Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis and chloroplast to nucleus retrograde signaling in Chlamydomonas, which has never been studied before.

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Identification and molecular characterization of a novel Chlamydomonas reinhardtii mutant defective in chlorophyll biosynthesis

F1000Research ◽

10.12688/f1000research.2-138.v2 ◽

2013 ◽

Vol 2 ◽

pp. 138

Author(s):

Phillip B Grovenstein ◽

Darryel A Wilson ◽

Cameron G Lennox ◽

Katherine P Smith ◽

Alisha A Contractor ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Insertional Mutagenesis ◽

Chlorophyll Biosynthesis ◽

Protoporphyrin Ix ◽

Model Organism ◽

Oxygenic Photosynthesis ◽

Functional Roles ◽

Protein Levels ◽

In The Wild ◽

Chl Biosynthesis

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms and are synthesized via a common branched tetrapyrrole biosynthetic pathway. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. MgChel is a multimeric enzyme that consists of three subunits designated CHLD, CHLI and CHLH. Plants have two isozymes of CHLI (CHLI1 and CHLI2) which are 70%-81% identical in protein sequences. Although the functional role of CHLI1 is well characterized, that of CHLI2 is not. We have isolated a non-photosynthetic light sensitive mutant 5A7 by random DNA insertional mutagenesis that is devoid of any detectable Chl. PCR based analyses show that 5A7 is missing the CHLI1 gene and at least eight additional functionally uncharacterized genes. 5A7 has an intact CHLI2 gene. Complementation with a functional copy of the CHLI1 gene restored Chl biosynthesis, photo-autotrophic growth and light tolerance in 5A7. We have identified the first chli1 (chli1-1) mutant of Chlamydomonas reinhardtii and in green algae. Our results show that in the wild type Chlamydomonas CHLI2 protein amount is lower than that of CHLI1 and the chli1-1 mutant has a drastic reduction in CHLI2 protein levels although it possesses the CHLI2 gene. Our chli1-1 mutant opens up new avenues to explore the functional roles of CHLI1 and CHLI2 in Chl biosynthesis in Chlamydomonas, which has never been studied before.

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Identification and molecular characterization of a Chlamydomonas reinhardtii mutant that shows a light intensity dependent progressive chlorophyll deficiency

F1000Research ◽

10.12688/f1000research.2-142.v1 ◽

2013 ◽

Vol 2 ◽

pp. 142

Author(s):

Phillip B Grovenstein ◽

Darryel A Wilson ◽

Kathryn D Lankford ◽

Kelsey A Gaston ◽

Surangi Perera ◽

...

Keyword(s):

Light Intensity ◽

Insertional Mutagenesis ◽

Protoporphyrin Ix ◽

Model Organism ◽

Genomic Region ◽

Oxygenic Photosynthesis ◽

Autotrophic Growth ◽

Wild Type ◽

Light Intensities ◽

In The Wild

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms. These tetrapyrroles are synthesized via a common branched pathway that involves mainly nuclear encoded enzymes. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4) protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m-2 s-1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m-2 s-1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.

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Identification and molecular characterization of the second Chlamydomonas gun4 mutant, gun4-II

F1000Research ◽

10.12688/f1000research.2-142.v2 ◽

2013 ◽

Vol 2 ◽

pp. 142

Author(s):

Phillip B Grovenstein ◽

Darryel A Wilson ◽

Kathryn D Lankford ◽

Kelsey A Gaston ◽

Surangi Perera ◽

...

Keyword(s):

Light Intensity ◽

Insertional Mutagenesis ◽

Protoporphyrin Ix ◽

Model Organism ◽

Genomic Region ◽

Oxygenic Photosynthesis ◽

Autotrophic Growth ◽

Wild Type ◽

Light Intensities ◽

In The Wild

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in photosynthesis and respiration. These tetrapyrroles are synthesized via a common branched pathway that involves mainly enzymes, encoded by nuclear genes. One of the enzymes in the pathway is Mg chelatase (MgChel). MgChel catalyzes insertion of Mg2+ into protoporphyrin IX (PPIX, proto) to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4) protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m-2 s-1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m-2 s-1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.

