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10 pages, 2200 KiB

Open AccessCommunication

Improvements for Tissue-Chopping-Based Immunofluorescence Staining Method of Chloroplast Proteins

by Lulu Wang, Yajuan Chen, Di Niu, Mingdong Tang, Jinjie An, Shanshan Xue, Xiaomin Liu and Hongbo Gao

Plants 2023, 12(4), 841; https://doi.org/10.3390/plants12040841 - 13 Feb 2023

Cited by 1 | Viewed by 1967

Abstract

Immunofluorescence staining is a very common method for the subcellular localization study of proteins. A tissue-chopping-based immunofluorescence staining method for chloroplast proteins overcomes the restriction of plant cell wall, makes the operation simpler, and uses less experimental materials. Here we provide some improvements [...] Read more.

Immunofluorescence staining is a very common method for the subcellular localization study of proteins. A tissue-chopping-based immunofluorescence staining method for chloroplast proteins overcomes the restriction of plant cell wall, makes the operation simpler, and uses less experimental materials. Here we provide some improvements for this method. We found that the stained tissues can be directly observed with a confocal microscope without tissue lysis. Samples maintained at a low temperature (0–4 °C) throughout the process can reduce the intensity of chlorophyll autofluorescence and the background signal. A low temperature is also good for the storage of the sample. Fluorescence signal of the stained samples can be kept for several weeks if they are stored at −20 °C. FtsZ is an essential component of the chloroplast division apparatus. We demonstrated this method with the immunofluorescence staining of FtsZ1 in wildtype Arabidopsis and some chloroplast division mutants. We also successfully tested this method by the immunofluorescence staining of FtsZ1 in many other plants, including woody plants. With these procedures, the performance of tissue-chopping-based immunofluorescence staining method are further improved. Full article

(This article belongs to the Special Issue Molecular Biology of Chloroplast: Structure, Function and Development)

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23 pages, 4474 KiB

Open AccessArticle

FKF1 Interacts with CHUP1 and Regulates Chloroplast Movement in Arabidopsis

by Ning Yuan, Lavanya Mendu, Kaushik Ghose, Carlie Shea Witte, Julia Frugoli and Venugopal Mendu

Plants 2023, 12(3), 542; https://doi.org/10.3390/plants12030542 - 25 Jan 2023

Cited by 2 | Viewed by 2258

Abstract

Plants have mechanisms to relocate chloroplasts based on light intensities in order to maximize photosynthesis and reduce photodamage. Under low light, chloroplasts move to the periclinal walls to increase photosynthesis (accumulation) and move to the anticlinal walls under high light to avoid photodamage, [...] Read more.

Plants have mechanisms to relocate chloroplasts based on light intensities in order to maximize photosynthesis and reduce photodamage. Under low light, chloroplasts move to the periclinal walls to increase photosynthesis (accumulation) and move to the anticlinal walls under high light to avoid photodamage, and even cell death (avoidance). Arabidopsis blue light receptors phot1 and phot2 (phototropins) have been reported to regulate chloroplast movement. This study discovered that another blue light receptor, FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), regulates chloroplast photorelocation by physically interacting with chloroplast unusual positioning protein 1 (CHUP1), a critical component of the chloroplast motility system. Leaf cross-sectioning and red-light transmittance results showed that overexpression of FKF1 compromised the avoidance response, while the absence of FKF1 enhanced chloroplast movements under high light. Western blot analysis showed that CHUP1 protein abundance is altered in FKF1 mutants and overexpression lines, indicating a potential regulation of CHUP1 by FKF1. qPCR results showed that two photorelocation pathway genes, JAC1 and THRUMIN1, were upregulated in FKF1-OE lines, and overexpression of FKF1 in the THRUMIN1 mutant weakened its accumulation and avoidance responses, indicating that JAC1 and THRUMIN1 may play a role in the FKF1-mediated chloroplast avoidance response. However, the precise functional roles of JAC1 and THRUMIN1 in this process are not known. Full article

(This article belongs to the Special Issue Molecular Biology of Chloroplast: Structure, Function and Development)

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21 pages, 5176 KiB

Open AccessArticle

Chloroplast Envelopes Play a Role in the Formation of Autophagy-Related Structures in Plants

by Makoto Yanagisawa and Simon D. X. Chuong

Plants 2023, 12(3), 443; https://doi.org/10.3390/plants12030443 - 18 Jan 2023

Viewed by 1695

Abstract

Autophagy is a degradation process of cytoplasmic components that is conserved in eukaryotes. One of the hallmark features of autophagy is the formation of double-membrane structures known as autophagosomes, which enclose cytoplasmic content destined for degradation. Although the membrane source for the formation [...] Read more.

