biostudies-arrayexpressAftab AhmadArabidopsis thalianahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15761Plant molecular improvement group at University of Shizuoka, Japan, previously identified 18 potential salt tolerance mutants using an activation-tagging approach at the cellular level. In one of these mutants, stc8, two genes AtbHLH106 and AtCRK1 were activated adjacent to the borders of T-DNA. AtbHLH106 is known to regulate salt stress by interacting with the G-box (CACGTG) present in the promoters of abiotic stress responsive genes. Here we report that AtCRK1 contributes to salt-stress regulation in Arabidopsis. In this study, TAIL-PCR revealed that AtCRK1 was present 5567 bp from the RB of the T-DNA and activated on both MS without salt and MS supplemented with 150 mM NaCl. Real-time PCR showed that AtCRK1 was enhanced 1.7 times on MS without salt and 2.7 times on MS with 150 mM NaCl. Quantitative expression in different tissues of plant and calli showed that AtCRK1 was highly expressed in leaves and flowers and least expressed in silique. In addition, expression analysis at different levels of NaCl showed that AtCRK1 was salt-inducible. Expression of AtCRK1 was significantly reduced in KO lines and the KO lines showed sensitive phenotype under salt stress, compared with Col-0 plants. We demonstrated that overexpressed calli of AtCRK1 were tolerant to NaCl and ABA, compared with control calli, and the survival percentage of overexpression lines was higher than in KO and wild-type plants. On vertical plates, shoots of overexpression lines did not show significantly stress-tolerant phenotypes, compared with wildtype and KO plants, but roots of overexpression plants were still longer than KO and wildtype plants. Furthermore, microarray analysis of stc8 mutant and KO lines showed that activation of AtCRK1 leads to upregulation of several heat shock proteins: myoinositol phosphate synthase gene, cold binding factor, ABC transporters, and CAT genes. Together, these findings indicate that AtCRK1 plays an important role during abiotic stress in plants through regulation of several abiotic responsive genes.biostudies-arrayexpressLabeling - We used the GeneChip IVT labelling kit for the labelling of cRNA, which involves the following steps: 1- Transfer the needed amount of template cDNA to RNase-free microfuge tubes and add the following reaction components in the order indicated in the table below. If more than one IVT reaction is to be performed, a master mix can be prepared by multiplying the reagent volumes by the number of reactions. Do not assemble the reaction on ice, since spermidine in the 10X IVT Labeling Buffer can lead to precipitation of the template cDNA. 2- Carefully mix the reagents and collect the mixture at the bottom of the tube by brief (~5 seconds) microcentrifugation. 3- Incubate at 37°C for 16 hours. To prevent condensation that may result from water bath-style incubators, incubations are best performed in oven incubators for even temperature distribution, or in a thermal cycler.Growth Protocol - For salt-stress evaluation, wildtype and back-transformed transgenic calli were screened on CIM containing hygromycin and 150 mM NaCl and kept in a growth chamber at 20℃. After three weeks of screening on CIM with 150 mM NaCl, growing calli were subsequently transferred to fresh CIM plates with the same stress level. For plants, seeds were sterilized, vernalized, and subsequently grown on MS containing 100 mM NaCl. Plants were photographed after 2–3 weeks of transfer on a salt-containing medium. For analysis of plants on vertical plates, seeds were grown on simple MS in vertical plates for four days at 20℃ under continuous light. Seedlings of equal length were transferred to MS plates for phenotypic analysis under NaCl, KCl, LiCl, or ABA, and photographs taken two weeks after transfer.Hybridization - The labelled and fragment cRNA was hybridized using the following protocol. The fragmented cRNA (10 microgram) was mixed with 2X hybridization mix 100 microliter, 3.3 ul control oligonuceleotide B2, 10ul 20X eukaryotic hybridization controls, DMSO 20ul and nuclease free water to make final reaction volume of 200ul. Incubate the probe array filled with Pre-Hybridization Mix at 45°C for 10 minutes with rotation. Transfer the hybridization cocktail that has been heated at 99°C, to