Genome-wide analysis of respiratory burst oxidase homolog (Rboh) genes in Aquilaria species and insight into ROS-mediated metabolites biosynthesis and resin deposition

Abstract

Respiratory burst oxidase homolog (Rboh) generates reactive oxygen species (ROS) as a defense response during biotic and abiotic stress. In Aquilaria plants, wounding and fungal infection result in biosynthesis and deposition of secondary metabolites as defense responses, which later form constituents of fragrant resinous agarwood. During injury and fungal invasion, Aquilaria tree generates ROS species via the Rboh enzymes. Despite the implication of Rboh genes in agarwood formation, no comprehensive genomic-level study of the Rboh gene family in Aquilaria is present. A systematic illustration of their role during stress and involvement in initiating signal cascades for agarwood metabolite biosynthesis is missing. In this study, 14 Rboh genes were retrieved from genomes of two Aquilaria species, A. agallocha and A. sinensis, and were classified into five groups. The promoter regions of the genes had abundant of stress-responsive elements. Protein-protein network and in silico expression analysis suggested their functional association with MAPK proteins and transcription factors such as WRKY and MYC2. The study further explored the expression profiles of Rboh genes and found them to be differentially regulated in stress-induced callus and stem tissue, suggesting their involvement in ROS generation during stress in Aquilaria. Overall, the study provides in-depth insight into two Rboh genes, AaRbohC and AaRbohA, highlighting their role in defense against fungal and abiotic stress, and likely during initiation of agarwood formation through modulation of genes involved in secondary metabolites biosynthesis. The findings presented here offer valuable information about Rboh family members, which can be leveraged for further investigations into ROS-mediated regulation of agarwood formation in Aquilaria species.

Keywords: Aquilaria; ROS generation; Rboh proteins; agarwood; secondary metabolites.

Figure 1

Phylogenetic relationship between the Rboh proteins of (A) agallocha, (A) sinensis, (A) thaliana, (G) max, (H) vulgare, and S. tuberosum. Molecular phylogenetic tree constructed using MEGA-X with NJ method–based P distance substitutions model. Bootstrap values used to assess the tree. Five group are shown as Groups 1–5 with different colors.

Figure 2

Schematic representation of structures of 14 putative Rboh genes in two Aquilaria species. The exons and introns are indicated with pink rectangles and black color lines, respectively, on the right of phylogenetic tree. The numbers (0, 1, and 2) on the gene structures indicate the intron phases.

Figure 3

Distribution of major cis-acting elements in the promoter of AaRboh and AsRboh genes. (A) Cis-acting regulatory elements predicted in the 2.4-kb upstream regions of AaRboh and AsRboh genes, indicating with different color rectangular boxes. (B) The number of hormone responsiveness and defense-related cis-acting regulatory elements of AaRboh and AsRboh genes.

Figure 4

Overview of evolutionary relationship of Rboh of A. agallocha, A. sinensis, A. thaliana, and S. tuberosum. (A) Synteny analysis of AaRboh. (B) Synteny analysis of AsRboh and AsRboh (green line shows duplicate genes). (C) Synteny analysis among A. agallocha and A. sinensis; A. agallocha and A. thaliana; A. agallocha and S. tuberosum; A. sinensis and A. thaliana; and A. sinensis and S. tuberosum. Gray lines represent all collinearity blocks, whereas red lines show orthologous gene pairs among two species.

Figure 5

Conserved motifs distribution of AaRboh and AsRboh protein sequences. (A) Ten types of conserved motifs of AsRboh and AaRboh. (B) Four particular characteristics of motif of AsRboh and AaRboh. (C) Sequence logos of the NADPH_Ox, Ferric_reduct, FAD_binding_8, and NAD_binding_6 motif.

Figure 6

Multiple protein sequence alignment and domain structure of Rboh proteins of A. agallocha and A. sinensis. Highly conserved amino acids indicate with red shading, and low amino acid levels represent with lighter shading. The NADPH_ox (PF08414), EF-hand domain, Ferric_reduct (PF01794), FAD_binding_8(PF08022), and NAD_binding_6 (PF08030) were indicated with blue, black, violet, brown, and yellow color, respectively.

