B. magnifica Cambium and Differentiating Xylem Transcriptome is Consistent with Model Tree Species
To understand the transcriptional regulation underlying the concomitant formation of distinct xylem phases in different parts of the same plant, we conducted an RNA-Seq analysis of the active cambium and differentiating xylem of portions producing the self-supporting or the lianescent xylem. We sequenced six biological samples for each phase, resulting in approximately 512 million paired-end reads (40 GB, Supporting Information Fig. S3, Table S1a). De novo assembly produced 133,883 transcripts, from which 54,207 longest isoforms were recovered (Supporting Information Fig. S3). Among these, 20,548 transcripts showed BLASTX hits against Viridiplantae sequences in the SwissProt database (downloaded on September 18, 2019). We identified 20,428 sequences associated with Gene Ontology (GO) terms, which were further analyzed (Supporting Information Fig. S3 and Tables S1a, S1b, S1c). Out of the 7,198 unique GO terms identified, 3,738, 2,714, and 746 were related to biological processes (BP), molecular functions (MF), and cellular components (CC), respectively (Supporting Information Tables S2).
The completeness of the assembled transcriptome was assessed using BUSCO v5.2.2 (Manni et al. , 2021), which showed 99% complete BUSCOs (number of BUSCOs: 425), indicating the high quality of our assembly. Among the 20,428 GO-associated sequences, we identified two transcripts homologous to the cambial markers PHLOEM INTERCALATED WITH XYLEM (PXY)/TDIF RECEPTOR (TDR; DN4695_c0_g1_i3 and DN19385_c0_g1_i1; transcript identifiers listed in Supporting Information Table S1b) and one homologous to WUSCHEL RELATED HOMEOBOX4 (WOX4; DN4743_c0_g1_i1) (Hirakawa et al. , 2010; Suer et al. , 2011; Shi et al. , 2019; Shi et al. , 2021). We also identified the phloem markers ALTERED PHLOEM DEVELOPMENT (APL; DN4491_c0_g1_i2 and DN5859_c0_g1_i6) and NAC domain-containing protein 86 (NAC086; DN12555_c0_g1_i1) (Froelich et al. , 2011; Furuta et al. , 2014; Shi et al. , 2021), indicating successful sampling of cambium and differentiating xylem tissues.
To check the quality of the assembled transcriptome, we compared the functional annotation of B. magnifica with those of the secondary xylem-forming tissues from model species Eucalyptus grandis andPopulus × euramericana, following the approach of Zinkgrafet al. (2017) using the data generated by Xu et al . (2014). The cambium and differentiating xylem transcriptome of B. magnifica (20,428 transcripts) exhibited a higher number of transcripts with associated GO terms compared to the other two species (15,127 and 14,186 for E. grandis and P. × euramericana , respectively). Interestingly, all the most significant BP present in the model species were also found in B. magnifica , with a similar number of associated transcripts, whether considering the total transcriptome (i.e. , from both phases combined, Fig. 5a) or each phase separately. Additionally, we identified 105 BP exclusively present inB. magnifica (Supporting Information Fig. S4), potentially encompassing transcripts with unique functions in B. magnifica.
The Transcriptome of Self-Supporting and Lianescent Phases of Xylem Development Differs in Their Most Relevant Gene Functions
To identify differentially expressed genes (DEGs) between the self-supporting and lianescent phases, we assessed the diversity of expression profiles within each phase. A multidimensional scaling (MDS) analysis showed a clear grouping of samples within each set, indicating their suitability for further analysis (Supporting Information Fig. S5).
Applying a significance cut-off of p < 0.05, false discovery rate (FDR) < 0.05, and logFC > |2| (Fig. 5b), we identified 140 upregulated transcripts in the self-supporting phase and 85 upregulated transcripts in the lianescent phases (Supporting Information Tables S3a, c, respectively; count of annotated transcripts shown in Supporting Information Table S1c). Enrichment analysis revealed that in the self-supporting phase, the most represented GO categories (BP, MF, and CC) could be grouped into cell wall-related processes (overrepresented), transcriptional regulation, and protein metabolism (both underrepresented) (Supporting Information Table S3b). In the lianescent phase, the BP category response to stimulus , which includesresponse to organic substance , response to hormone,and response to endogenous stimulus , was highly overrepresented, suggesting the involvement of hormone signaling in xylem differentiation. The MF L-glutamine transmembrane transporter activity was also overrepresented (Supporting Information Table S3d).