Strigolactones enhance apple drought resistance via the MsABI5-MsSMXL1-MsNAC022 cascade

Abstract

Drought stress limits plant growth, development, and yield in apple (Malus). Strigolactones (SLs) work with abscisic acid (ABA) to improve drought resistance in plants, but how this synergistic mechanism functions remains unclear. Here, we determined that SLs promote drought resistance in apple in an ABSCISIC ACID INSENSITIVE5 (MsABI5)-related manner. During drought stress of a wild apple species (Malus sieversii), SLs enhanced the expression of MsABI5, encoding a major transcription factor involved in ABA signaling. MsABI5 bound to the promoter of the gene encoding delta-1-pyrroline-5-carboxylate synthase (MsP5CS2.2), upregulating its expression and thereby enhancing proline accumulation and drought resistance. In addition, MsABI5 suppressed the expression of MsSMXL1, encoding a major transcriptional repressor involved in SL signaling. MsSMXL1 interacted with MsNAC022 instead of MsABI5 to repress the transactivation activity of MsNAC022. MsNAC022 was upregulated by MsABI5, and MsNAC022 directly promoted MsP5CS2.2 expression to enhance proline accumulation and drought resistance. These findings suggest that MsSMXL1 and MsNAC022 comprise a regulatory node downstream of MsABI5 during drought stress in apple. Together, our findings suggest that in apple, SLs increase drought resistance by activating the MsABI5-MsSMXL1-MsNAC022 cascade.

Figure 1

SLs treatment enhance drought resistance in apple. A and B Wild apple (Malus sieversii) plants at similar stages of growth (14–16 leaves, 18–21 cm height) were selected for treatment (A, phenotype before treatment). The plants were treated with rac-GR24 (5, 10, 20 μM) and grown without watering until soil volumetric water content reached 0% (rac-GR24/Drought). Plants subjected to normal watering with soil volumetric water content maintained at 40%–45% (Watering) or without watering until soil volumetric water content reached 0% (Drought) and plants treated with 5 μM rac-GR24 and normal watering with soil volumetric water content maintained at 40%–45% (rac-GR24/Watering) were used as controls (B, phenotypes of drought treatment). Scale bar, 10 cm. C and D Aboveground height and leaf number before and after treatment. The x-axis indicates days (d) after treatment. Three apple plants were used per treatment, an independent measurement of aboveground height and leaf number of M. sieversii plant from each treatment was used as one biological replicate. Three replicates were performed in this determination. Proline (E), MDA (F), and total chlorophyll contents (G) were measured after treatment. Three leaves each from the top, middle, and bottom of each plant were collected separately; a total of 27 leaves from three plants were collected for each treatment and evenly divided into three sets as three biological replicates per treatment. One independent proline, MDA, or chlorophyll measurement from each biological replicate was considered one technical replicate; three replicates were performed in this measurement. MDA, malondialdehyde. FW, fresh weight. Values represent means ± SD. Different letters (a–g) indicate significance differences as determined by LSD range test (P < 0.05).

