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1.
Plant Mol Biol ; 100(3): 231-246, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30911876

RESUMO

KEY MESSAGE: GluA and GluB-4/5 glutelin subfamilies are mainly localized to outer region of the endosperm, particularly in its ventral side, in rice grain, but GluC is localized to throughout the endosperm. The major seed storage protein in rice (Oryza sativa) is glutelin, which forms a vacuole-derived protein body type-II. Glutelins are encoded by multiple genes, and generally comprise four protein subfamilies, namely, GluA, GluB, GluC, and GluD: however, the localization pattern of glutelin in rice grains remains obscure. In this study, we investigated the localization pattern of five subtypes of the glutelin protein in rice grains using glutelin-subtype specific antibodies. Immunoblot analysis against sequentially polished rice flour fractions from three crop years and seven japonica rice varieties revealed that GluA was strongly localized in the outer region of the endosperm, including the subaleurone layer, whereas GluC was distributed throughout the endosperm. Among the glutelin subtypes, GluA was mostly found in the outer region of the rice grain, followed by GluB-4/5, GluB-1, GluD, and GluC. Immunofluorescence labeling microscopy analysis using immature rice seeds clearly revealed that the localization pattern of GluC and GluD was completely different from that of GluA and GluB. Expression levels of all glutelins, particularly GluA, GluB-1, and GluB-4/5, were stronger on the ventral than dorsal side in rice grains. These results provide strong and consistent evidence that glutelins localize to the rice grain in a subfamily-dependent manner.


Assuntos
Grão Comestível/metabolismo , Glutens/classificação , Glutens/metabolismo , Immunoblotting/métodos , Oryza/metabolismo , Anticorpos , Grão Comestível/genética , Endosperma/metabolismo , Epitopos/imunologia , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glutens/genética , Oryza/citologia , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transporte Proteico , Sementes/metabolismo
2.
Phytomedicine ; 57: 364-376, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30831485

RESUMO

BACKGROUND: Rice callus suspension culture (RCSC) has been shown to exhibit potent antiproliferative activity in multiple cancer cell lines. RCSC and its bioactive compounds can fill the need for drugs with no side effects. HYPOTHESIS/PURPOSE: The anti-inflammatory potential of RCSC and its bioactive fractions on normal colon epithelial cell lines, was investigated. STUDY DESIGN: Three cell lines, InEpC, NCM356 and CCD841-CoN were treated with proinflammatory cytokines followed by RCSC. Cytoplasmic and nuclear ROS were assayed with fluorescent microscopy and flow cytometer. Expression analysis of immune-related genes was performed in RCSC-treated cell lines. RCSC was fractionated using column chromatography and HPLC. Pooled fractions 10-18 was used to test for antiproliferative activity using colon adenocarcinoma cell line, SW620 and anti-inflammatory activity using CCD841-CoN. Mass spectrometric analysis was performed to identify candidate compounds in four fractions. RESULTS: RCSC treatment showed differential effects with higher cytoplasmic ROS levels in NCM356 and CCD841-CoN and lower ROS levels in InEpC. Nuclear generated ROS levels increased in all three treated cell lines. Flow cytometry analysis of propidium iodide stained cells indicated mitigation of cell death caused by inflammation in RCSC treated groups in both NCM356 and CCD841-CoN. Genes encoding transcription factors and cytokines were differentially regulated in NCM356 and CCD841-CoN cell lines treated with RCSC which provided insights into possible pathways. Analysis of pooled fractions 10-18 by HPLC identified 8 peaks. Cell viability assay with fractions 10-18 using SW620 showed that the number of viable cells were greatly reduced which was similar to 6X and 33X RCSC with very little effect on normal cells which similar to 1X RCSC. RCSC fractions increased nuclear and cytoplasmic ROS vs. both untreated and inflammatory control. Analysis of four fractions by mass spectrometry identified 4-deoxyphloridzin, 5'-methoxycurcumin, piceid and lupeol as candidate compounds which are likely to be responsible for the antiproliferative, anti-inflammatory and immune-regulating properties of RCSC. CONCLUSION: RCSC and its fractions showed anti-inflammatory activity on inflamed colon epithelial cells. Downstream target candidate genes which are likely to mediate RCSC effects were identified. Candidate compounds responsible for the antiproliferative and anti-inflammatory activity of RCSC and its fractions provide possible drug targets.


Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Fatores Biológicos/farmacologia , Fatores Imunológicos/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Oryza/citologia , Técnicas de Cultura de Tecidos/métodos , Adenocarcinoma , Anti-Inflamatórios/imunologia , Antineoplásicos/química , Fatores Biológicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Neoplasias Colorretais , Citocinas/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fatores Imunológicos/química , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Oryza/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genética
3.
Nat Plants ; 5(2): 204-211, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30737514

RESUMO

During establishment of arbuscular mycorrhizal symbioses, fungal hyphae invade root cells producing transient tree-like structures, the arbuscules, where exchange of photosynthates for soil minerals occurs. Arbuscule formation and collapse lead to rapid production and degradation of plant and fungal membranes, their spatiotemporal dynamics directly influencing nutrient exchange. We determined the ultra-structural details of both membrane surfaces and the interstitial apoplastic matrix by transmission electron microscopy tomography during growth and senescence of Rhizophagus irregularis arbuscules in rice. Invasive growth of arbuscular hyphae was associated with abundant fungal membrane tubules (memtubs) and plant peri-arbuscular membrane evaginations. Similarly, the phylogenetically distant arbuscular mycorrhizal fungus, Gigaspora rosea, and the fungal maize pathogen, Ustilago maydis, developed memtubs while invading host cells, revealing structural commonalities independent of the mutualistic or parasitic outcome of the interaction. Additionally, extracellular vesicles formed continuously in the peri-arbuscular interface from arbuscule biogenesis to senescence, suggesting an involvement in inter-organismic signal and nutrient exchange throughout the arbuscule lifespan.


Assuntos
Membrana Celular/ultraestrutura , Vesículas Extracelulares/metabolismo , Micorrizas/fisiologia , Oryza/microbiologia , Células Vegetais/microbiologia , Membrana Celular/microbiologia , Tomografia com Microscopia Eletrônica , Glomeromycota/fisiologia , Hifas/fisiologia , Micorrizas/citologia , Oryza/citologia , Oryza/genética , Folhas de Planta/citologia , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Raízes de Plantas/citologia , Raízes de Plantas/microbiologia , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , Simbiose , Ustilago/patogenicidade , Zea mays/microbiologia
4.
Nat Commun ; 10(1): 619, 2019 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-30728357

RESUMO

Axis formation is a fundamental issue in developmental biology. Axis formation and patterning in plant leaves is crucial for morphology and crop productivity. Here, we reveal the basis of proximal-distal patterning in rice leaves, which consist of a proximal sheath, a distal blade, and boundary organs formed between these two regions. Analysis of the three rice homologs of the Arabidopsis BLADE-ON-PETIOLE1 (BOP1) gene indicates that OsBOPs activate proximal sheath differentiation and suppress distal blade differentiation. Temporal expression changes of OsBOPs are responsible for the developmental changes in the sheath:blade ratio. We further identify that the change in the sheath:blade ratio during the juvenile phase is controlled by the miR156/SPL pathway, which modifies the level and pattern of expression of OsBOPs. OsBOPs are also essential for differentiation of the boundary organs. We propose that OsBOPs, the main regulators of proximal-distal patterning, control temporal changes in the sheath:blade ratio of rice leaves.


