Strawberry plants (Fragaria × ananassa ‘Benihoppe’) were grown on soil supplemented with nutrient soil, organic fertilizer, and vermiculite (7:2:1; v/v/v) in the greenhouse. The controlled condition of greenhouse was 12/12-h photoperiod at 450 μmol m^-2 s^-1 and 70% humidity, under day/night temperature of 25°C/15°C.

The cDNA sequences of full-length RLKs were obtained from the TAIR website¹ and NCBI². To identify CrRLK1-like RLK (CrRLK1L) genes in strawberry, the coding sequence of FERONIA (At3g51550) was used as query to BLAST the strawberry genome. Phylogenetic trees were constructed using the Neighbor-Joining (NJ) method in MEGA 4.0.2 software, with 1000 bootstrap replicates to evaluate the reliability of different phylogenetic groups. The deduced amino acid sequences of FaMRLKs were aligned using ClustalX 2.0.12 with default settings. The alignments were edited and marked using GeneDoc.

To isolate FaMRLK47 and FaMRLK50, total RNA was extracted from strawberry fruit using an E.Z.N.A.^®Total RNA Kit (OMEGA). The cDNA was synthesized from 1 μg of total RNA using M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s instruction. Full-length FaMRLK47 and FaMRLK50 were cloned by RT-PCR from cDNA using Q5 High-Fidelity DNA Polymerase (New England Biolabs) under the following conditions: 94°C/30 s for 1 cycle, 94°C/30 s, 56°C/25 s, and 72°C/5 min for 35 cycles, and a final extension of 72°C/5 min. The amplified fragments were subcloned into the pMD19-T vector and transformed into Escherichia coli DH5α. Then the selected positive colonies were sequenced by Invitrogen to confirm the full-length sequence. The Primer sequences and GenBank accession numbers are shown in Supplementary Table S1.

Quantitative reverse transcriptase PCR (RT-qPCR) was performed using SYBR Premix Ex TaqTM (TaKaRa) in a ABI7500 Real-Time PCR System. Primers used for RT-qPCR were designed using Primer3 Plus³. Three biological replicates were set up, and each sample was analyzed at least in triplicate. FaACTIN was used as an internal control and the 2^-ΔΔCT method (where ΔCT represents the difference between the cycle threshold values of the target and reference genes) was used to calculate the relative transcript levels (Schmittgen and Livak, 2008).

To construct vectors for overexpression of FaMRLK47 and FaMRLK50 (abbreviated hereafter as FaMRLK47-OE and FaMRLK50-OE), full-length FaMRLK47 and FaMRLK50 were cloned into the plant expression vector pCambia1304 using the XbaI and SacI restriction sites. To construct vectors for downregulating FaMRLK47 (abbreviated hereafter as FaMRLK47-RNAi), the plant expression vector pFGC5941 was used. pCambia1304, pFGC5941, FaMRLK47-OE, FaMRLK47-RNAi, and FaMRLK50-OE were transformed individually into Agrobacterium tumefaciens strain EHA105 (Lazo et al., 1991). The transformed strains were grown at 28°C in Luria-Bertani liquid medium containing 10 mM MES and 20 μM acetosyringone with appropriate antibiotics. When the culture reached an optical density at 600 nm of approximately 0.8, A. tumefaciens cells were harvested, resuspended in infection buffer [10 mM MgCl₂, 10 mM MES (pH 5.6), and 200 mM acetosyringone], and shaken for 2 h at room temperature before being used for infiltration. Pairs of fruits at 18 DPA (day past anthesis) and with similar phenotypes were selected and, for each pair of fruits, one was transfected with FaMRLK47-OE, FaMRLK50-OE, or FaMRLK47-RNAi and the other (the control) was transfected with pCambia1304, pCambia1304 empty vector, or pFGC5941, respectively. For transfection, A. tumefaciens suspension was evenly injected into the fruits with a syringe until the whole fruit became hygrophanous (Jia et al., 2013a), and for each gene, 25 pairs of fruit were injected. To examine the expression of ripening-related genes, FaMRLK47-OE and FaMRLK50-OE transformed fruits were collected 12 days after infiltration and FaMRLK47-RNAi fruit was collected 8 days after infiltration. After removing seeds, fruit samples were frozen in liquid nitrogen and kept at -80°C until used. To investigate the effect of FaMRLK47-OE or FaMRLK47-RNAi on ABA signaling in fruits, detached fruits were transfected with the FaMRLK47-OE, FaMRLK47-RNAi, or empty pCambia1304 vector, and then incubated at 22°C and 100% humidity. Three days after the transfection and incubation, fruits were treated with 100 μM ABA for 6 h and expression of the selected genes was analyzed as described above.

