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).