Assuntos
Padronização Corporal , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Oryza/crescimento & desenvolvimento , Oryza/genética , Desenvolvimento Vegetal/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular/genética , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Oryza/anatomia & histologia , Oryza/citologia , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/citologia , Proteínas de Plantas/genética , Caules de Planta/anatomia & histologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo
5.
Plant Reprod ; 32(1): 5-13, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30756188

RESUMO

KEY MESSAGE: A detailed, step-by-step protocol for isolation of rice gametes for transcriptional profiling, with a general workflow that includes controls for RNA contamination from surrounding cells and tissues is presented. Characterization of the transcriptome and other -omics studies of flowering plant gametes are challenging as a consequence of the small sizes and relative inaccessibility of these cells. Collecting such poorly represented cells is also complicated by potential contamination from surrounding sporophytic, adjacent gametophytic tissues and difficulties in extracting high-quality intact cells. Here we present detailed, step-by-step procedures for collecting intact, unfixed rice (Oryza sativa) egg cells and sperm cells without enzymatic treatments. In addition, we also present a general workflow for assessing sample purity by RT-PCR, using primers specific for marker genes preferentially expressed in surrounding cells and tissues. These protocols should facilitate future studies of genome-scale characterization of gametes in this important model crop.


Assuntos
Separação Celular/métodos , Células Germinativas Vegetais , Oryza/citologia , Separação Celular/instrumentação , Oryza/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real
6.
Mol Plant ; 12(2): 199-214, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30639120

RESUMO

Over the past few years, three photorespiratory bypasses have been introduced into plants, two of which led to observable increases in photosynthesis and biomass yield. However, most of the experiments were carried out using Arabidopsis under controlled environmental conditions, and the increases were only observed under low-light and short-day conditions. In this study, we designed a new photorespiratory bypass (called GOC bypass), characterized by no reducing equivalents being produced during a complete oxidation of glycolate into CO2 catalyzed by three rice-self-originating enzymes, i.e., glycolate oxidase, oxalate oxidase, and catalase. We successfully established this bypass in rice chloroplasts using a multi-gene assembly and transformation system. Transgenic rice plants carrying GOC bypass (GOC plants) showed significant increases in photosynthesis efficiency, biomass yield, and nitrogen content, as well as several other CO2-enriched phenotypes under both greenhouse and field conditions. Grain yield of GOC plants varied depending on seeding season and was increased significantly in the spring. We further demonstrated that GOC plants had significant advantages under high-light conditions and that the improvements in GOC plants resulted primarily from a photosynthetic CO2-concentrating effect rather than from improved energy balance. Taken together, our results reveal that engineering a newly designed chloroplastic photorespiratory bypass could increase photosynthetic efficiency and yield of rice plants grown in field conditions, particularly under high light.


Assuntos
Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Engenharia Genética , Luz , Oryza/citologia , Oryza/genética , Fotossíntese/genética , Dióxido de Carbono/metabolismo , Respiração Celular/genética , Respiração Celular/efeitos da radiação , Metabolismo Energético/genética , Metabolismo Energético/efeitos da radiação , Oryza/metabolismo , Oryza/efeitos da radiação , Fenótipo , Fotossíntese/efeitos da radiação , Plantas Geneticamente Modificadas
7.
Plant Mol Biol ; 99(1-2): 175-191, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30610522

RESUMO

KEY MESSAGE: OsMS1 functions as a transcriptional activator and interacts with known tapetal regulatory factors through its plant homeodomain (PHD) regulating tapetal programmed cell death (PCD) and pollen exine formation in rice. The tapetum, a hallmark tissue in the stamen, undergoes degradation triggered by PCD during post-meiotic anther development. This degradation process is indispensable for anther cuticle and pollen exine formation. Previous study has shown that PTC1 plays a critical role in the regulation of tapetal PCD. However, it remained unclear how this occurs. To further investigate the role of this gene in rice, we used CRISPR/Cas9 system to generate the homozygous mutant named as osms1, which showed complete male sterility with slightly yellow and small anthers, as well as invisible pollen grains. In addition, cytological observation revealed delayed tapetal PCD, defective pollen exine formation and a lack of DNA fragmentation according to a TUNEL analysis in the anthers of osms1 mutant. OsMS1, which encodes a PHD finger protein, was located in the nucleus of rice protoplasts and functioned as a transcription factor with transcriptional activation activity. Y2H and BiFC assays demonstrated that OsMS1 can interact with OsMADS15 and TDR INTERACTING PROTEIN2 (TIP2). It has been reported that TIP2 coordinated with TDR to modulate the expression of EAT1 and further regulated tapetal PCD in rice. Results of qPCR suggested that the expression of the genes associated with tapetal PCD and pollen wall biosynthesis, such as EAT1, AP37, AP25, OsC6 and OsC4, were significantly reduced in osms1 mutant. Taken together, our results demonstrate that the interaction of OsMS1 with known tapetal regulatory factors through its PHD finger regulates tapetal PCD and pollen exine formation in rice.


