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1.
J Plant Physiol ; 265: 153505, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34481359

RESUMO

Brassicaceae seeds consist of three genetically distinct structures: the embryo, endosperm and seed coat, all of which are involved in assimilate allocation during seed development. The complexity of their metabolic interrelations remains unresolved to date. In the present study, we apply state-of-the-art imaging and analytical approaches to assess the metabolic environment of the Brassica napus embryo. Nuclear magnetic resonance imaging (MRI) provided volumetric data on the living embryo and endosperm, revealing how the endosperm envelops the embryo, determining endosperm's priority in assimilate uptake from the seed coat during early development. MRI analysis showed higher levels of sugars in the peripheral endosperm facing the seed coat, but a lower sugar content within the central vacuole and the region surrounding the embryo. Feeding intact siliques with 13C-labeled sucrose allowed tracing of the post-phloem route of sucrose transfer within the seed at the heart stage of embryogenesis, by means of mass spectrometry imaging. Quantification of over 70 organic and inorganic compounds in the endosperm revealed shifts in their abundance over different stages of development, while sugars and potassium were the main determinants of osmolality throughout these stages. Our multidisciplinary approach allows access to the hidden aspects of endosperm metabolism, a task which remains unattainable for the small-seeded model plant Arabidopsis thaliana.


Assuntos
Brassica napus/crescimento & desenvolvimento , Brassica napus/metabolismo , Endosperma/crescimento & desenvolvimento , Endosperma/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Brassica napus/genética , Brassica napus/ultraestrutura , Endosperma/genética , Endosperma/ultraestrutura , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Sementes/genética , Sementes/ultraestrutura
2.
Plant Sci ; 311: 110986, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34482923

RESUMO

In recent years, the plant morphology has been well studied by multiple approaches at cellular and subcellular levels. Two-dimensional (2D) microscopy techniques offer imaging of plant structures on a wide range of magnifications for researchers. However, subcellular imaging is still challenging in plant tissues like roots and seeds. Here we use a three-dimensional (3D) imaging technology based on the X-ray microscope (XRM) and analyze several plant tissues from different plant species. The XRM provides new insights into plant structures using non-destructive imaging at high-resolution and high contrast. We also utilized a workflow aiming to acquire accurate and high-quality images in the context of the whole specimen. Multiple plant samples including rice, tobacco, Arabidopsis and maize were used to display the differences of phenotypes. Our work indicates that the XRM is a powerful tool to investigate plant microstructure in high-resolution scale. Our work also provides evidence that evaluate and quantify tissue specific differences for a range of plant species. We also characterize novel plant tissue phenotypes by the XRM, such as seeds in Arabidopsis, and utilize them for novel observation measurement. Our work represents an evaluated spatial and temporal resolution solution on seed observation and screening.


Assuntos
Arabidopsis/ultraestrutura , Imageamento Tridimensional , Organelas/ultraestrutura , Oryza/ultraestrutura , Sementes/ultraestrutura , Tabaco/ultraestrutura , Zea mays/ultraestrutura , Oryza/anatomia & histologia , Tomografia Computadorizada por Raios X
3.
Int J Mol Sci ; 22(18)2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34576106

RESUMO

We investigated low-temperature plasma effects on two Brassicaceae seeds (A. thaliana and C. sativa) using dielectric barrier discharge in air. Comparisons of plasma treatments on seeds showed distinct responses on germination rate and speed. Optimal treatment time giving optimal germination is 15 min for A. thaliana with 85% increase compared to control after 48 h of germination and 1 min for C. sativa with 75% increase compared to control after 32 h of germination. Such germination increases are associated with morphological changes shown by SEM of seed surface. For better understanding at the biochemical level, seed surfaces were analyzed using gas chromatography-mass spectrometry which underlined changes of lipidic composition. For both treated seeds, there is a decrease of saturated (palmitic and stearic) fatty acids while treated C. sativa showed a decrease of unsaturated (oleic and linoleic) acids and treated A. thaliana an increase of unsaturated ones. Such lipid changes, specifically a decrease of hydrophobic saturated fatty acids, are coherent with the other analyses (SEM, water uptake and contact angle). Moreover, an increase in A. thaliana of unsaturated acids (very reactive) probably neutralizes plasma RONS effects thus needing longer plasma exposure time (15 min) to reach optimal germination. For C. sativa, 1 min is enough because unsaturated linoleic acid becomes lower in treated C. sativa (1.2 × 107) compared to treated A. thaliana (3.7 × 107).