Figure 2

MsABI5 improves proline accumulation and osmotic stress resistance in apple. Effects of rac-GR24 on the expression of MsABF1 (A), MsABF2 (B) and MsABI5 (C) during drought stress. D Apple (Malus domestica) plants overexpressing MsABI5 (MsABI5-OE) or expressing a repressor version of MsABI5 (MsABI5-SRDX) and ‘GL-3’ plants were treated with mannitol or NaCl to induce osmotic stress for 14 days. Untreated plants were used as controls. Scale bars, 1 cm. E–G Proline, MDA, and chlorophyll contents were measured in MsABI5-OE, MsABI5-SRDX, or ‘GL-3’ plants. Nine apple plants were used per treatment, three apple plants were used as one biological replicate, and three biological replicates were performed for each treatment. Three independent measurements were performed for proline, MDA, and chlorophyll contents. The x-axis indicates the transgenic line numbers. FW, fresh weight. Values represent means ± SD. Different letters (a–f) indicate significant differences as determined by LSD range test (P < 0.05). H MsABI5 binds to the MsP5CS2.2 promoter. Y1H assays were performed by co-transforming yeast with a plasmid expressing MsABI5 and a plasmid driven by the MsP5CS2.2 promoter. The empty pB42AD vector (AD) and a plasmid containing the MsP5CS2.2 promoter were used as controls. I MsABI5 positively regulates MsP5CS2.2 promoter activity. MsABI5 effector and MsP5CS2.2 promoter-LUC reporter plasmids were co-expressed in N. benthamiana leaves to analyze LUC activity. Three independent replicates were performed. Values represent means ± SD. Different letters (a and b) indicate significant differences by LSD range test (P < 0.05). J MsABI5 binds to the MsP5CS2.2 promoter. Electrophoretic mobility shift assays (EMSAs) were conducted with a biotin-labeled MsP5CS2.2 promoter fragment containing an ABA-response element (ABRE). An unlabeled version of the same MsP5CS2.2 promoter fragment was used as an unlabeled competitor. For the mutant probe, the ABRE was mutated into AAAAAA and used as an unlabeled competitor.

Figure 3

SLs enhance osmotic stress resistance in apple in an MsABI5-related manner. A Apple plants expressing a repressor version of MsABI5 (MsABI5-SRDX) and ‘GL-3’ plants were treated with rac-GR24 and subjected to mannitol or NaCl treatment to induce osmotic stress for 14 days. The drought sensitivity among these apple plants was then compared. Apple plants not treated with rac-GR24 were used as controls. Scale bars, 1 cm. B–D Proline, MDA, and chlorophyll contents were measured in MsABI5-SRDX or ‘GL-3’ plants. Nine apple plants were used per treatment, three apple plants were used as one biological replicate, three biological replicates were performed for each treatment, and one representative apple plant from each treatment is pictured. Three independent measurements were performed for proline, MDA, and chlorophyll contents from three biological replicates. The x-axis indicates the transgenic line numbers. FW, fresh weight. Values represent means ± SD. Different letters (a-d) indicate significant differences as determined by LSD range test (P < 0.05).

Figure 4

MsABI5 inhibits MsSMXL1 expression by directly binding to its promoter to improve osmotic stress resistance in apple. A MsABI5 binds to the MsSMXL1 promoter. Y1H assays were conducted by co-transforming yeast cells with a plasmid expressing MsABI5 and a plasmid driven by the fragments from the MsSMXL1 promoter (P1–P3). The empty pB42AD vector (AD) and a plasmid containing the MsSMXL1 promoter were used as controls. P3-ACE is the ACGT element in P3 fragment of MsSMXL1 promoter, which is recognized by ABI5. P3-mACE, the ACGT element is mutated from TAACGTAC to TAAAAAAC in P3 fragment of MsSMXL1 promoter. The promoter fragments containing P3-ACE or P3-mACE were cloned into pLacZi vector. B MsABI5 negatively regulates MsSMXL1 promoter activity. MsABI5 effector and MsSMXL1 promoter-LUC reporter plasmids were co-expressed in N. benthamiana leaves to analyze LUC activity. EV, empty vector. Three independent infection replicates were performed. Values represent means ± SD. Different letters (a-c) indicate significant differences as determined by LSD range test (P < 0.05). C Apple plants overexpressing MsSMXL1 (MsSMXL1-OE) and ‘GL-3’ plants were treated with mannitol or NaCl to induce osmotic stress for 14 days. Drought sensitivity was compared among these apple plants (phenotype). Untreated plants were used as controls. Scale bars, 1 cm. D-F Proline, MDA, and chlorophyll contents were measured in MsSMXL1-OE and ‘GL-3’ plants. FW, fresh weight. Nine apple plants were used per treatment, three apple plants were used as one biological replicate, three biological replicates were performed for each treatment, and one representative apple plant for each treatment is pictured. Three independent measurements of proline, MDA, and chlorophyll contents were performed from three biological replicates. Values represent means ± SD. Different letters (a-e) indicate significant differences as determined by LSD range test (P < 0.05).

