... Symbiosis 21:4148 Burdman S, Vedder D, German M, Itzigsohn R, Kigel J, Jurke-vitch E, Okon Y... more ... Symbiosis 21:4148 Burdman S, Vedder D, German M, Itzigsohn R, Kigel J, Jurke-vitch E, Okon Y (1998) Legume crop yield promotion by ino-culation with Azospirillum. In: Elmerich C, Kondorosi A, Newton WE (eds) Biological nitrogen fixation for the 21st century. ...
Plants have two kinds of fructokinases (FRKs) that catalyze the key step of fructose phosphorylat... more Plants have two kinds of fructokinases (FRKs) that catalyze the key step of fructose phosphorylation, cytosolic and plastidic. The major cytosolic tomato FRK, SlFRK2, is essential for the development of xylem vessels. In order to study the role of SlFRK3, which encodes the only plastidic FRK, we generated transgenic tomato (Solanum lycopersicon) plants with RNAi suppression of SlFRK3 as well as plants expressing beta-glucoronidase (GUS) under the SlFRK3 promoter. GUS staining indicated SlFRK3 expression in vascular tissues of the leaves and stems, including cambium, differentiating xylem, young xylem fibers and phloem companion cells. Suppression of SlFRK3 reduced the stem xylem area, stem and root water conductance, and whole-plant transpiration, with minor effects on plant development. However, suppression of SlFRK3 accompanied by partial suppression of SlFRK2 induced significant growth-inhibition effects, including the wilting of mature leaves. Grafting experiments revealed that these growth effects are imposed primarily by the leaves, whose petioles had unlignified, thin-walled xylem fibers with collapsed parenchyma cells around the vessels. A cross between the SlFRK2-antisense and SlFRK3-RNAi lines exhibited similar wilting and anatomical effects, confirming that these effects are the result of the combined suppression of SlFRK3 and SlFRK2. These results demonstrate a role of the plastidic SlFRK3 in xylem development and hydraulic conductance.
A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Ly... more A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate
We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughpu... more We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse applications for the study of the RNA degradome. The method described here comprises a modified 5'-rapid amplification of cDNA ends, deep sequencing of 3' cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5'-RNA adapter that includes an MmeI recognition site is ligated to 5'-monophosphorylated products, which contain mRNA fragments generated through miRNA-induced cleavage. The ligated products are reverse-transcribed, slightly amplified and cleaved with MmeI. The 5' equally-sized fragments are gel-selected, ligated to a 3' double-stranded DNA adapter and PCR-amplified. Following gel purification, the products are subjected to deep sequencing. The data are then matched to cDNAs and analyzed through bioinformatics filters. We describe the high...
Methods in molecular biology (Clifton, N.J.), 2010
MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleo... more MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes.
The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and bi... more The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon. B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million dis...
... Symbiosis 21:4148 Burdman S, Vedder D, German M, Itzigsohn R, Kigel J, Jurke-vitch E, Okon Y... more ... Symbiosis 21:4148 Burdman S, Vedder D, German M, Itzigsohn R, Kigel J, Jurke-vitch E, Okon Y (1998) Legume crop yield promotion by ino-culation with Azospirillum. In: Elmerich C, Kondorosi A, Newton WE (eds) Biological nitrogen fixation for the 21st century. ...
Plants have two kinds of fructokinases (FRKs) that catalyze the key step of fructose phosphorylat... more Plants have two kinds of fructokinases (FRKs) that catalyze the key step of fructose phosphorylation, cytosolic and plastidic. The major cytosolic tomato FRK, SlFRK2, is essential for the development of xylem vessels. In order to study the role of SlFRK3, which encodes the only plastidic FRK, we generated transgenic tomato (Solanum lycopersicon) plants with RNAi suppression of SlFRK3 as well as plants expressing beta-glucoronidase (GUS) under the SlFRK3 promoter. GUS staining indicated SlFRK3 expression in vascular tissues of the leaves and stems, including cambium, differentiating xylem, young xylem fibers and phloem companion cells. Suppression of SlFRK3 reduced the stem xylem area, stem and root water conductance, and whole-plant transpiration, with minor effects on plant development. However, suppression of SlFRK3 accompanied by partial suppression of SlFRK2 induced significant growth-inhibition effects, including the wilting of mature leaves. Grafting experiments revealed that these growth effects are imposed primarily by the leaves, whose petioles had unlignified, thin-walled xylem fibers with collapsed parenchyma cells around the vessels. A cross between the SlFRK2-antisense and SlFRK3-RNAi lines exhibited similar wilting and anatomical effects, confirming that these effects are the result of the combined suppression of SlFRK3 and SlFRK2. These results demonstrate a role of the plastidic SlFRK3 in xylem development and hydraulic conductance.
A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Ly... more A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate
We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughpu... more We have developed a novel approach called parallel analysis of RNA ends (PARE) for high-throughput identification of microRNA (miRNA) targets and diverse applications for the study of the RNA degradome. The method described here comprises a modified 5'-rapid amplification of cDNA ends, deep sequencing of 3' cleavage products of mRNA and bioinformatic analysis. Following RNA extraction and isolation of polyadenylated RNA, a 5'-RNA adapter that includes an MmeI recognition site is ligated to 5'-monophosphorylated products, which contain mRNA fragments generated through miRNA-induced cleavage. The ligated products are reverse-transcribed, slightly amplified and cleaved with MmeI. The 5' equally-sized fragments are gel-selected, ligated to a 3' double-stranded DNA adapter and PCR-amplified. Following gel purification, the products are subjected to deep sequencing. The data are then matched to cDNAs and analyzed through bioinformatics filters. We describe the high...
Methods in molecular biology (Clifton, N.J.), 2010
MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleo... more MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes.
The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and bi... more The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon. B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million dis...
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Papers by Marcelo German