Assuntos
Ar , Arabidopsis/fisiologia , Brassicaceae/fisiologia , Eletricidade , Gases em Plasma/farmacologia , Sementes/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/ultraestrutura , Brassicaceae/efeitos dos fármacos , Brassicaceae/ultraestrutura , Ácidos Graxos/metabolismo , Germinação/efeitos dos fármacos , Lipidômica , Permeabilidade , Sementes/anatomia & histologia , Sementes/ultraestrutura , Fatores de Tempo , Água , Molhabilidade
4.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206400

RESUMO

Seeds of common bean (Phaseolus vulgaris L.), of the Etna variety, were treated with low-pressure oxygen plasma sustained by an inductively coupled radiofrequency discharge in the H-mode for a few seconds. The high-intensity treatment improved seed health in regard to fungal contamination. Additionally, it increased the wettability of the bean seeds by altering surface chemistry, as established by X-ray photoelectron spectroscopy, and increasing surface roughness, as seen with a scanning electron microscope. The water contact angle at the seed surface dropped to immeasurably low values after a second of plasma treatment. Hydrophobic recovery within a month returned those values to no more than half of the original water contact angle, even for beans treated for the shortest time (0.5 s). Increased wettability resulted in accelerated water uptake. The treatment increased the bean radicle length, which is useful for seedling establishment in the field. These findings confirm that even a brief plasma treatment is a useful technique for the disinfection and stimulation of radicle growth. The technique is scalable to large systems due to the short treatment times.


Assuntos
Germinação/efeitos dos fármacos , Phaseolus/efeitos dos fármacos , Phaseolus/crescimento & desenvolvimento , Gases em Plasma/farmacologia , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Espectroscopia Fotoeletrônica , Desenvolvimento Vegetal/efeitos dos fármacos , Sementes/ultraestrutura , Propriedades de Superfície , Água , Molhabilidade
5.
Plant Cell Rep ; 40(9): 1773-1787, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34181045

RESUMO

KEY MESSAGE: The present study showed that the heat stress (40 °C) caused changes in morphophysiological, biochemical, and ultrastructural parameters to the seeds Melanoxylon brauna, ultimately leading to loss of germination capacity. Temperature is an abiotic factor that influences seed germination. In the present study, we investigated morphophysiological, biochemical, and ultrastructural changes during the germination of Melanoxylon brauna seeds under heat stress. Seed germination was evaluated at constant temperatures of 25 and 40 °C. The samples consisted of seeds soaked in distilled and ionized water for 48 and 96 h at both temperatures. For the evaluation of internal morphology, the seeds were radiographed. Ultrastructural parameters were assessed using transmission electron microscopy (TEM). The production of reactive oxygen species (ROS), content of malondialdehyde (MDA) and glucose, carbonylated proteins, and activity of the enzymes (superoxide dismutase-SOD, ascorbate peroxidase-APX, catalase-CAT, peroxidase-POX, glucose-6-phosphate dehydrogenase-G6PDH, lipase, α- and ß-amylase, and protease) were measured by spectrophotometric analysis. An 82% reduction in the germination of M. brauna seeds was observed at 25 °C, and 0% at 40 °C. TEM showed that seeds submitted to heat stress (40 °C) had poorly developed mitochondria and significantly reduced respiration rates. The content of ROS and protein carbonylation in seeds subjected to 40 °C increased compared to that at 25 °C. The activity of antioxidant enzymes, namely SOD, APX, CAT, and POX, was significantly reduced in seeds subjected to heat stress. Glucose content, G6PDH, and lipase activity also decreased when the seeds were exposed to heat stress. Conversely, α- and ß-amylase enzymes and the protease increased due to the increase in temperature. Our data showed that the increase in temperature caused an accumulation of ROS, increasing the oxidative damage to the seeds, which led to mitochondrial dysfunction, ultimately leading to loss of germination.


