Plant materials and flower collection
The flower buds of J. curcas L. were collected fromZhenfeng, Guizhou Province, China (36°14050.2′N, 87°51047.8′E). Flower buds for morphological and microscopic observation were temporarily stored in the mixture of acetaldehyde acetic acid 50% alcohol (4: 6: 90, v/v), and for RNA extraction were temporarily stored in RNAlocker (Tiandz, Inc, Beijing China). All samples were placed on ice.
JcGASA6isolation and sequence analysis
The full-length cDNA of JcGASA6 was cloned from flower bud by RACE-pcr, then the obtained cDNA sequences were aligned in NCBI database (Accession number: KU500008).
Sequence of the GASA family of Arabidopsis thaliana was obtained from NCBI database (Table S1). Multiple sequence alignment used DNAMAN, phylogenetic tree constructed by MEGA 6.0, and signal peptide predicted by UniProt. (https://www.uniprot.org/peptidesearch/).
Subcellular localization, expression and identification of JcGASA6 protein
The full-length cDNA of JcGASA6 was fused with the pBWA(V)HS-osGFP. then recombinant plasmidp BWA(V)HS-JcGASA6-osGFPwas transfected transformed into rice protoplasts by PEG ( polyethylene glycol ). The protoplasts were observed by confocal laser microscope under the excitation of 480nm wavelength after dark culture at 28℃for 48 hours (FV10-ASWOLYMPUS, Japan)(Hichri et al., 2010). Gamatip protein located in the tonoplast was used as a marker(Besse et al., 2011). All primers used for subcellular localization are listed in Table S2.
The coding sequence of JcGASA6 was amplified by specific primers (JcGASA6ex-F and JcGASA6ex-R), then the coding sequence was connected to the expression vector pCold II after digesting with Nde I and Hind III. The sequence of recombinant plasmid JcGASA6-pCold II was examined using vector primers (pCold II-F and pCold II-R) (Table S2). The recombinant plasmid was transformed into E. coli competent cells (BL21 or ESLA) to overexpress JcGASA6, and the empty vector pCold II was transformed into BL21 or ESLA as the control. Then the BL21 or ESLA were cultured at 16℃, and isopropyl β-D-thiogalactoside (IPTG) was used as an expression inducer. The details of method same as previously reported (Li et al., 2020).
The expression products of JcGASA6 in E. coli were analyzed by SDS-PAGE electrophoresis. Cut off the SDS-PAGE glue containing JcGASA6 protein, and wash the SDS-PAGE glue with ultrapure water and decolorization with acetonitrile mixture. The decolorized SDS-PAGE glue was digested by trypsin overnight at 37 ℃ to form enzymolysis solution, then the enzymolysis solution was identified by LC-MS/MS method. The method was consistent with the previously reported (Liu et al., 2012). The three-dimensional structure of protein was analyzed by the SWISS-MODEL database (https://www.swissmodel.expasy.org/interactive).
Promoter isolation and analysis, construction of cDNA library for Yeast-Hybrid System
The promoter sequence of JcGASA6 was obtained from the published genomic database (ID: 105640538), and PlantCARE was used for promoter sequence analysis (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The cDNA library of Yeast-Hybrid System was constructed by mixed RNA from flowers at different developmental stages. The primary library was constructed using attB2 as linker and ATTB-A1, ATTB1-B and ATTB1-C as primers. The clone number of primary librarywas8.04×106 cfu. The plasmid of primary library was extracted and transferred into DH10B by the electrotransfer method, and then the secondary library was obtained. The clone number of secondary library was 1.31 × 107 cfu. The method is consistent with that previously reported(Mitsuda et al., 2010) (Table S3).
Yeast-one hybrid (Y1H) and dual-luciferase assay
The promoter of JcGASA6 was amplified using specific primer pro-JcGASA6-F/R, then fusion to the pHIS2 (Table S4). Co-transferred fusion plasmid and secondary library to Y187 yeast system, then screen the upstream regulator of JcGASA6. The screening process refers to the previous method (Shi et al., 2021). Three upstream regulators (JcFLX, JcERF1 and JcPYL9) were screened from the secondary library (Table S4). The interaction between JcGASA6 promoter and these three regulators was verified one by one with Y187 yeast system (Shi et al., 2021).pGADT7 and p53-pHis2 were co-transformed into Y187 as negative controls. pGADT7-53 and p53-pHis2were co-transformed into Y187 as positive controls. The promoter self-activation of JcGASA6 was detected using Y187 withplasmidspGADT7 and JcGASA6-Pro-pHis2.
