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1.
Molecules ; 27(7)2022 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-35408716

RESUMO

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.


Assuntos
Arabidopsis , Fosfolipases A1 , Proteínas de Plantas , Arabidopsis/enzimologia , Capsicum/enzimologia , Dimerização , Ésteres , Fosfolipases A1/química , Proteínas de Plantas/química
2.
J Biol Chem ; 298(4): 101749, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35189141

RESUMO

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain-containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain-containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD-Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3-STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Enxofre , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Liases de Carbono-Enxofre , Cisteína/metabolismo , Citosol/metabolismo , Domínios Proteicos , Enxofre/metabolismo , Sulfurtransferases/metabolismo , Tiossulfato Sulfurtransferase/genética , Tiossulfato Sulfurtransferase/metabolismo
3.
J Biol Chem ; 298(4): 101762, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35202657

RESUMO

The Absence of bc1 Complex (ABC1) is an ancient, atypical protein kinase family that emerged prior to the archaeal-eubacterial divergence. Loss-of-function mutants in ABC1 genes are linked to respiratory defects in microbes and humans and to compromised photosynthetic performance and stress tolerance in plants. However, demonstration of protein kinase activity remains elusive, hampering their study. Here, we investigate a homolog from Arabidopsis thaliana, AtABC1K6, and demonstrate in vitro autophosphorylation activity, which we replicate with a human ABC1 ortholog. We also show that AtABC1K6 protein kinase activity requires an atypical buffer composition, including Mn2+ as a divalent cation cofactor and a low salt concentration. AtABC1K6 associates with plastoglobule lipid droplets of A. thaliana chloroplasts, along with five paralogs. We show that the protein kinase activity associated with isolated A. thaliana plastoglobules was inhibited at higher salt concentrations, but could accommodate Mg2+ as well as Mn2+, indicating salt sensitivity, but not the requirement for Mn2+, may be a general characteristic of ABC1 proteins. Finally, loss of functional AtABC1K6 impairs the developmental transition from vegetative to reproductive growth. This phenotype was complemented by the wild-type sequence of AtABC1K6, but not by a kinase-dead point mutant in the unique Ala-triad of the ATP-binding pocket, demonstrating the physiological relevance of the protein's kinase activity. We suggest that ABC1s are bona fide protein kinases with a unique regulatory mechanism. Our results open the door to detailed functional and mechanistic studies of ABC1 proteins and plastoglobules.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Quinases , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Mutação , Fotossíntese , Proteínas Quinases/genética , Proteínas Quinases/metabolismo
4.
Plant Sci ; 317: 111192, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35193741

RESUMO

Reactive oxygen species (ROS) play essential roles as a second messenger in various physiological processes in plants. Due to their oxidative nature, ROS can also be harmful. Thus, the generation and homeostasis of ROS are tightly controlled by multiple enzymes. Membrane-localized NADPH oxidases are well known to generate ROS during developmental and stress responses, but the metabolic pathways of the superoxide (O2-) generated by them in the apoplast are poorly understood, and the identity of the apoplastic superoxide dismutase (SOD) is unknown in Arabidopsis. Here, we show that a putative manganese SOD, MSD2 is secreted and possesses a SOD activity that can be inhibited by nitration at tyrosine 68. The expression of MSD2 in roots is light condition-dependent, suggesting that MSD2 may act on ROS metabolism in roots during the light-to-dark transition. Root architecture is governed by ROS distribution that exhibits opposite gradient of H2O2 and O2-, which is indeed altered in etiolated msd2 mutants and accompanied by changes in the onset of differentiation. These results provide a missing link in our understanding of ROS metabolism and suggest that MSD2 plays a role in root skotomorphogenesis by regulating ROS distribution, thereby playing a pivotal role in plant growth and development.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Raízes de Plantas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio , Superóxido Dismutase , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Raízes de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
5.
Int J Mol Sci ; 23(3)2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35163124