Assuntos
Fabaceae/fisiologia , Resposta ao Choque Térmico/fisiologia , Proteínas de Plantas/metabolismo , Sementes/fisiologia , Sementes/ultraestrutura , Antioxidantes/metabolismo , Carotenoides/metabolismo , Enzimas/metabolismo , Fabaceae/ultraestrutura , Ácidos Graxos/metabolismo , Germinação , Glucose/metabolismo , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Estresse Oxidativo , Superóxidos/metabolismo
6.
Sci Rep ; 11(1): 9661, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33958620

RESUMO

Somatic embryogenesis (SE) is the most important plant biotechnology process for plant regeneration, propagation, genetic transformation and genome editing of coffee, Coffea arabica L. Somatic embryo (SEs) conversion to plantlets is the principal bottleneck for basic and applied use of this process. In this study we focus on the maturation of SEs of C. arabica var. Typica. SEs conversion to plantlet up to 95.9% was achieved under osmotic stress, using 9 g/L gelrite, as compared with only 39.34% in non-osmotic stress. Mature SEs induced in osmotic stress developed shoot and root apical meristems, while untreated SEs were unable to do it. C. arabica regenerated plants from osmotic stress were robust, with higher leaf and root area and internode length. To understand a possible regulatory mechanism, gene expression of key genes of C. arabica, homologous to sequences in the Arabidopsis thaliana genome, were analyzed. A set of two component system and cytokinin signaling-related coding genes (AHK1, AHK3, AHP4 and ARR1) which interact with WUSCHEL and WOX5 homedomains and morphogenic genes, BABY-BOOM, LEC1, FUS3 and AGL15, underwent significant changes during maturation of SEs of C. arabica var. Typica. This protocol is currently being applied in genetic transformation with high rate of success.


Assuntos
Coffea/crescimento & desenvolvimento , Meristema/crescimento & desenvolvimento , Pressão Osmótica , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimento , Coffea/embriologia , Coffea/ultraestrutura , Meristema/ultraestrutura , Pressão Osmótica/fisiologia , Raízes de Plantas/ultraestrutura , Brotos de Planta/ultraestrutura , Sementes/ultraestrutura , Transcriptoma
7.
Nat Genet ; 53(6): 906-915, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33927398

RESUMO

Phosphate (Pi) is essential to plant growth and crop yield. However, it remains unknown how Pi homeostasis is maintained during cereal grain filling. Here, we identified a rice grain-filling-controlling PHO1-type Pi transporter, OsPHO1;2, through map-based cloning. Pi efflux activity and its localization to the plasma membrane of seed tissues implicated a specific role for OsPHO1;2 in Pi reallocation during grain filling. Indeed, Pi over-accumulated in developing seeds of the Ospho1;2 mutant, which inhibited the activity of ADP-glucose pyrophosphorylase (AGPase), important for starch synthesis, and the grain-filling defect was alleviated by overexpression of AGPase in Ospho1;2-mutant plants. A conserved function was recognized for the maize transporter ZmPHO1;2. Importantly, ectopic overexpression of OsPHO1;2 enhanced grain yield, especially under low-Pi conditions. Collectively, we discovered a mechanism underlying Pi transport, grain filling and P-use efficiency, providing an efficient strategy for improving grain yield with minimal P-fertilizer input in cereals.


Assuntos
Membrana Celular/metabolismo , Grão Comestível/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfatos/metabolismo , Animais , Membrana Celular/ultraestrutura , Mapeamento Cromossômico , Grão Comestível/ultraestrutura , Regulação da Expressão Gênica de Plantas , Células HEK293 , Humanos , Mutação/genética , Oryza/enzimologia , Oryza/genética , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/genética , Sementes/ultraestrutura , Amido/biossíntese , Xenopus , Zea mays/genética
8.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467472

RESUMO

Melatonin priming is an effective strategy to improve the germination of aged oat (Avena sativa L.) seeds, but the mechanism involved in its time-course responses has remained largely unknown. In the present study, the phenotypic differences, ultrastructural changes, physiological characteristics, and proteomic profiles were examined in aged and melatonin-primed seed (with 10 µM melatonin treatment for 12, 24, and 36 h). Thus, 36 h priming (T36) had a better remediation effect on aged seeds, reflecting in the improved germinability and seedlings, relatively intact cell ultrastructures, and enhanced antioxidant capacity. Proteomic analysis revealed 201 differentially abundant proteins between aged and T36 seeds, of which 96 were up-accumulated. In melatonin-primed seeds, the restoration of membrane integrity by improved antioxidant capacity, which was affected by the stimulation of jasmonic acid synthesis via up-accumulation of 12-oxo-phytodienoic acid reductase, might be a candidate mechanism. Moreover, the relatively intact ultrastructures enabled amino acid metabolism and phenylpropanoid biosynthesis, which were closely associated with energy generation through intermediates of pyruvate, phosphoenolpyruvate, fumarate, and α-ketoglutarate, thus providing energy, active amino acids, and secondary metabolites necessary for germination improvement of aged seeds. These findings clarify the time-course related pathways associated with melatonin priming on promoting the germination of aged oat seeds.


