Temporal and metabolic overlap between lipid accumulation and programmed cell death due to nitrogen starvation in the unicellular chlorophyte <i>Chlamydomonas reinhardtii</i>

Sathe, Santosh; Orellana, Mónica V.; Baliga, Nitin S.; Durand, Pierre M.

doi:10.1111/pre.12368

Cited by 15 publications

(8 citation statements)

References 54 publications

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“…Photosynthetic capability is diminished, starch production increased, and the formation of mating competent gametes is induced [ 58 , 59 ]. Furthermore, programmed cell death is triggered in a small fraction of the population [ 60 ]. These effects are accompanied by growth arrest, which likely locks cells in G1 phase of the cell cycle.…”

Section: Resultsmentioning

confidence: 99%

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

Freudenberg

Wittemeier

Einhaus

et al. 2022

Cells

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Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.

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Section: Resultsmentioning

confidence: 99%

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

Freudenberg

Wittemeier

Einhaus

et al. 2022

Cells

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show abstract

“…Photosynthetic capability is diminished, starch production increased, and formation of mating competent gametes is induced [58,59]. Also, programmed cell death is triggered in a small fraction of the population [60]. These effects are accompanied by growth arrest, which likely locks cells in G1 phase of the cell cycle.…”

Section: Nitrogen Starvation Of the Pre-culture Substantially Increas...mentioning

confidence: 99%

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

Freudenberg¹,

Wittemeier²,

Einhaus³

et al. 2022

Preprint

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Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas9 allows directed exploration of new endogenous selectable markers. Since editing frequencies with CRISPR/Cas9 techniques are often low, the Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, increasing editing frequencies from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was targeted to either the cytosol or the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.

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“…The substances released during PCD are nutritionally and energetically expensive. Chemical energy is used in the architectural restructuring of the cell and its organelles as revealed by ultrastructural studies (Arnoult et al., 2002; Durand et al., 2016; Jiménez et al., 2009; Moharikar et al., 2006), active biosynthesis and secretion of volatile organic compounds (Zuo et al., 2012) and lipid molecules (Sathe et al., 2019). While the resources released are costly to produce, if their release involves cell death—as is the case with PCD—then the trait clearly bears a significant cost.…”

Section: Criteria For the Black Queen Hypothesismentioning

confidence: 99%

Programmed cell death as a black queen in microbial communities

2020

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Programmed cell death (PCD) in unicellular organisms is in some instances an altruistic trait. When the beneficiaries are clones or close kin, kin selection theory may be used to explain the evolution of the trait, and when the trait evolves in groups of distantly related individuals, group or multilevel selection theory is invoked. In mixed microbial communities, the benefits are also available to unrelated taxa. But the evolutionary ecology of PCD in communities is poorly understood. Few hypotheses have been offered concerning the community role of PCD despite its far‐reaching effects. The hypothesis we consider here is that PCD is a black queen. The Black Queen Hypothesis (BQH) outlines how public goods arising from a leaky function are exploited by other taxa in the community. Black Queen (BQ) traits are essential for community survival, but only some members bear the cost of possessing them, while others lose the trait In addition, BQ traits have been defined in terms of adaptive gene loss, and it is unknown whether this has occurred for PCD. Our conclusion is that PCD fulfils the two most important criteria of a BQ (leakiness and costliness), but that more empirical data are needed for assessing the remaining two criteria. In addition, we hold that for viewing PCD as a BQ, the original BQH needs to include social traits. Thus, despite some empirical and conceptual shortcomings, the BQH provides a helpful avenue for investigating PCD in microbial communities.

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Temporal and metabolic overlap between lipid accumulation and programmed cell death due to nitrogen starvation in the unicellular chlorophyte Chlamydomonas reinhardtii

Cited by 15 publications

References 54 publications

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

Programmed cell death as a black queen in microbial communities

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