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1.
BMC Plant Biol ; 21(1): 340, 2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-34273968

RESUMO

BACKGROUND: TLPs (Tubby-like proteins) are widespread in eukaryotes and highly conserved in plants and animals. TLP is involved in many biological processes, such as growth, development, biotic and abiotic stress responses, while the underlying molecular mechanism remains largely unknown. In this paper we characterized the biological function of cucumber (Cucumis sativus L.) Tubby-like protein 8 (CsTLP8) in Arabidopsis. RESULTS: In cucumber, the expression of the tubby-like protein CsTLP8 was induced by NaCl treatment, but reduced by PEG (Polyethylene Glycol) and ABA (Abscisic Acid) treatment. Subcellular localization and transcriptional activation activity analysis revealed that CsTLP8 possessed two characteristics of classical transcription factors: nuclear localization and trans-activation activity. Yeast two-hybrid assay revealed interactions of CsTLP8 with CsSKP1a and CsSKP1c, suggesting that CsTLP8 might function as a subunit of E3 ubiquitin ligase. The growth activity of yeast with ectopically expressed CsTLP8 was lower than the control under NaCl and mannitol treatments. Under osmotic and salt stresses, overexpression of CsTLP8 inhibited seed germination and the growth of Arabidopsis seedlings, increased the content of MDA (Malondialdehyde), and decreased the activities of SOD (Superoxide Dismutase), POD (Peroxidase) and CAT (Catalase) in Arabidopsis seedlings. Overexpression of CsTLP8 also increased the sensitivity to ABA during seed germination and ABA-mediated stomatal closure. CONCLUSION: Under osmotic stress, CsTLP8 might inhibit seed germination and seedling growth by affecting antioxidant enzymes activities. CsTLP8 acts as a negative regulator in osmotic stress and its effects may be related to ABA.


Assuntos
Ácido Abscísico/metabolismo , Cucumis sativus/metabolismo , Germinação , Pressão Osmótica , Proteínas de Plantas/metabolismo , Sementes , Transdução de Sinais , Antioxidantes/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/crescimento & desenvolvimento , Plântula/metabolismo , Sementes/embriologia , Cloreto de Sódio , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
2.
Methods Mol Biol ; 2309: 59-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028679

RESUMO

Strigolactones are a class of plant hormones involved in shoot branching, growth of symbiotic arbuscular mycorrhizal fungi, and germination of parasitic plant seeds. Assaying new molecules or compound exhibiting strigolactone-like activities is therefore important but unfortunately time-consuming and hard to implement because of the extremely low concentrations at which they are active. Seeds of parasite plants are natural integrator of these hormones since they can perceive molecule concentrations in the picomolar to nanomolar range stimulating their germination. Here we describe a simple and inexpensive method to evaluate the activity of these molecules by scoring the germination of parasitic plant seeds upon treatment with these molecules. Up to four molecules can be assayed from a single 96-well plate by this method. A comparison of SL-like bioactivities between molecules is done by determining the EC50 and the maximum percentage of germination.


Assuntos
Bioensaio , Germinação/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/metabolismo , Lactonas/metabolismo , Orobanche/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Sementes/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Orobanche/embriologia , Sementes/embriologia
3.
BMC Plant Biol ; 21(1): 178, 2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33849456

RESUMO

BACKGROUND: Apomixis is a form of asexual reproduction that produces offspring without the need for combining male and female gametes, and the offspring have the same genetic makeup as the mother. Therefore, apomixis technology has great application potential in plant breeding. To identify the apomixis types and critical period, embryonic development at different flower development stages of Zanthoxylum bungeanum was observed by cytology. RESULTS: The results show that the S3 stage is the critical period of apomixis, during which the nucellar cells develop into an adventitious primordial embryo. Cytological observations showed that the type of apomixis in Z. bungeanum is sporophytic apomixis. Furthermore, miRNA sequencing, miRNA-target gene interaction, dual luciferase reporter assay, and RT-qPCR verification were used to reveal the dynamic regulation of miRNA-target pairs in Z. bungeanum apomixis. The miRNA sequencing identified 96 mature miRNAs, of which 40 were known and 56 were novel. Additionally, 29 differentially expressed miRNAs were screened according to the miRNAs expression levels at the different developmental stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses showed that the target genes of the differentially expressed miRNAs were mainly enriched in plant hormone signal transduction, RNA biosynthetic process, and response to hormone pathways. CONCLUSIONS: During the critical period of apomictic embryonic development, miR172c significantly reduces the expression levels of TOE3 and APETALA 2 (AP2) genes, thereby upregulating the expression of the AGAMOUS gene. A molecular regulation model of miRNA-target pairs was constructed based on their interactions and expression patterns to further understand the role of miRNA-target pairs in apomixis. Our data suggest that miR172c may regulates AGAMOUS expression by inhibiting TOE3 in the critical period of apomixis.


