Diurnal O2 and carbohydrate levels in wheat kernels during embryony

In vitro zygotic and somatic embryogenesis procedures for wheat have been improved by simulating in ovulo nutritional, hormonal and dissolved oxygen (dO₂) conditions. However, diurnal fluctuations in these conditions during early embryony are not well characterized. In this study, dO₂ and water-soluble carbohydrate levels in wheat kernels were determined after 8 h of light and 8 h of dark at approximately 6, 12 and 18 day post anthesis (DPA). Clark style O₂ microelectrodes, having a tip diameter of approximately 115 μm, were inserted into intact kernels immediately distil to the developing embryo, and dO₂ levels were recorded at 50 μm intervals into the center of kernels. High-performance anion exchange chromatography with pulsed amperometric detection was used to quantify carbohydrate levels in endosperm sap. dO₂ levels in the chlorophyllous layer of the pericarp reached 190 mmol m⁻³ during the day, which probably represents, because of photosynthesis, a supersaturated O₂ condition relative to the external environment (21% O₂). At the embryo surface, dO₂ levels at 6 DPA ranged from 135 to 170 mmol m⁻³. At 12 and 18 DPA, dO₂ levels at the embryo axis ranged from 100 to 150 mmol m⁻³. At all three stages, dO₂ levels in the center of the endosperm were below 13 mmol m⁻³. Extreme fluctuations in carbohydrate levels were observed diurnally during rapid seed fill (12 DPA). Levels of sucrose and short-chain fructans were much higher during the day than during the night. In contrast, fructose, glucose, and myo-inositol levels were much higher during the night than during the day. By 18 DPA (hard dough stage), carbohydrate levels tended to be similar during the day and night. These dynamic fluctuations may assist in regulating embryony in ovulo, and their simulation might improve the development of somatic and zygotic embryos in vitro.     

Fiber development in preanthesis cotton ovules

A tissue culture method was developed to investigate the production of cotton (Gossypium hirsutum L. cv. Texas Marker-1) fibers in vitro. Ovules were excised from 3, 5, 7 and 9 days preanthesis ovaries and placed on an agar-solidified, modified Murashige and Skoog medium containing 2.3 μ M kinetin and 0.45 μ M –2,4–dichlo-rophenoxyacetic acid or 2.3 μ M kinetin and 10.7 μ M naphthaleneacetic acid. Ovules formed fibers and callus tissue. Fibers formed in vitro were up to 10 mm long, 10–22 μ wide and the cell wall was 1–3 μ M thick. Callus tissue cells were subcultured for over 25 weeks and their degree of elongation was monitored. The ability of ovule-derived cells to direct expansion in a longitudinal direction diminished, while lateral expansion increased with time in culture.     

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.     

Fluctuation in levels of endogenous plant hormones in ovules of normal and mutant cotton during flowering and their relation to fiber development

The contents of indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA), and cytokinins were determined in ovules of normal cotton (Tm-1) and a kind of fiber differentiation mutant (Xin) before and after flowering by enzyme-linked immunosorbent assays. It was found that 24 h before flowering, a peak of IAA content was observed in ovules of Tm-1, whereas in ovules of Xin, a low level of IAA was determined. From –1 day (1 day before flowering) to +3 days (3 days after flowering), GA₁₊₃ levels in ovules of Xin were 40–70% lower than those of Tm-1; GA₄₊₇ levels were very low, and there was no visible difference in GA₄₊₇ content between normal and mutant cotton. The ABA content in ovule of Tm-1 decreased by 70% 3 days after flowering, whereas that of Xin only decreased by 20%. The levels of cytokinins in ovules of Tm-1 decreased after flowering, and those of Xin kept up a steady increase.Abbreviations IAA indole-3-acetic acid - GA gibberellin - ABA abscisic acid - ELISA enzyme-linked immunosorbent assay - FW fresh weight - PBS phosphate-buffered saline - iPA isopentenyladenosine - ZR zeatin riboside - DHZR dihydrozeatin riboside - CTK cytokinin     

Identification of plant hormones from cotton ovules

An extract from 8-day-old cotton ovules (Gossypium hirsutum L.) was partitioned into three fractions and each fraction was derivatized and analyzed separately. Gas-liquid chromatography and computer-controlled gas-liquid chromatography-mass spectrometry were used to separate, measure, and identify the naturally occurring plant hormones. A single extract contained abscisic acid, indoleacetic acid, and gibberellins A(1), A(3), A(4), A(7), A(9), and A(13) in the first fraction; ethyl indole-3-acetate and indole-3-aldehyde in the second fraction; and the cytokinins 6-(3-methyl-4-hydroxybutylamino)purine (dihydrozeatin), 6-(4-hydroxy-3-methyl-2-trans-butenylamino) purine (zeatin), 6-(3-methyl-2-butenylamino)purine(2iP), 6-(3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine(2iPA), and 6-(4-hydroxy-3-methyl-2-trans-butenylamino)-9-beta-d- ribofuranosylpurine (zeatin riboside) in the third fraction.     

