RESUMO
Agrobacterium tumefaciens causes crown gall disease in plants by the horizontal transfer of oncogenic DNA. The conjugation is mediated by the VirB/D4 type 4 secretion system (T4SS) that assembles an extracellular filament, the T-pilus, and is involved in mating pair formation between A. tumefaciens and the recipient plant cell. Here, we present a 3 Å cryoelectron microscopy (cryo-EM) structure of the T-pilus solved by helical reconstruction. Our structure reveals that the T-pilus is a stoichiometric assembly of the VirB2 major pilin and phosphatidylglycerol (PG) phospholipid with 5-start helical symmetry. We show that PG head groups and the positively charged Arg 91 residues of VirB2 protomers form extensive electrostatic interactions in the lumen of the T-pilus. Mutagenesis of Arg 91 abolished pilus formation. While our T-pilus structure is architecturally similar to previously published conjugative pili structures, the T-pilus lumen is narrower and positively charged, raising questions of whether the T-pilus is a conduit for ssDNA transfer.
Assuntos
Agrobacterium tumefaciens , Proteínas de Bactérias , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Sistemas de Secreção Tipo IV , Microscopia Crioeletrônica , Proteínas de Fímbrias , Fímbrias Bacterianas , Fatores de VirulênciaRESUMO
GIGANTEA (GI) is a plant-specific nuclear protein that plays a pleiotropic role in the growth and development of plants. GI's involvement in circadian clock function, flowering time regulation, and various types of abiotic stress tolerance has been well documented in recent years. Here, the role of GI in response to Fusarium oxysporum (F. oxysporum) infection is investigated at the molecular level comparing Col-0 WT with the gi-100 mutant in Arabidopsis thaliana. Disease progression, photosynthetic parameters, and comparative anatomy confirmed that the spread and damage caused by pathogen infection were less severe in gi-100 than in Col-0 WT plants. F. oxysporum infection induces a remarkable accumulation of GI protein. Our report showed that it is not involved in flowering time regulation during F. oxysporum infection. Estimation of defense hormone after infection showed that jasmonic acid (JA) level is higher and salicylic acid (SA) level is lower in gi-100 compared to Col-0 WT. Here, we show that the relative transcript expression of CORONATINE INSENSITIVE1 (COI1) and PLANT DEFENSIN1.2 (PDF1.2) as a marker of the JA pathway is significantly higher while ISOCHORISMATE SYNTHASE1 (ICS1) and NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), the markers of the SA pathway, are downregulated in the gi-100 mutants compared to Col-0 plants. The present study convincingly suggests that the GI module promotes susceptibility to F. oxysporum infection by inducing the SA pathway and inhibiting JA signaling in A. thaliana.
RESUMO
Vigna mungo (L.) Hepper commonly known as blackgram is an important legume crop with good quality dietary proteins and vitamins. Low production of blackgram in the chromium rich soil of Odisha is a serious concern against its demand. Chromium (VI) was tested on V. mungo var. B3-8-8 at 100, 150, 200, 250 and 300 µM concentration on growth, anti-oxidative enzymes and chromium content at 15, 30 and 45 d of treatments. Seed germination and growth decreased with increase dose and duration. Cr uptake induced oxidative burst with significant increase of osmolytes was observed in cell at lower doses but failed to adjust homeostasis at higher dose. Increase of GPX and SOD and decrease of CAT was observed as dose dependent. Increased protein content was detected in < 200 µM Cr concentration whereas, significant decrease of protein was noted thereafter. Down regulation of proteins (29.2 kDa and 32.6 kDa) was observed at > 250 µM of Cr. Total Cr uptake was greater in root than in shoot which might be due to poor translocation of heavy metal or detoxification. Thus, blackgram was able to maintain homeostasis at lower concentrations of Cr by activating the cascade of enzymes following cellular detoxification mechanism. SUPPLEMENTARY INFORMATION: The online version of this article contains supplementary material available at (10.1007/s12298-021-00941-3).
