V-ATPase and V-PPase at the Tonoplast Affect NO3 − Content in Brassica napus by Controlling Distribution of NO3 − Between the Cytoplasm and Vacuole

Abstract

Nitrate, once taken up by plants, can either be stored in vacuoles or reduced by nitrate reductase in the cytoplasm. High accumulation of NO 3 -in the vacuole occurs when assimilation into the cytoplasm is saturated. This study elucidates how proton pumps at the tonoplast (V-ATPase and V-PPase) affect the NO 3 -content of Brassica napus by controlling the distribution of NO 3 -between the cytoplasm and vacuole. Pot experiments were conducted in a greenhouse under normal N (15.0 mM nitrate) conditions using B. napus genotypes that demonstrated either high (Xiangyou15) or low (814) nitrogen use efficiency (NUE). The NO 3 -content of the high NUE genotype was significantly lower than that of the low NUE genotype, whereas the total N per plant of the two genotypes was almost the same, suggesting that the different NUE between the two genotypes is not due to the difference of NO 3 -uptake. The relative expression levels of V-ATPase (vha-a3) and V-PPase (avp1) genes in the high NUE genotype were significantly lower than in the low NUE genotype, resulting in lower V-ATPase and V-PPase activities in the high NUE genotype. The transport of NO 3 -and protons from the cytoplasm to the vacuole is powered by V-ATPase and V-PPase, so their lower activities increase H ? efflux from and reduce NO 3 -influx into the vacuoles of the high NUE genotype. We conclude that the lower activity of proton pumps at the tonoplast is the main reason the high NUE genotype possesses lower NO 3 -content and higher N-use efficiency.

Nitrogen Use Efficiency Is Mediated by Vacuolar Nitrate Sequestration Capacity in Roots of Brassica napus

“…The energy required for vacuolar NO 3 2 sequestration is provided by the tonoplast proton pumps (Gaxiola et al, 2001;Brüx et al, 2008;Krebs et al, 2010), while the distribution channel CLCa enables transport of NO 3 2 across the vacuolar membrane (De Angeli et al, 2006;Wege et al, 2014). The activities of the tonoplast proton pumps (V-ATPase and V-PPase) in leaves of the H genotype were significantly lower than those of the L genotype at flowering stages (Han et al, 2015a(Han et al, , 2015b; thus, we expected similar differences in root tissues. Our results confirmed that the activities of tonoplast proton-pumps in root tissues of B. napus H genotype and Arabidopsis mutants were lower than those in the L genotype and Arabidopsis wild type, leading to less NO 3 2 influx into vacuoles and more in the cytosol ( Fig.…”

“…Our previous work identified high and low NUE B. napus genotypes (Han et al, 2015a;Han et al, 2015b). NUE of B. napus, whether based on biomass or on grain yield (Table I), was significantly higher for the high-NUE genotype (H: Xiangyou15) than for the low-NUE genotype (L: 814).…”

“…A lower proportion of NO 3 2 accumulating in vacuoles and a higher proportion retained in the cytosol will contribute to better NUE in crop plants (Han et al, 2015b). The energy required for vacuolar NO 3 2 sequestration is provided by the tonoplast proton pumps (Gaxiola et al, 2001;Brüx et al, 2008;Krebs et al, 2010), while the distribution channel CLCa enables transport of NO 3 2 across the vacuolar membrane (De Angeli et al, 2006;Wege et al, 2014).…”

“…The two oilseed rape (B.napus) cultivars used in this study have been characterized before as high (Xiangyou15, referred to as H genotype hereafter) and low (814, referred to as L genotype) NUE genotypes (Zhang et al, 2009;Han et al, 2015b). Here, we define NUE as the total biomass per unit of N uptake by the plant, with total biomass includes roots, shoots, and grains.…”

“…Because vacuoles are obviously the organelle for high NO 3 2 accumulation and storage in plant tissues, their function in NO 3 2 use efficiency cannot be ignored (Martinoia et al, 1981;Zhang et al, 2012;Han et al, 2015b). NO 3 2 assimilatory system in the cytoplasm is sufficient for its assimilation when it is transported out of the vacuoles.…”