Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters










Database
Language
Publication year range
1.
Plant Physiol ; 190(3): 1731-1746, 2022 10 27.
Article in English | MEDLINE | ID: mdl-35951755

ABSTRACT

In plant stems, secondary vascular development is established through the differentiation of cylindrical vascular cambium, producing secondary xylem (wood) and phloem (bast), which have economic importance. However, there is a dearth of knowledge on the genetic mechanism underlying this process. NAC with Transmembrane Motif 1-like transcription factor 9 (NTL9) plays a central role in abiotic and immune signaling responses. Here, we investigated the role of NTL9 in vascular cambium development in Arabidopsis (Arabidopsis thaliana) inflorescence stems by identifying and characterizing an Arabidopsis phloem circular-timing (pct) mutant. The pct mutant exhibited enhanced vascular cambium formation following secondary phloem production. In the pct mutant, although normal organization in vascular bundles was maintained, vascular cambium differentiation occurred at an early stage of stem development, which was associated with increased expression of cambium-/phloem-related genes and enhanced cambium activity. The pct mutant stem phenotype was caused by a recessive frameshift mutation that disrupts the transmembrane (TM) domain of NTL9. Our results indicate that NTL9 functions as a negative regulator of cambial activity and has a suppressive role in developmental transition to the secondary growth phase in stem vasculature, which is necessary for its precise TM domain-mediated regulation.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Cambium/metabolism , Arabidopsis Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Xylem/genetics , Xylem/metabolism , Plant Stems/genetics , Plant Stems/metabolism , Gene Expression Regulation, Plant
2.
Plant J ; 103(6): 2139-2150, 2020 09.
Article in English | MEDLINE | ID: mdl-32579240

ABSTRACT

We previously developed a large-scale genome restructuring technology called the TAQing system. It can induce genomic rearrangements by introducing transient and conditional formation of DNA double-strand breaks (DSBs) via heat activation of a restriction enzyme TaqI, which can cleave DNA at 5'-TCGA-3' sequences in the genome at higher temperatures (37-42°C). Such heat treatment sometimes confers lethal damage in certain plant species and TaqI cannot induce rearrangements in AT-rich regions. To overcome such problems we developed an extended TAQing (Ex-TAQing) system, which enables the use of a wider range of restriction enzymes active at standard plant-growing temperatures. We established the Ex-TAQing system using MseI that can efficiently cleave DNA at room temperature (at temperatures ranging from 22 to 25°C) and the 5'-TTAA-3' sequence which is highly abundant in the Arabidopsis genome. A synthetic intron-spanning MseI gene, which was placed downstream of a heat-shock-inducible promoter, was conditionally expressed upon milder heat treatment (33°C) to generate DSBs in Arabidopsis chromosomes. Genome resequencing revealed various types of genomic rearrangements, including copy number variations, translocation and direct end-joining at MseI cleavage sites. The Ex-TAQing system could induce large-scale rearrangements in diploids more frequently (17.4%, n = 23) than the standard TAQing system. The application of this system to tetraploids generated several strains with chromosomal rearrangements associated with beneficial phenotypes, such as high salinity stress tolerance and hypersensitivity to abscisic acid. We have developed the Ex-TAQing system, allowing more diverse patterns of genomic rearrangements, by employing various types of endonucleases and have opened a way to expand the capacity for artificial genome reorganization.


Subject(s)
Gene Editing/methods , Genome, Plant/genetics , Arabidopsis/genetics , Arabidopsis/metabolism , DNA Breaks, Double-Stranded , DNA, Plant/genetics , DNA, Plant/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , Gene Rearrangement/genetics , Hot Temperature , Introns/genetics , Ploidies , Tetraploidy
3.
Plant J ; 102(5): 1042-1057, 2020 06.
Article in English | MEDLINE | ID: mdl-31925982

