Temporal and tissue-specific expression of the tobacco ntf4 MAP kinase

The large number of mitogen-activated protein (MAP) kinase genes identified to date in plants suggests that their encoded proteins have a wide array of functions in development and physiological responses, as has been indicated by studies on the factors which lead to the activation of these kinases. Signalling pathways involving members of a multigene family employ a variety of mechanisms to ensure response specificity, one of which is via differential gene expression. We have performed detailed analyses of the expression of the tobacco ntf4 MAP kinase gene using a variety of approaches. The ntf4 gene promoter region was isolated and a chimeric ntf4 promoter-GUS fusion construct was introduced into plants. GUS expression was detected in pollen, in developing and mature embryos, and shortly after seed germination, but not in other floral tissues and tissues such as leaf, root, or stem. This expression pattern was confirmed by northern and western analyses. In situ hybridization and immunolocalization studies showed that the expression of the ntf4 gene and its encoded protein p45^Ntf4 occurred in embryos at least from the globular embryo stage until the mature seed, as well as in the seed endosperm. Taken together, the results show that the p45^Ntf4 MAP kinase has a very restricted expression pattern, being found only in pollen and seeds. These findings should be important when considering MAP kinase function in plants.     

A novel tobacco mitogen-activated protein (MAP) kinase kinase, NtMEK1, activates the cell cycle-regulated p43Ntf6 MAP kinase

Two-hybrid screening of a tobacco BY-2 cell suspension cDNA library using the p43(Ntf6) mitogen-activated protein (MAP) kinase as bait resulted in the isolation of a cDNA encoding a protein with features characteristic of a MAP kinase kinase (MEK), which has been called NtMEK1. Two-hybrid interaction analysis and pull-down experiments showed a physical interaction between NtMEK1 and the tobacco MAP kinases p43(Ntf6) and p45(Ntf4), but not p43(Ntf3). In kinase assays NtMEK1 preferentially phosphorylated p43(Ntf6). Functional studies in yeast showed that p43(Ntf6) could complement the yeast MAP kinase mutant mpk1 when co-expressed with NtMEK1, and that this complementation depended on the kinase activity of p43(Ntf6). Expression analysis showed that the NtMEK1 and ntf6 genes are co-expressed both in plant tissues and following the induction of cell division in leaf pieces. These data suggest that NtMEK1 is an MEK for the p43(Ntf6) MAP kinase.     

A homologue of the MAP/ERK family of protein kinase genes is expressed in vegetative and in female reproductive organs of Petunia hybrida

The mitogen activated protein (MAP) kinase pathway of eukaryotes is stimulated by many growth factors and is required for the integration of multiple cellular signals. In order to study the function of MAP kinases during plant ovule development we have synthesized a Petunia hybrida ovule-specific cDNA library and screened for MAP protein kinase-related sequences using a DNA probe obtained by PCR. A full-length cDNA clone was identified (PMEK for Petunia hybrida MAP/ERK-related protein kinase) and shown to encode a protein related to the family of MAP/ERK protein kinases. Southern blot analysis showed that PMEK is a member of a small multigene family in P. hybrida. The cDNA codes for a protein (PMEK1) of 44.4 kDa with an overall sequence identity of 44% to the products of the mammalian ERK/MAP kinase gene, and the budding yeast KSS1 and FUS3 genes. PMEK1 displays 96 and 80% identity respectively with the tobacco NTF3 and Arabidopsis ATMPK1 kinases, and only 50% to the more distantly related plant MAP kinase MsERK1 from alfalfa. The two phosphorylation sites found in the loop between subdomain VII and VIII in all the other MAP kinases are also present in PMEK1. RNA gel blot and RT-PCR analyses demonstrated that PMEK1 is expressed in vegetative organs and preferentially accumulated in female reproductive organs of P. hybrida. In situ hybridization experiments showed that in the reproductive organs PMEK1 is expressed only in the ovary and not in the stamen.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)⁺ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.     

