DOI: 10.5281/zenodo.18186873
title: Raw data for Insight into GABA shunt-associated aldehyde dehydrogenases (ALDH) and stress responses of ALDH superfamily in moss and barley

description: Description of the data and file structure

This supplementary dataset, integral to our research paper, contains data covering properties, interactions, and expression of studied aldehyde dehydrogenases (ALDHs). The dataset comprises gel permeation chromatography profiles of the studied plant ALDHs, microscale thermophoresis data used to describe the binding curves, as well as substrate screening profiles and saturation curves for the selected substrates, which were used to determine Km and kcat values. Thermal stability curves measured by nano-differential scanning fluorimetry are provided to show differences in stability. Quantitative PCR (qPCR) data are provided for the expression of barley ALDH gene superfamily and moss ALDH gene superfamily under various stress conditions and hormone treatments. Finally, for the aldh10A1, aldh21A1 and aldh5F2 mutants vs WT we provide RNA-seq data sets showing differentially expressed genes, phenotype and photosynthesis parameter data.

 

Files and variables

File: Gel_permeation_chromatography_dPpALDH10F1.csv

Description

Gel filtration of highly concentrated PpALDH10F1 (at 10 mg ml-1) on Superdex S200 column. The chromatogram shows the existence of a single peak corresponding to the dimer.

Variables



Elution time for dPpALDH10F1 (min): Elution time in minutes

dPpALDH10F1 (Abs at 280 nm): Absorbance of protein at 280 nm

Elution time for standards (min): Elution time in minutes

Bio-Rad standards (Abs at 280 nm): Absorbance of protein at 280 nm

Amy+Apo STD (Abs at 280 nm): Absorbance of protein at 280 nm


File: Gel_permeation_chromatography_PpALDH5F1.csv

Description

Gel filtration of highly concentrated PpALDH5F1 (at 10 mg ml-1) on Superdex S200 column. The chromatogram shows the existence of a single peak corresponding to the tetramer.

Variables



Elution time (min): Elution time in minutes

PpALDH5F1 (Abs at 280 nm): Absorbance of protein at 280 nm

Bio-Rad standards (Abs at 280 nm): Absorbance of protein at 280 nm


File: Moss_knockout_phenotype_mannitol.csv

Description

Growth after point inoculation on modified Knop medium and 1% plant agar. Cultivated for one month with mannitol (500 mOsm/kg) at 19 °C. Graph values indicate mean ± S.D. (n=15). Data were analyzed in a t-test (*, **, and *** correspond to P-values of 0.05 > p > 0.01, 0.01 > p > 0.001, and p < 0.001, respectively).

Variables



Day

WT colony diameter (cm)

WT colony diameter st deviation (cm)

ppaldh5F2 colony diameter (cm)

ppaldh5F2 colony diameter st deviation (cm)

ppaldh5F2 vs WT colony diameter t-test

ppaldh10A1 colony diameter (cm)

ppaldh10A1 colony diameter st deviation (cm)

ppaldh10A1 vs WT colony diameter t-test

ppaldh21A1 colony diameter (cm)

ppaldh21A1 colony diameter st deviation (cm)

ppaldh21A1 vs WT colony diameter t-test


File: Moss_knockout_phenotype_NaCl.csv

Description

Growth after point inoculation on modified Knop medium and 1% plant agar. Cultivated for one month with 300 mM NaCl at 19 °C. Graph values indicate mean ± S.D. (n=15). Data were analyzed in t-test (*, **, and *** correspond to P-values of 0.05 > p > 0.01, 0.01 > p > 0.001, and p < 0.001, respectively).

Variables



Day

WT colony diameter (cm)

WT colony diameter st deviation (cm)

ppaldh5F2 colony diameter (cm)

ppaldh5F2 colony diameter st deviation (cm)

ppaldh5F2 vs WT colony diameter t-test

ppaldh10A1 colony diameter (cm)

ppaldh10A1 colony diameter st deviation (cm)

ppaldh10A1 vs WT colony diameter t-test

ppaldh21A1 colony diameter (cm)

ppaldh21A1 colony diameter st deviation (cm)

ppaldh21A1 vs WT colony diameter t-test


File: Moss_knockout_phenotype_standard_conditions.csv

Description

Growth after point inoculation on modified Knop medium and 1% plant agar. Cultivated for one month under normal conditions at 19 °C. Graph values indicate mean ± S.D. (n=15). Data were analyzed in t-test (*, **, and *** correspond to P-values of 0.05 > p > 0.01, 0.01 > p > 0.001, and p < 0.001, respectively).

Variables



Day

WT colony diameter (cm)

WT colony diameter st deviation (cm)

ppaldh5F2 colony diameter (cm)

ppaldh5F2 colony diameter st deviation (cm)

ppaldh5F2 vs WT colony diameter t-test

ppaldh10A1 colony diameter (cm)

ppaldh10A1 colony diameter st deviation (cm)

ppaldh10A1 vs WT colony diameter t-test

ppaldh21A1 colony diameter (cm)

ppaldh21A1 colony diameter st deviation (cm)

ppaldh21A1 vs WT colony diameter t-test


File: Photosynthetic_parameters NPQ.csv

Description

Non-photochemical quenching (NPQ) in PSII in moss aldh mutants. Data were recorded in cultures dark-adapted for 30 min and then exposed to actinic light (278 µmol photons m–2 s–1). The parameters were calculated using saturating red-light pulses (300 ms, 10,000 µmol photons m–2 s–1) during actinic light exposure, followed by a dark relaxation phase. Values are means ± S.D. (n=7). Recorded on a Dual-PAM100 system.

Variables



time [s]

WT average

WT SD

ppaldh21A1 average

ppland21 SD

ppaldh5F2 average

ppaldh5F2 SD

ppaldh10A1 average

ppaldh10A1 SD


File: Photosynthetic_parameters YI.csv

Description

The effective quantum yield of photosystem I photochemistry Y(I) in moss aldh mutants. Data were recorded in cultures dark-adapted for 30 min and then exposed to actinic light (278 µmol photons m–2 s–1). The parameters were calculated using saturating red-light pulses (300 ms, 10,000 µmol photons m–2 s–1) during actinic light exposure, followed by a dark relaxation phase. Values are means ± S.D. (n=7). Recorded on a Dual-PAM100 system.

Variables



time [s]

WT average

WT SD

ppaldh21A1 average

ppland21 SD

ppaldh5F2 average

ppaldh5F2 SD

ppaldh10A1 average

ppaldh10A1 SD


File: Photosynthetic_parameters YNA.csv

Description

The effective quantum yield of non-photochemical energy dissipation in PSI due to donor (Y(ND)) side limitation in moss aldh mutants. Data were recorded in cultures dark-adapted for 30 min and then exposed to actinic light (278 µmol photons m–2 s–1). The parameters were calculated using saturating red-light pulses (300 ms, 10,000 µmol photons m–2 s–1) during actinic light exposure, followed by a dark relaxation phase. Values are means ± S.D. (n=7). Recorded on a Dual-PAM100 system.

Variables



time [s]

WT average

WT SD

ppaldh21A1 average

ppland21 SD

ppaldh5F2 average

ppaldh5F2 SD

ppaldh10A1 average

ppaldh10A1 SD


File: Photosynthetic_parameters YND.csv

Description

The effective quantum yield of non-photochemical energy dissipation in PSI due to acceptor (Y(ND)) side limitation in moss aldh mutants. Data were recorded in cultures dark-adapted for 30 min and then exposed to actinic light (278 µmol photons m–2 s–1). The parameters were calculated using saturating red-light pulses (300 ms, 10,000 µmol photons m–2 s–1) during actinic light exposure, followed by a dark relaxation phase. Values are means ± S.D. (n=7). Recorded on a Dual-PAM100 system.