RESUMO
Agrobacterium tumefaciens transfers DNA and proteins to a plant cell inciting crown gall tumor disease on most plants. VirD4 targets the DNA and protein substrates to a type IV secretion (T4S) apparatus for translocation into the plant cell. Several bacteria with VirD4 homologs use T4S for intercellular export of microbial macromolecules to eukaryotic and prokaryotic hosts. How the VirD4 proteins recognize the diverse substrates is not well understood. To identify functional domains of A. tumefaciens pTiA6 VirD4, we introduced random 19-codon and targeted 10-codon insertions throughout the coding region. Analysis of 21 mutants showed that only the carboxy-terminal end of VirD4 is tolerant of an insertion. Sequence comparison of VirD4 proteins of Agrobacterium spp. and their close relative, Rhizobium etli, showed that these proteins contain a highly conserved C-terminal end, but the immediate upstream regions share no discernible sequence similarity. The conserved region sequence is rich in the amino acid glutamine (6/13 Q). Using site-specific and deletion mutagenesis, we demonstrated that the conserved Q-rich region is required for VirD4 function and for the specific recognition of VirD2-linked T-strand DNA as a substrate for translocation to plants. The Q-rich region is not required for the transfer of a second A. tumefaciens substrate, VirE2, to plants or a promiscuous Escherichia coli IncQ plasmid to another A. tumefaciens strain. We identified Q-rich sequences at or near the C terminus of several VirD4 homologs, including the E. coli F plasmid TraD. In F TraD, the Q-rich sequence maps to a region required specifically for the conjugative transfer of the F plasmid.
Assuntos
Agrobacterium tumefaciens , Proteínas de Bactérias , Células Vegetais , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Células Vegetais/microbiologia , Plasmídeos/genética , Domínios ProteicosRESUMO
Finger millet (Eleusine coracana L. Gaertn., 2n=36) is one of the most important minor crops, commonly known as 'ragi' and used as a staple food grain in more than 25 countries including Africa and south Asia. Twenty-seven accessions of ragi were collected from different parts of India and were evaluated for morpho-genetic diversity studies. Simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) markers were used for assessment of genetic diversity among 27 genotypes of E. coracana. High degree of similarity (90%) was obtained between 'IC49979A' and 'IC49974B' genotypes, whereas low level of similarity (9.09%) was found between 'IC204141' and 'IC49985' as evident in morphological and DNA markers. A total of 64 SSR and 301 RAPD amplicons were produced, out of which 87.50% and 77.20% DNA fragments showed polymorphism, respectively. The clustering pattern obtained among the genotypes corresponded well with their morphological and cytological data with a monophyletic origin of this species which was further supported by high bootstrap values and principal component analysis. Cluster analysis showed that ragi accessions were categorised into three distinct groups. Genotypes IC344761, IC340116, IC340127, IC49965 and IC49985 found accession specific in RAPD and SSR markers. The variation among ragi accessions might be used as potential source of germplasm for crop improvement.
Assuntos
DNA de Plantas/genética , Eleusine/anatomia & histologia , Variação Genética , Eleusine/classificação , Eleusine/genética , Marcadores Genéticos , Genótipo , Índia , Repetições de Microssatélites , Filogenia , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
Agrobacterium tumefaciens transfers DNA and proteins to a plant cell through a type IV secretion apparatus assembled by the VirB proteins. All VirB proteins localized to a cell pole, although these conclusions are in dispute. To study subcellular location of the VirB proteins and to identify determinants of their subcellular location, we tagged two proteins, VirB4 and VirB11, with the visual marker green fluorescent protein (GFP) and studied localization of the fusion proteins by epifluorescence microscopy. Both GFP-VirB4 and GFP-VirB11 fusions localized to a single cell pole. GFP-VirB11 was also functional in DNA transfer. To identify the polar localization domains (PLDs) of VirB4 and VirB11, we analyzed fusions of GFP with smaller segments of the two proteins. Two noncontiguous regions in VirB4, residues 236-470 and 592-789, contain PLDs. The VirB11 PLD mapped to a 69 amino acid segment, residues 149-217, in the central region of the protein. These domains are probably involved in interactions that target the two proteins to a cell pole.
