Transcriptional and post-transcriptional PHOSPHATE 2 (PHO2) gene expression regulation in barley

Wydział BiologiiJedną z podstawowych strategii roślin przystosowanych do bytowania w zmiennych warunkach stężenia fosforu nieorganicznego (Pi) w glebie jest regulacja wykorzystująca proces degradacji białek determinujący liczbę transporterów fosforanowych (PHT) wysycających błony komórkowe. W tym procesie uczestniczy m.in. białko kodowane przez gen PHOSPHATE 2 (PHO2). PHO2 jest enzymem typu E2 (UBC24) sprzęgającym ubikwitynę, który razem z odpowiednią ligazą typu E3 kieruje białka docelowe na szlak degradacji. Czteroczęściowa praca doktorska w sposób wieloaspektowy poddaje analizie mechanizmy molekularne zaangażowane w homeostazę fosforanową jęczmienia. Wyniki uzyskane w ramach rozprawy doktorskiej pozwoliły na identyfikację oraz charakterystykę nowych czynników transkrypcyjnych, które nie były dotąd rozważane jako potencjalne białka regulujące ekspresję genu PHO2 w jęczmieniu. Przeprowadzone badania doprowadziły do odkrycia możliwej regulacji transkrypcji genu PHO2 poprzez wiązanie się białek PHR1/APL do motywów P1BS oraz P-responsive PHO element obecnych w obrębie rejonu 5’-UTR. Ponadto scharakteryzowano pulę małych RNA oraz mRNA zaangażowanych w odpowiedź jęczmienia na stres niedoboru fosforu za pomocą technik głębokiego sekwencjonowania. Informacje te mogą być niezwykle ważne z punktu widzenia zarówno ograniczenia zasobów fosforu na świecie jak i usuwania nadmiaru fosforu z gleby w celu zapobieżenia zanieczyszczeniu wód. Dodatkowo w znaczący sposób poszerzają obecny stan wiedzy na temat procesów warunkujących tolerancję roślin na niedobór fosforanów w podłożu.One of the basic strategies of plants adapted to living in conditions of inorganic phosphate (Pi) deficiency in soil is to regulate the level of proteins, including phosphate transporters (PHT), by molecular pathway, which results in a variable number of phosphate transporters saturating cell membranes. The gene encoding protein PHOSPHATE 2 (PHO2) is responsible for this process. PHO2 is an E2-type (UBC24) ubiquitin-conjugating enzyme which, together with the appropriate E3-type ligase, directs target proteins down the degradation pathway. The four-part doctoral dissertation analyzes the molecular mechanisms involved in barley Pi homeostasis in a complex manner. The results obtained during the doctoral dissertation allowed for the identification and characterization of new transcription factors that have not been considered as potential proteins regulating the expression of the PHO2 gene in barley. The conducted research led to the discovery of possible regulation of PHO2 gene transcription by binding of PHR1 / APL proteins to P1BS and P-responsive PHO element motifs present within the 5'-UTR region. In addition, the pool of small RNAs and mRNAs involved in the barley response to Pi deficiency stress were characterized using deep sequencing techniques. This information can be essential both in terms of reducing the world's phosphorus resources and removing excess phosphorus from the soil to prevent water pollution. In addition, the results significantly expand the current state of knowledge about the processes determining the tolerance of plants to the Pi deficiency

HorTILLUS - a rich and renewable source of induced mutations for forward/reverse genetics and pre-breeding programs in barley (Hordeum vulgare L.)

TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN3) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M2 plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M3 progeny of 3,481 M2 individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M2 plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platformis the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations.We offer the usage of this valuable resource to the interested barley researchers on cooperative basis

Transcriptional and post-transcriptional PHOSPHATE 2 (PHO2) gene expression regulation in barley

