Embryo and hip development in hybrid roses

Roses are known to produce seeds with high concentrations of abscisic acid (ABA), both in the pericarp and in the testa tissues of the seed coat. No studies on roses have documented embryo morphological differentiation or the concentration of ABA in the embryo, which is known to inhibit premature germination. In this study, hip and seed growth of two hybrid roses were characterised from 3 to 60 days after pollination (DAP). An increase of about five times the hip mass at 3 DAP was necessary to obtain fully developed seeds. Fully developed embryos were found at 15 DAP and completely developed seeds at 30 DAP. The same pattern of hip mass increase was shown in both genotypes. In parallel, quantification of ABA in the developing embryos was carried out by ELISA. An exponential decay in ABA concentration was found in embryos of both genotypes, with basal levels (<0.5 pmol mg^?1) registered at 30 DAP. These changes in ABA, during the embryo development, could be used to formulate time points for embryo rescue and understanding of the pollination to seedling stage.     

Regulation of abscisic acid translocation during embryo maturation of Phaseolus vulgaris

When R, S [2-¹⁴C] abscisic acid (ABA) was applied to the leaf of Phaseolus vulgaris , a part of the radioactivity was always found 24 h later in the only pod left on the plant. In early podfill, a large part of the labeled material was found in the maternal tissues while in late podfill, most had migrated to the embryos. During embryogenesis, embryo cells became more and more alkaline with respect to the seed coat cells. These results suggest that the distribution of ABA within the tissue is regulated by the pH differential between the two compartments.
The decrease of endogenous ABA level observed in situ during the second part of the embryo development therefore cannot be explained only in terms of the passive diffusion of the undissociated species (ABA-H). The empty-ovules technique revealed that ABA was only partly affected by an inversion of the pH gradient and that metabolic inhibitors influenced the release of ABA. These results indicate that in addition to a diffusive path, an energy-dependent component was involved.     

Abscisic acid and osmoticum prevent germination of developing alfalfa embryos,but only osmoticum maintains the synthesis of developmental proteins

Developing seeds of alfalfa (Medicago sativa L.) acquire the ability to germinate during the latter stages of development, the maturation drying phase. Isolated embryos placed on Murashige and Skoog medium germinate well during early and late development, but poorly during mid-development; however, when placed on water they germinate well only during the latter stage of development. Germination of isolated embryos is very slow and poor when they are incubated in the presence of surrounding seed structures (the endosperm or seed coat) taken from the mid-development stages. This inhibitory effect is also achieved by incubating embryos in 10^?5 M abscisic acid (ABA). Endogenous ABA attains a high level during mid-development, especially in the endosperm. Seeds developing in pods treated with fluridone (1-methyl-3-phenyl-5[3-(trifluoromethyl)-phenyl]-4(1H)-pyridinone) contain low levels of ABA during mid-development, and the endosperm and seed coat only weakly inhibit the germination of isolated embryos. However, intact seeds from fluridone-treated pods do not germinate viviparously, which is indicative that ABA alone is not responsible for maintaining seeds in a developing state. Application of osmoticum (e.g. 0.35 M sucrose) to isolated developing embryos prevents their germination. Also, in the developing seed in situ the osmotic potential is high. Thus internal levels of osmoticum may play a role in preventing germination of the embryo and maintaining development. Abscisic acid and osmoticum impart distinctly different metabolic responses on developing embryos, as demonstrated by their protein-synthetic capacity. Only in the presence of osmoticum do embryos synthesize proteins which are distinctly recognizable as those synthesized by developing embryos in situ, i.e. when inside the pod. Abscisic acid induces the synthesis of a few unique proteins, but these arise even in mature embryos treated with ABA. Thus while both osmoticum and ABA prevent precocious germination, their effects on the synthetic capacity of the developing embryo are quite distinct. Since seeds with low endogenous ABA do not germinate, osmotic regulation may be the more important of these two factors in controlling seed development.     

Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

Summary The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16–26 days after pollination (DAP) and desiccation tolerance was induced after 20–26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.Abbreviations ABA Abscisic acid - DAP Days after pollination     

Legume embryos develop in a hypoxic environment

Specific morphological and biochemical characteristics of seeds can cause oxygen deficiency within maternal and embryonic tissues. In this study, optical sensors were used to measure O(2) profiles across developing seeds of Vicia faba and Pisum sativum and developmental and environmental modulations of internal O(2) levels were studied. In addition, the metabolic state of developing embryos was analysed by monitoring adenylate energy charge, adenylate nucleotides and the levels of nucleotide sugars. Within the seed coat O(2) concentration decreased sharply to approximately 3% towards the inner border. Lowest O(2) levels were detected within the endospermal cavity between the seed coat and embryo. It is probable that low seed coat permeability provides an hypoxic environment for legume embryo development. The O(2) concentration in embryonic tissue changed during development with the lowest levels in the early stages. Measured in darkness, the levels were below 3%, but increased upon illumination indicating that photosynthesis significantly contributes to internal O(2) levels. Only in very young embryos were ATP levels and energy charge low. Otherwise they were maintained at a constant higher value. ADP-glucose and UDP-glucose did not show large fluctuations. Throughout embryo development fermentative activity did not play a major role. Obviously, specific mechanisms prevent seed tissues from becoming anoxic during development. The possible role of low oxygen on seed metabolism and on the control of seed development in legumes is discussed.     

