Sterols in Germinating Seeds and Developing seedlings of Longleaf Pine, Pinus palustris

Sterols in germinating embryos and young seedlings of longleaf pine (Pinus palustris Mill.) were identified and quantities determined for different periods after germination. Sterol analyses were performed by gas-liquid chromatography (GLC) and verified by combination of GLC-mass spectrometry. Campesterol and β-sitosterol were two major sterols which accounted for most of the sterol composition while stigmasterol was present in very small amounts. No cholesterol was revealed by GLC-mass spectrometry although there was a minor peak appearing on the sterol gas-liquid chromatograms with a retention time close to that of authentic cholesterol. By fractionation, three different forms of sterols were obtained: steryl esters, steryl glycosides, and free sterols. The sterols were mainly found in the esterified fraction, while steryl glycosides and free sterols only made up a small portion of the total sterol value. The total sterol content in general increased during seedling development, and this increase reflected mainly a change in steryl esters. The low levels of both free and glycosidic sterols remained nearly unchanged throughout the experimental germination period.     

Seasonal and geographic variations of the sterols from the starfish asterias vulgaris (verrill)

The levels of the major sterols of the starfish $\text{Asterias vulgaris}$ collected at one location in Nova Scotia varied considerably from month to month. After spawning in June the levels of the sterols in the starfish were very low, but a rapid assimilation of dietary sterols allowed the total sterol level to increase approximately two-hundred-fold to the annual maximum in July. The levels of a few minor sterols were unaffected by the spawning process, and during this period they emerged as the major components of the sterol mixture. The sterol mixtures from samples collected at different locations were compared.     

High level of glycosylated sterols in species of solanum and sterol changes during the development of the tomato

In the Solanaceae, leaves from various Solanum species show an exceptional abundance of glycosylated sterols essentially due to a high acylated steryl glycoside content. Changes in sterol composition during the development of tomato indicated a low proportion of glycosylated sterols in the ungerminated seeds with these compounds becoming progressively predominant compared with other sterols during the course of germination. This biochemical characteristic could be observed throughout the development of all parts of this plant. However, the high glycosidic sterol content tends to decrease when the organs become senescent.     

Variations in sterol and triterpene contents of developing Sorghum bicolor grains

The free, esterified and glycosylated sterols and the pentacyclic triterpene esters of developing Sorghum bicolor grains were analysed by GLC and GC-MS. All the pentacyclic triterpenes were completely esterified but were not detected until 24 days after anthesis. Lupanol, multiflorenol, α-amyrin and isoarborinol were identified in the mature grains as components of the triterpene fraction but no 4,4-dimethylsterols could be found at any stage of development. A sixfold increase in total sterol per grain occurred during development. At 8 days after anthesis, 28-isofucosterol was found to be the second most abundant steryl ester. Campesterol was the major steryl glycoside and obtusifoliol was the major 4-monomethylsterol.     

Annual Variation in the Effect of Red Light on Sterol Biosynthesis in Digitalis purpurea L

The effect of varying sequences of red and far red light on sterol biosynthesis in etiolated seedlings of Digitalis purpurea L. was examined. Red light caused a marked increase in the amounts of free and glycosidic sterols and a small decrease in esterified sterols during the first 4 hours after illumination. Far red light elicited the same response but to a lesser degree. Exposure to red followed by far red light or the reverse caused little or no increase in the amounts of free and glycosidic sterols. The magnitude of the increase in the amounts of sterols varied, depending upon the season in which the experiments were performed. The largest increments were obtained during the summer and fall, whereas the smallest were observed during the winter and spring. Correlation of these data with previous observations of an annual cycle in the sterol content of Digitalis seedlings showed that the maximum stimulation in sterol biosynthesis occurs when the endogenous level of sterols is minimal.

Sterol monoglycosides, acylmonoglycosides, and an unidentified sterol conjugate from the lipid extracts were quantitated. Changes in conjugated sterol content were related to the particular light conditions of each experiment. The results are discussed in terms of physiological cycles and the possible influence of hormones upon the control of sterol biosynthesis in Digitalis.

     

Changes in steryl lipids of oat root cells as a function of water-deficit stress

Five-day-old seedlings of oat ( Avena saliva L. cv. Seger) were subjected to water-deficit stress for two and four periods, each of 24h duration with interjacent rewatering periods of 24 h. After two and tour stress periods the fresh weight/dry weight ratio of the roots was 73 and 74% of the control value, respectively. Two stress periods did not affect the amount or composition of free, esterified and glycosylated sterols (desmethylsterols) or methylsterols (mono- and dimethylsterols). After four stress periods the amount of tree sterols increased by 25% on a dry-weight basis but that of free methylsterols only slightly. The most significant increase (by over 60%) occurred in esterified sterols and methylsterols. The amount of sterols bound as glycosides and acylaled glycosides decreased slightly (by 10%) after four stress periods. The amount of glycosylated methylsterols was negligible and did not respond to water-deficit stress. Within all component groups the proportions of individual compounds remained unaffected after two and four stress periods. The increase of the sterol levels caused by the stress is discussed in terms of a hormone-induced synthesis leading to a changed sterol/membrane acyl lipid ratio. This has implications for the chemo-physical properties of the root cell membranes.     

Changes in Sterol Composition during Greening of Etiolated Barley Shoots

The following sterols were identified in barley shoots: stigmasterol, β-sitosterol, campesterol, and cholesterol. The total sterol content of green and etiolated tissue was 2.84 and 3.20 milligrams per gram dry weight, respectively. The free sterols accounted for most of the difference in total sterol content. The sterol ester, sterol glycoside, and acylated sterol glycoside contents of green and etiolated barley shoots were essentially the same. Etiolated tissue had twice as much total β-sitosterol as stigmasterol, while green tissue had equal amounts of these two sterols. The campesterol and cholesterol content was the same in green and etiolated tissue. This same sterol composition pattern held true for the free, glycosidic, and acylated glycosidic sterols; however, the sterol ester fraction had a completely different composition pattern. The esterified stigmasterol content was quite low in green and etiolated tissue, and campesterol was the second largest esterfied sterol component in etiolated tissue. Etiolated barley seedlings exposed to light had a shift in the ratio of free stigmasterol to β-sitosterol in favor of stigmasterol; however, no correlation was observed between chlorophyll synthesis and shift in sterol composition.     

Sterol Changes during Germination of Nicotiana tabacum Seeds

The identity, composition, and concentration of the total, free, esterified, and glycosidic sterol fractions were determined during germination of tobacco seeds. The total, free, and esterified sterols increased, with stigmasterol and campesterol accounting for most of the increase. Steryl glycosides decreased during germination, and stigmasteryl and sitosteryl glycosides showed the largest decrease. During germination, sitosterol was the major sterol in all fractions but stigmasterol and campesterol showed the greatest changes. The fatty acid composition of the steryl esters and acylated steryl glycosides most closely resembled the di- and triglycerides.     

The pentacyclic triterpene Esters and the free,esterified and glycosylated sterols of Sorghum vulgare grain

The free, esterified and glycosylated sterols and triterpene esters of Sorghum vulgare grains were analysed by GLC. 4-Demethylsterols were found free, esterified and glycosidated. 4-Monomethyl-sterols were found completely free whilst the triterpenes were completely esterified. Three quarters of the total sterols in the grain were located in the embryo whilst the triterpenes were equally distributed between the embryo and endosperm.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Survival of Schismus arabicus seedlings exposed to desiccation depends on annual periodicity

Schismus arabicus, a desert annual grass, is one of the most common pasture annuals in the deserts of Israel and Asia. S. arabicus exhibits a unique set of adaptations and survival strategies, which enable it to germinate, develop and produce seeds even in years with annual rainfall of less than 100 mm. The current study examined whether an annual rhythm exists in the survival ability of S. arabicus seedlings exposed to desiccation. Our results indicate that survival of S. arabicus seedlings after six different periods of 7 to 42 days of desiccation depended on the month of germination of the caryopses (seeds). Seed germination was 80–100% in all experiments, regardless the month of germination; however, seedlings that germinated in different months varied in their root and shoot elongation rates. None of about 2,500 seedlings that germinated in July (in each of the 4 years) survived the desiccation treatment. The percentages of surviving seedlings in each month of June from 2002 to 2005 were less than 40%. In contrast, over 80% of the seedlings that germinated in each of the months of December and January survived after the desiccation periods of 7–42 days. Seedlings that survived were transferred to 5 L soil pots in which the seedlings developed into mature plants, completed their life cycle and produced seeds that germinated well. The current study demonstrated a novel phenomenon indicating that seedling survival in plants may depend on an annual periodicity according to the date of germination.     

