Seed coat morphology and its systematic implications in Cyanea and other genera of Lobelioideae (Campanulaceae).

Recent surveys of seed coat morphology in Lobelioideae (Campanulaceae) have demonstrated the systematic utility of such data in the subfamily and led to a revision of the supraspecific classification of Lobelia. Expanding upon these studies, we examined via scanning electron microscopy 41 seed accessions, emphasizing lobelioid genera in which only one or no species had been examined. Most conformed to previously described testal patterns. However, five species of the endemic Hawaiian genus Cyanea, comprising the molecularly defined Hardyi Clade, had a unique testal pattern (here termed Type F), characterized by laterally compressed, almost linear, areoles with rounded, knob-like protuberances on the radial walls at opposite ends. This offered a convenient synapomorphy for recognition of a clade originally defined on a molecular basis. A second unique testal pattern was found in the related Hawaiian endemics Brighamia and Delissea, thus supporting their close relationship. In this type (here termed Type G), the seed coat is irregularly wrinkled (rugose), creating broad, rounded ridges that run more-or-less perpendicular to the long axis of the seed and thus to the long axis of the testal cells. Seed coat morphology also supported the monophyly of all 124 species of Hawaiian Lobelioideae and their probable derivation from Asian species of Lobelia subg. Tupa. Additional studies supported close relationships between (1) the neotropical genera Centropogon and Siphocampylus; (2) the western American genera Legenere and Downingia; and (3) Jamaican Hippobroma and Lobelia sect. Tylomium, a group endemic to the West Indies.

Chilling-enhanced photooxidation: The production, action and study of reactive oxygen species produced during chilling in the light.

Chilling-enhanced photooxidation is the light- and oxygen-dependent bleaching of photosynthetic pigments that occurs upon the exposure of chilling-sensitive plants to temperatures below approximately 10 °C. The oxidants responsible for the bleaching are the reactive oxygen species (ROS) singlet oxygen ((1)O2), superoxide anion radical (O 2 (∸) ,hydrogen peroxide (H2O2), the hydroxyl radical (OH·), and the monodehydroascorbate radical (MDA) which are generated by a leakage of absorbed light energy from the photosynthetic electron transport chain. Cold temperatures slow the energy-consuming Calvin-Benson Cycle enzymes more than the energy-transducing light reactions, thus causing leakage of energy to oxygen. ROS and MDA are removed, in part, by the action of antioxidant enzymes of the Halliwell/Foyer/Asada Cycle. Chloroplasts also contain high levels of both lipid- and water-soluble antioxidants that act alone or in concert with the HFA Cycle enzymes to scavenge ROS. The ability of chilling-resistant plants to maintain active HFA Cycle enzymes and adequate levels of antioxidants in the cold and light contributes to their ability to resist chilling-enhanced photooxidation. The absence of this ability in chilling-sensitive species makes them susceptible to chilling-enhanced photooxidation. Chloroplasts may reduce the generation of ROS by dissipating the absorbed energy through a number of quenching mechanisms involving zeaxanthin formation, state changes and the increased usage of reducing equivalents by other anabolic pathways found in the stroma. During chilling in the light, ROS produced in chilling-sensitive plants lower the redox potential of the chloroplast stroma to such a degree that reductively-activated regulatory enzymes of the Calvin Cycle, sedohepulose 1,7 bisphosphatase (EC 3.1.3.37) and fructose 1,6 bisphosphatase (EC 3.1.3.11), are oxidatively inhibited. This inhibition is reversible in vitro with a DTT treatment indicating that the enzymes themselves are not permanently damaged. The inhibition of SBPase and FBPase may fully explain the inhibition in whole leaf gas exchange seen upon the rewarming of chilling-sensitive plants chilled in the light. Methods for the study of ROS in chilling-enhanced photooxidation and challenges for the future are discussed.

Spatial Distribution of Photosynthesis during Drought in Field-Grown and Acclimated and Nonacclimated Growth Chamber-Grown Cotton.

Inhomogeneous photosynthetic activity has been reported to occur in drought-stressed leaves. In addition, it has been suggested that these water stress-induced nonuniformities in photosynthesis are caused by "patchy" stomatal closure and that the phenomenon may have created the illusion of a nonstomatal component to the inhibition of photosynthesis. Because these earlier studies were performed with nonacclimated growth chamber-grown plants, we sought to determine whether such "patches" existed in drought-treated, field-grown plants or in chamber-grown plants that had been acclimated to low leaf water potentials (psi(leaf)). Cotton (Gossypium hirsutum L.) was grown in the field and subjected to drought by withholding irrigation and rain from 24 d after planting. The distribution of photosynthesis, which may reflect the stomatal aperture distribution in a heterobaric species such as cotton, was assayed by autoradiography after briefly exposing attached leaves of field-grown plants to (14)CO(2). A homogeneous distribution of radioactive photosynthate was evident even at the lowest psi(leaf) of -1.34 MPa. "Patchiness" could, however, be induced by uprooting the plant and allowing the shoot to air dry for 6 to 8 min. In parallel studies, growth chamber-grown plants were acclimated to drought by withholding irrigation for three 5-d drought cycles interspersed with irrigation. This drought acclimation lowered the psi(leaf) value at which control rates of photosynthesis could be sustained by approximately 0.7 MPa and was accompanied by a similar decline in the psi(leaf) at which patchiness first appeared. Photosynthetic inhomogeneities in chamber-grown plants that were visible during moderate water stress and ambient levels of CO(2) could be largely removed with elevated CO(2) levels (3000 muL L(-1)), suggesting that they were stomatal in nature. However, advanced dehydration (less than approximately 2.0 MPa) resulted in "patches" that could not be so removed and were probably caused by nonstomatal factors. The demonstration that patches do not exist in drought-treated, field-grown cotton and that the presence of patches in chamber-grown plants can be altered by treatments that cause an acclimation of photosynthesis leads us to conclude that spatial heterogeneities in photosynthesis probably do not occur frequently under natural drought conditions.

