Arabidopsis plasma membrane H+-ATPase genes AHA2 and AHA7 have distinct and overlapping roles in the modulation of root tip H+ efflux in response to low-phosphorus stress

Abstract

Phosphorus deficiency in soil is one of the major limiting factors for plant growth. Plasma membrane H+-ATPase (PM H+-ATPase) plays an important role in the plant response to low-phosphorus stress (LP). However, few details are known regarding the action of PM H+-ATPase in mediating root proton (H+) flux and root growth under LP. In this study, we investigated the involvement and function of different Arabidopsis PM H+-ATPase genes in root H+ flux in response to LP. First, we examined the expressions of all Arabidopsis PM H+-ATPase gene family members (AHA1-AHA11) under LP. Expression of AHA2 and AHA7 in roots was enhanced under this condition. When the two genes were deficient in their respective Arabidopsis mutant plants, root growth and responses of the mutants to LP were highly inhibited compared with the wild-type plant. AHA2-deficient plants exhibited reduced primary root elongation and lower H+ efflux in the root elongation zone. AHA7-deficient plants exhibited reduced root hair density and lower H+ efflux in the root hair zone. The modulation of H+ efflux by AHA2 or AHA7 was affected by the action of 14-3-3 proteins and/or auxin regulatory pathways in the context of root growth and response to LP. Our results suggest that under LP conditions, AHA2 acts mainly to modulate primary root elongation by mediating H+ efflux in the root elongation zone, whereas AHA7 plays an important role in root hair formation by mediating H+ efflux in the root hair zone.

Keywords: Arabidopsis; low phosphorus; plasma membrane H+-ATPase; proton flux; root growth; root hair..

Fig. 1.

Expression of PM H⁺-ATPase gene family members (AHA1–AHA11) in Arabidopsis plants in response to low phosphorus stress. Relative expression of various AHA genes in the whole plants of Arabidopsis grown in phosphorus-sufficient conditions (Control; 1 mM) or low phosphorus stress (LP; 1 μM) for 4 days. Values are first normalized to At-ACT2 (housekeeping gene; 100 REU) and then to control (+P) plants. Expression of these studied genes (AHA1–AHA11) under control was taken as 1-fold, and the expression level of these studied genes under LP is shown in this figure. The values are the means and SD of six replicates from two independent experiments. Changes in the relative expression levels of gene were checked for statistical significance according to Student’s t-test (P<0.05).

Fig. 2.

Expression of PM H⁺-ATPase gene family members (AHA1, AHA2, and AHA7) in the roots of Arabidopsis plants under phosphorus sufficient conditions (Control; 1 mM) or low phosphorus stress (LP; 1 μM) over 0, 1, 2, 4, 6, and 8 days. Relative expression levels were calculated and normalized with respect to At-ACT2 mRNA (=100 REU). Changes in the relative expression levels (REU) of gene mRNA were checked for statistical significance according to Student’s t-test (P<0.05). The values are the means and SD of six replicates from two independent experiments.

Fig. 3.

Proton (H⁺) flux in meristem zone (MZ), transition zone (TZ), elongation zone (EZ), and root hair zone (RHZ) in the root tip of wild-type Arabidopsis (Col-0), aha2 mutant lines (aha2-1 and aha2-2) and aha7 mutant lines (aha7-1 and aha7-2) under normal growth condition (Control; A) or low phosphorus stress (B). Arabidopsis plants were treated as described in Table 1.

Fig. 4.

Proton (H⁺) flux in meristem zone (MZ), transition zone (TZ), elongation zone (EZ) and root hair zone (RHZ) in the root tip of wild-type Arabidopsis (Col-0), OE-TFT7 (TFT7-overexpressing Arabidopsis plants) and mutant lines (aha2-2, aha7-1, OE-TFT7/aha2-2 and OE-TFT7/aha7-1) in response to low phosphorus stress. Arabidopsis plants were treated as described in Table 1.

Fig. 5.

Root response of wild-type Arabidopsis plants (Col-0), OE-TFT7 (TFT7-overexpressing Arabidopsis plants) and mutant lines (aha2-2, aha7-1, OE-TFT7/aha2-2 and OE-TFT7/aha7-1) to low phosphorus stress. Twenty-two-day-old Arabidopsis plants were treated with 1 μM phosphorus (LP), LP with 1 mM vanadate (VA; PM H⁺-ATPase inhibitor) or NPA (auxin flux inhibitor; 10 μM) for 8 days under a hydroponic system. (A) Elongation rate of primary root; (B) root hair density in the root tip of Arabidopsis (0–5000 μm from the root cap junction); (C) H⁺ flux in elongation zone (EZ); and (D) H⁺ flux in root hair zone (RHZ) in the root tip of Arabidopsis plants.