Assuntos
Regulação da Expressão Gênica de Plantas , Oryza/genética , Pólen/genética , Fatores de Transcrição/metabolismo , Mutação , Oryza/citologia , Oryza/crescimento & desenvolvimento , Fenótipo , Infertilidade das Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/citologia , Pólen/crescimento & desenvolvimento , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
8.
Plant Cell Rep ; 38(3): 345-359, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30649573

RESUMO

KEY MESSAGE: FLO15encodes a plastidic glyoxalase I protein, OsGLYI7, which affects compound starch granule formation and starch synthesis in rice endosperm. Starch synthesis in rice (Oryza sativa) endosperm is a sophisticated process, and its underlying molecular machinery still remains to be elucidated. Here, we identified and characterized two allelic rice floury endosperm 15 (flo15) mutants, both with a white-core endosperm. The flo15 grains were characterized by defects in compound starch granule development, along with decreased starch content. Map-based cloning of the flo15 mutants identified mutations in OsGLYI7, which encodes a glyoxalase I (GLYI) involved in methylglyoxal (MG) detoxification. The mutations of FLO15/OsGLYI7 resulted in increased MG content in flo15 developing endosperms. FLO15/OsGLYI7 localizes to the plastids, and the in vitro GLYI activity derived from flo15 was significantly decreased relative to the wild type. Moreover, the expression of starch synthesis-related genes was obviously altered in the flo15 mutants. These findings suggest that FLO15 plays an important role in compound starch granule formation and starch synthesis in rice endosperm.


Assuntos
Endosperma/enzimologia , Regulação da Expressão Gênica de Plantas , Lactoilglutationa Liase/metabolismo , Oryza/enzimologia , Amido/metabolismo , Grânulos Citoplasmáticos/metabolismo , Endosperma/citologia , Endosperma/genética , Genes Reporter , Lactoilglutationa Liase/genética , Mutação , Oryza/citologia , Oryza/genética , Fenótipo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastídeos/enzimologia , Sementes/citologia , Sementes/enzimologia , Sementes/genética , Técnicas do Sistema de Duplo-Híbrido
9.
Mol Plant ; 12(2): 263-277, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30578854

RESUMO

Histone H2B monoubiquitination (H2Bub1) plays important roles in several physiological and developmental processes, but its roles in the regulation of plant stress responses remain elusive. Here, we report that H2Bub1 is crucially involved in abscisic acid (ABA) signaling and drought response in rice. We found that rice HISTONE MONOUBIQUITINATION2 (OsHUB2), an E3 ligase for H2Bub1, interacted with OsbZIP46, a key transcription factor regulating ABA signaling and drought response in rice. Genetic analyses suggest that OsHUB2, upregulated by drought and ABA, positively modulates ABA sensitivity and drought resistance. The H2Bub1 levels were increased in the target genes of OsbZIP46 under the drought stress and ABA treatments, which were positively correlated with their increased expression levels. Interestingly, MODD, a reported suppressor of ABA signaling and drought resistance by mediating OsbZIP46 deactivation and degradation, could reduce the H2Bub1 levels in the target genes of OsbZIP46 by recruiting a putative deubiquitinase OsOTLD1. Suppression of OsOTLD1 in vivo resulted in increased H2Bub1 levels and expression of OsbZIP46 target genes. Collectively, these findings established an elaborate mechanism of histone monoubiquitination in the fine-turning of ABA signaling and drought response by balancing H2Bub1 deposition and removal.


