Roles of plasma membrane proton ATPases AHA2 and AHA7 in normal growth of roots and root hairs in Arabidopsis thaliana

Abstract

Plasma membrane H⁺ -ATPase pumps build up the electrochemical H⁺ gradients that energize most other transport processes into and out of plant cells through channel proteins and secondary active carriers. In Arabidopsis thaliana, the AUTOINHIBITED PLASMA MEMBRANE H⁺ -ATPases AHA1, AHA2 and AHA7 are predominant in root epidermal cells. In contrast to other H⁺ -ATPases, we find that AHA7 is autoinhibited by a sequence present in the extracellular loop between transmembrane segments 7 and 8. Autoinhibition of pump activity was regulated by extracellular pH, suggesting negative feedback regulation of AHA7 during establishment of an H⁺ gradient. Due to genetic redundancy, it has proven difficult to test the role of AHA2 and AHA7, and mutant phenotypes have previously only been observed under nutrient stress conditions. Here, we investigated root and root hair growth under normal conditions in single and double mutants of AHA2 and AHA7. We find that AHA2 drives root cell expansion during growth but that, unexpectedly, restriction of root hair elongation is dependent on AHA2 and AHA7, with each having different roles in this process.

Figure 1

Localization of AHA7 in root hairs is different from that in pollen tubes. AHA7 promoter‐driven expression of the GUS reporter gene shows specific expression in root hairs (A) and pollen grains (B). (C) AHA7 promoter‐driven expression of an AHA7::GFP fusion protein confirms expression in root hairs and shows strong fluorescence signal in the plasma membrane at the root hair bulging site. (D) In growing root hairs, AHA7::GFP localizes to the apex of the growing tip. (E) AHA7 promoter‐driven expression of an AHA7::GFP fusion protein in pollen tubes shows localization of AHA7 to the plasma membrane in the region behind the growing tip apex (z‐projection of max intensity). Scale bars in A, C and D represent 100 μm, in E 10 μm.

Figure 2

AHA7 activity is controlled by apoplastic pH. (A) Phylogenetic tree showing that AHA7 evolved with appearance of vascular plants with a root system. Two major clades of land plant sequences are highlighted (labeled a and b, respectively). Roman numerals indicate the previous nomenclature for angiosperm clades of plasma membrane H⁺‐ATPases (Palmgren 2001). In the tree, Bayesian probabilities are indicated at nodes. Black circles at nodes indicate maximum statistical support (Bayesian probability = 1). Accession codes for protein sequences used for building the tree are indicated. Abbreviations are: AHA, autoinhibited H⁺‐ATPase from Arabidopsis thaliana (Dicotyledonae); Orysa, Orysa sativa (Monocotyledonae); Ambtr, Amborella trichopoda (basal angiosperm); Picab, Picea abies (Gymnospermae), Selmo, Selaginella moellendorffii (Lycopodiophyta; basal vascular plants); Phypa, Physcomitrella patens (Bryophyta; land plants without a vascular system); Klebsormidium flaccidens (Charophyta; green algae). (B) AHA7 contains a stretch of charged amino acids in a loop on the extracellular side of the protein facing the apoplast (between transmembrane segments 7 and 8) which is absent from plasma membrane H⁺‐ATPases in clade a. A model of the AHA7 pump built on the AHA2 crystal structure is shown with the extracellular amino acid stretch marked in red. (C) Plasma membrane H⁺‐ATPases in clade b contain a stretch of charged amino acid residues between transmembrane segments 7 and 8. An exception is the S. moellendorffii sequence at the base of the clade. (D) Complementation of a yeast strain lacking the endogenous plasma membrane H⁺‐ATPase shows that the extracellular loop between transmembrane segments 7 and 8 is involved in restricting pumping of protons when apoplastic pH is more acidic than pH 6.0. The last 92 C‐terminal amino acid residues were truncated at the gene level to relieve the pump's autoinhibition in the heterologous expression system. (E) AHA7 has half maximal ATP hydrolytic activity at pH 7.0 (around cytoplasmic pH) whereas AHA2 is almost inactive at neutral pH. The ATP hydrolytic activity as a function of pH was measured in microsomal membranes of yeast expressing the recombinant protein. (F) When deprived of their regulatory autoinhibitory C‐terminal domains, AHA2 and AHA7 have similar ATPase activity profiles as a function of pH. SGAH, synthetic galactose medium supplied with adenine and histidine. SDAH, synthetic dextrose (glucose) medium supplied with adenine and histidine.

Figure 3

Root hairs of aha2 and aha2 aha7 mutants are longer. (A) Pictures of roots representative for the different Arabidopsis lines lacking AHA proteins and grown on solid Hoagland medium (scale bar = 0.5 mm). (B) The length of root hairs is significantly longer for the aha2 single and aha2 aha7 double mutants (n > 1000; ***, P < 0.0001; anova with Dunnett's multiple comparison test; error bars show sd). Similar results were obtained in three independent replicates. (C) The number of root hairs per 10 mm of primary root length was similar between lines (n = 30; anova with Dunnett's multiple comparison test; error bars show sd). (d) Growth curves show that root hairs of aha2 grow faster than wild‐type, and root hairs of aha2 aha7 grow as fast as wild‐type, but for a longer period (n ≥ 4; error bars show SEM).

Figure 4

Loss of aha2 leads to shorter roots and smaller cotyledons. (A) Representative seedlings grown for 7 days on Hoagland medium (scale bar 1 cm). (B) Root length of aha2 and aha2 aha7 double mutants is reduced compared to wild‐type, whereas aha7 single mutant lines are similar to wild‐type (n ≥ 35, **P < 0.001, ***P < 0.0001; anova with Dunnett's multiple comparison test; error bars show sd). (C) Cotyledon diameter of aha2 and aha2 aha7 double mutants is reduced compared to wild‐type, whereas aha7 single mutant lines are similar to wild‐type (n ≥ 35, *** P < 0.001; anova with Dunnett's multiple comparison test; error bars show sd). (D) Length of trichoblast cells is shorter in aha2 than wild‐type (n ≥ 7; Student's t test; error bars show sd). (E) ICP‐OES results show that K, Fe and P are unaltered in roots of hydroponically grown aha2 compared to wild‐type (pool of 16–22 plants, Student's t test; error bars show sd). (F) qPCR results show that in 7‐day‐old roots of aha2, AHA2 itself and HAK5, a high affinity potassium uptake protein, are significantly downregulated compared to wild‐type (**P < 0.001, ***P < 0.0001; Student's t test; error bars show sd). Results are from six biological replicates with around 20 roots each. Genes well known for their responsiveness under altered iron (FRO2, FRO3, FER1, BHLH39), phosphate conditions (PHT1;4, SPX1, SPX3), transcription factors controlling cell growth and size in root hairs (RSL1, RSL3, RSL4) and a transcription factor modulating plant response to low‐potassium conditions (RAP2.11) were unchanged in aha2.