymes of the terpene cyclase, cytochrome P450 mono-oxygenase and 2-oxoglutarate-dependent dioxygenase classes. We show that the N-terminal pre-sequences of the Arabidopsis thaliana terpene cyclases copalyl diphosphate synthase (AtCPS1) and ent-kaurene synthase (AtKS1) direct GFP to chloroplasts in transient assays following microprojectile bombardment of tobacco leaves. The AtKS1-GFP fusion is also imported by isolated pea chloroplasts. The N-terminal portion of the cytochrome P450 protein ent-kaurene oxidase (AtKO1) directs GFP to chloroplasts in tobacco leaf transient assays. Chloroplast import assays with 35S-labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. The leader sequences of the two ent-kaurenoic acid oxidases (AtKAO1 and AtKAO2) from Arabidopsis direct GFP to the endoplasmic reticulum. These data suggest that the AtKO1 protein links the plastid- and endoplasmic reticulum-located steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid.