README File

Dataset title: Effects of hydrogen peroxide and glutathione on recombinant beta-amylase1 (BAM1) from Arabidopsis thaliana and its behavior in plants with altered glutathione homeostasis.

Dataset authors:

Gurrieri, Libero. Department of Pharmacy and Biotechnology, University of Bologna, Italy, https://orcid.org/0000-0002-3537-2512
Capuzzi, Anna Clara. Department of Pharmacy and Biotechnology, University of Bologna, Italy, https://orcid.org/0009-0004-3376-2148
Müller-Schüssele, Stefanie. Department of Biology, University of Kaiserslautern-Landau, Germany, https://orcid.org/0000-0003-4061-1175
Trost, Paolo. Department of Pharmacy and Biotechnology, University of Bologna, Italy, https://orcid.org/0000-0002-6347-8701
Sparla, Francesca Department of Pharmacy and Biotechnology, University of Bologna, Italy, https://orcid.org/0000-0002-2103-4870

Dataset license: this data set is distributed under Creative Commons Attribution 4.0 International. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/

Dataset year: 2025

Funding: L.G. acknowledges financing of the Agritech National Research Center, Spoke1, and received funding from the European Union Next-GenerationEU (PIANO NAZIONALE DI RIPRESA E RESILIENZA [PNRR]—MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4—D.D. 1032 17/06/2022, CN00000022).

Abstract: The present dataset contains redox treatments of BAM1 with redox reagents like hydrogen peroxide and glutathione. The effect of glutathione has been monitored at several timepoints. Release of glutathione from BAM1 has been monitored and quantified. The inhibition following the release of glutathione has been detailed performing reactivations with thioredoxins, glutathioredoxins, dithiothreitol and glutathione. Arabidopsis thaliana plants with low levels of glutathione reductase 2 in chloroplasts were analyzed by in-gel amylolytic activity. In the same genotypes starch granule area in guard cells and stomata aperture have been quantified.

The present dataset is related to the research article "Dynamic regulation of Arabidopsis β-AMYLASE1 by glutathione and thioredoxins affects starch in guard cells", published on the journal Plant Physiology in open access (https://doi.org/10.1093/plphys/kiaf344). 

Abbreviations:
BAM1 - beta-amylase 1
H2O2 - Hydrogen peroxide
GSH - reduced glutathione
GSSG - oxidized glutathione
TRX - thioredoxin
GRX - glutaredoxin
DTT - dithiothreitol
IAM - iodoacetamide
NEM - N-ethylmaleimide

Files/folders:

- 01_Inhibition_1h_H2O2andGSH.csv, inhibition of BAM1 activity after 1h of incubation with 0.5 mM H2O2 or 0.5 mM H2O2 and GSH followed by recovery of activity with 60 mM DTT.

- 02_Inhibition_kinetics_control_H2O2_H2O2andGSH.csv, same condition of previous file, activity was monitored at different timepoints.

- 03_westernblot_antiglutathione_1hGSSGincubation
  2 files in the folder:
  - 03_blot_antiGSSG_1hGSSGincubation.jpg, raw data of the western blot with anti-glutathione antibodies. The lanes of
  interest are the first 3 from the left, marker (Biorad Precision Plus Dual Color), control BAM1 untreated and 1 mM GSSG treated
  BAM1.
  - 03_coomassie_antiGSSG_1hGSSGincubation.jpg

- 04_1mMGSSGincubation.csv, activity of BAM1 following the incubation with 1 mM GSSG.

- 05_westernblot_glutathionylationofthesamecysteinebyGSSGandGSH
  2 files in the folder:
  - 05_blot_glutathionylationofthesamecysteinebyGSSGandGSH.jpg, the lanes of interest are the last four from the left:
    GSSG treated BAM1, GSSG-treated BAM1 treated with IAM and NEM, GSSG,IAM,NEM-treated BAM1 treated then treated
    with DTT, desalted BAM1 after all the treatments then treated with H2O2 and GSH.
  - 05_ponceau_glutathionylationofthesamecysteinebyGSSGandGSH.tif, same loading, the marker is Biorad Precision Plus Dual Color.