Assuntos
Ácido Abscísico/metabolismo , Secas , Histonas/metabolismo , Oryza/citologia , Oryza/metabolismo , Transdução de Sinais , Ubiquitinação , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Estresse Fisiológico
10.
Int J Mol Sci ; 19(12)2018 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-30545137

RESUMO

In flowering plants, ideal male reproductive development requires the systematic coordination of various processes, in which timely differentiation and degradation of the anther wall, especially the tapetum, is essential for both pollen formation and anther dehiscence. Here, we show that OsGPAT3, a conserved glycerol-3-phosphate acyltransferase gene, plays a critical role in regulating anther wall degradation and pollen exine formation. The gpat3-2 mutant had defective synthesis of Ubisch bodies, delayed programmed cell death (PCD) of the inner three anther layers, and abnormal degradation of micropores/pollen grains, resulting in failure of pollen maturation and complete male sterility. Complementation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) experiments demonstrated that OsGPAT3 is responsible for the male sterility phenotype. Furthermore, the expression level of tapetal PCD-related and nutrient metabolism-related genes changed significantly in the gpat3-2 anthers. Based on these genetic and cytological analyses, OsGPAT3 is proposed to coordinate the differentiation and degradation of the anther wall and pollen grains in addition to regulating lipid biosynthesis. This study provides insights for understanding the function of GPATs in regulating rice male reproductive development, and also lays a theoretical basis for hybrid rice breeding.


Assuntos
Apoptose , Oryza/citologia , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Pólen/citologia , Pólen/crescimento & desenvolvimento , Sequência de Bases , Mapeamento Cromossômico , Fragmentação do DNA , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Estudos de Associação Genética , Teste de Complementação Genética , Mutação/genética , Oryza/genética , Fenótipo , Infertilidade das Plantas/genética , Proteínas de Plantas/genética , Pólen/metabolismo , Pólen/ultraestrutura , Reprodutibilidade dos Testes
11.
Genes Genomics ; 40(3): 233-241, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29892794

RESUMO

Cullin-RING E3 ubiquitin ligase (CRL) complex is known as the largest family of E3 ligases. The most widely characterized CRL, SCF complex (CRL1), utilizes CUL1 as a scaffold protein to assemble the complex components. To better understand CRL1-mediated cellular processes in rice, three CUL1 genes (OsCUL1s) were isolated in Oryza sativa. Although all OsCUL1 proteins exhibited high levels of amino acid similarities with each other, OsCUL1-3 had a somewhat distinct structure from OsCUL1-1 and OsCUL1-2. Basal expression levels of OsCUL1-3 were much lower than those of OsCUL1-1 and OsCUL1-2 in all selected samples, showing that OsCUL1-1 and OsCUL1-2 play predominant roles relative to OsCUL1-3 in rice. OsCUL1-1 and OsCUL1-2 genes were commonly upregulated in dry seeds and by ABA and salt/drought stresses, implying their involvement in ABA-mediated processes. These genes also showed similar expression patterns in response to various hormones and abiotic stresses, alluding to their functional redundancy. Expression of the OsCUL1-3 gene was also induced in dry seeds and by ABA-related salt and drought stresses, implying their participation in ABA responses. However, its expression pattern in response to hormones and abiotic stresses was somehow different from those of the OsCUL1-1 and OsCUL1-2 genes. Taken together, these findings suggest that the biological role and function of OsCUL1-3 may be distinct from those of OsCUL1-1 and OsCUL1-2. The results of expression analysis of OsCUL1 genes in this study will serve as a useful platform to better understand overlapping and distinct roles of OsCUL1 proteins and CRL1-mediated cellular processes in rice plants.


Assuntos
Proteínas Culina/genética , Oryza/genética , Sequência de Aminoácidos , Proteínas Culina/biossíntese , Proteínas Culina/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Genes Reguladores , Germinação , Hormônios/farmacologia , Oryza/citologia , Oryza/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/genética , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/metabolismo , Estresse Fisiológico/genética , Ubiquitina-Proteína Ligases/química , Ubiquitinação
12.
Carbohydr Polym ; 192: 273-281, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29691021

