Short heat shock factor A2 regulates heat resistance and growth balance in Arabidopsis

The target DNA fragments were obtained via PCR with the Taq enzyme (Takara, Japan) and subsequently cloned and inserted into the corresponding vectors with the ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China).

The S-HsfA2 and S-HsfA2L-A coding sequences lacking stop codons were amplified from Arabidopsis genomic DNA via S-HsfA2-Flag-F/-R and S-HsfA2-Flag-F/S-HsfA2mut-Flag-R, respectively. Similarly, the coding sequence of HSP17.6B lacking the stop codon ‘TGA’ was amplified via HSP17.6B-Flag-F/-R. The PCR products were cloned and inserted into the XbaI/BamHI site 35S:3×Flag (our laboratory) and sequenced via GUS-R to generate 35S:S-HsfA2-Flag (S-HsfA2-OE), 35S:S-HsfA2L-A-Flag (S-HsfA2L-A-OE), and 35S:HSP17.6B-Flag (HSP17.6B-OE).

We used a fusion PCR method to generate the 35S:S-HsfA2-RNAi construct via pUCCRNAi. First, the PCR1 products (forward intron 1a of HsfA2, 104 bp) were amplified from the BD-S-HsfA2 plasmid via the primers F1/R1. Second, the PCR2 products (intron 1 of potato GA20 oxidase, 219 bp) were amplified from pUCCRNAi via the primers F2/R2. Third, the PCR3 products (reverse intron 1a of HsfA2, 105 bp) were amplified from the BD-S-HsfA2 plasmid via the primers F3/R3. Fourth, fusion PCR was performed in a total volume of 50 μl containing 33 ng of each purified PCR product (template) for 5 cycles at an annealing temperature of 55°C (without primers) followed by 25 cycles (adding F1/R3) at 65°C. The fusion fragments were cloned and inserted into the BamHI/SacI site of pBI121 and sequenced via GUS-R.

For the expression vector constructs, HsfA2-RFP-F/-R, S-HsfB1-RFP-F/-R, and S-HsfB2a-RFP-F/-R were used to amplify the coding regions of HsfA2, S-HsfB1, and S-HsfB2a from cDNA, which were subsequently cloned and inserted into the BamHI/SpeI sites of pCAMBIA1300. The resulting 35S:HsfA2-RFP, 35S:S-HsfB1-RFP, and 35S:S-HsfB2a-RFP were sequenced via RFP-R. The expression vectors 35S:S-HsfB1 and 35S:S-HsfB2a were constructed via antisense RNA knockdown technology and reverse primers anti-S-HsfB1-F/-R and anti-S-HsfB2a-F/-R. The coding region of S-HsfA4c lacking the stop codon TAG was amplified from cDNA via the primers S-HsfA4c-GFP-F/-R and cloned and inserted into the BglII/SpeI sites of pCAMBIA1302 (Clontech) to generate 35S:S-HsfA4c-GFP. The coding region of S-HsfA2 lacking the stop codon TAG was amplified from genomic DNA via the primers S-HsfA2-GFP-F/-R and cloned and inserted into the NcoI/SpeI sites of pCAMBIA1302 (Clontech) to generate 35S:S-HsfA2-GFP.

For the AD constructs, the coding sequences of 19 HSFs, S-HsfA2, and S-HsfA4c were amplified from heat-treated Arabidopsis cDNA via the corresponding primers, inserted into the BamHI/BglII site of pGAD424 (Clontech) to generate corresponding AD-HSF fusion vectors and identified by sequencing.

For bait construction, 3×HRE, 3×mHRE, HSE (Enoki and Sakurai, 2011), and mHSE were synthesized by Shanghai Sangon Biotechnology and subsequently cloned and inserted into pHIS2.1 (Clontech). The PCR products were cloned and inserted into the EcoRI/SpeI site of the pHIS2.1 vector (Clontech) and sequenced via pHIS2.1 forward or reverse primers.

