The R2R3-MYB transcription factor FaMYB63 participates in regulation of eugenol production in strawberry

Abstract

The biosynthetic pathway of volatile phenylpropanoids, including 4-allyl-2-methoxyphenol (eugenol), has been investigated in petunia (Petunia hybrida). However, the regulatory network for eugenol accumulation in strawberry (Fragaria × ananassa Duch.) fruit remains unclear. Here, an R2R3-type MYB transcription factor (TF; FaMYB63) was isolated from strawberry by yeast one-hybrid (Y1H) screening using the promoter of the FaEGS1 (eugenol synthase 1 [EGS 1]) gene, which encodes the enzyme responsible for the last step in eugenol biosynthesis. FaMYB63 is phylogenetically distinct from other R2R3-MYB TFs, including FaEOBІІ (EMISSION OF BENZENOID II [EOBII]), which also participates in regulating eugenol biosynthesis in strawberry receptacles. Reverse transcription quantitative PCR (RT-qPCR) assays showed that the expression of FaMYB63 was tissue-specific and consistent with eugenol content through strawberry fruit development, was repressed by abscisic acid, and was activated by auxins (indole-3-acetic acid). Overexpression and RNA interference-mediated silencing of FaMYB63 resulted in marked changes in the transcript levels of the biosynthetic genes FaEGS1, FaEGS2, and FaCAD1 (cinnamyl alcohol dehydrogenase 1 [CAD1]) and, thereby, the accumulation of eugenol. Electrophoretic mobility shift, Y1H, GUS activity, and dual-luciferase activity assays demonstrated that the transcript levels of FaEOBІІ and FaMYB10 were regulated by FaMYB63, but not the other way around. Together, these results demonstrate that FaMYB63 directly activates FaEGS1, FaEGS2, FaCAD1, FaEOBІІ, and FaMYB10 to induce eugenol biosynthesis during strawberry fruit development. These findings deepen the understanding of the regulatory network that influences eugenol metabolism in an edible fruit crop.

Figure 1

Biosynthetic pathway of eugenol. CHS, chalcone synthase; Blue box: TF, Black arrows: previously known pathway components; red arrows represent results shown in this study.

Figure 2

FaMYB63 localization and expression and eugenol content in different stages and under different treatments. A, Subcellular localization of CaMV35S:FaMYB63–GFP fusions in transiently transformed N. benthamiana leaves. All experiments were assayed 72 h after infiltration. The same N. benthamiana leaves were stained with DAPI to show the locations of the nuclei. Bars = 20 μm. B, Analysis by RT-qPCR of FaMYB63 transcript levels (left axis) and the eugenol content (right axis) in fruit receptacles at different developmental stages of Fragaria × ananassa cv. ‘Benihoppe’. C, Analysis by RT-qPCR of FaMYB63 transcript levels in achenes at different developmental stages of F. × ananassa cv. ‘Benihoppe’. G1, small green fruit stage; G2, large green fruit stage; G3, green-white fruit stage; W, white fruit stage; T, red-turning fruit stage; R, red-ripening fruit stage; OR, over-ripening fruit stage. Relative expression values were relative to receptacles at the G1 stage in all cases, which was assigned an arbitrary value equal to one. In (D) and (E), achenes were removed from developing fruits in the G2 stage of F. × ananassa cv. ‘Benihoppe’ for analysis of (D) FaMYB63 expression by RT-qPCR (left axis) and eugenol content (right axis) and (E) IAA content. Control + G2, control, large green fruit receptacle; G2-A + L, G2 fruit receptacle without achenes for 5 d covered by a lanolin paste; G2-A + IAA + L, G2 fruit receptacle without achenes for 5 d treated with the synthetic auxin IAA (1 mM) in lanoline paste. F and G, Analysis by RT-qPCR of FaMYB63 and FaNCED1 transcript levels (in bars) and content of eugenol (in lines) and (H and I) ABA content. F and H, Control, green-white fruits were injected with sterile water; NDGA, green-white fruits were injected with 100-μM NDGA. G and I, Control, fruit pedicels were immersed in MS medium containing sucrose; Water Stress, fruit pedicels were exposed to air without MS medium. Values are the mean ± sd of three biological replicates. The different letters above the columns in (B) and (C) indicate significant differences at the 5% level (P < 0.05, Duncan’s test). Statistical significance with respect to the reference sample (control) was determined by the Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Transcript analysis of eugenol biosynthetic genes and eugenol content in transient FaMYB63-OVX and FaMYB63-silenced fruit receptacles. A, Analysis by RT-qPCR of transcript levels of genes involved in the eugenol biosynthesis pathway in FaMYB63-silenced fruit receptacles agro-infiltrated with the pHellsgate2-FaMYB63 construct, compared with control fruit receptacles agro-infiltrated with an empty vector (pHellsgate2). B, FaMYB63 silencing effect on eugenol content. FaMYB63-RNAiTA, FaMYB63-RNAiTB, and FaMYB63-RNAiTC represent three independent fruit receptacles (considered as three biological replicates) transiently injected with Agrobacterium containing FaMYB63-RNAi construct. RT-qPCR analysis of (C) FaMYB63, (D) FaEGS1, (E) FaEGS2, (F) FaCAD1 transcript levels and (G) eugenol content in strawberry fruit receptacles collected 5 d after infiltration with Agrobacterium carrying pCXSN-FLAG-FaMYB63 construct or an empty vector (pCXSN-FLAG) as a control. FaMYB63-OVX1, FaMYB63-OVX2, and FaMYB63-OVX3 represent three independent fruit receptacles (three biological replicates) transiently overexpressing FaMYB63, respectively. Error bars indicate the standard deviation of three technical replicates. Statistical significance with respect to the control (empty vector) was determined by the Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

