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1.
Plant Physiol Biochem ; 159: 372-382, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33444896

RESUMO

Calcium-dependent protein kinases (CDPKs) are essential regulators of plant growth and development, biotic and abiotic stress responses. Inactivation of the auto-inhibitory domain (AID) of CDPKs provides the constitutive activity. This study investigated the effect of overexpressed native and constitutive active (AtCPK1-Ca) forms of the AtCPK1 gene on abiotic stress tolerance and the ROS/redox system in Rubia cordifolia transgenic callus lines. Overexpression of the native AtCPK1 increased tolerance to salinity and cold almost in two times, when AtCPK1-Ca - in three times compare to control culture. A more interesting effect of overexpression of the AtCPK1 and AtCPK1-Ca was observed for heat resistance. The native form of AtCPK1 increased resistance to heating by 45%, while the AtCPK1-Ca increased by 80%. At the same time, another type of mutation of the AID (AtCPK1-Na, not active) did not affect the tolerance of the cell culture to stresses. We suppose, in this process, the ROS/redox system might be involved. Levels of intracellular ROS, ROS-generating enzymes expression and activities (Rbohs, Prx) and ROS-detoxifying enzymes (SOD, Cat, Apx and Prx) changed in a coordinated manner and in strict interconnection, depending of the callus growth phase and correlated with improved stress tolerance caused by AtCPK1. Because overexpression of both the AtCPK1 and AtCPK1-Ca did not significantly change callus growth, we propose that inactivation of AID of the AtCPK1 or its ortholog, might be an interesting instrument for improvement of plant cells resistance to abiotic stress.


Assuntos
Arabidopsis , Rubia , Tolerância ao Sal , Termotolerância , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultura de Células , Regulação da Expressão Gênica de Plantas , Oxirredutases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Domínios Proteicos/genética , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rubia/genética , Rubia/metabolismo , Tolerância ao Sal/genética , Estresse Fisiológico/genética , Termotolerância/genética
2.
J Agric Food Chem ; 69(2): 638-645, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33398988

RESUMO

With widespread applications of the latest neonicotinoid in agriculture, dinotefuran has gradually become a hazardous contaminant for plants through the generation of excessive reactive oxygen species. However, the potential toxic mechanisms of oxidative damages to plants induced by dinotefuran are still unknown. As a core component of the glutathione antioxidant enzyme system, glutathione peroxidases have been used as biomarkers to reflect excessive oxidative stress. In this study, the hazardous effects of dinotefuran on AtGPX6 were investigated at the molecular level. The intrinsic fluorescence intensity of AtGPX6 was quenched using the static quenching mechanism upon binding with dinotefuran. Moreover, a single binding site was predicted for AtGPX6 toward dinotefuran, and the complex formation was presumed to be driven by hydrogen bonds or van der Waals forces, which conformed with the molecular docking results. In addition, AtGPX6 exhibited moderate binding affinity with dinotefuran based on the bio-layer interferometry assay. In addition, the loosening and unfolding of the protein skeleton of AtGPX6 with the addition of dinotefuran were explored along with the increase of hydrophobicity around tryptophan residues. Lastly, the toxic effects of dinotefuran on the root growth of Arabidopsis seedlings were also examined. The exploration of the binding mechanism of dinotefuran with AtGPX6 at the molecular level would provide the toxicity assessment of dinotefuran on plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Guanidinas/farmacologia , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Nitrocompostos/farmacologia , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Inseticidas/química , Simulação de Acoplamento Molecular , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/química , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Plântula/química , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genética
3.
Plant Physiol Biochem ; 159: 28-36, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33321375