Flesh firmness was measured after removing fruit skin on opposite sides of the fruit using a GY-4 fruit hardness tester (Zhejiang Top Instrument). The contents of anthocyanins, flavonoid, and total phenol in the fruit were evaluated using described methods (Fuleki and Francis, 1968; Lees and Francis, 1971). The soluble sugar content was examined as described by Jia et al. (2011). The total titratable acidity calculated, expressed as percent malic acid, was measured using the acid–base titration method (Kafkas et al., 2007). Volatile organic components were analyzed by headspace solid-phase microextraction and gas chromatography–mass spectrometry as described by Dong et al. (2013). ABA content was measured by an indirect enzyme-linked immunosorbent assay (ELISA) (Zhang et al., 2009). The ELISA procedures were performed according to the instructions provided by the manufacturer (China Agricultural University, Beijing, China) and the assay plates were read by the Thermo Electron (Labsystems) Multiskan MK3 (PIONEER, Co., Beijing).

Yeast two-hybrid assays were performed using the Matchmaker GAL4-based Two-Hybrid System 3 (Clontech), according to the manufacturer’s instructions. The coding sequence of the FaMRLK47 kinase domain (540–892 aa) was fused in-frame with the GAL4 DNA-binding domain (BD) in the pGBKT7 vector to generate the FaMRLK47-BD plasmid. The full-length cDNA sequences of FaABI1 were inserted into the pGADT7 vector. Four different combinations, pGADT7/pGBKT7, FaABI1-AD/pGBKT7, pGADT7/FaMRLK47-BD, and FaABI1-AD /FaMRLK47-BD were respectively transformed into AH109 strains using the lithium acetate method. After transfection, strains were steaked on -Leu/-Trp medium and further selected on minimal -Leu/-Trp/-His/-Ade medium, and then treated with 20 μg/mL X-Gal for interaction validation. Combinations of pGADT7/pGBKT7, FaABI1-AD/pGBKT7, and pGADT7/FaMRLK47-BD were used as negative controls. The primers used for yeast two-hybrid assays are provided in Supplementary Table S4.

For the bimolecular fluorescence complementation (BiFC) assay, the full-length cDNA sequence of FaMRLK47 or FaABI1 was cloned into pCambia1300-YFP^n/c to generate the interaction vectors FaMRLK47-YFP^c or FaABI1-YFPⁿ. FaMRLK47-YFP^c or FaABI1-YFPⁿ plasmids were further transformed into A. tumefaciens strain EHA105, and cultured at 28°C. To examine the in vivo interaction, FaMRLK47-YFP^c and FaABI1-YFPⁿ were co-expressed in tobacco leaves (Nicotiana tabacum) by Agrobacterium-mediated infiltration (Schütze et al., 2009). The negative control was performed by co-expressing FaMRLK47-YFP^c and empty pCambia1300-YFPⁿ vector in tobacco leaves. Chimeric fluorescence was examined by confocal microscopy (Olympus Fluoview FV1000). For YFP and bright field imaging, excitation wavelengths of 488 and 543 nm were used, respectively.