Regulation of coupling factor in field-grown sunflower: A Redox model relating coupling factor activity to the activities of other thioredoxin-dependent chloroplast enzymes.

Simultaneous, non-invasive measurements were made of the rate of photosynthetic CO2 fixation and the state of activation of the chloroplast CF1CF0-ATP synthase (CF) in field-grown sunflower (Helianthus annuus L.) during the dark-to-light transition at sunrise. CO2 fixation showed a linear response with light intensity from zero to about 500-700 µE m(-2) s(-1). However, at light intensities of only 5-22 µE m(-2) s(-1), the energetic threshold for activation of the CF was found to be significantly lowered (as compared to the pre-dawn state), presumably through reduction of the regulatory sulfhdryl groups of the γ-subunit of the CF. When these studies were extended to chamber-grown plants, it was found that as little as 5 seconds of illumination at 4 µE m(-2) s(-1) caused apparently full CF reduction. It is clear, therefore, that the catalytic activation of CF is not rate limiting to the induction of carbon assimilation under field conditions during a natural dark-to-light transition at sunrise. A model, based on the redox properties of the regulatory sulfhydryls, was developed to examine the significance of sulfhydryl midpoint potential in explaining the differences in light sensitivity and oxidation and reduction kinetics, between the CF and other thioredoxin-modulated chloroplast enzymes. Computer simulations of the light-induced regulation of three representative thioredoxin-modulated enzymes are presented.

Photophosphorylation after chilling in the light : effects on membrane energization and coupling factor activity.

The response of in situ photophosphorylation in attached cucumber (Cucumis sativus L. cv Ashley) leaves to chilling under strong illumination was investigated. A single-beam kinetic spectrophotometer fitted with a clamp-on, whole leaf cuvette was used to measure the flash-induced electrochromic absorbance change at 518 minus 540 nanometers (DeltaA(518-540)) in attached leaves. The relaxation kinetics of the electric field-indicating DeltaA(518-540) measures the rate of depolarization of the thylakoid membrane. Since this depolarization process is normally dominated by proton efflux through the coupling factor during ATP synthesis, this technique can be used, in conjuction with careful controls, as a monitor of in situ ATP formation competence. Whole, attached leaves were chilled at 5 degrees C and 1000 microeinsteins per square meter per second for up to 6 hours then rewarmed in the dark at room temperature for 30 minutes and 100% relative humidity. Leaf water potential, chlorophyll content, and the effective optical pathlength for the absorption measurements were not affected by the treatment. Light- and CO(2)-saturated leaf disc oxygen evolution and the quantum efficiency of photosynthesis were inhibited by approximately 50% after 3 hours of light chilling and by approximately 75% after 6 hours. Despite the large inhibition to net photosynthesis, the measurements of DeltaA(518-540) relaxation kinetics showed photophosphorylation to be largely unaffected by the chilling and light exposure. The amplitude of the DeltaA(518-540) measures the degree of energization of the photosynthetic membranes and was reduced significantly by chilling in the light. The cause of the decreased energization was traced to impaired turnover of photosystem II. Our measurements showed that the chilling of whole leaves in the light caused neither an uncoupling of photophosphorylation from photosynthetic electron transport nor any irreversible inhibition of the chloroplast coupling factor in situ. The sizeable inhibition in net photosynthesis observed after chilling in the light cannot, therefore, be attributed to any direct effect on photophosphorylation competence.

Chilling-Enhanced Photooxidation : The Peroxidative Destruction of Lipids during Chilling Injury to Photosynthesis and Ultrastructure.