Assuntos
Apomixia/genética , Flores/crescimento & desenvolvimento , MicroRNAs/genética , RNA de Plantas/genética , Sementes/embriologia , Zanthoxylum/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Análise de Sequência de RNA , Zanthoxylum/embriologia , Zanthoxylum/genética
4.
Plant Physiol ; 185(2): 478-490, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33721907

RESUMO

The architecture of the seed is shaped by the processes of tissue partitioning, which determines the volume ratio of maternal and zygotic tissues, and nutrient partitioning, which regulates nutrient distribution among tissues. In angiosperms, early seed development is characterized by antagonistic development of the nucellus maternal tissue and the endosperm fertilization product to become the main sugar sink. This process marked the evolution of angiosperms and outlines the most ancient seed architectures. In Arabidopsis, the endosperm partially eliminates the nucellus and imports sugars from the seed coat. Here, we show that the nucellus is symplasmically connected to the chalaza, the seed nutrient unloading zone, and works as both a sugar sink and source alongside the seed coat. After fertilization, the transient nucellus accumulates starch early on and releases it in the apoplasmic space during its elimination. By contrast, the persistent nucellus exports sugars toward the endosperm through the SWEET4 hexose facilitator. Finally, we analyzed sugar metabolism and transport in the transparent testa 16 mutant, which fails to undergo nucellus cell elimination, which shed light on the coordination between tissue and nutrient partitioning. Overall, this study identifies a path of sugar transport in the Arabidopsis seed and describes a link between sugar redistribution and the nucellus cell-elimination program.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Magnoliopsida/embriologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Açúcares/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico , Endosperma/embriologia , Endosperma/genética , Endosperma/metabolismo , Magnoliopsida/genética , Magnoliopsida/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Sementes/embriologia , Sementes/genética , Sementes/metabolismo , Amido/metabolismo
5.
J Plant Physiol ; 258-259: 153333, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33581559

RESUMO

Zygotic and somatic embryogenesis in plants is a fascinating event that is finely regulated through the expression of a specific group of genes and dynamic levels of plant hormones whose concerted action determines the fate that specific cells follow towards zygotic or somatic embryo development. This work studied different stages of Capsicum chinense Jacq. zygotic and somatic embryogenesis. HPLC quantification determined that the levels of indole-3-acetic acid (IAA) increase as the zygotic or somatic embryogenesis progresses, being higher at maturity, thus supporting a positive correlation between embryo cell differentiation and IAA increase. A monoclonal anti-IAA-antibody was used to detect IAA levels. Findings revealed a dynamic pattern of auxin distribution along the different embryogenic embryonic stages. In the early stages of zygotic embryos, the IAA gradient was observed in the basal cells of the suspensor and the hypostases, suggesting that they are the initial source of the IAA hormone. As embryogenesis proceeds, the dynamic of the IAA gradient is displaced to the embryo and endosperm cells. In the case of induced somatic embryogenesis, the IAA gradient was detected in the dividing cells of the endodermis, from where pre-embryogenic cells emerge. However, the analysis of somatic embryos revealed that IAA was homogeneously distributed. This study shows evidence supporting a correlation between IAA levels during zygotic or somatic embryogenesis in Capsicum chinense species.


Assuntos
Capsicum/embriologia , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Sementes/embriologia , Zigoto/crescimento & desenvolvimento
6.
J Plant Physiol ; 258-259: 153364, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33465637

RESUMO

DEAD-box (DDX) proteins belong to the largest subfamily of RNA helicase SF2, which contributes to all biological processes of RNA metabolism in the plant kingdom. Till now, no significant data are available regarding studies on DDX in Somatic Embryogenesis (SE) of woody plants. It is important to investigate the biological function of the DlDDX family in longan SE. Thus, a comprehensive analysis of 58 longan DEAD-box (DlDDX) genes characterization was performed by genome-wide identification and transcript abundance validation analysis. Homologous evolution has revealed that some DlDDXs in longan had high sequence similarity with Mus musculus, Citrus and Saccharomyces cerevisiae, indicating that DlDDXs were highly conservative in the animal, plant, and microorganism. Remarkably, gene duplication, purifying selection, and alternative splicing events, and new auxiliary domains have likely contributed to the functional evolution of DlDDX, indicating that DlDDX appeared neofunctionalization in longan. Besides, DlDDX3, 15, 28, 36 might interact with protein complex (MAC3A, MAC3B, CDC5, CBP20) of miRNA biosynthesis. Notably, DlDDX28 contained a novel auxiliary domain (CAF-1 p150), which might contribute to DNA demethylation in longan early SE. 4 DlDDX genes significantly expressed not only in early SE and zygotic embryogenesis (ZE) but also up-regulated at high levels in 'Honghezi' and 'Quanlongbaihe' with abortive seeds, which are of great significance. Moreover, some DlDDXs presented abiotic stress-response dynamic expression patterns by ABA, SA, JA, and NaCl treatments during early SE. Hence, DEAD-box is essential to SE development and seed abortive in longan.


Assuntos
RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Sapindaceae/genética , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Plantas/metabolismo , Sapindaceae/embriologia , Sapindaceae/enzimologia , Sementes/embriologia
7.
Biochem J ; 477(19): 3743-3767, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33045058

RESUMO

Seeds are essential for human civilization, so understanding the molecular events underpinning seed development and the zygotic embryo it contains is important. In addition, the approach of somatic embryogenesis is a critical propagation and regeneration strategy to increase desirable genotypes, to develop new genetically modified plants to meet agricultural challenges, and at a basic science level, to test gene function. We briefly review some of the transcription factors (TFs) involved in establishing primary and apical meristems during zygotic embryogenesis, as well as TFs necessary and/or sufficient to drive somatic embryo programs. We focus on the model plant Arabidopsis for which many tools are available, and review as well as speculate about comparisons and contrasts between zygotic and somatic embryo processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Sementes/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sementes/genética
8.
Int J Mol Sci ; 21(21)2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33126660

RESUMO

Dry fruits consist of two types, dehiscent and indehiscent, whereby the fruit is splitting open or remains closed at maturity, respectively. The seed, the dispersal unit (DU) of dehiscent fruits, is composed of three major parts, the embryo and the food reserve, encapsulated by the maternally-derived organ, the seed coat. Indehiscent fruit constitutes the DU in which the embryo is covered by two protective layers (PLs), the seed coat and the fruit coat. In grasses, the caryopsis, a one-seeded fruit, can be further enclosed by the floral bracts to generate two types of DUs, florets and spikelets. All protective layers enclosing the embryo undergo programmed cell death (PCD) at maturation and are thought to provide mainly a physical shield for embryo protection and a means for dispersal. In this review article, I wish to highlight the elaborate function of these dead organs enclosing the embryo as unique storage structures for beneficial substances and discuss their potential role in seed biology and ecology.


Assuntos
Brassicaceae/embriologia , Brassicaceae/fisiologia , Frutas/fisiologia , Germinação , Sementes/embriologia , Sementes/fisiologia , Água
9.
Sci Rep ; 10(1): 15166, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938968