GERMINATION ECOLOGY OF PHACELIA DUBIA VAR. DUBIA IN TENNESSEE GLADES

The germination characteristics of a population of the winter annual Phacelia dubia (L.) Trel. var. dubia from the middle Tennessee cedar glades were investigated in an attempt to define the factor(s) regulating germination in nature. Factors considered were changes in physiological response of the seeds (after-ripening), temperature, age, light and darkness, and soil moisture. At seed dispersal (late May to early June), approximately 50 % of the seeds were non-dormant but, would germinate only at low temperatures (10–15 C). As the seeds aged from June to September, there was an increase in rate and total percent of germination at 10, 15, and 20 C, and the maximum temperature for germination increased to 25 C. Little or no germination occurred at the June, July, and August temperatures in 0- to 2-month-old seeds, even in seeds on soil that was kept continuously moist during this 3-month period. At the September, October, and November temperatures 3- to 5-month-old seeds germinated to high percentages. In all experiments seeds germinated better at a 14-hr photoperiod than in constant darkness. Inability of 0- to 2-month-old seeds to germinate at high summer temperatures allows P. dubia dubia to pass the dry summer in the seed stage, while increase in optimum and maximum temperatures for germination during the summer permits seeds to germinate in late summer and early fall when conditions are favorable for seedling survival and eventual maturation.     

Sterol Composition of the Corn Cyst Nematode,Heterodera zeae,and Corn Roots

Sterols from free sterol and steryl ester fractions from Heterodera zeae and from total lipids of Zea mays roots were analyzed by gas-liquid chromatography (GLC) and by GLC-mass spectrometry. The major free sterols of H. zeae were 24-ethylcholesterol (54.4% of total free sterol), 24-ethylcholesta-5,22-dien-3β-ol (13.3%), 24-methylcholesterol (12.5%), and cholesterol (7.2%). The same four sterols comprised 34.6%, 7.2%, 30.3%, and 18.6%, respectively, of the esterified sterols of H. zeae. Corn root sterols included 46.6% 24-ethylcholesta-5,22-dien-3β-ol, 16.7% methylcholesterol, 16.4% cycloartenol, 12.7% 24-ethylcholesterol, and 0.5% cholesterol. The sterol 24-composition of H. zeae differed greatly from that of the only other cyst nematode previously investigated, Globodera solanacearum.     

24-methylenepollinastanol and 24-dehydropollinastanol—two sterols of pollen

The esterified and unesterified sterol fractions of bee-gathered mixed pollens were examined, and total sterol composition was determined. Two new sterols of pollens, 14α-methyl-9β,19-cyclo-5α-cholest-24-en-3β-ol (24-dehydropollinastanol) and 14α-methyl-5α-ergost-24(28)-en-3β-ol (24-methylenepollinastanol) were isolated and identified. Both sterols were found primarily in the esterified sterol fraction, and 24-methylenepollinastanol accounted for 43% of the sterols of this fraction. 24-Dehydropollinastanol and four other sterols which also contain a 9β,19-cyclopropane ring were found only in the esterified sterol fraction. 24-Methylenecholesterol was the major sterol of the unesterified sterol fraction.     

Phenology and life cycle of the annual, <Emphasis Type="Italic">Chamaesyce maculata</Emphasis> (L.) Small (Euphorbiaceae), with multiple overlapping generations in Japan

The phenology of germination, vegetative growth and sexual reproduction in the annual Chamaesyce maculata (L.) Small (Euphorbiaceae) were investigated in a natural population in western Japan. Seedlings emerged from mid-June to early October, with three peaks: mid-June, late July and late August. Plants that emerged in June commenced sexual reproduction from late July, and thereafter both vegetative growth and sexual reproduction occurred together until early November, the plants showing no switching from vegetative growth to sexual reproduction. Seedlings that emerged in June and July suffered high mortality, but most seedlings that emerged from August onward survived until the reproductive stage. The minimum size for reproduction was largest for plants that emerged early in the season, and it decreased with a delay in seedling emergence. The late emergence of seedlings that resulted in low reproductive output may be to some extent compensated for by the increased probability of survival in the seedling stage. A transplant experiment clarified that C. maculata can repeat a maximum of three overlapping generations within a year. Multiple generations per year were attained by non-dormant seeds produced in the first and second generations and clearly resulted in an increased reproductive output per year. The life cycle with multiple overlapping generations may have been acquired in habitats where unpredictable disturbance results in temporally unsuitable conditions for germination, vegetative growth and sexual reproduction of annual plants, but where suitable conditions frequently continue over a period longer than the single generation time of annual plants.     

The biosynthesis of the free sterols and sterol esters of Neurospora crassa.

The composition of the free and esterified sterols in Neurospora crassa was examined as a function of incubation time in starvation medium containing [2-14C]mevalonic acid. The 14C incorporation was monitored in nuclear methylated and 4,14-desmethyl sterol fractions. After 7 h incubation, sterol esterification had increased from an initial 5% in the log phase culture to 48% of the total sterol pool, with a concomitant decrease in free sterols. The relationship of the free and esterified sterol components in ergosterol biosynthesis is discussed.     