RESUMO

Miscanthus is a leading bioenergy crop and rice provides enormous biomass for biofuels. Using Calcofluor White staining, this work in situ observed an initial lignocellulose hydrolysis in two distinct Miscanthus accessions, rice cultivar (NPB), and Osfc16 mutant after mild chemical pretreatments. In comparison, the M. sin and Osfc16 respectively exhibited weak Calcofluor fluorescence compared to the M. sac and NPB during enzymatic hydrolysis, consistent with the high biomass saccharification detected in vitro. Using xyloglucan-directed monoclonal antibodies (mAbs), xyloglucan deconstruction was observed from initial cellulose hydrolysis, whereas the M. sin and Osfc16 exhibited relatively strong immunolabeling using xylan-directed mAb, confirming previous findings of xylan positive impacts on biomass saccharification. Furthermore, the M. sin showed quick disappearance of RG-I immunolabeling with varied HG labelings between acid and alkali pretreatments. Hence, this study demonstrated a quick approach to explore wall polymer distinct deconstruction for enhanced biomass saccharification under chemical pretreatment in bioenergy crops.


Assuntos
Biomassa , Biopolímeros/metabolismo , Parede Celular/metabolismo , Oryza/citologia , Oryza/efeitos dos fármacos , Poaceae/citologia , Poaceae/efeitos dos fármacos , Metabolismo dos Carboidratos/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Celulose/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Hidróxidos/farmacologia , Oryza/metabolismo , Poaceae/metabolismo , Compostos de Potássio/farmacologia
13.
Plant Sci ; 270: 85-96, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29576089

RESUMO

Small signaling peptides play important roles in plant development and responses to abiotic and biotic stresses. We have identified a novel small peptide gene in rice, OsDSSR1, which is expressed mainly in the root, stem, node, leaf, and panicle. OsDSSR1 expression is also induced by drought, salinity, ABA, and H2O2 treatment. OsDSSR1 is localized in the nucleus and cytoplasm. Transgenic plants overexpressing OsDSSR1 exhibited enhanced drought stress tolerance and decreased ABA sensitivity as compared to the wild type. Overexpression of OsDSSR1 promoted the accumulation of compatible osmolytes, such as free proline and soluble sugars. OsDSSR1-overexpressing plants displayed enhanced OsSodCc2 and OscAPX expression and superoxide dismutase and ascorbate peroxidase activities under drought stress. RNA-sequencing data revealed that the expression of 72 abiotic stress-responsive genes was significantly altered in homozygous transgenic plants. These stress-responsive candidate genes will aid in expanding our understanding of the mechanisms by which small peptides mediate tolerance in crop species.


Assuntos
Ácido Abscísico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oryza/genética , Reguladores de Crescimento de Planta/metabolismo , Sequência de Aminoácidos , Secas , Expressão Gênica , Genes Reporter , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oryza/citologia , Oryza/efeitos dos fármacos , Oryza/fisiologia , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Prolina/metabolismo , Plântula/citologia , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/fisiologia , Alinhamento de Sequência , Estresse Fisiológico , Açúcares/metabolismo
14.
Plant Cell Physiol ; 59(5): 887-902, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29566164

RESUMO

Lesion mimic mutants are powerful tools for unveiling the molecular connections between cell death and pathogen resistance. Various proteins responsible for lesion mimics have been identified; however, the mechanisms underlying lesion formation and pathogen resistance are still unknown. Here, we identify a lesion mimic mutant in rice, lesion mimic leaf 1 (lml1). The lml1 mutant exhibited abnormal cell death and resistance to both bacterial blight and rice blast. LML1 is expressed in all types of leaf cells, and encodes a novel eukaryotic release factor 1 (eRF1) protein located in the endoplasmic reticulum. Protein sequences of LML1 orthologs are conserved in yeast, animals and plants. LML1 can partially rescue the growth delay phenotype of the LML1 yeast ortholog mutant, dom34. Both lml1 and mutants of AtLML1 (the LML1 Arabidopsis ortholog) exhibited a growth delay phenotype like dom34. This indicates that LML1 and its orthologs are functionally conserved. LML1 forms a functional complex with a eukaryotic elongation factor 1A (eEF1A)-like protein, SPL33/LMM5.1, whose mutant phenotype was similar to the lml1 phenotype. This complex was conserved between rice and yeast. Our work provides new insight into understanding the mechanism of cell death and pathogen resistance, and also lays a good foundation for studying the fundamental molecular function of Pelota/DOM34 and its orthologs in plants.