Assuntos
Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Polaridade Celular , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/citologia , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Prasanna K. Mohanty, a great scientist, a great teacher and above all a great human being, left us more than a year ago (on March 9, 2013). He was a pioneer in the field of photosynthesis research; his contributions are many and wide-ranging. In the words of Jack Myers, he would be a "photosynthetiker" par excellence. He remained deeply engaged with research almost to the end of his life; we believe that generations of researchers still to come will benefit from his thorough and enormous work. We present here his life and some of his contributions to the field of Photosynthesis Research. The response to this tribute was overwhelming and we have included most of the tributes, which we received from all over the world. Prasanna Mohanty was a pioneer in the field of "Light Regulation of Photosynthesis", a loving and dedicated teacher-unpretentious, idealistic, and an honest human being.
Assuntos
Botânica/história , Fotossíntese , História do Século XX , História do Século XXIRESUMO
Eleven proteins of the Agrobacterium tumefaciens virB operon are required for type IV secretion. All octopine Ti-plasmid pTiA6NC VirB proteins, except VirB8, could be expressed from a cloned monocistronic gene. Accumulation of VirB8 required translation of the upstream virB7 gene. Analysis of chimeric virB8 genes and a newly constructed virB7 deletion mutant Agrobacterium AD1275 showed that translation of virB7, and not the gene product, is required for VirB8 accumulation. Agrobacterium AD1275 accumulated VirB8 and other downstream virB gene products, and could be complemented with only virB7 in trans. In monocistronic virB8, sequences upstream of the virB8 ORF negatively controls virB8 expression possibly through the formation of a secondary structure that occludes both the ribosome binding site and translation start codon. Disruption of the structure through translation of the upstream gene ensures efficient translation of the virB8 mRNA in wild type bacteria. The pTiA6NC virB8 contains two potential translation start sites within the first eight codons. We show that the first AUG is used for virB8 translation initiation. The seven N-terminal residues resulting from translation initiation at the first AUG are required for both tumor formation and stabilization of VirB3. VirB8 and VirB4 are sufficient for the stabilization of VirB3, and VirB7 stabilizes VirB3 indirectly through its effect on virB8 expression.
Assuntos
Agrobacterium tumefaciens/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Plasmídeos Indutores de Tumores em Plantas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Códon de Iniciação/genética , Teste de Complementação Genética , Conformação de Ácido Nucleico , Óperon , Biossíntese de Proteínas , Estabilidade Proteica , RNA Mensageiro/genética , Deleção de SequênciaRESUMO
Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus and a T-pilus for secretion of DNA and proteins into plant cells. The pilin-like protein VirB3, a membrane protein of unknown topology, is required for the assembly of the T-pilus and for T-DNA secretion. Using PhoA and green fluorescent protein (GFP) as periplasmic and cytoplasmic reporters, respectively, we demonstrate that VirB3 contains two membrane-spanning domains and that both the N and C termini of the protein reside in the cytoplasm. Fusion proteins with GFP at the N or C terminus of VirB3 were fluorescent and, like VirB3, localized to a cell pole. Biochemical fractionation studies demonstrated that VirB3 proteins encoded by three Ti plasmids, the octopine Ti plasmid pTiA6NC, the supervirulent plasmid pTiBo542, and the nopaline Ti plasmid pTiC58, are inner membrane proteins and that VirB4 has no effect on membrane localization of pTiA6NC-encoded VirB3 (pTiA6NC VirB3). The pTiA6NC and pTiBo542 VirB2 pilins, like VirB3, localized to the inner membrane. The pTiC58 VirB4 protein was earlier found to be essential for stabilization of VirB3. Stabilization of pTiA6NC VirB3 requires not only VirB4 but also two additional VirB proteins, VirB7 and VirB8. A binary interaction between VirB3 and VirB4/VirB7/VirB8 is not sufficient for VirB3 stabilization. We hypothesize that bacteria use selective proteolysis as a mechanism to prevent assembly of unproductive precursor complexes under conditions that do not favor assembly of large macromolecular structures.
