Flowering plant genomes encode multiple cation/H+ exchangers (CHXs) whose functions are largely unknown. AtCHX17, AtCHX18, and AtCHX19 are membrane transporters that modulate K+ and pH homeostasis and are localized in the dynamic endomembrane system. Loss of function reduced seed set, but the particular phase(s) of reproduction affected was not determined. Pollen tube growth and ovule targeting of chx17chx18chx19 mutant pollen appeared normal, but reciprocal cross experiments indicate a largely male defect. Although triple mutant pollen tubes reach ovules of a wild-type pistil and a synergid cell degenerated, half of those ovules were unfertilized or showed fertilization of the egg or central cell, but not both female gametes. Fertility could be partially compromised by impaired pollen tube and/or sperm function as CHX19 and CHX18 are expressed in the pollen tube and sperm cell, respectively. When fertilization was successful in self-pollinated mutants, early embryo formation was retarded compared with embryos from wild-type ovules receiving mutant pollen. Thus CHX17 and CHX18 proteins may promote embryo development possibly through the endosperm where these genes are expressed. The reticulate pattern of the pollen wall was disorganized in triple mutants, indicating perturbation of wall formation during male gametophyte development. As pH and cation homeostasis mediated by AtCHX17 affect membrane trafficking and cargo delivery, these results suggest that male fertility, sperm function, and embryo development are dependent on proper cargo sorting and secretion that remodel cell walls, plasma membranes, and extracellular factors.

Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na(+)/H(+) antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 and aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. These results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates in ion recognition, coordination and/or translocation, similar to characterized cation/H(+) exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family.