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Structure of GUN4 fromChlamydomonas reinhardtii

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15012248 ◽

2015 ◽

Vol 71 (8) ◽

pp. 1094-1099 ◽

Cited By ~ 4

Author(s):

Shabnam Tarahi Tabrizi ◽

David B. Langley ◽

Stephen J. Harrop ◽

Anthony P. Duff ◽

Robert D. Willows

Keyword(s):

Crystal Structure ◽

Chlamydomonas Reinhardtii ◽

Chlorophyll Biosynthesis ◽

Conformational Dynamics ◽

Protoporphyrin Ix ◽

Biosynthesis Pathway ◽

Broad Agreement ◽

Binding Cleft ◽

Mg Chelatase

The genomes uncoupled 4 (GUN4) protein stimulates chlorophyll biosynthesis by increasing the activity of Mg-chelatase, the enzyme that inserts magnesium into protoporphyrin IX (PPIX) in the chlorophyll biosynthesis pathway. One of the roles of GUN4 is in binding PPIX and Mg-PPIX. In eukaryotes, GUN4 also participates in plastid-to-nucleus signalling, although the mechanism for this is unclear. Here, the first crystal structure of a eukaryotic GUN4, fromChlamydomonas reinhardtii, is presented. The structure is in broad agreement with those of previously solved cyanobacterial structures. Most interestingly, conformational divergence is restricted to several loops which cover the porphyrin-binding cleft. The conformational dynamics suggested by this ensemble of structures lend support to the understanding of how GUN4 binds PPIX or Mg-PPIX.

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Rapid and high efficiency transformation of Chlamydomonas reinhardtii by square-wave electroporation

Bioscience Reports ◽

10.1042/bsr20181210 ◽

2019 ◽

Vol 39 (1) ◽

Cited By ~ 4

Author(s):

Liang Wang ◽

Lijing Yang ◽

Xin Wen ◽

Zhuoya Chen ◽

Qiaoying Liang ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Insertional Mutagenesis ◽

Molecular Mechanisms ◽

High Efficiency ◽

Model Organism ◽

Dependent Manner ◽

Exogenous Dna ◽

Square Wave ◽

Physiological Processes ◽

Final Yield

Abstract Chlamydomonas reinhardtii, the unicellular green algae, is the model organism for studies in various physiological processes and for bioindustrial applications. To explore the molecular mechanisms underlying physiological processes or to establish engineered cell lines, the exogenous DNA needs to be integrated into the genome for the insertional mutagenesis or transgene expression. However, the amount of selected marker DNA is not seriously considered in the existing electroporation methods for mutants library construction. Here, we reported a rapid-and-high-efficiency transformation technique for cell-walled strains using square-wave electroporation system. The final yield with this electroporation method was 2–6 × 103 transformants per μg exogenous DNA for cell-walled strains in a strain-dependent manner. In general, this electroporation technique was the easy and applicable way to build a mutant library for screening phenotypes of interest.

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Mg-Protoporphyrin IX and Heme Control HEMA, the Gene Encoding the First Specific Step of Tetrapyrrole Biosynthesis, in Chlamydomonas reinhardtii

Eukaryotic Cell ◽

10.1128/ec.4.10.1620-1628.2005 ◽

2005 ◽

Vol 4 (10) ◽

pp. 1620-1628 ◽

Cited By ~ 19

Author(s):

Zinaida Vasileuskaya ◽

Ulrike Oster ◽

Christoph F. Beck

Keyword(s):