Polysaccharide and glycoprotein distribution in the epidermis of cotton ovules during early fiber initiation and growth

The cotton fiber is a model system to study cell wall biosynthesis because the fiber cell elongates (∼3 cm in ∼20 days) without mitosis. In this study, developing cotton ovules, examined from 1 day before anthesis (DBA) to 2 days post-anthesis (DPA), that would be difficult to investigate via classical carbohydrate biochemistry were probed using a battery of antibodies that recognize a large number of different wall components. In addition, ovules from these same stages were investigated in three fiberless lines. Most antibodies reacted with at least some component of the ovule, and several of the antibodies reacted specifically with the epidermal layer of cells that may give clues as to the nature of the development of the fibers and the neighboring, nonfiber atrichoblasts. Arabinogalactan proteins (AGPs) labeled the epidermal layers more strongly than other ovular tissue, even at 1 DBA. One of the AGP antibodies, CCRC-M7, which recognizes a 1➔6 galactan epitope of AGPs, is lost from the fiber cells by 2 DPA, although labeling in the atrichoblasts remained strong. In contrast, LM5 that recognizes a 1➔4 galactan RGI side chain is unreactive with sections until the fibers are produced and only the fibers are reactive. Dramatic changes also occur in the homogalacturonans (HGs). JIM5, which recognizes highly de-esterified HGs, only weakly labels epidermal cells of 1 DBA and 0 DPA ovules, but labeling increases in fibers cells, where a pectinaceous sheath is produced around the fiber cell and stronger reaction in the internal and external walls of the atrichoblast. In contrast, JIM7-reactive, highly esterifed HGs are present at high levels in the epidermal cells throughout development. Fiberless lines displayed similar patterns of labeling to the fibered lines, except that all of the cells had the labeling pattern of atrichoblasts. That is, CCRC-M7 labeled all cells of the fiberless lines, and LM5 labeled no cells at 2 DPA. These data indicate that a number of polysaccharides are unique in quantity or presence in the epidermal cell layers, and some of these might be critical participants in the early stages of initiation and elongation of cotton fibers.     

Brassinosteroid regulates fiber development on cultured cotton ovules

Our current understanding of the role of phytohormones in the development of cotton fibers is derived largely from an amenable culture system in which cotton ovules, collected on the day of anthesis, are floated on liquid media. Under these conditions, supplemental auxin and gibberellin were found to promote fiber initiation and elongation. More recently, addition of low concentrations of the brassinosteroid brassinolide (BL) were also found to promote fiber elongation while a brassinosteroid biosynthesis inhibitor brassinazole2001 (Brz) inhibited fiber development. In order to elucidate the role of brassinosteroid in cotton fiber development further, we have performed a more detailed analysis of the effects of these chemicals on cultured cotton ovules. Our results confirm that exogenous BL promotes fiber elongation while treatment with Brz inhibits it. Furthermore, treatment of cotton floral buds with Brz results in the complete absence of fiber differentiation, indicating that BR is required for fiber initiation as well as elongation. Expression of fiber genes associated with cell elongation increased in ovules treated with BL and was suppressed by Brz treatment, establishing a correlation between brassinosteroid-regulated gene expression and fiber elongation. These results establish a clear connection between brassinosteroid and fiber development and open the door for genetic analysis of cotton development through direct modification of the brassinosteroid signal transduction pathway.     

Independent control of fiber development and nitrate reduction in cultured cotton ovules

Several lines of evidence implicate ammonium as an important factor in the growth and development of cotton (Gossypium hirsutum L.) ovules cultured in vitro. For example, ovules cultured at 28 C require indoleacetic acid (IAA) and either ammonium or gibberellic acid (GA₃) in the medium for fiber development, whereas ovules cultured at 34 C require only IAA. Because of this effect of ammonium supply, it seemed possible that hormones or increased temperature were also promoting the availability of reduced nitrogen by induction of increased nitrate reductase activity in the ovules. This possibility was tested.     