- 06_westernblot_antiglutathione_deglutathionylationbyDTT_GSH_GRX
  2 files in the folder:
  - 06_blot_antiglutathione_deglutathionylationbyDTT_GSH_GRX.jpg, the lanes of interest are all but the last from the left. Loading is: GSSG treated-BAM1, GSSG
    treated-BAM1 treated with 0.5 mM DTT, GSSG treated-BAM1 treated with 1 uM GRX C5 in presence of 2.5 mM GSH, 
    GSSG treated-BAM1 treated with 1 uM GRX S12 in presence of 2.5 mM GSH, GSSG treated-BAM1 treated with 2.5 mM GSH.
  - 06_ponceau_antiglutathione_deglutathionylationbyDTT_GSH_GRX.tif, coomassie staining of gel with same samples loaded, same loading as previous file, the marker is Biorad Precision Plus Dual Color.
  
- 07_westernblot_antiglutathione_glutathionerelease_afterGSSG
  3 files in the folder:
  - 07_blot_antiglutathione_glutathionerelease_afterGSSG.tif, 
    first five lanes from the left are of interest. Marker Biorad Precision Plus Dual Color, Control BAM1 untreated, GSSG treated BAM1 desalted and samples
    at 0 min after desalting, GSSG treated BAM1 desalted and samples at 20 min after desalting, GSSG treated BAM1 desalted and samples at 60 min after 
    desalting.
  - 07_blotmarker_antiglutathione_glutathionerelease_afterGSSG.jpg, same loading as previous file, the marker is Biorad Precision Plus Dual Color.
  - 07_coomassie_antiglutathione_glutathionerelease_afterGSSG.tif, same loading as previous file.

- 08_westernblot_antiglutathione_glutathionerelease_afterH2O2GSH
  2 files in the folder:
  - 08_blot_antiglutathione_glutathionerelease_afterH2O2GSH.tif, first five lanes from the left are of interest. Marker Biorad Precision Plus Dual Color,
    Control BAM1 untreated, GSH+H2O2 treated BAM1 desalted and samples at 0 min after desalting, GSH+H2O2 treated BAM1 desalted and samples at 30 min after 
    desalting, GSH+H2O2 treated BAM1 desalted and samples at 60 min after desalting.
  - 08_coomassie_antiglutathione_glutathionerelease_afterH2O2GSH.tif, coomassie staining of gel with same samples loaded, same loading as previous file.
  - 08_marker_antiglutathione_glutathionerelease_afterH2O2GSH.tif, the marker is Biorad Precision Plus Dual Color.

- 09_inhibition_kinetics_control_GSSGdesalting_H2O2GSHdesalting.csv, activity of BAM1 after the incubation for 1 h with 1 mM GSSG or 0.5 mM H2O2 and 2.5 mM GSH and desalting to remove the reagents.

- 10_GSHreleasefromBAM1.csv, amount of soluble thiols released from BAM1.

- 11_inhibitedBAM1reactivation_DTT_TRX_GRX.csv, reactivation of inhibited BAM1 by different chemical and biological reducing agents.

- 12_westernblot_antiBAM1_genotypesWT_epc2_miao
  3 files in the folder:
  - 12_blot_antiBAM1_genotypesWT_epc2_miao.jpg, loading from left to right is: Biorad Precision Plus Dual Color, wildtype 9h darkness, epc-2 9h darkness, miao 9h darkness, wildtype 1h light, epc-2 1h light, miao 1h light, wildtype 6h light, epc-2 6h light, miao 6h light.
  - 12_coomassie_antiBAM1_genotypesWT_epc2_miao.tif, same loading as previous file.
  - 12_ponceau_antiBAM1_genotypesWT_epc2_miao.tif, same loading as previous file.
  
- 13_ingelctivities
  4 files in the folder, for loading please refer to the related article:
  - 13_ingelctivities_epc2.jpg
  - 13_ingelctivities_miao.tif
  - 13_ingelctivities_wildtype.jpg
  - 13_ingelctivities_WTepc2miao.jpg

- 14_guardcell_starchgranulearea.csv
  Granule area is expressed as micrometers and was measured on samples collected at 11 hours of darkness, 1 hour of light, 3 hours of light.

- 15_stomata_aperture.csv
  Pore aperture is expressed as micrometers and was measured on samples collected at 0 hours of light and 1 hour of light.

All activity data are expressed as percentage of the maximal activity of BAM1 obtained after 1h of incubation at 37°C in presence of 20 mM reduced DTT, generally ranging aroun 1.6 micromols/(minutes x mg of protein). For materials and methods and further description refer to the article https://doi.org/10.1093/plphys/kiaf344