Chilling-induced photooxidation was studied in detached leaves of chilling-sensitive (CS) cucumber (Cucumis sativus L.) and chilling resistant (CR) pea (Pisum sativum L.). The rates of photosynthesis and respiration, measured as O(2) exchange, were found to be comparable in the two species over a temperature range of 5 to 35 degrees C. Chilling at 5 degrees C for 12 hours in high light (1000 microeinsteins per square meter per second) decreased CO(2) uptake 75% in detached pea leaves whereas CO(2) uptake by cucumber was reduced to zero within 2 hours. Respiration was unaffected in either species by the chilling and light treatment. Although ultrastructural alterations were apparent in chloroplasts of both species, cucumber's were affected sooner and more severely. The mechanism of photooxidative lipid peroxidation was investigated by following the production of ethane gas under a variety of conditions. Maximum ethane production occurred in the CS cucumber at low temperature (5 degrees C) and high light (1000 microeinsteins per square meter per second). Atrazine, an inhibitor of photosynthetic electron transport, almost completely halted this chilling- and light-induced ethane production. These data, taken with those reported in an accompanying article (RR Wise, AW Naylor 1986 Plant Physiol 83: 278-282) suggest that the superoxide anion radical is generated in cucumber chloroplasts (probably via a Mehler-type reaction) during chilling-enhanced photooxidation. Parallel experiments were conducted on pea, a CR species. Detached pea leaves could only be made to generate ethane in the cold and light if they were pretreated with the herbicide parquat, a known effector of O(2) (-) production. Even so, pea showed no lipid peroxidation for 6 hours, at which time ethane production began and was at a rate equal to that for the chilled and irradiated cucumber leaves. The results indicate that pea has an endogenous mechanism(s) for the removal of toxic oxygen species prior to lipid peroxidation. This mechanism breaks down in pea after 6 hours in the cold, light, and the presence of paraquat.

Chilling-enhanced photooxidation : evidence for the role of singlet oxygen and superoxide in the breakdown of pigments and endogenous antioxidants.

Chilling temperatures (5 degrees C) and high irradiance (1000 microeinsteins per square meter per second) were used to induce photooxidation in detached leaves of cucumber (Cucumis sativus L.), a chilling-sensitive plant. Chlorophyll a, chlorophyll b, beta carotene, and three xanthophylls were degraded in a light-dependent fashion at essentially the same rate. Lipid peroxidation (measured as ethane evolution) showed an O(2) dependency. The levels of three endogenous antioxidants, ascorbate, reduced glutathione, and alpha tocopherol, all showed an irradiance-dependent decline. alpha-Tocopherol was the first antioxidant affected and appeared to be the only antioxidant that could be implicated in long-term protection of the photosynthetic pigments. Results from the application of antioxidants having relative selectivity for (1)O(2), O(2) (-), or OH indicated that both (1)O(2) and O(2) (-) were involved in the chilling- and light-induced lipid peroxidation which accompanied photooxidation. Application of D(2)O (which enhances the lifetime of (1)O(2)) corroborated these results. Chilling under high light produced no evidence of photooxidative damage in detached leaves of chilling-resistant pea (Pisum sativum L.). Our results suggest a fundamental difference in the ability of pea to reduce the destructive effects of free-radical and (1)O(2) production in chloroplasts during chilling in high light.

Calibration and use of a Clark-type oxygen electrode from 5 to 45 degrees C.

A calibration procedure for a Clark-type oxygen electrode over a wide range of temperatures is described. The autoxidation of duroquinol (2,3,5,6-tetramethyl-1,4-benzenediol) was used to verify the electrode's ability to accurately sense the total amount of dissolved O2 in an aqueous buffer. Electrode response time was measured by using oxygenated ethanol to deliver a rapid increase in O2 concentration to the reaction medium. An oxygen-producing system (spinach thylakoids) was utilized to test the range of O2-evolution rates able to be sensed. It was concluded that a Clark-type oxygen electrode has the absolute sensitivity, rapidity, and range necessary to accurately track rates of O2 production or consumption from 5 to 45 degrees C.

Chloroplast ultrastructure, chlorophyll fluorescence, and pigment composition in chilling-stressed soybeans.

Shoots of 16-day-old soybeans (Glycine max L. Merr. cv Ransom) were chilled to 10 degrees C for 7 days and monitored for visible signs of damage, ultrastructural changes, perturbations in fluorescence of chlorophyll (Chl), and quantitative changes in Chl a and b and associated pigments. Precautions were taken to prevent the confounding effects of water stress. A technique for the separation of lutein and zeaxanthin was developed utilizing a step gradient with the high performance liquid chromatograph. Visible losses in Chl were detectable within the first day of chilling, and regreening did not occur until the shoots were returned to 25 degrees C. Ultrastructurally, unstacking of chloroplast grana occurred, and the envelope membranes developed protrusions. Furthermore, the lipids were altered to the point that the membranes were poorly stabilized by a glutaraldehyde/osmium double-fixation procedure. Chl fluorescence rates were greatly reduced within 2 hours after chilling began and returned to normal only after rewarming. The rapid loss of Chl that occurred during chilling was accompanied by the appearance of zeaxanthin and a decline in violaxanthin. Apparently a zeaxanthin-violaxanthin epoxidation/de-epoxidation cycle was operating. When only the roots were chilled, no substantial changes were detected in ultrastructure, fluorescence rates, or pigment levels.

Citrus artifact interference in aflatoxin M1 determination in milk.

Dried citrus waste was fed to dairy cows, their milk was extracted, and aflatoxin M1 was quantitated by using both high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC). Results indicate that a compound from the citrus waste, which is excreted into the milk, interferes with the HPLC determination of aflatoxin M1 in milk and causes a false positive test. This interference can be overcome by using TLC with proper selection of developing solvents.