Carbohydrates and carbohydrate enzymes in developing cotton ovules

Patterns of carbohydrates and carbohydrate enzymes were investigated in developing cotton ovules to establish which of these might be related to sink strength in developing bolls. Enzymatic analysis of extracted tissue indicated that beginning 1 week following anthesis, immature cotton seeds (Gossypium hirsutum L. cv. Coker 100A glandless) accumulated starch in the tissues which surround the embryo. Starting at 15 days post anthesis (DPA), this starch was depleted and starch simultaneously appeared in the embryo. Sucrose entering the tissues surrounding the embryo was rapidly degraded, apparently by sucrose synthase; the free hexose content of these tissues reached a peak at about 20 DPA. During the first few weeks of development these tissues contained substantial amounts of hexose but little sucrose; the reverse was true for cotton embryos. Embryo sucrose content rose sharply from the end of the first week until about 20 DPA; it then remained roughly constant during seed maturation. Galactinol synthase (EC 2.4.1.x) appeared in the embryos approximately 25 days after flowering. Subsequently, starch disappeared and the galactosides raffinose and stachyose appeared in the embryo. Except near maturity, sucrose synthase (EC 2.4.1.13) activity in the embryos predominated over that of both sucrose phosphate synthase (EC 2.4.1.14) and acid invertase (EC 3.2.1.26). Activities of the latter enzymes increased during the final stages of embryo maturation. The ratio of sucrose synthase to sucrose phosphate synthase was found to be high in young cotton embryos but the ratio reversed about 45 DPA, when developing ovules cease being assimilate sinks. Insoluble acid invertase was present in developing cotton embryos, but at very low activities; soluble acid invertase was present at significant activities only in nearly mature embryos. From these data it appears that sucrose synthase plays an important role in young cotton ovule carbohydrate partitioning and that sucrose phosphate synthase and the galactoside synthesizing enzymes assume the dominant roles in carbohydrate partitioning in nearly mature cotton seeds. Starch was found to be an important carbohydrate intermediate during the middle stages of cotton ovule development and raffinose and stachyose were found to be important carbohydrate pools in mature cotton seeds.     

Expression of abundant mRNAs during somatic embryogenesis of white spruce [Picea glauca (Moench) Voss]

Embryogenic tissues of white spruce [Picea glauca (Moench) Voss] remain in an early developmental stage while cultured on 2,4-dichlorophenoxyacetic acid and N⁶-benzyladenine, but develop to cotyledonary embryos when these phytohormones are replaced by abscisic acid. Twenty-eight cDNAs were isolated from cotyledonary embryos by differential screening against immature embryo and non-embryonic tissues. Temporal expression patterns of these cDNAs during ABA-stimulated somatic embryo development were observed. This showed that clones could be allocated to various groups, including embryo-abundant, embryo-maturation-induced, and those whose expression was modulated during embryo development, germination or in non-embryogenic tissues. Expression corresponding to these cDNA clones showed that there were various responses to exogenous ABA or polyethylene glycol during a period of 48 h. Analyses of DNA and predicted amino acid sequence revealed that 12 of 28 cDNA clones had no known homologues, while others were predicted to encode different late-embryogenesis-abundant proteins, early methionine-labelled proteins, storage proteins, heat-shock proteins, glycine-rich cell wall protein, metallothionein-like protein and some other metabolic enzymes.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - BA N⁶-benzyladenine - cDNA complementary deoxyribonucleic acid - Em early methionine-labelled - HSP heat-shock protein - LEA late embryogenesis abundant - PEG polyethylene glycol The authors thank Mr. Terry Bethune for his assistance, and Dr. Larry Pelcher, Mr. Barry Panchuk and Mr. Don Schwab for DNA sequencing and primer synthesis. This is National Research Council of Canada publication number 38929.     

