ASK1, a SKP1 homolog, is required for nuclear reorganization, presynaptic homolog juxtaposition and the proper distribution of cohesin during meiosis in Arabidopsis

Nuclear reorganization and juxtaposition of homologous chromosomes at late leptotene/early zygotene are essential steps before chromosome synapsis at pachytene. We report the results of detailed studies, which demonstrate that nuclear reorganization and homolog juxtapositioning processes are defective in a null mutant, ask1-1. Our results from 4, 6-diamino-2-phenylindole (DAPI)-stained spreads showed that the “synizetic knot”, which is typically found in wild type (WT) meiosis during late leptotene and zygotene, was missing in the ask1-1 mutant. Furthermore, ask1-1 meiocytes exhibited only limited homolog juxtaposition at centromere regions at early zygotene. Immunodetection of the cohesin protein SYN1 identified ask1 defects in cohesin distribution from zygotene to anaphase I. Analysis of meiotic chromosomes in ask1-1 and syn1 single mutants, as well as an ask1-1 syn1 double mutant indicate that ASK1 is required for normal SYN1 distribution during meiotic prophase I and suggest that ask1 associated defects may be primarily related to SYN1 mislocalization.     

ASK1 physically interacts with COI1 and is required for male fertility inArabidopsis

Jasmonates are a new class of plant hormones that play important roles in plant development and plant defense. TheCOI1 gene was previously shown to be required for jasmonate-regulated plant fertility and defense. We demonstrated for the first time that COI1 interacts with theArabidopsis SKP1-LIKE1 (ASK1) to form a complex that is required for jasmonate action inplanta. Functional analysis by antisense strategy showed thatASK1 is involved in male fertility.     

Functional expression and molecular characterization of AtUBC2-1, a novel ubiquitin-conjugating enzyme (E2) from Arabidopsis thaliana

The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana. Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L. Sullivan and R.D. Vierstra, Proc. Natl. Acad. Sci. USA 86 (1989) 9861-9865). The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa. The protein encoded by AtUBC2-1 is shown to accept ¹²⁵I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions. It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme.     

Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response

The male component of the self-incompatibility response in Brassica has recently been shown to be encoded by the S locus cysteine-rich gene (SCR). SCR is related, at the sequence level, to the pollen coat protein (PCP) gene family whose members encode small, cysteine-rich proteins located in the proteo-lipidic surface layer (tryphine) of Brassica pollen grains. Here we show that the Arabidopsis genome includes two large gene families with homology to SCR and to the PCP gene family, respectively. These genes are poorly predicted by gene-identification algorithms and, with few exceptions, have been missed in previous annotations. Based on sequence comparison and an analysis of the expression patterns of several members of each family, we discuss the possible functions of these genes. In particular, we consider the possibility that SCR-related genes in Arabidopsis may encode ligands for the S gene family of receptor-like kinases in this species.     

Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine,serine and threonine

An intact cDNA fromArabidopsis thaliana for adenine phosphoribosyltransferase (APRT) was isolated and sequenced. The cDNA is 729 nucleotides in length and predicts a protein ofM _r 27140. The deduced amino acid sequence has been compared with those of other APRTs and shown to be most similar to theEscherichia coli protein. Construction of a molecular tree of the known APRT amino acid sequences indicates theA. thaliana andE. coli APRT sequences form one cluster and the currently available vertebrate and invertebrate sequences form a separate grouping. Since it is possible to select either for or against the expression of APRT, the isolation of this APRT cDNA clone will allow these selection schemes to be used in plant genetic experiments.     