Assuntos
Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/patogenicidade , Proteínas de Bactérias/genética , Kalanchoe/microbiologia , Proteínas de Membrana/genética , Microscopia de Fluorescência , Plasmídeos/genética , Virulência/genética , Virulência/fisiologiaRESUMO
One-year-old seedlings of Bruguiera gymnorrhiza (L) Savingay were exposed to 500 mM NaCl for 6d under hydroponic culture condition to characterize the changes in leaf and thylakoid protein profiles in response to short-term salt exposures. Significant changes in leaf dry mass, chlorophylls and soluble leaf proteins were observed in short term of salt exposures, as it happens under tidal situations in nature. Chlorophyll a/b ratio showed decrease of light harvesting efficiency in salt treatment. Total soluble proteins in leaves were extracted from control and NaCl-treated plants at 2d intervals and were analyzed by SDS-PAGE. Intensity of several protein bands of different molecular mass of leaf protein profile ranging from 10 to 86 kDa (10, 16, 23, 33, 37, 42, 44, 50 and 86 kDa) were decreased due to high salt treatment. Out of these, 16, 23 and 33 kDa protein bands decreased dramatically from 1-3 fold but recovered in 7d growth, except the 33 kDa band. SDSPAGE profile of thylakoid protein revealed that both number and the intensity of several protein bands got altered by salt concentration. However, 33 kDa protein band of thylakoid reappeared in recovery that might not be of the same characteristics with same molecular mass as shown in total leaf protein profile. The numbers of major bands found in SDS-PAGE were reduced when analyzed in urea-SDS-PAGE to minimize protein aggregations by high salt. It was noted that 47 kDa disappeared while some proteins of apparent molecular mass like 23 kDa, 33 kDa, 37 kDa and 50 kDa degraded to minor bands. Partial restoration of protein bands occurred when the salt-treated plants were brought back to initial growth condition. These results clearly demonstrate that short term high salt concentration could cause major alterations to photosynthetic apparatus of a true non salt-secreting tree mangrove Bruguiera gymnorrhiza and adapted against fluctuation of salinity by altering leaf protein pool relatively more than the thylakoid proteins.
RESUMO
Analysis of karyotype, nuclear DNA content and RAPD markers were performed in four species of Bruguiera (Rhizophoraceae) of Bhitarkanika mangrove forests, Orissa, India. Detailed karyotype analysis revealing 2n=34 in B. cylindrica and 2n=36 in B. gymnorrhiza was reported for the first time and 2n=34 in B. parviflora and B. sexangula was confirmed. On the basis of the common types of chromosomes present among Bruguiera, two distinct groups were found; one consists of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula. The symmetrical karyotype with same chromosome types grouped B. cylindrica and B. parviflora together and presence of Type E chromosomes placed B. gymnorrhiza and B. sexangula in a separate group, suggesting their closer affinity in their respective group. Analysis of chromosome length, volume, INV and 4C DNA content confirmed this division. Nuclear DNA content was two-fold higher (~17.0 pg) in the second group than in the first (~8.0 pg). The amplification products generated through RAPD revealed 1-9 amplicons with size variations from 600 bp to 2 500 bp with 49.31% genetic similarity between B. gymnorrhiza and B. sexangula and 47.10% in between B. cylindrica and B. parviflora. The high copy number marker band (~ 1 100 bp) yielded in OPN-15 primer in B. parviflora the characteristic DNA marker, which was cloned and used as probes for assessment of genetic diversity, and demonstrated its close genetic affinity to B. cylindrica. B. gymnorrhiza and B. sexangula also produced similar marker bands of ~600 bp and ~2 200 bp in the same primer. All of the cytological, 4C DNA content and RAPD data confirmed the existence of two taxonomically distinct groups of Bruguiera: one consisting of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula as placed earlier (1862) in the tribe Rhizophoreae by Bentham and Hooker, on the basis of the flowering habits of Bruguiera. Genetically, the B. sexangula and B. gymnorrhiza group was found to be very closely, rather than distantly, related to B. parviflora and B. cylindrica. Our results demonstrate that molecular markers together with cytological evidence provide an effective tool to access the existing interspecific genetic polymorphism in mangrove species, to solve the taxonomic problems and to design their conservation strategy. Rev. Biol. Trop. 55 (2): 437-448. Epub 2007 June, 29.