Effects of cycling temperatures on fiber metabolism in cultured cotton ovules

The effects of temperature on rates of cellulose synthesis, respiration, and long-term glucose uptake were investigated using cultured cotton ovules (Gossypium hirsutum L. cv Acala SJ1). Ovules were cultured either at constant 34°C or under cycling temperatures (12 h at 34°C/12 h at 15-40°C). Rates of respiration and cellulose synthesis at various temperatures were determined on day 21 during the stage of secondary wall synthesis by feeding cultured ovules with [¹⁴C]glucose. Respiration increased between 18 and approximately 34°C, then remained constant up to 40°C. In contrast, the rate of cellulose synthesis increased above 18°C, reached a plateau between about 28 and 37°C, and then decreased at 40°C. Therefore, the optimum temperature for rapid and metabolically efficient cellulose synthesis in Acala SJ1 is near 28°C. In ovules cycled to 15°C, respiration recovered to the control rate immediately upon rewarming to 34°C, but the rate of cellulose synthesis did not fully recover for several hours. These data indicate that cellulose synthesis and respiration respond differently to cool temperatures. The long-term uptake of glucose, which is the carbon source in the culture medium, increased as the low temperature in the cycle increased between 15 and 28°C. However, glucose uptake did not increase in cultures grown constantly at 34°C compared to those cycled at 34/28°C. These observations are consistent with previous observations on the responses of fiber elongation and weight gain to cycling temperatures in vitro and in the field.     

An ultrastructural study of early endosperm development and synergid changes in unfertilized cotton ovules

Excised, unfertilized cotton (Gossypium hirsutum L.) ovules were cultured for 1–5 days postanthesis and embryo-sac development was studied with the electron microscope. In some ovules the two polar nuclei fuse and the diploid endosperm nucleus goes through a limited number of free nuclear divisions after 2–3 days in culture. Each nucleus has two nucleoli, in contrast to nuclei of fertilized triploid endosperm which have three nucleoli. Precocious cell walls form between the endosperm nuclei on the 3rd day in culture. The morphology of the plastids, mitochondria, rough endoplasmic reticulum (RER), dictyosomes and microbodies, and the amount of starch and lipid in the diploid cellular endosperm are similar to those of the central cell. A few large helical polysomes appear close to plastids and mitochondria. After 2 days in culture, one of the two synergids in the unfertilized cultured ovules shows degenerative changes which in fertilized ovules are associated with the presence of the pollen tube, i.e., increase in electron density, collapse of vacuoles, irregular darkening and thickening of mitochondrial and plastid membranes, disappearance of the plasmalemma and the membranes of the plasmalemma and the membranes of the RER. The second synergid remains unchanged in appearance. The egg cell does not shrink or divide or show structural changes characteristic of the cotton zygote. Embryo-sac development is arrested on the 4th and 5th days in culture. The nucellus continues growth and at 14 days crushes the degenerate embryo sac.     

Integumenal embryony in CMS sunflower line

In ovules of cultural sunflower (Helianthus annuus L.) CMS line VIR 116, which were pollinated by wild perennial specie H. occidentalis, the integumentary embryos have been revealed. They are found out at 7-9 days after pollination in embryo sacs where there was no normal fertilization. The integumentary embryos arise from somatic cells of a parent organism and their occurrence can be one of the reasons matroclinal inheritances at interspecific hybrids.     

Integumentary embryony in CMS sunflower line

Formation of integumentary embryos has been revealed in the ovules of the VIR 116 CMS line of sunflower (Helianthus annuus L.) pollinated by H. occidentalis, a perennial wild sunflower species. These embryos are observed within 7–9 days after the pollination in embryo sacs, where the normal fertilization did not occur. The embryos develop from somatic cells of a maternal organism (an integument) and can be one of the reasons of a matroclinal inheritance in interspecies hybrids.     

Changes in thyroid hormone levels during zebrafish development

The zebrafish (Danio rerio) has been used as a model for the study of endocrine disrupting chemicals. This study set out to determine the profiles of whole-body thyroxine (T4) and 3,5,3'-triiodothyronine (T3) levels during the development of zebrafish from embryo to adult. Enzyme-linked immunoassay was used to analyze whole-body T4 and T3 contents. The results showed that whole-body T4 and T3 levels remained stable during the pre-hatching period (0-3 d) and increased significantly during early development after hatching. The T3 level peaked at 0.28 ± 0.01 ng g(-1) body weight at 10 days post-fertilization (dpf), and T4 peaked at 0.58 ± 0.09 ng g(-1) body weight at 21 dpf. Both thyroid hormones subsequently declined during later development. This study establishes a baseline for thyroid hormones in zebrafish, which will be vital for the understanding of thyroid hormone functions and in future studies of thyroid toxicants in this species.     