Effect of gibberellic acid on sterol production in Corylus avellana seeds

Gibberellic acid-induced germination of hazel seeds was accompanied by little change in the sterol content of the cotyledons. Dormant and germinating cotyledons rapidly incorporated [2-¹⁴C]MVA into squalene which was slowly converted to sterols. Gibberellin treatment induced an increase in the incorporation of [2-¹⁴C]MVA into cotyledon esterified sterols. An increase in free sterols occurred in the germinating embryonic axes, with increased relative amounts of stigmasterol and campesterol in the free 4-desmethylsterols. Germination was accompanied by increased incorporation of [2-¹⁴C]MVA into free and esterified sterols in the embryonic axes.     

Activities of isolated mitochondria and mitochondrial enzymes from aerobically and anaerobically germinated barnyard grass (echinochloa) seedlings

Activity of mitochondria isolated from whole seedlings of Echinochloa crus-galli (L.) Beauv. var oryzicola germinated under aerobic and anaerobic conditions for 5 to 7 days was investigated. Mitochondria from both treatments exhibited good respiratory control and ADP/O ratios. Although O₂ uptake was low in anaerobic mitochondria, activity rapidly increased when the seedlings were transferred to air. Mitochondria from both aerobically and anaerobically grown seedlings of E. crus-galli var oryzicola maintained up to 66% of their initial respiration rate in the presence of both cyanide and salicylhydroxamic acid, and the inhibitory effects of cyanide and azide were additive. In addition, antimycin A was not an effective inhibitor of respiration. Reduced-minus-oxidized absorption spectra revealed that cytochromes a, a₃, and b were reduced to a greater extent and cytochrome c was reduced to a lesser extent in anaerobically germinated seedlings relative to that in aerobically germinated seedlings. An absorption maximum in the cytochrome d region of the spectrum was reduced to the same extent under both germination conditions and an absorption maximum at 577 nm was present only in anaerobically germinated seedlings. Anaerobically germinated seedlings contained 70% of the cytochrome c oxidase activity found in air grown seedlings. Upon exposure to air, the developmental pattern of this enzyme in anaerobically germinated seedlings was similar to air controls. Succinate dehydrogenase activity in anaerobic seedlings was only 45% of the activity found in aerobically germinated seeds, but within 1 hour of exposure to air, the activity had increased to control levels. The results suggest that mitochondria isolated from E. crus-galli var oryzicola differ from other plants studied and that the potential for mitochondrial function during anaerobiosis exists.     

Sterol distribution in intracellular organelles isolated from tobacco leaves

All membrane-containing fractions isolated from tobacco leaves contained free sterols, sterol glycosides, and sterol esters. The three sterol forms increased, on a dry weight basis, with a decrease in particle size. The supernatant fraction contained only trace amounts of sterol. The major sterols in all cellular fractions, in the order of decreasing amounts, were: stigmasterol, β-sitosterol, campesterol, and cholesterol. The 500g pellet contained the largest percentage of free sterol, while the 46,000g pellet contained the largest percentage of esterified sterol. The individual sterol composition of the free sterol and sterol glycoside fraction was very similar; however, the composition of the sterol ester fraction varied widely among intracellular fraction. The intracellular distribution pattern of cholesterol-¹⁴C added to the isolation medium provided evidence that the intracellular sterol distribution pattern is not an artifact. These results support the suggestion that sterols in plant cells may have a physiological function associated with membranes.     

STEROLIC BIOMARKERS IN MARINE PHYTOPLANKTON. II. FREE AND CONJUGATED STEROLS OF SEVEN SPECIES USED IN MARICULTURE

The sterols of seven unicellular algae widely used in mariculture and belonging to different classes (Prasinophyceae, Haptophyceae, Eustigmatophyceae, and Diatomophyceae) were examined for free and combined forms. Under standard culture conditions, all synthesized free sterols and combined forms (steryl esters, acyl steryl glycosides, and steryl glycosides). Free sterols were dominant in five of the species. In contrast, sterols were mostly esterified in the eustigmatophyte Nannochloropsis oculata (Droop) Hibberd and the diatom Thalassiosira pseudonana Hasle et Heimdal, whereas in another diatom, Chaetoceros calcitrans (Paulsen) Takano, glycosylated forms represented over 60% of total sterols. The pennate diatom Haslea ostrearia (Gaillon) Simonsen synthesized large amounts of steryl glycosides consisting mainly of an unusual sterol, 23,24-dimethylcholest-5-en-3β-ol, occurring in some dinoflagellates.