For subcellular localization of FaMRLK47, full-length cDNA sequences were amplified by PCR using the forward primer 5′-TTAATTAAATGAAGTGTTTCTTTTTCTATATTTGGTTC-3′ and the reverse primer 5′-GGCGCGCCAACGTCCCTTTGGGTTCATGATTTGTGAG-3′. The PCR fragments were inserted into pMDC83 using Asc1 and Pac1 and the constructs were then introduced into A. tumefaciens strain EHA105 and transformed into tobacco leaves as described by Schütze et al. (2009). Transfected plants were grown in darkness for 24 h and in light for 48 h at 24°C. After 3 days, fluorescence was observed using a confocal laser-scanning microscope (Olympus Fluoview FV1000). The primers used for BiFC and localization are shown in Supplementary Table S5.

Samples were analyzed in triplicate, and the data were noted as the mean ± SD. Data were analyzed using Student’s t-test implemented in SAS software (version 8.1, United States), and the least significant difference at a 0.05 level of probability was used to explore the effect of P input on parameters. A P-value of ≤ 0.05 was considered to indicate a significant difference, and a P-value of ≤ 0.01 was considered to indicate a highly significant difference.

The FERONIA-like genes belong to a family of CrRLK1-like RLKs (CrRLK1Ls), which in turn belong to a super-family of malectin domain-containing RLKs. In a previous study (Zhang et al., 2016), we conducted a genome-wide screen of woodland strawberry, Fragaria vesica, for ‘Malectin domain-containing RLKs’ (accordingly designated as MRLKs) and identified 62 members (named FvMRLK1–62). In the present study, a screen of the F. vesca genome revealed a CrRLK1L family consisting of 20 members. Phylogenetic analysis of CrRLK1L homologs from various plant species revealed eight clades, with FERONIA, NAXURs, HERKs, and THESEUSs being distributed in different clades (Figure 1 and Supplementary Figure S1). Notably, FERONIA was located in Clade II and only one member of the FaCrRLK1L family, i.e., FaMRLK47, was located in this clade. FaMRLK47 showed a relatively high level of amino acid sequence identity (72.33%) with FERONIA, and can thus be viewed as a homolog of FER. Interestingly, clade I, which contains five members (FaMRLK50–54), was found to consist exclusively of proteins from the strawberry genome. Furthermore, members of clades I, II, and III exhibited relatively high levels of amino acid sequence identity with FERONIA (FER hereafter), and can thus be considered FEL-like receptor kinases. FaCrRLK1Ls and FER exhibited between 42.45 and 48.03% amino acid sequence identity for clade I proteins and between 38.02 and 50.66% for clade III proteins.

Strawberry fruit development, from fruit set to ripening, can be divided into several substages, i.e., the small green fruit (SG), middle green fruit (MG), large green fruit (LG), white fruit (W), initially reddening fruit (IR), and fully reddening fruit (FR) substages (Figure 2A). Given that FaMRLK47 is a homolog of FER and the only FaMRLK member in clade II (Figure 1), we focused on this protein in the present study. As the members of clade I only existed in the strawberry genome and members of clade II exhibited relatively high levels of amino acid sequence identity with FERONIA, we also evaluated their expression in relation to strawberry fruit development and ripening (Figure 2B). Whereas FaMRLK34, FaMRLK39, and FaMRLK6 transcripts were not detected in strawberry fruit, the relative levels of the other six members differed, all tending to decrease from the SG to W substages. Notably, FaMRLK47 expression was higher than that of other members from clade I and II. Furthermore, while the expression levels of FaMRLK47 started to drop during the MG substage, those of all other members examined started to decline during the SG substage, which suggests that FaMRLK47 is more tightly associated with the onset of fruit ripening.

