The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces

Jungnick, Nadine; Ma, Yunbing; Mukherjee, Bratati; Cronan, J.; Speed, DeQuantarius; Laborde, Susan M.; Longstreth, David J.; Moroney, James V.

doi:10.1007/s11120-014-0004-x

Cited by 40 publications

(39 citation statements)

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“…In the study in which the cells were screened for aberrant responses to sulfur limitation, a number of novels genes were discovered, including proteins involved in the responses of Chlamydomonas to sulfur deprivation [19, 21]. In the screen for mutants unable to grow photoautotrophically on low CO 2 , insertions in CIA6 [22], bestrophin, MITC11 and LCI9 [23] and CIA8 (Machingura, Bajsa-Hirschel and Moroney, unpublished) were identified using the forward genetics approach described by González-Ballester et al [5]. No large deletions were observed in these studies.…”

Section: Resultsmentioning

confidence: 99%

A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii

et al. 2017

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BackgroundRandom insertional mutagenesis of Chlamydomonas reinhardtii using drug resistance cassettes has contributed to the generation of tens of thousands of transformants in dozens of labs around the world. In many instances these insertional mutants have helped elucidate the genetic basis of various physiological processes in this model organism. Unfortunately, the insertion sites of many interesting mutants are never defined due to experimental difficulties in establishing the location of the inserted cassette in the Chlamydomonas genome. It is fairly common that several months, or even years of work are conducted with no result. Here we describe a robust method to identify the location of the inserted DNA cassette in the Chlamydomonas genome.ResultsInsertional mutants were generated using a DNA cassette that confers paromomycin resistance. This protocol identified the cassette insertion site for greater than 80% of the transformants. In the majority of cases the insertion event was found to be simple, without large deletions of flanking genomic DNA. Multiple insertions were observed in less than 10% of recovered transformants.ConclusionThe method is quick, relatively inexpensive and does not require any special equipment beyond an electroporator. The protocol was tailored to ensure that the sequence of the Chlamydomonas genomic DNA flanking the random insertion is consistently obtained in a high proportion of transformants. A detailed protocol is presented to aid in the experimental design and implementation of mutant screens in Chlamydomonas.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0170-x) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii

et al. 2017

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“…Intriguingly, these clusters also include newly identified CO 2 -responsive genes encoding membrane and soluble proteins with unknown functions, providing a potentially rich source of candidate genes that may function in Ci transport or Ci uptake or other CCM functional or regulatory processes. Together with on-going efforts to generate a large Chlamydomonas mutant library or a CCM-specific mutant library (Jungnick et al, 2014;Zhang et al, 2014;Dent et al, 2015), future analysis of these novel candidate genes should provide additional insight for understanding the CCM.…”

Section: Regulation Of the Ccmmentioning

confidence: 99%

The CO₂ concentrating mechanism and photosynthetic carbon assimilation in limiting CO₂: how Chlamydomonas works against the gradient

2015

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SUMMARYThe CO 2 concentrating mechanism (CCM) represents an effective strategy for carbon acquisition that enables microalgae to survive and proliferate when the CO 2 concentration limits photosynthesis. The CCM improves photosynthetic performance by raising the CO 2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), simultaneously enhancing carbon fixation and suppressing photorespiration. Active inorganic carbon (Ci) uptake, Rubisco sequestration and interconversion between different Ci species catalyzed by carbonic anhydrases (CAs) are key components in the CCM, and an array of molecular regulatory elements is present to facilitate the sensing of CO 2 availability, to regulate the expression of the CCM and to coordinate interplay between photosynthetic carbon metabolism and other metabolic processes in response to limiting CO 2 conditions. This review intends to integrate our current understanding of the eukaryotic algal CCM and its interaction with carbon assimilation, based largely on Chlamydomonas as a model, and to illustrate how Chlamydomonas acclimates to limiting CO 2 conditions and how its CCM is regulated.

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“…Several promising candidate genes and putative transporters have been identified as playing a role in the chloroplast membrane bound C i transport system, including LCIA (NAR1.2) (Miura et al 2004, Mariscal et al 2006 belonging to the formate/nitrite (FNT) family of transporters, unique LCIB protein family LCIC/LCID/LCIE (Miura et al 2004;Wang and Spalding 2006), Ccp1/Ccp2 belonging to the mitochondrial carrier protein family (Grossman et al 2007;Jungnick et al 2014), and ycf10 belonging to the chloroplast envelope membrane protein A (CemA) family (Rolland et al 1997). …”

Section: Chloroplast C I Transportermentioning

confidence: 99%

Photosynthetic Microorganisms

Singh

Sundaram

Kishor

2014

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The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces

Cited by 40 publications

References 62 publications

A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii

A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii

The CO₂ concentrating mechanism and photosynthetic carbon assimilation in limiting CO₂: how Chlamydomonas works against the gradient

Photosynthetic Microorganisms

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The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces

Cited by 40 publications

References 62 publications

A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii

A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii

The CO2 concentrating mechanism and photosynthetic carbon assimilation in limiting CO2: how Chlamydomonas works against the gradient

Photosynthetic Microorganisms

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Product

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The CO₂ concentrating mechanism and photosynthetic carbon assimilation in limiting CO₂: how Chlamydomonas works against the gradient