Assuntos
Avena/metabolismo , Germinação/efeitos dos fármacos , Melatonina/farmacologia , Proteômica/métodos , Sementes/metabolismo , Antioxidantes/metabolismo , Avena/genética , Avena/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Germinação/genética , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Melatonina/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo , Sementes/genética , Sementes/ultraestrutura , Fatores de Tempo
9.
BMC Plant Biol ; 21(1): 58, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33482732

RESUMO

BACKGROUND: Physical seed dormancy is an important trait in legume domestication. Although seed dormancy is beneficial in wild ecosystems, it is generally considered to be an undesirable trait in crops due to reduction in yield and / or quality. The physiological mechanism and underlying genetic factor(s) of seed dormancy is largely unknown in several legume species. Here we employed an integrative approach to understand the mechanisms controlling physical seed dormancy in common bean (Phaseolus vulgaris L.). RESULTS: Using an innovative CT scan imaging system, we were able to track water movements inside the seed coat. We found that water uptake initiates from the bean seed lens. Using a scanning electron microscopy (SEM) we further identified several micro-cracks on the lens surface of non-dormant bean genotypes. Bulked segregant analysis (BSA) was conducted on a bi-parental RIL (recombinant inbred line) population, segregating for seed dormancy. This analysis revealed that the seed water uptake is associated with a single major QTL on Pv03. The QTL region was fine-mapped to a 118 Kb interval possessing 11 genes. Coding sequence analysis of candidate genes revealed a 5-bp insertion in an ortholog of pectin acetylesterase 8 that causes a frame shift, loss-of-function mutation in non-dormant genotype. Gene expression analysis of the candidate genes in the seed coat of contrasting genotypes indicated 21-fold lower expression of pectin acetylesterase 8 in non-dormant genotype. An analysis of mutational polymorphism was conducted among wild and domesticated beans. Although all the wild beans possessed the functional allele of pectin acetylesterase 8, the majority (77%) of domesticated beans had the non-functional allele suggesting that this variant was under strong selection pressure through domestication. CONCLUSIONS: In this study, we identified the physiological mechanism of physical seed dormancy and have identified a candidate allele causing variation in this trait. Our findings suggest that a 5-bp insertion in an ortholog of pectin acetylesterase 8 is likely a major causative mutation underlying the loss of seed dormancy during domestication. Although the results of current study provide strong evidences for the role of pectin acetylesterase 8 in seed dormancy, further confirmations seem necessary by employing transgenic approaches.


Assuntos
Cromossomos de Plantas/genética , Esterases/metabolismo , Phaseolus/genética , Dormência de Plantas/genética , Locos de Características Quantitativas/genética , Mapeamento Cromossômico , Produtos Agrícolas , Domesticação , Ecossistema , Esterases/genética , Genótipo , Microscopia Eletrônica de Varredura , Mutagênese Insercional , Phaseolus/enzimologia , Phaseolus/fisiologia , Phaseolus/ultraestrutura , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Sementes/genética , Sementes/fisiologia , Sementes/ultraestrutura , Água/metabolismo
10.
Plant Signal Behav ; 16(4): 1872217, 2021 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-33446024

RESUMO

Sterols are essential lipids for plant growth, and the sterol content is tightly regulated by a fail-safe system consisting of two processes: 1) suppression of excess sterol production by a negative regulator of sterol biosynthesis (HIGR STEROL ESTER 1, HISE1), and 2) conversion of excess sterols to sterol esters by PHOSPHOLIPID STEROL ACYLTRANSFERASE 1 (PSAT1) in Arabidopsis thaliana. The hise1-3 psat1-2 double mutant has a 1.5-fold higher sterol content in leaves than the wild type; this upregulates the expression of stress-responsive genes, leading to disruption of cellular activities in leaves. However, the effects of excess sterols on seeds are largely unknown. Here, we show that excess sterols cause multiple defects in seeds. The seeds of hise1-3 psat1-2 plants had a higher sterol content than wild-type seeds and showed a deeper color than wild-type seeds because of the accumulation of proanthocyanidin. The seed coat in the hise1-3 psat1-2 mutant was abnormally wrinkled. Seed coat formation is accompanied by cell death-mediated shrinkage of the inner integument. In the hise1-3 psat1-2 mutant, transmission electron microscopy showed that shrinkage of the integument was impaired, resulting in a thick seed coat and delayed seed germination. Moreover, psat1-2 and hise1-3 psat1-2 seeds displayed defective imbibition. Taken together, the results suggest that excess sterols impair proper seed coat formation, thereby inhibiting seed germination.


