The AaCBF4-AaBAM3.1 module enhances freezing tolerance of kiwifruit (Actinidia arguta)

Abstract

Beta-amylase (BAM) plays an important role in plant resistance to cold stress. However, the specific role of the BAM gene in freezing tolerance is poorly understood. In this study, we demonstrated that a cold-responsive gene module was involved in the freezing tolerance of kiwifruit. In this module, the expression of AaBAM3.1, which encodes a functional protein, was induced by cold stress. AaBAM3.1-overexpressing kiwifruit lines showed increased freezing tolerance, and the heterologous overexpression of AaBAM3.1 in Arabidopsis thaliana resulted in a similar phenotype. The results of promoter GUS activity and cis-element analyses predicted AaCBF4 to be an upstream transcription factor that could regulate AaBAM3.1 expression. Further investigation of protein-DNA interactions by using yeast one-hybrid, GUS coexpression, and dual luciferase reporter assays confirmed that AaCBF4 directly regulated AaBAM3.1 expression. In addition, the expression of both AaBAM3.1 and AaCBF4 in kiwifruit responded positively to cold stress. Hence, we conclude that the AaCBF-AaBAM module is involved in the positive regulation of the freezing tolerance of kiwifruit.

Fig. 1. Phenotypes of Actinidia arguta plants under cold stress and the expression of AaBAM3.1 in A. arguta in response to cold stress.

a Chlorophyll fluorescence image. b Corresponding F_v/F_m over the course of a 5-day low-temperature treatment. c Electrolyte leakage of Actinidia arguta plants after being subjected to 5 days of low-temperature treatment. d The time course of expression levels in the leaves was analyzed via RT-qPCR in response to cold stress. e Tissue-specific expression in the vegetative growth stage was analyzed via RT-qPCR. f AaBAM3.1 expression in the shoots was analyzed between the growth stage and dormant stage under cold stress. S1: 15 April; S2: 15 November; S3: 7 December; S4: 23 December; S5: 15 January; S6: 15 March

Fig. 2. Subcellular localization of AaBAM3.1-GFP and BAM activity in pGEX4T-1.

a Tobacco leaves were injected with the control (35S-GFP) or recombinant plasmid (35S-AaBAM3.1-GFP) and visualized under a confocal microscope. GFP imaging and autofluorescence are shown. b SDS-PAGE analysis of the expression of the recombinant protein in E. coli. The total proteins from bacteria and the purified recombinant protein from the soluble crude extract were separated via 10% SDS-PAGE and stained with Coomassie brilliant blue. M, protein size marker (15–150 kDa). Lane 1 contains proteins from uninduced cells, and lane 2 contains proteins from induced cells. The OD540 results obtained using a BAM enzyme kit showed that the recombinant protein presented enzymatic activity

Fig. 3. Characterization of freezing tolerance of Arabidopsis harboring the 35 S::AaBAM3.1 construct.

a Phenotypes of the overexpression lines (#1, #2, and #3) and WT plants under cold stress (−2 °C for 2 h). b Diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining of the overexpression lines (#1, #2, and #3) and WT plants under cold stress (−2 °C for 2 h). c, m Fv/Fm of Arabidopsis over the course of 2 h of low-temperature treatment. Color barcodes are shown below the images. d–l Survival rates and relative electrolyte leakage (REL), proline (PRO), soluble sugars (SS), malondialdehyde (MDA), BAM activity, superoxide dismutase (SOD) activity, peroxidase (POD) activity and catalase (CAT) activity measured under cold stress. n Expression of AaBAM3.1 in Arabidopsis under cold stress

Fig. 4. Cold tolerance characterization of A. chinensis harboring the 35 S::AaBAM3.1 construct.

a Phenotypes of the overexpression lines (#1, #2, and #3) and WT plants under cold stress (2 °C for 2 h). b Diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining of the overexpression lines (#1, #2, and #3) and WT plants under cold stress (2 °C for 2 h). c, i Fv/Fm of kiwifruit over the course of a 2 h low-temperature treatment. A colored barcode is shown below the images. d–g Relative electrolyte leakage (REL), malondialdehyde (MDA), soluble sugars (SS) and BAM activity levels were measured under cold stress. (h) Expression of AaBAM3 in kiwifruit under cold stress

Fig. 5. Analysis of AaBAM3.1 promoter activity in transgenic Arabidopsis under cold stress.

a Schematic diagram of PpCAMBIA3301-P_BAM::GUS. b Analysis of GUS activity in transgenic Arabidopsis expressing the AaBAM3.1 promoter under cold stress (4 °C). I: WT; II: transgenic plant (4 °C for 0 min); III: transgenic plant (4 °C for 10 min); IV: transgenic plant (4 °C for 20 min); V: transgenic plant (4 °C for 30 min); VI: transgenic plant (4 °C for 60 min). c GUS mRNA expression was measured via RT-qPCR at 4 °C for 0 min, 10 min, 20 min, 30 min, and 60 min

Fig. 6. Analysis of AaCBF4 sequence and protein characterization.

a AaCBF expression was analyzed under cold stress. b Transactivation assay of CBFs in yeast. The fusion proteins of the GAL4 DNA-binding domain (BD) and full-length CBFs were expressed in yeast strain AH109. The empty vector pGBKT7 was used as a negative control. A culture solution of the transformed yeast was plated on SD/-Trp solid media, SD/-Trp/-Ade/-His solid media and SD/-Trp/-Ade/-His solid X-α-Gal solid media, as indicated. c. Subcellular localization of AaCBF4 in tobacco. Plant leaves were injected with Agrobacterium tumefaciens GV3101 containing the AaCBF4:eGFP vector. After 3 days, the leaves were observed for fluorescence with a confocal microscope (FV1000; Olympus, Tokyo, Japan). The nuclei were stained with DAPI

Fig. 7. AaCBF4 binds to the CRT/DRE cis-element in the promoter of AaBAM3.1.

a Structure of the AaBAM3.1 promoter regions. A CRT/DRE cis-element was identified at the -1166 bp promoter position. b Yeast one-hybrid analysis using pGADT7-AaCBF4 as prey, pPBAM3.1-AbAi, and p-AbAi (empty vector) as bait and pGADT7-p53 and p53-AbAi as positive controls. c Transient glucuronidase (GUS) expression analysis using 35S::AaCBF4 as an effector and PBAM3.1::GUS as reporters. GUS expression was visualized in representative tobacco leaves transformed with different combinations of the effector and reporter constructs. d Luciferase activity analysis using 35S::AaCBF4 as the effector and 35S::LUC and PBAM3.1-35S::LUC as the reporters. REN and LUC activities resulting from different combinations of effector and reporter constructs were measured