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.