Embryo Axis Research Articles

A hierarchical model for the development of cerebral arteriovenous malformations

ObjectiveCerebral arteriovenous malformations (AVMs) are vascular lesions whose pathogenesis, although not fully elucidated, is likely multifactorial. Recent research investigating vessel development suggests a potential hierarchical model in which capillary sprouts from higher-flow arteries give rise to lower-flow veins. It is possible that an embryologic structural vascular dysgenesis in this hierarchical development heavily contributes to the formation of AVMs. Subsequent genetic “second hits” may then allow development of a clinically significant cerebral AVM. We review this vascular developmental process and describe a novel proposal for the embryogenesis of AVMs and its implications in relation to recent research on polymorphisms and AVMs. MethodsA comprehensive literature search was performed using PubMed for recent research relative to cerebral AVMs, embryologic vascular development, and polymorphisms involved in AVM pathology. ResultsIt has recently been shown that both centrally, in the axial embryo, and peripherally, in the embryonic yolk sac, veins form via capillary sprouting from parent arteries. In developing intracranial vessels, a derangement in this embryonic process may lead to a primitive arteriovenous shunt. After this structural “first hit,” we suggest that single nucleotide polymorphisms (SNPs) are a major component in allowing AVM growth into symptomatic clinical lesions. ConclusionsThis is a novel theory for the embryologic formation of cerebral AVMs. Hierarchical vessel development, where higher-flow parent arteries give rise to lower-flow veins, provides a potential mechanism for the formation of primitive arteriovenous shunts that, with the influence of polymorphisms, allows AVMs to develop.

Effects of endogenous signals and Fusarium oxysporum on the mechanism regulating genistein synthesis and accumulation in yellow lupine and their impact on plant cell cytoskeleton.

The aim of the study was to examine cross-talk interactions of soluble sugars (sucrose, glucose and fructose) and infection caused by Fusarium oxysporum f.sp. lupini on the synthesis of genistein in embryo axes of Lupinus luteus L.cv. Juno. Genistein is a free aglycone, highly reactive and with the potential to inhibit fungal infection and development of plant diseases. As signal molecules, sugars strongly stimulated accumulation of isoflavones, including genistein, and the expression of the isoflavonoid biosynthetic genes. Infection significantly enhanced the synthesis of genistein and other isoflavone aglycones in cells of embryo axes of yellow lupine with high endogenous sugar levels. The activity of β-glucosidase, the enzyme that releases free aglycones from their glucoside bindings, was higher in the infected tissues than in the control ones. At the same time, a very strong generation of the superoxide anion radical was observed in tissues with high sugar contents already in the initial stage of infection. During later stages after inoculation, a strong generation of semiquinone radicals was observed, which level was relatively higher in tissues deficient in sugars than in those with high sugar levels. Observations of actin and tubulin cytoskeletons in cells of infected embryo axes cultured on the medium with sucrose, as well as the medium without sugar, showed significant differences in their organization.

Programmed cell death (PCD): an essential process of cereal seed development and germination.

The life cycle of cereal seeds can be divided into two phases, development and germination, separated by a quiescent period. Seed development and germination require the growth and differentiation of new tissues, but also the ordered disappearance of cells, which takes place by a process of programmed cell death (PCD). For this reason, cereal seeds have become excellent model systems for the study of developmental PCD in plants. At early stages of seed development, maternal tissues such as the nucellus, the pericarp, and the nucellar projections undergo a progressive degeneration by PCD, which allows the remobilization of their cellular contents for nourishing new filial tissues such as the embryo and the endosperm. At a later stage, during seed maturation, the endosperm undergoes PCD, but these cells remain intact in the mature grain and their contents will not be remobilized until germination. Thus, the only tissues that remain alive when seed development is completed are the embryo axis, the scutellum and the aleurone layer. In germinating seeds, both the scutellum and the aleurone layer play essential roles in producing the hydrolytic enzymes for the mobilization of the storage compounds of the starchy endosperm, which serve to support early seedling growth. Once this function is completed, scutellum and aleurone cells undergo PCD; their contents being used to support the growth of the germinated embryo. PCD occurs with tightly controlled spatial-temporal patterns allowing coordinated fluxes of nutrients between the different seed tissues. In this review, we will summarize the current knowledge of the tissues undergoing PCD in developing and germinating cereal seeds, focussing on the biochemical features of the process. The effect of hormones and redox regulation on PCD control will be discussed.

