Samuel J Gurr, PhD

TagSeq_prep_Pgenerosa_putative_genes

by Samuel J Gurr PhD 6 min read

Summary of Experiment in 2019

Table: Results of the repeat pCO2 stress exposure

Stress acclimation improves performance and oxidative status under subsequent stress encounter(s)
life stage age Stress acclimation (Y/N) intensity (uatm) Repeat exposure (Y/N) intensity (uatm) Respiration rate Shell growth Tissue growth (AFDW) Antioxidant capacity
postlarval ‘settlement’; pediveliger to juvenile 30 d to 5 mo post-fertilization N 900 µatm (ambient) N 900 µatm (ambient) - - ↓’’ ↑’’
- - N 900 µatm (ambient) Y 3000 and 4900 µatm (moderate & severe) ↓’ ↓’’ ↓’’ ↑’’
- - Y 3000 µatm (moderate) N 900 µatm (ambient) ↓’’ - ↑’’ ↓’’
- - Y 3000 µatm (moderate) Y 3000 µatm (moderate) ↑’ ↓’’ ↑’’ ↓’’
- - Y 3000 µatm (moderate) Y 3000 and 4900 µatm (moderate and severe) ↑’’ ↑’’ ↑’’ ↓’’
  • ↑ ↓ relative rates/analysis between treatments after a single repeat exposure (‘) and multiple exposures (‘’)

  • Table summary: acclimated phenotype had greater shell size/tissue biomass/respiration rate and reduced antioxidant protein activity*

Moving forward…

Questions:

  • Can moderate oxidative stress or mitchondrial dysfunction improve cellular stress response (i.e. anticipatory frontloading) and physiological performance?
    • What is frontloading?

      Figure from Barshis et al. 2012: Genomic basis for coral resilience to climate change

Barshis_etal2012

  • Is the alternative oxidase (AOX) mitchondrial pathway expressed by acclimatized phenotype to permit enhanced performance (continue ATP production) and decrease mtROS/mitchondrial dysfunction under acidification?

  • Does the early-life and/or paternal envrionment increase mitchondrial flexibility in under equal or greater stress intensity? - driven by non-genetic acclimation /inheritance? - is/are there stress-intensity-dependent effects of this pheonomenon? (i.e. magnitude, freqency, duration of stress encounters)

Objective:

  • investigate mitchondrial mechanism(s) and differnetial expression patterns driving stress-intensity-dependent phenotypic variation (table above)
    • TagSeq = characterize the expression of target GOIs
    • Metabolomics = analyze the relative importance of reduced cofactors (NAD+ and CoQH) and ATP between treatments
    • DNA sequencing methylation (downstream from prior measurements..) = are there patterns of differential methylation alongside treatments with greater phenotype variation (table above) and differential expressions? Can reveal a stress intensity dependent effect (timing, magnitude, frequency) to elicit epigenotypes on a intragenerational hormetic framework under hatchery timeline
  • Extract RNA/DNA from ~3 samples in ALL treatments at T0, Day 7, Day 14, and Day 21

Figure and Table: Experiment from 2019 and sampling plan

Repeat_exposure_TagSeq

  • Magenta circles highlight the target samples for extractions and sequencing

DNARNA_extraction_table

Mechanism Figures

Connections between the phenotype and proposed pathway(s) for further investigation

  • References in figure…
    • a few GOIs as numbers
    • metabolomics targets as M

Mechanism

How hypercapnia and acidosis may influence Fe-dependent activity (TET Jmj-c), retrograde-ROS signaling, and sirtuin-mediated response

FE2_OA_methyl

Current findings

  • Acclimated phenotype : = ↑ growth, ↑ metabolic rate, ↓ CSR
  • Not acclimated = ↓ growth, ↓ metabolic rate, ↑ CSR

Next Analysis

  • Acclimated phenotype : proposed pathway = Alternate oxidase (AOX)
  • Not acclimated : proposed pathway = Reverese electron transport (RET)
  • TagSeq & metabolomics : investigate a stress-acclimation-dependent effect on mitchondrial function(s) [targets: AOX, UCPs, sirtuins, complexes 1 & III, ATP, NAD+:NADH, CoQ:CoQH]

Future directions

  • Mitonuclear crosstalk :
    • What is the role of stress hormesis (via ocean acidification exposure(s)) on DNA/histone methylation (i.e. Jmj-C and TET activity) and anterograde and retrograde signaling (i.e. unfolded protein response)? Is there as stress timing- (i.e. paternal & early-life environment) and intensity-dependent (i.e. duration, magnitude, frequency) effect?

I) TagSeq

Identified target genes of interest (GOIs)

1. AOX

  • putative P. generosa seqID: PGEN_.00g108770
Why alternative oxidase (AOX)?
  • Function:
    • catalyzes the oxidation of ubiquinol and reduction of oxygen to water. Protons taken from uqiquinolnot the mitchondrial matrix (unlike the cytochrome c oxidase reaction; complex IV).
    • allows ATP production and reduces ROS under envrionmental disturbances.
  • AOX is theorized to be prevalent in animals that have frequent transitions from hypoxia to reoxygenation during thier life history - helps taxa survive transitions from anaerobic conditions without substantial damage to membrrane lipids and proteins by ROS (McDonald and Gospodaryov 2019).
    • Example: In Eastern oyster, Crassostrea virginica, there are two splice forms of AOX mRNA which are expressed in a number of tissues (Liu and Guo, 2017) and assist resitance to hypoxia-reoxygenation

