SEN1 Gene Research Articles

Effect of nitrogen fixation enhancing type SEN1 gene on soybean growth

ABSTRACT It is well known that the growth of legumes is greatly affected not only by fertilizer nitrogen but also by fixed nitrogen by symbiosis with rhizobia. We have reported that a polymorphism in SEN1, which is essential for nitrogen fixation in Lotus japonicus, could be used to enhance nitrogen fixation and plant growth. In this study, we examined whether the same strategy could be applied to Glycine max. Sequencing of orthologs of the LjSEN1 gene in 38 soybean cultivars revealed that the polymorphism occurred at Glyma.08g076300. We defined the standard sequence that most of the cultivars retained as Peking type SEN1, and the mutated sequence found in Enrei as Enrei type SEN1. To investigate the effect of genotype differences, we generated near-isogenic lines by crossing Enrei with Fukuyutaka, which has Peking type SEN1, and analyzed their characteristics. The results showed that Enrei type enhanced nitrogen fixation activity and promoted plant growth. Since soybean SEN1 has been reported to be a homolog of vacuolar iron transporter-like protein, we investigated the iron content in the nodules. In the result, the iron content in the nodules of Enrei type was significantly higher than that of Peking type. Iron is known to be present in the active center of nitrogenase, and the high iron content may have contributed to the increased nitrogen fixation activity. These results suggest that Enrei type SEN1 is a nitrogen fixation enhancing gene and that introduction of Enrei type SEN1 may reduce nitrogen fertilizer application and increase yield.

Molecular screening of potato varieties bred by Falenki Breeding station for resistance to phytopathogens

The genotypes of potatoes bred by Falenki Breeding station were studied for the presence of resistance genes markers to the following pathogens: Globodera rostochiensis, Globodera pallidа, Synchytrium endobioticum, potato virus X (PVХ) and potato virus Y (PVY). The method of multiplex PCR analysis was used. The varieties Shurminsky 2, Alisa, Viza, Chayka, Ognivo, Darik, Gloriya, Golubka, Virazh and a promising variety sample 56-09 were studied. In most (8 out of 10) genotypes, marker linked to the Sen1 gene of resistance to S. endobioticum was identified. DNA marker of the G. rostochiensis resistance gene (H1) and the G. pallida resistance gene marker (Gpa2) were found in six genotypes. The marker of the PVX resistance gene (Rx1) was detected in the varieties Shurminsky 2, Alisa, Chayka, Golubka, and Virazh. It has been established that none of the studied potato genotypes carries markers RYSC3, Ry186, YES3-3A linked to the PVY resistance genes. Although in the field, resistance was detected in the samples Chayka, Darik, Virazh, Alisa. Molecular markers linked to the largest number of resistance genes studied (H1, Gpa2, Sen1, and Rx1) were identified in the varieties Shurminsky 2, Golubka, and Virazh. Among the DNA markers used in the work, the data of potato genotype assessment using markers of virus resistance genes (PVX, RYSC3, Ry186, YES3-3A) were less consistent with field observations. The use of molecular markers makes it possible to determine the presence of resistance genes and assess the prospects of a sample in a short period of time, but, at the same time, requires careful choice of a DNA marker that is highly correlated with the manifestation of the trait.

SEN1 gene from Lotus japonicus MG20 improves nitrogen fixation and plant growth

ABSTRACT In Lotus japonicus, quantitative trait loci (QTLs) for nitrogen fixation activity (acetylene reduction activity, ARA) and seed weight are located in the same region on chromosome 4 as the gene responsible for the Fix− mutant sen1. The SEN1 gene of L. japonicus Gifu B129 has a deletion of three nucleotides in comparison with that of L. japonicus Miyakojima MG20, which results in a deletion of one amino acid. Here, we examined whether SEN1 is responsible for these QTLs. Because the MG20 allele of the QTL for ARA and seed weight has a positive effect, we generated a near-isogenic line (NIL) in which the SEN1 gene region of B129 was replaced with that of MG20. Growth parameters of this NIL (MG20-type) 21 days after inoculation with Mesorhizobium loti were significantly higher than those of B129-type, and ARA was also significantly higher. Nodules formed on hairy roots in which the sen1 mutant was complemented with the MG20 SEN1 gene had higher ARA than those of B129. The seeds of the MG20-type NIL were significantly heavier than those of B129-type. These results indicate that the SEN1 gene is a very likely candidate for the QTL for ARA and seed weight on chromosome 4.