Assuntos
Arabidopsis/anatomia & histologia , Arabidopsis/fisiologia , Sementes/anatomia & histologia , Sementes/fisiologia , Esteróis/metabolismo , Apoptose , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/metabolismo , Germinação , Mutação/genética , Sementes/ultraestrutura
11.
Plant Sci ; 302: 110692, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33288006

RESUMO

Human transforming growth factor-ß1 (hTGF-ß1) was produced in transgenic rice seeds. To boost its production yield and to extract it simply, it was expressed under the control of seed-specific promoters along with the simultaneous suppression of endogenous seed storage proteins (SSPs) through RNA interference (RNAi). When driven by the 26 kDa α-globulin endosperm-specific promoter, it accumulated up to the markedly high level of 452 µg/grain. However, exchange with other seed-specific promoters such as 18 kDa oleosin and AGPase promoters resulted in remarkable reduction to the levels of 62 and 48 µg/grain, respectively, even though endogenous SSPs were reduced to the similar level. These production levels were almost similar to those (42 and 108 µg/grain) produced by the glutelin GluB-1 endosperm-specific promoter and the maize ubiquitin constitutive promoter without reduction of SSPs, respectively. When extracted from these transgenic rice seeds with reduced SSPs with various buffers, it could be solubilized with denaturant solution, which was in remarkable contrast with those without depressed SSPs which required further supplementation of reducing agent for extraction. This difference was associated with the fact that it was mainly deposited to ER-derived structures though self-aggregation or interaction with remaining prolamin via intermolecular disulfide bonds.


Assuntos
Produção Agrícola/métodos , Endosperma/metabolismo , Oryza/crescimento & desenvolvimento , Proteínas de Armazenamento de Sementes/metabolismo , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/metabolismo , Endosperma/crescimento & desenvolvimento , Immunoblotting , Microscopia Confocal , Oryza/genética , Oryza/metabolismo , Oryza/ultraestrutura , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sementes/metabolismo , Sementes/ultraestrutura , Fator de Crescimento Transformador beta/metabolismo
12.
Nat Commun ; 11(1): 5346, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093471

RESUMO

The mechanism that creates vitreous endosperm in the mature maize kernel is poorly understood. We identified Vitreous endosperm 1 (Ven1) as a major QTL influencing this process. Ven1 encodes ß-carotene hydroxylase 3, an enzyme that modulates carotenoid composition in the amyloplast envelope. The A619 inbred contains a nonfunctional Ven1 allele, leading to a decrease in polar and an increase in non-polar carotenoids in the amyloplast. Coincidently, the stability of amyloplast membranes is increased during kernel desiccation. The lipid composition in endosperm cells in A619 is altered, giving rise to a persistent amyloplast envelope. These changes impede the gathering of protein bodies and prevent them from interacting with starch grains, creating air spaces that cause an opaque kernel phenotype. Genetic modifiers were identified that alter the effect of Ven1A619, while maintaining a high ß-carotene level. These studies provide insight for breeding vitreous kernel varieties and high vitamin A content in maize.


Assuntos
Carotenoides/metabolismo , Zea mays/metabolismo , Alelos , Mapeamento Cromossômico , Cruzamentos Genéticos , Endosperma/genética , Endosperma/metabolismo , Endosperma/ultraestrutura , Genes de Plantas , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Fenótipo , Melhoramento Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastídeos/genética , Plastídeos/metabolismo , Plastídeos/ultraestrutura , Locos de Características Quantitativas , Sementes/genética , Sementes/metabolismo , Sementes/ultraestrutura , Zea mays/genética , Zea mays/ultraestrutura
13.
Sci Rep ; 10(1): 11766, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678191