Assuntos
Sequência Conservada , Resistência à Doença , Oryza/citologia , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Morte Celular , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Mapeamento Cromossômico , Relógios Circadianos/efeitos da radiação , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Luz , Magnaporthe/fisiologia , Oryza/genética , Oryza/imunologia , Fenótipo , Fotoperíodo , Filogenia , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica , Temperatura Ambiente
15.
Photosynth Res ; 137(1): 53-67, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29392476

RESUMO

Isolation of leaf-color mutants is important in understanding the mechanisms of chloroplast biogenesis and development. In this study, we identified and characterized a rice (Oryza sativa) mutant, yellow leaf 2 (yl2), exhibiting pale yellow leaves with a few longitudinal white stripes at the early seedling stage then gradually turning yellow. Genetic analyses revealed that YL2 encodes a thylakoid membrane-localized protein with significant sequence similarity to UMP kinase proteins in prokaryotes and eukaryotes. Prokaryotic UMP kinase activity was subsequently confirmed, with YL2 deficiency causing a significant reduction in chlorophyll accumulation and photochemical efficiency. Moreover, YL2 is also light dependent and preferentially expressed in green tissues. Chloroplast development was abnormal in the yl2 mutant, possibly due to reduced accumulation of thylakoid membranes and a lack of normal stroma lamellae. 2D Blue-Native SDS-PAGE and immunoblot analyses revealed a reduction in several subunits of photosynthetic complexes, in particular, the AtpB subunit of ATP synthase, while mRNA levels of corresponding genes were unchanged or increased compared with the wild type. In addition, we observed a significant decrease (ca. 36.3%) in cpATPase activity in the yl2 mutant compared with the wild type. Taken together, our results suggest that UMP kinase activity plays an essential role in chloroplast development and regulating cpATPase biogenesis in rice.


Assuntos
Cloroplastos/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Oryza/citologia , Proteínas de Plantas/metabolismo , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Cloroplastos/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Mutação , Núcleosídeo-Fosfato Quinase/genética , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Tilacoides/metabolismo
16.
Methods Mol Biol ; 1675: 443-454, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29052206

RESUMO

Chromatin organization in eukaryotes is highly dynamic, playing fundamental roles in regulating diverse nuclear processes including DNA replication, transcription, and repair. Thus, the analysis of chromatin organization is of great importance for the elucidation of chromatin-mediated biological processes. Immunostaining coupled with imaging is one of the most powerful tools for chromatin analysis at the cellular level. However, in plants, it is sometimes technically challenging to apply this method due to the inaccessibility of certain cell types and/or poor penetration of the reagents into plant tissues and cells. To circumvent these limitations, we developed a highly efficient protocol enabling the analysis of chromatin modifications and nuclear organization at the single-cell level with high resolution in whole-mount plant tissues. The main procedure consists of five steps: (1) tissue fixation; (2) dissection and embedding; (3) tissue processing; (4) antibody incubation; and (5) imaging. This protocol has been simplified for the processing of multiple samples without the need for laborious tissue sectioning. Additionally, it preserves cellular morphology and chromatin organization, allowing comparative analyses of chromatin organization between different cell types or developmental stages. This protocol was successfully used for various tissues of different plant species, including Arabidopsis thaliana, Oryza sativa (rice), and Zea mays (maize). Importantly, this method is very useful to analyze poorly accessible tissues, such as female meiocytes, gametophytes, and embryos.


