Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

Characterization of eight new members of the calmodulin-like domain protein kinase gene family from Arabidopsis thaliana

A family of calcium-responsive protein kinases is abundant in plant cell extracts but has not been identified in animals and fungi. These enzymes have a unique structure consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains. In this report, we present the amino acid sequences for eight new Arabidopsis cDNA clones encoding isoforms of this enzyme. Three isoforms were expressed as fusion proteins in Escherichia coli and exhibited calcium-stimulated protein kinase activity. We propose CPK as the gene designation for this family of enzymes and describe a phylogenetic analysis for all known isoforms.     

Characterization of a cDNA encoding the 55 kDa B regulatory subunit of Arabidopsis protein phosphatase 2A

Type 2A serine/threonine protein phosphatases (PP2A) are key components in the regulation of signal transduction and control of cell metabolism. The activity of these protein phosphatases is modulated by regulatory subunits. While PP2A activity has been characterized in plants, little is known about its regulation. We used the polymerase chain reaction to amplify a segment of a cDNA encoding the B regulatory subunit of PP2A from Arabidopsis. The amplified DNA fragment of 372 nucleotides was used as a probe to screen an Arabidopsis cDNA library and a full-length clone (AtB) of 2.1 kbp was isolated. The predicted protein encoded by AtB is 43 to 46% identical and 53 to 56% similar to its yeast and mammalian counterparts, and contains three unique regions of amino acid insertions not present in the animal B regulatory subunit. Genomic Southern blots indicate the Arabidopsis genome contains at least two genes encoding the B regulatory subunit. In addition, other plant species also contain DNA sequences homologous to the B regulatory subunit, indicating that regulation of PP2A activity by the 55 kDa B regulatory subunit is probably ubiquitous in plants. Northern blots indicate the AtB mRNA accumulates in all Arabidopsis tissues examined, suggesting the protein product of the AtB gene performs a basic housekeeping function in plant cells.     

Molecular analysis of the myosin gene family in Arabidopsis thaliana

Myosin is believed to act as the molecular motor for many actin-based motility processes in eukaryotes. It is becoming apparent that a single species may possess multiple myosin isoforms, and at least seven distinct classes of myosin have been identified from studies of animals, fungi, and protozoans. The complexity of the myosin heavy-chain gene family in higher plants was investigated by isolating and characterizing myosin genomic and cDNA clones from Arabidopsis thaliana. Six myosin-like genes were identified from three polymerase chain reaction (PCR) products (PCR1, PCR11, PCR43) and three cDNA clones (ATM2, MYA2, MYA3). Sequence comparisons of the deduced head domains suggest that these myosins are members of two major classes. Analysis of the overall structure of the ATM2 and MYA2 myosins shows that they are similar to the previously-identified ATM1 and MYA1 myosins, respectively. The MYA3 appears to possess a novel tail domain, with five IQ repeats, a six-member imperfect repeat, and a segment of unique sequence. Northern blot analyses indicate that some of the Arabidopsis myosin genes are preferentially expressed in different plant organs. Combined with previous studies, these results show that the Arabidopsis genome contains at least eight myosin-like genes representing two distinct classes.     

Trehalose‐6‐phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant

It is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6°C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.     

Structure and molecular evolutionary analysis of a plant cytochrome c gene: Surprising implications forArabidopsis thaliana

Summary We have isolated a cytochrome c gene fromArabidopsis thaliana (cv. Columbia), which is the first cytochrome c gene to be cloned from a higher plant. Genomic DNA blot analysis indicates that there is only one copy of cytochrome c inArabidopsis. The gene consists of three exons separated by two introns. Gene features such as regulatory regions, codon usage, and conserved splicing-specific sequences are all present and typical of dicotyledonous plant nuclear genes. We have constructed phenograms and cladograms for cytochrome c amino acid sequences and histone H3, alcohol dehydrogenase, and actin DNA sequences. For both cytochrome c and histone H3,Arabidopsis clusters poorly with other higher plants. Instead, it clusters withNeurospora and/or the yeasts. We suggest that perhaps this observation should be considered when usingArabidopsis as a model system for higher plants.     

The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protistTrichomonas vaginalis

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones fromTrichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which containedT. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of theT. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists likeEntamoeba histolytica, Trypanosoma cruzi, andLeishmania infantum.     

