Aquaporins and lipids molecular analysis
The expression analysis of integral membrane proteins such as AQPs
(Figure 4 ) showed that B deficiency had the greatest effect on
the expression of these proteins in broccoli leaves. The B (-) treated
plants leaf presented generally lower levels of mRNA transcripts
compared to control leaves, particularly in PIP2 subfamily isoforms,
PIP2-1, PIP2-2/3, and PIP2-7. Similarly, B (+) leaves also resulted in
lower expression levels of PIP2-1. Out of the other treatments, only
salinity had an impact on the expression of PIP1-2, which showed an
almost threefold increase in mRNA transcript levels compared to control.
The vesicles obtained from microsomal fraction and plasma membrane
isolation were analyzed to characterize their size and shape (seeFigure 5 ), as well as their polydispersity index (PDI).
Overall, no differences were observed among the different samples with
respect to the treatments applied. However, differences were found
between the types of samples. Specifically, the plasma membrane vesicles
were more size-stable with a PDI ranging from 0.06 to 0.18 and a mean
size around 240-280 nm, while the microsomal fraction vesicles were
larger (830 nm) and more polydisperse (PDI of 0.7-0.9).
The immunoblotting analysis (Figure 6 ) displayed the
quantification of PIP1, PIP2 subfamilies, and PIP2-7 isoforms in
different samples of microsomal fraction and plasma membrane from
broccoli leaves of various treatments. In the case of microsomal
fractions, changes were only observed in the membrane extractions of the
Comb (+) treated plant leaves for PIP1 abundance (Figure 6A ).
However, the levels of PIP1 in plasma membrane increased in the NaCl and
B (-) treatments compared to the control, but not in the combination of
these two, Comb (-). There was a general increase in PIP2 subfamily
abundance compared to control, but only the Comb (-) treatment had the
same levels of PIP2 as the control leaves. The levels of PIP2-7
decreased in the plasma membranes of the two combinations, Comb (-) and
Comb (+), while they remained stable in other treatments compared to the
control leaves (Figure 6D-F ).
Finally, during lipids analysis, no changes were observed in fatty acid
composition (data not shown), but changes were found in sterol content
(Table 2 ). For instance, lower levels of campesterol were
observed in all treatments except B (+) when compared to the control in
the microsomal fraction samples. Also, in the same fraction, lower
levels of stigmasterol and sitosterol were found in salinity and B (-)
treated samples. When analyzed the total sterol content of MF, only the
combination treatment presented differences, with lower concentration in
total sterol content. In plasma membrane sterols, changes were mainly
seen in campesterol abundance, with lower concentrations in B (-), Comb
(-), and Comb (+) when compared to control leaves. All treatments,
except salinity, showed a reduction in sitosterol content, although no
changes were observed in stigmasterol. Overall, the total presence of
sterols was reduced in all treatment but salinity when compared to
control. However, no changes were observed in the
stigmasterol/sitosterol ratio in either the plasma membrane or
microsomal fraction sample leaves among all treatments and control.
Figure 1 . (A ) Graphical representation of dry weight
(g) root and upper part with the scale on the left and relative water
content of the plants with the scale on the right represented with
diamonds. Each measure is represented as the mean ± SE (n =6).
(B ) Leaf per mass area (LMA) (g m-2) of
treated plants after 15 days of treatment application. Data are
represented as box plot. Different letters show statistical differences,
the data corresponding both dry weight measures resulted in
non-parametric data and Kluscal wallis post hoc was selected, on
the case of relative water content and LMA statistical differences were
evaluated with one-way ANOVA using Duncan test as post hoc . Both
analyses were conducted with p < 0.05.
Figure 2. Stomatal conductance (mmol m-2s-1) of leaves from each treatment measured each 3-4
days starting the day of treatment application. Each measure is
represented as means ± SE (n = 4). Different letters in lower case
represent the statistical differences between treatments each separate
day, statistical differences were calculated with one-way ANOVA using aspost hoc Duncan test when the data were parametric and
non-parametric data was evaluated via Kluscal wallis test aspost hoc . Legend of the right represent statistical differences
in capital letters of each treatment using repeated measures ANOVA. All
analyses were conducted with p <0.05.
Figure 3. Analysis of mineral nutrients of broccoli leaves,
(A ) PCA analysis of micro and macronutrients, (B )
concentration of B, (C ) Na, (D ) and K in in treated
plants leaves. Data is represented as boxes (25-75%), error bars
represent range within 1.5 quartile, and median line (n = 4). Different
letters mean statistical differences of one-way ANOVA with Duncan test
as post hoc , p < 0.05.
Figure 4. Gene expression of different aquaporins in broccoli
leaves expressed as fold change (F. C) respect the control of isoforms
of PIP1 group (A-D ) and PIP2 group (E-I ). Data is
represented as means ± SE (n = 4). Different letters show statistical
differences one-way ANOVA and Duncan test as post hoc .
(E ) PIP2-1 and (F ) PIP2-2/3 statistical analyses for
non-parametric data, Kruskal–Wallis. p < 0.05 in all
cases.