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that inhibit pathogen and pest polygalacturonases (PGs). PGIPs are members of the leucine-rich repeat (LRR) protein family that play crucial roles in development, pathogen defense and recognition of beneficial microbes in plants. Two sugar beet PGIP genes, Bv(FC607)PGIP1 and Bv(FC607)PGIP2, were cloned from a breeding line FC607. Sequence analysis showed that both genes encoded about 380 amino acids and shared 74.8% sequence similarity. They were most closely matched to PGIPs of a sugar beet line KWS2320 and a subgroup of M. truncatula PGIP (GenBank No: XP_003621816). FCPGIPs exhibited characteristics of other plant PGIPs, including the presence of an N-terminal signal peptide and LRR repeats that were50-60 amino acid longer than has been reported for most other PGIPs. In 2-month old plants, RT-PCR analysis demonstrated that each PGIP gene was expressed constitutively, with maximum expression being observed in roots, followed by leaves then petioles and hypocotyls, suggesting that the gene is developmentally regulated. A study of PGIP inhibitory effect on pathogens and pests is ongoing. Introduction Polygalacturonase-inhibiting proteins (PGIPs), produced in most plant cell walls, effectively and specifically combine with pathogen and pest polygalacturonases (PGs) and inhibit their activity used for overcoming plant defense responses (D’Ovidio et al., 2004). Phytopathogenic fungi, bacteria, nematodes and insects are known to secrete PG enzymes for breaking down the polygalacturonate chain in plant cell walls. The interaction between PGs and plant PGIPs favors the accumulation of oligogalacturonides which elicit a wide range of plant defense responses (Gomathi and Gnanamanickam 2004; Schacht et al., 2011). Many plants possess more than one PGIP with differential abilities to inhibit different PGs of pathogens. Like the products of many resistance genes, PGIPs belong to the subclass of proteins containing leucine-rich repeats (LRRs) of the extracytoplasmic type (Jones and Jones 1997). In our laboratory, one of the sugar beet genes that codes for PGIP has been found to be induced by the sugar beet root maggot in roots (Puthoff and Smigocki, 2007). In order to characterize the structure and functional features of sugar beet PGIPs, several PGIP genes were cloned and characterized from several sugar beet breeding lines. Here we report on the cloning of two PGIP genes from sugar beet line FC607 and on their tissue-specific expression in 2-month old plants. These findings will facilitate further studies on PGIP inhibition of pathogens and pests and will advance the development of novel approaches for more effective disease and pest control in sugar beet. Materials and Methods Plant materials A sugar beet breeding line FC607 was used in this study. Seeds were germinated at room temperature in Pro-Mix (Professional Horticulture) soil. Seedlings were grown in the growth chamber at 24°C with a 16 h photoperiod. Full-length cDNA cloning and sequence analysis The full-length sequence of the sugar beet PGIP cDNA was obtained through rapid amplification of cDNA ends polymerase chain reaction (RACE PCR) using the SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA). Gene specific primers were designed and the RACE fragment was amplified as previously reported (Padmanaban et al., 2011). PCR amplified fragments were sequenced and nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches were performed using default parameters. Amino acid sequences and opening reading frames were determined by ExPASy translation tool (http://web.expasy.org/translate/). Using genomic DNA and gene specific primers, PGIP genomic DNA was cloned by PCR and the resulting DNA fragment was sequenced. The SignalP 4.1 server (Petersen et al., 2011) was used to predict the signal peptide. The LRR domains were predicted according to sequence alignment of BvPGIPs and the bean PvPGIP2 whose secondary crystallographic structure has been determined (Di Matteo et al., 2003). Amino acid sequences of BvPGIPs and other PGIPs were aligned by ClustalW through MEGA6 program (www.megasoftware.net). Phylogenetic tree was constructed with the neighbor-joining method (Tamura et al., 2013). Analysis of PGIP gene expression Total RNA was extracted from leaves, petioles, hypocotyls, and roots of 2-month old plants using RNeasy Plant Mini Kit (Qiagen, Valencia, CA). RT-PCR was carried out on 100 ng of total RNA using Titanium One-Step RT-PCR Kit (Clontech) under the following conditions: 50oC for 1 h, 94oC for 2 min, 30 cycles of 94oC for 30 sec, 60oC for 30 sec, 72oC for 1 min, followed by 72oC for 5 min. RT-PCR results were normalized to transcripts of the constitutively expressed plant actin gene (Smigocki et al., 2013). Gene expression was quantified by densitometry with an AlphaImager HP (Alpha Innotech, San Leandro, CA). RT-PCR analyses were repeated three times. Results and Discussion Cloning and analysis of Beta vulgaris PGIPs To investigate intergenotype variation in sugar beet PGIP genes, the full complements of PGIP genes of the B. vulgaris breeding line FC607 were cloned and characterized. To date, two cDNA fragments were cloned from FC607 using primers based on the PGIP EST sequence (GenBank No: DV501910) isolated from sugar beet root after insect feeding (Puthoff and Smigocki, 2007). Comparison of the nucleotide and deduced amino acid sequences using BLAST revealed that the cloned cDNAs shared the highest sequence homology with other known PGIP genes. Comparison of Bv(FC607)PGIP1 and Bv(FC607)PGIP2 cDNAs and PCR derived genomic DNA sequences revealed that neither gene had introns. The full-length cDNA sequences of 1,152 and 1,146 bp, encoded 384 and 382 amino acids, respectively, with a calculated molecular weight of about 49.0 kDa. These two peptides showed 74.8% similarity, and shared the typical PGIP topology, which included a signal peptide for secretion, a 75-amino acid N-terminal domain, a domain comprising leucine rich repeats (LRRs), and a 21-amino acid C-terminal domain (Fig. 1). A total of eleven imperfect LRR regions of about 24 amino acids each was predicted according to sequence alignment of Bv(FC607)PGIPs with the bean PvPGIP2 whose secondary crystallographic structure has been determined (Di Matteo et al., 2003). However, according to the LRR search program (http://lrrsearch.com/), only 10 LRR domains (excludes the first one) were predicted for Bv(FC607)PGIP1 and only 9 LRRs (excludes the second and the ninth LRRs) were predicted for Bv(FC607)PGIP2. Nevertheless, the LRR region of these two genes exhibited the identical consensus sequence (LxxLxLxxNxLxGxIPxxLGxLxx) and matched precisely the extracytoplasmic LRR consensus sequence of other resistance genes (Jones and Jones 1997). There were 4 potential Nglycosylation sites (NxS/T; where x can be any amino acid), three of which occurred in the LRR domain, where the third NxS/T site was different between these two genes. Both genes contained 5 cysteine residues at the N-terminal and C-terminal regions. Sequence alignment by ClustalW revealed that both Bv(FC607)PGIP1 and Bv(FC607)PGIP2 showed the closest homology with PGIP or PGIP-like proteins from a B. vulgaris line KWS2320, a recently released genome sequence (Dohm et al., 2014). A 97.4% similarity between Bv(FC607)PGIP1 and Bv(KWS2320)PGIP (accession No: XP_010675711) and an 80.0% similarity between Bv(FC607)PGIP2 and Bv(KWS2320)PGIP (XP_010676004) was observed (Fig. 2A, 2B). The second closest homology of our sugarbeet PGIPs was to Medicago truncatula PGIP (MtPGIP2, XP_003621816), followed by Nicotiana tabacum PGIP (NtPGIP, AIA22327), and Arabidopsis thaliana PGIP (AtPGIP2, AAF69828). Like most of the PGIPs from B. vulgaris KWS2320, Bv(FC607)PGIP1 and Bv(FC607)PGIP2 were approximately 50 amino acids longer than the PGIP structure model PvPGIP2 that represents most of other known plant PGIPs (Fig. 2A). The additional amino acids were found in the N-terminal and the LRR regions of Bv(FC607)PGIP1 and Bv(FC607)PGIP2 LRR but not found in most other plant PGIPs, thus potentially being unique to the sugar beet PGIPs .