Pea Marker Database (PMD) - A new online database combining known pea (Pisum sativum L.) gene-based markers.

Pea (Pisum sativum L.) is the oldest model object of plant genetics and one of the most agriculturally important legumes in the world. Since the pea genome has not been sequenced yet, identification of genes responsible for mutant phenotypes or desirable agricultural traits is usually performed via genetic mapping followed by candidate gene search. Such mapping is best carried out using gene-based molecular markers, as it opens the possibility for exploiting genome synteny between pea and its close relative Medicago truncatula Gaertn., possessing sequenced and annotated genome. In the last 5 years, a large number of pea gene-based molecular markers have been designed and mapped owing to the rapid evolution of “next-generation sequencing” technologies. However, the access to the complete set of markers designed worldwide is limited because the data are not uniformed and therefore hard to use. The Pea Marker Database was designed to combine the information about pea markers in a form of user-friendly and practical online tool. Version 1 (PMD1) comprises information about 2484 genic markers, including their locations in linkage groups, the sequences of corresponding pea transcripts and the names of related genes in M. truncatula. Version 2 (PMD2) is an updated version comprising 15944 pea markers in the same format with several advanced features. To test the performance of the PMD, fine mapping of pea symbiotic genes Sym13 and Sym27 in linkage groups VII and V, respectively, was carried out. The results of mapping allowed us to propose the Sen1 gene (a homologue of SEN1 gene of Lotus japonicus (Regel) K. Larsen) as the best candidate gene for Sym13, and to narrow the list of possible candidate genes for Sym27 to ten, thus proving PMD to be useful for pea gene mapping and cloning. All information contained in PMD1 and PMD2 is available at www.peamarker.arriam.ru.

Genetic diversity of potato varieties bred in Russia and its neighboring countries based on the polymorphism of SSR-loci and markers associated with resistance R-genes

The genetic diversity of potato varieties from the collection of Vavilov Institute of Plant Genetic Resources (VIR) was analyzed based on microsatellite analysis. These varieties have been bred in Russia and its neighboring countries since 1931. Application of 14 highly polymorphic nuclear microsatellites (SSRs) has enabled 113 varieties to be fully distinguished. Additionally, we have studied these varieties for the distribution of 8 DNA markers associated with three R-genes involved in controlling resistance to two quarantine objects: potato wart Synchytrium endobioticum and golden potato cyst nematode Globodera rostochiensis, occurring locally in some regions of the Russian Federation. All the analyzed varieties with resistance to the S. endobioticum pathotype 1 revealed the diagnostic marker Nl251400 of the Sen1 gene, while a few susceptible cultivars lost this diagnostic fragment. The tested markers of the H1 and Gro1-4 genes, which confer resistance to the G. rostochiensis pathotype Ro1 revealed different predictability. In the molecular screening of potato varieties, it is better to use several markers of these genes. The results of molecular screening using six markers of H1 and Gro1-4 genes allowed us to detect six haplotypes in the tested subset. Five haplotypes include varieties with different combinations of the markers tested; the majority (87.9%) of these varieties were highly resistant or moderately resistant to G. rostochiensis. The most numerous haplotype H1/0 included 76 varieties, which did not possess any marker of H1 and Gro1-4 genes; 96.1% of these varieties were susceptible to G. rostochiensis. Predictive associations between the haplotype content, wart and nematode resistance, pedigree, and the variety in their age are discussed.

Genetic diversity of potato varieties bred in Russia and near-abroad countries based on polymorphism of SSR-loci and markers associated with resistance R-genes