RESUMO

Holm oak trees (Quercus ilex L.) mortality is increasing worryingly in the Mediterranean area in the last years. To a large degree this mortality is caused by the oomycete Phytophthora spp., which is responsible for forest decline and dieback in evergreen oak forest areas of the southwestern Iberian Peninsula. This study is based on the possibility of applying chemical elicitors or filtered oomycete extracts to holm oak somatic embryos (SE) in order to induce epigenetic memory, priming, that may increase tolerance to the pathogen in future infections. To this end, we first examined the effect of priming treatments on SE development and its oxidative stress state, to avoid elicitors that may cause damage to embryogenic tissues. Both, the sterile oomycete extracts and the chemical elicitor methyl jasmonate (MeJA) did not produce any detrimental effect on SE growth and development, unlike the elicitors benzothiadiazole (BTH) and p-aminobenzoic acid (PABA) that reduced the relative weight gain and resulted in necrotic and deformed SE when were applied at high concentrations (25 µM BTH or 50 µM PABA) in accordance with their high malondialdehyde content. No significant differences among elicitation treatments were found in dual culture bioassays, although those SEs elicited with 50 µM MeJA increased H2O2 production after challenged against active oomycete indicating the activation of stress response. Since this elicitation treatment did not produce any adverse effect in the embryogenic process we suggest that could be used in further priming experiments to produce holm oak plants adapted to biotic stress.


Assuntos
Phytophthora/patogenicidade , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Quercus/embriologia , Quercus/microbiologia , Ácido 4-Aminobenzoico/toxicidade , Acetatos/farmacologia , Ciclopentanos/farmacologia , Florestas , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Oxilipinas/farmacologia , Phytophthora/química , Proteínas/farmacologia , Quercus/efeitos dos fármacos , Sementes/efeitos dos fármacos , Sementes/embriologia , Sementes/metabolismo , Espanha , Tiadiazóis/toxicidade
10.
Int J Mol Sci ; 21(14)2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32674459

RESUMO

During early plant embryogenesis, some of the most fundamental decisions on fate and identity are taken making it a fascinating process to study. It is no surprise that higher plant embryogenesis was intensively analysed during the last century, while somatic embryogenesis is probably the most studied regeneration model. Encoded by the MIRNA, short, single-stranded, non-coding miRNAs, are commonly present in all Eukaryotic genomes and are involved in the regulation of the gene expression during the essential developmental processes such as plant morphogenesis, hormone signaling, and developmental phase transition. During the last few years dedicated to miRNAs, analytical methods and tools have been developed, which have afforded new opportunities in functional analyses of plant miRNAs, including (i) databases for in silico analysis; (ii) miRNAs detection and expression approaches; (iii) reporter and sensor lines for a spatio-temporal analysis of the miRNA-target interactions; (iv) in situ hybridisation protocols; (v) artificial miRNAs; (vi) MIM and STTM lines to inhibit miRNA activity, and (vii) the target genes resistant to miRNA. Here, we attempted to summarise the toolbox for functional analysis of miRNAs during plant embryogenesis. In addition to characterising the described tools/methods, examples of the applications have been presented.


Assuntos
Desenvolvimento Embrionário/genética , MicroRNAs/genética , Técnicas de Embriogênese Somática de Plantas/métodos , RNA de Plantas/genética , Sementes/genética , Zigoto/fisiologia , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Genômica/métodos , Sementes/embriologia
11.
Int J Mol Sci ; 21(9)2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32403374

RESUMO

The effects of auxins 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (1-naphthaleneacetic acid) or picloram (4-amino-3,5,6-trichloropicolinic acid; 9 µM) and cytokinin BA (benzyloadenine; 4.5 µM) applied in the early stages of somatic embryogenesis (SE) on specific stages of SE in Picea abies and P. omorika were investigated. The highest SE initiation frequency was obtained after 2,4-D application in P. omorika (22.00%) and picloram application in P. abies (10.48%). NAA treatment significantly promoted embryogenic tissue (ET) proliferation in P. abies, while 2,4-D treatment reduced it. This reduction was related to the oxidative stress level, which was lower with the presence of NAA in the proliferation medium and higher with the presence of 2,4-D. The reduced oxidative stress level after NAA treatment suggests that hydrogen peroxide (H2O2) acts as a signalling molecule and promotes ET proliferation. NAA and picloram in the proliferation medium decreased the further production and maturation of P. omorika somatic embryos compared with that under 2,4-D. The quality of the germinated P. abies embryos and their development into plantlets depended on the auxin type and were the highest in NAA-originated embryos. These results show that different auxin types can generate different physiological responses in plant materials during SE in both spruce species.