Strawberry fruit development and ripening are regulated by both internal and external factors, including IAA, ABA, and temperature (Giovannoni, 2001; Seymour et al., 2013). While the application of IAA delays strawberry fruit ripening, application of ABA promotes it Given et al. (1988) and Jia et al. (2011, 2013a). In our previous studies, we found that ripening of strawberry fruit was stimulated by high temperatures and delayed by low temperatures (Han et al., 2015). We therefore studied the expression profiles of the FER-like FaMRLKs in response to IAA, ABA, and low/high temperature treatment. While the transcription of all of these genes was sensitive to IAA, ABA, and temperature treatments, their responses differed. FaMRLK47, FaMRLK50, and FaMRLK53 expression were inhibited by ABA treatment, whereas FaMRLK51 and FaMRLK52 expression were upregulated by ABA treatment. FaMRLK47, FaMRLK50, FaMRLK52, and FaMRLK53 expression were also sensitive to temperature stress; however, while FaMRLK47 expression was promoted by both low and high temperature treatment, FaMRLK52 expression was inhibited and promoted, respectively, by low and high temperature treatments (Figure 3).

To investigate a potential role of FaMRLK47 in the regulation of strawberry fruit development and ripening, we transiently manipulated its expression in strawberry fruits. As a representative member of the FaMRLK family that specifically exists in the strawberry genome, FaMRLK50 was also investigated. Since FaMRLK47 and FaMRLK50 transcript levels dramatically decreased from the SG to LG substages, we first sought to examine the function of FaMRLK47 and FaMRLK50 by transiently overexpressing these two genes in strawberry plants. As shown in Figure 4A, overexpression of FaMRLK47 and FaMRLK50 resulted in a great increase in their transcript levels in fruits. While overexpression of FaMRLK50 did not affect fruit development and ripening, overexpression of FaMRLK47 delayed fruit ripening, as reflected by pigment accumulation. Conversely, RNAi-mediated downregulation of FaMRLK47 accelerated fruit ripening (Figures 4A,B). Collectively, these experiments indicate that FaMRLK47 is an important regulator of strawberry fruit development and ripening.

To further explore the FaMRLK47-mediated mechanisms underlying the regulation of strawberry fruit development and ripening, we examined the effects of FaMRLK47 overexpression and downregulation on the expression of a series of ripening-related genes (Figure 4C). Most of these ripening-related genes are important structure genes and transcription factors that are involved in the formation of fruit qualities such as color, texture, aroma, and sugar (Jia et al., 2011, 2013a; Seymour et al., 2011; Lin-Wang et al., 2014; Han et al., 2015). Given that fruit ripening is highly affected by ABA, we also detected the expression of genes involved in ABA biosynthesis, metabolism, and signal transduction. As shown in Figure 4C, manipulating FaMRLK47 expression altered the expression patterns of both ripening-related and ABA-related genes, which implied that FaMRLK47 is an important regulator of diverse processes in fruit ripening, including fruit quality formation, ABA production, and signal transduction. Further detection of the ripening-related physiological parameters demonstrated that overexpression and downregulation of FaMRK47 resulted in a decrease and increase, respectively, in most of the fruit quality parameters that were expected to increase and decrease during fruit development and ripening (Table 1). Moreover, the ABA content showed a decline in FaMRLK47-OE fruit (Figure 5A). Comprehensive analysis of the changing patterns of gene expression and physiological parameters showed that FaMRLK47 mainly plays a role in the regulation of anthocyanins accumulation, flavonoid metabolism and fruit softness, and FaCHS, FaCHI, FaUFGT, FaPAL, FaPE, FaPG, FaXYL1, and FaEXP1 might function as the important downstream structure genes of FaMRLK47 in these processes. In addition, as shown in Figure 4C, FaNCED1-4, FaCYP707A, FaPYL1/12, FaABI1/5, and FaSnRK2.3/2.6 might participate in FaMRLK47- mediated ABA production and signal transduction.

