Extended Data Fig. 1: Generation of transgenic Arabidopsis lines expressing various forms of CBF1, Trx-h2, and their mutants in trx-h2 and cbfs backgrounds.
From: Redox-dependent structural switch and CBF activation confer freezing tolerance in plants
a, A schematic diagram of CBFs genomic structure in Col-0 Arabidopsis and the DNA construct for generating cbfs null-mutant using the CRISPR-Cas9 system. b, DNA constructs used to overexpress CBF1-Myc under the control the CaMV 35 S promoter (CBF1-MycOE) and to express CBF1-Myc under the control of its native promoter (PCBF1) (PCBF1:CBF1-Myc) in Col-0 and trx-h2 backgrounds; both constructs utilized the nopaline synthase (NOS) terminator. c,d, Expression analysis of CBF1 and Trx-h2 in the overexpression lines (CBF1-MycOE/Col-0 or CBF1-MycOE/trx-h2) (c) and transgenic lines (PCBF1:CBF1-Myc/Col-0 and PCBF1:CBF1-Myc/trx-h2) (d) by RT-PCR. e, Genomic structure of the Arabidopsis T-DNA insertion knockout mutant, trx-h2 (SALK_079507). The T-DNA insertion site and forward (For)/reverse (Rev) primer-binding sites are indicated. ATG and TAA represent the start and stop codons, respectively. f, Confirmation of Trx-h2 knockout in trx-h2 plant by PCR-based genotyping. g, Schematic diagrams of DNA constructs used for the overexpression of V5-tag fusions of Trx-h2, Trx-h2(G/A), and Trx-h2(C/S) under the control of the CaMV 35 S promoter and octopine synthase (OCS) terminator. Agrobacterium tumefaciens strain GV3101 carrying each construct was transformed into the trx-h2 mutant to generate Trx-h2-V5OE/trx-h2, Trx-h2(G/A)-V5OE/trx-h2, and Trx-h2(C/S)-V5OE/trx-h2 overexpression lines. h,i, Expression levels of Trx-h2 mRNA (h) and Trx-h2 protein (i) in various transgenic Arabidopsis analyzed by RT-PCR and western blotting. To quantitatively compare the expression levels of Trx-h2 mRNA and the corresponding protein among various genotypes, two different blots with short (middle panel) and long (upper panel) exposure times in the ChemiDoc™ MP System were shown. j–l, A schematic diagram of DNA construct for generating Trx-h2-HAOE/cbfs lines (j). Expression levels of Trx-h2 mRNA (k) and Trx-h2 protein (l) in Trx-h2-HAOE/cbfs (lines #1 and #2) plants were analyzed by RT-PCR and western blotting, respectively. In (h–l), Tubulin and Rbc L were used as loading controls.
