Deletion of the chloroplast-localized AtTerC gene product in Arabidopsis thaliana leads to loss of the thylakoid membrane and to seedling lethality

Early seedling development in plants depends on the biogenesis of chloroplasts from proplastids, accompanied by the formation of thylakoid membranes. An Arabidopsis thaliana gene, AtTerC , whose gene product shares sequence similarity with bacterial tellurite resistance C (TerC), is shown to be involved in a critical step required for the normal organization of prothylakoids and transition into mature thylakoid stacks. The AtTerC gene encodes an integral membrane protein, which contains eight putative transmembrane helices, localized in the thylakoid of the chloroplast, as shown by localization of an AtTerC–GFP fusion product in protoplasts and by immunoblot analysis of subfractions of chloroplasts. T-DNA insertional mutation of AtTerC resulted in a pigment-deficient and seedling-lethal phenotype under normal light conditions. Transmission electron microscopic analysis revealed that mutant etioplasts had normal prolamellar bodies (PLBs), although the prothylakoids had ring-like shapes surrounding the PLBs. In addition, the ultrastructures of mutant chloroplasts lacked thylakoids, did not have grana stacks, and showed numerous globular structures of varying sizes. Also, the accumulation of thylakoid membrane proteins was severely defective in this mutant. These results suggest that the AtTerC protein plays a crucial role in prothylakoid membrane biogenesis and thylakoid formation in early chloroplast development.     

Dual targeting and function of a protease in mitochondria and chloroplasts

Here we show, using the green fluorescent protein (GFP) fusion system, that an Arabidopsis thaliana zinc-metalloprotease (AtZn-MP) is targeted to both mitochondria and chloroplasts. A deletion mutant lacking the amino-terminal 28 residues, with translation initiation at the second methionine residue, was imported into chloroplasts only. However, a mutated form of the full-length targeting peptide, in which the second methionine residue is changed to leucine, was imported to both organelles. No GFP fluorescence was detected when a frame-shift mutation was introduced between the first and second ATG codons of the Zn-MP–GFP construct, suggesting no alternative translational initiation. Our results show that the dual targeting of the Zn-MP is due to an ambiguous targeting peptide. Furthermore, we show that the recombinant AtZn-MP degrades mitochondrial and chloroplastic targeting peptides, indicating its function as a signal peptide degrading protease in both mitochondria and chloroplasts.     

Subcellular localization and targeting of glucocorticoid receptor protein fusions expressed in transgenic Arabidopsis thaliana

An animal system of inducible activation of protein fusions with the binding domain of glucocorticoid receptor (BDGR) was tested in Arabidopsis thaliana by monitoring dexamethasone (DEX)-induced nuclear targeting of reporter constructs. Two constructs containing green fluorescent protein (GFP), human homeobox protein Hanf-1 and Xenopus laevis BDGR were used, GFP/Hanf-1/BDGR and GFP/BDGR. The control construct contained GFP alone. In the absence of DEX both fusion proteins were uniformly distributed in the cytoplasm of root cells, but showed strong association with plastids in plant aerial parts. DEX treatment of roots prompted a strong and reversible nuclear accumulation of GFP/Hanf-1/BDGR, but not GFP/BDGR. Thus, in roots, the specific nuclear translocation of GFP/Hanf-1/BDGR was driven by Hanf-1 and tightly regulated by BDGR. However, in plant aerial parts treated with DEX, nuclear translocation of GFP/Hanf-1/BDGR was observed only in a few cases, and most part of the fusion protein was incorrectly and irreversibly targeted to plastids. Protease X digestion of isolated chloroplasts showed that BDGR fusion proteins were translocated into the chloroplast envelope and bound to envelope membranes, probably due to association with the chloroplast import apparatus. Thus, for efficient use of the glucocorticoid-inducible system in plants, it will be necessary to modify BDGR structure to prevent incorrect targeting of fusion proteins.     

Chlorophyll a Fluorescence Transients in Mesophyll and Guard Cells : MODULATION OF GUARD CELL PHOTOPHOSPHORYLATION BY CO(2)

Chlorophyll fluorescence transients from mesophyll and guard cell chloroplasts of variegated leaves from Chlorophytum comosum were compared using high resolution fluorescence spectroscopy. Like their mesophyll counterparts, guard cell chloroplasts showed the OPS fluorescence transient indicating the operation of the linear electron transport and the possible generation of NADPH in these organelles. They also showed a slow fluorescence yield decrease, equivalent to the MT transition in mesophyll, suggesting the formation of the high energy state and photophosphorylation. Unlike the mesophyll chloroplasts, the fluorescence from guard cell chloroplasts lacked the increment of the SM transition, indicating that the two types of chloroplasts have some metabolic differences. The presence of CO₂ (supplied as bicarbonate, pH 6.7) specifically inhibited the MT-equivalent transition while its absence accelerated it. These observations constitute the first specific evidence of a guard cell chloroplast response to CO₂. Control of photosynthetic ATP levels in the guard cell cytoplasm by CO₂ may provide a mechanism regulating the availability of high energy equivalents at the guard cell plasmalemma, thus affecting stomatal opening.     

