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1.
Plant Mol Biol ; 101(3): 325-339, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31399934

RESUMO

KEY MESSAGE: Combining genetic engineering of MPK4 activity and quantitative proteomics, we established an in planta system that enables rapid study of MPK4 signaling networks and potential substrate proteins. Mitogen activated protein kinase 4 (MPK4) is a multifunctional kinase that regulates various signaling events in plant defense, growth, light response and cytokinesis. The question of how a single protein modulates many distinct processes has spurred extensive research into the physiological outcomes resulting from genetic perturbation of MPK4. However, the mechanism by which MPK4 functions is still poorly understood due to limited data on the MPK4 networks including substrate proteins and downstream pathways. Here we introduce an experimental system that combines genetic engineering of kinase activity and quantitative proteomics to rapidly study the signaling networks of MPK4. First, we transiently expressed a constitutively active (MPK4CA) and an inactive (MPK4IN) version of a Brassica napus MPK4 (BnMPK4) in Nicotiana benthamiana leaves. Proteomics analysis revealed that BnMPK4 activation affects multiple pathways (e.g., metabolism, redox regulation, jasmonic acid biosynthesis and stress responses). Furthermore, BnMPK4 activation also increased protein phosphorylation in the phosphoproteome, from which putative MPK4 substrates were identified. Using protein kinase assay, we validated that a transcription factor TCP8-like (TCP8) and a PP2A regulatory subunit TAP46-like (TAP46) were indeed phosphorylated by BnMPK4. Taken together, we demonstrated the utility of proteomics and phosphoproteomics in elucidating kinase signaling networks and in identification of downstream substrates.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteômica , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Brassica napus/enzimologia , Engenharia Genética , Sistema de Sinalização das MAP Quinases , Fosforilação , Imunidade Vegetal , Folhas de Planta/enzimologia , Proteoma , Transdução de Sinais , Tabaco/enzimologia
2.
Plant Mol Biol ; 101(1-2): 183-202, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31286324

RESUMO

KEY MESSAGE: Isoforms of 2-OGDH E1 subunit are not functionally redundant in plant growth and development of A. thaliana. The tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase (2-OGDH) converts 2-oxoglutarate (2-OG) to succinyl-CoA concomitant with the reduction of NAD+. 2-OGDH has an essential role in plant metabolism, being both a limiting step during mitochondrial respiration as well as a key player in carbon-nitrogen interactions. In Arabidopsis thaliana two genes encode for E1 subunit of 2-OGDH but the physiological roles of each isoform remain unknown. Thus, in the present study we isolated Arabidopsis T-DNA insertion knockout mutant lines for each of the genes encoding the E1 subunit of 2-OGDH enzyme. All mutant plants exhibited substantial reduction in both respiration and CO2 assimilation rates. Furthermore, mutant lines exhibited reduced levels of chlorophylls and nitrate, increased levels of sucrose, malate and fumarate and minor changes in total protein and starch levels in leaves. Despite the similar metabolic phenotypes for the two E1 isoforms the reduction in the expression of each gene culminated in different responses in terms of plant growth and seed production indicating distinct roles for each isoform. Collectively, our results demonstrated the importance of the E1 subunit of 2-OGDH in both autotrophic and heterotrophic tissues and suggest that the two E1 isoforms are not functionally redundant in terms of plant growth in A. thaliana.


Assuntos
Arabidopsis/enzimologia , Carbono/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Nitrogênio/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Complexo Cetoglutarato Desidrogenase/genética , Mitocôndrias/enzimologia , Mutagênese Insercional , Nitratos/metabolismo , Fenótipo , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Isoformas de Proteínas , Subunidades Proteicas , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento
3.
Planta ; 250(1): 381-390, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31062160

RESUMO

MAIN CONCLUSION: Ethylene receptor is crucial for PCD and aerenchyma formation in Typha angustifolia leaves. Not only does it receive and deliver the ethylene signal, but it probably can determine the cell fate during aerenchyma morphogenesis, which is due to the receptor expression quantity. Aquatic plant oxygen delivery relies on aerenchyma, which is formed by a programmed cell death (PCD) procedure. However, cells in the outer edge of the aerenchyma (palisade cells and septum cells) remain intact, and the mechanism is unclear. Here, we offer a hypothesis: cells that have a higher content of ethylene receptors do not undergo PCD. In this study, we investigated the leaf aerenchyma of the aquatic plant Typha angustifolia. Ethephon and pyrazinamide (PZA, an inhibitor of ACC oxidase) were used to confirm that ethylene is an essential hormone for PCD of leaf aerenchyma cells in T. angustifolia. That the ethylene receptor was an indispensable factor in this PCD was confirmed by 1-MCP (an inhibitor of the ethylene receptor) treatment. Although PCD can be avoided by blocking the ethylene receptor, excessive ethylene receptors also protect cells from PCD. TaETR1, TaETR2 and TaEIN4 in the T. angustifolia leaf were detected by immunofluorescence (IF) using polyclonal antibodies. The result showed that the content of ethylene receptors in PCD-unsusceptible cells was 4-14 times higher than that one in PCD-susceptible cells, suggesting that PCD-susceptible cells undergo the PCD programme, while PCD-unsusceptible cells do not due to the content difference in the ethylene receptor in different cells. A higher level of ethylene receptor content makes the cells insensitive to ethylene, thereby avoiding cell death and degradation.


