PLANT-SPECIFIC K+ TRANSPORTERS WITH DISTINCT PROPERTIES AND THEIR EMERGING ROLES IN ENDOMEMBRANE TRAFFICKING

Plant growth, development and survival are dependent on the dynamic endomembrane system that modulates the delivery of various cargos to their destination or removal of components for degradation in time and in space. Yet the molecular determinants that regulate this dynamic and intricate machinery in plants are poorly understood. The multiplicity of Cation/H+ eXchangers (CHX) genes in higher plants, but not in metazoans, raises questions about their transport activity and suggests roles characteristic to plant life. Five CHX proteins from Arabidopsis thaliana were implicated in pH homeostasis as each rescued growth of alkaline-sensitive yeast strains, though with distinct properties. Each CHX improved growth of K+-uptake deficient strain at different pHs. Moreover, CHX17, 18 and 19 conferred hygromycin B resistance when CHX20 could not. Although both CHX17 and CHX20 mediated K+ uptake when expressed in Escherichia coli; their properties suggested differential modes of transport. CHX20, but not CHX17, acidified cytoplasmic pH and alkalinized vacuolar pH in yeast. CHX17 was more effective than CHX20 in reducing secretion of a vacuolar lumen protein in yeast. As these CHXs were localized to yeast endomembranes, results suggested that CHXs differentially modulate pH and K+ homeostasis of intracellular compartments which affect protein sorting. CHX20-tagged with fluorescent protein was localized to ER, whereas CHX16, 17, 18 and 19 localized to prevacuolar compartments (PVC) and to plasma membrane (PM) in planta. Brefeldin A diminished PM-associated CHX17, whereas wortmannin caused formation of ring-like structures of CHX17-bound compartments. When full-length CHX17 (820 residues) was truncated, CHX17(1-472) partially restored yeast growth on alkaline medium. However, the truncated protein was localized to Golgi, suggesting a role of the C tail in sorting CHX17 from Golgi to PVC and PM. These results suggest that CHX17 is associated with a subset of endosomes that traffick among PVC, vacuole and PM in cells of whole plants. Together, the results support a model where CHX17 and its homologs modulate localized pH and K+ environment of distinct endomembrane compartments and so affect membrane trafficking and cargo sorting through the endocytic and/or secretory pathways. CHXs are proposed to facilitate cell wall modifications as plants adapted to dry land

Conserved and diversified gene families of monovalent cation/H+ antiporters from algae to flowering plants

All organisms have evolved strategies to regulate ion and pH homeostasis in response to developmental and environmental cues. One strategy is mediated by monovalent cation–proton antiporters (CPA) that are classified in two superfamilies. Many CPA1 genes from bacteria, fungi, metazoa, and plants have been functionally characterized; though roles of plant CPA2 genes encoding K+-efflux antiporter (KEA) and cation/H+ exchanger (CHX) families are largely unknown. Phylogenetic analysis showed that three clades of the CPA1 Na+–H+ exchanger (NHX) family have been conserved from single-celled algae to Arabidopsis. These are (i) plasma membrane-bound SOS1/AtNHX7 that share ancestry with prokaryote NhaP, (ii) endosomal AtNHX5/6 that is part of the eukaryote Intracellular-NHE clade, and (iii) a vacuolar NHX clade (AtNHX1–4) specific to plants. Early diversification of KEA genes possibly from an ancestral cyanobacterium gene is suggested by three types seen in all plants. Intriguingly, CHX genes diversified from three to four members in one subclade of early land plants to 28 genes in eight subclades of Arabidopsis. Homologs from Spirogyra or Physcomitrella share high similarity with AtCHX20, suggesting that guard cell-specific AtCHX20 and its closest relatives are founders of the family, and pollen-expressed CHX genes appeared later in monocots and early eudicots. AtCHX proteins mediate K+ transport and pH homeostasis, and have been localized to intracellular and plasma membrane. Thus KEA genes are conserved from green algae to angiosperms, and their presence in red algae and secondary endosymbionts suggest a role in plastids. In contrast, AtNHX1–4 subtype evolved in plant cells to handle ion homeostasis of vacuoles. The great diversity of CHX genes in land plants compared to metazoa, fungi, or algae would imply a significant role of ion and pH homeostasis at dynamic endomembranes in the vegetative and reproductive success of flowering plants. [EN]This work was support in part by National Science Foundation Grant IBN0209788 and US Department of Energy Grant BES DEFG0207ER15883 to Heven Sze, grant BIO2008-01691 from Spanish Plan Nacional I + D + I to Kees Venema, and a Royal Thai Government Graduate Fellowship to Salil Chanroj. Work of CFD was supported by NSF grants #MCB-0523719 and DEB1036506.Peer reviewe

Watermelon seeds and peels: fatty acid composition and cosmeceutical potential

Watermelon consumption results in generation of organic waste in the form of seeds and peels. We have evaluated the fatty acid profiles and antioxidant content of watermelon (Kinnaree cultivar) seed oil and peel wax. In addition, we assessed the potential use of these watermelon industry byproducts in the development of cosmeceuticals. The most abundant fatty acids in seed oil and peel wax were linoleic acid and arachidic acid, respectively. Fatty acids form an essential component in the cell membranes and have seen increased recognition in the cosmeceutical industry. Antioxidants also play a beneficial role in skincare in combating free-radicals resulting from sun damage and pollutants. The seed oil showed stronger antioxidant activity than the peel wax, as indicated by the DPPH radical scavenging ability of 0.894 mg α-tocopherol equivalent/g dried seeds versus 0.036 mg α-tocopherol equivalent/g dried peels. Therefore, the seed oil was formulated into skincare products, in the form of emulsions and nanoemulsions. The most effective formulae were stable at room temperature for seven days, or following repeated cycles of heating and cooling. This work demonstrates the potential for watermelon seed oil to be employed in skincare product formulations, which could maximize agricultural profit and minimize environmental waste