Wydział BiologiiJedną z podstawowych strategii roślin przystosowanych do bytowania w zmiennych warunkach stężenia fosforu nieorganicznego (Pi) w glebie jest regulacja wykorzystująca proces degradacji białek determinujący liczbę transporterów fosforanowych (PHT) wysycających błony komórkowe. W tym procesie uczestniczy m.in. białko kodowane przez gen PHOSPHATE 2 (PHO2). PHO2 jest enzymem typu E2 (UBC24) sprzęgającym ubikwitynę, który razem z odpowiednią ligazą typu E3 kieruje białka docelowe na szlak degradacji. Czteroczęściowa praca doktorska w sposób wieloaspektowy poddaje analizie mechanizmy molekularne zaangażowane w homeostazę fosforanową jęczmienia. Wyniki uzyskane w ramach rozprawy doktorskiej pozwoliły na identyfikację oraz charakterystykę nowych czynników transkrypcyjnych, które nie były dotąd rozważane jako potencjalne białka regulujące ekspresję genu PHO2 w jęczmieniu. Przeprowadzone badania doprowadziły do odkrycia możliwej regulacji transkrypcji genu PHO2 poprzez wiązanie się białek PHR1/APL do motywów P1BS oraz P-responsive PHO element obecnych w obrębie rejonu 5’-UTR. Ponadto scharakteryzowano pulę małych RNA oraz mRNA zaangażowanych w odpowiedź jęczmienia na stres niedoboru fosforu za pomocą technik głębokiego sekwencjonowania. Informacje te mogą być niezwykle ważne z punktu widzenia zarówno ograniczenia zasobów fosforu na świecie jak i usuwania nadmiaru fosforu z gleby w celu zapobieżenia zanieczyszczeniu wód. Dodatkowo w znaczący sposób poszerzają obecny stan wiedzy na temat procesów warunkujących tolerancję roślin na niedobór fosforanów w podłożu.One of the basic strategies of plants adapted to living in conditions of inorganic phosphate (Pi) deficiency in soil is to regulate the level of proteins, including phosphate transporters (PHT), by molecular pathway, which results in a variable number of phosphate transporters saturating cell membranes. The gene encoding protein PHOSPHATE 2 (PHO2) is responsible for this process. PHO2 is an E2-type (UBC24) ubiquitin-conjugating enzyme which, together with the appropriate E3-type ligase, directs target proteins down the degradation pathway. The four-part doctoral dissertation analyzes the molecular mechanisms involved in barley Pi homeostasis in a complex manner. The results obtained during the doctoral dissertation allowed for the identification and characterization of new transcription factors that have not been considered as potential proteins regulating the expression of the PHO2 gene in barley. The conducted research led to the discovery of possible regulation of PHO2 gene transcription by binding of PHR1 / APL proteins to P1BS and P-responsive PHO element motifs present within the 5'-UTR region. In addition, the pool of small RNAs and mRNAs involved in the barley response to Pi deficiency stress were characterized using deep sequencing techniques. This information can be essential both in terms of reducing the world's phosphorus resources and removing excess phosphorus from the soil to prevent water pollution. In addition, the results significantly expand the current state of knowledge about the processes determining the tolerance of plants to the Pi deficiency

Plant PHR Transcription Factors: Put on A Map

The phosphate starvation response (PHR) protein family exhibits the MYB and coiled-coil domains. In plants, within the either 5′ untranslated regions (UTRs) or promoter regions of phosphate starvation-induced (PSI) genes are characteristic cis-regulatory elements, namely PHR1 binding sequence (P1BS). The most widely studied PHR protein family members, such as AtPHR1 in Arabidopsis thaliana (L.) and OsPHR2 in Oryza sativa (L.), may activate the gene expression of a broad range of PSI genes by binding to such elements in a phosphate (Pi) dependent manner. In Pi signaling, PHR transcription factors (TFs) can be selectively activated or deactivated by other proteins to execute the final step of signal transduction. Several new proteins have been associated with the AtPHR1/OsPHR2 signaling cascade in the last few years. While the PHR TF transcriptional role has been studied intensively, here we highlight the recent findings of upstream molecular components and other signaling pathways that may interfere with the PHR final mode of action in plants. Detailed information about transcriptional regulation of the AtPHR1 gene itself and its upstream molecular events has been reviewed

Table2.DOCX

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Table3.DOCX

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Image2.tif

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Image3.tif

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Image1.tif

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p

Table1.DOCX

<p>TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN₃) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M₂ plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M₃ progeny of 3,481 M₂ individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M₂ plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2 Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platform is the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations. We offer the usage of this valuable resource to the interested barley researchers on cooperative basis.</p