Assuntos
Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/metabolismo , Receptores de Superfície Celular/metabolismo , Typhaceae/fisiologia , Aminoácido Oxirredutases/antagonistas & inibidores , Apoptose/genética , Diferenciação Celular/genética , Ciclopropanos/farmacologia , Etilenos/metabolismo , Compostos Organofosforados/farmacologia , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Pirazinamida/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Typhaceae/efeitos dos fármacos , Typhaceae/enzimologia , Typhaceae/crescimento & desenvolvimento
4.
Environ Sci Pollut Res Int ; 26(19): 19705-19718, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31089999

RESUMO

Leaf blast is the main rice disease in the world causing significant losses in productivity. Blast integrate management (BIM) requires the use of genetic resistance, cultural practices, and chemical control, although for sustainable BIM, the insertion of biological agents may be the fourth component for. The objective of this work was to test three formulations of Burkholderia pyrrocinia (BRM32113) previously selected and to verify the effectiveness in resistance induction and blast control in rice. Two experiments were carried out, in a completely randomized design with three replications, in the greenhouse (E1 and E2). E1 aimed to select the best treatment for suppressing leaf blast severity and activating plant defense mechanisms. It was composed of 8 treatments: (1) formulated 11+ B. pyrrocina × Magnaporthe oryzae; (2) formulated 17+ B. pyrrocina × M. oryzae; (3) formulated 32+ B. pyrrocina × M. oryzae; (4) formulated 11 × M. oryzae; (5) B. pyrrocinia 17 × M. oryzae; (6) formulated 32 × M. oryzae; (7) B. pyrrocina × M. oryzae; (8) M. oryzae; (9) control (water). E2 aimed to investigate the effect of the best treatments, for the promotion of plant growth and suppression of leaf blast by calculating AUDPC. It was composed of 6 treatments: (1) formulated 11+ B. pyrrocina × M. oryzae; (2) formulated 32+ B. pyrrocina × M. oryzae; (3) formulated 11 × M. oryzae; (4) formulated 32 × M. oryzae; (5) B. pyrrocina × M. oryzae; (6) water. And after, we did two assays aimed to localize this biological agent after application at seed, soil, and rice plant. In E1, formulated 11+ B. pyrrocinia and 32+ formulated and B. pyrrocina were the best, suppressing leaf blast by up to 97% and providing the significant increase of the enzymes ß-1,3-glucanase, chitinase, phenylalanine ammonia lyase, lipoxygenase, and salicylic acid at 24 h and 48 h after inoculation with M. oryzae. In E2, treatments formulated 11+ B. pyrrocinia, formulated 32+ B. pyrrocinia, and B. pyrrocina provided more significant increases in growth promotion and reduced area under disease progress curve. B. pyrrocinia was detected in the rice plant for 18 days, predominantly in the root system (internal and external). The use of B. pyrrocinia formulations based on sugarcane molasses and glycerol can be an essential strategy for sustainable management. Although all the benefits come from these sustainable formulations, the adoption by commercial biological segment depends on an established formulation process. It seems that all the results showed here by this research will be readily assimilated by startups of the organic segment.


Assuntos
Agentes de Controle Biológico/farmacologia , Burkholderia , Resistência à Doença/efeitos dos fármacos , Magnaporthe , Oryza/microbiologia , Doenças das Plantas/prevenção & controle , Agentes de Controle Biológico/isolamento & purificação , Burkholderia/metabolismo , Magnaporthe/crescimento & desenvolvimento , Oryza/enzimologia , Doenças das Plantas/microbiologia , Folhas de Planta/enzimologia , Folhas de Planta/microbiologia , Distribuição Aleatória , Ácido Salicílico/metabolismo
5.
Ecotoxicol Environ Saf ; 180: 179-184, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31082582

RESUMO

Selenium (Se) and zinc (Zn) are necessary mineral nutrients for human body but millions of people have an inadequate intake of them, and eat food enriched with Se and Zn may minimize these problems. Chinese cabbage is an important food in people's daily life. The aim of this study was to evaluate the effects of single Se, Zn and their combination treatment in soil on their accumulation, antioxidant system and lipid peroxidation in roots and leaves of Chinese cabbage using soil pot culture experiment. When 0.5 mg kg-1 Se +30 mg kg-1 Zn and 1.0 mg kg-1 Se +30 mg kg-1 Zn were spiked in soils, Zn concentrations in roots and leaves of Chinese cabbage were significantly increased (p < 0.05) by 20.2%, 37.8% and 17.9%, 34.1% respectively compared to the treatment of 30 mg kg-1 Zn added, and the latter was significantly higher (p < 0.05) than that of former, indicating Se significantly promoted Zn accumulation. Almost all physiological indexes including POD, SOD, CAT, APX, GR, Chlorophyll a, Chlorophyll b, Carotenoids, MDA and Free proline in the treatments of Se or Zn spiked were significantly improved (p < 0.05) or basically unaffected compared to the control without Se or Zn added. The biomass change trends were similar with these indexes either. These results showed that the addition in soil of Se and Zn significantly increased their accumulation in Chinese cabbage without affected its formal growth. Particularly, the addition of Se promoted Zn accumulation. The conclusions were more important reference for the production practice of cash crop enriched of Se and Zn either.


Assuntos
Brassica/efeitos dos fármacos , Selênio/farmacologia , Solo , Zinco/metabolismo , Antioxidantes/metabolismo , Brassica/enzimologia , Brassica/metabolismo , Carotenoides/metabolismo , Clorofila/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/metabolismo , Selênio/metabolismo
6.
BMC Plant Biol ; 19(1): 141, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987599

RESUMO

BACKGROUND: Tomato mutants altered in leaf morphology are usually identified in the greenhouse, which demands considerable time and space and can only be performed in adequate periods. For a faster but equally reliable scrutiny method we addressed the screening in vitro of 971 T-DNA lines. Leaf development was evaluated in vitro in seedlings and shoot-derived axenic plants. New mutants were characterized in the greenhouse to establish the relationship between in vitro and in vivo leaf morphology, and to shed light on possible links between leaf development and agronomic traits, a promising field in which much remains to be discovered. RESULTS: Following the screening in vitro of tomato T-DNA lines, putative mutants altered in leaf morphology were evaluated in the greenhouse. The comparison of results in both conditions indicated a general phenotypic correspondence, showing that in vitro culture is a reliable system for finding mutants altered in leaf development. Apart from providing homogeneous conditions, the main advantage of screening in vitro lies in the enormous time and space saving. Studies on the association between phenotype and nptII gene expression showed co-segregation in two lines (P > 99%). The use of an enhancer trap also allowed identifying gain-of-function mutants through reporter expression analysis. These studies suggested that genes altered in three other mutants were T-DNA tagged. New mutants putatively altered in brassinosteroid synthesis or perception, mutations determining multiple pleiotropic effects, lines affected in organ curvature, and the first tomato mutant with helical growth were discovered. Results also revealed new possible links between leaf development and agronomic traits, such as axillary branching, flower abscission, fruit development and fruit cracking. Furthermore, we found that the gene tagged in mutant 2635-MM encodes a Sterol 3-beta-glucosyltransferase. Expression analysis suggested that abnormal leaf development might be due to the lack-off-function of this gene. CONCLUSION: In vitro culture is a quick, efficient and reliable tool for identifying tomato mutants altered in leaf morphology. The characterization of new mutants in vivo revealed new links between leaf development and some agronomic traits. Moreover, the possible implication of a gene encoding a Sterol 3-beta-glucosyltransferase in tomato leaf development is reported.


Assuntos
Glucosiltransferases/genética , Lycopersicon esculentum/genética , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Frutas/enzimologia , Frutas/genética , Frutas/crescimento & desenvolvimento , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/crescimento & desenvolvimento , Mutação , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética
7.
BMC Plant Biol ; 19(1): 124, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940071

RESUMO

BACKGROUND: Acetolactate synthase (ALS)-inhibiting herbicide tribenuron-methyl (TBM) is an efficient gametocide that can cause rapeseed (Brassica napus L.) to become male sterile and outcrossing. To find the reason the TBM treatment leads to male sterility, an integrated study using cytological, physiological, and transcriptomic methods was conducted. RESULTS: Some temporary symptoms, including the discoloration of young leaves and a short halt of raceme elongation, were observed in the rapeseed plants exposed to TBM at an application rate of 1 µg per plant. Both chloroplasts in young leaves and plastids in anthers were deformed. TBM also reduced the leaf photosynthetic rate and the contents of chlorophyll, soluble sugar and pyruvate. Both the tapetal cells and uni-nucleate microspores in the treated plants showed large autophagic vacuoles, and the tissue degenerated quickly. A transcriptomic comparison with the control identified 200 upregulated and 163 downregulated differential expression genes in the small flower buds of the TBM treatment. The genes encoding functionally important proteins, including glucan endo-1,3-beta-glucosidase A6, QUARTET3 (QRT3), ARABIDOPSIS ANTHER 7 (ATA7), non-specific lipid-transfer protein LTP11 and LTP12, histone-lysine N-methyltransferase ATXR6, spermidine coumaroyl-CoA acyltransferase (SCT), and photosystem II reaction centre protein psbB, were downregulated by TBM exposure. Some important genes encoding autophagy-related protein ATG8a and metabolic detoxification related proteins, including DTX1, DTX6, DTX35, cytosolic sulfotransferase SOT12, and six members of glutathione S-transferase, were upregulated. In addition, several genes related to hormone stimulus, such as 1-aminocyclopropane-1-carboxylate synthase 8 (ACS8), ethylene-responsive factor ERF1A, ERF1, ERF71, CRF6, and RAP2-3, were also upregulated. The transcriptional regulation is in accordance with the functional abnormalities of pollen wall formation, lipid metabolism, chloroplast structure, ethylene generation, cell cycle, and tissue autophagy. CONCLUSION: The results suggested that except for ALS, the metabolic pathways related to lipid metabolism, pollen exine formation, photosynthesis and hormone response are associated with male sterility induced by TBM. The results provide new insight into the molecular mechanisms of inducing male sterility by sulfonylurea.