Morfologia do Fruto e da Semente de Três Espécies de Senna Mill. (Leguminosae - Caesalpinioideae)

Legume is the characteristic fruit of Leguminosae, but this botanical family has variability in fruits. The purpose of this study was to describe morphologycally fruits and seeds of Senna obtusifolia (L.) H.S.Irwin & Barneby, S. occidentalis (L.) Link. and S. tapajozensis (Ducke) Irwin and Barneby. General characteristics of fruits were described: morphology, classification, coloration, pericarp texture and consistence, dehiscence and indument. Morphological characters of seeds analyzed: a) external: shape, testa consistence, color and texture, hilum and micropyle position and form; b) internal: seed coat, endosperm and type, shape and color of the embryo. The studied species present legume and follicle fruit types, lineal, and broadly lineal, dry pericarp, cartaceous and subligneous, glabrous without magnifying glass, in brown tones and dehiscent. Seeds were obovate, suborbicular, elliptical with a straight side or irregularly rhombic, tests in brown and grayish tones, complete pleurogram, when present, punctiforme and orbicular hiulum, axial and foliaceous embryo, straight and oblique embryonic axis, and rudimentary plumule. The pericarp indument and pleurogram were the main external feature to separate the studied species. Keywords: Senna obtusifolia ; S. occidentalis ; S. tapajozensis ; Cassieae; Cassiinae.

Endoreduplication in the germinating embryo and young seedling is related to the type of seedling establishment but is not coupled with superoxide radical accumulation.

During germination, the embryo axis elongates and the radicle emerges through the surrounding structures of the seed. However, this elongation is not even along the axis, and it has been suggested that the region responsible for radicle protrusion is related to the type of subsequent seedling establishment. Eleven epigeal- and five hypogeal-type species were selected to study endoreduplication, a process coupled with cell elongation, in the radicle, hypocotyl-radicle transition zone, hypocotyl, and cotyledons of dry and germinating seeds, and in seedlings after radicle protrusion. Flow cytometry was used to establish the proportions of nuclei with different DNA contents, the mean C-value, and the (Σ>2C)/2C ratio. Additionally, a nitroblue tetrazolium chloride test was applied to the embryos/seedlings in the dry state and during and after germination to localize superoxide radical (O2(•-)) accumulation, which has been suggested to play a role in cell elongation. Endoreduplication intensity varied in different species, in the embryo/seedling regions, and with the type of seedling establishment. In most of the cases, it was highest in the transition zone of epigeal species and in the hypocotyl in hypogeal species. O2(•-) was invariably produced during germination in the radicle, and additionally in the transition zone at the time of radicle protrusion; thus, it was not coupled with endoreduplication, and most probably played a role in defence against biotic and abiotic environmental stresses. These results provide information to aid in the selection of the most suitable plant material for molecular research on germination and for monitoring seed priming.

Dormancy alleviation by NO or HCN leading to decline of protein carbonylation levels in apple (Malus domestica Borkh.) embryos

Deep dormancy of apple (Malus domestica Borkh.) embryos can be overcome by short-term pre-treatment with nitric oxide (NO) or hydrogen cyanide (HCN). Dormancy alleviation of embryos modulated by NO or HCN and the first step of germination depend on temporary increased production of reactive oxygen species (ROS). Direct oxidative attack on some amino acid residues or secondary reactions via reactive carbohydrates and lipids can lead to the formation of protein carbonyl derivatives. Protein carbonylation is a widely accepted covalent and irreversible modification resulting in inhibition or alteration of enzyme/protein activities. It also increases the susceptibility of proteins to proteolytic degradation. The aim of this work was to investigate protein carbonylation in germinating apple embryos, the dormancy of which was removed by pre-treatment with NO or HCN donors. It was performed using a quantitative spectrophotometric method, while patterns of carbonylated protein in embryo axes were analyzed by immunochemical techniques. The highest concentration of protein carbonyl groups was observed in dormant embryos. It declined in germinating embryos pre-treated with NO or HCN, suggesting elevated degradation of modified proteins during seedling formation. A decrease in the concentration of carbonylated proteins was accompanied by modification in proteolytic activity in germinating apple embryos. A strict correlation between the level of protein carbonyl groups and cotyledon growth and greening was detected. Moreover, direct in vitro carbonylation of BSA treated with NO or HCN donors was analyzed, showing action of both signaling molecules as protein oxidation agents.