RESUMO

Seed mucilage polysaccharide production, storage and release in Plantago ovata is strikingly different to that of the model plant Arabidopsis. We have used microscopy techniques to track the development of mucilage secretory cells and demonstrate that mature P. ovata seeds do not have an outer intact cell layer within which the polysaccharides surround internal columellae. Instead, dehydrated mucilage is spread in a thin homogenous layer over the entire seed surface and upon wetting expands directly outwards, away from the seed. Observing mucilage expansion in real time combined with compositional analysis allowed mucilage layer definition and the roles they play in mucilage release and architecture upon hydration to be explored. The first emergent layer of hydrated mucilage is rich in pectin, extremely hydrophilic, and forms an expansion front that functions to 'jumpstart' hydration and swelling of the second layer. This next layer, comprising the bulk of the expanded seed mucilage, is predominantly composed of heteroxylan and appears to provide much of the structural integrity. Our results indicate that the synthesis, deposition, desiccation, and final storage position of mucilage polysaccharides must be carefully orchestrated, although many of these processes are not yet fully defined and vary widely between myxospermous plant species.


Assuntos
Mucilagem Vegetal/metabolismo , Plantago/fisiologia , Sementes/fisiologia , Parede Celular/metabolismo , Imunofluorescência , Fenótipo , Desenvolvimento Vegetal , Plantago/ultraestrutura , Sementes/ultraestrutura
14.
Methods Mol Biol ; 2149: 365-382, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32617946

RESUMO

High-resolution imaging of the membranous intermediates and cytoskeletal arrays involved in the assembly of a new cell wall during plant cytokinesis requires state-of-the-art electron microscopy techniques. The combination of cryofixation/freeze-substitution methods with electron tomography (ET) has revealed amazing structural details of this unique cellular process. This chapter deals with the main steps associated with these imaging techniques: selection of samples suitable for studying plant cytokinesis, sample preparation by high-pressure freezing/freeze substitution, and ET of plastic sections. In addition, immunogold approaches for identification of proteins and polysaccharides during cell wall assembly are discussed.


Assuntos
Arabidopsis/citologia , Arabidopsis/ultraestrutura , Parede Celular/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Imuno-Histoquímica/métodos , Células Vegetais/ultraestrutura , Citoesqueleto de Actina/metabolismo , Linhagem Celular , Congelamento , Processamento de Imagem Assistida por Computador , Microtúbulos/ultraestrutura , Pressão , Resinas Vegetais/química , Ribossomos/ultraestrutura , Plântula/citologia , Plântula/ultraestrutura , Sementes/citologia , Sementes/ultraestrutura , Tabaco/citologia
15.
Plant Cell Environ ; 43(10): 2523-2539, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32519347

RESUMO

Seed longevity is a polygenic trait of relevance for agriculture and for understanding the effect of environment on the ageing of biological systems. In order to identify novel longevity genes, we have phenotyped the natural variation of 270 ecotypes of the model plant, Arabidopsis thaliana, for natural ageing and for three accelerated ageing methods. Genome-wide analysis, using publicly available single-nucleotide polymorphisms (SNPs) data sets, identified multiple genomic regions associated with variation in seed longevity. Reverse genetics of 20 candidate genes in Columbia ecotype resulted in seven genes positive for seed longevity (PSAD1, SSLEA, SSTPR, DHAR1, CYP86A8, MYB47 and SPCH) and five negative ones (RBOHD, RBOHE, RBOHF, KNAT7 and SEP3). In this uniform genetic background, natural and accelerated ageing methods provided similar results for seed-longevity in knock-out mutants. The NADPH oxidases (RBOHs), the dehydroascorbate reductase (DHAR1) and the photosystem I subunit (PSAD1) highlight the important role of oxidative stress on seed ageing. The cytochrome P-450 hydroxylase, CYP86A8, and the transcription factors, MYB47, KNAT7 and SEP3, support the protecting role of the seed coat during seed ageing.


Assuntos
Arabidopsis/genética , Genes de Plantas/genética , Longevidade/genética , Estresse Oxidativo/genética , Sementes/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Genes de Plantas/fisiologia , Estudo de Associação Genômica Ampla , Microscopia Confocal , Plantas Geneticamente Modificadas , Polimorfismo de Nucleotídeo Único/genética , Característica Quantitativa Herdável , Genética Reversa , Sementes/fisiologia , Sementes/ultraestrutura , Transcriptoma
16.
PLoS One ; 15(6): e0234627, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530960

RESUMO

Cuscuta (dodders) is a group of parasitic plants with tremendous economic and ecological significance. Their seeds are often described as "simple" or "unspecialized" because they do not exhibit any classical dispersal syndrome traits. Previous studies of seed morphology and/or anatomy were conducted on relatively few species. We expanded research to 101 species; reconstructed ancestral character states; investigated correlations among seed characters and explored allometric relationships with breeding systems, the size of geographical distribution of species in North America, as well as the survival of seedlings. Seed morphological and anatomical characters permit the separation of subgenera, but not of sections. Identification of Cuscuta species using seed characteristics is difficult but not impossible if their geographical origin is known. Seeds of subg. Monogynella species, exhibit the likely ancestral epidermis type consisting of elongated and interlocked cells, which are morphologically invariant, uninfluenced by dryness/wetness. Subgenera Cuscuta, Pachystigma and Grammica have evolved a seed epidermis with isodiametric cells that can alternate their morphology between two states: pitted when seeds are dry, and papillose after seed imbibition. A seed coat with double palisade architecture throughout the entire seed has also apparently evolved in subgenera Cuscuta, Pachystigma and Grammica, but several species in two clades of the latter subgenus reverted to a single palisade layer outside the hilum area. The same latter species also evolved a peculiar, globose embryo, likely having a storage role, in contrast to the ancestral filiform and coiled embryo present throughout the remainder of the genus. Autogamous species had on average the highest number of seeds per capsule, whereas fully xenogamous taxa had the lowest. No correlation was revealed between the size of the seeds and the size of their geographical distribution in North America, but seedlings of species with larger seeds survived significantly longer than seedlings resulted from smaller seeds. Diversity and evolution of seed traits was discussed in relationship with their putative roles in dormancy, germination and dispersal.


Assuntos
Biodiversidade , Evolução Biológica , Classificação , Cuscuta/anatomia & histologia , Cuscuta/classificação , Sementes/anatomia & histologia , Análise de Variância , Estimativa de Kaplan-Meier , Tamanho do Órgão , Filogenia , Melhoramento Vegetal , Análise de Regressão , Plântula/anatomia & histologia , Sementes/ultraestrutura
17.
Int J Mol Sci ; 21(9)2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32380646

RESUMO

Starch and prolamin composition and content are important indexes for determining the processing and nutritional quality of wheat (Triticum aestivum L.) grains. Several transcription factors (TFs) regulate gene expression during starch and protein biosynthesis in wheat. Storage protein activator (TaSPA), a member of the basic leucine zipper (bZIP) family, has been reported to activate glutenin genes and is correlated to starch synthesis related genes. In this study, we generated TaSPA-B overexpressing (OE) transgenic wheat lines. Compared with wild-type (WT) plants, the starch content was slightly reduced and starch granules exhibited a more polarized distribution in the TaSPA-B OE lines. Moreover, glutenin and ω- gliadin contents were significantly reduced, with lower expression levels of related genes (e.g., By15, Dx2, and ω-1,2 gliadin gene). RNA-seq analysis identified 2023 differentially expressed genes (DEGs). The low expression of some DEGs (e.g., SUSase, ADPase, Pho1, Waxy, SBE, SSI, and SS II a) might explain the reduction of starch contents. Some TFs involved in glutenin and starch synthesis might be regulated by TaSPA-B, for example, TaPBF was reduced in TaSPA-B OE-3 lines. In addition, dual-luciferase reporter assay indicated that both TaSPA-B and TaPBF could transactivate the promoter of ω-1,2 gliadin gene. These results suggest that TaSPA-B regulates a complex gene network and plays an important role in starch and protein biosynthesis in wheat.


Assuntos
Grão Comestível/genética , Grão Comestível/metabolismo , Expressão Gênica , Proteínas de Plantas/genética , Amido/metabolismo , Triticum/genética , Grão Comestível/química , Perfilação da Expressão Gênica , Ontologia Genética , Anotação de Sequência Molecular , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Sementes/metabolismo , Sementes/ultraestrutura , Amido/ultraestrutura , Triticum/química , Triticum/metabolismo
18.
Plant Cell Physiol ; 61(7): 1273-1284, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32374847

RESUMO

In this report, we describe studies on symplasmic communication and cellular rearrangement during direct somatic embryogenesis (SE) in the tree fern Cyathea delgadii. We analyzed changes in the symplasmic transport of low-molecular-weight fluorochromes, such as 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS) and fluorescein (delivered to cells as fluorescein diacetate, FDA), within stipe explants and somatic embryos originating from single epidermal cells and developing during 16-d long culture. Induction of SE is preceded by a restriction in fluorochrome distribution between certain explant cells. Microscopic analysis showed a series of cellular changes like a decrease in vacuole size, increase in vacuole numbers, and increased density of cytoplasm and deposition of electron-dense material in cell walls that may be related with embryogenic transition. In somatic embryos, the limited symplasmic communication between cells was observed first in linear tri-cellular embryos. Further development of the fern embryo was associated with the formation of symplasmic domains corresponding to the four segments of the plant body. Using symplasmic tracers, we provided evidence that the changes in plasmodesmata permeability are corelated with somatic-to-embryogenic transition and somatic embryo development.


