The absence of PIP1;1 and PIP1;3 leads to a lower N content in the leaves of Arabidopsis
In Arabidopsis, PIP aquaporins were recently shown to impact on nitrate acquisition by increasing the root hydraulic conductivity (Li et al., 2016). At the amino acid level, the Arabidopsis AtPIP1;3 and AtPIP1;1 share 87.2% and 85.9% of sequence identity with the potato StPIP1;3 and StPIP1;1, respectively. When Arabidopsis WT was grown underin-vitro based N-deficient conditions, we observed a decrease in the expression of both AtPIP genes (Fig. 5). Subsequently, the Arabidopsis T-DNA insertion mutants Atpip1;1 and Atpip1;3were used to elucidate the functional properties and potential significance of these two aquaporins in the N deficiency tolerance. The WT and the T-DNA insertion lines were cultivated on the soil substrate fertilized as described earlier (Pommerrenig et al., 2018) with the exception that N was supplemented either in limiting (30 mM) or sufficient amounts (60 mM). Under N-sufficient conditions, the shoot fresh weights of the Atpip1;1 and Atpip1;3 mutants were comparable to those of the WT. When the WT and the mutants were grown under N deficiency, a reduction in the leaf biomass was observed in all plants (Fig. 6) to a similar extent. The N content of the leaves, which is a representation of N assimilated by the plant, comprised around 5 to 6 % N of the leaf dry weight in all examined plants when N was supplied in sufficient amounts (Fig. 7A). When the level of N in the soil was reduced, the WT plants contained 3.4 % of N in the leaf dry weight. Interestingly, the accumulation of N was significantly lower in bothpip mutants, where the N content was as low as 2.4% inAtpip1;1 and 2.5% in Atpip1;3 plants. In contrast, the C content increased under N deficiency similarly in both, the leaves of WT and the pip mutants. In line with these results, the pipmutants displayed a purple coloration of the leaves when the level of N in the soil was reduced (Fig. 6). Since the purple coloration of Arabidopsis leaves was attributed to anthocyanin accumulation (Kovinich, Kayanja, Chanoca, Otegui, & Grotewold, 2015), we analyzed the soluble phenylpropanoids levels. The total soluble phenylpropanoid levels of the leaves were similar in all plants when N was sufficient. As shown in Fig. 7B, under N deficiency, the content of total phenylpropanoids increased in all plants, but this change was higher in the pipmutants compared to the WT. In agreement with the observed coloration, the same trend was observed in the anthocyanin content, with an average 2.5, 2.1 and 1.7-fold increase in the Atpip1;1 , Atpip1;3and WT plants under N deficiency, respectively. These results show that the lack of either AtPIP1;1 or AtPIP1;3 in Arabidopsis leads to an increased susceptibility to N deficiency, since low N typically results in increased C/N ratios and the accumulation of secondary metabolites, including phenylpropanoids and flavonoids (Fritz, Palacios-Rojas, Feil, & Stitt, 2006).