Research Article Genome-Wide Identification and ... · Research Article Genome-Wide Identification...

Research ArticleGenome-Wide Identification and Characterization of theLRR-RLK Gene Family in Two Vernicia Species

Huiping Zhu,1,2 Yangdong Wang,1,2 Hengfu Yin,1,2 Ming Gao,1,2

Qiyan Zhang,1,2 and Yicun Chen1,2

1State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China2Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China

Correspondence should be addressed to Yicun Chen; [email protected]

Received 15 October 2015; Accepted 17 November 2015

Academic Editor: Henry Heng

Copyright © 2015 Huiping Zhu et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) make up the largest group of RLKs in plants and play important roles inmany key biological processes such as pathogen response and signal transduction. To date, most studies on LRR-RLKs have beenconducted on model plants. Here, we identified 236 and 230 LRR-RLKs in two industrial oil-producing trees: Vernicia fordii andVernicia montana, respectively. Sequence alignment analyses showed that the homology of the RLK domain (23.81%) was greaterthan that of the LRR domain (9.51%) among the Vf/VmLRR-RLKs. The conserved motif of the LRR domain in Vf/VmLRR-RLKsmatched well the known plant LRR consensus sequence but differed at the third last amino acid (W or L). Phylogenetic analysisrevealed that Vf/VmLRR-RLKs were grouped into 16 subclades. We characterized the expression profiles of Vf/VmLRR-RLKs invarious tissue types including root, leaf, petal, and kernel. Further investigation revealed that Vf/VmLRR-RLK orthologous genesmainly showed similar expression patterns in response to tree wilt disease, except 4 pairs ofVf/VmLRR-RLKs that showed oppositeexpression trends. These results represent an extensive evaluation of LRR-RLKs in two industrial oil trees and will be useful forfurther functional studies on these proteins.

1. Introduction

Plants and animals respond to changes in their environmentvia cell surface receptors, which allow them to sense bothexternal and internal signals and adapt accordingly. Receptor-like protein kinases (RLKs) are one of the most importantgroups of cell surface receptors. These proteins have specialstructural features that make them particularly suitable forcell-to-cell signaling. Since the first RLK was identified inmaize [1], many studies have functionally characterized RLKsfrom various plants, including rice, poplar, soybean, andpotato, and have shown that the RLKsmake up a superfamilyin plants. A typical RLK usually includes three distinct parts:an extracellular N-terminal domain, a single transmembrane(TM) domain, and a C-terminal intracellular kinase domain.RLKs can be classified according to their extracellular N-terminal domain.The RLKs with a leucine-rich-repeat (LRR)N-terminal domain, the LRR-RLKs, are the largest group ofproteins in the RLK superfamily.

LRR-RLK proteins in various organisms contain a con-sensus motif of 20–30 amino acid residues [2] that is tan-demly repeated to build the domain [3]. The distinguishingfeature of an LRR motif is an 11-amino acid consensussequence, LxxLxLxxNxL, where x is any amino acid [4]. Thisdomain can bind to ligands or participate in protein-proteininteractions [4].

The protein kinase (PK) domain of LRR-RLKs usuallyconsists of approximately 250–300 amino acid residues [3]and has a cytoplasmic PK domain [5]. LRR-RLKs can beclassified into three types depending on their cytoplasmicPK domain: (1) protein Ser/Thr kinases, (2) protein tyrosinekinases, and (3) protein histidine kinases [6]. The Ser/Thrkinases have been well studied in plants.The Ser/Thr domaintransduces signals downstream via autophosphorylation andthen phosphorylates specific substrates [7].

Previous studies have shown that the LRR-RLK familyhas 216 members in Arabidopsis thaliana [7], 234 members

Hindawi Publishing CorporationInternational Journal of GenomicsVolume 2015, Article ID 823427, 17 pageshttp://dx.doi.org/10.1155/2015/823427

2 International Journal of Genomics

in Solanum lycopersicum [8], 379 members in Populus tri-chocarpa [9], and 309 members in Oryza sativa [3]. Thisextreme expansion in plant genomes reflects their functionalsignificance [10]. Members of the LRR-RLK family have beenshown to play critical and diverse roles in physiological proc-esses such as secondary wall formation [11], embryogen-esis [12], meristematic growth [13], maintaining vasculartissue polarity [14], germination speed [15], regulation oforgan shape [16], pollen self-incompatibility [17], negativeregulator-programmed cell death [18], signaling pathways[19], abscisic acid (ABA) early signaling [20], brassinosteroidsignaling [21], hormone regulation [22], pathogen defense[23], tolerance to oxidative stress [15], and tolerance to saltand heat stress [10].

To date, most LRR-RLK genes have been isolated frommodel plants and herbs, rather than woody oil plants.Tung oil tree (Vernicia fordii) and wood oil tree (Verniciamontana) are important industrial oil plants belonging to theEuphorbiaceae family.The oil extracted from tung seeds is anexcellent drying oil that is renewable, safe, and environmen-tally friendly. This oil is widely used in industrial productssuch as paints, plasticizers, resins,medicine, synthetic rubber,and printing ink [24], and as a raw material for biodieselproduction [25]. China produces approximately 70–80% ofthe tung oil on the global market. However, tung trees aresusceptible to Fusarium wilt disease. Interestingly, the twodifferent species of Vernicia show different degrees of resis-tance to this disease;V. fordii, which is themain oil-producingspecies, is susceptible to the disease, while wood oil tree (V.montana) is resistant. A previous study showed that manyLRR-RLKs are defense-related [10]; therefore, studies on theLRR-RLKs of these two Vernicia species may help to clarifywhy one species is more resistant than the other.

In this study, we identified the LRR-RLKs in two Verniciaspecies and conducted multiple sequence alignments, phylo-genetic analyses, and conservedmotif analyses of theVfLRR-RLK and VmLRR-RLK gene families. We selected severalLRR-RLK genes for gene expression analyses in varioustissues of V. fordii and V. montana. Finally, we investigatedthe changes in expression of 22 Vm/fLRR-RLK genes duringinfection with Fusarium oxysporum. These results will beuseful for further studies on the functions of LRR-RLKs inwoody oil trees.

2. Materials and Methods

2.1. Plant Materials. Samples of V. fordii and V. montanawere collected from Fuyang Urban Forest Park, Hangzhoucity, Zhejiang Province, China, and then separated into roots,stems, leaves, flower buds, ovaries, and kernels. No specificpermits were required to collect the samples from the park.Three replicates were collected for all samples. The sampleswere immediately frozen in liquid nitrogen and stored at−80∘C until use.

2.2. Total RNA Isolation and cDNA Synthesis. Total RNAwas extracted separately from each sample using an RN38-EASY Spin Plus Plant RNA kit (Aidlab Biotech, Beijing,

China) following the manufacturer’s instructions. The con-centration of purified RNA was determined by agarose gelelectrophoresis and spectrophotometry (NanoDrop 5000,Thermo Scientific, Waltham, MA, USA). Only RNA sampleswith a 260/280 wavelength ratio between 2.0 and 2.2 and a260/230wavelength ratio greater than 1.8were used for cDNAsynthesis. The cDNA was synthesized using Superscript IIIRT (Invitrogen, Carlsbad, CA, USA) following the manu-facturer’s instructions. All cDNA synthesis reactions wereperformed at the same time so that the efficiency of reversetranscription was approximately equal among the samples.The cDNAs were diluted 1 : 10 with nuclease-free water forRT-PCR and amplification.

2.3. Screening for LRR-RLK Genes in V. fordii and V. montana.The members of the LRR-RLK superfamily in the twoVernicia species were first identified from transcriptomedata using look-up function of computer and using “LRR”as the key word; then we sought the selected genes oneby one according to their descriptions of annotations. Allhit genes were considered to be the purpose genes. Then,the corresponding ORF and amino acid sequences wereidentified. For all of the obtained protein sequences, thepresence of characteristic domains (LRR, TM, and RLKdomains) was confirmed using the Conserved Domain Data-base of NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Controversial sequences were used as search que-ries at PBLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PRO-GRAM=blastp&PAGE TYPE=BlastSearch&LINK LOC=blasthom). Sequences not belonging to the LRR-RLK familywere rejected. The Simple Modular Architecture ResearchTool (SMART) (http://smart.embl-heidelberg.de/) [26] wasused as a secondary method to confirm the presence of thedomain(s). All of the obtained sequences were submitted tothe NCBI. Arabidopsis LRR-RLK amino acid sequences withknown functions were downloaded from the NCBI database.

2.4. Sequence Alignment and Construction of PhylogeneticTrees. Multiple sequence alignments of amino acid sequen-ces of RLK domains and full-length amino acid sequences ofVernicia LRR-RLKs with complete domains were performedusing ClustalX v.1.83 [27] using the default settings. DNA-MAN v.5.5.2 was used as a secondary method for aligningsequences and rechecking results.

There were some studies of LRR-RLKs in many plants,such as A. thaliana, S. lycopersicum, P. trichocarpa, and O.sativa; however,muchmore information about the functionalclassification was reported in A. thaliana. To compare theevolutionary relationships of LRR-RLKs between Verniciaspecies and A. thaliana and roughly predict the functionsof LRR-RLKs in Vernicia species, multiple sequence align-ments were performed for VmLRR-RLK, VfLRR-RLK, and35 AtLRR-RLKs with known functions using the amino acidsequences of the RLK domains.

The phylogenetic trees were constructed with the neigh-bor-joiningmethod usingMEGA 5.1 software [28] with posi-tion correction, pairwise deletion, and 1000 bootstrap repli-cates indicated at each node.

International Journal of Genomics 3

Table 1: Sequences of primers specific for VfLRR-RLK and VmLRR-RLK genes amplification used qRT-PCR.