Assuntos
Acetolactato Sintase/antagonistas & inibidores , Sulfonatos de Arila/farmacologia , Brassica napus/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Herbicidas/farmacologia , Infertilidade das Plantas/efeitos dos fármacos , Acetolactato Sintase/metabolismo , Brassica napus/enzimologia , Brassica napus/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/metabolismo
8.
Int J Mol Sci ; 20(8)2019 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-31010077

RESUMO

Purple acid phosphatase (PAP) encoding genes are a multigene family. PAPs require iron (Fe) to exert their functions that are involved in diverse biological roles including Fe homeostasis. However, the possible roles of PAPs in response to excess Fe remain unknown. In this study, we attempted to understand the regulation of PAPs by excess Fe in tea plant (Camellia sinensis). A genome-wide investigation of PAP encoding genes identified 19 CsPAP members based on the conserved motifs. The phylogenetic analysis showed that PAPs could be clustered into four groups, of which group II contained two specific cysteine-containing motifs "GGECGV" and "YERTC". To explore the expression patterns of CsPAP genes in response to excessive Fe supply, RNA-sequencing (RNA-seq) analyses were performed to compare their transcript abundances between tea plants that are grown under normal and high iron conditions, respectively. 17 members were shown to be transcribed in both roots and leaves. When supplied with a high amount of iron, the expression levels of four genes were significantly changed. Of which, CsPAP15a, CsPAP23 and CsPAP27c were shown as downregulated, while the highly expressed CsPAP10a was upregulated. Moreover, CsPAP23 was found to be alternatively spliced, suggesting its post-transcriptional regulation. The present work implicates that some CsPAP genes could be associated with the responses of tea plants to the iron regime, which may offer a new direction towards a further understanding of iron homeostasis and provide the potential approaches for crop improvement in terms of iron biofortification.


Assuntos
Fosfatase Ácida/genética , Camellia sinensis/enzimologia , Glicoproteínas/genética , Ferro/metabolismo , Proteínas de Plantas/genética , Fosfatase Ácida/classificação , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Camellia sinensis/genética , Genes de Plantas , Glicoproteínas/classificação , Glicoproteínas/metabolismo , Família Multigênica , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Processamento de RNA , Alinhamento de Sequência , Transcriptoma
9.
Molecules ; 24(8)2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999664

RESUMO

Melatonin can increase plant resistance to stress, and exogenous melatonin has been reported to promote stress resistance in plants. In this study, a melatonin biosynthesis-related SlCOMT1 gene was cloned from tomato (Solanum lycopersicum Mill. cv. Ailsa Craig), which is highly expressed in fruits compared with other organs. The protein was found to locate in the cytoplasm. Melatonin content in SlCOMT1 overexpression transgenic tomato plants was significantly higher than that in wild-type plants. Under 800 mM NaCl stress, the transcript level of SlCOMT1 in tomato leaf was positively related to the melatonin contents. Furthermore, compared with that in wild-type plants, levels of superoxide and hydrogen peroxide were lower while the content of proline was higher in SlCOMT1 transgenic tomatoes. Therefore, SlCOMT1 was closely associated with melatonin biosynthesis confers the significant salt tolerance, providing a clue to cope with the growing global problem of salination in agricultural production.


Assuntos
Lycopersicon esculentum , Melatonina , Metiltransferases , Proteínas de Plantas , Plantas Geneticamente Modificadas , Estresse Salino , Tolerância ao Sal , Frutas/enzimologia , Frutas/genética , Peróxido de Hidrogênio/metabolismo , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/genética , Melatonina/biossíntese , Melatonina/genética , Metiltransferases/biossíntese , Metiltransferases/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética
10.
Plant Sci ; 280: 355-366, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30824015

RESUMO

Glycinebetaine has been widely considered as an effective protectant against abiotic stress in plants, and also found to promote plant growth under normal growing conditions, especially during the reproductive stage. Betaine aldehyde dehydrogenase (BADH) and choline oxidase (COD) are two key enzymes which have been used to confer glycinebetaine synthesis in plant which normally does not synthesis glycinebetaine. In this study, we used the tomato (Solanum lycopersicum, cv 'Moneymaker') plants of wild-type and the transgenic lines codA (L1, L2) and BADH (2, 46), which were transformed with codA and BADH, respectively, to study the impact of glycinebetaine on tomato fruit development. Our results showed that the codA and BADH transgenes induced the formation of enlarged flowers and fruits in transgenic tomato plants. In addition, the transgenic tomato plants had a higher photosynthetic rate, higher assimilates content, and higher leaf chlorophyll content than the wild-type plants. We also found that the enlargement of fruit size was related to the contents of phytohormones, such as auxin, brassinolide, gibberellin, and cytokinin. Additionally, qPCR results indicated that the expressions levels of certain genes related to fruit growth and development were also elevated in transgenic plants. Finally, transcriptome sequencing results revealed that the differences in the levels of gene expression in tomato fruit between the transgenic and wild-type plants were observed in multiple pathways, predominantly those of photosynthesis, DNA replication, plant hormone signal transduction, and biosynthesis. Taken together, our results suggest that glycinebetaine promotes tomato fruit development via multiple pathways. We propose that genetic engineering of glycinebetaine synthesis offers a novel approach to enhance the productivity of tomato and other crop plants.


Assuntos
Oxirredutases do Álcool/metabolismo , Betaína-Aldeído Desidrogenase/metabolismo , Betaína/metabolismo , Lycopersicon esculentum/genética , Reguladores de Crescimento de Planta/metabolismo , Transcriptoma , Oxirredutases do Álcool/genética , Arthrobacter/enzimologia , Arthrobacter/genética , Betaína-Aldeído Desidrogenase/genética , Clorofila/metabolismo , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Frutas/enzimologia , Frutas/genética , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Engenharia Genética , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/crescimento & desenvolvimento , Fotossíntese , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Spinacia oleracea/enzimologia , Spinacia oleracea/genética , Transgenes
11.
Planta ; 249(6): 1963-1975, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30900084

RESUMO

MAIN CONCLUSION: Plastid genome engineering is an effective method to generate drought-resistant potato plants accumulating glycine betaine in plastids. Glycine betaine (GB) plays an important role under abiotic stress, and its accumulation in chloroplasts is more effective on stress tolerance than that in cytosol of transgenic plants. Here, we report that the codA gene from Arthrobacter globiformis, which encoded choline oxidase to catalyze the conversion of choline to GB, was successfully introduced into potato (Solanum tuberosum) plastid genome by plastid genetic engineering. Two independent plastid-transformed lines were isolated and confirmed as homoplasmic via Southern-blot analysis, in which the mRNA level of codA was much higher in leaves than in tubers. GB accumulated in similar levels in both leaves and tubers of codA-transplastomic potato plants (referred to as PC plants). The GB content was moderately increased in PC plants, and compartmentation of GB in plastids conferred considerably higher tolerance to drought stress compared to wild-type (WT) plants. Higher levels of relative water content and chlorophyll content under drought stress were detected in the leaves of PC plants compared to WT plants. Moreover, PC plants presented a significantly higher photosynthetic performance as well as antioxidant enzyme activities during drought stress. These results suggested that biosynthesis of GB by chloroplast engineering was an effective method to increase drought tolerance.


Assuntos
Oxirredutases do Álcool/metabolismo , Arthrobacter/enzimologia , Betaína/metabolismo , Solanum tuberosum/enzimologia , Oxirredutases do Álcool/genética , Arthrobacter/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cloroplastos/enzimologia , Cloroplastos/genética , Secas , Engenharia Genética , Fotossíntese , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/enzimologia , Plastídeos/genética , Solanum tuberosum/genética , Solanum tuberosum/fisiologia , Estresse Fisiológico
12.
Int J Mol Sci ; 20(6)2019 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-30884876

RESUMO

Artificial light at night (ALAN) is an increasing phenomenon worldwide that can cause a series of biological and ecological effects, yet little is known about its potential interaction with other stressors in aquatic ecosystems. Here, we tested whether the impact of lead (Pb) on litter decomposition was altered by ALAN exposure using an indoor microcosm experiment. The results showed that ALAN exposure alone significantly increased leaf litter decomposition, decreased the lignin content of leaf litter, and altered fungal community composition and structure. The decomposition rate was 51% higher in Pb with ALAN exposure treatments than in Pb without ALAN treatments, resulting in increased microbial biomass, ß-glucosidase (ß-G) activity, and the enhanced correlation between ß-G and litter decomposition rate. These results indicate that the negative effect of Pb on leaf litter decomposition in aquatic ecosystems may be alleviated by ALAN. In addition, ALAN exposure also alters the correlation among fungi associated with leaf litter decomposition. In summary, this study expands our understanding of Pb toxicity on litter decomposition in freshwater ecosystems and highlights the importance of considering ALAN when assessing environmental metal pollutions.


Assuntos
Água Doce/análise , Água Doce/microbiologia , Chumbo/toxicidade , Iluminação , Biomassa , Ecossistema , Poluição Ambiental/efeitos adversos , Poluição Ambiental/análise , Fungos/efeitos dos fármacos , Fungos/genética , Fungos/isolamento & purificação , Fungos/efeitos da radiação , Hidrólise/efeitos dos fármacos , Hidrólise/efeitos da radiação , Iluminação/efeitos adversos , Iluminação/métodos , Lignina/análise , Metagenômica , Folhas de Planta/química , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/efeitos da radiação
13.
Molecules ; 24(3)2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717241

RESUMO

Theanine, a unique amino acid in Camellia sinensis, accounts for more than 50% of total free amino acids in tea and has a significant contribution to the quality of green tea. Previous research indicated that theanine is synthesized from glutamic acid (Glu) and ethylamine mainly in roots, and that theanine accumulation depends on the availability of ethylamine which is derived from alanine (Ala) decarboxylation catalyzed by alanine decarboxylase (AlaDC). However, the specific gene encoding AlaDC protein remains to be discovered in tea plants or in other species. To explore the gene of AlaDC in tea plants, the differences in theanine contents and gene expressions between pretreatment and posttreatment of long-time nitrogen starvation were analyzed in young roots of two tea cultivars. A novel gene annotated as serine decarboxylase (SDC) was noted for its expression levels, which showed high consistency with theanine content, and the expression was remarkably high in young roots under sufficient nitrogen condition. To verify its function, full-length complementary DNA (cDNA) of this candidate gene was cloned from young roots of tea seedlings, and the target protein was expressed and purified from Escherichia coli (E. coli). The enzymatic activity of the protein for Ala and Ser was measured in vitro using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). The results illustrated that the target protein could catalyze the decarboxylation of Ala despite of its high similarity with SDC from other species. Therefore, this novel gene was identified as AlaDC and named CsAlaDC. Furthermore, the gene expression levels of CsAlaDC in different tissues of tea plants were also quantified with quantitative real-time PCR (qRT-PCR). The results suggest that transcription levels of CsAlaDC in root tissues are significantly higher than those in leaf tissues. That may explain why theanine biosynthesis preferentially occurs in the roots of tea plants. The expression of the gene was upregulated when nitrogen was present, suggesting that theanine biosynthesis is regulated by nitrogen supply and closely related to nitrogen metabolism for C. sinensis. The results of this study are significant supplements to the theanine biosynthetic pathway and provide evidence for the differential accumulation of theanine between C. sinensis and other species.


