Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K+ homeostasis in pollen development

Abstract

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 beta-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.

Figure 1.

Phylogenetic tree of predicted AtCHX proteins aligned by T-Coffee (A) and the chromosomal locations of the genes (B). A, Unrooted phylogenetic tree. Values shown indicate the number of times (in percent) that each branch topology was found in 1,000 replicates of the performed bootstrap analysis using PAUP^*, version 4.0b10. Five major branches are indicated as I to V. B, Multiple AtCHX genes result from segmental duplication and tandem duplication. Chromosomes I to V (top to bottom) are shown as horizontal bars. Duplicated segments are shown in the same gray shade and connected by bands that are twisted if corresponding segments have reversed orientation.

Figure 2.

AtCHX proteins share similarity with a putative K⁺/H⁺ antiporter from yeast. A, Members of the CPA superfamily have an amino-terminal hydrophobic domain and a hydrophilic tail of variable lengths. This scaled graphic representation of the TM regions for the selected protein sequences was created using information compiled from the Simple Modular Architecture Research Tool site (http://smart.embl-heidelberg.de). Each gray bar corresponds to a TM region of 17 to 22 amino acids. Accession numbers are RnNHE1, rat P26431; AtSOS1/AtNHX7, At2g01980; AtNHX1, At5g27150; KefB, E. coli AAC76375; ScKHA1, yeast P40309; NhaS4, Synechocystis PCC 6803 slr1595 or NP_440311; AtKEA1, At1g01790; and AtCHX17, At4g23700. Total residue number is given at the end of each protein. B, AtCHX proteins cluster with yeast KHA1, and Synechocystis NhaS4 in phylogenetic analyses of the TM domain. The hydrophobic domains, including the first Met to the end of the last TM span, from several cation/proton exchangers were aligned. This unrooted phylogenetic tree was created by using the multiple sequence alignment computed by the program T-Coffee, version 1.42. Values shown indicate the number of times (in percent) that each branch topology was found in 1,000 replicates of the performed bootstrap analysis using PAUP^*, version 4.0b10. Accession numbers are RnNHE1, rat P26431; AtSOS1, Arabidopsis At2g01980; AtNHX1, At5g27150; KefB, E. coli, AAC76375; ScKHA1, yeast P40309; ScNHX1, NP_010744; NhaS4 and NhaS1, Synechocystis PCC 6803 slr1595 or NP_440311 and NP_441245; AtKEA1, At1g01790; GerN, B. cereus AAF91326; and NapA, E. hirae P26235. Accession numbers of AtCHX02, 08, 13, 17, 25, and 28 are in Table I. C, The TM domain of AtCHX17 or OsCHX13 shares high identity with that of ScKHA1 and Synechocystis NhaS4. The TM domain of AtCHX17 (At4g23700), OsCHX13 (TIGR ID 3571.m00152), and ScKHA1 (P40309), including residues 1 to 427, 1 to 415, and 1 to 428, respectively, were aligned with the entire Synechocystis NhaS4 (NP_440311) of 410 residues using T-Coffee, version 1.83. TM5 is particularly conserved with 54% (77%) identity (similarity). Identical or similar residues are blocked as dark or light boxes, respectively. Gray underline marks the approximate TM region. •, Conserved residues in all CHX proteins; ⋄, residues conserved in all CHX and in CPA1 shown in B.

Figure 2.

AtCHX proteins share similarity with a putative K⁺/H⁺ antiporter from yeast. A, Members of the CPA superfamily have an amino-terminal hydrophobic domain and a hydrophilic tail of variable lengths. This scaled graphic representation of the TM regions for the selected protein sequences was created using information compiled from the Simple Modular Architecture Research Tool site (http://smart.embl-heidelberg.de). Each gray bar corresponds to a TM region of 17 to 22 amino acids. Accession numbers are RnNHE1, rat P26431; AtSOS1/AtNHX7, At2g01980; AtNHX1, At5g27150; KefB, E. coli AAC76375; ScKHA1, yeast P40309; NhaS4, Synechocystis PCC 6803 slr1595 or NP_440311; AtKEA1, At1g01790; and AtCHX17, At4g23700. Total residue number is given at the end of each protein. B, AtCHX proteins cluster with yeast KHA1, and Synechocystis NhaS4 in phylogenetic analyses of the TM domain. The hydrophobic domains, including the first Met to the end of the last TM span, from several cation/proton exchangers were aligned. This unrooted phylogenetic tree was created by using the multiple sequence alignment computed by the program T-Coffee, version 1.42. Values shown indicate the number of times (in percent) that each branch topology was found in 1,000 replicates of the performed bootstrap analysis using PAUP^*, version 4.0b10. Accession numbers are RnNHE1, rat P26431; AtSOS1, Arabidopsis At2g01980; AtNHX1, At5g27150; KefB, E. coli, AAC76375; ScKHA1, yeast P40309; ScNHX1, NP_010744; NhaS4 and NhaS1, Synechocystis PCC 6803 slr1595 or NP_440311 and NP_441245; AtKEA1, At1g01790; GerN, B. cereus AAF91326; and NapA, E. hirae P26235. Accession numbers of AtCHX02, 08, 13, 17, 25, and 28 are in Table I. C, The TM domain of AtCHX17 or OsCHX13 shares high identity with that of ScKHA1 and Synechocystis NhaS4. The TM domain of AtCHX17 (At4g23700), OsCHX13 (TIGR ID 3571.m00152), and ScKHA1 (P40309), including residues 1 to 427, 1 to 415, and 1 to 428, respectively, were aligned with the entire Synechocystis NhaS4 (NP_440311) of 410 residues using T-Coffee, version 1.83. TM5 is particularly conserved with 54% (77%) identity (similarity). Identical or similar residues are blocked as dark or light boxes, respectively. Gray underline marks the approximate TM region. •, Conserved residues in all CHX proteins; ⋄, residues conserved in all CHX and in CPA1 shown in B.