Pollen Tubes Lacking a Pair of K+ Transporters Fail to Target Ovules in Arabidopsis[C][W][OA]

How pollen tubes respond to female cues and precisely deliver sperm cells to the ovule is largely unknown. This article shows that two members of a cation transporter family are required in pollen tube navigation and in shifting polar tip growth

A Distinct Endosomal Ca2+/Mn2+ Pump Affects Root Growth through the Secretory Process1[C][W][OA]

Ca2+ is required for protein processing, sorting, and secretion in eukaryotic cells, although the particular roles of the transporters involved in the secretory system of plants are obscure. One endomembrane-type Ca-ATPase from Arabidopsis (Arabidopsis thaliana), AtECA3, diverges from AtECA1, AtECA2, and AtECA4 in protein sequence; yet, AtECA3 appears similar in transport activity to the endoplasmic reticulum (ER)-bound AtECA1. Expression of AtECA3 in a yeast (Saccharomyces cerevisiae) mutant defective in its endogenous Ca2+ pumps conferred the ability to grow on Ca2+-depleted medium and tolerance to toxic levels of Mn2+. A green fluorescent protein-tagged AtECA3 was functionally competent and localized to intracellular membranes of yeast, suggesting that Ca2+ and Mn2+ loading into internal compartment(s) enhanced yeast proliferation. In mesophyll protoplasts, AtECA3-green fluorescent protein associated with a subpopulation of endosome/prevacuolar compartments based on partial colocalization with the Ara7 marker. Interestingly, three independent eca3 T-DNA disruption mutants showed severe reduction in root growth normally stimulated by 3 mm Ca2+, indicating that AtECA3 function cannot be replaced by an ER-associated AtECA1. Furthermore, root growth of mutants is sensitive to 50 μm Mn2+, indicating that AtECA3 is also important for the detoxification of excess Mn2+. Curiously, Ateca3 mutant roots produced 65% more apoplastic protein than wild-type roots, as monitored by peroxidase activity, suggesting that the secretory process was altered. Together, these results demonstrate that the role of AtECA3 is distinct from that of the more abundant ER AtECA1. AtECA3 supports Ca2+-stimulated root growth and the detoxification of high Mn2+, possibly through activities mediated by post-Golgi compartments that coordinate membrane traffic and sorting of materials to the vacuole and the cell wall

Participation of Endomembrane Cation/H+ Exchanger AtCHX20 in Osmoregulation of Guard Cells1[W][OA]

Guard cell movement is induced by environmental and hormonal signals that cause changes in turgor through changes in uptake or release of solutes and water. Several transporters mediating these fluxes at the plasma membrane have been characterized; however, less is known about transport at endomembranes. CHX20, a member of a poorly understood cation/H+ exchanger gene family in Arabidopsis (Arabidopsis thaliana), is preferentially and highly expressed in guard cells as shown by promoter∷β-glucuronidase activity and by whole-genome microarray. Interestingly, three independent homozygous mutants carrying T-DNA insertions in CHX20 showed 35% reduction in light-induced stomatal opening compared to wild-type plants. To test the biochemical function of CHX20, cDNA was expressed in a yeast (Saccharomyces cerevisiae) mutant that lacks Na+(K+)/H+ antiporters (Δnhx1 Δnha1 Δkha1) and plasma membrane Na+ pumps (Δena1-4). Curiously, CHX20 did not enhance tolerance of mutants to moderate Na+ or high K+ stress. Instead, it restored growth of the mutant on medium with low K+ at slightly alkaline pH, but had no effect on growth at acidic pH. Green fluorescent protein-tagged CHX20 expressed in mesophyll protoplasts was localized mainly to membranes of the endosomal system. Furthermore, light-induced stomatal opening of the Arabidopsis mutants was insensitive to external pH and was impaired at high KCl. The results are consistent with the idea that, in exchanging K+ for H+, CHX20 maintains K+ homeostasis and influences pH under certain conditions. Together, these results provide genetic and biochemical evidence that one CHX protein plays a critical role in osmoregulation through K+ fluxes and possibly pH modulation of an active endomembrane system in guard cells

Arabidopsis KEA2, a homolog of bacterial KefC, encodes a K+/H+ antiporter with a chloroplast transit peptide

KEA genes encode putative K efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast; however, a truncated AtKEA2 protein (AtsKEA2) lacking the N-terminal domain complemented disruption of the Na(K )/H antiporter Nhx1p to confer hygromycin resistance and tolerance to Na or K stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H exchange with preference for K = Cs > Li > Na. When a conserved Asp in transmembrane helix 6 that aligns to the cation binding Asp of Escherichia coli NhaA was replaced with Ala, AtsKEA2 was completely inactivated. Mutation of a Glu between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K /H antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids