Additional file 1 of Metabolic activities and molecular investigations of the ameliorative impact of some growth biostimulators on chilling-stressed coriander (Coriandrum sativum L.) plant
Creators: name: Hassanein, Raifa A.
name: Hussein, Omaima S.
name: Abdelkader, Amal F.
name: Farag, Iman A.
name: Hassan, Yousra E.
name: Ibrahim, Mohamed
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: Plant Biology

description: Additional file 1: Suppl. Fig. 1. Impact of alleviation treatments on growth parameters of chilling-stressed coriander plants at the flowering stage. Suppl. Fig. 2. Impact of alleviation treatments on TCPs profiles of chilling-stressed (6 °C ± 0.5) coriander plants at the vegetative stage (75 days old). Protein extraction procedure for each physiological status (control, stress, etc.) was performed from the leaves of 3 biological and 3 technical replicates. Each technical replicate represented one biological replicate. Each biological replicate is composed of the collection of leaves of 10 plants. The latter collected leaves represented one technical replicate. The protein extraction was carried out from each technical replicate independently. Finally extracted proteins from the 3 technical replicates were pooled together. Pooled sample were quantified and equally loaded into 10% SDS-PAGE consequently after measuring its concentration. Aliquots of pooled sample were kept as − 80 °C after the short snap for 30 s in Liquid Nitrogen. Coriander control (Lane 1) seeds and chilling-stressed (Lane 2) ones were subjected to pre- soaking in 80 mM Pot. silicate (Lane 3), 50 mg l− 1 HA (Lane 4) or soaked in water after exposed to 50 Gy gamma irradiation (Lane 5). TCPs were then extracted, fractionated on 10% SDS-PAGE for season 1 (Panel a) and season 2 (Panel b), and finally stained with CoBB stain. The numbers shown on the left-handed side of the figures indicate molecular weight standards in kDa (High-Range SDS-PAGE Standards, GeneON, Ludwigshafen, Germany). Red arrowheads refer to induced upregulated polypeptides detected in “chilling+HA”, but not in “Chilling” and/or other chilling plus alleviation elements. The asterisk refers to approximate molecular weight of RuBisCOLS. ImageJ software (IJ 1.46r) was used for image processing and analysis of the electrophoretic running of ascending concentration series of BSA (as protein size standard) to quantify RuBisCOLS concentration in (μg) downward of every panel. Full-length gels are presented in Supplementary Fig. (2a and b). Black arrowhead refers to the protein band at approximate molecular weight of 180 kDa used for normalization of quantified RuBisCO LS protein expression. Suppl. Fig. 3 Full-length original and unprocessed Immunoblot analysis of the expression of chloroplast marker proteins. TCPs were extracted and fractionated by SDS-PAGE and immunodecorated against α-Toc34, α-Toc75, eHSP70, and actin primary antibody in a dilution of 1:10,000 as demonstrated in [51]. Cropping of the shown blots was performed properly for sake of clarity and focusing the information. Full-length blots are accompanied the manuscript as Supplementary Fig. 3. Protein extraction procedure for each physiological status (control, chilling stressed, etc.) was performed from the leaves of 3 biological replicates and 3 technical replicates. Each technical replicate represented one biological replicate. Each biological replicate comprises the collection of leaves of 10 plants. The protein extraction was carried out from each technical replicate independently. Finally extracted proteins from the 3 technical replicates were pooled together. Pooled sample were quantified, equally loaded into 10% SDS-PAGE, and blotted onto PVDF membrane as shown in methods section. Consequently, aliquots of pooled sample were kept as − 80 °C after the short snap for 30 s in Liquid Nitrogen. Suppl. Fig. 4 Fractionation of chloroplast protein complexes by HDN-PAGE technique profiles of chilling-stressed (6 °C ± 0.5) coriander plants at the vegetative stage. Panel (a) Coriander control (Lane 2, 7) seeds and chilling-stressed (Lane 3) ones were subjected to pre- soaking in 80 mM Pot. silicate (Lane 5), 50 mg l− 1 HA (Lane 5) or soaked in water after exposed to 50 Gy gamma irradiation (Lane 6). Extracted protein complexes (especially RuBisCO enzyme complex) were then fractionated on 4–12% gradient native HDN-PAGES, and finally stained with CoBB stain. Native molecular weight standards (HMW Native Marker kit, GE Healthcare) (lane 1) was loaded and denoted by numbers left-handed of the figure indicating molecular weight standards in kDa. ImageJ software (IJ 1.46r) was used for image processing and analysis of the electrophoretic running of ascending concentration series of BSA (as protein size standard) to quantify RuBisCOLS concentration and Toc complex bands in (μg) were depicted downward of the figure. Cropping of the HDN-PAGE shown was performed for sake of clarity and focusing the information. Full-length original HDN-PAGE gel is presented in Supplementary Fig. 4 (Repeat 1, 2). Black arrowhead refers to the protein band at approximate molecular weight of 300 kDa used for normalization of quantified Toc and RuBisCO protein complexes. Original full length HDN-PAGEs; Repeat 1 (Panel a) and Repeat 2 (Panel b) were shown without cropping for sake of clarity. Repeat 1 and 2 were executed to manifest the consistency and reproducibility of the given protein complexes. Panel (b) specifically showed also the loading of different concentrations of solubilized protein complexes of control sample for optimization and high-resolution purposes.