Quantitative determination of 13C-labeled and endogenous beta-carotene, lutein, and vitamin A in human plasma.

Quantitative procedures employing liquid-chromatography/particle beam-mass spectrometry (LC/PB-MS) and gas chromatography-mass spectrometry (GC-MS) were applied to the determination of the endogenous and 13C-labeled beta-carotene, lutein, and retinol in plasma of a subject who consumed kale (Brassica oleracea) that had been grown in a 13CO2-enriched atmosphere. All compounds were analyzed in the negative chemical ionization (NCI) mode using methane as the moderating reagent gas. Beta-carotene and lutein were analyzed using LC/PB-MS applying reversed-phase high-performance liquid chromatography (HPLC) separation procedures to resolve the analytes. The concentrations of the beta-carotene isotopomers in the plasma over a several-week period were determined using 2H8-beta-carotene as an internal standard. The total plasma concentrations of all trans-lutein were quantified by HPLC analysis with a photodiode array detector using beta-apo-8'-carotenal as an internal standard, and the ratio of the 13C:12C isotopomers of lutein was determined by PB-MS. The retinol isotopomers were collected from individual HPLC fractions of the plasma extract and then analyzed as the trimethylsilyl ethers by GC-MS in the NCI mode. The 13C- and 12C-retinol isotopomers were quantified using 2H4-retinol as an internal standard. These methods demonstrate the application of highly sensitive procedures employing NCI MS for the quantitative determination of carotenoids and vitamin A for the purpose of conducting metabolism studies of phytonutrients.

Tolerance of a field grown soybean cultivar to elevated ozone level is concurrent with higher leaflet ascorbic acid level, higher ascorbate-dehydroascorbate redox status, and long term photosynthetic productivity.

We examined the characteristics of ascorbic acid (ASC) level, dehydroascorbate (DHA) level, and the ASC-DHA redox status in the leaflets of two soybean cultivars grown in a field environment and exposed to elevated ozone (O(3)) levels. These two cultivars, one that preliminary evidence indicated to be O(3)-tolerant (cv Essex), and one that was indicated to be O(3)-sensitive (cv Forrest), were grown in open-top chambers during the summer of 1997. The plants were exposed daily to a controlled, moderately high O(3) level ( approximately 58 nl l(-1) air) in the light, beginning at the seedling stage and continuing to bean maturity. Concurrently, control plants were exposed to carbon-filtered, ambient air containing a relatively low O(3) level ( approximately 24 nl l(-1) air) during the same period. Elevated O(3) did not affect biomass per plant, mature leaf area accretion, or bean yield per plant of cv Essex. In contrast, elevated O(3) level decreased the biomass and bean yield per plant of cv Forrest by approximately 20%. Daily leaflet photosynthesis rate and stomatal conductance per unit area did not decrease in either cultivar as a result of prolonged O(3) exposure. A 10% lower mature leaflet area in O(3)-treated cv Forrest plants contributed to an ultimate limitation in long-term photosynthetic productivity (vegetative and bean yield). Possible factors causing cv Essex to be more O(3) tolerant than cv Forrest were: 1) mature leaflets of control and O(3)-treated cv Essex plants consistently maintained a higher daily ASC level than leaflets of cv Forrest plants, and 2) mature leaflets of cv Essex plants maintained a higher daily ASC-DHA redox status than leaflets of cv Forrest plants.

Photomorphogenesis and photoassimilation in soybean and sorghum grown under broad spectrum or blue-deficient light sources.

