Genome-Wide Identification and Expression Analysis of MRLK Family Genes Associated with Strawberry (Fragaria vesca) Fruit Ripening and Abiotic Stress Responses

Malectin-like domain-containing receptor-like kinases (MRLK) constitute a large and divergent family of proteins in plants; however, little is known about the role of MRLKs in fruit growth and development. In this study, we characterized MRLK family genes in diploid strawberry, Fragaria vesca. Based on an analysis of malectin-like domain and a search in the strawberry genome and NCBI database, we identified 62 FvMRLKs in the strawberry genome, and classified these genes into six subfamilies with distinct malectin domains in the extracellular regions of the encoded proteins. Gene expression analysis indicated that more than 80% of the FvMRLKs were expressed in various tissues, with higher levels in roots than in other organs. Thirty-three FvMRLKs were found to be expressed in fruits during the early stages of development, and over 60% of these exhibited dramatic decreases in expression during fruit growth and development. Moreover, the expression of some FvMRLKs was sensitive to both environmental and internal cues that play critical roles in regulating strawberry fruit development and ripening. Collectively, this study provides valuable insight into the FvMRLKs gene family and its role in regulating strawberry fruit development and ripening.


Introduction
Receptor-like kinases (RLKs) in plants comprise a large family of proteins with putative amino-terminal extracellular domains and carboxyl-terminal intracellular kinase domains [1,2]. A phylogenetic analysis of the model plant Arabidopsis thaliana identified more than 610 proteins containing RLK domain sequences. RLKs can be further divided into more than 44 subfamilies based on their extracellular domain structures [3,4]; plant RLKs were initially categorized into three major types, the S-domain type, the leucine-rich repeat type, and the and development, we conducted a genome-wide analysis of the MRLK subfamily in diploid strawberry, Fragaria vesca. We found that the F. vesca genome contains more than 60 MRLKs, about 50% of which are expressed in the fruit. Among the MRLKs expressed in fruit, more than 60% were expressed at relatively high levels only in the early stages of fruit development, with transcript levels declining dramatically as fruit development progressed. Furthermore, many of these genes were sensitive to both environmental and internal cues controlling fruit development and ripening. These findings provide valuable insight into the roles of FvMRLKs in the regulation of flesh fruit development and ripening.

Identification of MRLK proteins in Fragaria vesca
Annotated strawberry MRLK proteins were identified in the following three public databases: the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/), the Strawberry Genome Browser (https://strawberry.plantandfood.co.nz/index.html), and PLAZA (http://bioinformatics.psb.ugent.be/plaza/). Specifically, to identify the MRLK gene family, we first identified the HMM profile of the malectin domain (pfam11721) and malectin-like domain (pfam12819) from the protein families database (http://pfam.janelia.org); Based on the HMM profile of the malectin and malectin-like domains, we obtained the MRLK protein candidates by searching the database of strawberry genome (https://strawberry.plantandfood.co. nz/index.html) and the MRLK protein candidates were further identified and characterized in the NCBI database for a final confirmation of the MRLK proteins. The lengths of amino acid sequences, molecular weights, and isoelectric points of the putative proteins were calculated using tools provided at the ExPasy website (http://web.expasy.org/protparam/). The signal peptides of the strawberry MRLK proteins were predicted based on the SignalP 4.1 server (http:// www.cbs.dtu.dk/services/SignalP/) [34]. Information of the Arabidopsis MRLK gene family was obtained from The Arabidopsis Information Resource (http://www.arabidopsis.org/) and used for the comparative study.

Phylogenetic analysis and classification of the MRLK protein family in Fragaria vesca
All identified MRLK genes (FvMRLKs) in Fragaria vesca were classified into different groups based on the alignment of FvMRLK proteins using ClustalX 2.1with default settings [35]. The phylogenetic trees were constructed using the neighbor-joining method implemented in MEGA 5.0 [36]. Bootstrap values were calculated for 1000 iterations.

Chromosomal location of FvMRLKs
All FvMRLK genes were mapped to strawberry chromosomes based on information available at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). The map was drafted using Mapchart software (http://www.wageningenur.nl/en.htm) [37]. Tandem duplicated FvMRLK genes in the strawberry genome were identified by checking their physical locations on individual chromosomes and were defined as adjacent paralogous genes with no more than one intervening gene. Physical chromosomal locations were graphically represented to scale on the seven chromosomes. was used to illustrate the exon-intron structure of the FvMRLKs [38]. The Expectation Maximization for Motif Elicitation online program (http://meme-suite.org/) [39] for DNA and protein sequence analysis was used to identify conserved motifs in the 62 FvMRLK-deduced proteins. The optimized parameters of Expectation Maximization for Motif Elicitation were set as follows: the number of repetitions, any; the maximum number of motifs, 50; and the optimum width of each motif, between 6 and 300 residues.