In vitro and in vivo interaction of AtRma2 E3 ubiquitin ligase and auxin binding protein 1

E3 ubiquitin (Ub) ligases play diverse roles in cellular regulation in eukaryotes. Three homologous AtRmas (AtRma1, AtRma2, and AtRma3) were recently identified as ER-localized Arabidopsis homologs of human RING membrane-anchor E3 Ub ligase. Here, auxin binding protein 1 (ABP1), one of the auxin receptors in Arabidopsis, was identified as a potential substrate of AtRma2 through a yeast two-hybrid assay. An in vitro pull-down assay confirmed the interaction of full-length AtRma2 with ABP1. AtRma2 was transiently expressed in tobacco (Nicotiana benthamiana) plants through an Agrobacterium-mediated infiltration method and bound ABP1 in vivo. In vitro ubiquitination assays revealed that bacterially-expressed AtRma2 ubiquitinated ABP1. ABP1 was poly-ubiquitinated in tobacco cells and its stability was significantly increased in the presence of MG132, a 26S proteasome inhibitor. This suggests that ABP1 is controlled by the Ub/26S proteasome system. Therefore, AtRma2 is likely involved in the cellular regulation of ABP1 expression levels.     

Analysis of recombination rate in female and male gametogenesis in pearl millet (Pennisetum glaucum) using RFLP markers

Sex as a factor affecting recovered recombination in plant gametes was investigated in pearl millet, Pennisetum glaucum, by using reciprocal three-way crosses [(AxB)xCvCx(A x B)]. The two populations were mapped at 42 loci pre-selected to cover the majority of the genome. No differences in recombination distances were observed at the whole-genome level and only a few individual linkage intervals were found to differ, all in favour of increased recombination through the male. Distorted segregations found in the three-way crosses provide evidence of post-gametic selection for particular gene(s) or chromosome regions. The significance of these results for the design of pearl millet breeding programmes and inheritance experiments, as well as for other experimental strategies, is discussed.     

Homologues of wheat ubiquitin-conjugating enzymes - TaUBC1 and TaUBC4 are encoded by small multigene families in Arabidopsis thaliana

Covalent attachment of ubiquitin to other cellular proteins has been implicated in a multitude of diverse physiological processes in eukaryotes including selective protein degradation. This attachment is carried out by a multi-enzyme pathway consisting of three classes of enzymes: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin-protein ligases (E3s). E2s accept activated ubiquitin from E1 and conjugate it to target proteins with or without the participation of specific E3s. Previously, we have isolated wheat cDNAs encoding 16 and 23 kDa E2s, TaUBC1 and TaUBC4, respectively. TaUBC1 shows structural homology to the yeast RAD6 E2 that is essential for DNA repair whereas TaUBC4 is related to the yeast ScUBC8 E2, both of which effectively conjugate ubiquitin to histones in vitro but as yet are without a known in vivo function. Here, we report the isolation of genomic and cDNA homologues of these genes from Arabidopsis thaliana. In Arabidopsis, both of these E2s are encoded by three member gene families. Members of the AtUBC1 gene family, comprising AtUBC1, 2 and 3, encode 150–152 amino acid proteins that are 83–99% identical to each other and TaUBC1 and contain four introns that are conserved with respect to position. Members of the AtUBC4 gene family, comprising AtUBC4, 5 and 6, encode 187–191 amino acid proteins that are 73–88% identical to each other and TaUBC4 and contain five introns that are conserved with respect to position. In contrast, AtUBC1-3 gene products are only 31–36% identical to those derived from AtUBC4-6. mRNA for each family was detected in Arabidopsis roots, leaves, stems, and flowers indicating that members of each family are expressed in most if not all tissues.     

Primary structure and expression of a gene homologous to nifH (nitrogenase Fe protein) from the archaebacterium Methanococcus voltae