Abscisic acid biosynthesis during corn embryo development

In this study we examined the biosynthesis of abscisic acid (ABA) by developing corn (Zea mays L.) embryos. Three comparisons were made: ABA biosynthesis in embryos isolated from kernels grown in vitro with those grown in the field; the developmental profile of ABA content with that of biosynthesis; and ABA biosynthesis in corn embryos lacking carotenoid precursors with ABA biosynthesis in normal embryos. Embryos were harvested at various times during seed development and divided into two groups. Endogenous levels of ABA were measured in one group of embryos and ABA biosynthetic capacity was measured in the other group. The ABA biosynthetic capacity was measured with and without tetcyclacis (an inhibitor of ABA degradation) in embryos from both field-grown and in-vitro-grown corn kernels. Reduced-carotenoid (either fluridone-treated or genetically viviparous) embryos were also included in the study. Corn kernels developing under field and in-vitro conditions differed from each other in their responses to tetcyclacis and in their profiles of ABA biosynthesis during development. Therefore, in-vitro kernel culture may not be an appropriate substitute for field conditions for studies of embryo development. The developmental profiles of endogenous ABA content differed from those of ABA biosynthesis in isolated embryos of both in-vitro-and field-grown kernels. This indicated that ABA levels in the developing embryos were determined by import from the maternal tissues available to the embryos rather than by in-situ biosynthesis. In embryos with reduced levels of carotenoids, either fluridone-treated or genetically viviparous embryos, ABA biosynthesis was low or nonexistent. This result is expected for the presence of an indirect pathway of ABA biosynthesis and in the absence of ABA precursors.Abbreviations ABA abscisic acid - DAP days after pollination     

The molecular genetics of seed maturation in maize

The maturation phase of seed formation involves coordinated expression of multiple developmental pathways. These processes include abscisic acid regulated responses associated with the arrest of embryo development and induction of anthocyanin synthesis in embryo and aleurone tissues. Studies of the maturation defective vivaparous mutants of maize suggest that one gene, viviparous-1 ( vpl ), regulates both of these pathways in the developing seed. Mutations at vpl reduce the sensitivity of the developing embryo to abscisic acid. In addition, Vpl is required for expression of Cl , a regulatory gene for the anthocyanin pathway. This interaction is consistent with the idea that Vpl and Cl function as part of a regulatory hierarchy controlling seed development. Molecular studies of vpl mutations which separate control of embryo arrest and anthocyanin synthesis suggest that these functions map to discrete domains in the Vpl protein. Therefore, coordinate control of diverse maturation processes may be achieved through expression of a functionally complex regulatory molecule.     

The controls of late dicot embryogenesis and early germination

During seed formation, the embryo appears to be germinable as soon as cell division is completed; however, it continues development on the plant. This review describes the stages of development after cell division and provides a summary of important observations and recent use of molecular markers as they apply to the regulation of dicot seed formation. Genetic evidence suggests that abscisic acid may help initiate late embryogenesis, although no evidence firmly establishes that abscisic acid controls any other aspect of late dicot development. Previous studies utilizing cultured embryos have implicated abscisic acid and water potential as endogenous promoters of late embryogenesis and inhibitors of germination. However, these embryo culture experiments have been misinterpreted. The experiments show that both immature and mature embryos respond to environmental water stress by expressing a developmental program that is normally induced in late embryogenesis by abscission of the vascular connection. This postabscission program probably prepares the embryo for its forthcoming desiccation during normal development and is predicted to be important in protecting the embryo from water stress during germination.     

Abscisic Acid Accumulation in Developing Seeds of Phaseolus vulgaris L

Free and bound abscisic acid (ABA) in the pod, seed coat, and embryo were determined separately throughout seed development of Phaseolus vulgaris L. cv. `Taylor's Horticultural.' An internal standard method of gas-liquid chromatography was used for ABA quantification. In the embryo, two peaks of free ABA occurred at days 22 (1.18 micrograms per gram or 5.5 micromolar) and 28 (1.74 micrograms per gram or 12 micromolar); and a single peak of bound ABA at day 30. In the seed coat, there was one peak of free ABA at day 22 and only small amounts of bound ABA. Very small amounts of ABA were detected in the pod at any stage of development. In cv. PI 226895, in which seed development is more rapid than in `Taylor's Horticultural,' the embryo ABA peaks occur on days 20 and 26. The timing of the ABA peak in the embryo, and the concentration attained, are consistent with previous reports on the natural pattern of RNA synthesis and with ABA inhibition of RNA synthesis in developing bean fruit.     