Plant materials and treatments
The strawberry (Fragaria vesca'Ruegen') plants were cultivated at 22±1°C in a 13/11 h light/ dark photoperiod in the Tissue Culture Center at Beijing University of Agriculture. To investigate FvMRLK expression in different strawberry tissues, the roots, stems, leaves, and fruits were independently sampled and analyzed using qRT-PCR. Root samples were taken from white, new roots, stem samples were taken from new creeping stems, leaf samples were the leaves 15 days old, fruit samples were taken from medium green fruits. To study the expression of FvMRLKs at different developmental stages, strawberry fruits were divided into five developmental stages, i.e., SG: small green stage; MG: medium green stage; LG: large green stage; TN: turn stage; and FR: full red stage, and were independently sampled. At each developmental stage, ten representative fruits were sampled, snap-frozen in liquid nitrogen, and kept at -80°C for subsequent analysis. The expression of the FvMRLK genes in the five fruit developmental stages was analyzed using qRT-PCR. MG fruits were selected to study some of the high-expression genes of fruit in response to various environmental cues and internal signals, including heat treatment (40°C,4 h), cold treatment (4°C,48 h), dehydration treatment (25°C,8 h), ABA treatment (25°C,100μM, 6 h), IAA treatment (25°C,500μM, 6 h), and sucrose treatment (25°C,100mM, 6 h).The fruits were cut in half with one-half used for the treatment and the other half as a control. The controls were maintained at 25°C and100% relative humidity. Three biological replicates were used in all experiments. The expression data in the heat-map were calculated as log 2 fold change in treated vs. untreated samples.

RNA preparation and quantitative RT-PCR analysis
Total RNA was isolated using a Plant RNA Kit (Omega) according to the manufacturer's instructions. Quantitative RT-PCR was conducted on a LightCycler 96 Real Time PCR System (Roche Diagnostics GmbH, Mannheim City, Germany) using SYBR-Green (Takara, Dalian, China).The oligonucleotide primers were designed based on the predicted coding region using Beacon designer software and are listed in S1 Table. Primers were checked using the BLAST tool of NCBI and the dissociation curve was analyzed after the PCR reaction for primer specificity. In addition, the specificity of PCR products was verified by cloning the relative amplicons in the pMD19-T vector (Takara), sequencing, and aligning them onto the reference genome. Each reaction was carried out in a volume of 20μL, which contained 10μL SYBR--Green master mix, 8.6μL ddH 2 O, 1μL diluted template (1μL of the generated first-strand cDNA was diluted in 9 μL ddH 2 O), and 0.2μL of each gene-specific primer. The following program conditions were used: 95°C for 600 s (pre-denaturation) followed by 40 cycles at 95°C for 20 s (denaturation), 53°C for 20 s (primer annealing), and 72°C for 20 s (extension and data acquisition). Three technical replicates were performed for each biological replicate. The actin-7 gene (LOC101313051), which maintained almost constant expression levels under all experimental conditions, was used as an internal control.

Identification and annotation of MRLK genes in the strawberry genome
To identify FvMRLK subfamily genes, we first obtained the HMM profile of the malectin domain (pfam11721) and malectin-like domain (pfam12819) from the pfam protein families database. Using the malectin or malectin-like domain information, we searched three databases, the National Centre for Biotechnology Information, the Strawberry Genome Browser, and PLAZA. We identified a total of 100 candidate FvMRLK genes in the Fragaria vesca genome. The 100 FvMRLK candidates were further examined by searching the malectin or malectin-like domain in the NCBI database and 62 members were confirmed to be FvMRLKs, which were named FvMRLK1 to FvMRLK62 based on their chromosome location. The characteristics of the FvMRLK proteins are listed in Table 1 and include the deduced protein length, molecular weight, isoelectric point (PI), aliphatic index, and the grand average of hydropathicity. The deduced length of the FvMRLK proteins ranged from 132 (FvMRLK16) to 1,955 (FvMRLK 51) amino acid residues, whereas the PI ranged from 4.54 (FvMRLK33) to 8.88 (FvMRLK55). Thirty-one FvMRLK members contained a signal peptide (S2 Table).