Plasma hormone levels in beef heifers during prostaglandin-induced parturition

Plasma hormone levels were determined at frequent intervals in 12 two-year-old Hereford heifers after treatment with prostaglandin F₂α on day 267 of gestation to induce parturition. Of the 12 animals, six also received a daily injection of estradiol-17β until calving occurred. An additional six heifers received no treatment and therefore served as a control group. The treatment with PGF₂α was effective since the treated heifers calved a mean of 12 days sooner than the untreated heifers (P < .01). A metabolite of PGF₂α, 13, 14-dihydro-15-keto PGF₂α, rose to a peak level in the plasma within one hour after injection (im) of PGF₂α then declined to a baseline level within four hours. The plasma progesterone level had fallen significantly within two hours after injection, but no change in plasma estradiol-17β or estrone was observed. In the eight heifers that responded, plasma estrogen levels were greater at the time of treatment than in the four heifers that did not respond.     

Maternal corticotropin-releasing hormone levels during pregnancy and offspring adiposity

Objective: Animal models suggest that fetal exposure to glucocorticoids can program adiposity, especially central adiposity, later in life. We examined associations of maternal corticotropin‐releasing hormone (CRH) levels in the late 2nd trimester of pregnancy, a marker of fetal glucocorticoid exposure, with child adiposity at age 3 years. Research Methods and Procedures: We analyzed data from 199 participants in Project Viva, a prospective cohort study of pregnant women and their children, At age 3 years, the main outcomes were age‐sex‐specific BMI z score and the sum of subscapular (SS) and triceps (TR) skinfold thicknesses to represent overall adiposity, and ratio of SS to TR (SS:TR) to represent central adiposity. Results: Mean (standard deviation) maternal 2nd trimester log CRH was 4.94 (0.56) pg/mL. At age 3, mean (standard deviation) for BMI z score was 0.52 (1.02); for SS + TR, 16.51 (3.94) mm; and for SS:TR, 0.67 (0.17). Log CRH was mildly inversely correlated with birth weight (r = ?0.08), chiefly because of its association with length of gestation (r = ?0.21) rather than fetal growth (r = ?0.004). After adjustment for sociodemographic factors, maternal smoking, BMI, and gestational weight gain, fetal growth, length of gestation, breastfeeding duration, and (for SS:TR only) child's 3‐year BMI, each increment of 1 unit of log CRH was associated with a reduction in BMI z score [?0.43; 95% confidence interval (CI), ?0.73, ?0.14; p = 0.004] and possible reduction in SS + TR (?1.10; 95% CI, ?2.33, 0.14; p = 0.08). In contrast, log CRH was associated with higher SS:TR (0.07; 95% CI, 0.02, 0.13; p = 0.007). Discussion: Fetal exposure to glucocorticoids, although associated with an overall decrease in body size, may cause an increase in central adiposity.     

Interaction of boron with components of nucleic Acid metabolism in cotton ovules cultured in vitro

Cotton (Gossypium hirsutum L.) ovules grown in a defined nutrient medium undergo normal morphogenesis, including fiber production. In identical medium lacking boron, ovules callus and accumulate brown substances. Boron deficiency-like symptoms were induced by 6-azauracil and 6-azauridine in ovules growing in boron-sufficient media. Other nucleoside base analogs either reduced or had no effect on over-all growth, but did not cause typical boron-deficient callus growth of cotton ovules. Orotic acid and uracil countered the effects of 6-azauracil. Actinomycin D, fluorodeoxyuridine, and ethidium bromide reduced not only fiber production on ovules growing in boron-sufficient media but also callusing of ovules in boron-deficient media.     

Ethylene from alcohol lamps and natural gas burners: Effects on cotton ovules cultured in vitro

Summary In a transfer hood having positive pressure via flow of filtered air, momentary flaming of flask mouths via an alcohol lamp or natural gas burner introduced variable and often large amounts of ethylene into these culture vessels. Amounts of ethylene introduced depended upon where flask mouths were placed within the flame. Most ethylene diffused out of flasks within 2 hr if adequate fresh air exchange was provided, permitting cotton ovules to respond to hormones in ways reported in previous literature. However, it was concluded that flask-flaming could occasionally account for some variability in hormonal responses of cultured cotton ovules. Specifically, ethylene (assumed to be the most abundant and harmful substance admitted to the flasks via flaming) caused excessive ovular callus formation in combination with GA₃ and decreased the percentage of ovules forming fibers in response to IAA. This work was supported by the National Science Foundation Grant PCM 75-03944.