The role of blue light in plant growth and development was investigated in soybean (Glycine max [L.] Merr. cv Williams) and sorghum (Sorghum bicolor [L.] Moench. cv Rio) grown under equal photosynthetic photon fluxes (approximately 500 micromoles per square meter per second) from broad spectrum daylight fluorescent or blue-deficient, narrow-band (589 nanometers) low pressure sodium (LPS) lamps. Between 14 and 18 days after sowing, it was possible to relate adaptations in photosynthesis and leaf growth to dry matter accumulation. Soybean development under LPS light was similar in several respects to that of shaded plants, consistent with an important role for blue light photoreceptors in regulation of growth response to irradiance. Thus, soybeans from LPS conditions partitioned relatively more growth to leaves and maintained higher average leaf area ratios (mean LAR) that compensated lower net assimilation rates (mean NAR). Relative growth rates were therefore comparable to plants from daylight fluorescent lamps. Reductions in mean NAR were matched by lower rates of net photosynthesis (A) on an area basis in the major photosynthetic source (first trifoliolate) leaf. Lower A in soybean resulted from reduced leaf dry matter per unit leaf area, but lower A under LPS conditions in sorghum correlated with leaf chlorosis and reduced total nitrogen (not observed in soybean). In spite of a lower A, mean NAR was larger in sorghum from LPS conditions, resulting in significantly greater relative growth rates (mean LAR was approximately equal for both light conditions). Leaf starch accumulation rate was higher for both species and starch content at the end of the dark period was elevated two- and three-fold for sorghum and soybean, respectively, under LPS conditions. Possible relations between starch accumulation, leaf export, and plant growth in response to spectral quality were considered.

Regulation of photosynthate partitioning into starch in soybean leaves : response to natural daylight.

Studies conducted in controlled environments indicate that daylength affects the proportion of photosynthate stored in leaves as starch or sucrose. To examine the response of partitioning to natural daylight, soybeans (Glycine max [L.] Merr. cv Williams) were grown at 12 different times between May and November in a constant temperature greenhouse without supplemental lighting. Plants were transferred from the greenhouse to a controlled environment chamber at the end of civil twilight at a set developmental stage (expanding seventh trifoliolate leaf, counting acropetally). Net photosynthesis and the accumulation of starch and sugar in fully expanded fourth trifoliolate leaves were determined the following day under standard conditions in the chamber (lights-on synchronized with sunrise). Photosynthesis on a leaf area basis decreased about 10% between midsummer and early autumn. Leaf soluble sugar accumulation was low at all harvests. However, a twofold increase in photosynthate partitioning into starch occurred over the same time period, resulting in an 80% increase in absolute starch accumulation rate. Starch was responsible for about 78% of the increase in leaf dry matter during the light at all harvests, indicating that starch accumulation as affected by prior daylight conditions will alter export of photosynthate during the light period. Photosynthate partitioning into starch was linearly correlated with daylength at harvest, prior average peak solar irradiance, and other parameters that correlated with daylength and solar radiation such as harvested top dry matter. The relation between growth and seasonal changes in daylight (including daylength, irradiance, and light integral) are discussed in relation to photosynthate partitioning under field conditions.

Rhythms during extended dark periods determine rates of net photosynthesis and accumulation of starch and soluble sugars in subsequent light periods in leaves of Sorghum.

Photosynthesis and photosynthate partitioning in leaves of Sorghum bicolor (L.) Moench exhibited a cyclic dependence on the duration (10-62 h) of dark periods inserted prior to bright light test periods (550 µmol·s(-1)·m(-2), photosynthetic photon flux). Maximum rates of net photosynthesis and of accumulation of starch and soluble sugars were, in the order given, two-, three- and fourfold greater than minimum values. Between 14 and 53% of photosynthate was retained in leaves depending on the length of the dark period. These changes were sufficient to account for the previously described stimulatory effect of short daylengths (i.e., long nights) on carbohydrate accumulation in leaves (N.J. Chatterton and J.E. Silvius, 1980, Physiol. Plant. 49, 141-144). The freerunning periods for the rhythmic dependence on darkness, determined either directly or by curve fitting, were about 24 h for net photosynthesis, 23 h for starch accumulation, and 26 h for solublesugar cccumulation. The deviation from period lengths of 24 h for carbohydrate accumulation indicates that these rhythms are probably endogenous and circadian. Initial maxima were observed after 14 h of darkness for photosynthesis, after 18-22 h for starch, and after 26 h for soluble sugars. The differences in period length and phase indicate that at least three separate rhythms underlie the dependence of photosynthate partitioning in Sorghum on darkness. Periods of low leaf dry-matter accumulation coincided approximately with periods of high net photosynthesis. As a result, maximum photoassimination and maximum export were synchronized and, furthermore, occurred at about the same time as expected light periods.

Photoperiodic Regulation of Photosynthate Partitioning in Leaves of Digitaria decumbens Stent.