Estudiamos cuatro especies del mangle Bruguiera (Rhizophoraceae) en Orissa, India. Los cromosomas indican queB. cylindrica y B. parviflora son un grupo taxonómico, y que B. gymnorrhiza y B. sexangula son otro. Genéticamente, el par B. sexangula y B. gymnorrhiza está cercanamente emparentado con B. parviflora and B. cylindrica. Nuestros datos indican que el uso combinado de marcadores genéticos y evidencia citológica permiten discernir el polimorfismo genético interespecífico en los mangles, tanto para resolver problemas taxonómicos como para diseña estrategias eficaces de conservación.
Assuntos
Cromossomos de Plantas/genética , DNA de Plantas/análise , Filogenia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Rhizophoraceae/genética , Núcleo Celular/genética , Marcadores Genéticos , Cariotipagem , Rhizophoraceae/classificação , Especificidade da Espécie , Árvores/classificação , Árvores/genéticaRESUMO
Analysis of karyotype, nuclear DNA content and RAPD markers were performed in four species of Bruguiera (Rhizophoraceae) of Bhitarkanika mangrove forests, Orissa, India. Detailed karyotype analysis revealing 2n=34 in B. cylindrica and 2n=36 in B. gymnorrhiza was reported for the first time and 2n=34 in B. parviflora and B. sexangula was confirmed. On the basis of the common types of chromosomes present among Bruguiera, two distinct groups were found; one consists of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula. The symmetrical karyotype with same chromosome types grouped B. cylindrica and B. parviflora together and presence of Type E chromosomes placed B. gymnorrhiza and B. sexangula in a separate group, suggesting their closer affinity in their respective group. Analysis of chromosome length, volume, INV and 4C DNA content confirmed this division. Nuclear DNA content was two-fold higher (approximately 17.0 pg) in the second group than in the first (approximately 8.0 pg). The amplification products generated through RAPD revealed 1-9 amplicons with size variations from 600 bp to 2 500 bp with 49.31% genetic similarity between B. gymnorrhiza and B. sexangula and 47.10% in between B. cylindrica and B. parviflora. The high copy number marker band (approximately 1,100 bp) yielded in OPN-15 primer in B. parviflora the characteristic DNA marker, which was cloned and used as probes for assessment of genetic diversity, and demonstrated its close genetic affinity to B. cylindrica. B. gymnorrhiza and B. sexangula also produced similar marker bands of approximately 600 bp and approximately 2,200 bp in the same primer. All of the cytological, 4C DNA content and RAPD data confirmed the existence of two taxonomically distinct groups of Bruguiera: one consisting of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula as placed earlier (1862) in the tribe Rhizophoreae by Bentham and Hooker, on the basis of the flowering habits ofBruguiera. Genetically, the B. sexangula and B. gymnorrhiza group was found to be very closely, rather than distantly, related to B. parviflora and B. cylindrica. Our results demonstrate that molecular markers together with cytological evidence provide an effective tool to access the existing interspecific genetic polymorphism in mangrove species, to solve the taxonomic problems and to design their conservation strategy.