The activity of SnRK1 is increased in Phaseolus vulgaris seeds in response to a reduced nutrient supply.

Phaseolus vulgaris seeds can grow and develop at the expense of the pod reserves after the fruits have been removed from the plant (Fountain etal., 1989). Because this process involves sensing the reduction of nutrients and the remobilisation of pod reserves, we investigated the effect on sucrose non-fermenting related kinase 1 (SnRK1) activity during this process. Bean fruits removed from the plant at 20 days after flowering (DAF) demonstrated active remobilisation of nutrients from the pod to the seeds. After 5 days, the pod dry weight was reduced by 50%. The process was characterized by a rapid degradation of starch, with the greatest decrease observed on day 1 after the fruits were removed. The pod nutrients were insufficient for the needs of all the seeds, and only some seeds continued their development. Those seeds exhibited a transient reduction in sucrose levels on day 1 after the fruits were removed. However, the normal level of sucrose was recovered, and the rate of starch synthesis was identical to that of a seed developed under normal conditions. Removing the fruits from the plant had no effect on the activity of SnRK1 in the pods, whereas in the seeds, the activity was increased by 35%. Simultaneously, a large reduction in seed sucrose levels was observed. The increase in SnRK1 activity was observed in both the cotyledon and embryo axes, but it was higher in the cotyledon. At 20–25 DAF, cotyledons actively accumulate storage materials. It is possible that the increase in SnRK1 activity observed in seeds developed in fruits that have been removed from the plant is part of the mechanism required for nutrient remobilisation under conditions of stress.

Purification and identification of a nuclease activity in embryo axes from French bean

Plant nucleases are involved in nucleic acid degradation associated to programmed cell death processes as well as in DNA restriction, repair and recombination processes. However, the knowledge about the function of plant nucleases is limited. A major nuclease activity was detected by in-gel assay with whole embryonic axes of common bean by using ssDNA or RNA as substrate, whereas this activity was minimal in cotyledons. The enzyme has been purified to electrophoretic homogeneity from embryonic axes. The main biochemical properties of the purified enzyme indicate that it belongs to the S1/P1 family of nucleases. This was corroborated when this protein, after SDS-electrophoresis, was excised from the gel and further analysis by MALDI TOF/TOF allowed identification of the gene (PVN1) that codes this protein. The gene that codes the purified protein was identified. The expression of PVN1 gene was induced at the specific moment of radicle protrusion. The inclusion of inorganic phosphate to the imbibition media reduced the level of expression of this gene and the nuclease activity suggesting a relationship with the phosphorous status in French bean seedlings.

Effects of Environmental Temperature on the Regeneration Frequency of the Immature Embryos of Wheat (Triticum aestivum L.)

The immature embryos (IEs) of wheat are the most widely used tissues for in vitro culture and genetic transformation due to its high regeneration competency. However, this explant can only be maintained in 4°C daily cooler for a short period time for its use in plant tissue culture or transformation experiments. This study aimed to investigate the effects of environmental temperature, cryopreservation storage temperature, and heat shock culture (HSC) temperature on the regeneration frequency of wheat IEs. Results indicated that environmental temperature significantly affected the induction of embryonic calli. The optimum total accumulated temperature (TAT) during the time of anthesis and sampling for regeneration of these tissues was around 280°C for spring wheat type cv. CB037 and approximately 300°C for winter wheat type cv. Kenong 199. Regeneration ability obviously declined when the highest environmental temperature was over 35°C for 1 d or a high temperature between 30 and 33°C lasted for 5 d during anthesis and sampling. This finding was verified by culturing the freshly isolated IEs under different temperatures from 29 to 37°C in different controlled growth incubators for 5 d; the IEs almost completely lost regeneration ability when the temperature rose to 37°C. Cryopreservation of −20°C caused the wheat samples lost ability of producing callus or embryonic callus in a few days, and cryopreservation of −10°C more than 10 d made the regeneration potential of the tissues dramatically declined. Comparatively, the temperature that best maintained high regeneration ability was −5°C, at which the materials can be maintained for around 1 mon. In addition, the preservation of the immature samples at −5 or −10°C inhibited the direct germination of the IEs, avoiding the embryo axis removing process. Our results are useful for ensuring that field collection and cryopreservation of the wheat IEs are done correctly to enable tissue culture and genetic transformation.