Primer names Sequences (5-3) 𝑇𝑚

Amplicons length (bp)VfLRR-RLK2 GGCTCAATCCCAGAATCAT/CCAACAGAAACGAAACATC 51.9 166VfLRR-RLK6 TTTAGACTTGCTGCCTGAC/TAACACCACCAGTGACCTG 54.1 121VfLRR-RLK7 TCAGGCAGTGAGGTAGAAG/GAAGCACGAGAAAGATTAA 48.9 182VfLRR-RLK9 ATTTCGCCACCATAGAGTC/ATTTCATCCTGCGTAAGTG 51.9 167VfLRR-RLK11 AGGAGAACAGGAAGCCCACT/GAAGGTCCATAAATGTATCA 53.3 198VfLRR-RLK13 GACCTGAAAACTGGAAATG/TATGTAACCAATGGAGCCT 49.9 147VfLRR-RLK159 TTAGGATAAGCGACAACAA/GCTACTCAGATTGGGAAAA 49.75 197VfLRR-RLK172 GTCCAATTTCGGTCGGTTGC/AGGAATGGTGTTCGGGTTT 55.2 132VfLRR-RLK256 CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG 58.6 181VfLRR-RLK260 CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG 58.6 181VfLRR-RLK271 CAACAGTCTCAACGGAAGC/AATGGATGATGGAATGGGT 53.0 106VmLRR-RLK18 TTGCCTCATGGAAATCCGACA/GGCCTGTAAGATTGGTTAAT 53.45 156VmLRR-RLK17 GGACCAGCAGTTGTGAGT/CTTTCTGTTGGGTGGAGA 53.75 168VmLRR-RLK30 ATGTCAGGCAGTGAGGTAG/GAAGCACGAGAAAGATTAA 51.95 135VmLRR-RLK29 CGCCACCATAGAGTCATAG/CACTTACGCAGGATGAAAT 53.0 163VmLRR-RLK111 TTCTTCTGGAGATCCCATTT/GTAAACCATCCTTTGCCTC 52.15 151VmLRR-RLK241 TGACCTGAAACCTGGAAAT/GCCACCCATACCATACTCT 53 172VmLRR-RLK178 TTAGGATAAGCGACAACAA/GCTACTCAGATTGGGAAAA 49.75 197VmLRR-RLK164 CTATGGAGGGTCCTATTC/TTAAGCCAGTGATTGAGC 51.45 159VmLRR-RLK206 TCGCAAATCGCCTTTATTC/ATGGCTATGCTAGGGTCAA 51.9 118VmLRR-RLK202 CTTGAGCGGTGGGCGTATC/ACCCTGGAATGACGAAGGTATG 58.6 181VmLRR-RLK210 TCATAGGCCCAGAACACTC/TCCTGGTGCTTATGTGAGT 54.1 231ACT CGATGAAGCACAGTCCAAAAG/GTTGAGAGGAGCCTCAGTG 58.85 170

2.5. Motif Recognition of LRR-RLKs in Vernicia Species. Theconserved motifs of LRR-RLK protein sequences in twoVernicia species were identified using the motif-basedsequence analysis tool, Multiple Expectation Maximizationfor Motif Elicitation (MEME) Suite version 4.10.0 (http://meme.nbcr.net/meme90/tools/meme) [29], with the follow-ing parameters: any number of repetitions of a motif, maxi-mum number of motifs = 25.

2.6. Inoculation of V. fordii and V. montana with FusariumPathogen. The tung wilt disease pathogen F. oxysporum wascultivated in potato dextrose broth (PDB, 1/4 strength) on ashaker at 180 rpm (28∘C) for 4 days to reach a fungal titerof 106 spores/mL. Roots of 2-month-old seedlings were dugfrom the soil, rinsed with water, then soaked in 75% alcoholfor 1min, 0.5% sodium hypochlorite for 3min, 90% alcoholfor 30 s, and then rinsed three times in sterile water.The rootswere wounded with a sterile knife, dipped in 100mL sporeliquid, and then replanted in soil. After this infection process,the plants were cultivated in an artificial climate chamber(8 h light/16 h dark) at 26∘C with 95% relative humidity. Theplants were observed regularly and the disease incidence wasrecorded [30]. Roots of plants were collected, and the stage ofinfection was determined according to the symptoms of theseedlings.

2.7. Real-Time Quantitative PCR (RT-qPCR). The primersused for RT-qPCR were designed using Primer Premier

5.0 with the following criteria: product size between 100and 250 bp; melting temperature around 60∘C; 40–60% GCcontent; and primer length of 18–21 bp. Primers specific forACT7 (Actin7a) [31] were used to standardize the cDNA.Subsequently, LRR-RLK gene-specific primers (Table 1) wereused to amplify the corresponding genes. The qRT-PCRswere carried out using an SYBR Premix Ex Taq Kit (TaKaRa,Tokyo, Japan) according to the manufacturer’s protocol. EachPCR mixture (20 𝜇L) consisted of 2𝜇L 4-fold diluted 1st-strand cDNA, 10 𝜇L 2x SYBR Premix Ex Taq, 0.4 𝜇L 10 𝜇Mforward and reverse primers, 0.4 𝜇L 50x ROX reference dye,and 6.8 𝜇L DEPC-treated water. Reactions were performedon an ABI 7300 Real-Time quantitative instrument (AppliedBiosystems, Foster City, CA, USA). The cycling parameterswere as follows: 95∘C for 30 s, 40 cycles of 95∘C for 5 s, and60∘C for 31 s. A melting curve analysis was performed afterthe PCR cycling to verify the specificity of the amplification.

3. Results and Discussion

3.1. Identification of VfLRR-RLKs and VmLRR-RLKs in V.fordii and V. montana. A total of 286 and 260 candidategenes in the LRR-RLK superfamily were obtained based onannotations of RNA-seq data. Then, 236 and 230 sequencesin V. fordii and V. montana with at least one characteristicdomain were positively identified as members of the LRR-RLK superfamily. All of the 466 sequences were submitted tothe NCBI by our laboratory, and the accession numbers werelisted in Table 2.


Table 2: GenBank accession numbers of VfLRR-RLK and VmLRR-RLK genes.

Vernicia fordii Vernicia montanaGene ID GenBank accession number Gene ID GenBank accession numberVfLRR-RLK1-VfLRR-RLK14 c805427-KT805440 VmLRR-RLK1-VmLRR-RLK9 KT805663-KT805671VfLRR-RLK18-VfLRR-RLK19 KT805441-KT805442 VmLRR-RLK11-VmLRR-RLK18 KT805672-KT805679VfLRR-RLK22-VfLRR-RLK24 KT805443-KT805445 VmLRR-RLK20-VmLRR-RLK32 KT805680-KT805692VfLRR-RLK26-VfLRR-RLK28 KT805446-KT805448 VmLRR-RLK34 KT805693VfLRR-RLK30-VfLRR-RLK46 KT805449-KT805465 VmLRR-RLK36-VmLRR-RLK39 KT805694-KT805697VfLRR-RLK48 KT805466 VmLRR-RLK41-VmLRR-RLK67 KT805698-KT805724VfLRR-RLK50-VfLRR-RLK53 KT805467-KT805470 VmLRR-RLK69-VmLRR-RLK83 KT805725-KT805739VfLRR-RLK55-VfLRR-RLK57 KT805471-KT805473 VmLRR-RLK86-VmLRR-RLK90 KT805740-KT805744VfLRR-RLK59-VfLRR-RLK60 KT805474-KT805475 VmLRR-RLK92 KT805745VfLRR-RLK63-VfLRR-RLK65 KT805476-KT805478 VmLRR-RLK94-VmLRR-RLK108 KT805746-KT805760VfLRR-RLK68-VfLRR-RLK70 KT805479-KT805481 VmLRR-RLK110-VmLRR-RLK115 KT805761-KT805766VfLRR-RLK72 KT805482 VmLRR-RLK117-VmLRR-RLK118 KT805767-KT805768VfLRR-RLK74-VfLRR-RLK90 KT805483-KT805499 VmLRR-RLK120 KT805769VfLRR-RLK92-VfLRR-RLK93 KT805500-KT805501 VmLRR-RLK122-VmLRR-RLK123 KT805770-KT805771VfLRR-RLK95-VfLRR-RLK97 KT805502-KT805504 VmLRR-RLK125-VmLRR-RLK129 KT805772-KT805776VfLRR-RLK99-VfLRR-RLK104 KT805505-KT805510 VmLRR-RLK131-VmLRR-RLK142 KT805777-KT805788VfLRR-RLK106-VfLRR-RLK124 KT805511-KT805529 VmLRR-RLK144-VmLRR-RLK151 KT805789-KT805796VfLRR-RLK126 KT805530 VmLRR-RLK153-VmLRR-RLK157 KT805797-KT805801VfLRR-RLK128 KT805531 VmLRR-RLK159-VmLRR-RLK187 KT805802-KT805830VfLRR-RLK130-VfLRR-RLK140 KT805532-KT805542 VmLRR-RLK190 KT805831VfLRR-RLK143 KT805543 VmLRR-RLK192-VmLRR-RLK195 KT805832-KT805835VfLRR-RLK145-VfLRR-RLK146 KT805544-KT805545 VmLRR-RLK197-VmLRR-RLK199 KT805836-KT805838VfLRR-RLK148 KT805546 VmLRR-RLK202-VmLRR-RLK206 KT805839-KT805843VfLRR-RLK150-VfLRR-RLK156 KT805547-KT805553 VmLRR-RLK208-VmLRR-RLK222 KT805844-KT805858VfLRR-RLK158-VfLRR-RLK159 KT805554-KT805555 VmLRR-RLK225-VmLRR-RLK245 KT805859-KT805879VfLRR-RLK161-VfLRR-RLK170 KT805556-KT805565 VmLRR-RLK247-VmLRR-RLK257 KT805880-KT805890VfLRR-RLK172-VfLRR-RLK190 KT805566-KT805584 VmLRR-RLK259-VmLRR-RLK260 KT805891-KT805892VfLRR-RLK193-VfLRR-RLK197 KT805585-KT805589VfLRR-RLK199-VfLRR-RLK202 KT805590-KT805593VfLRR-RLK204-VfLRR-RLK208 KT805594-KT805598VfLRR-RLK210 KT805599VfLRR-RLK212-VfLRR-RLK214 KT805600-KT805602VfLRR-RLK216-VfLRR-RLK220 KT805603-KT805607VfLRR-RLK222-VfLRR-RLK223 KT805608-KT805609VfLRR-RLK225 KT805610VfLRR-RLK227-VfLRR-RLK230 KT805611-KT805614VfLRR-RLK232-VfLRR-RLK237 KT805615-KT805620VfLRR-RLK239-VfLRR-RLK246 KT805621-KT805628VfLRR-RLK248-VfLRR-RLK258 KT805629-KT805639VfLRR-RLK260-VfLRR-RLK264 KT805640-KT805644VfLRR-RLK266-VfLRR-RLK276 KT805645-KT805655VfLRR-RLK279-VfLRR-RLK281 KT805656-KT805658VfLRR-RLK283-VfLRR-RLK286 KT805659-KT805662

