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1.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360759

RESUMO

Salt and osmotic stress are the main abiotic stress factors affecting plant root growth and architecture. We investigated the effect of salt (100 mM NaCl) and osmotic (200 mM mannitol) stress on the auxin metabolome by UHPLC-MS/MS, auxin distribution by confocal microscopy, and transcript levels of selected genes by qRT-PCR in Arabidopsis thaliana ecotype Columbia-0 (Col-0) and DR5rev::GFP (DR5) line. During long-term stress (13 days), a stability of the auxin metabolome and a tendency to increase indole-3-acetic acid (IAA) were observed, especially during salt stress. Short-term stress (3 h) caused significant changes in the auxin metabolome, especially NaCl treatment resulted in a significant reduction of IAA. The data derived from auxin profiling were consistent with gene expressions showing the most striking changes in the transcripts of YUC, GH3, and UGT transcripts, suggesting disruption of auxin biosynthesis, but especially in the processes of amide and ester conjugation. These data were consistent with the auxin distribution observed in the DR5 line. Moreover, NaCl treatment caused a redistribution of auxin signals from the quiescent center and the inner layers of the root cap to the epidermal and cortical cells of the root elongation zone. The distribution of PIN proteins was also disrupted by salt stress; in particular, PIN2 was suppressed, even after 5 min of treatment. Based on our results, the DR5 line was more sensitive to the applied stresses than Col-0, although both lines showed similar trends in root morphology, as well as transcriptome and metabolome parameters under stress conditions.


Assuntos
Proteínas de Arabidopsis/biossíntese , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Estresse Salino/efeitos dos fármacos , Cloreto de Sódio/farmacologia
2.
Plant Mol Biol ; 102(4-5): 359-372, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31848919

RESUMO

KEY MESSAGE: Protein degradation is essential in plant growth and development. The stability of Cullin3 substrate adaptor protein BPM1 is regulated by multiple environmental cues pointing on manifold control of targeted protein degradation. A small family of six MATH-BTB genes (BPM1-6) is described in Arabidopsis thaliana. BPM proteins are part of the Cullin E3 ubiquitin ligase complexes and are known to bind at least three families of transcription factors: ERF/AP2 class I, homeobox-leucine zipper and R2R3 MYB. By targeting these transcription factors for ubiquitination and subsequent proteasomal degradation, BPMs play an important role in plant flowering, seed development and abiotic stress response. In this study, we generated BPM1-overexpressing plants that showed an early flowering phenotype, resistance to abscisic acid and tolerance to osmotic stress. We analyzed BPM1-GFP protein stability and found that the protein has a high turnover rate and is degraded by the proteasome 26S in a Cullin-dependent manner. Finally, we found that BPM1 protein stability is environmentally conditioned. Darkness and salt stress triggered BPM1 degradation, whereas elevated temperature enhanced BPM1 stability and accumulation in planta.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Flores/fisiologia , Estresse Fisiológico , Fatores de Transcrição/fisiologia , Ácido Abscísico , Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Plasmídeos/genética , Pólen/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteólise , Sementes/fisiologia , Ubiquitina-Proteína Ligases/fisiologia
3.
Front Plant Sci ; 10: 1469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824527

RESUMO

MATH-BTB proteins are known to act as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. Their BTB domain binds to CUL3 scaffold proteins and the less conserved MATH domain targets a highly diverse collection of substrate proteins to promote their ubiquitination and subsequent degradation. In plants, a significant expansion of the MATH-BTB family occurred in the grasses. Here, we report analysis of TaMAB2, a MATH-BTB protein transiently expressed at the onset of embryogenesis in wheat. Due to difficulties in studying its role in zygotes and early embryos, we have overexpressed TaMAB2 in Arabidopsis to generate gain-of-function mutants and to elucidate interaction partners and substrates. Overexpression plants showed severe growth defects as well as disorganization of microtubule bundles indicating that TaMAB2 interacts with substrates in Arabidopsis. In tobacco BY-2 cells, TaMAB2 showed a microtubule and ubiquitin-associated cytoplasmic localization pattern in form of foci. Its direct interaction with CUL3 suggests functions in targeting specific substrates for ubiquitin-dependent degradation. Although direct interactions with tubulin could not be confimed, tandem affinity purification of TaMAB2 interactors point towards cytoskeletal proteins including tubulin and actin as well as the translation initiation machinery. The idenification of various subunits of eucaryotic translation initiation factors eIF3 and eIF4 as TaMAB2 interactors indicate regulation of translation initiation as a major function during onset of embryogenesis in plants.