RESUMO

The two homologous genes, NIA1 and NIA2, encode nitrate reductases in Arabidopsis, which govern the reduction of nitrate to nitrite. This step is the rate-limiting step of the nitrate assimilation and utilization. Therefore, the regulation of NIA1 and NIA2 is important for plant development and growth. Although they are similar in sequence and structure, their regulations are different. Genetic analysis uncovers that NIA1, rather than NIA2, plays a predominant role in adopting to ABA stress. Although both long-term stress conditions can cause an improvement in NIA1 levels, a decrease in NIA1 levels under short-term treatments seems to be necessary for plants to switch from the growth status into the adopting status. Interestingly, the downregulation of the NR is distinct under different stress conditions. Under ABA treatment, the NR proteins are degraded via a 26S-proteasome dependent manner, while the transcriptional regulation is the main manner to rapidly reduce the NIA1 levels under nitrogen deficiency and NaCl stress conditions. These results indicate that under stress conditions, the regulation of NIA1 is complex, and it plays a key role in regulating the balance between growth and adaptation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Nitrato Redutase/metabolismo , Folhas de Planta/enzimologia , Estresse Fisiológico , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Nitrato Redutase/genética , Nitratos/metabolismo , Folhas de Planta/genética , Folhas de Planta/fisiologia
6.
BMC Plant Biol ; 22(1): 18, 2022 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-34991468

RESUMO

BACKGROUND: Nuclear endosperm development is a common mechanism among Angiosperms, including Arabidopsis. During nuclear development, the endosperm nuclei divide rapidly after fertilization without cytokinesis to enter the syncytial phase, which is then followed by the cellularized phase. The endosperm can be divided into three spatial domains with distinct functions: the micropylar, peripheral, and chalazal domains. Previously, we identified two putative small invertase inhibitors, InvINH1 and InvINH2, that are specifically expressed in the micropylar region of the syncytial endosperm. In addition, ectopically expressing InvINH1 in the cellularized endosperm led to a reduction in embryo growth rate. However, it is not clear what are the upstream regulators responsible for the specific expression of InvINHs in the syncytial endosperm. RESULTS: Using protoplast transient expression system, we discovered that a group of type I MADS box transcription factors can form dimers to activate InvINH1 promoter. Promoter deletion assays carried out in the protoplast system revealed the presence of an enhancer region in InvINH1 promoter, which contains several consensus cis-elements for the MADS box proteins. Using promoter deletion assay in planta, we further demonstrated that this enhancer region is required for InvINH1 expression in the syncytial endosperm. One of the MADS box genes, AGL62, is a key transcription factor required for syncytial endosperm development. Using promoter-GFP reporter assay, we demonstrated that InvINH1 and InvINH2 are not expressed in agl62 mutant seeds. Collectively, our data supports the role of AGL62 and other type I MADS box genes as the upstream activators of InvINHs expression in the syncytial endosperm. CONCLUSIONS: Our findings revealed several type I MADS box genes that are responsible for activating InvINH1 in the syncytial endosperm, which in turn regulates embryo growth rate during early stage of seed development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Endosperma/metabolismo , Proteínas de Domínio MADS/genética , beta-Frutofuranosidase/antagonistas & inibidores , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Endosperma/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica de Plantas , Proteínas de Domínio MADS/metabolismo , Regiões Promotoras Genéticas , Protoplastos/metabolismo , Fatores de Transcrição/metabolismo
7.
Microb Cell Fact ; 21(1): 8, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-35012528