Chromosomal distribution and exon-intron organization of the FvMRLK genes
The FvMRLK genes were mapped across the chromosomes of the strawberry genome. As shown in Fig 1, 60 of the 62 FvMRLK genes were distributed on chromosomes 1 through 7. Chromosome 3 (22 genes) contained the largest number of FvMRLK genes and chromosome 5 (2 genes) contained the smallest number of FvMRLK genes. We were unable to map two FvMRLKs (FvMRLK61 and FvMRLK62) to any of the chromosomes. Based on the definition of a gene cluster by Holub [40], the FvMRLKs were placed into ten clusters. Among the ten clusters, five were found on chromosome 3 and two on chromosomes 6, whereas only one cluster was located on chromosomes 1, 4, and 7. Tandem and segmental duplications have been suggested as two main causes for gene family expansion in plants [41]. Five clusters of FvMRLKs with tandem duplications were found to be located on chromosome 3. To characterize the evolution of the FvMRLK family genes in strawberry, we analyzed the exon-intron structures of all FvMRLKs. We found that the intron numbers varied greatly (from 0 to 47) (Fig 2) amongst members of this gene family. For example, while FvMRLK5 had no introns, FvMRLK62 had 47. Interestingly, it appeared that the FvMRLKs within the same group generally had a similar exon-intron structure.

Phylogenetic analysis, classification, and motifs of the strawberry MRLK gene family
To determine the phylogenetic relationship between strawberry and Arabidopsis MRLKs, we constructed an unrooted phylogenetic tree based on alignments of the full-length protein sequences. The phylogenetic analyses showed that FvMRLKs could be classified into six large groups. As shown in Fig 3, groupI, II, III, IV, V, and VI contained 10, 12, 7, 14, 7, and 4 members, respectively. A major difference in the protein structure among the different groups was the number of malectin and malectin-like domains. For example, while most of the members of group I contained two malectin domains and one malectin-like domain, most of the members of group II contained one malectin domain and one malectin-like domain. Furthermore, while most of the members of group III and V contained one malectin-like domain, most of the members of group IV and VI contained only one malectin domain. Using the Multiple Expectation Maximization for Motif Elicitation online tool (http://meme-suite.org/), we identified 20 conserved motifs in the FvMRLK subfamily (Fig 4 and Table 2). Motif 6,8,9,10,14,15,16,17,18,19, and 20 represented pivotal motifs of the malectin domains in strawberry and these conserved motifs were distributed widely amongst all FvMRLK groups. As shown in Fig 4, the FvMRLKs within the same group were found to share a similar motif composition. For example, whereas motif 19 was only found in group III, motif 10,15, and 20 were found mainly in groupI and II.  that FvMRLKs is a regulator of root growth and development. Although the expression levels of most of the genes were lower in fruits than in roots, 33 members genes were found to be expressed in fruits (Fig 6a). More than 60% of these 33 genes were expressed at high levels only in the early stages of fruit development and exhibited dramatic decreases in expression as fruit development progressed (Fig 6b).  The phylogenetic trees were constructed using the neighbour-joining method implemented in MEGA 5.0. Reliability of the predicted tree was tested using bootstrapping with 1000 replicates. Numbers at the nodes indicate how often the group to the right appeared among bootstrap replicates. The motif composition related to each FvMRLK protein is displayed on the right-hand side. The motifs, numbered 1-20, are displayed in different colored boxes. The sequence information for each motif is provided in Table 2. doi:10.1371/journal.pone.0163647.g004 The Malectin-Like Domain-Containing Receptor-Like Kinases in Strawberry

Expression of FvMRLKs in response to environmental and internal cues
Strawberry fruit development and ripening are regulated by environmental cues, such as temperature and drought stress, and internal signals, such as auxin (IAA), abscisic acid (ABA), and sucrose. To establish whether FvMRLKs are indeed involved in strawberry fruit development and ripening, we examined the expression of selected FvMRLKs in response to IAA, ABA, and sucrose treatment as well as to temperature and dehydration stresses. As shown in Fig 7a, the expression of nearly all of the FvMRLK genes examined were sensitive to low temperature. The gene expression of some FvMRLKs increased more than 25-fold in response to low temperature treatment. Some FvMRLKs were also sensitive to the dehydration treatment, but the dehydration-induced changes in gene expression were generally smaller than those following low temperature treatment. As a negative regulator, IAA plays critical roles in the regulation of strawberry fruit development and ripening, whereas both ABA and sucrose have been demonstrated to be positive regulators of strawberry fruit ripening [43]. Consistent with this, IAA treatment was found to result in a dramatic increase in gene expression for most of the  FvMRLK genes examined. By contrast, ABA and sucrose treatments resulted in a decrease in expression of some of the FvMRLK genes examined (Fig 7b). Collectively, these results suggest that FvMRLKs play important roles, and moreover, most of them function as negative regulators in the regulation of strawberry fruit development and ripening.

