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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Plant Sciences".

Deadline for manuscript submissions: closed (30 December 2021) | Viewed by 28589

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Prof. Dr. Bartolome Sabater

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Department of Life Sciences (Ciencias de la Vida), University of Alcalá, Alcalá de Henares, 28805 Madrid, Spain
Interests: genetic machinery of chloroplasts; molecular mechanisms of plant senescence; molecular mechanisms of stress responses; organism entropy; regulation of photosynthetic electron transport
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

This Special Issue is the continuation of our previous Special Issue, “Chloroplast 3.0”.

Chloroplasts are at the front line of many advances in molecular biology, ranging from evolutionary biology to the mechanism of energy transduction, and also including stress responses and programmed leaf death. In addition to the relevance of basic knowledge, advances are unveiling promising insights to improve plant productivity, disease resistance, and environmental control. The production of secondary metabolites and proteins by transformed chloroplasts adds further excitement to applied investigations of chloroplasts.

The new issue focused on chloroplasts is intended to continue with the successful quantity and quality of papers in the previous issue. Papers submitted to the second Special Issue must report high novelty results and/or plausible and testable new models. The molecular basis of the conversion of light to chemical energy in photosystems is a reference topic. In addition, the structure and function of other components of photosynthesis machinery, plastid genomics, plastid biogenesis and senescence, metabolism, reactive oxygen species, and membrane transport are also of interest, with a special emphasis on the gene level and evolutionary comparisons. Significant advances in chloroplast transformation are also welcome.

Prof. Dr. Bartolome Sabater
Guest Editor

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Keywords

electron transport
endosymbiosis
photosynthesis
photosystems
plants
plastid DNA
reactive oxygen species (ROS)
thylakoid

Published Papers (11 papers)

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Research

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16 pages, 2237 KiB

Open AccessArticle

Three-Dimensional Envelope and Subunit Interactions of the Plastid-Encoded RNA Polymerase from Sinapis alba

by Rémi Ruedas, Soumiya Sankari Muthukumar, Sylvie Kieffer-Jaquinod, François-Xavier Gillet, Daphna Fenel, Grégory Effantin, Thomas Pfannschmidt, Yohann Couté, Robert Blanvillain and David Cobessi

Int. J. Mol. Sci. 2022, 23(17), 9922; https://doi.org/10.3390/ijms23179922 - 31 Aug 2022

Cited by 4 | Viewed by 2766

Abstract

RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of the plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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14 pages, 31784 KiB

Open AccessArticle

PAP8/pTAC6 Is Part of a Nuclear Protein Complex and Displays RNA Recognition Motifs of Viral Origin

by Louise Chambon, François-Xavier Gillet, Maha Chieb, David Cobessi, Thomas Pfannschmidt and Robert Blanvillain

Int. J. Mol. Sci. 2022, 23(6), 3059; https://doi.org/10.3390/ijms23063059 - 11 Mar 2022

Cited by 7 | Viewed by 2580

Abstract

Chloroplast biogenesis depends on a complex transcriptional program involving coordinated expression of plastid and nuclear genes. In particular, photosynthesis-associated plastid genes are expressed by the plastid-encoded polymerase (PEP) that undergoes a structural rearrangement during chloroplast formation. The prokaryotic-type core enzyme is rebuilt into a larger complex by the addition of nuclear-encoded PEP-associated proteins (PAP1 to PAP12). Among the PAPs, some have been detected in the nucleus (PAP5 and PAP8), where they could serve a nuclear function required for efficient chloroplast biogenesis. Here, we detected PAP8 in a large nuclear subcomplex that may include other subunits of the plastid-encoded RNA polymerase. We have made use of PAP8 recombinant proteins in Arabidopsis thaliana to decouple its nucleus- and chloroplast-associated functions and found hypomorphic mutants pointing at essential amino acids. While the origin of the PAP8 gene remained elusive, we have found in its sequence a micro-homologous domain located within a large structural homology with a rhinoviral RNA-dependent RNA polymerase, highlighting potential RNA recognition motifs in PAP8. PAP8 in vitro RNA binding activity suggests that this domain is functional. Hence, we propose that the acquisition of PAPs may have occurred during evolution by different routes, including lateral gene transfer. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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18 pages, 2428 KiB

Open AccessArticle

Reduction in Phosphoribulokinase Amount and Re-Routing Metabolism in Chlamydomonas reinhardtii CP12 Mutants

by Cassy Gérard, Régine Lebrun, Erwan Lemesle, Luisana Avilan, Kwang Suk Chang, EonSeon Jin, Frédéric Carrière, Brigitte Gontero and Hélène Launay

Int. J. Mol. Sci. 2022, 23(5), 2710; https://doi.org/10.3390/ijms23052710 - 28 Feb 2022