Plant aldolase: cDNA and deduced amino-acid sequences of the chloroplast and cytosol enzyme from spinach

We report the sequences of full-length cDNAs for the nuclear genes encoding the chloroplastic and cytosolic fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) from spinach. A comparison of the deduced amino-acid sequences with one another and with published cytosolic aldolase sequences of other plants revealed that the two enzymes from spinach share only 54% homology on their amino acid level whereas the homology of the cytosolic enzyme of spinach with the known sequences of cytosolic aldolases of maize, rice and Arabidopsis range from 67 to 92%. The sequence of the chloroplastic enzyme includes a stroma-targeting N-terminal transit peptide of 46 amino acid residues for import into the chloroplast. The transit peptide exhibits essential features similar to other chloroplast transit peptides. Southern blot analysis implies that both spinach enzymes are encoded by single genes.     

Molecular characterization of catalytic-subunit cDNA sequences encoding protein phosphatases 1 and 2A and study of their roles in the gibberellin-dependent Osamy-c expression in rice

To understand the molecular mechanism of gibberellin-dependent gene regulation, the effect of three phosphatase inhibitors on the germination of rice seeds and the expression of a target gene, the -amylase gene, Osamy-c, were measured. We found that okadaic acid, microcystin-LR, and calyculin A, which are known to specifically inhibit Ser/Thr phosphatases 1 and 2A, strongly inhibit the expression of the Osamy-c and may be involved in the germination of rice seeds.The protein phosphatase enzyme activity assays showed that there is no obvious effect of GA3 on total PP1/PP2A activities. To further understand the possible role of protein phosphatases 1 and 2A in the GA-dependent expression of Osamy-c, we isolated cDNA clones encoding protein phosphatase 1 and protein phosphatase 2A from a rice aleurone cDNA library. These were designated OsPP1c and OsPP2Ac, respectively. Comparison of the deduced amino acid sequences of OsPP1c and OsPP2Ac with the catalytic subunits of PP1 or PP2A of rabbit skeletal muscle, Arabidopsis thaliana, maize and Brassica napus showed that the catalytic subunit sequences of PP1 or PP2A among these organisms are highly conserved (73% to 90% similarity). Genomic Southern blot analysis indicated that there are only one or two copies of OsPP1c genes and more than two copies of OsPP2Ac genes in the rice genome. Northern blot analysis showed that OsPP1c and OsPP2Ac genes are expressed in several organs of rice, including seed, shoot and root. We also showed by using 3 gene-specific probes of OsPP1c and OsPP2Ac cDNA, that the expression of neither gene is regulated by GA. Taken together, our results suggest that protein phosphatases PP1 or PP2A are involved in the GA-dependent expression of the rice Osamy-c gene, though the PP1 or/and PP2A enzymatic activities as well as mRNA levels do not increase upon GA3 treatment.     

Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

The Arabidopsis thaliana PPX/PP4 phosphatases: molecular cloning and structural organization of the genes and immunolocalization of the proteins to plastids

The PPX/PP4 Ser/Thr protein phosphatases belong to the type 2A phosphatase subfamily and are present in most eukaryotic organisms. We have previously isolated two closely related DNAs encoding PPX isoforms (PPX-1 and PPX-2) of Arabidopsis thaliana. Here we report the molecular cloning of the genes encoding these proteins. The genes PPX-1 and PPX-2 are composed of eight exons and seven introns located at equivalent positions related to the coding sequences. Whereas the intron-exon organization of the PPX genes is completely different from that of the PP2A-3/PP2A-4 A. thaliana family, specific intron-exon boundaries are conserved among PPX genes from distantly related organisms. Based on GUS expression, both PPX genes show the same spatial and temporal pattern of expression: they are expressed in all the organs and tissues analyzed, and from the earliest stage of development. When PPX proteins were localized to the root in semi-thin methacrylate sections by immunofluorescence, staining was predominantly confined to small organelles, shown to be plastids by co-localization of PPX and ferredoxin. Interestingly, only some ferredoxin-positive plastids were also PPX-positive, and PPX staining was consistently brighter in the epidermis. The localization was confirmed with immunogold and electron microscopy. Our results suggest that, despite its strong sequence conservation, PPX in plants functions differently than in animals.     

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II.The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2–3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3 untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.     