4.
Food Technol Biotechnol ; 53(1): 3-10, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27904326

RESUMO

Rosmarinic acid is one of the main active components of Coleus blumei and is known to have numerous health benefits. The pharmacological significance of rosmarinic acid and its production through in vitro culture has been the subject of numerous studies. Here, the ability of different tissues to accumulate rosmarinic acid and sustainability in production over long cultivation have been tested. Calli, tumours, normal roots and hairy roots were established routinely by application of plant growth regulators or by transformation with agrobacteria. The differences among the established tumour lines were highly heterogeneous. Hairy root lines showed the highest mean growth rate and consistency in rosmarinic acid production. Although some tumour lines produced more rosmarinic acid than the hairy root lines, over a long cultivation period their productivity was unstable and decreased. Further, the effects of plant growth regulators on growth and rosmarinic acid accumulation were tested. 2,4-Dichlorophenoxyacetic acid significantly reduced tumour growth and rosmarinic acid production. 1-Naphthaleneacetic acid strongly stimulated hairy root growth whilst abscisic acid strongly enhanced rosmarinic acid production. Hairy roots cultured in an airlift bioreactor exhibited the highest potential for mass production of rosmarinic acid.

5.
Plant Sci ; 199-200: 18-28, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23265315

RESUMO

The accumulation of phenolic compounds in plants is often part of the defense response against stress and pathogen attack, which can be triggered and activated by elicitors. Oomycetal proteinaceous elicitor, ß-cryptogein, induces hypersensitive response and systemic acquired resistance against some pathogens. In order to test the effect of endogenously synthesized cryptogein protein on phenolic compounds accumulation in tissue, and secretion into the culture medium, Coleus blumei hairy roots were generated. Agrobacterium rhizogenes was employed to insert synthetic crypt gene, encoding ß-cryptogein, under the control of alcohol-inducible promoter. The expression of ß-cryptogein, in C. blumei hairy roots, was controlled by application of 1% and 2% ethanol, during 21 days induction period. Ethanol-induced expression of ß-cryptogein caused significant decrease of soluble phenolics and rosmarinic acid (RA) in hairy root lines and increase of phenolics, RA and caffeic acid in culture medium. These data suggest that ß-cryptogein might be a potential regulatory factor for phenolics secretion from the roots.


Assuntos
Proteínas de Algas/genética , Coleus/metabolismo , Oomicetos/genética , Fenóis/metabolismo , Raízes de Plantas/metabolismo , Proteínas de Algas/efeitos dos fármacos , Proteínas de Algas/metabolismo , Transporte Biológico , Ácidos Cafeicos/metabolismo , Cromatografia Líquida de Alta Pressão , Cinamatos/metabolismo , Coleus/efeitos dos fármacos , Coleus/genética , DNA de Plantas/genética , Depsídeos/metabolismo , Etanol/farmacologia , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA de Plantas/genética , Transgenes
6.
PLoS One ; 7(12): e51184, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23251450

RESUMO

BPM1 belongs to the MATH-BTB family of proteins, which act as substrate-binding adaptors for the Cullin3-based E3 ubiquitin ligase. MATH-BTB proteins associate with Cullin3 via the BTB domain and with the substrate protein via the MATH domain. Few BPM1-interacting proteins with different functions are recognized, however, specific roles of BPM1, depending on its cellular localization have not been studied so far. Here, we found a novel bipartite nuclear localization signal at the C-terminus of the BPM1 protein, responsible for its nuclear and nucleolar localization and sufficient to drive the green fluorescent protein and cytoplasmic BPM4 protein into the nucleus. Co-localization analysis in live Nicotiana tabacum BY2 cells indicates a Cullin3 independent function since BPM1 localization is predominantly nucleolar and thus devoid of Cullin3. Treatment of BY2 cells with the proteasome inhibitor MG132 blocks BPM1 and Cullin3 degradation, suggesting turnover of both proteins through the ubiquitin-proteasome pathway. Possible roles of BPM1 in relation to its in vivo localization are discussed.


