Discussion
In the present work, we characterize how the attachment to physical support leads to the production of the lianescent xylem in the liana species Bignonia magnifica . We have also demonstrated that the more complex anatomy of the lianescent xylem results from a more complex transcriptional regulation involving the upregulation of transcriptional factors and hormone-responsive genes.
Our finding that the attachment to physical supports signals the onset of lianescent xylem production in B. magnifica (Bignoniaceae), a species that employs leaflets modified into tendrils to climb, is similar to that recently observed in the also tendrilate lianaSerjania Mexicana (Sapindaceae; Rajput et al. 2021) and in the stem twinner Condylocarpon guianense (Apocynaceae; Soffiatti et al., 2022). This mechanical and functional convergence in lianas from different families and with different climbing strategies highlights the relevance of the environmental perception in the habit. The attachment to external supports relaxes the initial mechanical function of the xylem, representing an important resource economy in the construction of supportive tissue for lianas and allowing the specialization in hydraulic conduction. The phenotypic plasticity represented by the retention of the mechanical function in young searcher stems and the control of lianescent xylem formation only by stems attached to external supports, in turn, allows to better explore the environment and can be viewed as an important ecological and evolutionary strategy, considering the high prevalence of this character among lianas (Caballé, 1993, 1998).
The ca. 30% reduction of fibers relative area represented the replacement of this cell type by large vessels, stressing the hydraulic conduction specialization (Ewerset al. , 1989, 1990; Ewers and Fisher 1991; Gasson and Dobbins, 1991; Fisher and Ewers, 1995, Lens et al. ,2011) over the mechanical function. Pit diameter increase might enhance hydraulic conductivity as well, as pit conductivity resistance accounts for about 80% of the total resistance (Choat et al., 2006), while it might also increase the probability of embolism spread (Wheeler et al. 2005; Choat et al. 2005; Hacke et al. 2006). Similarly, the connectivity of the vessel network, as indicated by the vessel grouping index, increases pathway redundancy and conductivity safety (Carlquist, 1985; Ewerset al. , 2007; Wason et al. , 2021). However, this trend is limited, as enhanced redundancy can be counteracted by the facilitated spread of embolism through the interconnected vessels above a certain threshold (Mrad et al. 2021). Finally, the increase in fiber wall thickness was in contrast to what was found in other lianas (Gartner, 1991; Ménard et al. , 2009), but it may have a role in preventing embolisms, since volume allocation to fiber walls increases embolism resistance (Janssen et al. , 2020). Our findings highlight the complex relationship between anatomical characteristics, hydraulic conductivity, and gene expression, providing insights into the differentiation of self-supporting and lianescent xylems.
The self-supporting and lianescent xylems showed distinct expression patterns. We have visually summarized the main findings of our differential expression analysis, establishing correlations between them and the observed anatomical changes during the transition from the self-supporting to lianescent phase in B. magnifica xylem (Fig. 6). The self-supporting xylem shows a higher level of cellular division, evidenced by the increased secondary xylem production and a composition comprising fibers and small diameter vessels, resulting in a greater cell density per unit area. This pattern is in accordance with the upregulation of homologs of the mitotic-specific cyclin CYCB2;3(Van Leene et al. 2010) and of the cell cycle regulatorTCP20 (Guan et al. 2017), increasing cambial cell divisions in this phase. In this sense, the upregulation of homologs of mitotic-specific kinesins and chromatin segregation-related proteins were also identified. On the other hand, the lianescent-phase transcriptome showed the upregulation of a homolog of the cambial cell proliferation repressor PTL (Zhang et al. , 2019), and four different CK catabolism/deactivation homolog transcripts, CKX1/5 and UGT85A1/85A3. CKs have long been known to induce cambial activity (Torrey & Loomis, 1967; Aloni et al. , 1990), and reduced concentrations of bioactive CKs result in decreased secondary growth in both Arabidopsis and Populus (Matsumoto-Kitano et al. , 2008; Nieminen et al. , 2008).
The higher number of cells in the self-supporting phase is expected to correlate with more abundant cell wall biosynthesis. In this sense, homologs of master MYB TFs, MYB26 and MYB52 , involved in the regulation of SCW biosynthesis (Yang et al. , 2007, 2017; Cassan-Wang et al. , 2013), were found to be upregulated in the self-supporting phase. This upregulation was accompanied by the higher expressions of cellulose synthase A homologs and four fasciclin-like arabinogalactan proteins that have been proposed to contribute to stem strength and stiffness in Eucalyptus and A. thaliana by increasing cellulose deposition and affecting cell-wall matrix integrity (Macmillan et al. , 2010). This expression pattern is compatible with the higher stiffness of young liana stems and with the colonizing role of searcher branches in nature (Caballé, 1993, 1998; Rowe & Speck, 1996, 2005; Soffiatti et al. , 2022). Interestingly, twoSPR1 homolog transcripts required for the anisotropic cell growth (Nakajima et al ., 2004) were upregulated in the self-supporting phase. SPR1 overexpression increases cell elongation, in accordance with the longer fibers found in the self-supporting xylem, while spr loss-of-function mutants show helical growth of epidermal cells and entire organs (Smyth, 2016), a characteristic of twining vine stems that has long been known (Darwin, 1875; Isnard & Silk, 2009).
The most striking feature of the lianescent xylem is the production of large vessel elements, which dramatically increased potential specific conductivity and vessel relative area. The transcriptional regulation of cell death is an essential aspect of the overall xylem maturation program, which also encompasses cell expansion and SCW deposition, and emphasizes the crucial role of inhibiting cell death during this process (Bollhöneret al ., 2012). Besides the upregulation of NC104/XND1, which represses cell wall production and PCD (Zhang et al ., 2020; Zhonget al. , 2021), ACAULIS5 (ACL5), another gene responsible for preventing premature PCD (Muñiz et al ., 2008), was upregulated in the lianescent phase. The increase in cell expansion period before PCD was linearly correlated with the lumen area in Picea trees tracheids (Anfodillo et al ., 2013; Buttò et al. , 2019). Yet, a homolog of MYBH TF, which was shown to trigger cell growth through IAA accumulation (Kwon et al ., 2013, Lu et al ., 2014), was also upregulated in the lianescent phase and might also be involved in the differentiation of larger vessels.