Autophagy is a degradation process of cytoplasmic components that is conserved in eukaryotes. One of the hallmark features of autophagy is the formation of double-membrane structures known as autophagosomes, which enclose cytoplasmic content destined for degradation. Although the membrane source for the formation of autophagosomes remains to be determined, recent studies indicate the involvement of various organelles in autophagosome biogenesis. In this study, we examined the autophagy process in Bienertia sinuspersici: one of four terrestrial plants capable of performing C₄ photosynthesis in a single cell (single-cell C₄ species). We demonstrated that narrow tubules (stromule-like structures) 30–50 nm in diameter appear to extend from chloroplasts to form the membrane-bound structures (autophagosomes or autophagy-related structures) in chlorenchyma cells of B. sinuspersici during senescence and under oxidative stress. Immunoelectron microscopic analysis revealed the localization of stromal proteins to the stromule-like structures, sequestering portions of the cytoplasm in chlorenchyma cells of oxidative stress-treated leaves of B. sinuspersici and Arabidopsis thaliana. Moreover, the fluorescent marker for autophagosomes GFP-ATG8, colocalized with the autophagic vacuole maker neutral red in punctate structures in close proximity to the chloroplasts of cells under oxidative stress conditions. Together our results implicate a role for chloroplast envelopes in the autophagy process induced during senescence or under certain stress conditions in plants. Full article

(This article belongs to the Special Issue Molecular Biology of Chloroplast: Structure, Function and Development)

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12 pages, 10823 KiB

Open AccessArticle

Variation in Chloroplast Genome Size: Biological Phenomena and Technological Artifacts

by Ante Turudić, Zlatko Liber, Martina Grdiša, Jernej Jakše, Filip Varga and Zlatko Šatović

Plants 2023, 12(2), 254; https://doi.org/10.3390/plants12020254 - 5 Jan 2023

Cited by 1 | Viewed by 1766

Abstract

The development of bioinformatic solutions is guided by biological knowledge of the subject. In some cases, we use unambiguous biological models, while in others we rely on assumptions. A commonly used assumption for genomes is that related species have similar genome sequences. This [...] Read more.

The development of bioinformatic solutions is guided by biological knowledge of the subject. In some cases, we use unambiguous biological models, while in others we rely on assumptions. A commonly used assumption for genomes is that related species have similar genome sequences. This is even more obvious in the case of chloroplast genomes due to their slow evolution. We investigated whether the lengths of complete chloroplast sequences are closely related to the taxonomic proximity of the species. The study was performed using all available RefSeq sequences from the asterid and rosid clades. In general, chloroplast length distributions are narrow at both the family and genus levels. In addition, clear biological explanations have already been reported for families and genera that exhibit particularly wide distributions. The main factors responsible for the length variations are parasitic life forms, IR loss, IR expansions and contractions, and polyphyly. However, the presence of outliers in the distribution at the genus level is a strong indication of possible inaccuracies in sequence assembly. Full article

(This article belongs to the Special Issue Molecular Biology of Chloroplast: Structure, Function and Development)

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22 pages, 5245 KiB

Open AccessArticle

Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (Solanum lycopersicum)

by Qi Wang, Jiang Yue, Chaozhong Zhang and Jianmin Yan

Plants 2022, 11(21), 2923; https://doi.org/10.3390/plants11212923 - 30 Oct 2022

Cited by 1 | Viewed by 2124

Abstract

The post-translational import of nuclear-encoded chloroplast preproteins is critical for chloroplast biogenesis, and the Toc159 family of proteins is the receptor for this process. Our previous work identified and analyzed the Toc GTPase in tomato; however, the tomato-specific transport substrate for Toc159 is [...] Read more.