The genetic diversity of potato varieties from the VIR collection was analyzed based on microsatellite analysis. These varieties have been bred in Russia and near-abroad countries since the 1931. Application of 14 highly polymorphic nuclear microsatellites (SSR) enabled the complete discrimination of all 113 varieties. Additionally, we have studied these varieties for the distribution of 8 DNA markers associated with three R-genes involved in the control of resistance to two quarantine objects: the potato wart Synchytrium endobioticum and golden potato cyst nematode Globodera rostochiensis, which occur locally in some regions of the Russian Federation. All the analyzed varieties with resistance to S. endobioticum pathotype 1 revealed the diagnostic marker Nl-251400 of the Sen1 gene and a few susceptible cultivars lost this diagnostic fragment. The tested markers of the H1 and Gro1-4 genes, which confer resistance to G. rostochiensis pathotype Ro1 revealed different predictiveness. In the molecular screening of potato varieties, it is better to use several markers of these genes. Results of molecular screening using six markers of the H1 and Gro1-4 genes allowed us to detect 6 haplotypes in the tested subset. Five haplotypes include varieties with different combinations of the markers tested, the majority (87.9 %) of these varieties were highly resistant or moderately resistant to G. rostochiensis. The most numerous haplotype H1/0 included 76 varieties, which did not possess any marker; 96.1 % of these varieties were susceptible to G. rostochiensis. Predictive associations between haplotype content, wart and nematode resistance, pedigree and ‘variety age’ are discussed.

Correlación fenotípica y genotípica-molecular de la resistencia a enfermedades en variedades mejoradas de papa (Solanum tuberosum L.)

In two consecutive agricultural campaigns (2010-2011 and 2011-2012), 18 improved potato varieties were planted in the PROINPA Foundation greenhouses as well as two witness varieties (Waych’a and Desiree) with the aim to study: i) late blight resistance ( Phytophthora infestans ), wart ( Synchytrium endobioticum) an virus PVX and ii) validate molecular markers GP94, RB, CP60 and NL25 for co-localizing with major resistance genes of diseases mentioned. The results showed significant differences among varieties for AUDPCrel (late blight), severity (Wart) and severity and absorbance (PVX). Different levels of resistance were observed in varieties, such varieties with high resistance and moderate resistance. Molecular analysis were highly significant to Pr<0.01 of probability for three of the market used. It was observed that the allele for marker GP94 was co-located with the resistance gene Rpi-phu1 for late blight at a distance of 350 bp, showing the resistance allele in all varieties except the Waych'a variety. The allele for RB marker that is co-localizes with the RB gene for resistance to blight was not found in any of the varieties tested. The allele for marker CP60 was co-located with Rx1 gene to PVX resistance at a distance of 350 bp and it was observed in almost all varieties except Chota Nawi variety. Finally, the allele for marker NL25 is co-located with Sen1 gene resistance to wart at a distance of 1000 bp and it was observed in all varieties.

The Integral Membrane Protein SEN1 is Required for Symbiotic Nitrogen Fixation in Lotus japonicus Nodules

Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.

Sen1p Performs Two Genetically Separable Functions in Transcription and Processing of U5 Small Nuclear RNA inSaccharomyces cerevisiae

The Saccharomyces cerevisiae SEN1 gene codes for a nuclear-localized superfamily I helicase. SEN1 is an ortholog of human SETX (senataxin), which has been implicated in the neurological disorders ataxia-ocular apraxia type 2 and juvenile amyotrophic lateral sclerosis. Pleiotropic phenotypes conferred by sen1 mutations suggest that Sen1p affects multiple steps in gene expression. Sen1p is embedded in a protein-protein interaction network involving direct binding to multiple partners. To test whether the interactions occur independently or in a dependent sequence, we examined interactions with the RNA polymerase II subunit Rpb1p, which is required for transcription, and Rnt1p, which is required for 3'-end maturation of many noncoding RNAs. Mutations were identified that impair one of the two interactions without impairing the other interaction. The effects of the mutants on the synthesis of U5 small nuclear RNA were analyzed. Two defects were observed, one in transcription termination and one in 3'-end maturation. Impairment of the Sen1p-Rpb1p interaction resulted in a termination defect. Impairment of the Sen1p-Rnt1p interaction resulted in a processing defect. The results suggest that the Sen1p-Rpb1p and Sen1p-Rnt1p interactions occur independently of each other and serve genetically separable purposes in targeting Sen1p to function in two temporally overlapping steps in gene expression.