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.     

GFP-mTn融合蛋白对蓝猪耳生活细胞微丝骨架的标记

用农杆菌介导法将嵌合基因GFP-mTn(mTn是微丝结合蛋白Talin的微丝结合域,可以显示活体细胞中微丝的结构)导入蓝猪耳。经激光共聚焦显微镜观察了转基因植株的各种不同组织中融合蛋白的表达和分布情况。在叶片的表皮细胞、保卫细胞、根部的皮层细胞中有融合蛋白的不同程度表达。但仅在保卫细胞中微丝标记状况良好,显示基因表达的组织特异性。经光诱导处于开放态的气孔的保卫细胞微丝呈网状结构,在细胞内无规则分布;经黑暗诱导处于关闭态的气孔保卫细胞中微丝束沿保卫细胞纵轴排列,呈卷曲状分布,并观察到螺旋和环状的微丝结构。在转基因植株的其他部位,例如茎表皮细胞、根毛细胞和花粉粒中,未检测到目的基因的表达。本研究获得的转基因植株为研究气孔运动过程中微丝动态变化提供了有用的材料。     

GFP-mTn融合蛋白对蓝猪耳生活细胞微丝骨架的标记

用农杆菌介导法将嵌合基因GFP-mTn(mTn是微丝结合蛋白Talin的微丝结合域,可以显示活体细胞中微丝的结构)导入蓝猪耳.经激光共聚焦显微镜观察了转基因植株的各种不同组织中融合蛋白的表达和分布情况.在叶片的表皮细胞、保卫细胞、根部的皮层细胞中有融合蛋白的不同程度表达.但仅在保卫细胞中微丝标记状况良好,显示基因表达的组织特异性.经光诱导处于开放态的气孔的保卫细胞微丝呈网状结构,在细胞内无规则分布;经黑暗诱导处于关闭态的气孔保卫细胞中微丝束沿保卫细胞纵轴排列,呈卷曲状分布,并观察到螺旋和环状的微丝结构.在转基因植株的其他部位,例如茎表皮细胞、根毛细胞和花粉粒中,未检测到目的基因的表达.本研究获得的转基因植株为研究气孔运动过程中微丝动态变化提供了有用的材料.     

Chloroplast unusual positioning1 is essential for proper chloroplast positioning

The intracellular distribution of organelles is a crucial aspect of effective cell function. Chloroplasts change their intracellular positions to optimize photosynthetic activity in response to ambient light conditions. Through screening of mutants of Arabidopsis defective in chloroplast photorelocation movement, we isolated six mutant clones in which chloroplasts gathered at the bottom of the cells and did not distribute throughout cells. These mutants, termed chloroplast unusual positioning (chup), were shown to belong to a single genetic locus by complementation tests. Observation of the positioning of other organelles, such as mitochondria, peroxisomes, and nuclei, revealed that chloroplast positioning and movement are impaired specifically in this mutant, although peroxisomes are distributed along with chloroplasts. The CHUP1 gene encodes a novel protein containing multiple domains, including a coiled-coil domain, an actin binding domain, a Pro-rich region, and two Leu zipper domains. The N-terminal hydrophobic segment of CHUP1 was expressed transiently in leaf cells of Arabidopsis as a fusion protein with the green fluorescent protein. The fusion protein was targeted to envelope membranes of chloroplasts in mesophyll cells, suggesting that CHUP1 may localize in chloroplasts. A glutathione S-transferase fusion protein containing the actin binding domain of CHUP1 was found to bind F-actin in vitro. CHUP1 is a unique gene identified that encodes a protein required for organellar positioning and movement in plant cells.     

Role of N-terminal 28-amino-acid region of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lipase in directing proteins to secretory pathway of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL-green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL-GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.     

Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein

The translocation of proteins to cyanobacterial cell envelope is made complex by the presence of a highly differentiated membrane system. To investigate the protein translocation in cyanobacterium Synechococcus PCC 7942 using the truncated ice nucleation protein (InpNC) from Pseudomonas syringae KCTC 1832, the green fluorescent protein (GFP) was fused in frame to the carboxyl-terminus of InpNC. The fluorescence of GFP was found almost entirely as a halo in the outer regions of cells which appeared to correspond to the periplasm as demonstrated by confocal laser scanning microscopy, however, GFP was not displayed on the outermost cell surface. Western blotting analysis revealed that InpNC-GFP fusion protein was partially degraded. The N-terminal domain of InpNC may be susceptible to protease attack; the remaining C-terminal domain conjugated with GFP lost the ability to direct translocation across outer membrane and to act as a surface display motif. The fluorescence intensity of cells with periplasmic GFP was approximately 6-fold lower than that of cells with cytoplasmic GFP. The successful translocation of the active GFP to the periplasm may provide a potential means to study the property of cyanobacterial periplasmic substances in response to environmental changes in a non-invasive manner.     

Control of leaf and chloroplast development by the Arabidopsis gene pale cress.

Leaf plastids of the Arabidopsis pale cress (pac) mutant do not develop beyond the initial stages of differentiation from proplastids or etioplasts and contain only low levels of chlorophylls and carotenoids. Early in development, the epidermis and mesophyll of pac leaves resemble those of wild-type plants. In later stages, mutant leaves have enlarged intercellular spaces, and the palisade layer of the mesophyll can no longer be distinguished. To study the molecular basis of this phenotype, we cloned PAC and determined that this gene is regulated by light and has the capacity to encode an acidic, predominantly alpha-helical protein. The PAC gene appears to be a novel component of a light-induced regulatory network that controls the development of leaves and chloroplasts.     

Visualisation of plastids in endosperm,pollen and roots of transgenic wheat expressing modified GFP fused to transit peptides from wheat SSU RubisCO,rice FtsZ and maize ferredoxin III proteins

The ability to target marker proteins to specific subcellular compartments is a powerful research tool to study the structure and development of organelles. Here transit sequences from nuclear-encoded, plastid proteins, namely rice FtsZ, maize non-photosynthetic ferredoxin III (FdIII) and the small subunit of RubisCO were used to target a modified synthetic GFP (S65G, S72A) to plastids. The localisations of the fusion proteins expressed in transgenic wheat plants and under the control of the rice actin promoter were compared to an untargeted GFP control. GFP fluorescence was localised to non-green plastids in pollen, roots and seed endosperm and detected in isolated leaf chloroplasts using a GFP-specific antibody. Transit peptides appeared to influence the relative fluorescence intensities of plastids in different tissues. This is consistent with differential targeting and/or turnover of GFP fusion proteins in different plastid types. Replacement of GFP sequences with alternative coding regions enables immediate applications of our vectors for academic research and commercial applications.     

Differential coloring reveals that plastids do not form networks for exchanging macromolecules

Stroma-filled tubules named stromules are sporadic extensions of plastids. Earlier, photobleaching was used to demonstrate fluorescent protein diffusion between already interconnected plastids and formed the basis for suggesting that all plastids are able to form networks for exchanging macromolecules. However, a critical appraisal of literature shows that this conjecture is not supported by unequivocal experimental evidence. Here, using photoconvertible mEosFP, we created color differences between similar organelles that enabled us to distinguish clearly between organelle fusion and nonfusion events. Individual plastids, despite conveying a strong impression of interactivity and fusion, maintained well-defined boundaries and did not exchange fluorescent proteins. Moreover, the high pleomorphy of etioplasts from dark-grown seedlings, leucoplasts from roots, and assorted plastids in the accumulation and replication of chloroplasts5 (arc5), arc6, and phosphoglucomutase1 mutants of Arabidopsis thaliana suggested that a single plastid unit might be easily mistaken for interconnected plastids. Our observations provide succinct evidence to refute the long-standing dogma of interplastid connectivity. The ability to create and maintain a large number of unique biochemical factories in the form of singular plastids might be a key feature underlying the versatility of green plants as it provides increased internal diversity for them to combat a wide range of environmental fluctuations and stresses.     

Searching for new cell wall protein genes in Arabidopsis

The objective of this study was to test an approach that combines bioinformatic and subcellular localization analysis to identify novel cell wall protein genes in Arabidopsis. Proteins with unknown function in the Arabidopsis genome were first identified and scanned for the presence of N-terminal signal peptides. The signal peptide-containing function-unknown proteins were further analyzed to eliminate the ones containing other sequences, such as endoplasmic reticulum and vacuole retention signals, that may prevent a protein from secretion into cell walls. The top ten genes passing the bioinformatic analysis were selected for protein subcellular localization using green fluorescence protein (GFP) as a reporter. A vector was constructed for high throughput gene-GFP fusion protein generation and overexpression in Arabidopsis for gene function analysis. Transformants of six genes showed reasonable expression of GFP fusion protein. However, none of the transformants showed GFP localization in cell walls. The low rate of new cell wall protein discovery suggests that the number of unidentified cell wall proteins in the Arabidopsis genome may be small.     