Chlamydomonas Reinhardtii ◽

Posttranscriptional Regulation ◽

Higher Plants ◽

Chlorophyll Biosynthesis ◽

Single Gene ◽

Protoporphyrin Ix ◽

Tetrapyrrole Biosynthesis ◽

Gene Encoding ◽

Mrna Accumulation ◽

Specific Step

ABSTRACT HEMA encodes glutamyl-tRNA reductase (GluTR), which catalyzes the first step specific for tetrapyrrole biosynthesis in plants, archaea, and most eubacteria. In higher plants, GluTR is feedback inhibited by heme and intermediates of chlorophyll biosynthesis. It plays a key role in controlling flux through the tetrapyrrole biosynthetic pathway. This enzyme, which in Chlamydomonas reinhardtii is encoded by a single gene (HEMA), exhibits homology to GluTRs of higher plants and cyanobacteria. HEMA mRNA accumulation was inducible not only by light but also by treatment of dark-adapted cells with Mg-protoporphyrin IX (MgProto) or hemin. The specificity of these tetrapyrroles as inducers was demonstrated by the absence of induction observed upon the feeding of protoporphyrin IX, the precursor of both heme and MgProto, or chlorophyllide. The HEMA mRNA accumulation following treatment of cells with light and hemin was accompanied by increased amounts of GluTR. However, the feeding of MgProto did not suggest a role for Mg-tetrapyrroles in posttranscriptional regulation. The induction by light but not that by the tetrapyrroles was prevented by inhibition of cytoplasmic protein synthesis. Since MgProto is synthesized exclusively in plastids and heme is synthesized in plastids and mitochondria, the data suggest a role of these compounds as organellar signals that control expression of the nuclear HEMA gene.

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Insertional Mutagenesis of Chlamydomonas reinhardtii

BIO-PROTOCOL ◽

10.21769/bioprotoc.1680 ◽

2015 ◽

Vol 5 (24) ◽

Author(s):

Chia-Hong Tsai ◽

Christoph Benning

Keyword(s):

Chlamydomonas Reinhardtii ◽

Insertional Mutagenesis

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Transcription Profile Analysis of Chlorophyll Biosynthesis in Leaves of Wild-Type and Chlorophyll b-Deficient Rice (Oryza sativa L.)

Agriculture ◽

10.3390/agriculture11050401 ◽

2021 ◽

Vol 11 (5) ◽

pp. 401

Author(s):

Minh Khiem Nguyen ◽

Tin-Han Shih ◽

Szu-Hsien Lin ◽

Jun-Wei Lin ◽

Hoang Chinh Nguyen ◽

...

Keyword(s):

Oryza Sativa ◽

Core Protein ◽

Profile Analysis ◽

Oryza Sativa L ◽

Electron Flow ◽

Chlorophyll Biosynthesis ◽

Wild Type ◽

Transcription Analysis ◽

Chl A ◽

Chl Biosynthesis

Photosynthesis is an essential biological process and a key approach for raising crop yield. However, photosynthesis in rice is not fully investigated. This study reported the photosynthetic properties and transcriptomic profiles of chlorophyll (Chl) b-deficient mutant (ch11) and wild-type rice (Oryza sativa L.). Chl b-deficient rice revealed irregular chloroplast development (indistinct membranes, loss of starch granules, thinner grana, and numerous plastoglobuli). Next-generation sequencing approach application revealed that the differential expressed genes were related to photosynthesis machinery, Chl-biosynthesis, and degradation pathway in ch11. Two genes encoding PsbR (PSII core protein), FtsZ1, and PetH genes, were found to be down-regulated. The expression of the FtsZ1 and PetH genes resulted in disrupted chloroplast cell division and electron flow, respectively, consequently reducing Chl accumulation and the photosynthetic capacity of Chl b-deficient rice. Furthermore, this study found the up-regulated expression of the GluRS gene, whereas the POR gene was down-regulated in the Chl biosynthesis and degradation pathways. The results obtained from RT-qPCR analyses were generally consistent with those of transcription analysis, with the exception of the finding that MgCH genes were up-regulated which enhance the important intermediate products in the Mg branch of Chl biosynthesis. These results indicate a reduction in the accumulation of both Chl a and Chl b. This study suggested that a decline in Chl accumulation is caused by irregular chloroplast formation and down-regulation of POR genes; and Chl b might be degraded via the pheophorbide b pathway, which requires further elucidation.