Cited by 6 | Viewed by 2334

Abstract

The chloroplast protein CP12 is involved in the dark/light regulation of the Calvin–Benson–Bassham cycle, in particular, in the dark inhibition of two enzymes: glyceraldehyde−3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), but other functions related to stress have been proposed. We knocked out the unique CP12 gene to prevent its expression in Chlamydomonas reinhardtii (ΔCP12). The growth rates of both wild-type and ΔCP12 cells were nearly identical, as was the GAPDH protein abundance and activity in both cell lines. On the contrary, the abundance of PRK and its specific activity were significantly reduced in ΔCP12, as revealed by relative quantitative proteomics. Isolated PRK lost irreversibly its activity over-time in vitro, which was prevented in the presence of recombinant CP12 in a redox-independent manner. We have identified amino acid residues in the CP12 protein that are required for this new function preserving PRK activity. Numerous proteins involved in redox homeostasis and stress responses were more abundant and the expressions of various metabolic pathways were also increased or decreased in the absence of CP12. These results highlight CP12 as a moonlighting protein with additional functions beyond its well-known regulatory role in carbon metabolism. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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15 pages, 14975 KiB

Open AccessArticle

Mitochondrial and Plastid Genomes of the Monoraphid Diatom Schizostauron trachyderma

by Ewa Górecka, Romain Gastineau, Nikolai A. Davidovich, Olga I. Davidovich, Matt P. Ashworth, Jamal S. M. Sabir, Claude Lemieux, Monique Turmel and Andrzej Witkowski

Int. J. Mol. Sci. 2021, 22(20), 11139; https://doi.org/10.3390/ijms222011139 - 15 Oct 2021

Cited by 6 | Viewed by 1912

Abstract

We provide for the first time the complete plastid and mitochondrial genomes of a monoraphid diatom: Schizostauron trachyderma. The mitogenome is 41,957 bp in size and displays two group II introns in the cox1 gene. The 187,029 bp plastid genome features the typical quadripartite architecture of diatom genomes. It contains a group II intron in the petB gene that overlaps the large single-copy and the inverted repeat region. There is also a group IB4 intron encoding a putative LAGLIDADG homing endonuclease in the rnl gene. The multigene phylogenies conducted provide more evidence of the proximity between S. trachyderma and fistula-bearing species of biraphid diatoms. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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13 pages, 4093 KiB

Open AccessArticle

Ulva compressa from Copper-Polluted Sites Exhibits Intracellular Copper Accumulation, Increased Expression of Metallothioneins and Copper-Containing Nanoparticles in Chloroplasts

by Daniela Espinoza, Alberto González, Jaime Pizarro, Rodrigo Segura, Daniel Laporte, Fernanda Rodríguez-Rojas, Claudio A. Sáez and Alejandra Moenne

Int. J. Mol. Sci. 2021, 22(19), 10531; https://doi.org/10.3390/ijms221910531 - 29 Sep 2021

Cited by 5 | Viewed by 1475

Abstract

In order to analyze the mechanisms involved in copper accumulation in Ulva compressa, algae were collected at control sites of central and northern Chile, and at two copper-polluted sites of northern Chile. The level of intracellular copper, reduced glutathione (GSH), phytochelatins (PCs), PC2 and PC4, and transcripts encoding metallothioneins (MTs) of U. compressa, UcMT1, UcMT2 and UcMT3, were determined. Algae of control sites contained around 20

μ

g of copper g⁻¹ of dry tissue (DT) whereas algae of copper-polluted sites contained 260 and 272

μ

g of copper g⁻¹ of DT. Algae of control sites and copper-polluted sites did not show detectable amounts of GSH, the level of PC2 did not change among sites whereas PC4 was increased in one of the copper-polluted sites. The level of transcripts of UcMT1 and UcMT2 were increased in algae of copper-polluted sites, but the level of UcMT3 did not change. Algae of a control site and a copper-polluted site were visualized by transmission electron microscopy (TEM) and the existence of copper in electrodense particles was analyzed using energy dispersive x-ray spectroscopy (EDXS). Algae of copper-polluted sites showed electrodense nanoparticles containing copper in the chloroplasts, whereas algae of control sites did not. Algae of a control site, Cachagua, were cultivated without copper (control) and with 10

μ

M copper for 5 days and they were analyzed by TEM-EDXS. Algae cultivated with copper showed copper-containing nanoparticles in the chloroplast whereas control algae did not. Thus, U. compressa from copper-polluted sites exhibits intracellular copper accumulation, an increase in the level of PC4 and expression of UcMTs, and the accumulation of copper-containing particles in chloroplasts. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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13 pages, 2768 KiB

