Cation/H⁺ (NHX-type) antiporters are important regulators of intracellular ion homeostasis and are critical for cell expansion and plant stress acclimation. In Arabidopsis (Arabidopsis thaliana), four distinct NHX isoforms, named AtNHX1 to AtNHX4, locate to the tonoplast. To determine the concerted roles of all tonoplast NHXs on vacuolar ion and pH homeostasis, we examined multiple knockout mutants lacking all but one of the four vacuolar isoforms and quadruple knockout plants lacking any vacuolar NHX activity. The nhx triple and quadruple knockouts displayed reduced growth phenotypes. Exposure to sodium chloride improved growth while potassium chloride was deleterious to some knockouts. Kinetic analysis of K⁺ and Na⁺ transport indicated that AtNHX1 and AtNHX2 are the main contributors to both vacuolar pH and K⁺ and Na⁺ uptake, while AtNHX3 and AtNHX4 differ in Na⁺/K⁺ selectivity. The lack of any vacuolar NHX activity resulted in no K⁺ uptake, highly acidic vacuoles, and reduced but not abolished vacuolar Na⁺ uptake. Additional K⁺/H⁺ and Na⁺/H⁺ exchange activity assays in the quadruple knockout indicated Na⁺ uptake that was not H⁺ coupled, suggesting the existence of an alternative, cation/H⁺-independent, Na⁺ conductive pathway in vacuoles. These results highlight the importance of NHX-type cation/H⁺ antiporters in the maintenance of cellular cation homeostasis and in growth and development.

See also the Commentary on this article by McGrath, 226: 1225–1227.

Demand for all-natural vanilla flavor is increasing, but its botanical source, Vanilla planifolia, faces critical challenges arising from a narrow germplasm base and supply limitations. Genomics tools are the key to overcoming these limitations by enabling advanced genetics and plant breeding for new cultivars with improved yield and quality. The objective of this work was to establish the genomic resources needed to facilitate analysis of diversity among Vanilla accessions and to provide a resource to analyze other Vanilla collections. A V. planifolia draft genome was assembled and used to identify 521,732 single nucleotide polymorphism (SNP) markers using Genotyping-By-Sequencing (GBS). The draft genome had a size of 2.20 Gb representing 97% of the estimated genome size. A filtered set of 5,082 SNPs was used to genotype a living collection of 112 Vanilla accessions from 23 species including native Florida species. Principal component analysis of the genetic distances, population structure, and the maternally inherited rbcL gene identified putative hybrids, misidentified accessions, significant diversity within V. planifolia, and evidence for 12 clusters that separate accessions by species. These results validate the efficiency of genomics-based tools to characterize and identify genetic diversity in Vanilla and provide a significant tool for genomics-assisted plant breeding.

Na⁺/H⁺ antiporters (NHXs) are integral membrane transporters that catalyze the electroneutral exchange of K⁺ or Na⁺ for H⁺ and are implicated in cell expansion, development, pH and ion homeostasis and salt tolerance. Arabidopsis contains four vacuolar NHX isoforms (NHX1–NHX4), but only the functional roles for NHX1 and NHX2 have been assessed thus far. Colocalization studies indicated that NHX3 and NHX4 colocalize to the tonoplast. To investigate the role of all vacuolar NHX isoforms, a quadruple knockout nhx1nhx2nhx3nhx4, lacking all vacuolar NHXs, was generated. Seedlings of nhx1nhx2nhx3nhx4 displayed significantly reduced growth, with markedly shorter hypocotyls. Under high K⁺, but not Na⁺, pronounced root skewing occurred in nhx1nhx2nhx3nhx4, suggesting that the organization of the cytoskeleton might be perturbed. Whole mount immunolabeling of cortical microtubules indicated that high K⁺ caused significant microtubule reorganization in nhx1nhx2nhx3nhx4 root cells of the elongation zone. Using microtubule stabilizing (Taxol) and destabilizing (propyzamide) drugs, we found that the effect of K⁺ on nhx1nhx2nhx3nhx4 root growth was antagonistic to that of Taxol, whereas elevated K⁺ exacerbated the endogenous effect of propyzamide on root skewing. Collectively, our results suggest that altered K⁺ homeostasis leads to an increase in the dynamics of cortical microtubule reorganization in nhx1nhx2nhx3nhx4 root epidermal cells of the elongation zone.