Identification of a signal that distinguishes between the chloroplast outer envelope membrane and the endomembrane system in vivo

Certain small outer envelope membrane proteins of chloroplasts are encoded by the nuclear genome without a cleavable N-terminal transit peptide. We investigated in vivo the targeting mechanism of AtOEP7, an Arabidopsis homolog of the small outer envelope membrane protein. AtOEP7 was expressed as a fusion protein with the green fluorescent protein (GFP) either transiently in protoplasts or stably in transgenic plants. In either case, fluorescence microscopy of transformed cells and protein gel blot analysis of fractionated proteins confirmed that the AtOEP7:GFP fusion protein was targeted to the chloroplast outer envelope membrane. In vivo targeting experiments revealed that two regions, the transmembrane domain (TMD) and its C-terminal neighboring seven-amino acid region, were necessary and sufficient for targeting to the chloroplast outer membrane. Substitution of aspartic acid or lysine residues with glycine residues or scrambling of the amino acid sequence of the seven-amino acid region caused mistargeting to the plasma membrane. Although the amino acid sequence of the TMD is not important for targeting, amino acid residues with large side chains inhibited targeting to the chloroplasts and resulted in the formation of large aggregates in the protoplasts. In addition, introduction of a proline residue within the TMD resulted in inhibition of targeting. Finally, a fusion protein, AtOEP7:NLS:GFP, was targeted efficiently to the chloroplast envelope membranes despite the presence of a nuclear localization signal. On the basis of these results, we conclude that the seven-amino acid region and the TMD are determinants for targeting to the chloroplast outer envelope membrane. The seven-amino acid region plays a critical role in AtOEP7 evading the endomembrane system and entering the chloroplast pathway, and the TMD plays critical roles in migration to the chloroplasts and/or subsequent insertion into the membrane.     

A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells

The actin cytoskeleton coordinates numerous cellular processes required for plant development. The functions of this network are intricately linked to its dynamic arrangement, and thus progress in understanding how actin orchestrates cellular processes relies on critical evaluation of actin organization and turnover. To investigate the dynamic nature of the actin cytoskeleton, we used a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. The GFP-fABD2 fusion protein labeled highly dynamic and dense actin networks in diverse species and cell types, revealing structural detail not seen with alternative labeling methods, such as the commonly used mouse talin GFP fusion (GFP-mTalin). Further, we show that expression of the GFP-fABD2 fusion protein in Arabidopsis, unlike GFP-mTalin, has no detectable adverse effects on plant morphology or development. Time-lapse confocal microscopy and fluorescence recovery after photobleaching analyses of the actin cytoskeleton labeled with GFP-fABD2 revealed that lateral-filament migration and sliding of individual actin filaments or bundles are processes that contribute to the dynamic and continually reorganizing nature of the actin scaffold. These new observations of the dynamic actin cytoskeleton in plant cells using GFP-fABD2 reveal the value of this probe for future investigations of how actin filaments coordinate cellular processes required for plant development.     

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.     

Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment

The green fluorescent protein gene ( gfp ) is a widely used reporter in both animals and plants. Fusions between the plastid rrn promoter or the Escherichia coli trc promoter and the gfp coding region have been delivered to chloroplasts using gold or tungsten microprojectiles, and fluorescence from GFP was visible in individual tobacco chloroplasts and in the abnormally large chloroplasts of the arc 6 mutant of Arabidopsis thaliana 2–4 days after bombardment. The fusion of the gfp coding region to the bacterial trc promoter demonstrated that a bacterial promoter is active in chloroplasts in vivo . GFP was also detectable in amyloplasts of potato tubers and in chromoplasts of marigold petals, carrot roots and pepper fruits 4 days after bombardment. This demonstrates that GFP can be used as a reporter for transient gene expression in chloroplasts and in non-photosynthetic plastids in a range of higher plants.     

Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure

The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca(2+) ([Ca(2+)](ext))-induced [Ca(2+)](cyt) transients and stomatal closure in Arabidopsis. CAS (Ca(2+) sensing receptor) is a plant-specific putative Ca(2+)-binding protein that was originally proposed to be a plasma membrane-localized external Ca(2+) sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca(2+)-binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca(2+). In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca(2+). Furthermore, using the transgenic aequorin system, we showed that [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients and stomatal closure.