ABSTRACT

Plant cells have acquired chloroplasts (plastids) with a unique genome (ptDNA), which developed during the evolution of endosymbiosis. The gene content and genome structure of ptDNAs in land plants are considerably stable, although those of algal ptDNAs are highly varied. Plant cells seem, therefore, to be intolerant of any structural or organizational changes in the ptDNA. Genome rearrangement functions as a driver of genomic evolutionary divergence. Here, we aimed to create various types of rearrangements in the ptDNA of Arabidopsis genomes using plastid-targeted forms of restriction endonucleases (pREs). Arabidopsis plants expressing each of the three specific pREs, i.e., pTaqI, pHinP1I, and pMseI, were generated; they showed the leaf variegation phenotypes associated with impaired chloroplast development. We confirmed that these pREs caused double-stranded breaks (DSB) at their recognition sites in ptDNAs. Genome-wide analysis of ptDNAs revealed that the transgenic lines exhibited a large number of rearrangements such as inversions and deletions/duplications, which were dominantly repaired by microhomology-mediated recombination and microhomology-mediated end-joining, and less by non-homologous end-joining. Notably, pHinP1I, which recognized a small number of sites in ptDNA, induced drastic structural changes, including regional copy number variations throughout ptDNAs. In contrast, the transient expression of either pTaqI or pMseI, whose recognition site numbers were relatively larger, resulted in small-scale changes at the whole genome level. These results indicated that DSB frequencies and their distribution are major determinants in shaping ptDNAs.


Subject(s)
DNA Restriction Enzymes/metabolism , Plastids/genetics , DNA Copy Number Variations/genetics , DNA Copy Number Variations/physiology , DNA Restriction Enzymes/genetics , Evolution, Molecular , Genome, Chloroplast/genetics , Genome, Plastid/genetics
4.
Nat Commun ; 9(1): 1995, 2018 05 18.
Article in English | MEDLINE | ID: mdl-29777105

ABSTRACT

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.


Subject(s)
Arabidopsis/genetics , DNA Breaks, Double-Stranded , Genome, Fungal , Genome, Plant , Saccharomyces cerevisiae/genetics , Arabidopsis/metabolism , DNA Repair , Diploidy , Gene Rearrangement , Genomic Instability , Saccharomyces cerevisiae/metabolism , Tetraploidy
5.
Biotechnol Biofuels ; 10: 203, 2017.
Article in English | MEDLINE | ID: mdl-28852424

ABSTRACT

BACKGROUND: The yeast Saccharomyces cerevisiae, a promising host for lignocellulosic bioethanol production, is unable to metabolize xylose. In attempts to confer xylose utilization ability in S. cerevisiae, a number of xylose isomerase (XI) genes have been expressed heterologously in this yeast. Although several of these XI encoding genes were functionally expressed in S. cerevisiae, the need still exists for a S. cerevisiae strain with improved xylose utilization ability for use in the commercial production of bioethanol. Although currently much effort has been devoted to achieve the objective, one of the solutions is to search for a new XI gene that would confer superior xylose utilization in S. cerevisiae. Here, we searched for novel XI genes from the protists residing in the hindgut of the termite Reticulitermes speratus. RESULTS: Eight novel XI genes were obtained from a cDNA library, prepared from the protists of the R. speratus hindgut, by PCR amplification using degenerated primers based on highly conserved regions of amino acid sequences of different XIs. Phylogenetic analysis classified these cloned XIs into two groups, one showed relatively high similarities to Bacteroidetes and the other was comparatively similar to Firmicutes. The growth rate and the xylose consumption rate of the S. cerevisiae strain expressing the novel XI, which exhibited highest XI activity among the eight XIs, were superior to those exhibited by the strain expressing the XI gene from Piromyces sp. E2. Substitution of the asparagine residue at position 337 of the novel XI with a cysteine further improved the xylose utilization ability of the yeast strain. Interestingly, introducing point mutations in the corresponding asparagine residues in XIs originated from other organisms, such as Piromyces sp. E2 or Clostridium phytofermentans, similarly improved xylose utilization in S. cerevisiae. CONCLUSIONS: A novel XI gene conferring superior xylose utilization in S. cerevisiae was successfully isolated from the protists in the termite hindgut. Isolation of this XI gene and identification of the point mutation described in this study might contribute to improving the productivity of industrial bioethanol.