Variables



time [s]

WT average

WT SD

ppaldh21A1 average

ppland21 SD

ppaldh5F2 average

ppaldh5F2 SD

ppaldh10A1 average

ppaldh10A1 SD


File: qPCR_barley.csv

Description

Gene expression screening in barley upon hormone or stress treatments. Barley was grown in Hoagland's solution and expression was determined by qPCR using dual-labeled FAM-TAM probes and RT Luna Universal Probe qPCR Master Mix on a QuantStudio 5 Real-Time PCR System. Fold change in relative expression vs control in six technical replicates was statistically analyzed with a t-test; *, **, and *** correspond to p-values of 0.05 > p > 0.01, 0.01 > p > 0.001, and p < 0.001, respectively.

Variables



Gene name

PEG (Fold change in relative expression vs control)

PEG SD (Fold change)

PEG t-test

NaCl (Fold change in relative expression vs control)

NaCl SD (Fold change)

NaCl t-test

ABA (Fold change in relative expression vs control)

ABA SD (Fold change)

ABA t-test

MeJA (Fold change in relative expression vs control)

MeJA SD (Fold change)

MeJA t-test

BA (Fold change in relative expression vs control)

BA SD (Fold change)

BA t-test

2,4-D (Fold change in relative expression vs control)

2,4-D SD (Fold change)

2,4-D t-test

Excess of nitrogen (Fold change in relative expression vs control) 

Excess of nitrogen SD (Fold change)

Excess of nitrogen t-test

Lack of nitrogen (Fold change in relative expression vs control)

Lack of nitrogen SD (Fold change)

Lack of nitrogen t-test

Heat (Fold change in relative expression vs control)

Heat SD (Fold change)

Heat t-test

Heat Recovery for 1h (Fold change in relative expression vs control)

Heat Recovery for 1h SD (Fold change)

Heat Recovery for 1h t-test

Heat Recovery for 4h (Fold change in relative expression vs control)

Heat Recovery for 4h SD (Fold change)

Heat Recovery for 4h t-test

Cold for 5h (Fold change in relative expression vs control)

Cold for 5h SD (Fold change)

Cold for 5h t-test


File: qPCR_moss.csv

Description

Gene expression screening in moss upon hormone or stress treatments. Moss was grown in Knop's medium and expression was determined by qPCR using dual-labeled FAM-TAM probes and RT Luna Universal Probe qPCR Master Mix on a QuantStudio 5 Real-Time PCR System. Fold change in relative expression vs control in six technical replicates was statistically analyzed with a t-test; *, **, and *** correspond to p-values of 0.05 > p > 0.01, 0.01 > p > 0.001, and p < 0.001, respectively.

Variables



Gene name

Mannitol (Fold change in relative expression vs control)

Mannitol SD (Fold change)

Mannitol t-test

NaCl (Fold change in relative expression vs control)

NaCl SD (Fold change)

NaCl t-test

ABA (Fold change in relative expression vs control)

ABA SD (Fold change)

ABA t-test

MeJA (Fold change in relative expression vs control)

MeJA SD (Fold change)

MeJA t-test

BA (Fold change in relative expression vs control)

BA SD (Fold change)

BA t-test

2,4-D (Fold change in relative expression vs control)

2,4-D SD (Fold change)

2,4-D t-test

Excess of nitrogen (Fold change in relative expression vs control)

Excess of nitrogen SD (Fold change)

Excess of nitrogen t-test

Lack of nitrogen (Fold change in relative expression vs control)

Lack of nitrogen SD (Fold change)

Lack of nitrogen t-test

Heat (Fold change in relative expression vs control)

Heat SD (Fold change)

Heat t-test

Heat Recovery for 1h (Fold change in relative expression vs control)

Heat Recovery for 1h SD (Fold change)

Heat Recovery for 1h t-test

Heat Recovery for 4h (Fold change in relative expression vs control)

Heat Recovery for 4h SD (Fold change)

Heat Recovery for 4h t-test

Cold for 5h (Fold change in relative expression vs control)

Cold for 5h SD (Fold change)

Cold for 5h t-testFile: a.csv


File: RNA seq Fig6.csv

Description

Differentially expressed genes in three moss aldh mutants (aldh21A1, aldh10A1 and aldh5F2 vs WT in RNA-seq data (n=3). The reference index was constructed from the P. patens CDS dataset version 6.1. Raw reads were quality-filtered using Rcorrector and Trim Galore scripts, levels of transcript expression (transcript abundances quantified as transcripts per million – TPM) were determined using Salmon tool with parameters --posBias, --seqBias, --gcBias, --numBootstraps 30. q-value (qval) represents an adjusted p-value, while b value stands for the logarithm base 2 of the fold change in a gene's expression between two conditions (mutant vs WT). SE b stands for the standard error of b value (n=3).