The LRR-RLK family proteins contained at least one fullor partial characteristic domain (LRR, TM, and/or RLKdomains). According to the structural characteristics of theLRR-RLKs in the two Vernicia species, the proteins wereclassified into seven groups (Table 3): group 1 with an LRRdomain; group 2 with a TM domain; group 3 with an RLK

domain; group 4 with LRR and TM domains; group 5 withLRR and RLK domains; group 6 with TM and RLK domains;and group 7 with LRR, TM, and RLK domains. As shown inTable 3, groups 1, 3, and 5 had the most members and group4 had the fewest members (three in V. fordii and five in V.montana).The number ofmembers in each groupwas similar


Table 3: The number of LRR-RLK genes containing different conserved domains in V. fordii and V. montana.

Species Number of Total Number ofLRRNumber of

TMNumber of

RLKNumber ofLRR-TM

Number ofLRR-RLK

Number ofTM-RLK

Number ofLRR-TM-

RLKV. fordii 236 75 12 73 3 52 11 10V. montana 229 75 6 68 5 55 10 10

between V. fordii and V. montana, possibly because of theclose genetic relationship between these two species.

Approximately 223, 234, 309, and 379 LRR-RLK geneswere identified in the A. thaliana, O. sativa, S. Lycopersicum,and P. trichocarpa genomes, respectively [7]. Our resultsshowed that there were fewer LRR-RLK members in Verniciaspecies than in O. sativa and P. trichocarpa. This may berelated to interspecific differences or functional differenti-ation of LRR-RLKs. The genome sequences also providedinformation on the different ratios of Vernicia homologuesto LRR-RLK genes in other species.

3.2. Alignment and Evolutionary Analysis of VfLRR-RLKs andVmLRR-RLKs. Because of the large differences in length andcomplexity among the sequences, it was difficult to conductalignments for all of the LRR-RLKs identified in these twoVernicia species. Therefore, we conducted alignments for theprotein groups with the most members. First, we analyzedproteins with the LRR domain, since these were the mostabundant. When the LRR domain was selected for the align-ment the consistency was approximately 3.90%. Therefore,we selected different sequences, trimmed both ends of thesequences, and tried the alignment again. The consistencyreached 9.51%, which was still too low to build a phylogenetictree. The low consistency of LRR domains suggested a highdegree of sequence complexity and diversity between VfLRR-RLKs and VmLRR-RLKs. Therefore, we selected the RLKdomain amino acids sequence containing 53–394 aminoacids from 201 LRR-RLK genes in Vernicia species for align-ment. The consistency among these sequences was 23.81%(Supplementary Figure 1 in SupplementaryMaterial availableonline at http://dx.doi.org/10.1155/2015/823427). To analyzethe evolutionary relationships of the LRR-RLK superfamily inthese two Vernicia species, an unrooted NJ phylogenetic treewas constructed based on the multiple sequence alignmentsof 106 VfLRR-RLKs and 95 VmLRR-RLKs containing theRLK domain (Figure 1). There is no standard classificationmethod for LRR-RLKs. In previous studies, these proteinswere usually classified into different subfamilies according toclades in the phylogenetic tree. Therefore, we grouped theVfLRR-RLKs and VmLRR-RLKs into 16 subclades accordingto the phylogenetic tree (Figure 1, subclades 1–16). Subclades14, 15, and 16 had only one member, indicating that thesesubfamilies had few members or their members were toodifferent to group into the same subclade in the tree.

To confirm the reliability of the phylogenetic tree, a phy-logenetic tree was constructed for each of the two species,using the sequences of 106 VfLRR-RLK (SupplementaryFigure 2) and 95 VmLRR-RLK RLK (Supplementary Figure3) proteins. The evolutionary relationships were generally

consistent among the three trees. The genes showing closerelationships in the tree constructed for a single speciesalso showed close relationships in the tree combining bothspecies. Some VfLRR-RLK or VmLRR-RLK proteins classi-fied into the same clade in the tree for each single speciesgrouped into different clades in the tree combining the twospecies, possibly because of the more elaborate classificationin the larger tree.

To predict the function of LRR-RLKs in Vernicia species,35 Arabidopsis LRR-RLKs with known functions (Table 4)were compared with VfLRR-RLKs and VmLRR-RLKs(Figure 2). Almost every LRR-RLK subfamily in A. thalianacorresponded to an LRR-RLK subclade in Vernicia species.The members of subclade 7 in V. fordii and V. montana(Figures 1 and 2) grouped together with members ofsubfamily II in A. thaliana, suggesting that they may sharethe same function. These proteins may participate in brass-inosteroid signaling, pathogen responses, cell death, andmale sporogenesis. Similarly, members of subclade 6 in Ver-nicia species may be related to the plant brassinosteroidreceptor, vascular differentiation, abscisic acid signaling,embryonic pattern formation, another development, celldeath, and innate immunity. Subclade 10members inV. fordiiand V. montana may play a role in the pathogen response.Interestingly, the members of subclade 9 in Vernicia speciescorresponded to two different subclades in A. thaliana:AtLRR-RLKXIII and AtLRR-RLKXI. This may reflect func-tional differentiation of LRR-RLKs in A. thaliana. Based onthe roles of AtLRR-RLKXIII and AtLRR-RLKXI proteins inArabidopsis, the members of subclade 9 in Vernicia speciesmay be involved in meristem differentiation, epidermal sur-face formation during embryogenesis, floral organ abscission,determination of seed size, cell wall biosynthesis, organgrowth, and stomatal patterning and differentiation.

3.3. Motif Analysis of Vf/VmLRR-RLKs. To further revealthe diversification and potential functions of LRR-RLKs inVernicia, we selected 20 Vf/VmLRR-RLKs (Table 5) with fullcharacteristic domain and investigated their conservedmotifs using MEME version 4.10.0. A total of 25 conservedmotifs were identified and numbered 1–25 (Figure 3).

Among the 20 Vf/VmLRR-RLKs, there were six differentmotifs at the N-terminal and six at the C-terminal. The sixmotifs at the N-terminal were Motifs 19, 1, 8, 22, 17, and23. Ten of the 20 LRR-RLKs (50%) had Motif 19 at the N-terminal, and most of these LRR-RLKs were in subclades 1and 4 (Figure 4). The other five motifs were present in one tothree of the 20 LRR-RLKs. Interestingly, Motif 17 was presentat the N-terminal of two LRR-RLKs, both of which werein subclade 2. This may indicate that Motif 17 is specific to