Assuntos
Cromossomos de Plantas/genética , DNA de Plantas/análise , Filogenia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Rhizophoraceae/genética , Núcleo Celular/genética , Marcadores Genéticos , Cariotipagem , Rhizophoraceae/classificação , Especificidade da Espécie , Árvores/classificação , Árvores/genéticaRESUMO
The VirB proteins of Agrobacterium tumefaciens assemble a T-pilus and a type IV secretion (T4S) apparatus for the transfer of DNA and proteins to plant cells. VirB6 is essential for DNA transfer and is a polytopic integral membrane protein with at least four membrane-spanning domains. VirB6 is postulated to function in T-pilus biogenesis and to be a component of the T4S apparatus. To identify amino acids required for VirB6 function, random mutations were introduced into virB6, and mutants that failed to complement a deletion in virB6 in tumour formation assays were isolated. Twenty-one non-functional mutants were identified, eleven of which had a point mutation that led to a substitution in a single amino acid. Characterization of the mutants indicated that the N-terminal large periplasmic domain and the transmembrane domain TM3 are required for VirB6 function. TM3 has an unusual sequence feature in that it is rich in bulky hydrophobic amino acids. This feature is found conserved in the VirB6 family of proteins. Studies on the effect of VirB6 on other VirB proteins showed that the octopine Ti-plasmid VirB6, unlike its nopaline Ti-plasmid counterpart, does not affect accumulation of VirB3 and VirB5, but has a strong negative effect on the accumulation of the VirB7-VirB7 dimer. Using indirect immunofluorescence microscopy the authors recently demonstrated that VirB6 localizes to a cell pole in a VirB-dependent manner. Mutations identified in the present study did not affect polar localization of the protein or the formation of the VirB7-VirB7 dimer. A VirB6-GFP fusion that contained the entire VirB6 ORF did not localize to a cell pole in either the presence or the absence of the other VirB proteins. IMF studies using dual labelling demonstrated that VirB6 colocalizes with VirB3 and VirB9, and not with VirB4, VirB5 and VirB11. These results support the conclusion that VirB6 is a structural component of the T4S apparatus.
Assuntos
Agrobacterium tumefaciens/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Kalanchoe/microbiologia , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutação , Doenças das Plantas/microbiologia , Alinhamento de Sequência , Análise de Sequência de DNA , VirulênciaRESUMO
Type IV secretion is used by pathogenic microorganisms to transfer effector macromolecules to eukaryotic target cells. The VirB/D4 apparatus of Agrobacterium tumefaciens transfers DNA and proteins to plant cells. We postulated that the cell pole is the site of assembly of the A. tumefaciens type IV apparatus. Using immunofluorescence microscopy, we now demonstrate that 10 of the VirB proteins localized primarily to one cell pole and a macromolecular VirB complex is assembled at the pole. Neither the assembly of the complex nor polar localization of a VirB protein requires ATP utilization by the VirB ATPases. The requirement of other VirB proteins for the polar localization of at least six VirB proteins indicates an essential role of protein-protein interaction in polar targeting. Four proteins (VirB3, VirB4, VirB8, and VirB11) could target themselves to a cell pole independent of a VirB protein. We provide evidence that VirB6-VirB10 are the structural components of the type IV apparatus. Using strains that express defined subsets of the virB genes, we demonstrate that VirB7-VirB10 are the minimum components sufficient for the assembly of a polar VirB complex. VirB6 associates with this complex to form the type IV secretion apparatus. VirB8 functions as the assembly factor and targets the apparatus to the cell pole.
Assuntos
Agrobacterium tumefaciens/citologia , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Complexos Multiproteicos/biossíntese , Agrobacterium tumefaciens/fisiologia , Proteínas de Bactérias/fisiologia , Transporte Biológico/fisiologia , Microscopia de FluorescênciaRESUMO
Plants exposed to salt stress undergo changes in their environment. The ability of plants to tolerate salt is determined by multiple biochemical pathways that facilitate retention and/or acquisition of water, protect chloroplast functions, and maintain ion homeostasis. Essential pathways include those that lead to synthesis of osmotically active metabolites, specific proteins, and certain free radical scavenging enzymes that control ion and water flux and support scavenging of oxygen radicals or chaperones. The ability of plants to detoxify radicals under conditions of salt stress is probably the most critical requirement. Many salt-tolerant species accumulate methylated metabolites, which play crucial dual roles as osmoprotectants and as radical scavengers. Their synthesis is correlated with stress-induced enhancement of photorespiration. In this paper, plant responses to salinity stress are reviewed with emphasis on physiological, biochemical, and molecular mechanisms of salt tolerance. This review may help in interdisciplinary studies to assess the ecological significance of salt stress.