The primer JcCASA6-luc-F/R was used to construct pGreenII 0800-JcGASA6-luc, and three primers (AP2-F/R, FLX-F/R and PYL9-F/R) was used to construct regulators (pGreenII 62-JcFLX-SK, pGreenII 62-JcERF1-SK and pGreenII 62-PYL9-SK). Co-transferred pGreenII 0800-proJcGASA6-luc and regulator into tobacco leaves, then detected the fluorescence value (Dual-Luciferase Assay System, Promega) (Table S4) (Shi et al., 2021). pGreenII 62-SK and pGreenII0800-Luc were co-transformed into tobacco leaves as negative controls.
Yeast-two hybrid(Y2H) and bimolecular fluorescence complementation (BiFC) assay
Full-cDNA JcGASA6 fusion with pGBKT7 by using GASA6-GBK-F/R primer, then both of pGBKT7-JcGASA6and pGADT7-AD were transferred into AH109 by LiAc method, then the AH109was cultured for the detection of self-activation activity. Co-transferredpGBKT7-JcGASA6 and secondary library to AH109 for screening the interaction proteins of JcGASA6. Five proteins were screened, including JcCNR8, JcAMs, JcAPRR2, JcFRI and JcSIZ1. The interaction between the five proteins andJcGASA6 were verified by one-to-one in AH109(Liu et al., 2021) (Table S5). pGADT7 and pGBKT7 were co-transformed into AH109 as negative controls. pGADT7-53 andpGBKT7-T were co-transformed into AH109 as positive controls.
Full-cDNA JcCNR8, JcSIZ1,JcAMs, and JcAPRR2were fused with PSPYCE-35Srespectively by using specific primer, and Full-cDNA JcGASA6was fused with PSPYNE-35S. Co-transferred PSPYNE-35S-JcGASA6 and PSPYCE-35S-JcCNR8/PSPYCE-35S-JcSIZ1/PSPYCE-35S-JcAMs/PSPYCE-35S- JcAPRR2 into EHA105. The five types of EHA105 infected tobacco leaves respectively, then the fluorescence signal was detected 72 hours after infection(Liu et al., 2021)(Table S5).PSPYCE-35S and PSPYNE-35S were co-transformed into tobacco leavesas negative controls. PSPYCE-35S-bZIP63 and PSPYNE-35S-bZIP63 were co-transformed into tobacco leaves as positive control.
Morphology and microscopic observation of flower
Flower buds were classified according to their length, dissected under stereoscope, observed and photographed. Since the flower bud of undifferentiated stage is too small to be observed clearly under stereoscope, it was observed under the scanning electron microscope. The undifferentiated flower were fixed in the mixture of formaldehyde-acetic acid-50% ethanol, and then dissected and observed by electron microscope (Xu et al., 2016). According to the classification, paraffin sections of these flower buds were made (Chen et al., 2016) (Table 1).
Expression of JcGASA6 during flower development determined by qRT-PCR
The total RNA of flower was extracted by RNA isolation kit (Omega Bio-Tek, Beijing, China), qualified RNA was used to synthesize the first strand cDNA (TaKaRa, Beijing, China). PCR amplification was used Bio-Rad CFX system (Bio-Rad, USA). Beta-tubulin and actin as internal control. The 2 - ΔΔ CT method was used to calculate the relative expression of JcGASA6 (Shen et al., 2019). All primers used for PCR amplification are listed in Table S6 and each sample reaction was repeated three times.
Overexpression of JcGASA6 in Nicotiana tabacum L.
Fusion full-length cDNA of JcGASA6 with the pBWA(V)KS-GUS. The recombinant plasmid (pBWA(V)KS-JcGASA6-GUS) was transformed into agrobacterium tumefaciens (GV3101), then positive agrobacterium tumefaciens were screened. Tobacco (K326) leaves infected by GV3101 were cultured in dark at 25℃ for 2 days. After tobacco leaves were differentiated into seedlings, positive tobacco was detected (Fig. S1), and wild-type tobacco was used as control (Gomez et al., 2020) (Table S6).