Summary In Methanococcus voltae, a 3.0 kbp HindIII fragment carrying homology to nifH was recently cloned. In Escherichia coli maxicells, the fragment directed the synthesis of a 30 K polypeptide encoded by the region homologous to nifH. Plasmids carrying the fragment did not complement Klebsiella pneumoniae nifH mutants and did not inhibit the nitrogen fixation of a Nif⁺ strain. The complete nucleotide sequence of the nifH homologous region was determined. It contained an open reading frame (ORFnifH) of 834 bp encoding 278 amino acid residues (mol. wt. 30,362). The ORFnifH was surrounded by regions of very high A+T content as observed with other mc. voltae genes. The region upstream from ORFnifH contained potential prokaryotic-like promoters and a potential ribosome binding site located 5 bp preceding the translation initiation codon. Using a translational fusion to lacZ of a DNA fragment carrying the putative promoter region and the 5 end of ORFnifH, it was shown in E. coli that (i) a promoter activity was effectively carried by the cloned fragment and (ii) this activity was not significantly modified by the presence of nifA or ntrC products provided by multicopy plasmids. Though the codon usage was characteristic of Mc. voltae, ORFnifH was very similar to eubacterial nifH genes, in particular the position of the cysteine residues was highly conserved. These data confirmed the high conservation of nifH sequences. S_AB values (binary matching coefficients) of 0.5 were found with eubacterial nifH genes at the nucleotide or amino acid level suggesting that the mc. voltae ORFnifH sequence was distantly related to eubacterial nifH sequences.     

Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana

We analyzed the Arabidopsis thaliana genome sequence to detect Late Embryogenesis Abundant (LEA) protein genes, using as reference sequences proteins related to LEAs previously described in cotton or which present similar characteristics. We selected 50 genes representing nine groups. Most of the encoded predicted proteins are small and contain repeated domains that are often specific to a unique LEA group. Comparison of these domains indicates that proteins with classical group 5 motifs are related to group 3 proteins and also gives information on the possible history of these repetitions. Chromosomal gene locations reveal that several LEA genes result from whole genome duplications (WGD) and that 14 are organized in direct tandem repeats. Expression of 45 of these genes was tested in different plant organs, as well as in response to ABA and in mutants (such as abi3, abi5, lec2 and fus3) altered in their response to ABA or in seed maturation. The results demonstrate that several so-called LEA genes are expressed in vegetative tissues in the absence of any abiotic stress, that LEA genes from the same group do not present identical expression profile and, finally, that regulation of LEA genes with apparently similar expression patterns does not systematically involve the same regulatory pathway.     

The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases

Polygalacturonase-inhibiting proteins (PGIPs) selectively inhibit polygalacturonases (PGs) secreted by invading plant pathogenic fungi. PGIPs display differential inhibition towards PGs from different fungi, also towards different isoforms of PGs originating from a specific pathogen. Recently, a PGIP-encoding gene from Vitis vinifera (Vvpgip1) was isolated and characterised. PGIP purified from grapevine was shown to inhibit crude polygalacturonase extracts from Botrytis cinerea, but this inhibitory activity has not yet been linked conclusively to the activity of the Vvpgip1 gene product. Here we use a transgenic over-expression approach to show that the PGIP encoded by the Vvpgip1 gene is active against PGs of B. cinerea and that over-expression of this gene in transgenic tobacco confers a reduced susceptibility to infection by this pathogen. A calculated reduction in disease susceptibility of 47–69% was observed for a homogeneous group of transgenic lines that was statistically clearly separated from untransformed control plants following infection with Botrytis over a 15-day-period. VvPGIP1 was subsequently purified from transgenic tobacco and used to study the specific inhibition profile of individual PGs from Botrytis and Aspergillus. The heterologously expressed and purified VvPGIP1 selectively inhibited PGs from both A. niger and B.␣cinerea, including BcPG1, a PG from B. cinerea that has previously been shown to be essential for virulence and symptom development. Altogether our data confirm the antifungal nature of the VvPGIP1, and the in vitro inhibition data suggest at least in part, that the VvPGIP1 contributed to the observed reduction in disease symptoms by inhibiting the macerating action of certain Botrytis PGs in planta. The ability to correlate inhibition profiles to individual PGs provides a more comprehensive analysis of PGIPs as antifungal genes with biotechnological potential, and adds to our understanding of the importance of PGIP:PG interactions during disease and symptom development in plants.Dirk A. Joubert and Ana R. Slaughter contributed equally to this work.     

Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and <Emphasis Type="Italic">Arabidopsis</Emphasis>

Erythropoietin (EPO) is a glycoprotein used for curing human anemia by regulating the differentiation of erythroid progenitors and the production of red blood cells. To examine the expression of recombinant EPO in plants, pPEV-EP21, in which human epo cDNA under the control of the CaMV 35S promoter, was introduced into tobacco and Arabidopsisvia Agrobacterium tumefaciens-mediated transformation. The RNA expression level of epo in the transgenic lines was initially estimated by Northern blot analysis. Two transgenic lines, which exhibited a high expression level of epo mRNA determined by Northern analysis, were chosen for Western blot analysis to examine the production of EPO proteins. Those two lines, EP21-12 and EP21-14, revealed detectable bands on the immunoblot. Interestingly, constitutive expression of the human epo gene affected the morphologies in transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and ArabidopsisThese authors contributed equally to this work     

The C. elegans sex determination protein MOG-3 functions in meiosis and binds to the CSL co-repressor CIR-1

In the germ line of the Caenorhabditis elegans hermaphrodite, nuclei either proliferate through mitosis or initiate meiosis, finally differentiating as spermatids or oocytes. The production of oocytes requires repression of the fem-3 mRNA by cytoplasmic FBF and nuclear MOG proteins. Here we report the identification of the sex determining gene mog-3 and show that in addition to its role in gamete sex determination, it is necessary for meiosis by acting downstream of GLP-1/Notch. Furthermore, we found that MOG-3 binds both to the nuclear proteins MEP-1 and CIR-1. MEP-1 is necessary for oocyte production and somatic differentiation, while the mammalian CIR-1 homolog counters Notch signaling. We propose that MOG-3, MEP-1 and CIR-1 associate in a nuclear complex which regulates different aspects of germ cell development. While FBF triggers the sperm/oocyte switch by directly repressing the fem-3 mRNA in the cytoplasm, the MOG proteins play a more indirect role in the nucleus, perhaps by acting as epigenetic regulators or by controlling precise splicing events.     

The Saccharomyces cerevisiae genes ( CMP1 and CMP2 ) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B

Summary Saccharomyces cerevisiae genomic clones that encode calmodulin-binding proteins were isolated by screening a λgt11 expression library using¹²⁵I-labeled calmodulin as probe. Among the cloned yeast genes, we found two closely related genes (CMP1 andCMP2) that encode proteins homologous to the catalytic subunit of phosphoprotein phosphatase. The presumed CMP1 protein (62999 Da) and CMP2 protein (68496 Da) contain a 23 amino acid sequence very similar to those identified as calmodulin-binding sites in many calmodulin-regulated proteins. The yeast genes encode proteins especially homologous to the catalytic subunit of mammalian phosphoprotein phosphatase type 213 (calcineurin). The products of theCMP1 andCMP2 genes were identified by immunoblot analysis of cell extracts as proteins of 62000 and 64000 Da, respectively. Gene disruption experiments demonstrated that elimination of either or both of these genes had no effect on cell viability, indicating that these genes are not essential for normal cell growth.     

The yeast RNA1 protein, necessary for RNA processing, is homologous to the human ribonuclease/angiogenin inhibitor (RAI)

Summary Mutations in theRNA1 gene ofSaccharomyces cerevisiae, which encodes an essential cytosolic protein, affect the production and processing of all major classes of RNA. The mechanisms underlying these effects are not at all understood. Detailed comparative sequence analyses revealed that the RNA1 protein belongs to a superfamily, the members of which contain repetitive leucine-rich motifs (LRM). Within this superfamily RNA1 is most closely related to the ribonuclease/angiogenin inhibitor (RAI), which is a tightly binding inhibitor of ribonucleolytic activities in mammals. These results not only provide important clues to the structure, function and evolution of the RNAI protein, but also have intriguing implications for possible novel functions of RAI.