Ovule morphology, embryogenesis and seed development in three Hylocereus species (Cactaceae)

Pre-embryonic and embryonic stages and seed developments were studied in the diploids Hylocereus monacanthus and Hylocereus undatus and the tetraploid Hylocereus megalanthus. Ovule morphology was similar among species except for micropyle entrance. H. monacanthus had the thickest and most robust suspensor. Embryo developmental time, measured from fertilization to maturity, was significantly more prolonged in H. megalanthus. Typical to Cactaceae, the seed coat was formed by one layer of sclerenchymatous cells, but was more lignified in H. megalanthus. Morphological features common to all species included (1) cellular type endosperm with independent patterns of development in the chalazal and micropylar zones, forming a haustorium layer from the chalazal zone to the embryo; (2) an endothelial layer surrounding the embryo sac almost complete; (3) a nucellar summit growing into the micropyle; and (4) a placental obturator and a funicle connecting the ovarian tissue to the ovule. Seed development was typically endospermic (exendospermic orthodox seeds). Anomalies included two egg cells in the same embryo sac, two embryos developing in the same ovule, and embryos developing from the chalazal pole region. Total seed number and seed viability were significantly lower in H. megalanthus than in the other two taxa. Embryos at different developmental stages were observed in aborted H. megalanthus seeds.     

Sequential steps for developmental arrest in Arabidopsis seeds

The continuous growth of the plant embryo is interrupted during the seed maturation processes which results in a dormant seed. The embryo continues development after germination when it grows into a seedling. The embryo growth phase starts after morphogenesis and ends when the embryo fills the seed sac. Very little is known about the processes regulating this phase. We describe mutants that affect embryo growth in two sequential developmental stages. Firstly, embryo growth arrest is regulated by the FUS3/LEC type genes, as mutations in these genes cause a continuation of growth in immature embryos. Secondly, a later stage of embryo dormancy is regulated by ABI3 and abscisic acid; abi3 and aba1 mutants exhibit premature germination only after embryos mature. Mutations affecting both developmental stages result in an additive phenotype and double mutants are highly viviparous. Embryo growth arrest is regulated by cell division activities in both the embryo and the endosperm, which are gradually switched off at the mature embryo stage. In the fus3/lec mutants, however, cell division in both the embryo and endosperm is not arrested, but rather is prolonged throughout seed maturation. Furthermore ectopic cell division occurs in seedlings. Our results indicate that seed dormancy is secured via at least two sequential developmental processes: embryo growth arrest, which is regulated by cell division and embryo dormancy.     

A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

Concentrations of sucrose and nitrogenous compounds in the apoplast of developing soybean seed coats and embryos

The apoplast of developing soybean (Glycine max cv Hodgson) embryos and seed coats was analyzed for sucrose, amino acids, ureides, nitrate, and ammonia. The apoplast concentration of amino acids and nitrate peaked during the most rapid stage of seed filling and declined sharply as the seed attained its maximum dry weight. Amino acids and nitrate accounted for 80 to 95% of the total nitrogen, with allantoin and allantoic acid either absent or present in only very small amounts. Aspartate, asparagine, glutamate, glutamine, serine, alanine, and γ-aminobutyric acid were the major amino acids, accounting for over 70% of the total amino acids present. There was a nearly quantitative conversion of glutamine to glutamate between the seed coat and embryo, most likely resulting from the activity of glutamate synthase found to be present in the seed coat tissue. This processing of glutamine suggests a partly symplastic route for solutes moving from the site of phloem unloading in the seed coat to the embryo.     

Development and regulation of three glyoxysomal enzymes during cotton seed maturation and growth

The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton (Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady-state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s).Abbreviations ABA abscisic acid - bp base pairs - DPA days post anthesis - HPI hours post imbibition - kb kilobase (pairs) - M _r relative molecular weight - S Svedberg unit (10^-13s)     

Seed dormancy in Acer: The role of abscisic acid in the regulation of seed development in Acer platanoides L.

Germinability of isolated embryos from developing fruits of Acer platanoides was high at the earliest developmental stage assessed (90 dpa), but fell subsequently and at seed maturity was very low. These observations showed an inverse correlation with changes in endogenous free abscisic acid (ABA) levels in the embryo, which were low during early ontogeny, but reached maximum levels late in development (150–160 dpa). These observations suggest the possibility that dormancy may be induced during development as a result of ABA accumulation in the embryo, an argument strengthened by the obvious inhibitory effect of added ABA on the germinability of isolated embryos. The cotyledons appear to exert an inhibitory influence on embryo germinability that may result from their free ABA content although the embryonic axis itself possesses an innate dormancy that may reflect its own free ABA content. The increased germinability of isolated embryos resulting form added kinetin serves only to emphasise the complexity of the system and the dangers of simplistic interpretation.The correlation between germinability and ABA content is not complete, however, since much of the reduction in germinability had occurred before any appreciable increase in free ABA levels in the embryo was observed. Indeed the failure of the intact seed to respond to endogenous changes in embryonal ABA levels suggests that even though free ABA in the embryo may influence embryo germinability, it has little effect in the intact seed, where the presence of an intact testa may be a more important factor.The absence of a desiccation phase in the embryo during the late stages of development suggests that the large increases in endogenous free ABA did not cause dormancy by inhibiting water uptake, nor did they result from water stress in the embryonal tissues.