Abscisic acid-induced nitric oxide and proline accumulation in independent pathways under water-deficit stress during seedling establishment in Medicago truncatula

Nitric oxide (NO) production and amino acid metabolism modulation, in particular abscisic acid (ABA)-dependent proline accumulation, are stimulated in planta by most abiotic stresses. However, the relationship between NO production and proline accumulation under abiotic stress is still poorly understood, especially in the early phases of plant development. To unravel this question, this work investigated the tight relationship between NO production and proline metabolism under water-deficit stress during seedling establishment. Endogenous nitrate reductase-dependent NO production in Medicago truncatula seedlings increased in a time-dependent manner after short-term water-deficit stress. This water-deficit-induced endogenous NO accumulation was mediated through a ABA-dependent pathway and accompanied by an inhibition of seed germination, a loss of water content, and a decrease in elongation of embryo axes. Interestingly, a treatment with a specific NO scavenger (cPTIO) alleviated these water-deficit detrimental effects. However, the content of total amino acids, in particular glutamate and proline, as well as the expression of genes encoding enzymes of synthesis and degradation of proline were not affected by cPTIO treatment under water-deficit stress. Under normal conditions, exogenous NO donor stimulated neither the expression of P5CS2 nor the proline content, as observed after PEG treatment. These results strongly suggest that the modulation of proline metabolism is independent of NO production under short-term water-deficit stress during seedling establishment.

In vitro Plant Regeneration System for Two Middle East Cultivars of Chickpea (Cicer arietinum L.)

This study surveyed a rapid, efficient and reproducible protocol for in vitro shoot regeneration and rooting of two Middle East chickpea cultivars (Balila and Wady). Organogenic ability of various explants (decapitated embryo axes, fragments of embryo axes, fragments of embryo axes with basal part of cotyledons, entire and half of cotyledons) was tested on MS media containing different concentrations and nature of cytokinins (BAP and zeatin) or compound with cytokinin-like activity (TDZ). The highest level of shoot regeneration was achieved in Balila genotype using embryo axes fragments including the basal part of cotyledon on medium with 0.25 mg/L BAP. Higher BAP concentrations than 0.25 mg/L for Balila and 0.5 mg/L for Wady caused a decrease of shoot regeneration. On MS media supplemented with zeatin (1, 3 and 5 mg/L) and thidiazuron (1, 3 and 5 mg/L), the highest level of shoot differentiation was obtained from calli derived from fragments of embryo axes. Organogenic ability of calli from Wady genotype was enhanced by increasing zeatin and TDZ concentrations, up to 5 mg/L. Conversely, a progressive decrease of shoot frequency for Balila and root development for both genotypes, were observed with increasing concentrations of BAP, zeatin and TDZ.

Somatic Embryogenesis Media Optimization Study of Physic Nut (Jatropha curcas) as Biodiesel Feedstock

Jatropha curcas as potential biodiesel feedstock is difficult to propagate through tissue culture. The research development for Jatropha is massive nowadays and propagation can be conducted through conventional or non- conventional techniques in biotechnology. Biotechnology approach through organogenesis and embryogenesis pathways is needed. The objectives of the research were to obtain in vitro culture media optimization through embryogenesis pathway. Plant materials used in this experiment were embryo axis and cotyledon to obtain somatic embryo. The result of the experiment showed that somatic embryos were obtained only from MS medium supplemented with picloram 1.0mg/l for both embryo axis and cotyledon explant.