RESUMO

Salt stress is a serious abiotic stressor impeding plant growth and crop production around the world. Plant glycosyltransferases are thought to serve important roles in dealing with stress conditions, however, the functional role of how UGTs cope with salt stress is not well understood. Carex rigescens (Franch.) V. Krecz, is a widely distributed species of turfgrass with strong salinity tolerance found in northern China. To investigate how the glycosyltransferase gene, CrUGT87A1, functions in C. rigescens, we performed analyses of cloning, transcriptional expression, subcellular localization, and overexpression. The full-length sequence of CrUGT87A1 is 1455 bp with a 1338 bp length ORF, which encodes 445 amino acids, while CrUGT87A1 was found to be a nuclear and plasmalemma-localized protein. We found that the transcriptional expression of CrUGT87A1 was up-regulated under ABA, heat, salt, and drought treatments in leaf tissues. CrUGT87A1 overexpression in Arabidopsis plants had a significantly higher germination rate, better growth and physiology, and a higher expression levels of transcripts related to salt stress-related genes under high-salinity conditions, suggesting that CrUGT87A1 is involved in salt tolerance. The transcriptional expression of genes related to flavonoid-synthesis related and the flavonoid content reflected higher accumulations of flavonoids in transgenic plants. Our study demonstrated that CrUGT87A1 could play an important role in resisting salt stress due to increased flavonoid accumulation, which can promote antioxidation when dealing with high-salinity conditions. This study advances our collective understanding of the functional role of UGTs and can be used to improve the salt tolerance and breeding of crops and plants.


Assuntos
Arabidopsis , Carex (Planta) , Flavonoides , Glicosiltransferases , Proteínas de Plantas , Proteínas Recombinantes , Tolerância ao Sal , Arabidopsis/enzimologia , Arabidopsis/genética , Carex (Planta)/genética , Carex (Planta)/metabolismo , China , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosiltransferases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tolerância ao Sal/genética
4.
Methods Mol Biol ; 2213: 131-144, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33270199

RESUMO

Auxin plays important roles in almost all aspects of plant growth and development. Chemical genetics is an effective approach to understand auxin action, especially in nonmodel plant species, in which auxin-related mutants are not yet available. Among auxin-related chemical tools, we present approaches to utilize auxin biosynthesis inhibitors. The inhibitors are effective not only to understand auxin biosynthesis but also to understand auxin action. The effectiveness of the inhibitors can be assessed based on in vitro or in vivo assays. The in vitro assay employs enzyme inhibition assays. The in vivo assay employs UPLC-MS/MS-based analysis of endogenous IAA and its intermediates or metabolites.


Assuntos
Vias Biossintéticas , Ácidos Indolacéticos/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Ensaios Enzimáticos , Ácidos Indolacéticos/química , Oryza/enzimologia , Oryza/metabolismo , Proteínas Recombinantes/metabolismo , Padrões de Referência , Plântula/crescimento & desenvolvimento , Plântula/metabolismo
5.
PLoS One ; 15(9): e0238179, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881902

RESUMO

Carotenoid cleavage dioxygenase (CCD), a key enzyme in carotenoid metabolism, cleaves carotenoids to form apo-carotenoids, which play a major role in plant growth and stress responses. CCD genes had not previously been systematically characterized in Brassica napus (rapeseed), an important oil crop worldwide. In this study, we identified 30 BnCCD genes and classified them into nine subgroups based on a phylogenetic analysis. We identified the chromosomal locations, gene structures, and cis-promoter elements of each of these genes and performed a selection pressure analysis to identify residues under selection. Furthermore, we determined the subcellular localization, physicochemical properties, and conserved protein motifs of the encoded proteins. All the CCD proteins contained a retinal pigment epithelial membrane protein (RPE65) domain. qRT-PCR analysis of expression of 20 representative BnCCD genes in 16 tissues of the B. napus cultivar Zhong Shuang 11 ('ZS11') revealed that members of the BnCCD gene family possess a broad range of expression patterns. This work lays the foundation for functional studies of the BnCCD gene family.


Assuntos
Brassica napus/enzimologia , Dioxigenases/genética , Genoma de Planta , Proteínas de Plantas/genética , Arabidopsis/enzimologia , Brassica napus/genética , Carotenoides/metabolismo , Mapeamento Cromossômico , Dioxigenases/classificação , Dioxigenases/metabolismo , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas
6.
PLoS One ; 15(7): e0227466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678822

RESUMO

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.