Variables



gene ID

TPM average WT

TPM average ppaldh21A1

TPM average ppaldh5F2

TPM average ppaldh10A1

qval ppaldh21A1

b ppaldh21A1

SE b ppaldh21A1

qval ppaldh5F2

b ppaldh5F2

SE b ppaldh5F2

qval ppaldh10A1

b ppaldh10A1

SE b ppaldh10A1


File: RNAseq_PpALDH5_knockout.csv

Description

Differentially expressed genes in moss aldh5F2 mutant vs WT in RNA-seq data (n=3). The reference index was constructed from the P. patens CDS dataset version 6.1. Raw reads were quality-filtered using Rcorrector and Trim Galore scripts, levels of transcript expression (transcript abundances quantified as transcripts per million – TPM) were determined using Salmon tool with parameters --posBias, --seqBias, --gcBias, --numBootstraps 30. q-value (qval) represents an adjusted p-value and b value stands for the logarithm base 2 of the fold change in a gene's expression between two conditions (mutant vs WT). Upregulated and downregulated genes are identified by the following criteria: q ≤ 0.05 and a fold-change ≥ 2.

Variables



gene_ID

#pacId

Panther ID

EC number

KOG ID

KO ID

TPM average in WT

TPM average in ppaldh5F2 knockout

qval

b

Gene expression in ppaldh5F2 knockout vs WT


File: RNAseq_PpALDH10_knockout.csv

Description

Differentially expressed genes in moss aldh10A1 mutant vs WT in RNA-seq data (n=3). The reference index was constructed from the P. patens CDS dataset version 6.1. Raw reads were quality-filtered using Rcorrector and Trim Galore scripts, levels of transcript expression (transcript abundances quantified as transcripts per million – TPM) were determined using Salmon tool with parameters --posBias, --seqBias, --gcBias, --numBootstraps 30. q-value (qval) represents an adjusted p-value and b value stands for the logarithm base 2 of the fold change in a gene's expression between two conditions (mutant vs WT). Upregulated and downregulated genes are identified by the following criteria: q ≤ 0.05 and a fold-change ≥ 2.

Variables



gene_ID

#pacId

Panther ID

EC number

KOG ID

KO ID

TPM average in WT

TPM average in ppaldh10A1 knockout

qval

b

Gene expression in ppaldh10A1 knockout vs WT


File: RNAseq_PpALDH21_knockout.csv

Description

Differentially expressed genes in moss aldh21A1 mutant vs WT in RNA-seq data (n=3). The reference index was constructed from the P. patens CDS dataset version 6.1. Raw reads were quality-filtered using Rcorrector and Trim Galore scripts, levels of transcript expression (transcript abundances quantified as transcripts per million – TPM) were determined using Salmon tool with parameters --posBias, --seqBias, --gcBias, --numBootstraps 30. q-value (qval) represents an adjusted p-value and b value stands for the logarithm base 2 of the fold change in a gene's expression between two conditions (mutant vs WT). Upregulated and downregulated genes are identified by the following criteria: q ≤ 0.05 and a fold-change ≥ 2.

Variables



gene_ID

#pacId

Panther ID

EC number

KOG ID

KO ID

TPM average in WT

TPM average in ppaldh21A1 knockout

qval

b

Gene expression in ppaldh21A1 knockout vs WT


File: Substrate_specificity_ALDH5.csv

Description

Screening of substrate specificity of moss and barley ALDH5. Measured with 200 µM aldehyde in 150 mM HEPES buffer (pH 8.0) and 1.5 mM NAD+ at 30 °C. Error bars stand for S.D. (n=4). 