Qualitative changes and dynamics of protein synthesis during cold and warm stratification of Norway maple (Acer platanoides L.) seeds

Protein synthesis in cotyledons and embryo axes of Norway maple (<em>Acer platanoides</em> L.) was studied in seeds stratified at 3 or 15<sup>o</sup>C. At 3<sup>o</sup>C stratification, germination of seeds starts after 9 weeks, at 15<sup>o</sup>C stratification germination does not occur. The changes of protein synthesis level in both temperatures had two phasic character. In embryo axes (3 and 15<sup>o</sup>C) protein synthesis grow up, but in cotyledons (3<sup>o</sup>C) synthesis of proteins decrease. Generally, activity of protein synthesis was higher at 3<sup>o</sup>C, as like as DNA level was higher. After imbibition, in cold and warm stratification, in embryo axes, many new protein are become visible. We expect, that two from this proteins, determined as A and B my be connected with the releasing from dormancy. In seeds stratified at 15<sup>o</sup>C, these proteins are occuring in vestigal amount or are completely absent.

Rape embryogenesis. III. Embryo development in time

It was found that the growth curve of the rape embryo axis is of triple sigmoid type. Embryo growth occurs in 3 phases corresponding to 3 different periods of development. Phase I includes growth of the apical cell up to it's division into two layers of octants. Phase II comprises the increase of the spherical proembryo to the change of its symmetry from radial to bilateral. Phase III includes, growth of the embryo from the heart stage up to the end of embryogenesis. In each phase the relative growth rate increases drastically and then diminishes. The differences in growth intensity during the same phase are several-fold. The growth intensity maximum of the embryo axis occurs in phase II. The phasic growth intensity maxima occur: in phase I during apical cell elongation, :before its division, and in phases II and III in the periods of cell division ;growth in globular and torpedo-shaped -shaped embryos.

Activation of rape (Brassica napus L.) embryo during seed germination. V. The first zones of ultrastructural changes and their expansion

In the germinating rape embryo the columella and basal part of hypocotyl undergo earliest activation. Its first ultrastructural symptom is the appearance of numerous ER vesicles after 3-6 h of seed swelling. Their number is the highest in the external layers of the columella and decreases in basipetal direction. Dermatogen cells in the basal zone of the hypocotyl contain the greatest amount of ER structures, whereas decreasing amounts are found in both directions along the embryo axis and centripetally. Further changes in the ER spread in a similar order. The vesicles merge and form a tubular and plate-like ER. Then, they disappear and are replaced by tubular and vesicular forms. The changes in the ER are gradually followed by ultrastructural symptoms of activation of mitochondria, plastids and dictyosomes. The highest number of ER structures and other organelles accumulate in root cells shortly before piercing of the seed coat. After germination their amount decreases and remains almost stable.

Rape embryogenesis. V. Accumulation of lipid bodies

In embryo of winter rape var. Górczański lipid bodies have been observed in the light microscope starting from two-celled proembryo. Their number and size increase in the course of embryogenesis, especially since greening of endosperm and embryo. Lipid bodies, either single or in clusters, are present in all embryo cells, the clusters occur in various parts of the cytoplasm. During embryo maturation lipid bodies move and locate in a number of layers by the cell wall. At the same time their appearance change. Typical storage lipids originate. Lipid bodies are unevenly distributed within the embryo: their number and size decrease along the embryo axis from its part below cotyledons towards root apex. Moreover, they are histologically diversified: the biggest are located in epidermis and cortex, whereas the smallest -in central cylinder. In columella there are fewest lipid bodies.

Activation of rape (Brassica napus L.) embryo during seed germination. IV. Germinating embryo. The first zones of mitoses, starch and DNA synthesis and their expansion pattern

The rape radicle is completely covered by the root cap. The successive lateral cell layers of the root cap are terminated by T-forming walls in the dermatogen layer in a statistically constant position. T-walls in dermatogen were utilized for delimitation of successive root sectors on longitudinal microtome sections at the succeeding germination stages. The length and the cell number of the corresponding sectors were studied and the starch and DNA synthesis sites as well as cell divisions localized. All these processes are initiated in a constant sequence and in specific embryo zones. The first symptom of activation of the embryo is starch synthesis. It begins in two centres: in the apical part of the radicle columella and in the hypoctyl dermalogen it moves deep into the cortex and in both directions along the columella the activation of starch synthesis shifts basipetally into the whole columella and the initial centre as well as the lateral parts of the root cap. From the hypocotyl dermatogen it moves into the cortex and in both directions along the embryo axis. In the root dermatogen and periblem the activation zone is first located in the basal sector and, then, gradually, in the lower ones. Just before germination the basal and apical zones of activation meet. Starch can then, be found throughout the root. During starch synthesis, in the basal part of the radicle. DNA synthesis and cell growth begin. In the root sectors which have already begun to grow and synthesize DNA, cell divisions start. The boundary of the dividing cells zone shifts acropetally at some distance above the lower boundary of DNA synthesis and the zone of cell premitotic growth. The acropetal shift of the mitotic activation zones can be described as wave expansion. Before the first mitotic wave reaches the promeristem it is followed by at least three acropetal waves, arising in the already activated basal sectors. and then the mitoses are asynchronised. Mitotic.: activation of the root cap is partly independent of the acropetal wave of cell activation and results from the expanding weaker basipetal wave.

Activity ATPases during dormancy breaking in Norway maple (Acer platanoides L.) seeds

The activity of ATPases was studied in embryo axes and cotyledons of Norway maple seeds stratified at 3<sup>o</sup>C (dormancy broken) and 15<sup>o</sup>C (dormancy not broken). The activity of mitochondrial (F. III) and activity connected with all cellular membranes (F. Il) and soluble fraction (F. I) ATPases was investigated. It was found that mitochondrial ATPases are the most active. The activity of all types of ATPases is greater in seeds stratified at 3<sup>o</sup>C. It increases during seed dormancy breaking, particularly in the case of mitochondrial ATPases. The rise in the activity of mitochondrial and others ATPases in embryo axes of seeds stratified at 3<sup>o</sup>C is stepwise. In seeds stratified at 15<sup>o</sup>C ATPase activity generally decreases.

Rape embryogenesis. IV. Appearance and disappearance of starch during embryo development

Starch appears first in the suspensor of the proembryo with two-cell apical part. It is observed in the embryo proper from the octant stage. At first it is visible in all the embryo cells in the form of minute transient grains which disappear during cell divisions. But the columella mother cells and their derivatives have persistent large grains. When the embryo turns green in the heart stage a gradual accumulation of storage starch begins and lasts to the end of embryogenesis. Storage starch grains appear first in the auter cortex layers of the hypocotyl where the largest grains are to be found later, and afterwards in all the other tissues. Starch is usually absent in the frequently dividing cells, but even there it appears in the form of minute grains after the end of cell divisions. Disappearance of starch starts when the intensive green colour of the seed coat begins to fade. The first to disappear are the smallest granules in the regions where they were noted latest. In the embryo axis the starch grains remain deposited longest in dermatogen and cortex cells in the lower hypocotyl part. They are visible there, still when the seed turns brown. In black seeds starch may be only found in the columella the cells of which throughout embryogenesis contain amyloplasts filled with starch. These grains disappear completely at the time when the seeds become dry.

Two ways of plant regeneration from immature cotyledons of pea

Two different systems of plant regeneration via organogenesis and embryogenesis from immature cotyledons of pea (<i>Pisum sativum</i> L.) were developed. The first system was direct multiple shoot regeneration from the proximal to the embryo axis, injured part of cotyledon. The ability of six Polish cultivars to shoot formation was very high. The percent of regenerating cotyledons was from 73 to 86 and mean number of shoots from 3.5 to 9.9 after seven weeks of culture. This multiplification could be prolonged for next several months. The second system was somatic embryogenesis, initiating from the same part of cotyledon simultaneously with slowly proliferating callus. Only three out of six cultivars formed embryoids. The differences of ability to embryo formation ranged between 43% of responding explants for Heiga cultivar to only 6% for Cud Ameryki. The mean number of embryoids was from 4.2 to 2.3.