Discussion
The malectin and malectin-like domain containing RLKs family in relation to the CrRLK1L gene family In Arabidopsis, CrRLK1Ls are a unique subfamily of RLKs comprised of 17 members. Given that CrRLK1Ls were found to contain malectin or malectin-like domains in their extracellular sequence [31], the CrRLK1Ls family has been commonly thought to be the same in terminology with the family of proteins containing malectin and malectin-like domain. In the present study, to compare FvMRLKs with the MRLKs in Arabidopsis, we searched the Arabidopsis genmone for the identification of the proteins containing the malectin and malectin-like domain.
The results indicated that the Arabidopsis genome harbors 35 proteins containing malectin or malectin-like domains (S3 Table), which is greater than the 17 Arabidopsis CrRLK1L genes. Since the number of the malectin and malectin-like domain-containing proteins is much larger than that of the CrRLK1L family, it is clearly not reasonable to regard the CrRLK1L family as the family of proteins containing malectin and malectin-like domain. Because of this, in the present study, we categorized the malectin and malectin-like domain containing proteins into a unique family, and thus named as the MRLK gene family.

Phylogenetic characteristics of FvMRLKs
Strawberry genome size varies by species. With 34,809 genes resulted from gene prediction modeling, the genome of diploid strawberry, F. vesca, is about twice size of the Arabidosis genome (240 Mb for F. vesca versus 125 Mb for Arabidopsis) [44]. In the present study, we identified 62 FvMRLKs in the strawberry genome database and 35 MRLKs members in the Arabidopsis genmone. Based on a phylogenetic tree generated by an alignment of the MRLK protein sequences from strawberry and Arabidopsis, we classified the 62 FvMRLKs into six major groups (Fig 3). Members within the same group shared a similar gene structure, length, and amino acid motif composition, indicating their close evolutionary relationship. FvMRLK21, 22,23,24, and 25, which were similar in motif type and the distribution and contained tandem duplications, were clustered on chromosome 3. The change in motif composition from group I to group VI may reflect the evolutionary process of the FvMRLK gene family. Tandem duplications and segmental/whole-genome duplications represent two of the major mechanisms for the expansion of the RLK gene family in plants [25].The existence of ten clustered FvMRLK genes indicated that gene duplications had occurred. Notably, group III was strawberry specific; no members from Arabidopsis were classified into this group, suggesting that this group of genes may have special roles in the regulation of growth and development in strawberry. As shown in Fig 5, among the 62 FvMRLK genes, 33 were found to be expressed primarily in fruits, and these fruit-expressed genes included some homologs of FER, HERK1/2, and THE1 (i.e., FvMRLK10, FvMRLK30, and FvMRLK47). Except for FvMRLK7 and FvMRLK40, nine genes from group III were not expressed in the fruits, possibly implying a special role of group III genes in the regulation of vegetative growth and development in strawberry.

