Arabidopsis CAMTA family proteins enhance V-PPase expression in pollen

The pollen-specific cis-acting region of the AVP1 gene is involved in the expression of the Arabidopsis V-PPase gene during pollen development. We isolate AtCAMTA5, which binds to the 38-bp pollen-specific cis-acting region, by one-hybrid screening using the cis-acting region as a probe. The green fluorescent protein-fused AtCAMTA5 is specifically localized to the nucleus in Arabidopsis suspension cultured cells. The promoter-beta-glucuronidase reporter experiment shows the expression not only of AtCAMTA5 but also of AtCAMTA1 in pollen. In particular, AtCAMTA1 is specifically expressed in pollen. Both the one-hybrid analysis in the reporter yeast and in vivo transient effector-reporter analysis in Arabidopsis suspension cultured cells revealed that AtCAMTA1 could regulate gene expression depending on the CGCG-box within the 38-bp pollen-specific cis-acting region. These results indicate that AtCAMTA1 as well as AtCAMTA5 possibly enhance AVP1 expression in pollen.     

AVP2, a sequence-divergent, K(+)-insensitive H(+)-translocating inorganic pyrophosphatase from Arabidopsis

Plant vacuolar H(+)-translocating inorganic pyrophosphatases (V-PPases; EC 3.6.1.1) have been considered to constitute a family of functionally and structurally monotonous intrinsic membrane proteins. Typified by AVP1 (V. Sarafian, Y. Kim, R.J. Poole, P.A. Rea [1992] Proc Natl Acad Sci USA 89: 1775-1779) from Arabidopsis, all characterized plant V-PPases share greater than 84% sequence identity and catalyze K(+)-stimulated H(+) translocation. Here we describe the molecular and biochemical characterization of AVP2 (accession no. AF182813), a sequence-divergent (36% identical) K(+)-insensitive, Ca(2+)-hypersensitive V-PPase active in both inorganic pyrophosphate hydrolysis and H(+) translocation. The differences between AVP2 and AVP1 provide the first indication that plant V-PPases from the same organism fall into two distinct categories. Phylogenetic analyses of these and other V-PPase sequences extend this principle by showing that AVP2, rather than being an isoform of AVP1, is but one representative of a novel category of AVP2-like (type II) V-PPases that coexist with AVP1-like (type I) V-PPases not only in plants, but also in apicomplexan protists such as the malarial parasite Plasmodium falciparum.     

Identification of a novel cis-acting element conferring sulfur deficiency response in Arabidopsis roots

SULTR1;1 high-affinity sulfate transporter is highly regulated in the epidermis and cortex of Arabidopsis roots responding to sulfur deficiency (-S). We identified a novel cis-acting element involved in the -S-inducible expression of sulfur-responsive genes in Arabidopsis. The promoter region of SULTR1;1 was dissected for deletion and gain-of-function analysis using luciferase (LUC) reporter gene in transgenic Arabidopsis. The 16-bp sulfur-responsive element (SURE) from -2777 to -2762 of SULTR1;1 promoter was sufficient and necessary for the -S-responsive expression, which was reversed when supplied with cysteine and glutathione (GSH). The SURE sequence contained an auxin response factor (ARF) binding sequence (GAGACA). However, SURE was not responsive to naphthalene acetic acid, indicating its specific function in the sulfur response. The base substitution analysis indicated the significance of a 5-bp sequence (GAGAC) within the conserved ARF binding site as a core element for the -S response. Microarray analysis of early -S response in Arabidopsis roots indicated the presence of SURE core sequences in the promoter regions of -S-inducible genes on a full genome GeneChip array. It is suggested that SURE core sequences may commonly regulate the expression of a gene set required for adaptation to the -S environment.     

Characterization of the PHO1 gene family and the responses to phosphate deficiency of Physcomitrella patens