VMLR

R-RL

K209

VFLR

R-RL

K261

19

VFLR

R-RL

K258

65

VMLR

R-RL

K215

99

VFLR

R-RL

K172

93

VFLR

R-RL

K271

VMLR

R-RL

K208

VFLR

R-RL

K269

VMLR

R-RL

K210

2729

99

61

VFL

RR-R

LK26

2

VFL

RR-R

LK25

5

VM

LRR-

RLK1

84

VM

LRR-

RLK2

04

9756

93

36

VM

LRR-

RLK1

72

VFL

RR-R

LK23

4V

MLR

R-RL

K153

9799

99

VFL

RR-R

LK24

8V

MLR

R-RL

K137

69V

FLRR

-RLK

220

99V

MLR

R-RL

K222

VFL

RR-R

LK27

5V

MLR

R-RL

K221

6899

9972

VM

LRR-

RLK1

01V

MLR

R-RL

K132

99

VFL

RR-R

LK18

5

99

VFL

RR-R

LK24

1

99

VFL

RR-R

LK14

3V

MLR

R-RL

K48

VFLR

R-RL

K130

VFLR

R-RL

K114

VMLR

R-RL

K156

8999

9972

43

97

VMLR

R-RL

K216

VMLR

R-RL

K220

99

VFLR

R-RL

K274

99

VFLR

R-RL

K254

VMLR

R-RL

K170

99

VFLR

R-RL

K193

99

VMLR

R-RL

K202

VFLR

R-RL

K256

VMLR

R-RL

K207

VFLR

R-RL

K260

3232999999

63

VFLR

R-RL

K140

VFLR

R-RL

K176

99

VFLR

R-RL

K145

49

VMLR

R-RLK

122

VMLR

R-RLK

133

53VF

LRR-R

LK251

VFLR

R-RLK

249

41VM

LRR-R

LK176

99

VFLR

R-RLK

119

VFLRR

-RLK13

4

9999

VFLRR

-RLK10

4

VFLRR-

RLK182

VMLRR

-RLK5

977528

VFLRR-

RLK77

VFLRR-

RLK222

VMLRR-R

LK166

99 VFLRR-R

LK190

99 VMLRR-

RLK194

VMLRR-R

LK203

99 VMLRR-RLK

103

74 VMLRR-RLK

179

VFLRR-RLK19

7

VMLRR-RLK13

4

994411

97

6

00

0

VFLRR-RLK156

VFLRR-RLK24463

VFLRR-RLK243

VFLRR-RLK246

99

66

VFLRR-RLK170VMLRR-RLK47

34

VFLRR-RLK10199

VMLRR-RLK56VMLRR-RLK97

32

VFLRR-RLK155

99

VMLRR-RLK187VFLRR-RLK257VMLRR-RLK175VMLRR-RLK21333

9663

9899

6

0

VFLRR-RLK245VMLRR-RLK98VMLRR-RLK135

36

VFLRR-RLK187

99


99


VFLRR-RLK167

3499

99

99

13

0

VFLRR-RLK148

VMLRR-RLK104

76

VMLRR-RLK78

99

VMLRR-RLK70

31

VFLRR-RLK219

VMLRR-RLK114

99

9

VMLRR-RLK51

VMLRR-RLK141

99

VFLRR-RLK18

VFLRR-RLK59

88

VMLRR-RLK74

99

VFLRR-RLK174

VMLRR-RLK106

99VFLRR-RLK175

99

VFLRR-RLK194

VFLRR-RLK195

53VM

LRR-RLK218

99VFLRR-RLK218

VFLRR-RLK273

VMLRR-RLK192

VMLRR-RLK197

9999

7737

3076

77

2

0

VFLRR-RLK166

VMLRR-RLK161

70VFLRR-RLK189

99VM

LRR-RLK128

VMLRR-RLK139

VFLRR-RLK230

3599

99V

MLRR-RLK229

VM

LRR-RLK237

99

12V

FLRR-RLK250

VM

LRR-RLK16099

VFLRR-RLK206

VM

LRR-RLK155

99

48

VFLRR-RLK150

VM

LRR-RLK899

VFLRR-RLK131

VM

LRR-RLK15V

FLRR-RLK19

9930

VFLRR-RLK138

VM

LRR-RLK86

99

VM

LRR-RLK90V

FLRR-RLK41V

MLRR-RLK60

VFLRR-RLK123

5580

9944

1328

27

1

0

VFL

RR-R

LK24

0

VM

LRR-

RLK1

74

99

VFL

RR-R

LK16

8

73

VFLR

R-RL

K48

VFLR

R-RL

K109

65

VMLR

R-RL

K148

99

VMLR

R-RL

K165

VFLR

R-RL

K164

VMLR

R-RL

K167

99

VFLR

R-RL

K205

VMLR

R-RL

K159

89

VFLR

R-RL

K204

99

VMLR

R-RL

K198

VFLR

R-RL

K208

VMLR

R-RL

K212

5399 76

VMLR

R-RL

K225

VFLR

R-RL

K161

VMLR

R-RL

K205

57

VMLR

R-RL

K144

94

VFLR

R-RLK

186

VMLR

R-RLK

145

99 98

VFLR

R-RLK

84

55

VMLR

R-RLK

81

VMLR

R-RLK

131

56

VFLR

R-RLK

111

51

VFLRR

-RLK31

99

VFLRR-

RLK177

VMLRR

-RLK11

8

96

VFLRR-

RLK178

99

VFLRR-

RLK99

VFLRR-

RLK100

99VFL

RR-RLK1

63VFL

RR-RLK18

8

99 78 6951

4436 47 29 65

16

20

0

VFLRR-RL

K3VML

RR-RLK11

99

VFLRR-RLK

121

53

VFLRR-RLK1

18VFLRR-RLK25

2VMLRR-RLK16

3

99 95

18VFLRR-RLK200VM

LRR-RLK14 66VFLRR-R

LK199 99VMLRR-RLK147

VFLRR-RLK201VFLRR-RLK225

74 99

99


93

VFLRR-RLK108

59

VMLRR-RLK252

99

VMLRR-RLK55

VMLRR-RLK67

99

VFLRR-RLK228

99

VFLRR-RLK154

VMLRR-RLK59

99

VMLRR-RLK108

VFLRR-RLK212

VMLRR-RLK77

5299

9989

59

34

10

5

VFLRR-RLK183

VMLRR-RLK136

99

VMLRR-RLK142

VFLRR-RLK242

VMLRR-RLK99

9999

99

16

VFLRR-RLK266

VMLRR-RLK195

99

VFLRR-RLK270

VMLRR-RLK154

VMLRR-RLK168

9994

99

37

VMLRR-RLK66

42

VFLRR-RLK213

VFLRR-RLK232

Figure 1: Phylogenetic tree based on the RLK sequences of Vf/VmLRR-RLKs. The phylogenetic tree was constructed by MEGA package v5.1using neighbor-joining method. The numbers at each branch point represent the bootstrap scores (1,000 replicates). The VfLRR-RLKs weresigned by circle filled with green, and the VmLRR-RLKs were signed by circle filled with yellow. Amino acid sequences of RLK domain usedwere listed in supplementary Data Set 1.

subclade 2.Therewere too fewmembers of subfamilies 16 and5 to make accurate predictions about their motif structure.

The six motifs at the C-terminal were Motifs 6, 20, 16, 4,9, and 7. Motif 6 was present in 11 of the 20 LRR-RLKs (55%),and in almost every subclade. All members of subclade 4 hadMotif 6 at their C-terminal. Subclade 5 had only onemember,which had Motif 4 at its C-terminal. Motif 16 was present infour of the 20 LRR-RLKs, all of which were in subclade 1.Theother C-terminal motifs were detected in only one or two ofthe 20 LRR-RLKs.

The motifs of different domains were detected accordingto their sequences and sites. The most obvious motif was thatof the LRR domain, characterized by repeated “L” residues.This motif was present in Motifs 22, 8, and 1. Amongthem, Motif 1 was the most representative of the basic LRRstructural skeleton, with the sequence LxxLxLxxNxLxGx-IPxxLxxW/Lxx. This sequence matched well the plant LRRconsensus sequence (LxxLxLxxNxLxGxIPxxLxxLxx) butdiffered at the third last amino acid (W or L). Motifs 12, 15,3, and 5 corresponded to the TM domain, andMotifs 10, 4, 9,


Table4:Subclassificatio

nof

LRR-RL

KgenesinA.

thaliana

,V.fordii,andV.

Montana

.

Subgroup

inA.

thaliana

Genen

ame(accessionnu

mberin

GenBa

nk)

Functio

nsRe

ference

Subgroup

inV.

fordii

Subgroup

inV.

montana

LRRI

LRRP

K(At4g29990)

Lightsignaltransdu

ction

[1]

VfLR

R-RL

K182

(sub

clade

5)Vm

LRR-RL

K5(sub

clade

5)

LRRII

BAK1

/AtSER

K3(At4g33430);

BKK1

/AtSER

K4(At2g13790);

AtSE

RK1(At1g71830);A

tSER

K2(At1g

34210);N

IK1(At5g1600

0);

NIK

2(At3g25560);NIK

3(At1g

60800)

Antivira

ldefense

respon

se;B

Rsig

nalin

g;celldeath;male

sporogenesis;

andpathogen

respon

se

[32–34]

VfLR

R-RL

K187

(sub

clade

7)Vm

LRR-RL

K135

(sub

clade

7);

VmLR

R-RL

K98(sub

clade

7)

LRRV

SRF4

(At3g13065);

Scrambled/SRF

9/SU

B/Strubb

elig

(At1g

11130)

Cellm

orph

ogenesis;

leafsiz

econtrol;organdevelopm

ent;

positionalsignalin

g;androot

epidermispatte

rning

[13,21,35,36]

VfLR

R-RL

K232

(sub

clade

16)

VmLR

R-RL

K122

(sub

clade

5)Vm

LRR-RL

K133

(sub

clade

5)

LRRX

BRI1(At4g39400);BR

L1(At1g

55610);B

RL2/VH1

(At2g01950);BR

L3(At3g13380);

RPK1

/TOAD1(At1g69270);

RPK2

/TOAD2(At3g02130);

EMS1/EXS

(At5g07280);BIR1

(At5g48380)

Abscisica

cidsig

nalin

g;anther

developm

ent;brassin

osteroid

receptor;celld

eath

andinnate

immun

ity;embryonicp

attern

form

ation;

andvascular

different

[20,22,37–40

]

VfLR

R-RL

K155

(sub

clade

6);

VfLR

R-RL

K257

(sub

clade

6);

VfLR

R-RL

K244

(sub

clade

6);

VfLR

R-RL

K3(sub

clade

9);

VfLR

R-RL

K121

(sub

clade

9);

VfLR

R-RL

K118

(sub

clade

11);

VfLR

R-RL

K252

(sub

clade

11)

VmLR

R-RL

K56(sub

clade

6)Vm

LRR-RL

K97(sub

clade

6)Vm

LRR-RL

K187

(sub

clade

6)Vm

LRR-RL

K175

(sub

clade

6)Vm

LRR-RL

K213

(sub

clade

6)Vm

LRR-RL

K11(subclade

9)Vm

LRR-RL

K163

(sub

clade

11)

LRRXI

GSO

1(At4g20140);G

SO2

(At5g44700);CL

V1(At1g75820);

BAM1(At5g65700);B

AM2

(At3g49670);BA

M3(At4g20270);

SOBIR1

(At2g31880);HAES

A(At4g28490);IK

U2(At3g19700);

PXY/TD

Rv(At5g61480)