The SEN1 gene of Arabidopsis is regulated by signals that link plant defence responses and senescence

Plant defence and senescence share many similarities as evidenced by extensive co-regulation of many genes during these responses. To better understand the nature of signals that are common to plant defence and senescence, we studied the regulation of SEN1 encoding a senescence-associated protein during plant defence responses in Arabidopsis. Pathogen inoculations and treatments with defence-related chemical signals, salicylic acid and methyl jasmonate induced changes in SEN1 transcript levels. Analysis of transgenic plants expressing the SEN1 promoter fused to uidA reporter gene confirmed the responsiveness of the SEN1 promoter to defence- and senescence-associated signals. Expression analysis of SEN1 in a number of defence signalling mutants indicated that activation of this gene by pathogen occurs predominantly via the salicylic and jasmonic acid signalling pathways, involving the functions of EDS5, NPR1 and JAR1. In addition, in the absence of pathogen challenge, the cpr5/hys1 mutant showed elevated SEN1 expression and displayed an accelerated senescence response following inoculation with the necrotrophic fungal pathogen Fusarium oxysporum. Although the analysis of the sen1-1 knock-out mutant did not reveal any obvious role for this gene in defence or senescence-associated events, our results presented here show that SEN1 is regulated by signals that link plant defence and senescence responses and thus represents a useful marker gene to study the overlap between these two important physiological events.

The Lotus japonicus Sen1 gene controls rhizobial differentiation into nitrogen-fixing bacteroids in nodules.

A Lotus japonicus mutant, Ljsym75, which forms ineffective symbiotic nodules and defines a new locus involved in the process of nitrogen fixation, was characterized in detail in order to identify the stage of developmental arrest of the nodules. No nitrogen-fixing activity was detectable in Ljsym75 nodules at any stage during plant development, and plant growth was markedly retarded. Ljsym75 plants formed twice as many nodules as the wild-type Gifu, and this phenotype was not influenced by the application of low concentrations of nitrate. Although the ineffective nodules formed on Ljsym75 were anatomically similar to effective Gifu nodules, Ljsym75 nodules senesced prematurely. Microscopic examination revealed that bacteria endocytosed into Ljsym75 nodules failed to differentiate into bacteroids. Moreover, the bacteria contained no nitrogenase proteins, whereas leghemoglobin was detected in the cytosol of the nodules. These results indicate that Ljsym75 is required for bacterial differentiation into nitrogen-fixing bacteroids in nodules, and thus the Ljsym75 gene was renamed sen1 (for stationary endosymbiont nodule). Linkage analysis using DNA markers showed that Sen1 is located on chromosome 4.

The yeast SEN1 gene is required for the processing of diverse RNA classes.

A single base change in the helicase superfamily 1 domain of the yeast Saccharomyces cerevisiae SEN1 gene results in a heat-sensitive mutation that alters the cellular abundance of many RNA species. We compared the relative amounts of RNAs between cells that are wild-type and mutant after temperature-shift. In the mutant several RNAs were found to either decrease or increase in abundance. The affected RNAs include tRNAs, rRNAs and small nuclear and nucleolar RNAs. Many of the affected RNAs have been positively identified and include end-matured precursor tRNAs and the small nuclear and nucleolar RNAs U5 and snR40 and snR45. Several small nucleolar RNAs co-immunoprecipitate with Sen1 but differentially associate with the wild-type and mutant protein. Its inactivation also impairs precursor rRNA maturation, resulting in increased accumulation of 35S and 6S precursor rRNAs and reduced levels of 20S, 23S and 27S rRNA processing intermediates. Thus, Sen1 is required for the biosynthesis of various functionally distinct classes of nuclear RNAs. We propose that Sen1 is an RNA helicase acting on a wide range of RNA classes. Its effects on the targeted RNAs in turn enable ribonuclease activity.

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence.

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5'-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2-3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl2 which delayed loss of chlorophyll but not that of photochemical efficiency.

Inactivation of the yeast Sen1 protein affects the localization of nucleolar proteins.

A mutation in the Saccharomyces cerevisiae SEN1 gene causes accumulation of end-matured, intron-containing pre-tRNAs. Cells containing the thermosensitive sen1-1 mutation exhibit reduced tRNA splicing endonuclease activity. However, Sen1p is not the catalytic subunit of this enzyme. We have used Sen1p-specific antibodies for cell fractionation studies and immunofluorescent microscopy and determined that Sen1p is a low abundance protein of about 239 kDa. It localizes to the nucleus with a granular distribution. We verified that a region in SEN1 containing a putative nuclear localization signal sequence (NLS) is necessary for nuclear targeting. Furthermore, we found that inactivation of Sen1p by temperature shift of a strain carrying sen1-1 leads to mislocalization of two nucleolar proteins, Nop1 and Ssb1. Possible mechanisms are discussed for several related nuclear functions of Sen1p, including tRNA splicing and the maintenance of a normal crescent-shaped nucleolus.

SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.