The importance of sorting proteins and wall materials to their destination is critical for plant growth and development, though the machinery orchestrating membrane trafficking is poorly understood. Transporters that alter the environment across endomembrane compartments are thought to be important players. Using Escherichia coli and yeast, we previously showed that several Arabidopsis Cation/H(+) eXchanger (AtCHX) members were K(+) transporters with a role in pH homeostasis, though their subcellular location and biological roles in plants are unclear. Co-expression of markers with CHX16, CHX17, CHX18, or CHX19 tagged with a fluorescent protein indicated these transporters associated with plasma membrane (PM) and post-Golgi compartments. Under its native promoter, AtCHX17(1-820)-GFP localized to prevacuolar compartment (PVC) and to PM in roots. Brefeldin A diminished AtCHX17-GFP fluorescence at PM, whereas wortmannin caused formation of GFP-labeled ring-like structures, suggesting AtCHX17 trafficked among PVC, vacuole and PM. AtCHX17(1-472) lacking its carboxylic tail did not associate with PVC or PM in plant cells. Single chx17 mutant or higher-order mutants showed normal root growth and vegetative development. However, quadruple (chx16chx17chx18chx19) mutants were reduced in frequency and produced 50%-70% fewer seeds, indicating overlapping roles of several AtCHX17-related transporters in reproduction and/or seed development. Together, our results suggest that successful reproduction and seed development depend on the ability to regulate cation and pH homeostasis by AtCHX17-like transporters on membranes that traffic in the endocytic and/or secretory pathways.

Several sugar beet (Beta vulgaris L.) insect pests and fungal pathogens specifically target the tissues of the taproot. The use of root tissue-specific promoters to confer expression of pest and disease resistance genes in a targeted manner has the potential for genetic improvement of commercial sugar beet varieties. Using suppressive subtractive hybridization (SSH), our laboratory has identified several sugar beet root genes responding to infestations by the sugar beet root maggot (Tetanops myopaeformis). This sugar beet EST library has been subjected to macroarray analyses using RNA isolated from various sugar beet tissues. Several genes were identified that exhibit high levels of expression in the root peel, crown and cortex tissues as compared to young roots and vegetative tissues. Based on the macroarray and RT-PCR analysis, we are in the process of cloning several of the most interesting promoters by genome walking. Properties of promoters will be determined by expression of promoter reporter (Pro:: GUS-GFP) constructs in sugar beet hairy roots. Identification of root tissue-specific promoters will facilitate expression of resistance genes in root tissues as a first line of defense targeting root pests and diseases.