FaMYB63 directly interacts with the FaEGS1, FaEGS2, and FaCAD1 promoters. EMSA between FaMYB63 and the promoters of FaEGS1 (A), FaEGS2 (C), and FaCAD1 (E). Mutated nucleotides indicated with red letters. Purified GST-tagged FaMYB63 protein was incubated with unlabeled probe (cold) or biotin-labeled probe, and DNA–protein complexes were separated on native polyacrylamide gels, then photographed. The presence or absence of specific probes is marked by the symbol “+” or “−.” Specific binding of FaMYB63 to promoters of FaEGS1 (B), FaEGS2 (D), and FaCAD1 (F) in Y1H system with bait and either prey or negative control (bait/pGADT7) constructs simultaneously co-transformed into competent cells using selective growth medium (SD/-Leu) without (left) or with (right) AbA.

Figure 5

FaMYB63 regulates the transcription of FaEGS1, FaEGS2, and FaCAD1. A, Schematic diagrams of the effector (35S:FaMYB63) and reporter vectors (FaEGS1, FaEGS2, and FaCAD1 promoters fused to GUS) that were used for transient expression analysis, GUS staining, and GUS activity assays in transgenic N. benthamiana leaves. pFaEGS1:GUS, pFaEGS2:GUS, or pFaCAD1:GUS with or without the 35S:FaMYB63 effector were co-transformed into N. benthamiana leaves. The blue color indicates activated GUS activity. Data are means ± sd of nine biological replicates. B, Reporter and effector constructs used in the dual-LUC assay. LUC activity analysis, using 35S:FaMYB63 as the effector and P_EGS1:LUC, P_EGS2:LUC, or P_CAD1:LUC as reporters. Promoter activities of FaEGS1, FaEGS2, and FaCAD1 genes activated by FaMYB63 in dual-LUC assays were expressed as the ratio of LUC to REN in N. benthamiana leaves co-transformed with the effector and the reporter combinations. Data are means ± sd of nine biological replicates. Statistical significance was determined using Student’s t test: ***P < 0.001.

Figure 6

RT-qPCR analysis of FaEOBІІ and FaMYB10 transcript levels in strawberry FaMYB63-overexpressing and FaMYB63-silenced fruit receptacles (F. × ananassa cv. ‘Benihoppe’). A and B, FaMYB63-OVX1, FaMYB63-OVX2, and FaMYB63-OVX3 represent three independent FaMYB63 OVX fruit receptacles (three biological replicates), respectively. C and D, FaMYB63-RNAiTA, FaMYB63-RNAiTB, and FaMYB63-RNAiTC represent three independent fruit receptacles (three biological replicates) transiently injected with Agrobacterium containing FaMYB63-RNAi construct. All samples were collected 5 d after transient transformation. The transcript levels of both genes studied in FaMYB63-silenced fruit receptacles are expressed as a percentage against their expression levels in fruit receptacles injected with Agrobacterium containing empty vector. Error bars indicate the standard deviation of three technical replicates. Statistical significance was determined using Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

FaMYB63 binds to the FaEOBІІ and FaMYB10 promoters. EMSA analysis of the binding of recombinant FaMYB63 protein to the promoters of FaEOBІІ (A) and FaMYB10 (C). The presence or absence of specific probes is marked by the symbol “+” or “−”. Specific binding of FaMYB63 to promoters of FaEOBІІ (B) and FaMYB10 (D) by one-hybrid system using bait and either prey or negative control (bait/pGADT7) constructs co-transformed into yeast competent cells using selective growth medium (SD/-Leu) without (left) or with (right) AbA.

Figure 8

FaMYB63 regulates the transcription of FaMYB10 and FaEOBІІ. A, Schematic diagrams of effector (35S:FaMYB63, 35S:FaMYB10, and 35S:FaFaEOBІІ) and reporter vectors (promoters of FaMYB63, FaMYB10, or FaEOBІІ:GUS) that were used for transient expression. GUS activity in transgenic N. benthamiana leaves infiltrated with labeled constructs. Data are means ± sd of nine biological replicates (right side). B, Schematic diagram of the reporter and effector constructs used in the dual-LUC assay. Promoter activities were expressed as the ratio of LUC to REN in N. benthamiana leaves co-transformed with effector and the reporter combinations as listed. Data are means ± sd of nine biological replicates. Statistical significance was determined using Student’s t test: ***P < 0.001.

Figure 9

Analysis of the expression of eugenol-related genes and eugenol contents in stable FaMYB63 transgenic strawberry fruits. RT-qPCR analysis of MYB63, EGS1, CAD1, EGS2, EOBII, and MYB10 transcript levels (A) and GC–MS quantification of eugenol (B) in stable FaMYB63 transgenic strawberry plants. WT, G1 stage fruits of diploid wild strawberry (F. vesca) ‘Ruegen’; FaMYB63-RNAiS1, FaMYB63-RNAiS2, and FaMYB63-RNAiS3 represent G1 stage fruits from three independent FaMYB63-silenced transgenic strawberry lines, respectively. The expression levels of the different genes studied in stable FaMYB63 transgenic strawberry ‘Ruegen’ were expressed as a percentage against their expression levels in WT strawberry plants. Error bars indicate the standard deviation of three biological replicates for each line. Statistical significance was determined using Student’s t test. ***P < 0.001.