Assuntos
Adenosil-Homocisteinase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Evolução Molecular , Adenosil-Homocisteinase/classificação , Adenosil-Homocisteinase/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Eletroforese em Gel Bidimensional , Focalização Isoelétrica , Luz , Filogenia , Folhas de Planta/enzimologia , Processamento de Proteína Pós-Traducional/efeitos da radiação , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Estresse Fisiológico
7.
Proc Natl Acad Sci U S A ; 117(24): 13792-13799, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32471952

RESUMO

DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis thaliana Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when coexpressed in Nicotiana benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/química , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Repressoras/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
8.
Ecotoxicol Environ Saf ; 199: 110727, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32446101

RESUMO

Sulfonamides (SAs) are antibiotics widely used in clinical practice, livestock and poultry production, and the aquaculture industry. The compounds enter the soil environment largely through livestock and poultry manure application to farmland. SAs not only affect plant growth, but also pose a potential threat to human health through SA residues in plant tissues. In particular, sulfamethoxazole (SMZ) has been classified as a Category 3 carcinogen by the World Health Organization, and thus its soil ecological toxicity and possible health risks are of concern. Using A. thaliana as a model plant, stress responses and biological residues of sulfadiazine (SD), sulfametoxydiazine (SMD), and SMZ were investigated in the present study. Root length and aboveground plant biomass were significantly inhibited by the three types of SA, whereas lateral roots exposed to SMD grew vigorously. The contents of chlorophyll a and chlorophyll b and photosystem II maximum photochemical quantum yield declined with increase in drug concentration, which indicated that exposure to SAs affected photosynthesis and inhibited chlorophyll synthesis in A. thaliana. With increase in drug concentration, reactive oxygen species (ROS) accumulation in the leaves increased significantly. Activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were activated at low SA concentrations, but increased lipid peroxidation occurred with increase in SA concentration. Of the three compounds, SMZ was the most toxic to A. thaliana, followed by SD, and SMD was the least toxic. The results indicated that the risk of SMD entering an organism through the food chain is greater than that for SMZ and SD.


Assuntos
Antibacterianos/toxicidade , Arabidopsis/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Poluentes do Solo/toxicidade , Sulfanilamidas/toxicidade , Antioxidantes/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Clorofila/metabolismo , Clorofila A/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Nature ; 581(7807): 199-203, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32404997

RESUMO

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens1-3. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Imunidade Vegetal/imunologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Arabidopsis/enzimologia , Endocitose , Ligantes , Padrões Moleculares Associados a Patógenos/imunologia , Fosforilação , Proteínas Quinases/metabolismo
10.
Virology ; 546: 133-140, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32452413

RESUMO

Auxin has profound effects on plant growth and development. In addition to participating in plant growth and development, the auxin signaling pathway is involved in plant defense against pathogens. In this study, we investigated the molecular mechanism by which helper-component protease (HCPro) encoded by the Tobacco vein banding mosaic virus (TVBMV) activates auxin biosynthesis genes (YUCs) and interferes with the auxin signaling pathway. Our results demonstrated that the viral suppressor HCPro decreased the DNA methylation of dispersed repeats (DRs) within the promoters of YUC1, YUC5 and YUC10 and transcriptional activated these YUC genes targeted by RNA-directed DNA methylation (RdDM), leading to an increase in auxin accumulation in plants. Furthermore, we found that the induction of these YUCs by HCPro was attenuated in ros1 mutant plants, suggesting that HCPro-mediated transcriptional activation of the genes was partly dependent on ROS1-mediated DNA demethylation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Ácidos Indolacéticos/metabolismo , Oxigenases/genética , Doenças das Plantas/virologia , Potyvirus/enzimologia , Arabidopsis/genética , Arabidopsis/virologia , Proteínas de Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno , Oxigenases/metabolismo , Doenças das Plantas/genética , Potyvirus/genética , Regiões Promotoras Genéticas
11.
PLoS One ; 15(5): e0233493, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453778

RESUMO

Glyoxalase pathway is the major pathway of methylglyoxal detoxification and is ubiquitously present in all organisms ranging from prokaryotes to eukaryotes. Glyoxalase I (GLYI) and Glyoxalase II (GLYII), the two core enzymes of this pathway work together to neutralize methylglyoxal (MG), a dicarbonyl molecule with detrimental cytotoxicity at higher concentrations. The first step towards the detoxification of MG is catalyzed by GLYI, a metalloenzyme that requires divalent metal ions (either Zn2+ as seen in eukaryotes or Ni2+ as in prokaryotes). However, both Zn2+ and Ni2+ dependent GLYIs have been shown to co-exist in a higher eukaryote i.e. Arabidopsis thaliana. In the present study, we determine the role of both Zn2+ dependent (AtGLYI2) and Ni2+ dependent (AtGLYI3, AtGLYI6) GLYIs from Arabidopsis in salinity stress tolerance. AtGLYI2 overexpressing Arabidopsis plants showed better growth rate while maintaining lower levels of MG under high saline conditions. They were taller with more number of silique formation with respect to their Ni2+ dependent counterparts. Further, lack in germination of Arabidopsis AtGLYI2 mutants in presence of exogenous MG indicates the direct involvement of Zn2+ dependent GLYI in MG detoxification, suggesting Zn2+ dependent GLYI as the main enzyme responsible for MG detoxification and salinity stress tolerance.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação , Mutação , Aldeído Pirúvico/metabolismo , Estresse Salino , Zinco/metabolismo
12.
PLoS Genet ; 16(5): e1008681, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463832

RESUMO

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/metabolismo , Transposases/fisiologia , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Domínio Catalítico/genética , Células Cultivadas , Domesticação , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Proteínas do Grupo Polycomb/genética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Células Sf9 , Spodoptera , Transposases/genética
13.
Biochem Biophys Res Commun ; 526(4): 1036-1041, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32305137

RESUMO

Pollen wall characteristics are dramatically changed during pollen maturation. Many genes have been identified as regulators of such changes in pollen wall characteristics, but mechanisms of such changes have not been completely understood. Here, a GDSL-type esterase/lipase gene, GELP77, is shown to regulate such changes in Arabidopsis thaliana. GELP77-deficient (gelp77) plants exhibited male sterility, and this phenotype was suppressed by introduction of a GELP77 genomic fragment. Mature pollen grains of wild-type Arabidopsis plants have an organized reticulate surface structure and are dissociated from each other. In contrast, pollen grains of gelp77 lacked such a structure and were shrunken and stuck to each other. Nuclei were not detectable in gelp77 microspores at a putative uninucleate stage, suggesting that GELP77 is required as early as this stage. In plants that have the GELP77 promoter-GELP77-GFP transgene, the GELP77-GFP fusion protein was detected in microspores, tapetal cells and middle layer cells in anthers at post-meiotic stages, whereas not anthers at pre-meiotic stages. Analysis of amino acid sequences suggests that GELP77 is phylogenetically distant from the other 104 GDSL-type esterase/lipase genes in Arabidopsis and that GELP77 orthologs are present in various plant species. Together, these results indicate that GELP77 regulates pollen wall characteristics in Arabidopsis.


Assuntos
Arabidopsis/genética , Arabidopsis/fisiologia , Genes de Plantas , Lipase/genética , Pólen/fisiologia , Arabidopsis/enzimologia , Arabidopsis/ultraestrutura , Sequência Conservada/genética , Fertilidade/fisiologia , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Lipase/metabolismo , Filogenia , Infertilidade das Plantas/genética , Pólen/ultraestrutura , Via Secretória
14.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 4): 182-191, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254052

RESUMO

Casein kinase 2 (CK2) is a ubiquitous pleiotropic enzyme that is highly conserved across eukaryotic kingdoms. CK2 is singular amongst kinases as it is highly rigid and constitutively active. Arabidopsis thaliana is widely used as a model system in molecular plant research; the biological functions of A. thaliana CK2 are well studied in vivo and many of its substrates have been identified. Here, crystal structures of the α subunit of A. thaliana CK2 in three crystal forms and of its complex with the nonhydrolyzable ATP analog AMppNHp are presented. While the C-lobe of the enzyme is highly rigid, structural plasticity is observed for the N-lobe. Small but significant displacements within the active cleft are necessary in order to avoid steric clashes with the AMppNHp molecule. Binding of AMppNHp is influenced by a rigid-body motion of the N-lobe that was not previously recognized in maize CK2.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Caseína Quinase II/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Estrutura Terciária de Proteína
15.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 357-365, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254060

RESUMO

Starch is a key energy-storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. ß-Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small-angle X-ray scattering data were collected from AtBAM2 and its N-terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteínas Serina-Treonina Quinases/química , Amido/metabolismo , beta-Amilase/química , Sequência de Aminoácidos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Espalhamento de Radiação , Alinhamento de Sequência , Raios X
16.
Plant Biol (Stuttg) ; 22(4): 563-572, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32233097

RESUMO

A balance between the synthesis and degradation of active proteins governs diverse cellular processes in plants, spanning from cell-cycle progression and circadian rhythm to the outcome of several hormone signalling pathways. Ubiquitin-mediated post-translational modification determines the degradative fate of the target proteins, thereby altering the output of cellular processes. An equally important, and perhaps under-appreciated, aspect of this pathway is the antagonistic process of de-ubiquitination. De-ubiquitinases (DUBs), a group of processing enzymes, play an important role in maintaining cellular ubiquitin homeostasis by hydrolyzing ubiquitin poly-proteins and free poly-ubiquitin chains into mono-ubiquitin. Further, DUBs rescue the cellular proteins from 26S proteasome-mediated degradation to their active form by cleaving the poly-ubiquitin chain from the target protein. Any perturbation in DUB activity is likely to affect proteostasis and downstream cellular processes. This review illustrates recent findings on the biological significance and mechanisms of action of the DUBs in Arabidopsis thaliana, with an emphasis on ubiquitin-specific proteases (UBPs), the largest family among the DUBs. We focus on the putative roles of various protein-protein interaction interfaces in DUBs and their generalized function in ubiquitin recycling, along with their pre-eminent role in plant development.


Assuntos
Botânica , Endopeptidases , Plantas , Ubiquitina , Arabidopsis/enzimologia , Botânica/tendências , Endopeptidases/metabolismo , Plantas/enzimologia , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Ubiquitinação
17.
Nat Commun ; 11(1): 1417, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184398

RESUMO

Holliday junctions (HJs) are key DNA intermediates in genetic recombination and are eliminated by nuclease, termed resolvase, to ensure genome stability. HJ resolvases have been identified across all kingdoms of life, members of which exhibit sequence-dependent HJ resolution. However, the molecular basis of sequence selectivity remains largely unknown. Here, we present the chloroplast resolvase MOC1, which cleaves HJ in a cytosine-dependent manner. We determine the crystal structure of MOC1 with and without HJs. MOC1 exhibits an RNase H fold, belonging to the retroviral integrase family. MOC1 functions as a dimer, and the HJ is embedded into the basic cleft of the dimeric enzyme. We characterize a base recognition loop (BR loop) that protrudes into and opens the junction. Residues from the BR loop intercalate into the bases, disrupt the C-G base pairing at the crossover and recognize the cytosine, providing the molecular basis for sequence-dependent HJ resolution by a resolvase.


Assuntos
Arabidopsis/enzimologia , Cloroplastos/enzimologia , DNA Cruciforme/metabolismo , Oryza/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Recombinases/química , Recombinases/metabolismo , Soja/enzimologia , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Cloroplastos/química , Cloroplastos/genética , DNA Cruciforme/química , DNA Cruciforme/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/metabolismo , Oryza/química , Oryza/genética , Oryza/metabolismo , Recombinases/genética , Soja/química , Soja/genética , Soja/metabolismo
18.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 3): 109-115, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32133996

RESUMO

Citrate synthase (CS) catalyzes the conversion of oxaloacetate and acetyl coenzyme A into citrate and coenzyme A in the mitochondrial tricarboxylic acid (TCA) cycle. In plants, mitochondrial metabolism, including the TCA cycle, occurs in interaction with photosynthetic metabolism. The controlled regulation of several enzymes in the TCA cycle, such as CS, is important in plants. Here, the first crystal structure of a plant mitochondrial CS, CSY4 from Arabidopsis thaliana (AtCSY4), has been determined. Structural comparison of AtCSY4 with mitochondrial CSs revealed a high level of similarity. Inhibition analysis showed a similar manner of inhibition as in mitochondrial CSs. The effect of oxidation on one of a pair of cysteine residues in AtCSY4 was speculated upon based on the folded structure.