6.
J Exp Bot ; 65(18): 5385-400, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25038254

ABSTRACT

In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Arabidopsis/metabolism , Inflorescence/growth & development , Inflorescence/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Biomass , Cell Proliferation/physiology , Gene Expression Regulation, Plant , Inflorescence/genetics
7.
Plant Cell Rep ; 31(6): 987-97, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22212462

ABSTRACT

Black rot of sweet potato caused by pathogenic fungus Ceratocystis fimbriata severely deteriorates both growth of plants and post-harvest storage. Antimicrobial peptides from various organisms have broad range activities of killing bacteria, mycobacteria, and fungi. Plant thionin peptide exhibited anti-fungal activity against C. fimbriata. A gene for barley α-hordothionin (αHT) was placed downstream of a strong constitutive promoter of E12Ω or the promoter of a sweet potato gene for ß-amylase of storage roots, and introduced into sweet potato commercial cultivar Kokei No. 14. Transgenic E12Ω:αHT plants showed high-level expression of αHT mRNA in both leaves and storage roots. Transgenic ß-Amy:αHT plants showed sucrose-inducible expression of αHT mRNA in leaves, in addition to expression in storage roots. Leaves of E12Ω:αHT plants exhibited reduced yellowing upon infection by C. fimbriata compared to leaves of non-transgenic Kokei No. 14, although the level of resistance was weaker than resistance cultivar Tamayutaka. Storage roots of both E12Ω:αHT and ß-Amy:αHT plants exhibited reduced lesion areas around the site inoculated with C. fimbriata spores compared to Kokei No. 14, and some of the transgenic lines showed resistance level similar to Tamayutaka. Growth of plants and production of storage roots of these transgenic plants were not significantly different from non-transgenic plants. These results highlight the usefulness of transgenic sweet potato expressing antimicrobial peptide to reduce damages of sweet potato from the black rot disease and to reduce the use of agricultural chemicals.


Subject(s)
Antimicrobial Cationic Peptides/genetics , Ascomycota/physiology , Disease Resistance/genetics , Ipomoea batatas/genetics , Plant Diseases/microbiology , Plant Leaves/microbiology , Plant Proteins/genetics , Plant Roots/microbiology , Antifungal Agents/pharmacology , Antimicrobial Cationic Peptides/metabolism , Antimicrobial Cationic Peptides/pharmacology , Ascomycota/drug effects , Ascomycota/growth & development , Disease Resistance/drug effects , Gene Expression/drug effects , Gene Expression Regulation, Plant/drug effects , Genetic Vectors/genetics , Hordeum/drug effects , Hordeum/metabolism , Ipomoea batatas/drug effects , Ipomoea batatas/microbiology , Microbial Sensitivity Tests , Plant Diseases/immunology , Plant Leaves/drug effects , Plant Leaves/genetics , Plant Proteins/metabolism , Plant Proteins/pharmacology , Plant Roots/drug effects , Plant Roots/genetics , Plants, Genetically Modified , Plasmids/genetics , Promoter Regions, Genetic/genetics , Transformation, Genetic/drug effects , beta-Amylase/genetics
8.
Appl Environ Microbiol ; 75(17): 5536-43, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19592534

ABSTRACT

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter(-1)) rather than GGOH (0.2 mg liter(-1)) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter(-1) GGOH and 6.5 mg liter(-1) squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter(-1) GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.


Subject(s)
Biosynthetic Pathways/genetics , Diterpenes/metabolism , Genetic Engineering/methods , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Farnesyltranstransferase/genetics , Farnesyltranstransferase/metabolism , Geranyltranstransferase/genetics , Geranyltranstransferase/metabolism , Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent/genetics , Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Biological , Pyrophosphatases/genetics , Pyrophosphatases/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Squalene/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...