4.2. Arabidopsis Genotypes Growth Conditions and Salt Stress Induction

Arabidopsis seed sterilization, germination, and growth

We are sharing this detailed protocol as requested by Maryam Almazrouei at Iowa State University, USA). This is an elaborated version of the description of materials and methods 4.2. Arabidopsis Genotypes Growth Conditions and Salt Stress Induction (in: “Salinity Stress-Mediated Suppression of Expression of Salt Overly Sensitive Signaling Pathway Genes Suggests Negative Regulation by AtbZIP62 Transcription Factor in Arabidopsis thaliana”) published in the International Journal of Molecular Science (IJMS) by Rolly et al. (2020).

Seed sterilization

The Arabidopsis seed handling, sterilization, planting, growth, maintenance, and harvest were done following the protocol proposed by Rivero-Lepinckas, et al. [1]. In essence, seeds were sterilized with a mixture of 1 mL 50% commercial bleach and a drop (single drop from 1 mL tip) of 100X Triton solution in a 1.5 mL Eppendorf tube (e-tube), followed by a short vortex. Allow the seed to precipitate for a few seconds, remove the supernatant, and add 1 mL of distilled water to the 1.5 mL e-tubes containing seeds. Then, rinse three times by pipetting up and down for about a minute with distilled water immediately after vortexing. Prechill the seeds by incubating at 4°C overnight to break the dormancy. 

Seed sowing and growth conditions

Arabidopsis seeds were sown on the surface of peat moss substrate mixed with perlite and vermiculite (using a 1-mL tip and pipet) in 50-well trays, and treated with a preventive fungicide (before sowing). The watering regime was reduced to twice a week when plants developed their two true leaves. Arabidopsis seeds were grown for two weeks under growth chamber conditions (average temperature of 22°C and a 16h/8h light and dark cycle). Two-week-old Arabidopsis seedlings were carefully transplanted (1 seedling per well) using forceps (Note: avoid damaging the roots during transplanting) and grown for two to three more weeks. Water (tap water) was provided by sub-irrigation regularly to maintain adequate soil moisture during the first few days after transplanting.

Initial Screening for Selection of NaCl Concentration

Prior to exposing Arabidopsis plants to NaCl-induced salt stress at the rosette stage, an initial screening of genotypes was conducted to evaluate their response to gradient NaCl concentrations (50, 100, 150, or 200 mM) as described earlier [2]. We assessed the germination ability of each Arabidopsis mutant line and the wild-type (WT) Columbia zero (Col-0) for five days. We found that the germination of atbzip62 seeds was inhibited significantly under 150 mM NaCl compared to WT, atnced3, and atcat2. Likewise, 50 mM and 100 mM NaCl also showed a differential reduction in germination rate, after which recovery was observed. However, 200 mM NaCl severely inhibited the germination of atbzip62 seeds. Therefore, 150 mM NaCl was selected for downstream experiments. We then cross-evaluated the germination frequency data and the shoot and root growth patterns under 150 mM NaCl treatment. In addition, we calculated the cotyledon development frequency (CDF) by counting only the developed green seedlings (considering that some seeds might germinate but do not survive on stress media with time) [3,4] of the targeted genotypes on media with different concentrations of salt. We identified 150 mM NaCl as the most effective concentration for downstream experiments, due to the inhibitory effect recorded.

Salt stress induction and sampling

Arabidopsis plants were subjected to salinity stress at the rosette stage (the vegetative stage before the plant transitions to the bolting and flowering stage), and plants were treated with 150 mM sodium chloride (NaCl) solution by sub-irrigation. The control treatment was supplemented with tap water only of the same volume. For each Arabidopsis line (mutant lines and the Col-0 wild-type), a total of 30 plants were sown, 10 plants per replication. Samples for gene expression analysis by qPCR and physiological and biochemical analyses were collected at 3h and 6h after 150 mM NaCl treatment in triplicate. Another set of plants was kept under NaCl treatment for 10 days for phenotyping. The phenotypic response of genotypes was monitored daily to identify the early sensitivity response of target Arabidopsis mutant lines. The major symptoms are leaf wilting chlorosis, and necrosis.

1.            Rivero-Lepinckas, L.; Crist, D.; Scholl, R. Growth of plants and preservation of seeds. Arabidopsis Protocols 2006, 3-12.

2.            Rolly, N.K.; Imran, Q.M.; Lee, I.-J.; Yun, B.-W. Salinity stress-mediated suppression of expression of salt overly sensitive signaling pathway genes suggests negative regulation by AtbZIP62 transcription factor in Arabidopsis thaliana. International Journal of Molecular Sciences 2020, 21, 1726.

3.            Yun, B.-W.; Feechan, A.; Yin, M.; Saidi, N.B.; Le Bihan, T.; Yu, M.; Moore, J.W.; Kang, J.-G.; Kwon, E.; Spoel, S.H. S-nitrosylation of NADPH oxidase regulates cell death in plant immunity. Nature 2011, 478, 264-268.

4.            Imran, Q.M.; Hussain, A.; Lee, S.-U.; Mun, B.-G.; Falak, N.; Loake, G.J.; Yun, B.-W. Transcriptome profile of NO-induced Arabidopsis transcription factor genes suggests their putative regulatory role in multiple biological processes. Scientific Reports 2018, 8, 771.