Assuntos
Arabidopsis/metabolismo , Cromatina/metabolismo , Imunofluorescência/métodos , Oryza/metabolismo , Zea mays/metabolismo , Arabidopsis/citologia , Núcleo Celular , Cromatina/imunologia , Regulação da Expressão Gênica de Plantas , Especificidade de Órgãos , Oryza/citologia , Fixação de Tecidos , Zea mays/citologia
17.
Methods Mol Biol ; 1696: 81-90, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29086397

RESUMO

The integrity of a subcellular proteomics is largely dependent on purity of the isolated compartment away from other contaminants. If high-purity nuclei is isolated, nuclear proteomics is a useful approach for investigating the mechanisms underlying plant physiological function. Although the isolation of high-purity nuclei from tissue or organ in plant is a difficult task, successful purification has been achieved through fractionation processes. For purification, there are five protocols such as (1) differential centrifugation, (2) discontinuous Percoll gradients, (3) continuous sucrose gradients, (4) combined continuous Percoll/sucrose gradients, and (5) continuous Percoll gradients. Furthermore, because purity assessment of purified nuclei is an important step, it is also described in this chapter.


Assuntos
Proteínas Nucleares/análise , Oryza/citologia , Proteômica/métodos , Soja/citologia , Fracionamento Celular , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Oryza/metabolismo , Proteínas de Plantas/análise , Soja/metabolismo
18.
Methods Mol Biol ; 1696: 91-105, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29086398

RESUMO

The Golgi apparatus is an endomembrane system organelle and has roles in glycosylation, sorting, and secretion of proteins in the secretory pathway. It has a central function in living organism and is also essential for plant growth. Proteomic approaches to identify the Golgi membrane proteins have been performed in cell suspension cultures and many Golgi membrane-associated proteins were found, whereas it has well established in rice seedling yet. In this chapter, our recent improving published methods for isolated rice Golgi membranes by floating through a discontinuous sucrose density gradient are provided in detail with proteomic analyses.


Assuntos
Fracionamento Celular/métodos , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Oryza/citologia , Centrifugação com Gradiente de Concentração , Oryza/metabolismo , Proteínas de Plantas/análise , Proteômica/métodos
19.
Plant Physiol ; 176(1): 946-959, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29180380

RESUMO

Genetic improvement of plant architecture is one of the strategies for increasing the yield potential of rice (Oryza sativa). Although great progress has been made in the understanding of plant architecture regulation, the precise mechanism is still an urgent need to be revealed. Here, we report that over-expression of OsMIR396d in rice results in semidwarf and increased leaf angle, a typical phenotype of brassinosteroid (BR) enhanced mutant. OsmiR396d is involved in the interaction network of BR and gibberellin (GA) signaling. In OsMIR396d over-expression plants, BR signaling was enhanced. In contrast, both the signaling and biosynthesis of GA were impaired. BRASSINAZOLE-RESISTANT1, a core transcription activator of BR signaling, directly promoted the accumulation of OsmiR396d, which controlled BR response and GA biosynthesis by regulating the expression of different target genes respectively. GROWTH REGULATING FACTOR 6, one of OsmiR396d targets, participated in GA biosynthesis and signal transduction but was not directly involved in BR signaling. This study provides a new insight into the understanding of interaction between BR and GA from multiple levels on controlling plant architecture.


Assuntos
Brassinosteroides/metabolismo , Giberelinas/metabolismo , MicroRNAs/metabolismo , Oryza/anatomia & histologia , Oryza/metabolismo , Transdução de Sinais , Vias Biossintéticas , Divisão Celular , Tamanho Celular , MicroRNAs/genética , Modelos Biológicos , Mutação/genética , Oryza/citologia , Oryza/genética , Folhas de Planta/anatomia & histologia , Folhas de Planta/ultraestrutura , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas
20.
Int J Mol Sci ; 18(12)2017 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-29232920

RESUMO

Leaf senescence, the final stage of leaf development, is a complex and highly regulated process that involves a series of coordinated actions at the cellular, tissue, organ, and organism levels under the control of a highly regulated genetic program. In the last decade, the use of mutants with different levels of leaf senescence phenotypes has led to the cloning and functional characterizations of a few genes, which has greatly improved the understanding of genetic mechanisms underlying leaf senescence. In this review, we summarize the recent achievements in the genetic mechanisms in rice leaf senescence.


Assuntos
Redes Reguladoras de Genes , Oryza/fisiologia , Folhas de Planta/fisiologia , Senescência Celular , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutação , Oryza/citologia , Oryza/genética , Folhas de Planta/citologia , Proteínas de Plantas/genética
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