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The complexity of intracellular compartments in eukaryotic cells evolved to provide distinct environments to regulate processes necessary for cell proliferation and survival. A large family of predicted cation/proton exchangers (CHX), represented by 28 genes in Arabidopsis thaliana, are associated with diverse endomembrane compartments and tissues in plants, although their roles are poorly understood. We expressed a phylogenetically related cluster of CHX genes, encoded by CHX15–CHX20, in yeast and bacterial cells engineered to lack multiple cation-handling mechanisms. Of these, CHX16–CHX20 were implicated in pH homeostasis because their expression rescued the alkaline pH-sensitive growth phenotype of the host yeast strain. A smaller subset, CHX17–CHX19, also conferred tolerance to hygromycin B. Further differences were observed in K+- and low pH-dependent growth phenotypes. Although CHX17 did not alter cytoplasmic or vacuolar pH in yeast, CHX20 elicited acidification and alkalization of the cytosol and vacuole, respectively. Using heterologous expression in Escherichia coli strains lacking K+ uptake systems, we provide evidence for K+ (86Rb) transport mediated by CHX17 and CHX20. Finally, we show that CHX17 and CHX20 affected protein sorting as measured by carboxypeptidase Y secretion in yeast mutants grown at alkaline pH. In plant cells, CHX20-RFP co-localized with an endoplasmic reticulum marker, whereas RFP-tagged CHX17–CHX19 co-localized with prevacuolar compartment and endosome markers. Together, these results suggest that in response to environmental cues, multiple CHX transporters differentially modulate K+ and pH homeostasis of distinct intracellular compartments, which alter membrane trafficking events likely to be critical for adaptation and survival.

Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation: proton antiporters (CHX), which may mediate K+ transport; however, the vast majority of these transporters remain uncharacterized. Here, we show that AtCHX13 (At2g30240) has a role in K+ acquisition. AtCHX13 suppressed the sensitivity of yeast (Saccharomyces cerevisiae) mutant cells defective in K+ uptake. Uptake experiments using Rb-86(+) as a tracer for K+ demonstrated that AtCHX13 mediated high-affinity K+ uptake in yeast and in plant cells with a K-m of 136 and 196 mu M, respectively. Functional green fluorescent protein-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K+ deficiency conditions, whereas overexpression of AtCHX13 reduced the sensitivity to K+ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K+ uptake, although the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K+-deficient conditions. These results indicate that one role of AtCHX13 is to promote K+ uptake into plants when K+ is limiting in the environment.

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:: beta-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 (Dnhx1 Dnha1 Dkha1) and plasma membrane Na 1 pumps (Dena1-4). 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.

Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::beta-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742–5815 (K.W.B., S.P., E.P.N., H.S.); Laboratory of Pollen Biology, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, 16502 Prague 6, Czech Republic (D.H.); Department of Plant Physiology, Charles University, 128 44 Praha 2, Czech Republic (D.H.); Department Plant Biology, University of Minnesota, St. Paul, Minnesota 55108 (J.M.W.); Department of Pediatrics and Department of Human and Molecular Genetics, Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Texas 77030 (K.D.H.); and Department of Biology, University of Leicester, Leicester LE1 7RH, United Kingdom (D.T.)