Antherd

evelo

pment;celldeathand

innateim

mun

ity;epiderm

alsurfa

ceEm

bryogenesis

;formationdu

ring

floralorgan

abscission;

meristem

differentiatio

n;andseed

size

[40–

44]

VfLR

R-RL

K243

(sub

clade

6);

VfLR

R-RL

K246

(sub

clade

6);

VfLR

R-RL

K156

(sub

clade

6);

VfLR

R-RL

K206

(sub

clade

9);

VfLR

R-RL

K19(sub

clade

9)

VmLR

R-RL

K229

(sub

clade

9)Vm

LRR-RL

K237

(sub

clade

9)Vm

LRR-RL

K155

(sub

clade

9)Vm

LRR-RL

K15(sub

clade

9)

LRRXII

FLS2

(At5g46330);EF

R(At5g204

80)

Pathogen

respon

se[45]

VfLR

R-RL

K164

(sub

clade

10)

VfLR

R-RL

K48(sub

clade

10)

VfLR

R-RL

K109

(sub

clade

10)

VmLR

R-RL

K167

(sub

clade

10)

VmLR

R-RL

K148

(sub

clade

10)

LRRXIII

FEI1(At1g

31420);FEI2(At2g35620);

EREC

TA(At2g26330);ER

L1(At5g62230);ER

L2(At5g07180)

Cellw

allbiosynthesis;organ

grow

th;and

stomatalpatte

rning

anddifferentiatio

n[46–

48]

VfLR

R-RL

K230

(sub

clade

9)Vm

LRR-RL

K139

(sub

clade

9)Vm

LRR-RL

K128

(sub

clade

9)


VMLR

R-RL

K81

VMLR

R-RL

K131

58

VFLR

R-RL

K111

45

VFLR

R-RL

K31

99

VFLR

R-RL

K178

VFLR

R-RL

K177

VMLR

R-RL

K118

95100

18

VFLR

R-RL

K99

VFLR

R-RL

K100

99

VFLR

R-RL

K163

VFL

RR-R

LK18

8

10083

39

VFL

RR-R

LK84

VFL

RR-R

LK18

6

VM

LRR-

RLK1

45

100

VM

LRR-

RLK1

44

VFL

RR-R

LK16

1

VM

LRR-

RLK2

05

539698

63

28

VM

LRR-

RLK2

25

21

VFL

RR-R

LK20

5V

MLR

R-RL

K159

92

402KLR- RRLFV100

891KLR-RRLMV V

FLRR

-RLK

208

VM

LRR-

RLK2

1249100

08

13

At5g

2048

0V

FLRR

-RLK

164

VM

LRR-

RLK1

6710

0

1712

VM

LRR-

RLK1

65

23

VM

LRR-

RLK1

94V

MLR

R-RL

K203

100

VM

LRR-

RLK1

03

27

At5g

4633

0V

MLR

R-RL

K148

VFLR

R-RL

K48

VFLR

R-RL

K109

81100

99

510

VMLR

R-RL

K51

VMLR

R-RL

K141

100

VFLR

R-RL

K18

VFLR

R-RL

K59

88

VMLR

R-RL

K74

100

VFLR

R-RL

K174

VMLR

R-RL

K106

100 V

FLRR

-RLK

175

100

VFLR

R-RL

K194

VFLR

R-RL

K195

65

VMLR

R-RL

K218

100

VFLR

R-RL

K218

VFLR

R-RL

K273

VMLR

R-RL

K192

VMLR

R-RL

K197

99100

774835

70812

VFLR

R-RL

K170

VMLR

R-RLK

47

VFLR

R-RLK

101

100

At1G6

9270

VMLR

R-RLK

56

VMLR

R-RLK

97

VFLR

R-RLK

155

100 VMLR

R-RLK

187

At3g02

130

VFLRR

-RLK25

7

VMLRR

-RLK17

5

VMLRR

-RLK21

3

428073617

69698

0 At2g3

1880

VFLRR-

RLK243

VFLRR-

RLK246

100 VFLRR-

RLK156

At5g0728

0

At2g0195

0

At4g3940

0

VFLRR-RL

K244

At1g55610

At3g13380

10010098100

57333421

0

VFLRR-RLK2

45

VMLRR-RLK

207

VFLRR-RLK26026

VFLRR-RLK25624

VMLRR-RLK202100

VFLRR-RLK193

VFLRR-RLK254

VMLRR-RLK1709910

098

VFLRR-RLK274VMLRR-RLK216VMLRR-RLK220100100

99


90

VFLRR-RLK130

100

VMLRR-RLK48

100

VFLRR-RLK143

74

VFLRR-RLK241VFLRR-RLK185VMLRR-RLK101VMLRR-RLK13299

10099

48


63

VFLRR-RLK220

100

VMLRR-RLK222VFLRR-RLK275

VMLRR-RLK221

58100

100


97

VMLRR-RLK172

100

VMLRR-RLK184

VMLRR-RLK204

99

VFLRR-RLK255

58

VFLRR-RLK262

92

VFLRR-RLK269

VMLRR-RLK210

30

VMLRR-RLK208

25

VFLRR-RLK271

100

VFLRR-RLK172

VMLRR-RLK215

VFLRR-RLK261

VFLRR-RLK258

VMLRR-RLK209

296199

95

63

45

100

70

99

67

1

0

VMLRR-RLK139

VFLRR-RLK230

VMLRR-RLK128

100At1g31420

At2g35620

73100

VFLRR-RLK189

VFLRR-RLK166

VMLRR-RLK161

69100

100

At2g26330

At5g62230At5g07180

99

9933

VMLRR-RLK229

VMLRR-RLK237

100At4g20140

At5g44700

99100

17

VFLRR-RLK3

VMLRR-RLK11

100VFLRR-RLK121

78

At5g48380

VFLRR-RLK118V

FLRR-RLK252

VM

LRR-RLK163

100

7549

5

0V

FLRR-RLK240

VM

LRR-RLK174100

VFLRR-RLK168

64V

FLRR-RLK250

VM

LRR-RLK160

10021

At3g197005

VFLRR-RLK206

VM

LRR-RLK155100

At4g2849053

131K

LR-R

RLFVVM

LRR-RLK1569

At4g20270

93

At1g75820At5g65700At3g49670

10062

66

VFLRR-RLK19

43

At5g61480

40

VFLRR-RLK150

VM

LRR-RLK8

100

VFLRR-RLK138

VM

LRR-RLK86

100VMLRR-RLK90

VMLRR-RLK60

VFLRR-RLK41VFLRR-RLK123

52 87100

70

61

54

26

1

0

0

VFLR

R-RL

K140

VFLR

R-RL

K176

100

VFLR

R-RL

K145

54

VMLR

R-RL

K98

VMLR

R-RL

K135

VFLR

R-RL

K187

100

At3g

2556

0

94

At1g

6080

0

97

At1g

7183

0

At1g

3421

0

89

At4g

3343

0

97

At2g

1379

0

100

VFLR

R-RL

K158

VMLR

R-RL

K87

100

VFLR

R-RL

K165

VMLR

R-RLK

140

VFLR

R-RLK

167

100

100

56 100

25

0

VMLR

R-RLK

122

VMLR

R-RLK

133

56

At1g11

130

23

VFLR

R-RLK

222

VMLR

R-RLK

166

100

VFLRR

-RLK19

0

100

VMLRR

-RLK17

9

VFLRR-

RLK197

VMLRR

-RLK13

4

100

56 30

VFLRR-

RLK77

14

VFLRR-

RLK182

VMLRR

-RLK5

85

At4g2999

0

80

VFLRR-R

LK104

92

VFLRR-RL

K119VFLR

R-RLK134

100

VMLRR-R

LK176

VFLRR-RLK

251VFLRR-RLK

249

47 100 100

4817

2

0

VFLRR-RLK2

19VMLRR-RLK11

4 100

VMLRR-RLK70

VMLRR-RLK78VFL

RR-RLK148VMLR

R-RLK10474 100

32 11

0

VFLRR-RLK200VMLRR-RLK14 56

VFLRR-RLK199 100

VMLRR-RLK147VFLRR-RLK201VFLRR-RLK225

73100

100


91

VMLRR-RLK252

69

VFLRR-RLK108

100

VMLRR-RLK55

VMLRR-RLK67

100

VFLRR-RLK228

100

VFLRR-RLK154

VMLRR-RLK59

100

VMLRR-RLK108

VFLRR-RLK212

VMLRR-RLK77 57100

100

88

36

16

17

VFLRR-RLK266

VMLRR-RLK195

100

VFLRR-RLK270

VMLRR-RLK154

VMLRR-RLK168

100

96

100

20

VFLRR-RLK183

VMLRR-RLK136

100

VMLRR-RLK142

VFLRR-RLK242

VMLRR-RLK99

100

100

99

34

VMLRR-RLK66

65

VFLRR-RLK213

96

At5g16000

VFLRR-RLK232At3g13065

69

96

0.2

Figure 2: Phylogenetic tree based on the RLK sequences of LRR-RLK gene family both in V. fordii, V. montana, and A. thaliana. Thephylogenetic tree was constructed by MEGA package v5.1 using neighbor-joining method. The numbers at each branch point representthe bootstrap scores (1,000 replicates). The LRR-RLKs of V. Fordii were signed by circle filled with green, the LRR-RLKs of V. montana weresigned by circle filled with yellow, and the LRR-RLKs ofArabidopsis thalianawere signed by circle filled with blue.The accession number andthe amino acid sequences of the A. thaliana used were listed in supplementary Data Set 1.