Assuntos
Sinais de Localização Nuclear , Proteínas de Plantas/metabolismo , Tabaco/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo
7.
J Plant Physiol ; 168(16): 1943-51, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21807439

RESUMO

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.


Assuntos
Cucurbita/metabolismo , Nitratos/farmacologia , Técnicas de Embriogênese Somática de Plantas , Compostos de Amônio Quaternário/farmacologia , Cucurbita/efeitos dos fármacos , Cucurbita/embriologia , Cucurbita/enzimologia , Esterases/metabolismo , Glucanos/metabolismo , Glutamato-Amônia Ligase/efeitos dos fármacos , Glutamato-Amônia Ligase/metabolismo , Concentração de Íons de Hidrogênio , Ácidos Indolacéticos/farmacologia , Nitratos/metabolismo , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Estresse Oxidativo , Peroxidase/metabolismo , Fenilalanina Amônia-Liase/efeitos dos fármacos , Fenilalanina Amônia-Liase/metabolismo , Estresse Fisiológico , Superóxido Dismutase/metabolismo , Fatores de Tempo
8.
Mol Genet Genomics ; 281(2): 207-21, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19052776

RESUMO

18S-5.8S-26S rDNA family comprises tandemly arranged, repeating units separated by an intergenic spacer (IGS) that contains transcription initiation/termination signals and usually repeating elements. In this study, we performed for the first time thorough sequence analysis of rDNA IGS region in two dominant European oaks, Quercus petraea and Q. robur, in order to investigate (1) if IGS sequence composition allows discrimination between these two species, and (2) if there is an rDNA length heterogeneity arising from IGS sequence. Two spacer length variants (slvs), 2 and 4 kb in length, were found in the genomes of both species. Inter-comparison of both slvs revealed no species-specificity in sequence or structural organization. Both slvs could be divided into four subregions; (1) the subrepeat region containing three repeated elements, (2) the AT-rich region containing matrix attachment sites and putative origin of replication, (3) the promoter region containing putative transcription initiation site and (4) the 5'ETS region. In the 4-kb slvs all four subregions are extended, and the subrepeat, AT-rich and promoter regions are duplicated. This is unique compared to other known IGS sequences where the variation in number of subrepeats is responsible for slvs creation. We also propose a possible evolutionary scenario to explain the formation of the subrepeat region in oak IGS. Results obtained in this work add to the previous picture of low-genetic differentiation of the two oaks and provide important data for further analyses of the function of IGS in control of rRNA gene expression.


Assuntos
DNA Ribossômico/genética , Quercus/genética , Animais , Sequência de Bases , Metilação de DNA , Primers do DNA , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie
9.
Plant Cell Rep ; 23(3): 120-7, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15221278

RESUMO

Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 m M NH(4)Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 micro M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.


Assuntos
Cucurbita/embriologia , Cucurbita/metabolismo , Metilação de DNA/efeitos dos fármacos , DNA de Plantas/metabolismo , Sementes/embriologia , Sementes/metabolismo , Ácido 2,4-Diclorofenoxiacético/farmacologia , Cloreto de Amônio/farmacologia , Azacitidina/farmacologia , Cucurbita/genética , Meios de Cultura/farmacologia , DNA de Plantas/genética , Inibidores Enzimáticos/farmacologia , Herbicidas/farmacologia , Ácidos Indolacéticos/farmacologia , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Fixação de Nitrogênio/efeitos dos fármacos , Fixação de Nitrogênio/fisiologia , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Sementes/genética
10.
J Plant Physiol ; 161(2): 229-36, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15022838

RESUMO

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.


Assuntos
Cucurbita/crescimento & desenvolvimento , Compostos de Nitrogênio/farmacologia , Técnicas de Cultura de Células/métodos , Cucurbita/citologia , Cucurbita/efeitos dos fármacos , Cucurbita/embriologia , Meios de Cultura , Concentração de Íons de Hidrogênio , Compostos de Nitrogênio/metabolismo , Zigoto
11.
Z Naturforsch C J Biosci ; 59(7-8): 554-60, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15813379

RESUMO

Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days.


Assuntos
Cinamatos/metabolismo , Coleus/metabolismo , Células Cultivadas , Coleus/citologia , Coleus/crescimento & desenvolvimento , Depsídeos , Cinética , Fatores de Tempo
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