PHO1 was previously identified in Arabidopsis (Arabidopsis thaliana) as a protein involved in loading inorganic phosphate (Pi) into the xylem of roots and its expression was associated with the vascular cylinder. Seven genes homologous to AtPHO1 (PpPHO1;1-PpPHO1;7) have been identified in the moss Physcomitrella patens. The corresponding proteins harbor an SPX tripartite domain in the N-terminal hydrophilic portion and an EXS domain in the conserved C-terminal hydrophobic portion, both common features of the plant PHO1 family. Northern-blot analysis showed distinct expression patterns for the PpPHO1 genes, both at the tissue level and in response to phosphate deficiency. Transgenic P. patens expressing the beta-glucuronidase reporter gene under three different PpPHO1 promoters revealed distinct expression profiles in various tissues. Expression of PpPHO1;1 and PpPHO1;7 was specifically induced by Pi starvation. P. patens homologs to the Arabidopsis PHT1, DGD2, SQD1, and APS1 genes also responded to Pi deficiency by increased mRNA levels. Morphological changes associated with Pi deficiency included elongation of caulonemata with inhibition of the formation of side branches, resulting in colonies with greater diameter, but reduced mass compared to Pi-sufficient plants. Under Pi-deficient conditions, P. patens also increased the synthesis of ribonucleases and of an acid phosphatase, and increased the ratio of sulfolipids over phospholipids. These results indicate that P. patens and higher plants share some common strategies to adapt to Pi deficiency, although morphological changes are distinct, and that the PHO1 proteins are well conserved in bryophyte despite the lack of a developed vascular system.     

Isolation and characterization of TgVP1, a type I vacuolar H+-translocating pyrophosphatase from Toxoplasma gondii. The dynamics of its subcellular localization and the cellular effects of a diphosphonate inhibitor

Here we report the isolation and characterization of a type I vacuolar-type H(+)-pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation. The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category. Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T. gondii. Providing that the coding sequence for the entire N-terminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmid-borne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K(+)-activated pyrophosphate (PP(i)) hydrolysis and PP(i)-energized H(+) translocation. Immunofluorescence microscopy of both free and intracellular T. gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme. Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow. Although treatment of T. gondii with AMDP concentrations as high as 100 microm had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I(50) for the inhibition of TgVP1 activity in vitro (0.9 microm) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisome-like vesicular structures. A dynamic association of TgVP1 with the host cell invasion apparatus is invoked, one in which the effects of inhibitory V-PPase substrate analogs are exerted after rather than during host cell invasion.     

Roles of basic residues and salt-bridge interaction in a vacuolar H+-pumping pyrophosphatase (AVP1) from Arabidopsis thaliana

To investigate the possible role of basic residues in H+ translocation through vacuolar-type H+-pumping pyrophosphatases (V-PPases), conserved arginine and lysine residues predicted to reside within or close to transmembrane domains of an Arabidopsis thaliana V-PPase (AVP1) were subjected to site-directed mutagenesis. One of these mutants (K461A) exhibited a "decoupled" phenotype in which proton-pumping but not hydrolysis was inhibited. Similar results were reported previously for an E427Q mutant, resulting in the proposal that E427 might be involved in proton translocation. However, the double mutant E427K/K461E has a wild type phenotype, suggesting that E427 and K461 form a stabilising salt bridge, but that neither residue plays a critical role in proton translocation.     

A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps.

Vacuolar-type H(+)-translocating pyrophosphatases (V-PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists. Here, we describe PVP, a thermostable, sequence-divergent V-PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum. PVP shares only 38% sequence identity with both the prototypical V-PPase from Arabidopsis thaliana and the H(+)-PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V-PPases. Heterologous expression of PVP in Saccharomyces cerevisiae yields a M(r) 64? omitted?000 membrane polypeptide that specifically catalyzes Mg(2+)-dependent PPi hydrolysis. The existence of PVP implies that PPi-energized H(+)-translocation is phylogenetically more deeply rooted than previously thought.     

The Arabidopsis thaliana plasma membrane H(+)-ATPase multigene family. Genomic sequence and expression of a third isoform

The plasma membrane of higher plants contains a H(+)-ATPase as its major ion pump. This enzyme belongs to the P-type family of cation-translocating enzymes and generates the proton-motive force that drives solute uptake across the plasma membrane. In Arabidopsis thaliana the plasma membrane H(+)-ATPase is encoded by a multigene family (Harper, J. F., Surowy, T. K., and Sussman, M. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1234-1238). The complete genomic sequence of a third Arabidopsis H(+)-ATPase isoform (referred to as AHA2) is presented here, and the predicted protein sequence is compared with previously published AHA1, AHA3, and tobacco Nicotiana plumbaginifolia NP1 isoforms. The AHA2 gene is most similar to AHA1, with predicted proteins containing 95% amino acid identity. The mRNA start site and 5'-untranslated sequence for AHA2 were determined from cDNA amplified by the polymerase chain reaction. The 5' region contains a 23-base pair (bp) polypyrimidine sequence and a short upstream reading frame. In comparison with the 16 introns reported in AHA3, AHA2 is missing one intron in the 5'-untranslated region and a second intron in the C-terminal coding region. An unusually large intron for Arabidopsis (greater than 1000 bp) is present at the beginning of the coding sequence of both AHA2 and AHA3. In the 3'-untranslated sequence of AHA1 and AHA2 but not AHA3, there is a 65-bp region of 85% identity and a second shorter region of 16-bp identity harboring an unusual putative poly(A) addition signal (dTTTGAAGAAACAAGGC). Northern blot analysis indicates that AHA2 mRNA relative to total cellular RNA is expressed at significantly higher levels in root tissue as compared with shoot tissue.     