RESUMO

BACKGROUND: Phenylpropanoids represent a diverse class of industrially important secondary metabolites, synthesized in plants from phenylalanine and tyrosine. Cyanobacteria have a great potential for sustainable production of phenylpropanoids directly from CO2, due to their photosynthetic lifestyle with a fast growth compared to plants and the ease of generating genetically engineered strains. This study focuses on photosynthetic production of the starting compounds of the phenylpropanoid pathway, trans-cinnamic acid and p-coumaric acid, in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). RESULTS: A selected set of phenylalanine ammonia lyase (PAL) enzymes from different organisms was overexpressed in Synechocystis, and the productivities of the resulting strains compared. To further improve the titer of target compounds, we evaluated the use of stronger expression cassettes for increasing PAL protein levels, as well as knock-out of the laccase gene slr1573, as this was previously reported to prevent degradation of the target compounds in the cell. Finally, to investigate the effect of growth conditions on the production of trans-cinnamic and p-coumaric acids from Synechocystis, cultivation conditions promoting rapid, high density growth were tested. Comparing the different PALs, the highest specific titer was achieved for the strain AtC, expressing PAL from Arabidopsis thaliana. A subsequent increase of protein level did not improve the productivity. Production of target compounds in strains where the slr1573 laccase had been knocked out was found to be lower compared to strains with wild type background, and the Δslr1573 strains exhibited a strong phenotype of slower growth rate and lower pigment content. Application of a high-density cultivation system for the growth of production strains allowed reaching the highest total titers of trans-cinnamic and p-coumaric acids reported so far, at around 0.8 and 0.4 g L-1, respectively, after 4 days. CONCLUSIONS: Production of trans-cinnamic acid, unlike that of p-coumaric acid, is not limited by the protein level of heterologously expressed PAL in Synechocystis. High density cultivation led to higher titres of both products, while knocking out slr1573 did not have a positive effect on production. This work contributes to capability of exploiting the primary metabolism of cyanobacteria for sustainable production of plant phenylpropanoids.


Assuntos
Cinamatos/metabolismo , Ácidos Cumáricos/metabolismo , Engenharia Metabólica , Fenilalanina Amônia-Liase/biossíntese , Fenilalanina Amônia-Liase/genética , Synechocystis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Expressão Gênica , Fenilalanina Amônia-Liase/metabolismo , Fotossíntese , Synechocystis/genética , Synechocystis/crescimento & desenvolvimento
8.
Biochem Biophys Res Commun ; 587: 113-118, 2022 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-34871998

RESUMO

Receptor-like cytoplasmic kinase (RLCK) subfamily VII members are involved in diverse biological processes, like reproduction, immunity, growth and development. Ubiquitination and proteasomal degradation of a RLCK VII member, BOTRYTIS-INDUCED KINASE1 (BIK1) play important roles in regulating immune signaling. It remains largely unknown whether most other RLCK VII members undergo ubiquitination and proteasomal degradation. Here, we select the 6-member RLCK VII-4 to examine the potential proteasomal degradation of its members. We find that three closely related RLCK VII-4 members, PBL38 (AvrPphB SUSCEPTIBLE1-LIKE38), PCRK1 (PTI-COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE1), and PCRK2 are under proteasomal control, while the other members in this group are not. Moreover, we demonstrate that PCRK2 undergoes ubiquitination and proteasomal in a kinase activity-dependent manner. However, the plasma membrane (PM) localization of PCRK2 is not required for its degradation. Our work suggests that many other RLCK VII members may undergo ubiquitination and proteasomal degradation to modulate their homeostasis and cellular functions.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Fosforilação , Folhas de Planta/enzimologia , Folhas de Planta/genética , Ligação Proteica , Proteólise , Protoplastos/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ubiquitinação
9.
Plant J ; 109(1): 196-214, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34741366

RESUMO

The importance of the alternative donation of electrons to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex has been demonstrated. However, the functional significance of this pathway during seed development and germination remains to be elucidated. To assess the function of this pathway, we performed a detailed metabolic and transcriptomic analysis of Arabidopsis mutants to test the molecular consequences of a dysfunctional ETF/ETFQO pathway. We demonstrate that the disruption of this pathway compromises seed germination in the absence of an external carbon source and also impacts seed size and yield. Total protein and storage protein content is reduced in dry seeds, whilst sucrose levels remain invariant. Seeds of ETFQO and related mutants were also characterized by an altered fatty acid composition. During seed development, lower levels of fatty acids and proteins accumulated in the etfqo-1 mutant as well as in mutants in the alternative electron donors isovaleryl-CoA dehydrogenase (ivdh-1) and d-2-hydroxyglutarate dehydrogenase (d2hgdh1-2). Furthermore, the content of several amino acids was increased in etfqo-1 mutants during seed development, indicating that these mutants are not using such amino acids as alternative energy source for respiration. Transcriptome analysis revealed alterations in the expression levels of several genes involved in energy and hormonal metabolism. Our findings demonstrated that the alternative pathway of respiration mediated by the ETF/ETFQO complex affects seed germination and development by directly adjusting carbon storage during seed filling. These results indicate a role for the pathway in the normal plant life cycle to complement its previously defined roles in the response to abiotic stress.