Figure 5

MsSMXL1 interacts with MsNAC022 that is regulated by MsABI5. A Interaction between MsSMXL1 and MsNAC022 analyzed using a yeast two-hybrid assay. Yeast cells were co-transformed with plasmids expressing MsSMXL1 and MsNAC022. Yeast cells containing empty pGADT7-T and pGBKT7-lam vector or pGADT7-T and pGBKT7–53 were used as negative and positive controls, respectively. B Apple plants overexpressing MsNAC022 and ‘GL-3’ plants were treated with mannitol or NaCl to induce osmotic stress for 14 days. MdP5CS2.2 expression was analyzed using RT-qPCR. Untreated plants were used as controls. Nine apple plants were used per treatment, three apple plants were used as one biological replicate, and three biological replicates were performed for each treatment. Three independent RNA extractions were performed from three biological replicates. Values represent means ± SD. Different letters (a-c) indicate significant differences by LSD range test (P < 0.05). C MsNAC022 binds to the MsP5CS2.2 promoter. EMSAs were performed with a biotin-labeled MsP5CS2.2 promoter fragment containing a TCACGA motif. An unlabeled version of the same MsP5CS2.2 promoter was used as an unlabeled competitor. In the mutant probe, the TCACGA motif was mutated to AAAAAA and used as an unlabeled competitor. D MsNAC022 positively regulates MsP5CS2.2 promoter activity. The MsNAC022 and MdSMXL1 effectors or the MsNAC022 and MdSMXL1^△EAR effectors were co-expressed with the MsP5CS2.2 promoter-LUC reporter in N. benthamiana leaves to analyze LUC activity. The 62-SK empty vector was used as a negative control. Three independent replicates were performed. Values represent means ± SD. Different letters (a and b) indicate significant differences by LSD range test (P < 0.05). E MdNAC022 expression in MsABI5-OE and MsABI5-SRDX transgenic apple plants and in ‘GL-3’ plants treated with mannitol or NaCl to induce osmotic stress for 14 days, as analyzed using RT-qPCR. Untreated plants were used as controls. The biological replicates were set up the same as Fig. 5B. Values represent means ± SD. Different letters (a-f) indicate significant differences by LSD range test (P < 0.05). F MsABI5 positively regulates MsNAC022 promoter activity. MsABI5 effector and MsNAC022 promoter-LUC reporter plasmids were co-expressed in N. benthamiana leaves to analyze LUC activity. Three independent replicates were performed. Values represent means ± SD. Different letters (a and b) indicate significant differences by LSD range test (P < 0.05).

Figure 6

Proposed model for the role of the MsABI5-MsSMXL1-MsNAC022 cascade in enhancing drought resistance. SLs activate MsABI5 expression during drought stress in Malus sieversii. MsABI5 directly binds to the MsP5CS2.2 promoter and enhances its expression to stimulate proline accumulation and drought resistance. MsABI5 upregulates MsNAC022 expression but negatively regulates MsSMXL1 expression. MsNAC022 directly binds to the MsP5CS2.2 promoter to enhance proline accumulation during drought stress, and MsSMXL1 interacts with MsNAC022 to repress its transactivation activity, thus attenuating drought tolerance in apple. The → indicates activation. The indicates repression. MsABI5 → MsP5CS2.2 indicates MsP5CS2.2 is activated by MsABI5. MsABI5 → MsNAC022 indicates MsNAC022 is activated by MsABI5. MsNAC022 → MsP5CS2.2 indicates MsP5CS2.2 is activated by MsNAC022. MsABI5 MsSMXL1 indicates MsSMXL1 is repressed by MsABI5. MsSMXL1 MsNAC022 indicates the transactivation of MsNAC022 to MsP5CS2.2 expression is repressed by MsSMXL1.