Cloning, expression and purification of orthologous membrane proteins: a general protocol for preparation of the histidine sensor kinase ETR1 from different species

Orthologous proteins do not necessarily share the same function in all species and those sharing the same function might employ a modified catalytic mechanism. Thus, comparative analysis of homologous or orthologous proteins from different organisms can provide detailed information on the function and the mechanism of an entire protein family. The sensor kinase ETR1 from Arabidopsis thaliana has been well characterized by genetic, physiological and biochemical studies. However, as further model plants are coming into focus for plant hormone research, a general protocol for isolation and purification of orthologous ETR1 proteins seems instrumental for a detailed molecular analysis of this protein family. In this study, we describe the native purification of recombinant ETR1 from Arabidopsis thaliana by mild solubilization with the zwitter-ionic detergent Fos-Choline-14 and single-step purification by immobilized metal ion affinity chromatography. The same protocol was successfully applied for the purification of the orthologous proteins from the moss Physcomitrella patens subsp. patens and the tomato Lycopersicon esculentum. The successful transfer of the purification protocol to proteins of the same family which share sequence identity of 63-80% only suggests that this protocol presents a general purification strategy which is likely to apply also to the purification of other members of the sensor histidine kinase family.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

Molecular characterization of PAB2, a member of the multigene family coding for poly(A)-binding proteins in Arabidopsis thaliana.

The poly(A) tail of eukaryotic mRNAs associates with poly(A)-binding (PAB) proteins whose role in mRNA translation and stability is being intensively investigated. Very little is known about the structure and function of the PAB genes in plants. We have cloned multiple PAB-related sequences from Arabidopsis thaliana. Results suggest that PAB proteins are encoded by a multigene family. One member of this family (PAB2) is expressed in root and shoot tissues. The complete nucleotide sequence of PAB2 was determined. Study of the predicted PAB2 protein reveals a similarity in structure among vertebrate, insect, yeast, and plant PAB proteins. All contain two highly conserved domains: an amino-terminal sequence formed by four RNA recognition motifs and an uncharacterized carboxyl-terminal region of 69 to 71 amino acids. Possible roles for the carboxyl-terminal conserved domain are discussed in view of recently published data concerning the structure and function of PAB proteins.     

水稻MYB转录因子OsDUO1的cDNA克隆及其表达模式初步分析

我们利用RT-PCR方法成功从水稻中克隆了R2R3类MYB转录因子OsDUO1（Oryza sativa duo pollen1）的全长为1032bp的cDNA,该基因编码一个343个氨基酸残基的蛋白。RT-PCR分析结果表明OsDUO1只在水稻的花粉发育后期表达,说明OsDUO1可能对水稻花粉发育具有生物学功能。生物信息学分析表明,OsDUO1在短柄草、高粱、玉米、拟南芥、烟草、葡萄、蓖麻、杨毛果、小立碗藓植物中有相近同源序列,暗示该基因在进化中具有保守的生物学功能。     

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.     

The Arabidopsis microtubule-associated protein AtMAP65-1: molecular analysis of its microtubule bundling activity

The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle.     

The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase (PAL) catalyzes the first step in the biosynthesis of phenylpropanoids, and is usually encoded by a multi-gene family. Genomic clones for three Arabidopsis thaliana PAL genes containing the entire protein-coding region and upstream and downstream sequences have been obtained and completely sequenced. Two A. thaliana PAL genes (PAL1 and PAL2) are structurally similar to PAL genes that have been cloned from other plant species, with a single intron at a conserved position, and a long highly conserved second exon. Previously identified promoter motifs plus several additional sequence motifs were found in the promoter regions of PAL1 and PAL2. Expression of PAL1 and PAL2 is both qualitatively and quantitatively similar in different plant organs and under various inductive conditions. A third A. thaliana PAL gene, PAL3, differs significantly from PAL1 and PAL2 and other sequenced plant PAL genes. PAL3 contains an additional intron, and its deduced amino acid sequence is less homologous to other PAL proteins. The PAL3 promoter region lacks several sequence motifs conserved between A. thaliana PAL1 and PAL2, as well as motifs described in other genes involved in phenylpropanoid metabolism. A. thaliana PAL3 was expressed at very low levels under the conditions examined.