Motif 1

Motif 13

Motif 19

Motif 14

Motif 15 Motif 16

Motif 17

Motif 10

Motif 11 Motif 12

Motif 18

Motif 20

Motif 7 Motif 8

Motif 2

Motif 3

Motif 5 Motif 6

Motif 9

Motif 4

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

41

39

37

35

33

31

29

27

25

23

21

19

17

15

13

1197531

37

35

33

31

29

27

25

23

21

19

17

15

13

1197531

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

41

43

45

47

49

39

37

35

33

31

29

27

25

23

21

19

17

15

13

1197531

41

39

37

35

33

31

29

27

25

23

21

19

17

15

13

1197531

29

27

25

23

21

19

17

15

13

1197531

39

37

35

33

31

29

27

25

23

21

19

17

15

13

1197531

29

27

25

23

21

19

17

15

13

1197531

29

27

25

23

21

19

17

15

13

1197531

29

27

25

23

21

19

17

15

13

1197531

29

27

25

23

21

19

17

15

13

1197531

27

25

23

21

19

17

15

13

1197531

29

27

25

23

21

19

17

15

13

1197531

29

27

25

23

21

19

17

15

13

1197531

29

27

25

23

21

19

17

15

13

1197531

21

19

17

15

13

1197531

21

19

17

15

13

1197531

21

19

17

15

13

1197531

41

43

45

47

49

39

37

35

33

31

29

27

25

23

21

19

17

15

13

1197531

15

13

1197531

1197531

Figure 3: Continued.


Motif 23

Motif 22

Motif 25

Motif 21

Motif 24

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

4

2

0

(bits

)

1197531

15

13

1197531

41

43

45

47

49

39

37

35

33

31

29

27

25

23

21

19

17

15

13

1197531

21

19

17

15

13

1197531

15

13

1197531

Figure 3: Display of conserved motifs of Vf/VmLRR-RLK gene family. The conserved motifs were searched in 20 Vf/VmLRR-RLKswhich contained full characteristic domains (the amino acid sequences were listed in supplementary Data Set 2) by Multiple ExpectationMaximization for Motif Elicitation (MEME) Suite version 4.10.0. Overall height in each stack indicates the sequence conservation at thatposition; height of each residue letter indicates relative frequency of the corresponding residue.

Table 5: Basic information of some VfLRR-RLK and VmLRR-RLK family genes.

Gene name Amino acids length L content (%) PI Molecular mass (KD) LRR-Domain TM-Domain RLK-DomainBasic information of some VfLRR-RLK family genes

VfLRR-RLK159 567 11.88 9.06 62669.6 25–204 269–453 534–564VfLRR-RLK220 782 11.75 8.29 86377.1 187–333 384–569 656–781VfLRR-RLK233 836 10.16 7.01 93442.0 389–446 5–312 566–780VfLRR-RLK248 1002 11.93 6.71 110540.3 187–333 384–569 655–922VfLRR-RLK255 719 13.04 8.41 79622.9 6–220 297–481 564–708VfLRR-RLK256 991 12.10 6.91 111107.1 155–311 445–624 701–967VfLRR-RLK258 986 10.48 6.15 108664.5 248–319 380–561 644–913VfLRR-RLK261 984 10.62 6.05 108287.2 248–319 380–561 644–913VfLRR-RLK271 1010 11.04 5.93 112529.3 159–302 378–553 634–901VfLRR-RLK275 1036 11.08 5.03 113890.5 292–351 428–612 700–963

Basic information of some VmLRR-RLK family genesVmLRR-RLK172 1028 11.97 8.31 112996.4 116–342 413–600 683–950VmLRR-RLK178 567 11.87 9.19 62741.8 269–453 25–205 518–558VmLRR-RLK179 659 11.08 7.02 74326.8 389–446 5–312 566–651VmLRR-RLK202 935 12.04 6.95 105218.5 155–311 390–569 646–912VmLRR-RLK208 1018 10.94 5.42 113540.2 280–302 378–561 642–909VmLRR-RLK209 986 10.69 6.19 108573.5 259–319 380–561 644–913VmLRR-RLK210 1010 10.83 5.50 112606.3 280–302 378–553 634–901VmLRR-RLK215 742 9.60 7.49 82247.5 2–60 99–280 363–669VmLRR-RLK216 1061 13.01 8.29 117955.4 147–276 514–695 772–1050VmLRR-RLK220 847 12.16 8.51 94738.0 19–221 293–474 551–829

20, 2, 13, and 6 corresponded to the RLK domain. Among allof the motifs, the most conserved structure of LRR-RLKs inVernicia species was the RLK domain containing Motifs 4, 9,20, 2, 13, and 6.

3.4. Expression of VfLRR-RLKs and VmLRR-RLKs in Responseto Fusarium Infection. Fusarium wilt disease of tung oil treeis a devastating fungal soil-borne disease that severely affects

tree growth.V. fordii, which is themain oil-producing species,is susceptible to this disease, whileV.montana (wood oil tree)is resistant. To investigate the responses of Vm/VfLRR-RLKsto the Fusariumwilt pathogen, we collected roots from plantsbefore infection (stage 0), at an early stage of F. oxysporuminfection (stage 1), and at a late stage of F. oxysporuminfection (stage 2). We randomly selected 22 Vm/VfLRR-RLK orthologous genes and monitored their transcript


Subclade 1VMLRR-RLK21076

100100

100100

100

34

56

100

VMLRR-RLK208VFLRR-RLK271VFLRR-RLK159VMLRR-RLK178VFLRR-RLK255VMLRR-RLK172VMLRR-RLK215VMLRR-RLK209VFLRR-RLK258VFLRR-RLK261

Subclade 2

Subclade 4

Subclade 5

Subclade 16

Motif 1

Motif 23Motif 22

Motif 25Motif 13

Motif 19

Motif 21

Motif 14Motif 15Motif 16Motif 17

Motif 10Motif 11Motif 12

Motif 24

Motif 18

Motif 20Motif 7Motif 8

Motif 2Motif 3

Motif 5Motif 6

Motif 9

Motif 4

VFLRR-RLK233

VMLRR-RLK179

VMLRR-RLK220

VMLRR-RLK216

VMLRR-RLK202VFLRR-RLK256

VFLRR-RLK248

VFLRR-RLK220

VFLRR-RLK275

76

100

100

Figure 4: Conserve motifs of different subclades of LRR-RLKs in Vernicia species. The conserve motifs of each LRR-RLK gene weresearched by Multiple Expectation Maximization for Motif Elicitation (MEME) Suite version 4.10.0. Different colors and different lengthsboxes represent different motifs.

levels by RT-PCR. The 22 orthologous genes were VfLRR-RLK2/VmLRR-RLK18, VfLRR-RLK6/VmLRR-RLK17, VfLRR-RLK7/VmLRR-RLK30,VfLRR-RLK9/VmLRR-RLK29,VfLRR-RLK11/VmLRR-RLK111, VfLRR-RLK13/VmLRR-RLK241,VfLRR-RLK159/VmLRR-RLK178, VfLRR-RLK172/VmLRR-RLK164, VfLRR-RLK256/VmLRR-RLK206, VfLRR-RLK260/VmLRR-RLK202, and VfLRR-RLK271/VmLRR-RLK210. Allgenes were amplified reliably.

The qRT-PCR results showed that although there weresome differences in transcript levels between pairs of orthol-ogous genes, most of them showed similar transcription pro-files in response to Fusarium wilt disease in both V. fordiiand V. montana during the infection period (Figure 5). This

result suggests that many Vf/VmLRR-RLKs have similarfunctions during pathogen infection. Four pairs of orthol-ogous genes (VfLRR-RLK7/VmLRR-RLK30, VfLRR-RLK159/VmLRR-RLK178, VfLRR-RLK256/VmLRR-RLK206, andVfLRR-RLK271/VmLRR-RLK210) showed opposite expres-sion patterns between V. montana and V. fordii. In V. mon-tana, the transcript levels of VmLRR-RLK30, 178, 206, and210 increased at the early stage of infection, whereas thoseof the corresponding orthologous genes in V. fordii, VfLRR-RLK7, 159, 256, and 271, decreased.This finding suggests thatthese four VmLRR-RLK genes participate in resistance to F.oxysporum in V. montana.


0

0.2

0.4

0.6

0.8

1

1.2

00.010.020.030.040.050.060.070.08

VfLRR-RLK13VmLRR-RLK241

0

1

2

3

4

5

0

0.1

0.2

0.3

0.4

0 1 2


0246810121416

0

2

4

6

8

10

0 1 2


0

5

10

15

20

0

1

2

3

4

5

0 1 2


0

0.1

0.2

0.3

0.4

0.5

0.6


0 1 20

0.1

0.2

0.3

0.4

0.5

0.6

0

0.2

0.4

0.6

0.8

1

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0 1 2


0

5

10

15

20

0

0.2

0.4

0.6

0.8

1

0 1 2


0 1 2

Relat

ive l

evel

s of e

xpre

ssio

nRe

lativ

e lev

els o

f exp

ress

ion

Relat

ive l

evel

s of e

xpre

ssio

nRe

lativ

e lev

els o

f exp

ress

ion

Relat

ive l

evel

s of e

xpre

ssio

nRe

lativ

e lev

els o

f exp

ress

ion

Relat

ive l

evel

s of e

xpre

ssio

nRe

lativ

e lev

els o

f exp

ress

ion



0

0.2

0.4

0.6

0.8

1

1.2

0

0.01

0.02

0.03

0.04

0.05

0.06

0 1 2


012345678910

0

0.2

0.4

0.6

0.8

0 1 2


0102030405060708090100

0

1

2

3

4

5

6

0 1 2


Rela

tive l

evels

of e

xpre

ssio

n

Rela

tive l

evels

of e

xpre

ssio

n

Relat

ive l

evel

s of e

xpre

ssio

n

Figure 5: Expression analysis of 22 Vm/Vf LRR-RLK genes in roots of Vernicia during infection with Fusarium. Vertical axis representsgene transcript levels. Primary axis represents transcript levels of Vf LRR-RLKs; secondary axis represents transcript levels of VmLRR-RLKs.Standard errors are shown (𝑛 = 3 biological samples). Each sample was analyzed by real-time PCR in triplicate. 0, before infection; 1, earlystage of infection; 2, late stage of infection.

3.5. Transcription Patterns of VfLRR-RLKs and VmLRR-RLRsin Various Tissues. To investigate the tissue specificity ofVfLRR-RLKs and VmLRR-RLRs expression and further ana-lyze genes related to Fusarium wilt disease, we analyzed thetranscript levels of the 22 genes described above in seventissues of V. fordii and V. montana by qRT-PCR (Figure 6).Among them, VfLRR-RLK260 and VfLRR-RLK159 showedsimilar expression patterns in all seven tissues of V. fordii.Both showed higher transcript levels in leaves and kernelsand lower transcript levels in roots, stems, buds, and ovaries.However, compared with VfLRR-RLK260, VfLRR-RLK159was more strongly expressed in petals, suggesting that itmay have a special function in floral development. VfLRR-RLK2 was expressed in roots, stems, and leaves and stronglyexpressed in petals, but not in vascular tissues. VfLRR-RLK172 was expressed most strongly in petals, followed byleaves, but expressed at low levels in the other tissues.VfLRR-RLK13 showed the highest transcript level in ovaries, followedby leaves. VfLRR-RLK271 showed similar expression patternsin all tissues. The other five genes showed tissue-specificexpression patterns. VfLRR-RLK6 was specifically expressedin petals,VfLRR-RLK9 in ovaries, andVfLRR-RLK11 in roots.Both VfLRR-RLK7 and VfLRR-RLK256 were specificallyexpressed in kernels. Together, these results suggest that

VfLRR-RLKs play various roles in the development of tungtree.

Compared with VfLRR-RLKs, most VmLRR-RLKsshowed higher transcript levels in the seven tissues analyzed.Six VmLRR-RLKs (VmLRR-RLK18, VmLRR-RLK29, VmLRR-RLK202, VmLRR-RLK30, VmLRR-RLK178, and VmLRR-RLK210) showed the same expression patterns as theirorthologous genes in V. fordii. This may indicate that theyshare the same function inV. fordii andV.montana.Theotherfive VmLRR-RLKs showed different expression patterns in V.montana.VmLRR-RLK111wasmainly expressed in leaves andhad similar transcript levels in other tissues. VmLRR-RLK164and VmLRR-RLK241 showed peak expression in kernels,but VmLRR-RLK164 was also expressed at high levels in thestems. Both VmLRR-RLK17 and VmLRR-RLK206 showedthe highest transcript levels in roots and lower levels in othertissues. The different expression patterns in V. montana mayreflect functional differentiation during evolution.

Among the seven pairs of orthologous genes showingsimilar trends in gene expression in V. montana and V.fordii in response to Fusarium infection, three pairs alsoshowed similar expression patterns in the tissues (VfLRR-RLK2/VmLRR-RLK18, VfLRR-RLK9/VmLRR-RLK29, andVfLRR-RLK260/VmLRR-RLK202). The other four pairs


0

1

2

3

4

5

Root Stem Leaf Bud Petal Ovary Kernel


05101520253035

0

0.002

0.004

0.006

0.008

0.01


0

1

2

3

4

5

0

5

10

15

20

25



00.010.020.030.040.050.06

0

0.5

1

1.5

2


0

0.2

0.4

0.6

0.8

1

0

0.1

0.2

0.3

0.4



00.050.10.150.20.250.3

0

0.2

0.4

0.6

0.8

1


0

0.002

0.004

0.006

0.008

00.511.522.533.54

00.10.20.30.40.50.6



012345678

0

0.01

0.02

0.03

0.04

0.05



00.20.40.60.811.2

00.20.40.60.8

11.2



00.20.40.60.811.21.41.6

0

0.002

0.004

0.006

0.008






Relat

ive l

evel

s of

expr

essio

nRe

lativ

e lev

els o

f ex

pres

sion

Relat

ive l

evel

s of

expr

essio

nRe

lativ

e lev

els o

f ex

pres

sion

Relat

ive l

evel

s of

expr

essio

n

Relat

ive l

evel

s of

expr

essio

n

Relat

ive l

evel

s of

expr

essio

nRe

lativ

e lev

els o

f ex

pres

sion

Relat

ive l

evel

s of

expr

essio

nRe

lativ

e lev

els o

f ex

pres

sion



0

10

20

30

40

50

0

0.2

0.4

0.6

0.8

1



Relat

ive l

evel

s of

expr

essio

nFigure 6: Transcript levels of 22Vm/Vf LRR-RLK genes in various tissues. Columnheight shows gene transcript levels. Primary axis representstranscript levels of Vf LRR-RLKs; secondary axis represents transcript levels of VmLRR-RLKs. Standard errors are shown (𝑛 = 3 biologicalsamples). Each sample was analyzed by real-time PCR in triplicate.

showed different expression patterns in the seven tissuesanalyzed. Of the four pairs of orthologous genes showingopposite responses to Fusarium infection in V. montana andV. fordii (Figure 5), three pairs showed similar expressionpatterns in the tissues, and one pair (VfLRR-RLK256 andVmLRR-RLK206) showed different expression patterns inthe tissues (Figure 6). There were high transcript levels ofVfLRR-RLK256 in kernels and VmLRR-RLK206 in the roots.Given that the Fusarium pathogen invades via the roots oftung tree, these results suggest that VmLRR-RLK206 mayplay a role in resistance to Fusarium wilt disease.

4. Conclusion

This is the first extensive evaluation of the LRR-RLK super-family in tung oil tree and wood tung tree. Phylogeneticanalyses, conserved motif analyses, and expression analysesof VfLRR-RLKs and VmLRR-RLKs in different tissues and inresponse to Fusarium infection were conducted. Characteri-zation of LRR-RLK genes in a ligneous oil plant will improveour understanding of the evolutionary processes and func-tions of this gene superfamily.The results of this study provideimportant information for further research on the diversityand functions of the LRR-RLK gene family in tung tree.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Authors’ Contribution

Huiping Zhu and YangdongWang contributed equally to thepaper.

Acknowledgment

The work was supported by the National Natural ScienceFoundation of China (31200485).

References

[1] J. C. Walker and R. Zhang, “Relationship of a putative receptorprotein kinase from maize to the S-locus glycoproteins ofBrassica,” Nature, vol. 345, no. 6277, pp. 743–746, 1990.

[2] J. Bella, K. L. Hindle, P. A. McEwan, and S. C. Lovell, “Theleucine-rich repeat structure,” Cellular and Molecular Life Sci-ences, vol. 65, no. 15, pp. 2307–2333, 2008.

[3] X. Sun and G.-L. Wang, “Genome-wide identification, charac-terization and phylogenetic analysis of the rice LRR-kinases,”PLoS ONE, vol. 6, no. 3, Article ID e16079, 2011.

[4] A. V. Kajava, “Structural diversity of leucine-rich repeat pro-teins,” Journal of Molecular Biology, vol. 277, no. 3, pp. 519–527,1998.

[5] S. H. Shiu and A. B. Bleecker, “Plant receptor-like kinase genefamily: diversity, function, and signaling,” Science Signaling, vol.2001, no. 113, article re22, 2001.

[6] P. W. Becraft, P. S. Stinard, and D. R. McCarty, “Crinkly4:a TNFR-like receptor kinase involved in maize epidermaldifferentiation,” Science, vol. 273, no. 5280, pp. 1406–1409, 1996.

[7] X. Gou, K. He, H. Yang et al., “Genome-wide cloning andsequence analysis of leucine-rich repeat receptor-like proteinkinase genes in Arabidopsis thaliana,” BMC Genomics, vol. 11,article 19, 2010.

[8] Z.Wei, J.Wang, S. Yang, Y. Song, andM. Francki, “Identificationand expression analysis of the LRR-RLK gene family in tomato(Solanum lycopersicum) Heinz 1706,”Genome, vol. 58, no. 4, pp.121–134, 2015.

[9] Y. Zan, Y. Ji, Y. Zhang, S. Yang, Y. Song, and J. Wang, “Genome-wide identification, characterization and expression analysis ofpopulus leucine-rich repeat receptor-like protein kinase genes,”BMC Genomics, vol. 14, article 318, 2013.

[10] S. Park, J.-C. Moon, Y. C. Park, J.-H. Kim, D. S. Kim, and C. S.Jang, “Molecular dissection of the response of a rice leucine-richrepeat receptor-like kinase (LRR-RLK) gene to abiotic stresses,”Journal of Plant Physiology, vol. 171, no. 17, pp. 1645–1653, 2014.

[11] J. Wang, M. Kucukoglu, L. Zhang et al., “The ArabidopsisLRR-RLK, PXC1, is a regulator of secondary wall formationcorrelatedwith the TDIF-PXY/TDR-WOX4 signaling pathway,”BMC Plant Biology, vol. 13, no. 1, article 94, 2013.

[12] E. D. L. Schmidt, F. Guzzo, M. A. J. Toonen, and S. C. De Vries,“A leucine-rich repeat containing receptor-like kinase marks


somatic plant cells competent to form embryos,” Development,vol. 124, no. 10, pp. 2049–2062, 1997.

[13] S. E. Clark, R. W.Williams, and E. M. Meyerowitz, “The CLAV-ATA1 gene encodes a putative receptor kinase that controlsshoot and floral meristem size in Arabidopsis,” Cell, vol. 89, no.4, pp. 575–585, 1997.

[14] K. Fisher and S. Turner, “PXY, a receptor-like kinase essentialfor maintaining polarity during plant vascular-tissue develop-ment,” Current Biology, vol. 17, no. 12, pp. 1061–1066, 2007.

[15] L. De Lorenzo, F. Merchan, P. Laporte et al., “A novel plantleucine-rich repeat receptor kinase regulates the response ofMedicago truncatula roots to salt stress,”The Plant Cell, vol. 21,no. 2, pp. 668–680, 2009.

[16] E. D. Shpak, M. B. Lakeman, and K. U. Torii, “Dominant-negative receptor uncovers redundancy in the ArabidopsisERECTA leucine-rich repeat receptor-like kinase signalingpathway that regulates organ shape,”The Plant Cell, vol. 15, no.5, pp. 1095–1110, 2003.

[17] J. Muschietti, Y. Eyal, and S. McCormick, “Pollen tube localiza-tion implies a role in pollen-pistil interactions for the tomatoreceptor-like protein kinases LePRK1 and LePRK2,” The PlantCell, vol. 10, no. 3, pp. 319–330, 1998.

[18] M.-H. Oh, X. Wang, X. Wu, Y. Zhao, S. D. Clouse, and S.C. Huber, “Autophosphorylation of Tyr-610 in the receptorkinase BAK1 plays a role in brassinosteroid signaling and basaldefense gene expression,” Proceedings of the National Academyof Sciences of the United States of America, vol. 107, no. 41, pp.17827–17832, 2010.

[19] N. Shimizu, T. Ishida, M. Yamada et al., “BAM 1 and RECEP-TOR−LIKE PROTEIN KINASE 2 constitute a signaling path-way and modulate CLE peptide−triggered growth inhibition inArabidopsis root,”New Phytologist, vol. 208, no. 4, pp. 1104–1113,2015.

[20] Y. Osakabe, K. Maruyama, M. Seki, M. Satou, K. Shinozaki,and K. Yamaguchi-Shinozaki, “Leucine-rich repeat receptor-like kinase1 is a key membrane-bound regulator of abscisic acidearly signaling inArabidopsis,” Plant Cell, vol. 17, no. 4, pp. 1105–1119, 2005.

[21] J. Li and J. Chory, “A putative leucine-rich repeat receptor kinaseinvolved in brassinosteroid signal transduction,” Cell, vol. 90,no. 5, pp. 929–938, 1997.

[22] S.W.Hong, J. H. Jon, J.M. Kwak, andH.G.Nam, “Identificationof a receptor-like protein kinase gene rapidly induced byabscisic acid, dehydration, high salt, and cold treatments inArabidopsis thaliana,” Plant Physiology, vol. 113, no. 4, pp. 1203–1212, 1997.

[23] J. Wang, S. Tan, L. Zhang, P. Li, and D. Tian, “Co-variationamong major classes of LRR-encoding genes in two pairs ofplant species,” Journal of Molecular Evolution, vol. 72, no. 5-6,pp. 498–509, 2011.

[24] J.-Y. Park, D.-K. Kim, Z.-M. Wang, P. Lu, S.-C. Park, and J.-S.Lee, “Production and characterization of biodiesel from tungoil,” Applied Biochemistry and Biotechnology, vol. 148, no. 1–3,pp. 109–117, 2008.

[25] Y.-H. Chen, J.-H. Chen, C.-Y. Chang, and C.-C. Chang, “Biod-iesel production from tung (Verniciamontana) oil and its blend-ing properties in different fatty acid compositions,” BioresourceTechnology, vol. 101, no. 24, pp. 9521–9526, 2010.

[26] J. Schultz, R. R. Copley, T. Doerks, C. P. Ponting, and P. Bork,“SMART: a web-based tool for the study of genetically mobiledomains,” Nucleic Acids Research, vol. 28, no. 1, pp. 231–234,2000.

[27] R. Chenna, H. Sugawara, T. Koike et al., “Multiple sequencealignment with the Clustal series of programs,” Nucleic AcidsResearch, vol. 31, no. 13, pp. 3497–3500, 2003.

[28] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, andS. Kumar, “MEGA5: molecular evolutionary genetics analysisusing maximum likelihood, evolutionary distance, and max-imum parsimony methods,” Molecular Biology and Evolution,vol. 28, no. 10, pp. 2731–2739, 2011.

[29] T. L. Bailey, N. Williams, C. Misleh, and W. W. Li, “MEME:discovering and analyzing DNA and protein sequence motifs,”Nucleic Acids Research, vol. 34, pp. W369–W373, 2006.

[30] S. Yang, Y. Wang, H. Yin et al., “Identification and characteri-zation of NF-YB family genes in tung tree,” Molecular Geneticsand Genomics, vol. 290, no. 6, pp. 2187–2198, 2015.

[31] X.Han,M. Lu, Y. Chen, Z. Zhan,Q.Cui, andY.Wang, “Selectionof reliable reference genes for gene expression studies using real-time PCR in tung tree during seed development,” PLoS ONE,vol. 7, no. 8, Article ID e43084, 2012.

[32] P.W. Becraft, “Receptor kinase signaling in plant development,”Annual Review of Cell and Developmental Biology, vol. 18, pp.163–192, 2002.

[33] S. A. Morillo and F. E. Tax, “Functional analysis of receptor-like kinases in monocots and dicots,” Current Opinion in PlantBiology, vol. 9, no. 5, pp. 460–469, 2006.

[34] S.-H. Shiu and A. B. Bleecker, “Receptor-like kinases fromArabidopsis form a monophyletic gene family related to animalreceptor kinases,” Proceedings of the National Academy ofSciences of theUnited States of America, vol. 98, no. 19, pp. 10763–10768, 2001.

[35] K. U. Torii, N. Mitsukawa, T. Oosumi et al., “The ArabidopsisERECTA gene encodes a putative receptor protein kinase withextracellular leucine-rich repeats,” The Plant Cell, vol. 8, no. 4,pp. 735–746, 1996.

[36] J. Li, J. Wen, K. A. Lease, J. T. Doke, F. E. Tax, and J. C. Walker,“BAK1, an Arabidopsis LRR receptor-like protein kinase, inter-acts with BRI1 and modulates brassinosteroid signaling,” Cell,vol. 110, no. 2, pp. 213–222, 2002.

[37] K. H. Nam and J. Li, “BRI1/BAK1, a receptor kinase pair medi-ating brassinosteroid signaling,”Cell, vol. 110, no. 2, pp. 203–212,2002.

[38] N. K. Clay and T. Nelson, “VH1, a provascular cell-specificreceptor kinase that influences leaf cell patterns in Arabidopsis,”The Plant Cell, vol. 14, no. 11, pp. 2707–2722, 2002.

[39] C. Albrecht, E. Russinova, V. Hecht, E. Baaijens, and S. deVries, “TheArabidopsis thaliana SOMATICEMBRYOGENESISRECEPTOR-LIKEKINASES1 and 2 controlmale sporogenesis,”The Plant Cell, vol. 17, no. 12, pp. 3337–3349, 2005.

[40] R. Tsuwamoto, H. Fukuoka, and Y. Takahata, “GASSHO1and GASSHO2 encoding a putative leucine-rich repeat trans-membrane-type receptor kinase are essential for the normaldevelopment of the epidermal surface in 𝐴𝑟𝑎𝑏𝑖𝑑𝑜𝑝𝑠𝑖𝑠 embryos,”Plant Journal, vol. 54, no. 1, pp. 30–42, 2008.

[41] M. Gao, X. Wang, D. Wang et al., “Regulation of cell death andinnate immunity by two receptor-like kinases in Arabidopsis,”Cell Host and Microbe, vol. 6, no. 1, pp. 34–44, 2009.

[42] D. Chinchilla, C. Zipfel, S. Robatzek et al., “A flagellin-inducedcomplex of the receptor FLS2 and BAK1 initiates plant defence,”Nature, vol. 448, no. 7152, pp. 497–500, 2007.

[43] A. Heese, D. R. Hann, S. Gimenez-Ibanez et al., “The receptor-like kinase SERK3/BAK1 is a central regulator of innate immu-nity in plants,” Proceedings of the National Academy of Sciences


of the United States of America, vol. 104, no. 29, pp. 12217–12222,2007.

[44] B. Kemmerling, A. Schwedt, P. Rodriguez et al., “The BRI1-associated kinase 1, BAK1, has a brassinolide-independent rolein plant cell-death control,” Current Biology, vol. 17, no. 13, pp.1116–1122, 2007.

[45] M. Seki, M. Narusaka, A. Kamiya et al., “Functional annotationof a full-length Arabidopsis cDNA collection,” Science, vol. 296,no. 5565, pp. 141–145, 2002.

[46] B. J. Haas, N. Volfovsky, C. D. Town et al., “Full-length mes-senger RNA sequences greatly improve genome,” Genome Bio-logy, vol. 3, no. 6, Article ID research0029, 12 pages, 2002.

[47] D.-X. Zhou, Y.-J. Kim, Y.-F. Li, P. Carol, and R. Mache, “COP1b,an isoform of COP1 generated by alternative splicing, has anegative effect on COP1 function in regulating light-dependentseedling development in Arabidopsis,” Molecular and GeneralGenetics, vol. 257, no. 4, pp. 387–391, 1998.

[48] K. Iida, M. Seki, T. Sakurai et al., “Genome-wide analysis ofalternative pre-mRNA splicing inArabidopsis thaliana based onfull-length cDNAsequences,”Nucleic Acids Research, vol. 32, no.17, pp. 5096–5103, 2004.

Submit your manuscripts athttp://www.hindawi.com

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation http://www.hindawi.com

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttp://www.hindawi.com

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research


International Journal of

Microbiology

Research Article Genome-Wide Identification and ... · Research Article Genome-Wide Identification...

Documents

Transcript of Research Article Genome-Wide Identification and ... · Research Article Genome-Wide Identification...