Assuntos
Adaptação Fisiológica , Fenômenos Fisiológicos Vegetais , Cloreto de Sódio , Fenômenos Fisiológicos Vegetais/efeitos dos fármacos , Cloreto de Sódio/farmacologiaRESUMO
Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus for the transfer of DNA and proteins to plant cells. To study the role of the VirB6 protein in the assembly and function of the type IV apparatus, we determined its subcellular location by immunofluorescence microscopy. In wild-type bacteria VirB6 localized to the cell poles but in the absence of the tumour-inducing plasmid it localized to random sites on the cell membranes. Five of the 11 VirB proteins, VirB7-VirB11, are required for the polar localization of VirB6. We identified two regions of VirB6, a conserved tryptophan residue at position 197 and the extreme C-terminus, that are essential for its polar localization. Topology determination by PhoA fusion analysis placed both regions in the cell cytoplasm. Alteration of tryptophan 197 or the deletion of the extreme C-terminus led to the mislocalization of the mutant protein. The mutations abolished the DNA transfer function of the protein as well. The C-terminus of VirB6, in silico, can form an amphipathic helix that may encode a protein-protein interaction domain essential for targeting the protein to a cell pole. We previously reported that another DNA transfer protein, VirD4, localizes to a cell pole. To determine whether VirB6 and VirD4 localize to the same pole, we performed colocalization experiments. Both proteins localized to the same pole indicating that VirB6 and VirD4 are in close proximity and VirB6 is probably a component of the transport apparatus.
Assuntos
Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Sinais Direcionadores de Proteínas/genética , Agrobacterium tumefaciens/genética , Fosfatase Alcalina , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Polaridade Celular , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Citoplasma/metabolismo , DNA Bacteriano/metabolismo , Microscopia de Fluorescência , Mutação , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A significant decrease in the amount of a protein, whose migration in two-dimensional gel electrophoresis corresponds to an apparent molecular mass of 23 kDa and pI = 6.5, was observed in leaves of NaCl-treated Bruguiera parviflora (Roxb.) Wt. & Arn. ex Griff. seedlings. This particular salt-sensitive protein, designated as SSP-23, almost disappeared after 45 days of treatment in 400 mM NaCl as compared to untreated seedlings (0 mM NaCl) where the presence of the protein was significant. A polyclonal antibody raised against the 23-kDa protein was used to determine the subcellular localization of this protein in leaves by cross-reaction with proteins from isolated chloroplasts, mitochondria, peroxisomes and cytosol fractions on Western blots. SSP-23 was confirmed to be localized in the cytosol by immunoblotting. The disappearance of SSP-23 as a result of high NaCl treatment suggests that this protein is salt-sensitive and has a possible role in salt adaptation.
Assuntos
Proteínas de Plantas/metabolismo , Rhizophoraceae/metabolismo , Cloreto de Sódio/farmacologia , Western Blotting , Cloroplastos/química , Cloroplastos/metabolismo , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Concentração Osmolar , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Proteínas de Plantas/isolamento & purificação , Rhizophoraceae/efeitos dos fármacosRESUMO
The influence of varying levels of salinity (0, 100, 200 and 400 mM) on the activities of nitrate reductase (NR, E.C. 1.6.6.1), acid phosphatase (ACP, E.C. 3.1.3.2), and alkaline phosphatase (ALP, EC 3.1.3.1 ) as well as on nitrate and phosphate uptake and total nitrogen levels in leaves of a true mangrove Bruguiera parviflora was investigated under hydroponic culture conditions. NR activity increased in 100mM NaCl treated plants, whereas it decreased gradually in 200 and 400 mM treated plants, relative to the controls. Decreased activity of NR by NaCl stress was also accompanied by a decrease in total nitrogen level and nitrate uptake. Decreases in NR activity, nitrate (NO3-), and total nitrogen level due to high salinity may be responsible for a decrease in growth and biomass production in this plant. However, salinity caused an increase in both ACP and ALP activity. Activity staining of ACP by native polyacrylamide gel electrophoresis revealed three isoforms: ACP-1, ACP-2, and ACP-3. We observed a preferential enhancement in the ACP-3 isoform by salinity. In order to understand whether the salinity-induced increase in phosphatase activity was due to inhibition in phosphate uptake, we monitored phosphate (Pi) levels in leaves and noted that phosphate levels decreased significantly under salinity. These results suggest that the induction of acid and ALP under salt stress may be due to a phosphorous deficiency.