a 45°C heat block for 5 minutes. Spin the hybridization cocktail at maximum speed in a microcentrifuge for 5 minutes to collect any insoluble material from the hybridization mixture. Remove the array from the hybridization oven. Vent the array with a clean pipette tip and extract the Pre-Hybridization Mix from the array with a micropipettor. Refill the array with the appropriate volume of the clarified hybridization cocktail, avoiding any insoluble matter at the bottom of the tube. Place probe array into the hybridization oven, set to 45°C. To avoid stress to the motor, load probe arrays in a balanced configuration around the axis. Rotate at 60. Hybridize for 16 hours.Sample Collection - For plants, seeds were sterilized, vernalized, and subsequently grown on MS containing 100 mM NaCl. Plants were photographed after 2–3 weeks of transfer on a salt-containing medium. For analysis of plants on vertical plates, seeds were grown on simple MS in vertical plates for four days at 20℃ under continuous light.Nucleic Acid Extraction - Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA).Scaning - 1. On the back of the probe array cartridge, clean excess fluid from around septa. 2. Carefully apply one Tough-Spot to each of the two septa. Press to ensure that the spots remain flat. If the Tough-Spots do not apply smoothly; that is, if you observe bumps, bubbles, tears or curled edges, do not attempt to smooth out the spot. Remove the spot and apply a new spot. 3. Insert the cartridge into the scanner and test the autofocus to ensure that the Tough-Spots do not interfere with the focus. If you observe a focus error message, remove the spot and apply a new spot. Ensure that the spots lie flat.MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsData Transformation - Data of Knockout lines and overexpression plants at 0 mM NaCl and 100 mM NaCl were normalized with Col-0 expression at 0mM NaCl and 100 mM NaCl.UnknownTranscriptomicsGenomicsProteomicsAffymetrix GeneChiptranscription profiling by arrayArabidopsis thalianaAftab AhmadfalseAtCRK1 positively regulates salt stress tolerance in Arabidopsis through the modulation of stress-responsive genesPlant molecular improvement group at University of Shizuoka, Japan, previously identified 18 potential salt tolerance mutants using an activation-tagging approach at the cellular level. In one of these mutants, stc8, two genes AtbHLH106 and AtCRK1 were activated adjacent to the borders of T-DNA. AtbHLH106 is known to regulate salt stress by interacting with the G-box (CACGTG) present in the promoters of abiotic stress responsive genes. Here we report that AtCRK1 contributes to salt-stress regulation in Arabidopsis. In this study, TAIL-PCR revealed that AtCRK1 was present 5567 bp from the RB of the T-DNA and activated on both MS without salt and MS supplemented with 150 mM NaCl. Real-time PCR showed that AtCRK1 was enhanced 1.7 times on MS without salt and 2.7 times on MS with 150 mM NaCl. Quantitative expression in different tissues of plant and calli showed that AtCRK1 was highly expressed in leaves and flowers and least expressed in silique. In addition, expression analysis at different levels of NaCl showed that AtCRK1 was salt-inducible. Expression of AtCRK1 was significantly reduced in KO lines and the KO lines showed sensitive phenotype under salt stress, compared with Col-0 plants. We demonstrated that overexpressed calli of AtCRK1 were tolerant to NaCl and ABA, compared with control calli, and the survival percentage of overexpression lines was higher than in KO and wild-type plants. On vertical plates, shoots of overexpression lines did not show significantly stress-tolerant phenotypes, compared with wildtype and KO plants, but roots of overexpression plants were still longer than KO and wildtype plants. Furthermore, microarray analysis of stc8 mutant and KO lines showed that activation of AtCRK1 leads to upregulation of several heat shock proteins: myoinositol phosphate synthase gene, cold binding factor, ABC transporters, and CAT genes. Together, these findings indicate that AtCRK1 plays an important role during abiotic stress in plants through regulation of several abiotic responsive genes.2025-08-30T00:00:00Z2026-05-27T16:36:26.431Z2025-10-29T05:23:27.128ZE-MTAB-15761EFO_0002768EFO_0002944EFO_0003814EFO_0003813EFO_0003789EFO_0005518EFO_0003816EFO_0003815