Open AccessArticle

Extreme Enlargement of the Inverted Repeat Region in the Plastid Genomes of Diatoms from the Genus Climaconeis

by Romain Gastineau, Nikolaï A. Davidovich, Olga I. Davidovich, Claude Lemieux, Monique Turmel, Rafał J. Wróbel and Andrzej Witkowski

Int. J. Mol. Sci. 2021, 22(13), 7155; https://doi.org/10.3390/ijms22137155 - 02 Jul 2021

Cited by 8 | Viewed by 2256

Abstract

We sequenced the plastid genomes of three diatoms from the genus Climaconeis, including two strains formerly designated as Climaconeis scalaris. At 208,097 and 216,580 bp, the plastid genomes of the latter strains are the largest ever sequenced among diatoms and their increased size is explained by the massive expansion of the inverted repeat region. Important rearrangements of gene order were identified among the two populations of Climaconeis cf. scalaris. The other sequenced Climaconeis chloroplast genome is 1.5 times smaller compared with those of the Climaconeis cf. scalaris strains and it features an usual quadripartite structure. The extensive structural changes reported here for the genus Climaconeis are compared with those previously observed for other algae and plants displaying large plastid genomes. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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23 pages, 4866 KiB

Open AccessArticle

Using Diatom and Apicomplexan Models to Study the Heme Pathway of Chromera velia

by Jitka Richtová, Lilach Sheiner, Ansgar Gruber, Shun-Min Yang, Luděk Kořený, Boris Striepen and Miroslav Oborník

Int. J. Mol. Sci. 2021, 22(12), 6495; https://doi.org/10.3390/ijms22126495 - 17 Jun 2021

Cited by 4 | Viewed by 3117

Abstract

Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway’s enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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11 pages, 3790 KiB

Open AccessArticle

Starch Granules in Arabidopsis thaliana Mesophyll and Guard Cells Show Similar Morphology but Differences in Size and Number

by Qingting Liu, Xiaoping Li and Joerg Fettke

Int. J. Mol. Sci. 2021, 22(11), 5666; https://doi.org/10.3390/ijms22115666 - 26 May 2021

Cited by 6 | Viewed by 3152

Abstract

Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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15 pages, 4322 KiB

Open AccessArticle

Fine Mapping and Identification of BnaC06.FtsH1, a Lethal Gene That Regulates the PSII Repair Cycle in Brassica napus

by Kai Xu, Yujin Wu, Jurong Song, Kaining Hu, Zengxiang Wu, Jing Wen, Bin Yi, Chaozhi Ma, Jinxiong Shen, Tingdong Fu and Jinxing Tu

Int. J. Mol. Sci. 2021, 22(4), 2087; https://doi.org/10.3390/ijms22042087 - 19 Feb 2021

Cited by 5 | Viewed by 2089

Abstract

Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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Review

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13 pages, 1323 KiB

Open AccessReview

On the Edge of Dispensability, the Chloroplast ndh Genes

by Bartolomé Sabater

Int. J. Mol. Sci. 2021, 22(22), 12505; https://doi.org/10.3390/ijms222212505 - 19 Nov 2021

Cited by 13 | Viewed by 2296

Abstract

The polypeptides encoded by the chloroplast ndh genes and some nuclear genes form the thylakoid NADH dehydrogenase (Ndh) complex, homologous to the mitochondrial complex I. Except for Charophyceae (algae related to higher plants) and a few Prasinophyceae, all eukaryotic algae lack ndh genes. Among vascular plants, the ndh genes are absent in epiphytic and in some species scattered among different genera, families, and orders. The recent identification of many plants lacking plastid ndh genes allows comparison on phylogenetic trees and functional investigations of the ndh genes. The ndh genes protect Angiosperms under various terrestrial stresses, maintaining efficient photosynthesis. On the edge of dispensability, ndh genes provide a test for the natural selection of photosynthesis-related genes in evolution. Variable evolutionary environments place Angiosperms without ndh genes at risk of extinction and, probably, most extant ones may have lost ndh genes recently. Therefore, they are evolutionary endpoints in phylogenetic trees. The low number of sequenced plastid DNA and the long lifespan of some Gymnosperms lacking ndh genes challenge models about the role of ndh genes protecting against stress and promoting leaf senescence. Additional DNA sequencing in Gymnosperms and investigations into the molecular mechanisms of their response to stress will provide a unified model of the evolutionary and functional consequences of the lack of ndh genes. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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13 pages, 1331 KiB

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The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants

by Marco Larrea-Álvarez and Saul Purton

Int. J. Mol. Sci. 2021, 22(16), 8806; https://doi.org/10.3390/ijms22168806 - 16 Aug 2021

Cited by 3 | Viewed by 2982

Abstract

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N₂) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N₂ by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme’s sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle’s genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an ‘Fe-only’ nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops. Full article

(This article belongs to the Special Issue Chloroplast 3.0)

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