Molecular cloning and expression of a MAP kinase homologue from pea

The cdc2 kinases are important cell cycle regulators in all eukaryotes. MAP kinases, a closely related family of protein kinases, are involved in cell cycle regulation in yeasts and vertebrates, but previously have not been documented in plants. We used PCR to amplify Brassica napus DNA sequences using primers corresponding to amino sequences that are common to all known protein kinases. One sequence was highly similar to KSS1, a MAP kinase from Saccharomyces cerevisiae. This sequence was used to isolate a full-length MAP kinase-like clone from a pea cDNA library. The pea clone, called D5, shared approximately 50% amino acid identity with MAP kinases from yeasts and vertebrates and about 41% identity with plant cdc2 kinases. An expression protein encoded by D5 was recognized by an antiserum specific to human MAP kinases (ERKs). Messenger RNA corresponding to D5 was present at similar levels in all tissues examined, without regard to whether cell division or elongation were occurring in those tissues.     

A cDNA from pea petals with sequence similarity to pollen allergen, cytokinin-induced and genetic tumour-specific genes: identification of a new family of related sequences

A cDNA isolated from pea petals exhibits extensive similarity to pollen allergen genes, a cytokinin-regulated cDNA from soybean suspension cultures, a partial cDNA preferentially expressed in tobacco genetic tumours, four Arabidopsis expressed sequence tags (ESTs) and fifteen rice ESTs. This diverse family of pollen allergen-likes genes may have a common ancestor or at least share common functional domains. Possession of a putative signal peptide and a presumed extracellular location is a common aspect of this family of sequences.     

Characterization of a pollen-expressed gene encoding a putative pectin esterase ofPetunia inflata

From a pollen tube cDNA library ofPetunia inflata, we isolated cDNA clones encoding a protein, PPE1, which exhibits sequence similarity with plant, bacterial, and fungal pectin esterases. Genomic clones containing thePPE1 gene were isolated using cDNA for PPE1 as a probe, and comparison of the cDNA and genomic sequences revealed the presence of a single intron in thePPE1 gene. During pollen development,PPE1 mRNA was first detected in anthers containing uninucleate microspores; it reached the highest level in mature pollen and persisted at a high level inin vitro germinated pollen tubes. The observed expression pattern of thePPE1 gene suggests that its product may play a role in pollen germination and/or tube growth.     

Isolation and characterization of a cDNA clone encoding the anti-viral protein from Phytolacca americana

Phytolacca anti-viral protein (PAP) was purified from Phytolacca leaves and the N-terminal was sequenced. A cDNA library was made from mRNAs isolated from Phytolacca leaves and cDNA clones for PAP were identified using oligonucleotide probes derived from the N-terminal amino acid sequence. The PAP-cDNA clone was sequenced from both directions. The predicted amino acid sequence of PAP was compared with the amino acid sequences of other ribosome-inactivating proteins. The identities of these proteins to PAP ranged from 29 to 38%, and a region was found in each with a sequence similar to the PAP sequence (AIQMVSEAARFKYI). Southern blot analysis indicates that PAP is encoded by a multi-gene family.Abbreviations MAP Mirabilis jalapa anti-viral protein - PAP Phytolacca anti-viral protein - SO6 30 kDa ribosome-inactivating protein from the seeds of Saponaria officinalis     

Isolation and Characterization of a cDNA That Encodes a Novel Proteinase Inhibitor I from a Tobacco Genetic Tumor

We have isolated a cDNA clone, designated GTI, by screeninga tobacco genetic tumor cDNA library with a tumor-specific "subtracted"cDNA probe. The cDNA contained the entire coding sequence fora 94-amino-acid polypeptide that exhibited significant homologyto members of the proteinase inhibitor I family from tomatoand potato. The predicted protein has a pre-sequence of 22 aminoacids but lacks a pro-sequence, unlike genes for proteinaseinhibitor I isolated to date. Furthermore, the protein encodedby GTI cDNA has a novel reactive site, having glutamine as theP₁ reactive residue. These results suggest that the GTI proteinis a novel member of the proteinase inhibitor I family. ThemRNA for GTI accumulated at a high level but only transientlyafter the wounding of tobacco plants. Thus, it appears thatthe GTI protein has a function that is related to the protectionof tissues against damage due to wounding. (Received July 22, 1992; Accepted November 3, 1992)     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

Cloning and expression of a Xenopus gene that prevents mitotic catastrophe in fission yeast