Assuntos
Aminoácidos/metabolismo , Arabidopsis/genética , Carbono/metabolismo , Flavoproteínas Transferidoras de Elétrons/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flavoproteínas Transferidoras de Elétrons/genética , Germinação , Proteínas Ferro-Enxofre/genética , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
10.
Plant J ; 109(1): 164-181, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34726315

RESUMO

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.


Assuntos
Arabidopsis/enzimologia , Galactanos/metabolismo , Mucoproteínas/metabolismo , Polissacarídeos/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Ácido Glucurônico/metabolismo , Mucoproteínas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/enzimologia , Pólen/genética , Pólen/fisiologia , Tubo Polínico/enzimologia , Tubo Polínico/genética , Tubo Polínico/fisiologia , Reprodução
11.
Ann Bot ; 129(1): 37-52, 2022 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-34549262

RESUMO

BACKGROUND AND AIMS: ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic. This study focuses on the four major cytosolic isoforms of PFK in vegetative tissues of A. thaliana. METHODS: We isolated homozygous knockout individual mutants (pfk1, pfk3, pfk6 and pfk7) and two double mutants (pfk1/7 and pfk3/6), and characterized their growth and metabolic phenotypes. KEY RESULTS: In contrast to single mutants and the double mutant pfk3/6 for the hypoxia-responsive isoforms, the double mutant pfk1/7 had reduced PFK activity and showed a clear visual and metabolic phenotype with reduced shoot growth, early flowering and elevated hexose levels. This mutant also has an altered ratio of short/long aliphatic glucosinolates and an altered root-shoot distribution. Surprisingly, this mutant does not show any major changes in short-term carbon flux and in levels of hexose-phosphates. CONCLUSIONS: We conclude that the two isoforms PFK1 and PFK7 are important for sugar homeostasis in leaf metabolism and apparently in source-sink relationships in A. thaliana, while PFK3 and PFK6 only play a minor role under normal growth conditions.


Assuntos
Arabidopsis , Fosfofrutoquinases , Folhas de Planta/enzimologia , Açúcares , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citosol/enzimologia , Homeostase , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Açúcares/metabolismo
12.
Plant Cell ; 34(1): 302-332, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34010411

RESUMO

Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regiões de Interação com a Matriz , Antígenos de Histocompatibilidade Menor/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
13.
Plant Cell ; 34(1): 287-301, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34524446

RESUMO

DNA-protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2-DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair. TDP1 and TDP2 function nonredundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative nonhomologous end joining (NHEJ) pathways. Double-mutant analysis indicates that "clean" DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , DNA Topoisomerases/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , DNA Topoisomerases/metabolismo
14.
Biochem Biophys Res Commun ; 589: 16-22, 2022 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-34883285

RESUMO

Gretchen Hagen 3 (GH3) amido synthetases conjugate amino acids to a carboxyl group of small molecules including hormones auxin, jasmonate, and salicylic acid. The Arabidopsis genome harbors 19 GH3 genes, whose exact roles in plant development have been difficult to define because of genetic redundancy among the GH3 genes. Here we use CRISPR/Cas9 gene editing technology to delete the Arabidopsis group II GH3 genes, which are able to conjugate indole-3-acetic acid (IAA) to amino acids. We show that plants lacking the eight group II GH3 genes (gh3 octuple mutants) accumulate free IAA and fail to produce IAA-Asp and IAA-Glu conjugates. Consequently, gh3 octuple mutants have extremely short roots, long and dense root hairs, and long hypocotyls. Our characterization of gh3 septuple mutants, which provide sensitized backgrounds, reveals that GH3.17 and GH3.9 play prominent roles in root elongation and seed production, respectively. We show that GH3 functions correlate with their expression patterns, suggesting that local deactivation of auxin also contributes to maintaining auxin homeostasis. Moreover, this work provides a method for elucidating functions of individual members of a gene family, whose members have overlapping functions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Ligases/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/enzimologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Homeostase , Ligases/genética , Família Multigênica , Mutação/genética , Fenótipo , Desenvolvimento Vegetal/genética , Raízes de Plantas/enzimologia
15.
Plant J ; 109(6): 1416-1426, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34913539

RESUMO

Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDP-galactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L-1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L-1 galactose versus 1.5 mmol L-1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.


