MaMAPK3-MaICE1-MaPOD P7 pathway, a positive regulator of cold tolerance in banana

Abstract

Background: Banana is a tropical fruit with a high economic impact worldwide. Cold stress greatly affects the development and production of banana.

Results: In the present study, we investigated the functions of MaMAPK3 and MaICE1 involved in cold tolerance of banana. The effect of RNAi of MaMAPK3 on Dajiao (Musa spp. 'Dajiao'; ABB Group) cold tolerance was evaluated. The leaves of the MaMAPK3 RNAi transgenic plants showed wilting and severe necrotic symptoms, while the wide-type (WT) plants remained normal after cold exposure. RNAi of MaMAPK3 significantly changed the expressions of the cold-responsive genes, and the oxidoreductase activity was significantly changed in WT plants, while no changes in transgenic plants were observed. MaICE1 interacted with MaMAPK3, and the expression level of MaICE1 was significantly decreased in MaMAPK3 RNAi transgenic plants. Over-expression of MaICE1 in Cavendish banana (Musa spp. AAA group) indicated that the cold resistance of transgenic plants was superior to that of the WT plants. The POD P7 gene was significantly up-regulated in MaICE1-overexpressing transgenic plants compared with WT plants, and the POD P7 was proved to interact with MaICE1.

Conclusions: Taken together, our work provided new and solid evidence that MaMAPK3-MaICE1-MaPOD P7 pathway positively improved the cold tolerance in monocotyledon banana, shedding light on molecular breeding for the cold-tolerant banana or other agricultural species.

Keywords: Antioxidant capacity; Cold tolerance; MaICE1; MaMAPK3; MaPOD P7.

Fig. 1

(A) Phenotypes of transgenic ‘Dajiao’ plants (MRi-15 and MRi-22) and wild type (WT) subjected to cold treatment (10 °C for 5 and 7 days), followed by recovery at the normal growth temperature for 2 days. (B) MDA content in WT and transgenic Dajiao (RNAi-22 and RNAi-15) before and after chilling treatment. Data represent the means ± SE of at least three replicates. Letters indicate significant differences from 0 h chilling treatment according to the Student–Newman–Keuls test (P < 0.05). (RE means recovery for 2 days)

Fig. 2

Analysis of relative expression levels of MAPK3 and cold-relative genes by quantitative real-time PCR in wild type and transgenic ‘Dajiao’ lines (MRi-15 and MRi-22) before and after cold treatment. Data represent the means ± SE of at least three replicates. Letters indicate significant differences from 0 h cold treatment according to the Student–Newman–Keuls test (P < 0.05) (RE means recovery for 2 days)

Fig. 3

(A) Physical interactions between MaICE1 and MaMAPK3 using Y2H Assays. (The sequences of MaICE1 and MaMAPK3 were cloned from ‘Dajiao’) (B) Biomolecular fluorescence complementation visualization of the interaction between MaMAPK3 and MaICE1. (B-a) Schematic diagrams of the constructs used for biomolecular fluorescence complementation assay. The WT1 and WT2 were two negative controls, respectively. (B-b) The images indicate an interaction between MaMAPK3 and MaICE1 by biomolecular fluorescence complementation

Fig. 4

Cold tolerance assay of transgenic Cavendish banana plants. (A) Phenotypes of transgenic Cavendish banana plants (#11 and #13), 'Dajiao' and the wild-type (WT) before and after cold treatment (10 °C for 48 h), followed by recovery in an ambient environment for 3 days (B) Injured area of WT banana and transgenic plants analyzed after cold treatment (bar: 3.5 cm). Effect of MaICE1 expression in banana on levels of (C) electrolyte leakage, (D) pro and (E) MDA. Two-month-old banana plants of WT, ‘Dajiao’ and transgenic Cavendish banana plants (#11 and #13) were exposed to 10 °C for 48 h. Leaves were collected before and after cold treatment. FW, fresh weight. Each value represents the means of three biological replicates, and vertical bars indicate the SE. **P < 0.01. ***P < 0.001

Fig. 5

(A) Physical interactions between MaICE1 and MaPOD P7 using Y2H assays. (B) Biomolecular fluorescence complementation visualization of the interaction betweenMaICE1 and MaPOD7. In the BiFC assay, MaICE1 and MaPOD7 DNA fragments were fused with the split mVenus N-terminal and C-terminal in the vectors of pRTVnVN and pRTVnVC, which the marker genes ECFP and mCherry were added into the pRTVnVN and pRTVnVC vectors, respectively. After co-transfection, we detected clear yellow fluorescence signals on the nucleus of the transfected protoplasts, indicating the formation of reconstituted mVenus through the MaICE1-MaPOD interaction

Fig. 6

Nitroblue tetrazolium (NBT), and 3,3′-diaminobenzidine (DAB) to analyze the accumulation of O₂⁻ and H²O² in the transgenic Cavendish banana (#11and #13) and wild type plants

Fig. 7

(A) POD activity, (B) the ratio of POD activity,(C) DAB straining, (D) NBT straining to analyze the accumulation of O₂⁻ and H²O² in wild type and transgenic ‘Dajiao’ (RNAi-22 and RNAi-15) before and after cold treatment. Data represent the means ± SE of at least three replicates. Letters indicate significant differences from 0 h cold treatment according to the Student–Newman–Keuls test (P < 0.05) (RE means recovery for 2 days)

Fig. 8

A working model showing the roles of MAPK3-ICE1-POD P7 pathway in ‘Dajiao’ cold stress response