Assuntos
Alanina/metabolismo , Camellia sinensis/genética , Carboxiliases/genética , Regulação da Expressão Gênica de Plantas , Glutamatos/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/genética , Camellia sinensis/enzimologia , Carboxiliases/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Etilaminas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Nitrogênio/deficiência , Nitrogênio/farmacologia , Especificidade de Órgãos , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Plântula/enzimologia , Plântula/genética , Serina/metabolismo , Chá
14.
Plant Biol (Stuttg) ; 21(4): 595-603, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30734982

RESUMO

Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1-aminocyclopropane-1-carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO-associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence. Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography-tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms. Our observations showed that the loss-of-function acs1-1 mutant ameliorated age- or dark-induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1-1 reduced ACC accumulation mainly in mature leaves and that acs1-1-promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO-deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence. These findings suggest that NOA1-dependent NO accumulation blocked the ACS1-induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.


Assuntos
Aminoácidos Cíclicos/metabolismo , Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Liases/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Glucuronidase/metabolismo , Liases/fisiologia , Óxido Nítrico Sintase/fisiologia , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Transcriptoma
15.
Phytochemistry ; 159: 137-147, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30611873

RESUMO

Stilbene phytoalexins derived from grapevine can be rapidly accumulated when exposed to an artificial UV-C treatment. However, the underlying mechanisms involved in this accumulation and translocation are unclear. Here, we describe an investigation of the influence of UV-C treatment on the dynamic subcellular distribution of a member of a stilbene synthase family VpSTS29 derived from Chinese wild Vitis pseudoreticulata W.T. Wang when over-expressed in V. vinifera L. cv. Thompson Seedless. Our results show that VpSTS29-GFP was accumulated at a relatively high level in roots and mature leaves of transgenic grape lines, and was predominantly distributed in the cytoplasm. When exposed to UV-C irradiation, VpSTS29 displayed UV-induced feature coupled with the accumulation of stilbene compounds. Notably, VpSTS29-GFP can be translocated from the cytoplasm into chloroplasts upon UV-irradiation. Leaves from the two VpSTS29-GFP-expressing lines displayed more serious UV damage, showing withering and marginal scorching phenotype, and decreased content of H2O2, compared to the untransformed plant. Also, overexpression of VpSTS29 altered the expression of genes related to redox regulation, stilbene biosynthesis and light stimulus. Co-expression of VpSTS29-GFP with Glycolate oxidase 1 (myc-VpGLO1) confirmed the ability of stilbenes to decrease the content of H2O2 in Arabidopsis mesophyll protoplasts. These results provide new insight into the biological functions and properties of stilbene synthase and its product in response to environmental stimulus.


Assuntos
Aciltransferases/metabolismo , Raios Ultravioleta , Vitis/efeitos da radiação , Aciltransferases/genética , Citosol/enzimologia , Regulação para Baixo , Oxirredução , Estresse Oxidativo/efeitos da radiação , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Plantas Geneticamente Modificadas , Transporte Proteico , Estilbenos/metabolismo , Vitis/enzimologia
16.
Plant Physiol Biochem ; 135: 322-330, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30599309

RESUMO

In this study, we investigated how 6.6 mM thiram induces to stress response in tomato and evaluated the possible protective role of different concentration of salicylic acid (0.01, 0.1 and 1 mM SA) against thiram toxicity by analyzing tomato leaf samples taken on the 1st, 5th, 11th day of the treatment. The thiram treatment resulted in oxidative stress through an increase in hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels in a time-dependent manner and led to a decline in the total chlorophyll and carotenoid levels. However, thiram-treated plants induced antioxidant enzyme activities, including catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2), and ascorbate peroxidase (APX; EC 1.11.1.11), as well as pesticide detoxification enzymes such as peroxidase (POX; EC 1.11.1.7) and glutathione S-transferase (GST; EC.2.5.1.18). In addition, three genes (GST1, GST2, GST3) that encode for glutathione S-transferase and one gene (P450) that encodes for cytochrome P-450 monooxygenases were upregulated. SA showed a positive effect on the plants treated with thiram thanks to the decrease in the H2O2 and MDA levels, the enhancement of photosynthetic pigments, and the regulation in antioxidant enzyme activities in the tomato leaves. In addition, the SA-pretreatment triggered the activity and expression of pesticide detoxification enzymes in the thiram-treated leaves. Particularly the pretreatment with 1 mM SA significantly improved the activity of GST and led to the upregulation of GST1, GST2, GST3, and P450 expression levels. These results indicate that the application of thiram fungicide causes toxicity; however, the damaging effect could be mitigated through pretreatment with SA.