In leaves of pangolagrass (Digitaria decumbens Stent.), the proportion of photosynthate partitioned into starch adjusts to a change in daylength within 24 hours. After a single 14-hour long day, the relative starch accumulation rate is approximately 50% of that under 7-hour short days. This rapid response was exploited to study the light requirement for the perception of changes in daylength. It was found for short day-grown plants that: (a) 7-hour daylength extensions with dim white light (below the light compensation point for photosynthesis); (b) 7-hour daylength extensions with dim far red light (wavelengths greater than 690 nanomoles); or (c) 0.5-hour night-break irradiations with bright white light were all capable of producing about one-half of the effect of a 7-hour daylength extension with bright light. However, long periods of bright light were not required for a complete effect, since a 7-hour shifted short day (i.e. beginning 7 hours later than usual) was as effective as a 14-hour-long day itself. There was also a critical daylength between 11 and 12 hours for the transition between short-day and long-day partitioning patterns. Photoperiod determination depends, at least in part, on a nonphotosynthetic photoreceptor sensitive to both visible and far red irradiation. The duration of the photosynthetic period, as shown in experiments with low-pressure sodium lamps, does not by itself determine the response to daylength.

Rhythmic chloroplast migration in the green alga Ulva: dissection of movement mechanism by differential inhibitor effects.

The chloroplasts of the green alga, Ulva lactuca L., migrate rhythmically between the outer (periclinal) cell walls in the daytime ("face" position) and the anticlinal cell walls at night ("profile" position). Both NaN3 and colchicine inhibit chloroplast movement mainly in the direction from profile towards face position. Differential drug sensitivity is suggestive of different mechanisms for the two directions of chloroplast migration. UV light reverses the inhibition by colchicine, presumably through the formation of lumicolchicine, the non-tubulin-binding isomer of colchicine. This result is indicative of microtubule involvement. Interpretation of the effects of azide is complicated by changes in the biological clock (phase delay and lengthening of the period). Cytochalasin B has no effect on chloroplast movement when added alone, but when added with colchicine it prevents colchicine inhibition of movement. We hypothesize that chloroplast position is controlled by a balance between two opposing movement systems with differential drug sensitivity.

Physiology of Movements in the Stems of Seedling Pisum sativum L. cv Alaska : III. Phototropism in Relation to Gravitropism, Nutation, and Growth.

Phototropic response in etiolated pea (Pisum sativum L. cv Alaska) seedlings is poor. However, the curvature induced by unilateral blue light can be hastened and increased in magnitude by a previously administered red light pulse followed by several hours of darkness. Phytochrome is involved in the red light effect. Phototropic response was almost completely inhibited by removal of the apical bud and hook, but it was restored if exogenous indole-3-acetic acid was applied apically to the cut stump. Therefore, the stem contains both the phototropic photoreceptor and response mechanism. Perception of gravity and gravitropic response were also localized in the stem, but gravitropism was scarcely inhibited by decapitation. It was also observed that the kinetics and curvature pattern of gravitropism differed greatly from those of phototropism. Like phototropism, stem nutation required auxin and was promoted by red light. Unlike phototropism, photoenhanced nutational curvature required the apical hook and was propagated as a wave down the stem. Naphthylphthalamic acid inhibited, in order of decreasing effect, nutation, phototropism/gravitropism, and growth. Phototropism, gravitropism, and nutation appear to represent distinct forms of stem movement with fundamental differences in the mechanisms of curvature development.

Physiology of Movements in Stems of Seedling Pisum sativum L. cv Alaska : II. The Role of the Apical Hook and of Auxin in Nutation.

The relationship between the apical hook and stem nutation in etiolated Alaska pea (Pisum sativum L. cv Alaska) seedlings was explored. The hook and maximum nutational displacement have the same plane of symmetry, and both are affected by light acting through phytochrome. However, the two processes do not appear to be obligatorily coupled. Light effects on nutation involve at least two components, an increase in amplitude as well as an increase in frequency. These components can be separated from one another on the basis of developmental time course or red light fluence. Excision of the plumule, leaving the hook attached to the stem, inhibits photostimulated nutation. This inhibition can be overcome by application of indole-3-acetic acid to the remaining stem. If the hook is also excised, then nutation in the stem cannot be restored by indole-3-acetic acid. It is possible, although not yet proven, that the oscillatory process regulating nutation in the stem is itself localized in the hook and that rhythms in the transport of indole-3-acetic acid are involved.