Assuntos
Arabidopsis/química , Arabidopsis/enzimologia , Citrato (si)-Sintase/química , Citrato (si)-Sintase/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Citrato (si)-Sintase/genética , Cristalografia por Raios X/métodos , Mitocôndrias/genética , Estrutura Secundária de Proteína
19.
Plant Physiol Biochem ; 149: 286-293, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32097847

RESUMO

Serine protease inhibitors (SPIs) play an important role in cell survival, development and host defense. In plants, serine protease inhibitors such as the Kunitz-type inhibitor (KTI) and the Bowman-Birk inhibitor (BBI) have been shown to be induced in response to abiotic stress such as salinity and drought resulting in tolerance to these stresses. In this study, Arabidopsis thaliana (T3) plants overexpressing the BBI gene from maize were generated and subjected to drought stress in order to study the role of BBI protease inhibitor in drought tolerance. Drought treatment of four-week-old Arabidopsis plants was performed by withholding water from plants for nine days and harvested plant material was used for physiological and biochemical analysis. The transgenic lines exhibited normal growth after nine days of drought as compared to the wild-type. The results also showed a higher leaf relative water content (RWC) in transgenic lines when compared to the wild-type (WT), with line 2 having the highest RWC of 72% and the WT having the lowest RWC of 32%. Trypsin-inhibitor activity indicated that the total protein of the positive transgenic plants had stronger protease inhibitory activity than the wild-type. Transgenic lines overexpressing BBI also showed reduced lipid peroxidation (MDA content) as well as enhanced activity of antioxidants glutathione-s-transferase (GST) and ascorbate peroxidase (APX). These results suggest that BBI protease inhibitor leads to drought tolerance associated with reduction in drought-induced oxidative stress.


Assuntos
Arabidopsis , Secas , Inibidores de Serino Proteinase , Estresse Fisiológico , Arabidopsis/enzimologia , Arabidopsis/genética , Plantas Geneticamente Modificadas , Inibidores de Serino Proteinase/genética , Inibidores de Serino Proteinase/metabolismo , Estresse Fisiológico/genética
20.
Plant Mol Biol ; 103(1-2): 173-184, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32100164

RESUMO

KEY MESSAGE: Arabidopsis LONG-CHAIN BASE KINASE 1 (LCBK1) interacts with MEDEA, a component of PCR2 complex that negatively regulates immunity. LCBK1 phosphorylates phytosphingosine and thereby promotes stomatal immunity against bacterial pathogens. Arabidopsis polycomb-group repressor complex2 (PRC2) protein MEDEA (MEA) suppresses both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). MEA represses the expression of RPS2 and thereby attenuates AvrRpt2 effector-mediated ETI. However, the mechanism of MEA-mediated PTI diminution was not known. By screening the Arabidopsis cDNA library using yeast-2-hybrid interaction, we identified LONG-CHAIN BASE KINASE1 (LCBK1) as an MEA-interacting protein. We found that lcbk1 mutants are susceptible to virulent bacterial pathogens, such as Pseudomonas syringae pv maculicola (Psm) and P. syringae pv tomato (Pst) but not the avirulent strain of Pst that carries AvrRpt2 effector. Pathogen inoculation induces LCBK1 expression, especially in guard cells. We found that LCBK1 has a positive regulatory role in stomatal closure after pathogen inoculation. WT plants close stomata within an hour of Pst inoculation or flg22 (a 22 amino acid peptide from bacterial flagellin protein that activates PTI) treatment, but not lcbk1 mutants. LCBK1 phosphorylates phytosphingosine (PHS). Exogenous application of phosphorylated PHS (PHS-P) induces stomatal closure and rescues loss-of-PTI phenotype of lcbk1 mutant plants. MEA overexpressing (MEA-Oex) plants are defective, whereas loss-of-function mea-6 mutants are hyperactive in PTI-induced stomatal closure. Exogenous application of PHS-P rescues loss-of-PTI in MEA-Oex plants. Results altogether demonstrate that LCBK1 is an interactor of MEA that positively regulates PTI-induced stomatal closure in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Fosfotransferases/metabolismo , Estômatos de Plantas/imunologia , Arabidopsis/enzimologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Fosfotransferases/genética , Doenças das Plantas/imunologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo
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