For the BD constructs, the S-HsfA2 and S-HsfA2L-A coding sequences were amplified from Arabidopsis genomic DNA via BD-S-HsfA2-F/-R and BD-S-HsfA2-F/BD-S-HsfA2mut-R, respectively. The S-HsfA4c, S-HsfA4c△LRD, S-HsfB1, and S-HsfB2a coding sequences were amplified from cDNA via BD-S-HsfA4c-F/-R, BD-S-HsfA4c-F/BD-S-HsfA4cΔLRD-R, BD-S-HsfB1-F/-R, and BD-S-HsfB2a-F/-R, respectively. The corresponding PCR products were subsequently cloned and inserted into the EcoRI/PstI sites of pGBKT7 (Clontech) to generate the BD-S-HsfA2, BD-S-HsfA2L-A, BD-S-HsfA4c, BD-S-HsfA4cΔLRD, BD-S-HsfB1, and BD-S-HsfB2a constructs. T7 primers were used to sequence the BD fusion vectors.

To generate the glutathione S-transferase (GST)-tagged expression plasmid pGEX4T-S-HsfA2. Using the BD-S-HsfA2 plasmid as a template, the PCR product was amplified with GST–S-HsfA2-F/-R and subsequently cloned and inserted into the BamHI/SalI sites of pGEX-4T.

For the His6-tagged expression plasmids, the HsfA2, S-HsfA2, S-HsfA4c, S-HsfB1, and S-HsfB2a coding sequences were amplified from cDNA via His6-HsfA2-F/-R, His6-HsfA2-F/His6-S-HsfA2-R, His6-S-HsfA4c-F/-R, His6-S-HsfB1-F/-R, and His6-S-HsfB2a-F/-R, respectively. The PCR products were inserted into the BamHI/SalI sites of pET-28a(+) to generate His6-HsfA2, His6-S-HsfA2, His6-S-HsfA4c, His6-S-HsfB1 and His6-S-HsfB2a. Then, using His6-HsfA2 and His6-S-HsfA2 as templates, the DBD and tDBD coding sequences were amplified with His6-DBD-F/-R and His6-DBD-F/His6-tDBD-R. The PCR products were also cloned and inserted into the BamHI/SalI sites of pET-28a(+) to obtain His6-DBD and His6-tD. The plasmids were subsequently sequenced via the T7 primer.

We used an asymmetric overlap extension PCR method to construct the HRE-35Sm:GUS and mHRE-35Sm:GUS plasmids via 3×HRE-35Sm-F or 3×mHRE-35Sm-F and the common reverse primer HRE-35Sm-R. The PCR products were subsequently cloned and inserted into the HindIII/XbaI sites of pBI121 instead of the 35S promoter. The recombinant reporter plasmids were subsequently sequenced via GUS-R primers for validation.

The WT HSP17.6B promoter (HSP17.6Bp, –637/+1) was amplified from Arabidopsis genomic DNA via HSP17Bp-F/-R. The resulting PCR product was subsequently cloned and inserted into the HindIII/BamHI sites of pBI121 and subsequently sequenced via GUS-R to generate HSP17.6Bp:GUS. On the basis of the HSP17.6Bp:GUS, fusion PCR was performed to generate HSP17.6BpΔHRE:GUS, HSP17.6BpΔHSE:GUS, and HSP17.6BpΔHREΔHSE:GUS via HSP17BpΔHRE-F/-R, HSP17.6BpΔHSE-F/-R, and HSP17.6BpΔHREΔHSE-R, respectively. The above PCR products were cloned and inserted into the HindIII/BamHI sites of pBI121 and sequenced by GUS-R.

A short, truncated 135-bp HSP17.6B promoter fragment (sHSP17.6Bp) was amplified from Arabidopsis genomic DNA via sHSP17.6Bp-LUC-F/-R and inserted into the HindIII/SpeI sites of pGreenII0800-LUC (LUC vectors containing the REN gene under the control of the 35S promoter as an internal control) to generate the reporter vector sHSP17.6Bp:LUC.

For the BiFC constructs, the coding regions of S-HsfA2 and HsfA2 and a series of HsfA2 genes progressively truncated from the N-terminus were amplified from AD-S-HsfA2 and AD-HsfA2 via cYFP-S-HsfA2-F/-R, nYFP-HsfA2-F/-R, nYFP-HsfA2ΔN-F and nYFP-HsfA2ΔNΔDBD-F, respectively. The above PCR products were cloned and inserted into the XbaI/BamHI sites of modified pCAMBIA1300 containing the cYFP or nYFP coding sequence to generate the corresponding constructs S-HsfA2-cYFP, HsfA2-nYFP, HsfA2ΔN-nYFP, and HsfA2ΔN-DBD-nYFP. The above vectors were identified by sequencing nYFP-R or cYFP-R.