Exploration of the B3 transcription factor superfamily in Aquilaria sinensis reveal their involvement in seed recalcitrance and agarwood formation

Abstract

The endangered tree species of the Aquilaria genus produce agarwood, a high value material produced only after wounding; however, conservation of Aquilaria seeds is difficult. The B3 transcription factor family has diverse important functions in plant development, especially in seed development, although their functions in other areas, such as stress responses, remain to be revealed. Here germination tests proved that the seeds of A. sinensis were recalcitrant seeds. To provide insights into the B3 superfamily, the members were identified and characterized by bioinformatic approaches and classified by phylogenetic analysis and domain structure. In total, 71 members were identified and classified into four subfamilies. Each subfamily not only had similar domains, but also had conserved motifs in their B3 domains. For the seed-related LAV subfamily, the B3 domain of AsLAV3 was identical to that of AsVALs but lacked a typical zf-CW domain such as VALs. AsLAV5 lacks a typical PHD-L domain present in Arabidopsis VALs. qRT-PCR expression analysis showed that the LEC2 ortholog AsLAV4 was not expressed in seeds. RAVs and REMs induced after wound treatment were also identified. These findings provide insights into the functions of B3 genes and seed recalcitrance of A. sinensis and indicate the role of B3 genes in wound response and agarwood formation.This is the first work to investigate the B3 family in A. sinensis and to provide insights of the molecular mechanism of seed recalcitrance.This will be a valuable guidance for studies of B3 genes in stress responses, secondary metabolite biosynthesis, and seed development.

Fig 1. Phylogenetic tree of B3 proteins from A. sinensis and Arabidopsis.

The phylogenetic tree was constructed with the IQ-TREE program by the maximum likelihood method.

Fig 2

Gene structure analysis of 71 B3 genes (a) and conserved motif analysis of B3 proteins (b) in A. sinensis. For gene structures, exons are represented by purple boxes, and introns are represented by black lines. For conserved motifs, each motif is represented by a colored box, and the box length corresponds to the motif length.

Fig 3. Distribution of 71 B3 genes in the chromosomes of A. sinensis.

The tandem duplication genes are indicated in red.

Fig 4. Expression profiles of B3 genes in eight tissues of A. sinensis.

All gene expression levels were transformed into scores ranging from −2 to 2. For each row, blue and orange correspond to low and high values of expression, respectively.

Fig 5. Recalcitrance of A. sinensis seeds, and expression patterns of candidate AFL genes in different tissues.

(a) Germination rate of peeled and unpeeled seeds; (b) germination rate of seeds after desiccation treatment; and (c) expression pattern of candidate AFL genes in different tissues assessed via qRT-PCR. AsLAV7 is the ortholog of ABI3, AsLAV6 is the ortholog of FUS3, and AsLAV4 is the ortholog of LEC2. Significant analysis was performed by SPSS software using two-tailed unpaired Student’s t-tests (** indicates p< 0.01), or one-way analysis of variance (ANOVA) showed with lower cases a, b, and c in figure. Different letters indicate significant differences between two samples.

Fig 6. Expression profiles of B3 genes in different layers at 2, 6, and 24 h, 3, 5, and 15 d, and 21, and 34 m after wound treatment.

H, healthy layer; A, agarwood layer; T, transition layer; B, blocked layer; D, dead layer after Agar-Wit treatment; AT, agarwood and transition layer (at the early timepoints, the agarwood layer and transition layer could not be clearly divided). The expression data of all wound-treated samples were standardized and then processed to generate the Heat Map with Heatmapper with the y = log₂ + [(Trpkm + 1)/(Crpkm + 1)] as the ordinate, Trpkm indicates the rpm value of the treated group, and Crpkm indicates the rpm value of the control group H0H (untreated sample, H layer at 0 h after treatment). All gene expression levels were transformed into scores ranging from −4 to 4. For each row, blue and orange correspond to low and high values of expression, respectively.

Fig 7. A summary of the B3 superfamily and their possible involvement in seed recalcitrance and agarwood formation.