The post-translational import of nuclear-encoded chloroplast preproteins is critical for chloroplast biogenesis, and the Toc159 family of proteins is the receptor for this process. Our previous work identified and analyzed the Toc GTPase in tomato; however, the tomato-specific transport substrate for Toc159 is still unknown, which limits the study of the function of the TOC receptor in tomato. In this study, we expand the number of preprotein substrates of slToc159 receptor family members using slToc159-1 and slToc159-2 as bait via a split-ubiquitin yeast two-hybrid membrane system. Forty-one specific substrates were identified in tomato for the first time. Using slToc159-1GM and slToc159-2GM as bait, we compared the affinity of the two bait proteins, with and without the A domain, to the precursor protein, which suggested that the A domain endowed the proproteins with subclass specificity. The presence of the A domain enhanced the interaction intensity of slToc159-1 with the photosynthetic preprotein but decreased the interaction intensity of slToc159-2 with the photosynthetic preprotein. Similarly, the presence of the A domain also altered the affinity of slToc159 to non-photosynthetic preproteins. Bimolecular fluorescence complementation (BiFC) analysis showed that A domain had the ability to recognize the preprotein, and the interaction occurred in the chloroplast. Further, the localization of the A domain in Arabidopsis protoplasts showed that the A domain did not contain chloroplast membrane targeting signals. Our data demonstrate the importance of a highly non-conserved A domain, which endows the slToc159 receptor with specificity for different protein types. However, the domain containing the information on targeting the chloroplast needs further study. Full article

(This article belongs to the Special Issue Molecular Biology of Chloroplast: Structure, Function and Development)

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15 pages, 2425 KiB

Open AccessArticle

A Transcriptomic Analysis of Tobacco Leaf with the Functional Loss of the Plastid rpoB Operon Caused by TALEN-Mediated Double-Strand Breakage

by Yu-Chang Liu, Chih-Hao Huang and Ching-Chun Chang

Plants 2022, 11(21), 2860; https://doi.org/10.3390/plants11212860 - 26 Oct 2022

Cited by 5 | Viewed by 1252

Abstract

At least two sets of RNA polymerase (RNAP), nucleus (NEP)- and plastid (PEP)-encoded polymerases, recognizing distinct promoters exist in the plastids of land plants. Most plastid genes are regulated by multiple promoters with different strengths in their response to developmental stages and environmental [...] Read more.

At least two sets of RNA polymerase (RNAP), nucleus (NEP)- and plastid (PEP)-encoded polymerases, recognizing distinct promoters exist in the plastids of land plants. Most plastid genes are regulated by multiple promoters with different strengths in their response to developmental stages and environmental cues. Recently, we applied chloroplast-targeted transcription activator-like effector nuclease (cpTALEN) technology to site-specifically cause double-strand DNA breaks in the rpoB gene of tobacco, which encodes the β-subunit of PEP. The repair of damaged chloroplast DNA (cpDNA) through microhomology-mediated recombination caused the functional loss of the rpoB operon and resulted in the heterotrophic growth of an albino plant. We conducted a genome-wide analysis of the steady state of gene expression in the leaf tissue of PEP-deficient tobacco by RNA-Seq and compared it with that of wild-type plants. The expression of NEP genes was up-regulated in PEP-deficient tobacco; in particular, the level of RpoT3 transcripts encoding the specifically plastid-targeted NEP was significantly increased. Alongside most housekeeping genes, NEP also plays an important role in the regulation of gene expression involved in photosynthesis. In contrast, alongside the photosynthesis-related genes, PEP also plays an important role in the regulation of gene expression involved in some housekeeping functions. Furthermore, the mitochondrial DNA copy number and the level of most mitochondrial protein-coding transcripts were slightly increased in PEP-deficient tobacco. The disruption of PEP function not only affected plastid gene expression, but also nuclear and mitochondrial gene expression. This study demonstrated the intercompartmental retrograde signaling in the regulation of gene expression. Full article

(This article belongs to the Special Issue Molecular Biology of Chloroplast: Structure, Function and Development)

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28 pages, 9869 KiB

Open AccessArticle

Cold Resistance of Euonymus japonicus Beihaidao Leaves and Its Chloroplast Genome Structure and Comparison with Celastraceae Species

by Hongyu Cai, Xiaozheng Gu, Yongtan Li, Yachao Ren, Shufang Yan and Minsheng Yang

Plants 2022, 11(19), 2449; https://doi.org/10.3390/plants11192449 - 20 Sep 2022

Cited by 1 | Viewed by 1548

Abstract

Euonymus japonicus Beihaidao is one of the most economically important ornamental species of the Euonymus genus. There are approximately 97 genera and 1194 species of plants worldwide in this family (Celastraceae). Using E. japonicus Beihaidao, we conducted a preliminary study of the cold [...] Read more.