Figure 3.

Many AtCHX genes are preferentially expressed in the male gametophyte according to whole-genome ATH1 microarray. A, AtCHX genes are differentially expressed during microgametogenesis. RNA isolated from microspores (UNM), bicellular pollen (BCP), tricellular pollen (TCP), or mature pollen (MPG) was used for microarray hybridization. Data represent the mean signal of two independent experiments that showed reliable expression signals (Supplemental Table I). B, Multiple Arabidopsis CHX genes show pollen-specific expression, whereas KEA1 and NHX1 are highly expressed in sporophytic and gametophytic tissues. Gene expression in pollen was compared with that in sporophytic tissues, including cotyledons (COT); leaves (LEF); whole sporophyte (green tissues) at rosette stage (SPR); petioles (PET); stem, top (STT); stem, base (STB); root hair zone (RHR); roots (ROT); and suspension cell cultures (SUS; Supplemental Table I). The same amount of total RNA was used in all Affychip hybridizations. Data represent normalized mean of two to three datasets, except for data of SPR, which came from four replicates.

Figure 4.

RT-PCR demonstrates additional AtCHX genes expressed in pollen, including CHX3, 4, 5, 6a, 6b, 11, and 12. RNA (1 μg) isolated from mature pollen, leaf, or root of wild-type Arabidopsis plants (ecotype Columbia) was reverse transcribed to cDNA. Each CHX gene was amplified for 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 90 s. Amplified products came from cDNA as their sizes were similar to the predicted length, and one-half of the primer sets spanned an intron (Supplemental Table II). Actin 11 (At3g12110) and VHA-c1 (At4g34720) fragments amplified by PCR are 1,130 and 482 bp long, respectively. Result is representative of two to three experiments.

Figure 5.

Promoter::GUS activity shows CHX expression in pollen and in vegetative tissues of transgenic Arabidopsis plants. AtCHX08::GUS (A) and AtCHX23::GUS (B) expression in pollen grains. GUS activity was detected after an overnight reaction period in mature flowers from 6-week-old T1 transgenic plants harboring either a 715-bp AtCHX08 or a 979-bp AtCHX23 promoter region fused transcriptionally to GUS. Scale bars = 100 μm. C, AtCHX17::GUS expression in epidermal and cortical cells of root. GUS-staining signals were detected in roots, but not leaves, of 6-week-old transgenic Arabidopsis plants harboring the 2.0-kb AtCHX17 promoter region transcriptionally fused to GUS. Scale bar = 50 μm. D to F, AtCHX13::GUS expression in reproductive organs. GUS staining was only seen in anthers and pollen grains of mature flowers from 6-week-old transgenic Arabidopsis plants harboring the 2.0-kb AtCHX13 promoter region transcriptionally fused to GUS. D, Whole flower; E, transverse section of anthers; F, longitudinal section of stigma showing growing pollen tubes expressing AtCHX13::GUS. Scale bar represents 400 μm (D), and 50 μm (E and F). G to I, AtCHX14::GUS expression in flowers and vegetative tissues. GUS-staining signals were detected in whole flowers (including anthers and pollen grains) from 6-week-old plants (G), leaf trichomes (H), and root vascular tissues (I) from 20-d-old transgenic Arabidopsis plants harboring the 774-kb AtCHX14 promoter region transcriptionally fused to GUS. Images in G to I are magnified seven times.

Figure 6.

Arabidopsis CHX are orthologous to rice CHX, except for a clade of 15 AtCHX. Accession and identification numbers for Arabidopsis and rice proteins are listed in Tables I and III, respectively. The full revised protein sequences from Arabidopsis and rice were aligned using T-Coffee, version 1.83, and PAUP^*, version 4.0b10, was used for bootstrap analysis. The number of times (in percent) that each branch topology was found in 1,000 replicates of the performed bootstrap analysis for clades I, II and III, IV, and V are 63%, 81%, 53%, and 98%, respectively.