PIP1;1 and PIP1;3 are indirectly involved in the N uptake
Interestingly, two aquaporins, StPIP1;1 and StPIP1;3 were induced under
N deficiency in the tolerant cultivar ‘Topas’. PIPs are channel proteins
located in the endoplasmic reticulum and the PM, involved in promoting
the transport of water and small neutral solutes such as hydrogen
peroxide, carbon dioxide or arsenite across the PM (Dynowski, Schaaf,
Loque, Moran, & Ludewig, 2008; Fetter et al., 2004; Mosa et al., 2012;
Uehlein, Lovisolo, Siefritz, & Kaldenhoff, 2003). Previously, it was
shown that certain aquaporin isoforms of the Tonoplast Intrinsic Protein
(TIP) family facilitate the transport of ammonia across biological
membranes (Jahn et al., 2004; Kirscht et al., 2016; Loque et al., 2005).
Here, we tested whether StPIP1;1 and StPIP1;3 are directly involved in N
uptake by sharing the selectivity properties with TIPs. The expression
of StPIP1;1 and StPIP1;3 did not increase the growth of yeast mutants
under low N supply (Fig. 4), strongly suggesting that these isoforms do
not facilitate the transport of ammonia and do not contribute directly
to N uptake in plants.
PIPs were described to be efficient water channels (Maurel et al., 2015)
and high PIP expression correlates with high root hydraulic conductivity
(L pr) (Gambetta et al., 2013; Perrone et al.,
2012). Remarkably, it was shown that the local and/or overall nitrate
availability influences the L pr and thereby, N
uptake by impacting the diffusion of nitrate towards the root surface
(Gorska, Ye, Holbrook, & Zwieniecki, 2008; Ishikawa-Sakurai, Hayashi,
& Murai-Hatano, 2014; G. Li, Tillard, Gojon, & Maurel, 2016).
Generally, it is observed that N limitation decreases whereas N resupply
stimulates, L pr and PIP expression. In contrast
to these observations, StPIP1;1 and StPIP1;3 protein abundance increased
along with StNRT2.1 in the N deficiency tolerant cultivar ‘Topas’,
whereas the transcript levels decreased for StPIP1;1 and remained
unchanged for StPIP1;3 (Fig. 3). Discrepancies between aquaporin
transcript and protein levels are not uncommon and might be reasoned by
more dynamic mRNA turnover, protein stability or by regulating protein
activity by post-translational modifications (Hachez et al., 2012;
Velez-Bermudez & Schmidt, 2014).
In order to further elucidate the possible function of these PIP
isoforms in respect to N deficiency tolerance, we used T-DNA knock-out
mutants of the homologous genes of StPIP1;1 and StPIP1;3in Arabidopsis (AtPIP1;1 and AtPIP1;3 ) and investigated
these plants in more detail. In WT plants, AtPIP1;1 andAtPIP1;3 transcript levels were downregulated in roots under
N-deficient conditions (Fig. 5), which is in line with observations in
other plant species in which the transcript levels of PIPs were
studied under N limitation (Ishikawa-Sakurai et al., 2014).
Mass flow of N towards the root surface, which is stimulated by the
close interrelation between transpiration, convection andL pr, is one of the factors contributing to
efficient N uptake (Tyerman, Wignes, & Kaiser, 2017). Working with
hydroponic- or in vitro -based plant cultivation systems, limits
the impact of these parameters on the mass flow towards the roots and
might mask the contribution of PIPs to these processes. We established a
soil substrate-based cultivation system to overcome this limitation.
Interestingly, both insertion lines displayed a higher sensitivity to N
deficiency reflected in their lower N content in the leaves, as well as
in their larger total anthocyanins content (Fig. 6). This indicates that
despite their transcriptional downregulation under N limiting conditions
in Arabidopsis, the complete lack of these proteins leads to an increase
in N deficiency sensitivity.
Recently, it was proposed that in Arabidopsis NRT2.1 in its action as a
nitrate sensor/transducer, impacts on PIP expression and thereby, on theL pr (G. Li et al., 2016). It was shown thatPIP1 and PIP2 expression and protein abundance were
largely unaffected under both high and low N treatments in the WT,
suggesting regulatory events on the post-translational level. PIP
expression in the Atnrt2;1 mutant was lower than in the WT, which
co-occurred with a decrease in L pr (G. Li et al.,
2016). In respect to N availability, it was shown that despite of a
decrease in PIP expression under N deficiency,L pr was rapidly recovered under N resupply
(Gloser, Zwieniecki, Orians, & Holbrook, 2007; Gorska, Ye, et al.,
2008; Gorska, Zwieniecka, Holbrook, & Zwieniecki, 2008). This is
probably due to a high degree of post-translational modifications, such
as phosphorylation, which enables a rapid response towards changing N
availabilities (di Pietro et al., 2013). It was already argued that
aquaporins activity is locally regulated by the N availability of roots
(Gorska, Ye, et al., 2008; Ishikawa-Sakurai et al., 2014). Therefore, it
is conceivable, that irrespectively of an overall transcriptional
downregulation of AtPIP1;1 and AtPIP1;3 under N limitation
in Arabidopsis, the protein is of importance to sustain local or overallL pr at places with higher N availability or under
prolonged N deficiency, respectively. Furthermore, a higher level of
certain PIP isoforms might be of advantage under N-deficient conditions
to enable rapid changes in protein activity.
We observed a concurrently increased abundance of StNRT2.1 with StPIP1;1
and StPIP1;3 in the tolerant potato cultivar. This indicates that in the
cultivar ‘Topas’, these two PIP aquaporins might be in their inactive
state, awaiting their local and rapid activation, to ensure efficient N
delivery to the root surface. Another explanation might be, that
StPIP1;1 and StPIP1;3 are important isoforms which ensure a steady state
mass flow of water towards the root under N limiting conditions, which
points towards a function of these PIPs in contributing to the N
deficiency tolerance in cultivar ‘Topas’.
In conclusion, N limitation has been shown to modify the potato root PM
proteome, and clear differences have been identified between the two
varieties contrasting in their response to N deficiency. It has been
possible to identify several candidate proteins likely involved in the
mechanisms underlying N deficiency tolerance. Our data suggest that the
increased abundance of StPIP1;1 and StPIP1;3 together with StNRT2.1 is
one of the factors contributing to N deficiency tolerance in potato. In
addition, evidence is presented that AtPIP1;1 and AtPIP1;3 interplay
with efficient N uptake under N limiting conditions in Arabidopsis,
which is in accordance with the working model proposed previously (G. Li
et al., 2016; Tyerman et al., 2017). To the best of our knowledge, it
has not been reported previously, that the lack of individual PIP
aquaporins results in a decrease in N deficiency tolerance.