Variables



Substrate

Relative activity of PpALDH5F1 (%)

Standard deviation of relative activity of PpALDH5F1 (%)

Relative activity of PpALDH5F2 (%)

Standard deviation of relative activity of PpALDH5F2 (%)

Relative activity of HvALDH5A (%)

Standard deviation of relative activity of HvALDH5A (%)


File: Substrate_specificity_PpALDH5_mutants.csv

Description

Screening of substrate specificity of moss ALDH5 variants. Measured with 200 µM aldehyde in 150 mM HEPES buffer (pH 8.0) and 1.5 mM NAD+ at 30 °C. Error bars stand for S.D. (n=4). 

Variables



Substrate

Relative rate with PpALDH5F1-WT (%)

St deviation of relative rate with PpALDH5F1-WT (%)

Relative rate with PpALDH5F1-Y115A (%)

St deviation of relative rate with PpALDH5F1-Y115A (%)

Relative rate with PpALDH5F1-R169A (%)

St deviation of relative rate with PpALDH5F1-R169A (%)

Relative rate with PpALDH5F1-R287A (%)

St deviation of relative rate with PpALDH5F1-R287A (%)

Relative rate with PpALDH5F1-S450A (%)

St deviation of relative rate with PpALDH5F1-S450A (%)


File: Substrate_specificity_PpALDH10.csv

Description

Substrate screening for major substrates of ΔPpALDH10A1. Measured with 1 mM substrate in 150 mM Tris-HCl buffer (pH 8.5) containing 1 mM NAD+ at 30 °C. Error bars stand for S.D. (n=4). 

Variables



Substrate at 1 mM

Relative activity (%)

Standard deviation  (%)


File: Thermal_stability_nanoDSF_dPpALDH10F1.csv

Description

Thermal stability was measured using nano-differential scanning fluorimetry on Tycho NT.6 and Prometheus Panta instruments (Nanotemper) with a heating rate of 1 °C min-1, and using Panta control software. Five different conditions were tested for PpALDH10A1.

Variables



Temperature (degrees C)

dPpALDH10F1 HEPES pH 7.5 (First derivative F350/F330)

dPpALDH10F1 in HEPES + 5 mM NAD+ (First derivative F350/F330)

dPpALDH10F1 in HEPES + 5% GOL (First derivative F350/F330)

dPpALDH10F1 in HEPES + 100 mM NaCl + 5% GOL (First derivative F350/F330)

dPpALDH10F1 in HEPES + 100 mM NaCl (First derivative F350/F330)


File: Thermal_stability_nanoDSF_PpALDH5F1.csv

Description

Thermal stability was measured using nano-differential scanning fluorimetry on Tycho NT.6 and Prometheus Panta instruments (Nanotemper) with a heating rate of 1 °C min-1, and using Panta control software. Five different conditions were tested for PpALDH5F1.

Variables



Temperature (degrees C)

PpALDH5F1 in HEPES pH 8.5 (First derivative F350/F330)

PpALDH5F1 in HEPES + 1 mM NAD+ (First derivative F350/F330)

PpALDH5F1 in HEPES + 1 mM NADP+ (First derivative F350/F330)

Temperature  (degrees C)

PpALDH5F1 in HEPES + 5% GOL (First derivative F350/F330)

PpALDH5F1 in HEPES + 100 mM NaCl (First derivative F350/F330)


File: Binding_curves_of_PpALDH5F1_ligands.csv

Description

Binding affinity measurements of PpALDH5F1 for NAD+, succinate and sulfate ions. Data were measured by MST in 50 mM Tris-HCl at pH 8.0, 100 mM NaCl, supplemented with 0.05% Tween on a Monolith X instrument (NanoTemper Technologies) at 25 °C. Fitted curves were obtained using MO Affinity Analysis software from Nanotemper.