Euonymus japonicus Beihaidao is one of the most economically important ornamental species of the Euonymus genus. There are approximately 97 genera and 1194 species of plants worldwide in this family (Celastraceae). Using E. japonicus Beihaidao, we conducted a preliminary study of the cold resistance of this species, evaluated its performance during winter, assembled and annotated its chloroplast genome, and performed a series of analyses to investigate its gene structure GC content, sequence alignment, and nucleic acid diversity. Our objectives were to understand the evolutionary relationships of the genus and to identify positive selection genes that may be related to adaptations to environmental change. The results indicated that E. japonicus Beihaidao leaves have certain cold resistance and can maintain their viability during wintering. Moreover, the chloroplast genome of E. japonicus Beihaidao is a typical double-linked ring tetrad structure, which is similar to that of the other four Euonymus species, E. hamiltonianus, E. phellomanus, E. schensianus, and E. szechuanensis, in terms of gene structure, gene species, gene number, and GC content. Compared to other Celastraceae species, the variation in the chloroplast genome sequence was lower, and the gene structure was more stable. The phylogenetic relationships of 37 species inferred that members of the Euonymus genus do not form a clade and that E. japonicus Beihaidao is closely related to E. japonicus and E. fortunei. A total of 11 functional positive selected genes were identified, which may have played an important role in the process of Celastraceae species adapting to environmental changes. Our study provides important genetic information to support further investigations into the phylogenetic development and adaptive evolution of Celastraceae species. Full article

(This article belongs to the Special Issue Molecular Biology of Chloroplast: Structure, Function and Development)

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19 pages, 5494 KiB

Open AccessArticle

Dissecting the Chloroplast Proteome of the Potato (Solanum Tuberosum L.) and Its Comparison with the Tuber Amyloplast Proteome

by Shengxuan Liu, Tengfei Liu, Enshuang Wang, Yunxia Cheng, Tiantian Liu, Guogang Chen, Minrui Guo and Botao Song

Plants 2022, 11(15), 1915; https://doi.org/10.3390/plants11151915 - 24 Jul 2022

Cited by 4 | Viewed by 2011

Abstract

The chloroplast, the energy organelle unique to plants and green algae, performs many functions, including photosynthesis and biosynthesis of metabolites. However, as the most critical tuber crop worldwide, the chloroplast proteome of potato (Solanum tuberosum) has not been explored. Here, we [...] Read more.

The chloroplast, the energy organelle unique to plants and green algae, performs many functions, including photosynthesis and biosynthesis of metabolites. However, as the most critical tuber crop worldwide, the chloroplast proteome of potato (Solanum tuberosum) has not been explored. Here, we use Percoll density gradient centrifugation to isolate intact chloroplasts from leaves of potato cultivar E3 and establish a reference proteome map of potato chloroplast by bottom-up proteomics. A total of 1834 non-redundant proteins were identified in the chloroplast proteome, including 51 proteins encoded by the chloroplast genome. Extensive sequence-based localization prediction revealed over 62% of proteins to be chloroplast resident by at least one algorithm. Sixteen proteins were selected to evaluate the prediction result by transient fluorescence assay, which confirmed that 14 were distributed in distinct internal compartments of the chloroplast. In addition, we identified 136 phosphorylation sites in 61 proteins encoded by chloroplast proteome. Furthermore, we reconstruct the snapshots along starch metabolic pathways in the two different types of plastids by a comparative analysis between chloroplast and previously reported amyloplast proteomes. Altogether, our results establish a comprehensive proteome map with post-translationally modified sites of potato chloroplast, which would provide the theoretical principle for the research of the photosynthesis pathway and starch metabolism. Full article

(This article belongs to the Special Issue Molecular Biology of Chloroplast: Structure, Function and Development)

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