Assuntos
Ascorbato Peroxidases/metabolismo , Catalase/metabolismo , Glutationa Redutase/metabolismo , Lycopersicon esculentum/efeitos dos fármacos , Ácido Salicílico/farmacologia , Tiram/antagonistas & inibidores , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Genes de Plantas/efeitos dos fármacos , Glutationa Transferase/metabolismo , Peróxido de Hidrogênio/metabolismo , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/genética , Lycopersicon esculentum/metabolismo , Malondialdeído/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Ácido Salicílico/administração & dosagem , Tiram/toxicidade
17.
Arch Environ Contam Toxicol ; 76(4): 605-616, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30652214

RESUMO

Particulate matters deposition on the leaves of S. robusta were investigated during three different seasons in two tropical forests: Barjora forest, situated adjacent to heavy pollution sources, and the control, Ballavpur Wildlife Sanctuary, West Bengal, India. The purpose of this study is to measure the dust fall and foliar transfer of heavy metals (viz., Pb, Cd, Cu, Cr, Fe, Ni, Zn, and Mn) and antioxidant enzyme activities (peroxidase, catalase) in S. robusta, including the measurement of heavy metals present in the suspended particulate matter in ambient air. Dust fall on leaves and the total metal accumulation capacity of the plant were the highest during winter season with metal accumulation index of 9.82. Based on two-way ANOVA, it has been shown that there is a statistically significant difference in dust fall between the two forests and in different seasons. From cluster analysis, correlation results, and principal component analysis, it was suggested that heavy metals in Barjora may be due to the traffic emission and various industrial activities. Increased levels of peroxidase and catalase activities and the presence of high levels of reactive oxygen species in the leaves of the Barjora forest was an indication of stress state in this forest. On the basis of these findings, controlling the emission of pollutants from industrial and vehicular activities in that area is highly encouraged.


Assuntos
Poluentes Atmosféricos/análise , Antioxidantes/metabolismo , Dipterocarpaceae/enzimologia , Monitoramento Ambiental/métodos , Metais Pesados/análise , Material Particulado/análise , Dipterocarpaceae/química , Florestas , Índia , Indústrias , Folhas de Planta/química , Folhas de Planta/enzimologia , Estações do Ano
18.
Sci Total Environ ; 658: 626-637, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30580217

RESUMO

Titanium (Ti) is considered an essential element for plant growth; however, its role in crop performance through stimulating the activities of certain enzymes, enhancing chlorophyll content and photosynthesis, and improving crop morphology and growth requires more study. We therefore conducted a laboratory experiments to study the effects of ionic Ti application on morphology, growth, biomass distribution, chlorophyll fluorescence performance and Rubisco activity of soybean (Glycine max L.) under normal light (NL) and shade conditions (SC). In this study, we sprayed soybean plants with five different levels of ionic Ti (T1 = 0, T2 = 1.25, T3 = 2.5, T4 = 5 and T5 = 10 mg Ti Plant-1) through foliar application method. Our results show that with increasing moderate (2.5 mg Ti Plant-1) Ti concentration, the chlorophyll pigments (chlorophyll [Chl] a, b, carotenoid [Car]), plant biomass, photochemical efficiency of photosystem II (Fv/Fm), and electron transport rate (ETR) of soybean increased, but higher levels (5-10 mg Ti Plant-1), resulted in leaf anatomical and chloroplast structural disruptions under both NL and SC. Soybean plants showed maximum biomass, leaf area, leaf thickness, Chl a, b, Car, Rubisco activity, Fv/Fm and ETR for T3 at 2.5 mg Ti Plant-1; however, declined significantly for T5 at high concentration of 10 mg Plant-1. In NL, the application of 2.5 mg Ti Plant-1 (T3) increased the Chl a, b, and total Chl contents 40, 20, and 27% as compared to control treatment (T1). In SC, the application of 1.25 mg Ti mg Plant-1 (T2) increased the Chl a, b, and total Chl contents 38, 19, and 14% as compared to control treatment. In NL, the Fv/Fm, qP, PSII, and ETR were higher in the T3 treatment over the T1 (control) by 7, 0.3, 16, and 16%, respectively. In SC, the Fv/Fm, qP, PSII, and ETR were higher in the T3 treatment over the T1 (control) by 5, 5, 19, and 19%, respectively. Moreover, Rubisco activity was at peak (55 and 6% increase under NL and SC) at 2.5 mg Ti Plant-1and decreased with increasing Ti concentration, reaching the lowest at 10 mg Ti Plant-1, which indicates that leaf cells were damaged as observed in the leaf anatomy. We concluded that ionic Ti expresses a hormesis effect: at lower concentrations, promoting soybean growth, however, at higher concentrations, suppressing soybean growth both under NL and SC. We therefore suggest that under different light stress conditions, Ti application could serve to mitigate abiotic stresses, especially in intercropping systems.