Assuntos
Ácidos Indolacéticos/farmacologia , Picea/efeitos dos fármacos , Técnicas de Embriogênese Somática de Plantas/métodos , Sementes/efeitos dos fármacos , Ácido 2,4-Diclorofenoxiacético/farmacologia , Células Cultivadas , Citocininas/farmacologia , Peróxido de Hidrogênio/metabolismo , Ácidos Indolacéticos/classificação , Morfogênese/efeitos dos fármacos , Ácidos Naftalenoacéticos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Picea/classificação , Picea/embriologia , Picloram/farmacologia , Reguladores de Crescimento de Plantas/classificação , Reguladores de Crescimento de Plantas/farmacologia , Sementes/citologia , Sementes/embriologia , Especificidade da Espécie
12.
Methods Mol Biol ; 2124: 163-176, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32277453

RESUMO

Genetic improvement of rice is crucial to achieve global food security as rice is an important staple crop for more than half of the global population. One of the methodologies for genetic improvement is biolistic delivery of genetic components into plant cells. In this chapter, we describe steps involved in introducing plasmid DNA carrying gene of interest into rice mature embryos using Biolistic® PDS-1000/He particle delivery system. We also provide information required for recovery of transformed plants and production of transgenic seed for next generation analysis. Using this protocol, typical 50-70 putative independent transgenic callus lines can be generated from 100 bombarded embryos. Transgenic rice plantlets can be produced within 2 months after the initiation of seed germination for transformation.


Assuntos
Biolística/métodos , Oryza/genética , Transformação Genética , Ouro/química , Padrões de Herança/genética , Oryza/embriologia , Osmose , Plantas/genética , Plasmídeos/genética , Regeneração , Sementes/embriologia , Sementes/genética , Esterilização , Transgenes
13.
Int J Mol Sci ; 21(4)2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32054063

RESUMO

Sweet cherry (Prunus avium L.) is a delicious nutrient-rich fruit widely cultivated in countries such as China, America, Chile, and Italy. However, the yield often drops severely due to the frequently-abnormal fruitlet abscission, and few studies on the metabolism during its ripening process at the proteomic level have been executed so far. To get a better understanding regarding the sweet cherry abscission mechanism, proteomic analysis between the abscising carpopodium and non-abscising carpopodium of sweet cherry was accomplished using a newly developed Liquid chromatography-mass spectrometry/mass spectrometry with Tandem Mass Tag (TMT-LC-MS/MS) methodology. The embryo viability experiments showed that the vigor of the abscission embryos was significantly lower than that of retention embryo. The activity of cell wall degrading enzymes in abscising carpopodium was significantly higher than that in non-abscising carpopodium. The anatomy results suggested that cells in the abscission zone were small and separated. In total, 6280 proteins were identified, among which 5681 were quantified. It has been observed that differentially accumulated proteins (DAPs) influenced several biological functions and various subcellular localizations. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that plenty of metabolic pathways were notably enriched, particularly those involved in phytohormone biosynthesis, cell wall metabolism, and cytoskeletal metabolism, including 1-aminocyclopropane-1-carboxylate oxidase proteins which promote ethylene synthesis, and proteins promoting cell wall degradation, such as endoglucanases, pectinase, and polygalacturonase. Differential expression of proteins concerning phytohormone biosynthesis might activate the shedding regulation signals. Up-regulation of several cell wall degradation-related proteins possibly regulated the shedding of plant organs. Variations of the phytohormone biosynthesis and cell wall degradation-related proteins were explored during the abscission process. Furthermore, changes in cytoskeleton-associated proteins might contribute to the abscission of carpopodium. The current work represented the first study using comparative proteomics between abscising carpopodium and non-abscising carpopodium. These results indicated that embryo abortion might lead to phytohormone synthesis disorder, which effected signal transduction pathways, and hereby controlled genes involved in cell wall degradation and then caused the abscission of fruitlet. Overall, our data may give an intrinsic explanation of the variations in metabolism during the abscission of carpopodium.


Assuntos
Proteínas de Plantas/metabolismo , Prunus avium/embriologia , Prunus avium/metabolismo , Lignina/metabolismo , Redes e Vias Metabólicas , Reguladores de Crescimento de Plantas/metabolismo , Biossíntese de Proteínas , Proteômica , Prunus avium/ultraestrutura , Sementes/embriologia , Sementes/metabolismo , Sementes/ultraestrutura
14.
F1000Res ; 92020.
Artigo em Inglês | MEDLINE | ID: mdl-32055398

RESUMO

The zygotic embryos of angiosperms develop buried deep within seeds and surrounded by two main extra-embryonic tissues: the maternally derived seed coat tissues and the zygotic endosperm. Generally, these tissues are considered to play an important role in nurturing the developing embryo by acting as conduits for maternally derived nutrients. They are also critical for key seed traits (dormancy establishment and control, longevity, and physical resistance) and thus for seed and seedling survival. However, recent studies have highlighted the fact that extra-embryonic tissues in the seed also physically and metabolically limit embryonic development and that unique mechanisms may have evolved to overcome specific developmental and genetic constraints associated with the seed habit in angiosperms. The aim of this review is to illustrate how these studies have begun to reveal the highly complex physical and physiological relationship between extra-embryonic tissues and the developing embryo. Where possible I focus on Arabidopsis because of space constraints, but other systems will be cited where relevant.


Assuntos
Arabidopsis/embriologia , Endosperma/embriologia , Magnoliopsida/embriologia , Sementes/embriologia
15.
Methods Mol Biol ; 2122: 3-14, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31975291

RESUMO

The major tissue types and stem-cell niches of plants are established during embryogenesis, and thus knowledge of embryo development is essential for a full understanding of plant development. Studies of seed development are also important for human health, because the nutrients stored in both the embryo and endosperm of plant seeds provide an essential part of our diet. Arabidopsis and maize have evolved different types of seeds, opening a range of experimental opportunities. Development of the Arabidopsis embryo follows an almost invariant pattern, while cell division patterns of maize embryos are variable. Embryo-endosperm interactions are also different between the two species: in Arabidopsis, the endosperm is consumed during seed development, while mature maize seeds contain an enormous endosperm. Genetic screens have provided important insights into seed development in both species. In the genomic era, genetic analysis will continue to provide important tools for understanding embryo and endosperm biology in plants, because single gene functional studies can now be integrated with genome-wide information. Here, we lay out important factors to consider when designing genetic screens to identify new genes or to probe known pathways in seed development. We then highlight the technical details of two previous genetic screens that may serve as useful examples for future experiments.


Assuntos
Arabidopsis/embriologia , Endosperma/embriologia , Zea mays/embriologia , Arabidopsis/genética , Endosperma/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutagênese , Sementes/embriologia , Sementes/genética , Zea mays/genética
16.
Methods Mol Biol ; 2122: 49-61, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31975295

RESUMO

Virtually all growth, developmental, physiological, and defense responses in plants are accompanied by reorganization of subcellular structures to enable altered cellular growth, differentiation or function. Visualizing cellular reorganization is therefore critical to understand plant biology at the cellular scale. Fluorescently labeled markers for organelles, or for cellular components are widely used in combination with confocal microscopy to visualize cellular reorganization. Early during plant embryogenesis, the precursors for all major tissues of the seedling are established, and in Arabidopsis, this entails a set of nearly invariant switches in cell division orientation and directional cell expansion. Given that these cellular reorganization events are genetically regulated and coupled to formative events in plant development, they offer a good model to understand the genetic control of cellular reorganization in plant development. Until recently, it has been challenging to visualize subcellular structures in the early Arabidopsis embryo for two reasons: embryos are deeply embedded in seed coat and fruit, and in addition, no dedicated fluorescent markers, expressed in the embryo, were available. We recently established both an imaging approach and a set of markers for the early Arabidopsis embryo. Here, we describe a detailed protocol to use these new tools in imaging cellular reorganization.


Assuntos
Arabidopsis/embriologia , Corantes Fluorescentes/análise , Microscopia Confocal/métodos , Sementes/embriologia , Arabidopsis/citologia , Arabidopsis/ultraestrutura , Microscopia Confocal/instrumentação , Sementes/citologia , Sementes/ultraestrutura
17.
Methods Mol Biol ; 2122: 101-111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31975298