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Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02268-x ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Ke Zhang ◽

Zhuoying Li ◽

Yunyang Lu ◽

Linyi Xiang ◽

Jiadong Sun ◽

...

Keyword(s):

Western Blotting ◽

Planar Cell Polarity ◽

Type Ii Collagen ◽

Mapk Signaling ◽

Dependent Manner ◽

Inflammatory Microenvironment ◽

Functional Roles ◽

Protein Levels ◽

Oa Chondrocytes

Abstract Background The Wnt planar cell polarity (PCP) pathway is implicated in osteoarthritis (OA) both in animals and in humans. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the orientation and alignment of chondrocytes in the growth plate. However, its functional roles in OA still remain undefined. Here, we explored the effects of Vangl2 on OA chondrocyte in vitro and further elucidated the molecular mechanism of silencing Vangl2 in Wnt5a-overexpressing OA chondrocytes. Methods Chondrocytes were treated with IL-1β (10 ng/mL) to simulate the inflammatory microenvironment of OA. The expression levels of Vangl2, Wnt5a, MMPs, and related proinflammatory cytokines were measured by RT-qPCR. Small interfering RNA (siRNA) of Vangl2 and the plasmid targeting Wnt5a were constructed and transfected into ATDC5 cells. Then, the functional roles of silencing Vangl2 in the OA chondrocytes were investigated by Western blotting, RT-qPCR, and immunocytochemistry (ICC). Transfected OA chondrocytes were subjected to Western blotting to analyze the relationship between Vangl2 and related signaling pathways. Results IL-1β induced the production of Vangl2, Wnt5a, and MMPs in a time-dependent manner and the significantly increased expression of Vangl2. Vangl2 silencing effectively suppressed the expression of MMP3, MMP9, MMP13, and IL-6 at both gene and protein levels and upregulated the expression of type II collagen and aggrecan. Moreover, knockdown of Vangl2 inhibited the phosphorylation of MAPK signaling molecules (P38, ERK, and JNK) and P65 in Wnt5a-overexpressing OA chondrocytes. Conclusions For the first time, we demonstrate that Vangl2 is involved in the OA process. Vangl2 silencing can notably alleviate OA progression in vitro by inhibiting the expression of MMPs and increasing the formation of the cartilage matrix and can inhibit the proinflammatory effects of Wnt5a via MAPK and NF-κB pathway. This study provides new insight into the mechanism of cartilage inflammation.

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To Divide or Not to Divide? How Deuterium Affects Growth and Division of Chlamydomonas reinhardtii

Biomolecules ◽

10.3390/biom11060861 ◽

2021 ◽

Vol 11 (6) ◽

pp. 861

Author(s):

Veronika Kselíková ◽

Vilém Zachleder ◽

Kateřina Bišová

Keyword(s):

Cell Cycle ◽

Chlamydomonas Reinhardtii ◽

Cell Cycle Progression ◽

Model Organism ◽

Cycle Progression ◽

Synchronous Cultures ◽

Multiple Fission ◽

First Choice ◽

High Doses

Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division in various organisms. Microalgae, including Chlamydomonas reinhardtii, a well-established model organism in cell cycle studies, are no exception. Chlamydomonas reinhardtii, a green unicellular alga of the Chlorophyceae class, divides by multiple fission, grows autotrophically and can be synchronized by alternating light/dark regimes; this makes it a model of first choice to discriminate the effect of deuterium on growth and/or division. Here, we investigate the effects of high doses of deuterium on cell cycle progression in C. reinhardtii. Synchronous cultures of C. reinhardtii were cultivated in growth medium containing 70 or 90% D2O. We characterize specific deuterium-induced shifts in attainment of commitment points during growth and/or division of C. reinhardtii, contradicting the role of the “sizer” in regulating the cell cycle. Consequently, impaired cell cycle progression in deuterated cultures causes (over)accumulation of starch and lipids, suggesting a promising potential for microalgae to produce deuterated organic compounds.

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