Samples for Differential Expression Analysis
To
comprehend the transcriptional control mechanism responsible for the
concomitant formation of the self-supporting and lianescent xylem in
different portions of the same plant of B. magnifica ,
we sampled the cambium and
differentiating xylem of entire internodes samples (approximately 30 cm
long) collected from 60 cm from
the shoot apex to the base of the stem (from around the
10th to the 20th internodes), along
different
stems
of a single plant. After two
years of cultivation, the sampling was conducted in April 2018 during
the period of active cambium by peeling the bark and gently scraping the
exposed xylem (Allona et
al. , 1998; Sterky et al. ,
1998; Perry et al. , 2021).
The cambial activity was assessed by anatomical analysis (Supporting
Information Fig. S1) and the ease with which the bark was peeled
(Wilcox, 1962; Melder et al. , 2015). We chose this sampling
strategy to avoid that the observed differences could be a product of,
or be masked by, allelic variations, as B. magnifica is a wild,
highly heterozygous species with no available sequence data. Given our
focus on cambium and differentiating xylem, we also avoided sampling
tissues below the peeled bark to prevent collecting other tissues, such
as phloem and cortex, as the bark in the species was fragile and wrapped
immediately after removal. This also prevented sampling the cambium and
phloem from the phloem wedges (Supporting Information Fig. S2), what
would add complexity to our analysis and was out of the scope of the
present work. The innermost position of the variant cambium in the stem
facilitated the collection of only regular portions for differential
expression analysis, which were the only ones analyzed for this purpose.
Tissues were immediately frozen in liquid nitrogen and stored at -80 °C.
Before tissue harvest, each stem segment had its base cut and stored in
50 % alcohol to determine the xylem phase, self-supporting or
lianescent, and any segment showing dubious or transitional anatomy, and
the segment next to it were discarded. Six pools of self-supporting and
six pools of lianescent cambium and differentiating xylem, composed of
8-19 segments each, were collected for sequencing and differential
expression analysis.