Assuntos
Arabidopsis/enzimologia , Galactose , Açúcares , UDPglucose 4-Epimerase , UTP-Glucose-1-Fosfato Uridililtransferase , Galactose/toxicidade , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/genética , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Difosfato de Uridina
16.
PLoS One ; 16(12): e0259365, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34871322

RESUMO

Glutamate decarboxylase (GAD; EC 4.1.1.15) catalyzes the irreversible decarboxylation of glutamate to produce γ-aminobutyric acid (GABA); a ubiquitous non-protein amino acid involved in the regulation of several aspects of plant metabolism and physiology. To study the function of GAD and GABA in maize, we have; 1) introduced native and deregulated forms of AtGAD1 into maize with the intent of increasing the synthesis of GABA and 2) introduced constructs into maize designed to suppress the activity of several GABA shunt, GABA transport and GABA pathway genes. Maize plants expressing the deregulated AtGAD1 exhibit a severe chlorosis and retarded growth phenotype and have high levels of GABA, and Ca++/CaM-independent GAD activity. Plants expressing the suppression constructs for GABA biosynthetic and transport pathway genes had no observable phenotype whereas a knockout of GABA catabolic pathway genes led to growth and developmental defects under standard growth conditions. The implications of this study to our understanding of the action and function of GABA and GAD in crops are discussed.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Glutamato Descarboxilase/genética , Zea mays/genética , Ácido gama-Aminobutírico/biossíntese , Animais , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Genótipo , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Redes e Vias Metabólicas/genética , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Transgenes , Zea mays/enzimologia
17.
Int J Mol Sci ; 22(23)2021 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-34884915

RESUMO

In Yarrowia lipolytica, expression of the genes encoding the enzymes of the N-acetylglucosamine (NAGA) utilization pathway (NAG genes) becomes independent of the presence of NAGA in a Ylnag5 mutant lacking NAGA kinase. We addressed the question of whether the altered transcription was due to a lack of kinase activity or to a moonlighting role of this protein. Glucosamine-6-phosphate deaminase (Nag1) activity was measured as a reporter of NAG genes expression. The NGT1 gene encoding the NAGA transporter was deleted, creating a Ylnag5 ngt1 strain. In glucose cultures of this strain, Nag1 activity was similar to that of the Ylnag5 strain, ruling out the possibility that NAGA derived from cell wall turnover could trigger the derepression. Heterologous NAGA kinases were expressed in a Ylnag5 strain. Among them, the protein from Arabidopsis thaliana did not restore kinase activity but lowered Nag1 activity 4-fold with respect to a control. Expression in the Ylnag5 strain of YlNag5 variants F320S or D214V with low kinase activity caused a repression similar to that of the wild-type protein. Together, these results indicate that YlNag5 behaves as a moonlighting protein. An RNA-seq analysis revealed that the Ylnag5 mutation had a limited transcriptomic effect besides derepression of the NAG genes.


Assuntos
Perfilação da Expressão Gênica/métodos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Yarrowia/crescimento & desenvolvimento , Arabidopsis/enzimologia , Arabidopsis/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Mutação , Análise de Sequência de RNA , Yarrowia/enzimologia , Yarrowia/genética
18.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34903670

RESUMO

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , RNA de Plantas/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , DNA de Plantas , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Modelos Moleculares , Conformação Proteica , RNA de Plantas/genética , RNA Polimerase Dependente de RNA/genética , Transcrição Genética
19.
Science ; 374(6575): 1579-1586, 2021 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-34941388