Interestingly, in contrast to the changes in expression profiles of other ripening-related genes following FaMRLK47 manipulation, overexpression and downregulation of FaMRLK47 respectively promoted and inhibited the expression of FaSS and FaSPS1, two key genes in the sucrose biosynthesis pathway (Figure 4C). Further analysis of sugar metabolism showed that, while RNAi-mediated downregulation of FaMRLK47 expression resulted in a dramatic increase in the major sugar components of the fruit, i.e., sucrose, fructose and glucose, overexpression of FaMRLK47 caused a significant decrease in sucrose and fructose content (Figure 5B). Additionally, overexpression and RNAi-mediated downregulation of FaMRLK47 resulted in a significant decrease and increase in starch content, respectively. Collectively, these results suggest that FaMRLK47 is an important regulator of sugar metabolism.

FaMYB10 was reported to be an important positive regulator of anthocyanin accumulation, whereas FaMADS9 was shown to regulate fruit development and ripening (Seymour et al., 2011; Lin-Wang et al., 2014). In this study, we also monitored the expression of FaMYB10 and FaMADS9 (Figure 4C), and found that the expression of FaMADS9 was repressed in FaMRLK47-OE fruit and upregulated in FaMRLK47-RNAi fruit. Although FaMYB10 expression was not expected to be altered by changes in FaMRLK47 expression, overexpression of FaMRLK47 resulted in an increase in FaMYB10 expression, and downregulation of FaMRLK47 resulted in a decrease in FaMYB10 expression. These results imply that FaMRLK47 and FaMYB10 are regulatory proteins with diverse functions in fruit ripening. Furthermore, their regulatory mechanisms are more complex than previously expected.

In Arabidopsis, FER is involved in ABA signaling (Yu et al., 2012). As FaMRLK47 shares 74% amino acid sequence identity with FER, which is known to modulate ABA signaling (Yu et al., 2012), we were interested in establishing whether FaMRLK47 was associated with ABA signaling in strawberry fruit. To investigate a possible role for FaMRLK47 in the modification of the fruit’s response to ABA treatment, we overexpressed and downregulated FaMRLK47 in strawberry fruit for a short time (72 h), and then examined the expression of a series of ripening-related genes following ABA treatment. As shown in Figure 6, overexpression of FaMRLK47 resulted in a great decrease in the ability of ABA to induce the expression of these genes in comparison with control fruits, and conversely, RNAi-mediated downregulation of FaMRLK47 resulted in a great increase in the ability of ABA to induce the expression of these genes. These results indicate that FaMRLK47 functions as a negative regulator of the ABA signaling cascade.

Given that FaMRLK47 is capable of modifying ABA signaling, we examined whether FaMRLK47 could physically interact with important signal proteins in the ABA signaling pathway. As FaABI1 is a key signaling protein in the ABA signaling pathway and negative regulator of strawberry fruit development and ripening (Jia et al., 2013a), we examined the interaction between FaMRLK47 and FaABI1. Yeast two-hybrid analysis showed that by co-transformed of FaMRLK47 and FaABI1 into AH109, the transformed strain grew well on auxotrophic medium (SD-Ade-Leu-Trp-His), which indicated that FaMRLK47 interacts with FaABI1 (Figure 7A). To test whether FaMRLK47 could interact with ABI1 in living plant cells, we first observed the localization of FaMRLK47 in tobacco leaf cells by fusing it with eGFP. We found that FaMRLK47 localized to the membrane in tobacco leaf cells (Figure 7B). Furthermore, we co-transformed the BiFC vectors FaMRLK47-YFP^c and FaABI1-YFPⁿ into tobacco leaves, using co-transformation of FaMRLK47-YFP^c and pCambia1300-YFPⁿ as a control. The results showed that, while fluorescence was not observed in the control transformed leaves, strong fluorescence appeared when FaABI1-YFPⁿ was combined with FaMRLK47-YFP^c (Figure 7C), indicating that FaABI1 and FaMRLK47 indeed physically interact.