RESUMO

Zygotic embryogenesis is one of key processes for fertile seed development and therefore has gained great attention for decades in the field of plant developmental biology. However, this process is deeply embedded in the maternal tissues. The inaccessibility of tiny early embryos has greatly hindered the study of early embryogenesis, especially limits direct observation and accurate omics investigations. In order to investigate the molecular mechanism regulating embryo development with modern technologies, it is necessary to develop a reliable method to isolate living embryos at different stages. For this purpose, plant scientists have been trying to develop different methods for isolating zygotes and early embryos in different plants such as maize, wheat, rice, and tobacco during past decades. Nicotiana tabacum has long been considered as an ideal model eudicot for the study of embryogenesis, which displays a traceable and predictable cell division pattern, spanning from the first zygotic division to the mature embryo formation. Here, we provide a detailed protocol for isolating living embryos from zygote to cotyledon embryo. Isolated living zygotes and early embryos could be used for several important studies such as cell type-specific transcriptome construction and clear GFP observation.


Assuntos
Sementes/embriologia , Tabaco/embriologia , Zigoto/citologia , Separação Celular/métodos , Sobrevivência Celular , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Microscopia/métodos , Polinização , Sementes/genética , Sementes/fisiologia , Tabaco/genética , Tabaco/fisiologia , Transcriptoma , Zigoto/metabolismo
18.
Methods Mol Biol ; 2122: 113-126, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31975299

RESUMO

Genome-wide characterization of RNA populations in early flowering plant embryos can yield insights into the gene regulatory processes functioning during this formative phase of development. However, early embryonic transcriptomes are technically challenging to profile because of the low amount of RNA obtainable and potential RNA contamination from surrounding nonembryonic tissues. Here we provide a detailed protocol for collecting early Arabidopsis thaliana (Arabidopsis) embryos, generating mRNA sequencing (mRNA-seq) libraries, and basic data processing and quality controls of the resulting mRNA-seq data.


Assuntos
Arabidopsis/embriologia , Perfilação da Expressão Gênica/métodos , Sementes/embriologia , Transcriptoma , Arabidopsis/genética , Dissecação/métodos , Regulação da Expressão Gênica de Plantas , Sementes/genética
19.
Methods Mol Biol ; 2122: 127-139, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31975300

RESUMO

Transcriptomic studies have proven powerful and effective as a tool to study the molecular underpinnings of plant development. Still, it remains challenging to disentangle cell- or tissue-specific transcriptomes in complex structures like the plant seed. In particular, the embryo of flowering plants is embedded in the endosperm, a nurturing tissue, which, in turn, is enclosed by the maternal seed coat. Here, we describe laser-assisted microdissection (LAM) to isolate highly pure embryo tissue from whole seeds. This technique is applicable to virtually any plant seed, and we illustrate the use of LAM to isolate embryos from species of the Boechera and Solanum genera. LAM is a tool that will greatly help to increase the repertoires of tissue-specific transcriptomes, including those of embryos and parts thereof, in nonmodel plants.


Assuntos
Brassicaceae/genética , Perfilação da Expressão Gênica/métodos , Microdissecção e Captura a Laser/métodos , Sementes/genética , Solanum/genética , Brassicaceae/embriologia , Brassicaceae/ultraestrutura , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Microscopia/métodos , Sementes/embriologia , Sementes/ultraestrutura , Solanum/embriologia , Solanum/ultraestrutura , Transcriptoma
20.
Methods Mol Biol ; 2122: 141-150, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31975301

RESUMO

Cells differentiate from undifferentiated precursors in order to establish the tissues of vascular plants. The different cell types and stem cells are first specified in the early embryo. How cell type specification is instructed by transcriptional control on a genome-wide level is poorly understood. A major hurdle has been the technical challenge associated with obtaining cellular transcriptomes in this inaccessible tissue. Recently, we adapted a two-component genetic labeling system called INTACT to isolate nuclei and generate a microarray-based expression atlas of the cell types in the early Arabidopsis thaliana embryo. Here we present a step-by-step description of the adapted INTACT protocol, as well as the approach to generate transcriptomic profiles. This protocol has been adapted to account for using seeds with embryos of various developmental stages as a starting material, and the relatively few cell type-specific nuclei that can be isolated from embryos.


Assuntos
Arabidopsis/genética , Perfilação da Expressão Gênica/métodos , Sementes/genética , Transcriptoma , Arabidopsis/embriologia , Regulação da Expressão Gênica de Plantas , Sementes/embriologia
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