RESUMO

DNA methylation affects gene expression and maintains genome integrity. The DNA-dependent RNA polymerase IV (Pol IV), together with the RNA-dependent RNA polymerase RDR2, produces double-stranded small interfering RNA precursors essential for establishing and maintaining DNA methylation in plants. We determined the cryo­electron microscopy structures of the Pol IV­RDR2 holoenzyme and the backtracked transcription elongation complex. These structures reveal that Pol IV and RDR2 form a complex with their active sites connected by an interpolymerase channel, through which the Pol IV­generated transcript is handed over to the RDR2 active site after being backtracked, where it is used as the template for double-stranded RNA (dsRNA) synthesis. Our results describe a 'backtracking-triggered RNA channeling' mechanism underlying dsRNA synthesis and also shed light on the evolutionary trajectory of eukaryotic RNA polymerases.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/química , RNA de Cadeia Dupla/biossíntese , RNA de Plantas/biossíntese , RNA Polimerase Dependente de RNA/química , Motivos de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Metilação de DNA , DNA de Plantas/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Holoenzimas/química , Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , Domínios Proteicos , RNA Polimerase II/química , RNA Interferente Pequeno/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo
20.
BMC Plant Biol ; 21(1): 590, 2021 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-34903166

RESUMO

BACKGROUND: Arabinogalactan-proteins (AGPs) are structurally complex hydroxyproline-rich cell wall glycoproteins ubiquitous in the plant kingdom. AGPs biosynthesis involves a series of post-translational modifications including the addition of type II arabinogalactans to non-contiguous Hyp residues. To date, eight Hyp-galactosyltransferases (Hyp-GALTs; GALT2-GALT9) belonging to CAZy GT31, are known to catalyze the addition of the first galactose residues to AGP protein backbones and enable subsequent AGP glycosylation. The extent of genetic redundancy, however, remains to be elucidated for the Hyp-GALT gene family. RESULTS: To examine their gene redundancy and functions, we generated various multiple gene knock-outs, including a triple mutant (galt5 galt8 galt9), two quadruple mutants (galt2 galt5 galt7 galt8, galt2 galt5 galt7 galt9), and one quintuple mutant (galt2 galt5 galt7 galt8 galt9), and comprehensively examined their biochemical and physiological phenotypes. The key findings include: AGP precipitations with ß-Yariv reagent showed that GALT2, GALT5, GALT7, GALT8 and GALT9 act redundantly with respect to AGP glycosylation in cauline and rosette leaves, while the activity of GALT7, GALT8 and GALT9 dominate in the stem, silique and flowers. Monosaccharide composition analysis showed that galactose was decreased in the silique and root AGPs of the Hyp-GALT mutants. TEM analysis of 25789 quintuple mutant stems indicated cell wall defects coincident with the observed developmental and growth impairment in these Hyp-GALT mutants. Correlated with expression patterns, galt2, galt5, galt7, galt8, and galt9 display equal additive effects on insensitivity to ß-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, galt7, galt8, and galt9 contributed more to primary root growth and root tip swelling under salt stress, whereas galt2 and galt5 played more important roles in seed morphology, germination defects and seed set. Pollen defects likely contributed to the reduced seed set in these mutants. CONCLUSION: Additive and pleiotropic effects of GALT2, GALT5, GALT7, GALT8 and GALT9 on vegetative and reproductive growth phenotypes were teased apart via generation of different combinations of Hyp-GALT knock-out mutants. Taken together, the generation of higher order Hyp-GALT mutants demonstrate the functional importance of AG polysaccharides decorating the AGPs with respect to various aspects of plant growth and development.


Assuntos
Arabidopsis/genética , Galactanos/metabolismo , Galactosiltransferases/metabolismo , Mucoproteínas/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Flores/enzimologia , Flores/genética , Flores/fisiologia , Flores/ultraestrutura , Galactosiltransferases/genética , Pleiotropia Genética , Germinação , Glucosídeos/química , Glicosilação , Hidroxiprolina/metabolismo , Meristema/enzimologia , Meristema/genética , Meristema/fisiologia , Meristema/ultraestrutura , Mucoproteínas/genética , Mutação , Especificidade de Órgãos , Floroglucinol/análogos & derivados , Floroglucinol/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/enzimologia , Caules de Planta/genética , Caules de Planta/fisiologia , Caules de Planta/ultraestrutura , Biossíntese de Proteínas , Estresse Salino , Sementes/enzimologia , Sementes/genética , Sementes/fisiologia , Sementes/ultraestrutura
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