Expression Profiling of Mitogen-Activated Protein Kinase Genes Reveals Their Evolutionary and Functional Diversity in Different Rubber Tree (Hevea brasiliensis) Cultivars

Abstract

Rubber tree (Heveabrasiliensis) is the only commercially cultivated plant for producing natural rubber, one of the most essential industrial raw materials. Knowledge of the evolutionary and functional characteristics of kinases in H. brasiliensis is limited because of the long growth period and lack of well annotated genome information. Here, we reported mitogen-activated protein kinases in H.brasiliensis (HbMPKs) by manually checking and correcting the rubber tree genome. Of the 20 identified HbMPKs, four members were validated by proteomic data. Protein motif and phylogenetic analyses classified these members into four known groups comprising Thr-Glu-Tyr (TEY) and Thr-Asp-Tyr (TDY) domains, respectively. Evolutionary and syntenic analyses suggested four duplication events: HbMPK3/HbMPK6, HbMPK8/HbMPK9/HbMPK15, HbMPK10/HbMPK12 and HbMPK11/HbMPK16/HbMPK19. Expression profiling of the identified HbMPKs in roots, stems, leaves and latex obtained from three cultivars with different latex yield ability revealed tissue- and variety-expression specificity of HbMPK paralogues. Gene expression patterns under osmotic, oxidative, salt and cold stresses, combined with cis-element distribution analyses, indicated different regulation patterns of HbMPK paralogues. Further, Ka/Ks and Tajima analyses suggested an accelerated evolutionary rate in paralogues HbMPK10/12. These results revealed HbMPKs have diverse functions in natural rubber biosynthesis, and highlighted the potential possibility of using MPKs to improve stress tolerance in future rubber tree breeding.

Keywords: Hevea brasiliensis; abiotic stress; functional diversity; gene family evolution; mitogen-activated protein kinase; natural rubber biosynthesis.

Figure 1

Proteomic validation of Hevea brasiliensis mitogen-activated protein kinases (HbMPK) expression patterns in rubber tree latex before and after ethephon treatment. Representative mass spectra indicate the signal intensities of isobaric tags for the validated MPKs. The spectra correspond to the peptides of “APELCGSFFSK”, “DYVHQLR”, “DLKPSNLLLNANCDLK” and “SFDFEQNALTEEQMK” for HbMPK12, HbMPK14, HbMPK16 and HbMPK19, respectively.

Figure 2

Scaffold distribution of MPK genes in Hevea. HbMPK family members were distributed onto 20 individual scaffolds. Scaffolds are represented by vertical columns, and scaffold numbers are shown at the bottom. Scale bars are on the left. Red lines connect paralogues.

Figure 3

Multiple sequence alignment of the MPK proteins in Hevea. Conserved regions are shadowed. (A) Ten HbMPK members that contain a Thr-Glu-Tyr (TEY) activation site and a CD-domain; (B) Ten HbMPK members that contain a Thr-Asp-Tyr (TDY) active site. The alignment regions range from amino acids 100 to 500; each line consists of 100 amino acids. The four conserved domains (P-loop, C-loop, activation loop and CD-domain) are marked in red boxes.

Figure 4

Motif analysis of the MPK family members in Hevea. (A) Motif distributions for 20 HbMPKs. Rectangles with different colour represent 12 recognised conserved motifs; (B) Detailed information (amino acid sequence) of the 12 motifs. The heights of each letter represent the frequency of amino acids at that position, and the black boxes indicate the key sites of the four conserved motifs.

Figure 5

Phylogenetic tree of the MPK family in Arabidopsis thaliana, Oryza sativa, Manihot esculenta, and Hevea brasiliensis. The phylogenetic tree was constructed by ClustalX 2.0 and MEGA5.0 software using the neighbour-joining (NJ) method and 1000 bootstrap replicates. The names of the MPKs from the four species are shown in different colours. Four distinct clades (groups A–D) are highlighted. The vertical bar indicates evolutionary distance.

Figure 6

Phylogenetic and exon-intron structure analyses of MPK genes in Arabidopsis (At) and Hevea (Hb). The cladogram (left) was constructed using MEGA5.0 as described above. The exon-intron distribution (right) was analysed using GSDS software. Introns and exons are represented by lines and yellow boxes. The scale bar for genomic length is indicated at the bottom.

Figure 7

Segmental duplication of HbMPK genes and syntenic analysis of Hevea, M. esculenta, and A. thaliana MPKs. Chromosomes and scaffolds are shown in different colour in circular form. The positions of the MPK genes are marked by black lines on the circle. Duplicated MPK pairs in Hevea are connected by red lines. Syntenic relationships between Hevea and the other two species are connected by purple lines. Blue lines indicate MPK pairs between or inside M. esculenta and A. thaliana.

Figure 8

Expression profiles of HbMPKs in different tissues and stresses obtained from three cultivated varieties of rubber tree. Expression patterns of HbMPKs in different tissues from different cultivated varieties were determined using quantitative real-time PCR (qRT-PCR) (A), and semi-quantitative PCR (B). Moreover, heat map of qRT-PCR (C) and electrophoretogram of semi-quantitative PCR results (D) for 20 HbMPKs at 0 h, 3 h, 12 h, and 24 h under osmotic (polyethylene glycol, PEG), oxidative (H₂O₂), salt (1 M NaCl), and cold (4 °C) stresses are shown. The colour bar at the bottom indicates the log₂ value of the expression fold change compared with that of the HbActin reference gene. The genes were ordered by the cluster of their expression patterns.

Figure 9

Distribution of cis-elements in the promoter regions of HbMPK genes. The HbMPKs were separated into four groups according to phylogenetic subfamily (groups A–D). The numerals in two parallel columns represent the cluster in the HbMPKs expression profiles in Figure 8. Coloured capital letters represent different cis-elements at corresponding positions. Red, green, purple, and blue letters represent the transcription initiation, phytohormone response, stress response, and high transcription classifications of cis-elements, respectively. Detailed information for each cis-element provided (Supplementary Table S2).