Novel antimicrobial peptides identified from an endoparasitic wasp cDNA library

We screened an endoparasitic wasp (Pteromalus puparum) cDNA library for DNA sequences having antimicrobial activity using a vital dye exclusion assay. Two dozens of clones were isolated that inhibited the growth of host Escherichia coli cells due to expression of the cloned genes. Three peptides (PP13, PP102 and PP113) were synthesized chemically based on the amino acid sequences deduced from these clones and assayed for their antimicrobial activity. These peptides have net positive charges and are active against both Gram‐negative and ‐positive bacteria, but are not active against fungi tested. Their hemolytic activity on human red blood cells was measured, and no hemolytic activity was observed after 1‐h incubation at a concentration of 62.5 µM or below. A Blast search indicated that the three peptides have not been previously characterized as antimicrobial peptides (AMPs). Salt‐dependency studies revealed that the biocidal activity of these peptides against E. coli decreased with increasing concentration of NaCl. Transmission electron microscopic (TEM) examination of PP13‐treated E. coli cells showed extensive damage of cell membranes. The CD spectroscopy studies noted that the enhanced α‐helical characteristics of PP13 strongly contribute to its higher antimicrobial properties. These results demonstrate the feasibility to identify novel AMPs by screening the expressional cDNA library. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Processed Pseudogenes, Processed Genes, and Spontaneous Mutations in the Arabidopsis Genome

We identified 411 processed sequences in the Arabidopsis thaliana genome based on the fact that they have lost their intron(s) and have a length that is at least 95% of the length of the gene that gave rise to them. These sequences were generated by 230 different genes and clearly originated from retrotranspositons events because most of them (91%) have a poly(A)-tail. They are composed of 376 sequences with frame shifts and/or premature stop codons (processed pseudogenes) and 35 sequences without disablements (processed genes). Eleven of these processed genes are likely functional retrotransposed genes because they have low Ka/Ks ratios and high Ks values, and their sequences match numerous Arabidopsis ESTs. Processed sequences are mostly randomly distributed in the Arabidopsis genome and their rate of accumulation has steadily been decreasing since it peaked some 50 MYA. In contrast with the situation observed in mammals, the processed sequences found in the Arabidopsis genome originate from genes with high copy numbers and not from highly expressed genes. The patterns of spontaneous mutations in Arabidopsis are slightly different than those of mammals but are similar to those observed in Drosophila. This suggests that methylated cytosine deamination is less frequent in Arabidopsis than in mammals. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Juergen Brosius]     

Tomato contains homologues of Arabidopsis cryptochromes 1 and 2

Cryptochromes are blue light photoreceptors found in both plants and animals. They probably evolved from photolyases, which are blue/UV-light-absorbing photoreceptors involved in DNA repair. In seed plants, two different cryptochrome (CRY) genes have been found in Arabidopsis and one in Sinapis, while three genes have been found in the fern Adiantum. We report the characterisation of tomato CRY genes CRY1 and CRY2. They map to chromosomes 4 and 9, respectively, show relatively constitutive expression and encode proteins of 679 and 635 amino acids, respectively. These proteins show higher similarity to their Arabidopsis counterparts than to each other, suggesting that duplication between CRY1 and CRY2 is an ancient event in the evolution of seed plants. The seed plant cryptochromes form a group distinct from the fern cryptochromes, implying that only one gene was present in the common ancestor between these two groups of plants. Most intron positions in CRY genes from plants and ferns are highly conserved. Tomato cry1 and cry2 proteins carry C-terminal domains 210 and 160 amino acids long, respectively. Several conserved motifs are found in these domains, some of which are common to both types of cryptochromes, while others are cryptochrome-type-specific.     

A serine/threonine protein phosphatase-like protein, CaPTC8, from Candida albicans defines a new PPM subfamily

Protein phosphatase M family (PPM; Mg²⁺-dependent protein phosphatases), which specifically dephosphorylates serine/threonine residues, consists of pyruvate dehydrogenase phosphatases, SpoIIE, adenylate cyclase and protein phosphatase type 2Cs (PP2Cs). To identify Candida albicans PP2Cs, the archetype of the PPM Ser/Thr phosphatases, we thoroughly searched the public C. albicans genome database and identified seven PP2C members. One of the PP2Cs in C. albicans, designated as CaPTC8 gene, represents a new member of PP2C genes. Northern blot analysis showed that the expression of CaPTC8 was positively responsive to high osmolarity, temperature or serum-stimulated filamentous growth. Gene disruption further demonstrated that deletion of CaPTC8 gene caused the defect of hyphal formation. Sequence analysis revealed that two conserved amino acids His and Asn in the prototypical members of the PPM family were substituted by Val and Asp in the PTC8p-like proteins. In addition, posterior analysis for site-specific profile showed that seven more sites are under the selection of functional divergence between these two groups of proteins. Three-dimensional homology modeling illustrated the signatures of the two groups in the conserved catalytic region of the protein phosphatases. Hence, CaPTC8 plays a role in stress responses and is required for the yeast-hyphal transition, and the CaPTC8-related genes are evolutionarily conserved. The phylogenetic relationships of all members of the PPM family strongly support the existence of a distinct and new subfamily of the PP2C-related proteins, PP2CR.     

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.