In fission yeast the Weel kinase and the functionally redundant Mikl kinase provide a regulatory mechanism to ensure that mitosis is initiated only after the completion of DNA synthesis. Yeast in which both Weel and Mik1 kinases are defective exhibit a mitotic catastrophe phenotype, presumably due to premature entry into mitosis. Because of the functional conservation of cell cycle control elements, the expression of a vertebrate weel or mikl homolog would be expected to rescue such lethal mutations in yeast. A Xenopus total ovary cDNA library was constructed in a fission yeast expression vector and used to transform a yeast temperature-dependent mitotic catastrophe mutant defective in both weel and mikl. Here we report the identification of a Xenopus cDNA clone that can rescue several different yeast mitotic catastrophe mutants defective in Weel kinase function. The expression of this clone in a weel/mikl-deficient mutant causes an elongated cell phenotype under non-permissive growth conditions. The 2.0 kb cDNA clone contains an open reading frame of 1263 nucleotides, encoding a predicted 47 kDa protein. Bacterially expressed recombinant protein was used to raise a polyclonal antibody, which specifically recognizes a 47 kDa protein from Xenopus oocyte nuclei, suggesting the gene encodes a nuclear protein in Xenopus. The ability of this cDNA to complement mitotic catastrophe mutations is independent of Weel kinase activity.     

Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants.

Summary Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 andcdc2Os-2) encoding structural homologues of thecdc2 ⁺/CDC28(cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62 % identical toCDC28 ofSaccharomyces cerevisiae and much more similar to the yeast and mammalian p34^cdc2 kinases than to riceR2, acdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1,cdc2Os-2 andR2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings.cdc2Os-1 could complement a temperature-sensitive yeast mutant ofcdc28. However, despite the similarity in structure, bothcdc2Os-2 andR2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of thecdc2 cell cycle kinase in rice.The nucleotide sequence data in this paper have been deposited in the EMBL database under accession number X60374 (cdc2Os-1) and X60375 (cdc2Os-2)     

Molecular cloning of a cDNA fromBrassica napus L. for a homologue of acyl-CoA-binding protein

A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.). The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly. Southern blot analysis ofBrassica napus genomic DNA revealed the presence of 6 genes, 3 derived from theBrassica rapa parent and 3 fromBrassica oleracea. Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots.     

A catalytic subunit of the sugar beet protein kinase CK2 is induced by salt stress and increases NaCl tolerance in Saccharomyces cerevisiae

Salinity is an important limiting factor in plant growth and development. We have cloned a catalytic subunit of the sugar beet protein kinase CK2 (BvCKA2) by functional expression in yeast of a NaCl-induced cDNA library. BvCKA2 was able to increase the yeast tolerance to NaCl and to functionally complement the cka1 cka2 yeast double mutant upon over-expression. Southern blot analysis indicated that, in sugar beet, the BCKA2 gene is a member of a multigene family. The mRNA levels of BvCKA2 were up-regulated in response to NaCl stress which suggests that protein kinase CK2 may be involved in the plant response to salt stress.     

Characterization and functional analysis of a pollen-specific gene st901 in Solanum tuberosum

A pollen-specific gene, sb401, which was isolated from a cDNA library of in vitro geminated pollen of the diploid potato species Solanum berthaultii, belongs to the class of genes expressed late during pollen development. Using sb401 as a probe, a pollen-specific gene st901 was isolated from the genomic library of a potato species Solanum tuberosum cv. Desiree. Sequencing and RT-PCR analysis showed that the st901 genomic gene is 2,889 bp long, contains three exons and two introns, and encodes a putative polypeptide of 217 residues. The predicted protein sequence contains four imperfect repeated motifs of V–V–E–K–K–N/E–E; the core sequence of the repeats (K–K–N/E–E) resembles a microtubule-binding domain of the microtubule-associated protein MAP1B from mouse. The examination of a promoter–reporter construct in transgenic potato plants revealed that the st901 is expressed exclusively in mature pollen grains, which is consistent with the results of Northern blot and RT-PCR. For analysis of the function of st901, transgenic plants harboring antisense copies of st901 cDNA driven by a native st901 promoter were generated. Suppression of st901 gene in potato resulted in aberrant pollen at maturation and pollen viability of transgenic plants ranged from 4.4 to 14.8%, while that of control plants were more than 90%. These results strongly suggest that st901 has an essential role in pollen development.The st901 gene sequence has been deposited in GenBank under accession number AY526087. Accession number for SB401 is X95984.1