Table below from: McDonald et al. 2009, Alternative oxidase in animals : unique characteristics and taxonomic distribution

AOX_McDonald_2009

What is mitchondrial dysfunction?
  • Mitochondrial dysfunction is defined as electron leak or increase of ROS production due to an alteration of normal electron transport.
  • Causes:
    • Reverese electron transport (RET): can occur under (i) high pools of reduced metabolites NADH:NAD+ and CoQH:CoQ; well-chracterized under dietary restriction (ii) pH gradient ([H+]) and/or protonmotive force of the inner mitchondrial membrane
In summary…
  • AOX is an alterantive mitchondrial pathway known as an adaptive response in bivalve taxa under envrionmnental stress. Thus, the lower antioxidant response and greater performance of the stress-acclimated phenotype in 2019 (table above) suggests higher ATP and lower ROS – this may be driven by enhanced mitchondrial flexibility (i.e. via AOX) due to prior stress priming!

Fig. below from: Saha et al. 2016, Alternative oxidase and plant stress tolerance

AOX_Saha2016

2. Sirtuins (SIR1, SIR2, SIR3, SIR5)

  • putative P. generosa seqID: PGEN_.00g048200 (SRT1)

  • putative P. generosa seqID: PGEN_.00g012340 & PGEN_.00g012350 (SRT2)

  • putative P. generosa seqID: PGEN_.00g153700 (SRT5)

  • putative P. generosa seqID: PGEN_.00g033970 (SRT7)

About:
  • sirtuins are NAD+ dehydrogenases involved in post-traslational modification of histones and proteins in the cytosol (Figure below)
  • Several sirtuins:
    • Mitochondria: SRTs 3, 4, and 5
    • Cytsol: SRT2
    • Nucleus: SRTs 1, 6,7
  • deacetylation of proteins can increase proteostasis and alleviate dysfunction - i.e. sirtuins can increase the antioxidant response (SRT 3 and 5) and promote forward electron transport/beta oxidation in the mitchondria (SRT 1); both suggest a sirtuin-mediated cellular response under mitchondrial dysfunction (Figure below)

sirtuin_mitonuclear

3. Mitchondrial carrier protein

  • putative P. generosa seqID: PGEN_.00g063670
About:
  • uncoupling proteins are activated by lipid peroxidation - thus they are a response of oxidative stress and are increased during dysfunction
  • uncoupling proteins reduce the protonmotive force (and pH gradient) of the inner mitchondrial membrane reducing ROS production from mitchondrial dysfunction at the expense of reduced potential for ATP

UCP_cartoon

4. NADH dehydrogenase

  • putative P. generosa seqID: PGEN_.00g299160
About:
  • complex 1 of mitochondrial electron transport chain.

  • major source of mithcondrial ROS production and increases activity under dysfunction - particularly knwon to increase activity under reverse electron transport under increase proton and pH gradient of the inner mitochondrial membrance

  • Note: This suggests mitchondrial dysfunction under intracellular acidosis and hypercapnia!

5. Cytochrome c reductase

  • putative P. generosa seqID: PGEN_00g275780 (Pfam: Ubiquinol-cytochrome C reductase complex 14kD subunit)
About:
  • complex III of mitochonrial electron transport chain
  • major source of mithcondrial ROS production; may reduce abundance/activity under mitchondrial dysfunction - Example: high ratio complexI:complexIII is an indicator of reverese electron transport

DNA and histone Methylation

6 & 7. Jumonji C and Ten-eleven translocation (TET)

  • putative P. generosa seqID: PGEN_.00g257110 (Jumonji-C)

  • putative P. generosa seqID: ? (TET)

8. DNMT 3a & DNMT 3b

  • putative P. generosa seqID: PGEN_.00g029420 (DNMT 3a; Sam White’s Pgenerosa annotation)

  • putative P. generosa seqID: PGEN_.00g067800 (Pfam: C-5 cytosine-specific DNA methylase; Cysteine rich ADD domain in DNMT3)

About:
  • Jumonji-C and TET function in the demethylation of histones and DNA, respectively - a post-translational (histones) and DNA modification that effects transcriptional regulation
  • Why is this interesting in response to OA stress?
    • Jumonji-C and TET activity is dependent on Fe2+. Chelated Fe2+ is released during intracellular acidosis - further Fe2+ is reduced to Fe3+ from the Fenton reaction also catalyzed by acidosis.
  • Altogether, an iron-acidosis interaction may have downstream effects on non-genetic transcriptional regulation - this is an interesting direction to investigate the role of epigentics in mitonucluear crosstalk and hormetic conditioning under OA conditions!

II) Metabolomics

1. ADP/ATP

  • Expected ATP: **AOX pathway ** RET/dysfunction pathway

2. NAD+:NADH

  • Expected NAD+/NADH: AOX pathway < RET/dysfunction pathway
  • Note: although dysfunction can be casued by a high pool of NADH, the high activity of complex I oxidizes NADH to NAD+.

3. CoQH

  • Expected CoQH: AOX pathway < RET/dysfunction pathway

Resources:

Panopea generosa (Pacific geoduck) draft genome

  • link: http://dx.doi.org/10.17605/OSF.IO/YEM8N

Repository of target genes

note: repo originally in preparation for qPCR - contains blast hits and primer3 outputs for several target GOIs and normalization genes

  • link: https://github.com/SamGurr/Pgenerosa_primers