Fig. 5. (a) Comparative functional annotation of cambial transcriptomes fromB. magnifica (Bma) and the model species Eucalyptus grandis (Egr) and Populus × euramericana (Pop) (using data generated by Xu et al. 2014). (b) Volcano plot showing transcript expression p-value versus log2 of fold-change of 240,428 annotated transcripts associated with GO terms. A total of 225 differentially expressed genes (DEGs) were recovered using a cutoff of p < 0.05 and log2 fold change > |2|: 140 up-regulated in self-supporting xylem forming tissue (blue dots) and 85 up-regulated in lianescent xylem forming tissue (green dots). (c) The five most represented categories of manually curated annotation of the 225 DEGs. In total, 154 (68%) DEGs were annotated in these five categories. Histograms in (d), (e), and (f) show the expression fold changes of genes related to cell wall, cell division, and hormone response, respectively. MYB26/52 : MYB DOMAIN PROTEIN 26/52;NC104 : NAC DOMAIN-CONTAINING PROTEIN 104/ XYLEM NAC DOMAIN 1;MYBH : MYB HYPOCOTYL ELONGATION-RELATED; CESA7 : CELLULOSE SYNTHASE CATALYTIC SUBUNIT 7; FLA1/9/11/12 : FASCICLIN-LIKE ARABINOGALACTAN 1/9/11/12; SP1L2/5 : SPIRAL1-like 2/5; CAD6/7 : CINNAMYL ALCOHOL DEHYDROGENASE 6/7;CYP75B1 : FLAVONOID 3’-MONOOXYGENASE; CHS : CHALCONE SYNTHASE; CYCB2;3 : CYCLIN B2;3; TCP20 : TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20; MAIL1 : MAINTENANCE OF MERISTEMS-LIKE1; KNAT6 : HOMEOBOX PROTEIN KNOTTED-1-LIKE6; PTL : PETAL LOSS; CKX1/5 : CYTOKININ OXIDASE/DEHYDROGENASE 1/5; UGT85A1/85ª3 : UDP-GLUCOSYL TRANSFERASE 85ª1/85ª3; GAOX1 : GA20 OXIDASE 1; CYP90B1 : CYTOCHROME P450 90B1; KIN14Q/7E/5B/12D : KINESIN-LIKE-14Q/7E/5B/12D; JASON ; ASY1 : ASYNAPTIC 1;SGO2 : SHUGOSHIN 2; HMGB13 : 3XHIGH MOBILITY GROUP-BOX1;OS1 : PARALLEL SPINDLE 1; PIN1 : PIN-FORMED 1;IAA19 ; ERF1B : ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 1B; LSH10 : LIGHT-DEPENDENT SHORT HYPOCOTYLS 10; HB17 : HOMEOBOX-LEUCINE ZIPPER PROTEIN 17; BLH1 : BEL1-LIKE HOMEODOMAIN; EDL3 : EID1-LIKE F-BOX PROTEIN 3; NPF4 : NRT1/ PTR FAMILY 4; HAB2 : PROTEIN PHOSPHATASE 2C;GAT22 : GATA TRANSCRIPTION FACTOR 22.
Upregulation of Cell Division and Cell Wall-Related Transcripts Characterizes the Self-Supporting Phase Transcriptome
To better understand the gene functions that determined the differences between self-supporting and lianescent xylem differentiation, we performed a manually-curated comprehensive annotation of all DEGs against the literature (Supporting Information Table S3a, c, column J). In line with the higher number of cells per area and the consequent greater cell wall deposition in self-supporting xylem, 11 % and 30 % of the DEGs are associated with cell division and cell wall, contrasting with only 4 % and 18 % in the lianescent counterpart, respectively (Fig. 5c, Supporting Information Table S4).
Among the DEGs associated with cell division in the self-supporting phase, we found two transcripts homologous to positive regulators of the cell cycle, the mitotic-specific cyclin CYCB2;3 and the transcription factor (TF) TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20 (TCP20 ) (DN860_c0_g2_i2 and DN10604_c0_g1_i2, respectively; Fig. 5e). We also found two transcripts whose homologs are related to meristem identity and activity maintenance, a homolog of the nuclear protein MAINTENANCE OF MERISTEMS-LIKE1 (MAIL1, DN8697_c0_g1_i3), and a homolog to the TF HOMEOBOX PROTEIN KNOTTED-1-LIKE6 (KNAT6, DN15272_c0_g1_i2), besides four mitotic specific kinesin homologs (KIN14Q, KIN7E, KIN5B, KIN12D; DN13653_c1_g1_i1, DN12012_c0_g1_i1, DN11506_c0_g1_i1, and DN13654_c0_g1_i2, respectively) and five chromatin segregation related proteins (JASON, ASY1, SGO2, HMGB13, PS1; DN1459_c0_g2_i1, DN5155_c0_g1_i1, DN19359_c0_g1_i1, DN5075_c0_g1_i1, and DN2177_c0_g1_i1, respectively; Fig. 5e).