Assuntos
Gleiquênias/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimento , Gleiquênias/ultraestrutura , Corantes Fluorescentes , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Epiderme Vegetal/crescimento & desenvolvimento , Sementes/ultraestrutura
19.
Plant Cell Environ ; 43(8): 1925-1943, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32406163

RESUMO

Salt stress activates defence responses in plants, including changes in leaf surface structure. Here, we showed that the transcriptional activation of cutin deposition and antioxidant defence by the R2R3-type MYB transcription factor AtMYB49 contributed to salt tolerance in Arabidopsis thaliana. Characterization of loss-of-function myb49 mutants, and chimeric AtMYB49-SRDX-overexpressing SRDX49 transcriptional repressor and AtMYB49-overexpressing (OX49) overexpressor plants demonstrated a positive role of AtMYB49 in salt tolerance. Transcriptome analysis revealed that many genes belonging to the category "cutin, suberin and wax biosyntheses" were markedly up-regulated and down-regulated in OX49 and SRDX49 plants, respectively, under normal and/or salt stress conditions. Some of these differentially expressed genes, including MYB41, ASFT, FACT and CYP86B1, were also shown to be the direct targets of AtMYB49 and activated by AtMYB49. Biochemical analysis indicated that AtMYB49 modulated cutin deposition in the leaves. Importantly, cuticular transpiration, chlorophyll leaching and toluidine blue-staining assays revealed a link between increased AtMYB49-mediated cutin deposition in leaves and enhanced salt tolerance. Additionally, increased AtMYB49 expression elevated Ca2+ level in leaves and improved antioxidant capacity by up-regulating genes encoding peroxidases and late embryogenesis abundant proteins. These results suggest that genetic manipulation of AtMYB49 may provide a novel way to improve salt tolerance in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Tolerância ao Sal/fisiologia , Fatores Genéricos de Transcrição/metabolismo , Antioxidantes/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Lipídeos/fisiologia , Lipídeos de Membrana/biossíntese , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas , Sementes/metabolismo , Sementes/ultraestrutura , Fatores Genéricos de Transcrição/genética
20.
Sci Rep ; 10(1): 6786, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321974

RESUMO

Aiming to understand Nature´s strategies that inspire new composite materials, the hierarchical levels of organization of the Brazil nut (Bertholletia excelsa) mesocarp were investigated. Optical microscopy, scanning electron microscopy (SEM), microtomography (MicroCT) and small-angle X-ray scattering (SAXS) were used to deeply describe the cellular and fibrillary levels of organization. The mesocarp is the middle layer of the fruit which has developed several strategies to avoid its opening and protect its seed. Fibers have a different orientation in the three layers of the mesocarp, what reduces the anisotropy of the structure. Sclereids cells with thick cell walls fill the spaces between the fibers resembling a foam-filled structural composite. The mesocarp has several tubular channels and fractured surfaces which may work as sites for crack trapping and increase toughness. The thick and lignified cell wall of sclereids and fibers and the weak interface between cells can promote a longer and tortuous intercellular crack path. Additionally, fibers with high strength and stiffness due to microfibrils oriented along the main cell axis (µ = 0° to 17°) were identified in the innermost layer of the mesocarp. Such an understanding of each hierarchical level can inspire the development of new cellular composites with improved mechanical behavior.


Assuntos
Bertholletia/metabolismo , Parede Celular/metabolismo , Frutas/metabolismo , Microfibrilas/metabolismo , Sementes/metabolismo , Algoritmos , Bertholletia/anatomia & histologia , Bertholletia/ultraestrutura , Parede Celular/ultraestrutura , Frutas/anatomia & histologia , Frutas/ultraestrutura , Humanos , Microfibrilas/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Espalhamento a Baixo Ângulo , Sementes/anatomia & histologia , Sementes/ultraestrutura , Difração de Raios X/métodos , Microtomografia por Raio-X/métodos
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