Acidification of intracellular compartments by the vacuolar-type H+-ATPases (VHA) is known to energize ion and metabolite transport, though cellular processes influenced by this activity are poorly understood. At least 26 VHA genes encode 12 subunits of the V1Vo-ATPase complex in Arabidopsis, and how the expression, assembly, and activity of the pump are integrated into signaling networks that govern growth and adaptation are largely unknown. The role of multiple VHA-c genes encoding the 16-kD subunit of the membrane Vo sector was investigated. Expression of VHA-c1, monitored by promoter-driven β-glucuronidase (GUS) activity was responsive to light or dark in an organ-specific manner. VHA-c1 expression in expanding cotyledons, hypocotyls of etiolated seedlings, and elongation zone of roots supported a role for V-ATPase in cell enlargement. Mutants reduced in VHA-c1 transcript using dsRNA-mediated interference showed reduction in root growth relative to wild-type seedlings. In contrast, VHA-c3 promoter::GUS expression was undetectable in most organs of seedlings, but strong in the root cap. Interestingly, dsRNA-mediated mutants of vha-c3 also showed reduced root length and decreased tolerance to moderate salt stress. The results suggest that V-ATPase functions in the root cap influenced root growth. Expression of VHA-c1 and VHA-c3 in tissues with active membrane flow, including root cap, vascular strands, and floral style would support a model for participation of the Vo sector and V1Vo-ATPase in membrane trafficking and fusion. Two VHA-c genes are thus differentially expressed to support growth in expanding cells and to supply increased demand for V-ATPase in cells with active exocytosis.

A combined bioinformatic and experimental approach is being used to uncover the functions of a novel family of cation/H+ exchanger (CHX) genes in plants using Arabidopsis as a model. The predicted protein (85–95 kD) of 28 AtCHX genes after revision consists of an amino-terminal domain with 10 to 12 transmembrane spans (approximately 440 residues) and a hydrophilic domain of approximately 360 residues at the carboxyl end, which is proposed to have regulatory roles. The hydrophobic, but not the hydrophilic, domain of plant CHX is remarkably similar to monovalent cation/proton antiporter-2 (CPA2) proteins, especially yeast (Saccharomyces cerevisiae) KHA1 and Synechocystis NhaS4. Reports of characterized fungal and prokaryotic CPA2 indicate that they have various transport modes, including K+/H+ (KHA1), Na+/H+-K+ (GerN) antiport, and ligand-gated ion channel (KefC). The expression pattern of AtCHX genes was determined by reverse transcription PCR, promoter-driven β-glucuronidase expression in transgenic plants, and Affymetrix ATH1 genome arrays. Results show that 18 genes are specifically or preferentially expressed in the male gametophyte, and six genes are highly expressed in sporophytic tissues. Microarray data revealed that several AtCHX genes were developmentally regulated during microgametogenesis. An exciting idea is that CHX proteins allow osmotic adjustment and K+ homeostasis as mature pollen desiccates and then rehydrates at germination. The multiplicity of CHX-like genes is conserved in higher plants but is not found in animals. Only 17 genes, OsCHX01 to OsCHX17, were identified in rice (Oryza sativa) subsp. japonica, suggesting diversification of CHX in Arabidopsis. These results reveal a novel CHX gene family in flowering plants with potential functions in pollen development, germination, and tube growth.

The vacuolar-type H(+)-ATPase acidifies intracellular compartments and is essential for many processes, including cotransport, guard cell movement, development, and tolerance to environmental stress. We have identified at least 26 genes encoding subunits of the vacuolar-type H(+)-ATPase in the Arabidopsis thaliana genome, although inconsistent nomenclature of these genes is confusing. The pump consists of subunits A through H of the peripheral V(1) complex, and subunits a, c, c" and d of the V(o) membrane sector. Most V(1) subunits are encoded by a single gene, whereas V(o) subunits are encoded by multiple genes found in duplicated segments of the genome. We propose to name these genes VHA-x, where x represents the letter code for each subunit. Applying a consistent nomenclature will help us to understand how the expression, assembly and activity of this pump are integrated with plant growth, signaling, development and adaptation.