Two transcripts homologs to MYB TFs involved in the regulation of SCW biosynthesis, MYB26, and MYB52 , were upregulated in the self-supporting phase (DN7293_c0_g2_i4 and DN35518_c0_g1_i1 respectively; Fig. 5d). Forty upregulated transcripts are directly involved in the SCW biosynthesis and structural modification, among which we highlight homologs of cellulose synthase A (CESA7 , DN8977_c0_g2_i4), four fasciclin-like arabinogalactan homolog proteins (FLA1, FLA9, FLA11, FLA12; DN7969_c0_g1_i2, DN10033_c0_g1_i1, DN49576_c0_g1_i1, and DN52901_c0_g1_i1, respectively), and two SPIRAL1-like (SP1L2 and SP1L5, DN1885_c0_g1_i2 and DN7226_c0_g1_i1 respectively; Fig. 5d).
Upregulation of Response to Stimulus, Hormone-Responsive, and Defense/Cell Death-Related Transcripts Characterizes Lianescent Phase Transcriptome
Manually-curated annotation of the lianescent phase transcriptome showed a higher proportion of TF and hormone-responsive genes, respectively, 17 % and 30 % of all DEGs of this phase, in contrast to only 11 % and 4 % in the self-supporting counterpart. Additionally, 25 % of the DEGs identified in the lianescent phase correspond to genes involved in defense/cell death mechanisms, compared to only 18 % of the DEGs in this category in the self-supporting phase (Fig. 5c, Supporting Information Table S4).
Our analysis revealed important cell differentiation regulators homologs among the upregulated DEGs that may be responsible for the fewer cell divisions and the production of larger vessels in the lianescent xylem. The repressor of cambial cell proliferation PETAL LOSS (PTL; Fig. 5e) and NAC DOMAIN-CONTAINING PROTEIN 104/ XYLEM NAC DOMAIN 1 (NC104; Fig. 5d), which delay SCW production and programmed cell death (PCD), had homologs upregulated in this phase (DN52789_c0_g1_i1 and DN10071_c0_g1_i1, respectively). ACAULIS5 (ACL5 ) was another gene responsible for preventing premature PCD with an upregulated homolog in the lianescent phase, although with a logFC of 1.1. Another candidate TF that may be related to large vessel formation was DN31217_c0_g1_i1, a homolog of the A. thaliana MYB HYPOCOTYL ELONGATION-RELATED (MYBH; Fig. 5d), that induces cellular growth triggered by auxin accumulation.
Fourteen upregulated transcripts have been annotated as TF homologs, six described as hormone-responsive: IAA19 (DN53387_c0_g1_i1), GATA TRANSCRIPTION FACTOR 22 (GAT22, DN16098_c0_g1_i1), ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 1B (ERF1B, DN35952_c0_g1_i1), LIGHT-DEPENDENT SHORT HYPOCOTYLS 10 (LSH10, DN507_c0_g1_i1), HOMEOBOX-LEUCINE ZIPPER PROTEIN 17 (HB17, DN11122_c0_g1_i1), and BEL1-LIKE HOMEODOMAIN (BLH1, DN13026_c0_g1_i2). These TFs are responsive to indole-3-acetic acid (IAA), cytokinin (CK), ethylene (ET), brassinosteroid (BR), and abscisic acid (ABA, HB17 and BLH1), respectively (Fig. 5f).
Not only hormone-responsive TF homologs were upregulated, but also homologs to transcripts related to hormone metabolism and signaling pathways. CK was the hormone with the highest number of annotated transcripts related to its catabolism/deactivation: two cytokinin dehydrogenases and two UDP-glycosyltransferases homologs (CKX1, CKX5, UGT85A1, UGT85A3; DN20233_c0_g1_i1, DN9178_c0_g1_i1, DN17065_c0_g1_i1, DN49211_c0_g1_i1 respectively). Upregulation of homologs to BR and gibberellin (GA) biosynthetic genes were also identified, CYTOCHROME P450 90B1 and GA20 OXIDASE 1 (GAOX1) respectively (DN4579_c0_g1_i1, DN11261_c0_g1_i1; Fig. 5e), as well as three genes that participate in the ABA signaling pathway, EID1-LIKE F-BOX PROTEIN 3 (EDL3 ), ABA-IMPORTING TRANSPORTER 1 (AIT1 ) and HOMOLOGY TO ABI2 (HAB2 ) (DN45297_c0_g1_i1, DN10749_c0_g1_i1, and DN10194_c0_g1_i5, respectively). Another highlight was the upregulation of the auxin efflux carrier PIN1 homolog (DN10775_c0_g1_i1; Fig. 5f), showing the relevance of all major plant hormones in the lianescent xylem differentiation process.
Together, these results show the prevalence of cell division and SCW biosynthesis during the formation of self-supporting xylem and the more complex regulation of the lianescent xylem formation, with the predominance of TF and hormone-responsive genes in this phase.