Figure 7

Protein interaction network of AaRboh in A. agallocha based on Arabidopsis orthologs. The potential AaRboh with their functional partners [MPK3 (mitogen-activated protein kinase 3), CPK28 (calcium-dependent protein kinase 28), CDPK1, WRKY33 (WRKY transcription factor 33), ERF (EF-TU receptor), BRI1 (brassinosteroid-insensitive 1), ABI1(abscisic acid–insensitive 1), ABI2, OST1(open stomata 1), ABF2 (abscisic acid–responsive element–binding factor 2), HAB (hypersensitive to ABA1), and PP2CA (protein phosphatase 2CA)] in each enriched pathway are displayed in a network model of proteins where the lines of various colors indicate the type of interactions between the potential AaRboh and their functional partners. The solid and dotted lines represent connections within the same and different clusters.

Figure 8

Expression profile of AaRboh and AsRboh genes of different types tissues of A. agallocha and A. sinensis. (A) AaRboh gene expression patterns in agarwood tissue. X-axis represents the AaRboh members, and Y-axis represents the log2 fold change value. (B) Expression patterns of genes involved in terpenoid biosynthesis genes where DXS indicates 1-deoxy-D-xylose-5-phosphate synthase, HMGR indicates 3-Hydroxy-3-methylglutaryl-coenzyme A reductase, MVK indicates mevalonate kinase, GGPS indicates geranylgeranyl diphosphate synthase, and FPS indicates farnesyl pyrophosphate synthase.(C) Expression patterns of mitogen-activated protein kinase (MAPK) signaling cascades genes. (D) Expression pattern of transcription factors. (E) AsRboh gene expression patterns in the six different tissues compared to aril tissue. * indicates p-value less than 0.05, and ** indicates p-value less than 0.01.

Figure 9

Relative expression levels of AaRboh genes in treated calli of A. agallocha. Relative transcripts abundance of seven AaRboh genes were measured in calli tissue transferred to MS media with H₂O₂, H₂O₂ + DMTU, DMTU, respectively, and calli without any treatment considered as control condition and samples harvested at 0 h, 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h. Transcript abundances were measured using A. agallocha GAPDH as internal control. Asterisk (*) denotes a significant difference compared with healthy samples at 0.05 or **P < 0.01 (Student’s t-test). Data represent means ± SE off three independent experiments.

Figure 10

Relative expression levels of AaRboh genes in H₂O₂-treated stem of A. agallocha. The stems were cut, and the apical end of each cut stem was placed in distilled H₂O, H₂O₂, DMTU, respectively, as appropriate. The pre-treating solution was thrown away after 2 h, and the stems were left exposed to air. The samples were taken at 0 h, 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h following air exposure. The samples without any treatments are considered as healthy. Asterisks (*) denotes a significant difference compared with healthy samples at 0.05 or **P < 0.01 (Student’s t-test). Data represent means ± SE off three independent experiments.

Figure 11

Endogenous H₂O₂ content in calli and stem of (A) agallocha. (A) Content of endogenous H₂O₂ in calli treated with H₂O₂, DMTU, and H₂O₂ + DMTU, respectively, for 0, 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h. (B) Content of endogenous H₂O₂ in the 1-year-old stems after pruning, the cut ends were immersed in distilled H₂O, H₂O₂, and DMTU. The pruned stems were exposed to air after 2 h, and the pretreating solution was discarded. The healthy condition indicates the samples without any treatment and served as control. Following air exposure, samples were collected at 0 h, 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h. Asterisks (*) indicate a statistically significant difference from healthy samples at *P < 0.05 or **P < 0.01 (Student’s t-test). The data represent the means and standard deviations of three independent experiment.

Figure 12

qRT-PCR analysis of two selected AaRboh genes. The −2^ΔΔCT method was used to determine relative gene expression value. The house keeping gene GAPDH was used to normalized the data. The * symbol indicates transcript levels that differ statistically significantly based on the student t test, and the P-value (**P < 0.01). The mean SE of three technical replicates is used to calculate each expression value. The infected and non-infected plants from Hoollongapar Gibbon Sanctuary.