Universal roles of FvMRLKs in the regulation of plant growth and development
Ripening of fleshy fruits is a complex process that involves dramatic changes in color, firmness, flavor, and aroma [43,45]. Strawberry is a model plant for the research of the non-climacteric fruits. Limited information is available on the mechanisms controlling fruit growth and development, especially for the non-climacteric fruits. A decrease in fruit firmness is a critical event in fruit ripening. Fruit firmness is essentially controlled by cell wall metabolism and the decrease in fruit firmness primarily results from the breakdown of cellulose and pectin. The CrRLK1L protein kinases have been shown to function in cell wall metabolism [46][47][48]. Given the close relationship between cell wall metabolism and fruit ripening and the relationship between CrRLK1Ls and cell metabolism, CrRLK1Ls may be important regulators of fruit development and ripening. Consist with this inference, in the present study, we had found that many MRLK genes were specifically and highly expressed in strawberry fruits (Fig 5). Interestingly, among the MRLKs expressed in the fruits, the expression profiles of more than twothirds of the MRLKs were found to be tightly correlated with the progress of strawberry fruit development and ripening (Fig 6). It has been well established that strawberry fruit development and ripening is regulated by both internal and environmental cues [49]. We examined the expression of MRLKs in response to these cues, such as IAA, ABA, sucrose, temperature and dehydration stresses. As shown in Fig 7A, most of the genes examined were sensitive to low temperature treatment, for example, in response to low temperate treatment, the expression of FvMRLK13 and FvMRLK21 increased to more than 25 folds compared with the control. The expression of FvMRLKs were also found to be strongly regulated by the internal cues. Furthermore, whereas treatments of ABA and sucrose, two positive signals controlling strawberry fruit ripening [42,49,50], caused an increase in the expression of some genes, the treatment of IAA [51], negative signal of fruit ripening, contrarily caused a dramatic increase in the gene expression ( Fig 7B). Collectively, these data implies a potential role of MRLKs in the regulation of strawberry fruit development and ripening. FER, HERK1/2, ANX1/2, and THE1 are several representative proteins that have been functionally elucidated in the CrRLK1L gene family [31]. FER together with its two closest homologs ANX1/2, controls polarized cell growth, thereby playing a critical role in the regulation of pollen tube and root hair elongation [52,53]. THE1 functions to mediate the responses of plant cells to the perturbation of cellulose synthesis [24]. Acting redundantly with THE1, HERK1/2 also functions to regulate cell elongation. FER, HERK1/2, ANX1/2, and THE1 may have important roles in universal biological processes. For example, FER might be implicated in ethylene biosynthesis [16], starch accumulation [17], seed development [18],vegetative growth [21,23], mechanical signal transduction [54], and pathogen resistance [19,20]. Given the importance of FER, HERK1/2, ANX1/2, and THE1, identification of their homologs in the strawberry genome is important for understanding the roles of FvMRLKs in regulating strawberry plant growth and development. As shown in Fig 3, phylogenetic analysis showed that the homologs of AXN1/2, FvMRLK33, and FvMRLK34, and the homologs of FER and FvMRLK47, are in group I. Three homologs of THE1, FvMRLK10, FvMRLK26, and FvMRLK58, and one homolog of HERK1, are in group II, implying the importance of members of group I and group II in the regulation of strawberry growth and development.
Analysis of the expression profiles of the FvMRLK genes indicated that more than 80% of the FvMRLKs were expressed in roots with transcript levels much higher than in other tissues, suggesting that FvMRLKs play a major role in the regulation of root growth and development (Fig 5). Further analysis showed that among the 62 FvMRLK genes, only 33 were expressed in the early stages of fruit development. Interestingly, among these 33 genes, the transcript levels of more than 20 genes were found to be dramatically decreased at advanced stages of fruit development. Strawberry fruit ripening is sensitive to both environmental and internal cues. Low temperature inhibits ripening, whereas high temperature promotes it. IAA, ABA, and sucrose are key signals controlling strawberry fruit ripening; thus, we examined the responses of some FvMRLKs to these compounds. As expected, the expression of many FvMRLKs was found to be sensitive to the environmental cues and the internal signals. These observations suggest that FvMRLKs play important roles in the regulation of strawberry fruit development and ripening.
Specific expression of a gene in a tissue or cell may imply that the gene plays a specific role in that tissue or cell. To identify the FvMRLKs implicated in strawberry fruit development and ripening, we hoped to identify FvMRLKs that were only expressed in fruits but not in other tissues. However, nearly all of the FvMRLKs that were expressed in fruits were also found to be expressed in other organs or tissues, especially in roots, although only about half of the FvMRLKs were expressed in the fruits. Assuming that some FvMRLKs are critical regulators of fruit development and ripening, these FvMRLKs may play different roles in different tissues or cells. This was demonstrated in a recent study. For example, OST1 is a key protein controlling stomatal movement and the ortholog of OST1 also plays a critical role in the regulation of strawberry fruit ripening. Similarly, while FER was found to play a critical role in the regulation of pollen tube growth and sperm release, it also plays a critical role in the regulation of root hair elongation as well as many other biological processes as described above. Based on the expression profile of the FvMRLK genes, further investigation is needed to establish how different FvMRLKs coordinately regulate a common biological process, and by contrast, how individual FvMRLK genes regulate distinct biological processes. Collectively, the present study provides valuable information about FvMRLKs in strawberry, and thereby casts light on the mechanisms underlying the regulation of fruit development and ripening in this model nonclimacteric fruit.

Conclusions
Sixty-two FvMRLKs were characterized in the strawberry genome, and classified these genes into six subfamilies with distinct malectin domains in the extracellular regions of the encoded proteins. More than 80% of the FvMRLKs were expressed in various tissues, with higher levels in roots than in other organs. Thirty-three FvMRLKs were expressed in fruits during the early stages of development, and over 60% of these exhibited dramatic decreases in expression during fruit growth and development. Moreover, the expression of some FvMRLKs was sensitive to both environmental and internal cues that play critical roles in regulating strawberry fruit development and ripening.
Supporting Information S1