Variables



Concentration of NAD in M

Fraction Bound for NAD bound to PpALDH5F1

Standard deviation of Fraction Bound for NAD bound to PpALDH5F1

Concentration of Succinate in M

Fraction Bound for Succinate bound to PpALDH5F1

Standard deviation of Fraction Bound for Succinate bound to PpALDH5F1

Concentration of Sulfate in M

Fraction Bound for Sulfate bound to PpALDH5F1

Standard deviation of Fraction Bound for Sulfate

Fit of concentration of NAD

Fitting curve of Fraction bound of NAD bound to PpALDH5F1

Fit of concentration of Succinate

Fitting curve of Fraction bound of Succinate bound to PpALDH5F1

Fit of concentration of Sulfate

Fitting curve of Fraction bound of Sulfate bound to PpALDH5F1


File: Enzyme_kinetics_PpALDH5.csv

Description

Saturation curves for four ligands with PpALDH5F1. Data were measured in 150 mM HEPES buffer pH 8.0, using 1.5 mM NAD+ (for SSAL) or 40 μM SSAL (for coenzymes).The data set comprises measured values (s-1) and std deviation, plus calculated curves fitted to the Michaelis-Menten equation or substrate inhibition equation by GraphPad Prism software version 8.0.

Variables



NAD+ concentration (Micromolar)

Reaction rate of PpALDH5F1 with NAD+ (1/s)

Standard deviation of reaction rate of PpALDH5F1 with NAD+ (1/s)

NADP+ concentration (Micromolar)

Reaction rate of PpALDH5F1 with NADP+ (1/s)

Standard deviation of reaction rate of PpALDH5F1 with NADP+ (1/s)

SSAL concentration (Micromolar)

Reaction rate of PpALDH5F1 with SSAL (1/s)

Standard deviation of reaction rate of PpALDH5F1 with SSAL (1/s)

Pentanal concentration (Micromolar)

Reaction rate of PpALDH5F1 with Pentanal (1/s)

Standard deviation of reaction rate of PpALDH5F1 with Pentanal (1/s)

Curve fit of NAD+ concentration (Micromolar)

Curve fit of Reaction rate of PpALDH5F1 with NAD+ (1/s)

Curve fit of NADP+ concentration (Micromolar)

Curve fit of Reaction rate of PpALDH5F1 with NADP+ (1/s)

Curve fit of SSAL concentration (Micromolar)

Curve fit of Reaction rate of PpALDH5F1 with SSAL (1/s)

Curve fit of Pentanal concentration (Micromolar)

Curve fit of Reaction rate of PpALDH5F1 with Pentanal (1/s)


File: Enzyme_kinetics_PpALDH10.csv

Description

Saturation curves for five aminoaldehydes with PpALDH10A1. Measured in 150 mM Tris-HCl buffer (pH 8.5) containing 1 mM NAD+ at 30 °C. Dataset comprises measured values (s-1) and std deviation plus calculated curves fitted to Michaelis-Menten equation or substrate inhibition equation by GraphPad Prism software version 8.0.

Variables



APAL concentration (Micromolar)

Reaction rate with APAL (1/s)

St deviation of reaction rate with APAL (1/s)

ABAL concentration (Micromolar)

Reaction rate with ABAL (1/s)

St deviation of reaction rate with ABAL (1/s)

TMABAL concentration (Micromolar)

Reaction rate with TMABAL (1/s)

St deviation of reaction rate with TMABAL (1/s)

GBAL concentration (Micromolar)

Reaction rate with GBAL (1/s)

St deviation of reaction rate with GABAL (1/s)

AcAPAL concentration (Micromolar)

Reaction rate with AcAPAL (1/s)

St deviation of reaction rate with AcAPAL (1/s)

Curve fit of APAL concentration (Micromolar)

Curve fit of Reaction rate with APAL (1/s)

Curve fit of ABAL concentration (Micromolar)

Curve fit of Reaction rate with ABAL (1/s)

Curve fit of TMABAL concentration (Micromolar)

Curve fit of Reaction rate with TMABAL (1/s)

Curve fit of GBAL concentration (Micromolar)

Curve fit of Reaction rate with GBAL (1/s)

Curve fit of AcAPAL concentration (Micromolar)

Curve fit of Reaction rate with AcAPAL (1/s)