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.