Assuntos
Luz , Folhas de Planta/efeitos dos fármacos , Soja/efeitos dos fármacos , Titânio/metabolismo , Biomassa , Clorofila/fisiologia , Cloroplastos/metabolismo , Relação Dose-Resposta a Droga , Fluorescência , Folhas de Planta/anatomia & histologia , Folhas de Planta/enzimologia , Folhas de Planta/fisiologia , Raízes de Plantas/anatomia & histologia , Ribulose-Bifosfato Carboxilase/metabolismo , Soja/anatomia & histologia , Soja/enzimologia , Soja/fisiologia , Titânio/administração & dosagem
19.
Ecotoxicol Environ Saf ; 167: 345-353, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30359901

RESUMO

Large amounts of heavy metals end up in the environment as a result of ever-increasing anthropogenic activities and economic development. At least two specific types of heavy metals occur in the soil sub-ecosystem in most regions of China, especially in farmland. The morphological and physiological performance of wheat play a vital role in its growth and development, but heavy metals, both occurring independently and combined, may affect wheat growth. Thus, this study examined different concentrations of two types of heavy metals (copper (Cu), lead (Pb), and Cu and Pb combined) on the morphological and physiological performance of wheat. The number of tillers, plant height, ground diameter, single-leaf fresh and dry weights, leaf thickness, single-plant fresh and dry weights, leaf chlorophyll and N contents, and plant peroxidase and catalase activities of wheat significantly decreased when treated with two types of heavy metals. Thus, the two types of heavy metals significantly reduced the morphological and physiological performance of wheat; the ecotoxicological effects of heavy metals on the morphological and physiological performance of wheat increased with increasing heavy metal concentrations, especially on the leaf chlorophyll and N contents of wheat. The ground diameter and single-plant fresh weight of wheat in the Pb treatment were significantly lower than those under the independent Cu treatment with same concentration. Thus, the Pb treatment exerted more toxic effects on the morphological performance of wheat than the independent Cu treatment. The ground diameter, single-leaf dry weight, leaf thickness, single-plant fresh weight, and plant proline content of wheat under the combined Cu and Pb were significantly lower than those in the Cu and/or the Pb treatments. The combined Cu and Pb treatments addressed synergistic effects on the morphological and physiological performance of wheat. Accordingly, the growth performance of wheat will be significantly reduced in the combined Cu and Pb treatments compared with the independent Cu or Pb treatments.


Assuntos
Cobre/toxicidade , Chumbo/toxicidade , Poluentes do Solo/toxicidade , Triticum/efeitos dos fármacos , Triticum/crescimento & desenvolvimento , Catalase/metabolismo , Clorofila/análise , Ecotoxicologia , Nitrogênio/análise , Peroxidase/metabolismo , Folhas de Planta/química , Folhas de Planta/enzimologia , Prolina/análise , Triticum/química
20.
BMC Plant Biol ; 18(1): 251, 2018 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-30348089

RESUMO

BACKGROUND: Nitrilases are nitrile-converting enzymes commonly found within the plant kingdom that play diverse roles in nitrile detoxification, nitrogen recycling, and phytohormone biosynthesis. Although nitrilases are present in all higher plants, little is known about their function in trees. Upon herbivory, poplars produce considerable amounts of toxic nitriles such as benzyl cyanide, 2-methylbutyronitrile, and 3-methylbutyronitrile. In addition, as byproduct of the ethylene biosynthetic pathway upregulated in many plant species after herbivory, toxic ß-cyanoalanine may accumulate in damaged poplar leaves. In this work, we studied the nitrilase gene family in Populus trichocarpa and investigated the potential role of the nitrilase PtNIT1 in the catabolism of herbivore-induced nitriles. RESULTS: A BLAST analysis revealed three putative nitrilase genes (PtNIT1, PtNIT2, PtNIT3) in the genome of P. trichocarpa. While PtNIT1 was expressed in poplar leaves and showed increased transcript accumulation after leaf herbivory, PtNIT2 and PtNIT3 appeared not to be expressed in undamaged or herbivore-damaged leaves. Recombinant PtNIT1 produced in Escherichia coli accepted biogenic nitriles such as ß-cyanoalanine, benzyl cyanide, and indole-3-acetonitrile as substrates in vitro and converted them into the corresponding acids. In addition to this nitrilase activity, PtNIT1 showed nitrile hydratase activity towards ß-cyanoalanine, resulting in the formation of the amino acid asparagine. The kinetic parameters of PtNIT1 suggest that the enzyme utilizes ß-cyanoalanine and benzyl cyanide as substrates in vivo. Indeed, ß-cyanoalanine and benzyl cyanide were found to accumulate in herbivore-damaged poplar leaves. The upregulation of ethylene biosynthesis genes after leaf herbivory indicates that herbivore-induced ß-cyanoalanine accumulation is likely caused by ethylene formation. CONCLUSIONS: Our data suggest a role for PtNIT1 in the catabolism of herbivore-induced ß-cyanoalanine and benzyl cyanide in poplar leaves.


Assuntos
Aminoidrolases/metabolismo , Nitrilos/metabolismo , Populus/enzimologia , Alanina/análogos & derivados , Alanina/metabolismo , Aminoidrolases/genética , Herbivoria , Folhas de Planta/enzimologia , Folhas de Planta/genética , Populus/genética
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