Fig. 6.Transcriptional regulatory network model for the self-supporting and lianescent xylem formation in the stems of the liana B. magnificabased on differential gene expression results and comprehensive anatomical analysis. The self-supporting phase has a higher growth in thickness and is composed mainly of fibers and small vessels arranged in radial files in front of protoxylem poles. Self-supporting xylem formation is associated with a higher expression of cell division and secondary cell wall biosynthesis-related transcripts. The lianescent phase has slower growth in thickness and is composed of a smaller fraction of fibers and a higher proportion of vessels. This change is due to the production of large vessels, which are produced throughout the cambium circumference. The formation of lianescent xylem is linked to a much more complex transcription network involving transcription factors, hormone-responsive genes, delayed programmed cell death, and redistribution of auxin-mediated by PIN proteins. Capital letters: observed morphoanatomical characters; in red: deduced characters.CYCB2;3 : CYCLIN B2;3; TCP20 : TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1-20; MAIL1 : MAINTENANCE OF MERISTEMS-LIKE1; KNAT6 : HOMEOBOX PROTEIN KNOTTED-1-LIKE6; KIN14Q/7E/5B/12D : KINESIN-LIKE-14Q/7E/5B/12D;JASON ; ASY1 : ASYNAPTIC 1; SGO2 : SHUGOSHIN 2;HMGB13 : 3XHIGH MOBILITY GROUP-BOX1; PS1 : PARALLEL SPINDLE 1; MYB26/52 : MYB DOMAIN PROTEIN 26/52; CESA7 : CELLULOSE SYNTHASE CATALYTIC SUBUNIT 7; FLA1/9/11/12 : FASCICLIN-LIKE ARABINOGALACTAN 1/9/11/12; SP1L2/5 : SPIRAL1-LIKE2/5; PTL : PETAL LOSS; CKX1/5 : CYTOKININ OXIDASE/DEHYDROGENASE 1/5; UGT85A1/85A3 : UDP-GLUCOSYL TRANSFERASE 85A1/85A3; GAOX1 : GA20 OXIDASE 1; C90B1 : CYTOCHROME P450 90B1; PIN1 : PIN-FORMED 1; ACL5 : ACAULIS5; NC104/XND1 : NAC DOMAIN-CONTAINING PROTEIN 104/ XYLEM NAC DOMAIN 1; MYBH : MYB HYPOCOTYL ELONGATION-RELATED;HB17 : HOMEOBOX-LEUCINE ZIPPER PROTEIN 17; BLH1 : BEL1-LIKE HOMEODOMAIN; IAA19 ; GAT22 : GATA TRANSCRIPTION FACTOR 22; ERF1B : ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 1B; LSH10 : LIGHT-DEPENDENT SHORT HYPOCOTYLS 10.
IAA polar flow also seems to be modified in the lianescent phase, as indicated by the upregulation of the polar auxin transporter PINFORMED1 (PIN1). Various processes take place along a PIN1-driven auxin flow, including bundle differentiation (Sachs, 1981; Scarpella et al ., 2006), cambium formation and maintenance (Snow,1935; Uggla et al ., 1996; Ko et al ., 2004; Mazur et al ., 2014; Ye & Zhong, 2015), and the differentiation of vessels bypassing wounds (Mazuret al ., 2016). Hence, vascular differentiation can be used as a marker of auxin flow (Sachs, 2000). Similarly, vessels in the secondary xylem are formed by the coordinated differentiation of thousands of cells, i.e. the vessel elements, in a continuous longitudinal series that can reach several meters in lianas (Zimmermann & Jeje, 1981; Ewers et al. , 1990). PIN1 upregulation is likely related to the higher number of xylem mother cell derivatives differentiating into vessel elements, leading to the broader distribution of vessels observed in the lianescent xylem. This result reinforces those previously reported suggesting that polar auxin transport defines vessel distribution and size in the secondary xylem (Johnson et al ., 2018; Novitskaya et al. , 2020).
Transcription factors are central nodes in the transcriptional network responsible for cell identity and differentiation in the xylem (Xieet al. , 2021). Here we found 14 TF upregulated in the lianescent phase, six of which participate in hormone-response pathways: two homologs to ABA response regulators, HB17 and BLH1 , which also responds to CK (Hoth et al. , 2003; Park et al. , 2013); a CYTOKININ-RESPONSIVE GATA FACTOR 1 (GAT22 ) homolog, which was shown to have a regulatory role downstream from both IAA and GA signaling pathways (Richter et al. , 2013); a homolog to ethylene-responsive transcription factor ERF1B, which was found to increase IAA production (Mao et al . 2016) and integrates ET and JA pathways (Lorenzo et al. 2003); a homolog to the IAA repressor IAA19; and a homolog to the BR responsive TF LSH10 , which was recently found to be a co-repressor of target genes by its epigenetic regulation (Goda et al. , 2004; Vo Phan et al. , 2023).
The crosstalk between TFs and various plant hormones in lianescent xylem differentiation was further supported by the upregulation of gene homologs associated with hormone biosynthesis and signaling pathway. Homologs to GAOX1 and C90B1, responsible for GA and BR biosynthesis, respectively, were upregulated in this phase. GA is known to act synergistically with IAA, increasingPIN1 expression and polar auxin transport (Willige et al. , 2011; Mäkilä et al. , 2023), conserving cambium homeostasis and xylem differentiation (Ben-Targem et al. , 2021), and induces the expression of the same genes as auxin feeding experiments inPopulus (Bjorklund et al ., 2007). Similarly, BR regulates PIN protein expression and high levels of IAA signaling in the cambium (Li et al. , 2005; Lee et al. , 2021), cellular differentiation and SCW biosynthesis (Yamamoto et al. , 1997; Caño-Delgado et al. , 2004; Du et al. , 2020), and affects CK biosynthesis and catabolism genes (Wang et al. , 2022). ABA, which is known as a crucial hormone to drought stress response, was recently shown to regulate the SCW formation under drought conditions (Yu et al. , 2021; Liu et al. , 2021), besides modulating the expression of target genes that affect vessel traits by inducing the expression of the AREB1 TF (Li et al , 2019). Two ABA signaling pathway regulators, HAB2 and EDL3 (Saez et al. , 2004; Yoshidaet al. , 2006; Koops et al. , 2011) were upregulated in the lianescent phase, what may indicate the cooption of the drought response pathway for the higher range of vessel diameters produced in the lianescent phase.
Concluding Remarks
In the present study, we verified the profound impacts of physical support on the liana B. magnifica wood anatomy, promoting the formation of the lianescent xylem. The detailed characterization of xylem anatomy showed that the onset of the lianescent phase is characterized by the beginning of vessel production also by the interfascicular cambium, previously restricted to the fascicular cambium; by the formation of large vessels of a new diameter class, that drastically increases potential specific conductivity; and by a lesser amount of cambial divisions. The comprehensive integration of anatomical and differential expression analysis data allowed us to propose a model to characterize the molecular control of the lianescent vascular syndrome establishment (Fig. 6). Our model shows that the more complex lianescent xylem reflects a more intricate transcriptional regulation network, involving a more diverse repertoire of TFs and hormone-responsive genes. We hope the analysis of the transcriptional control of the vascular system differentiation in lianas helps to better understand the differentiation of the vascular system in other habits, in addition to shedding light on the formation of the structural diversity present in tropical forests, widely recognized as a center of global diversity.