Physiology of Movements in Stems of Seedling Pisum sativum L. cv. Alaska : I. Experimental Separation of Nutation from Gravitropism.

Gravitropism and nutation in the stems of dark-grown, seedling peas (Pisum sativum L. cv. Alaska) were recorded on time-lapse photographs made with photomorphogenetically inactive light. Although gravitropism and nutation have been connected by several different theories in the past, our experiments indicate that the two processes are in fact dissociable. The evidence is as follows: (a) Nutational patterns are asymmetric. There is much greater amplitude of oscillation in the plane parallel ( parallel) to the plane of the apical hook than in the plane perpendicular ( perpendicular), yet the average gravitropic response is equal in these two planes. (b) Brief red light irradiation given 16 to 24 hours before observation greatly increases the amplitude of nutation in the parallel-plane, but has no influence on the kinetics of gravitropic response. (c) An inhibitor of auxin transport, alpha-naphthylphthalamic acid, strongly inhibits nutation at 5 micromolar but affects gravitropism only at higher concentrations. (d) Nutation is also strongly inhibited by removal of the apical bud, but gravitropism is unaffected. (e) The period of nutation does not exhibit a constant relationship to the response time of gravitropism. The above evidence is inconsistent with theories that gravitropism is an asymmetrically modified nutation or, alternatively, that nutational oscillations result in a simple fashion from gravitropic overshoots. The evidence is consistent with, although not proof of, autonomous factors such as an endogenous rhythm of growth as the cause of nutation in pea stems. However, gravity and nutation do interact. Nutation in a population of seedlings can be synchronized and brought into phase by a single gravitropic induction. Furthermore, the response time and initial rate of gravitropic curvature depend to some extent on the phase of nutational curvature at which gravitropic induction is begun.

Light-enhanced perception of gravity in stems of intact pea seedlings.

Dark-grown, 6-d-old pea seedlings (Pisum sativum L. cv. Alaska) do not respond gravitropically to brief (approx. 3 min) horizontal presentations, but seedlings given a pulse of red light (R) 16-24 h earlier respond to such stimuli by vigorous curvature of the epicotyl. With continuous horizontal stimulation (approx. 100 min), the kinetics and extent of the gravitropic response are almost identical in irradiated and dark-control plants. Prior R thus increases graviperception without altering the rate-limiting steps underlying the generation of curvature. This effect of R on graviperception develops slowly; seedlings studied only a few hours after R show differences in the kinetics of the gravitropic response, but not in presentation time. Neither the kinetics nor the extent of gravitropic curvature should be used as criteria for establishing changes in primary processes in gravitropism.

Automatic monitoring of a circadian rhythm of change in light transmittance in ulva.

Ulva lactuca L. var. latissima (L.) DeCandolle has a circadian rhythm of visible light transmittance change which is caused by chloroplast orientation. With a continuously recording microphotometer system, clear rhythms could be monitored for up to 10 days. Measuring beam intensity effects on the free running period were seen down to 10(-7) w cm(-2). While these effects complicate the measuring process, they demonstrate that Ulva is very sensitive to light. The free running period in constant darkness at 20 C is 24 to 25 hours. The position in the cell occupied by the chloroplasts when the rhythm damps out can be influenced by light. A method is described by which the times of rhythm maxima can be calculated accurately and objectively from a relatively small number of points.

Circadian rhythms of chloroplast orientation and photosynthetic capacity in ulva.

Ulva lactuca L. var. latissima (L.) Decandolle and var. rigida (C. Agardh) Le Jolis and U. mutabilis Foyn have a circadian rhythm of chloroplast orientation which results in large changes in the light-absorption properties of the thallus. During the day, the chloroplasts cover the outer face of the cells and absorbance is high. At night, the chloroplasts are along the side walls and absorbance is low. Enteromorpha linza (L.) J. Agardh, E. intestinalis (L.) Link, E. sp., and Monostroma grevillei (Thuret) Wittrock, members of the Ulvales, were not observed to have this rhythmic movement. Chloroplasts, when in the face position, could not be induced to move to the sides by high intensity light up to 80,000 lux. Unrelated to chloroplast position per se and light-absorption efficiency, there is a rhythm of photosynthetic capacity which peaks just before midday and which continues in constant darkness.