Ammonium Accumulation Caused by Reduced Tonoplast V-ATPase Activity in Arabidopsis thaliana

Abstract

Plant vacuoles are unique compartments that play a critical role in plant growth and development. The vacuolar H⁺-ATPase (V-ATPase), together with the vacuolar H⁺-pyrophosphatase (V-PPase), generates the proton motive force that regulates multiple cell functions and impacts all aspects of plant life. We investigated the effect of V-ATPase activity in the vacuole on plant growth and development. We used an Arabidopsisthaliana (L.) Heynh. double mutant, vha-a2 vha-a3, which lacks two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a. The mutant is viable but exhibits impaired growth and leaf chlorosis. Nitrate assimilation led to excessive ammonium accumulation in the shoot and lower nitrogen uptake, which exacerbated growth retardation of vha-a2 vha-a3. Ion homeostasis was disturbed in plants with missing VHA-a2 and VHA-a3 genes, which might be related to limited growth. The reduced growth and excessive ammonium accumulation of the double mutant was alleviated by potassium supplementation. Our results demonstrate that plants lacking the two tonoplast-localized subunits of V-ATPase can be viable, although with defective growth caused by multiple factors, which can be alleviated by adding potassium. This study provided a new insight into the relationship between V-ATPase, growth, and ammonium accumulation, and revealed the role of potassium in mitigating ammonium toxicity.

Keywords: ammonium; ion homeostasis; nitrate; potassium; proton pump; vacuole.

Figure 1

Loss of tonoplast vacuolar H⁺-ATPase (V-ATPase) caused severe growth inhibition and senescence. (a) phenotype comparison between Arabidopsis thaliana wild type (Col-0) and the vha-a2 vha-a3 double mutant. (b) the biomass of Col-0 and vha-a2 vha-a3 was determined. (c) gene expression of senescence-associated gene 29 (SAG29) in wild type and vha-a2 vha-a3 four-week-old leaves. Wild type was set to 100% and error bars were defined as S.E. of n = 3 biological replicates. Pictures show four-week-old plants in hydroponics. Error bars in (b) indicate S.D. of n = 3 technical replicates. Asterisks (*) and (**) indicate significant differences at p < 0.05 and p < 0.01, respectively.

Figure 2

Nitrogen absorption in wild type and the vha-a2 vha-a3 double mutant. Total nitrogen accumulation (a) and total nitrogen content (b) were measured of wild type and vha-a2 vha-a3 mutant. Data in (a) and (b) were mean ± S.D. (n = 3). (c) Relative expression of nitrate transporter 1.1 (NRT1.1) in Col-0 and vha-a2 vha-a3 was measured and Col-0 was set to 100%. Data in are mean ± S.E. (n = 3). Asterisks (*) and (**) indicate significant differences at p < 0.05 and p < 0.01, respectively. DW, Dry weight.

Figure 3

Nitrogen metabolism was measured using wild type and vha-a2 vha-a3 mutant four-week-old seedlings in hydroponics. Nitrate concentration (a), nitrate reductase (NR) activity (b), ammonium concentration (c), and glutamine synthetase (GS) activity (d) of Col-0 and vha-a2 vha-a3 mutant were determined in the shoots and roots. Error bars represent S.D. of three biological replicates. Asterisks (*) and (**) indicate significant differences at p < 0.05 and p < 0.01, respectively. FW, Fresh weight.

Figure 4

Nitrogen transport over long distance between shoots and roots. Relative expression of NRT1.5 (a), NRT1.8 (b), and ammonium transporter (AMT)2;1 (c) was determined from the roots of wild type and vha-a2 vha-a3 mutant. The wild type was set to 100%. (d) higher xylem sap NH₄⁺ concentration in vha-a2 vha-a3 mutant. Error bars in (a) to (c) were mean ± S.E. (n = 3) and in (d), mean ± S.D. (n = 3). Asterisks (*) and (**) indicate significant differences at p < 0.05 and p < 0.01, respectively.

Figure 5

Impaired tonoplast V-ATPase activity prejudiced ion homeostasis. The concentration of K⁺, Ca²⁺, Na⁺, Mg²⁺, Fe²⁺, Mn²⁺, Cu²⁺, and Zn²⁺ in the shoots of wild type and vha-a2 vha-a3 mutant. Asterisks (*) and (**) indicated significant differences at p < 0.05 and p < 0.01, respectively.

Figure 6

The growth performance of vha-a2 vha-a3 mutant was improved with the additional K⁺ supplied. (a) the phenotype of wild type and vha-a2 vha-a3 mutant with additional K⁺ supplied. Pictures show four-week-old plants with different K⁺ supply levels. (b) the biomass of Col-0 and vha-a2 vha-a3 mutant was determined. (c) gene expression levels of SAG29 in four-week-old leaves of wild type and vha-a2 vha-a3 mutant with different levels of K⁺. Wild type with an additional supply of 2 mM K⁺ was set to 100% and error bars were defined as S.E. of n = 3 biological replicates. Pictures show four-week-old plants with different K⁺ supply levels. Error bars in (b) indicate S.D. of n = 3 technical replicates. Asterisks (**) indicate significant differences at p < 0.01.

Figure 7

Promotion of nitrogen absorption and reduction of ammonium accumulation with an additional K⁺ supply in mutant. The total nitrogen accumulation (a), total nitrogen content (b), ammonium concentration (c), and chlorophyll concentration (d) were measured with different K⁺ doses for wild type and vha-a2 vha-a3 mutant grown in hydroponics for four-week-old plants. Error bars indicated S.D. of n = 3 technical replicates. Asterisks (*) and (**) indicate significant differences at p < 0.05 and p < 0.01, respectively.