Influence of chloroplast development on the activation of the diphenyl ether herbicide acifluorfen-methyl

The activity of acifluorfen-methyl (AFM); methyl 5-(2-chloro-4-[trifluoromethyl] phenoxy)-2-nitrobenzoate in excised cucumber cotyledons (Cucumis sativus L.) was examined. AFM induced membrane disruption, was significantly greater when etiolated cotyledons were illuminated 16 hours at 150 microeinsteins per square meter per second photosynthetically active radiation versus incubation under illumination of 4-fold greater intensity. These results were unexpected since the loss of membrane integrity is initiated by photodynamic reactions. Untreated, etiolated cotyledons were not able to accumulate chlorophyll under the higher light intensity while control and herbicide treated cotyledons greened significantly under the lower intensity illumination suggesting that some process associated with greening stimulated AFM activity. Inhibition of greening by cycloheximide also reduced AFM activity. Intermittent lighting induced greening in AFM treated cotyledons without causing any detectable loss of plasmalemma integrity. Utilization of this system for pretreatment of cotyledons prior to continuous illumination revealed that activity was greater when tissue was greened in the presence of AFM than when herbicide treatments were made after a greening period of the same duration. The results indicate that the pigments in situ in etiolated tissue are sufficient, without greening, to initiate membrane disruption by AFM. However, greening increases the herbicidal efficacy greatly. Furthermore, the stimulation appears to be due to specific interactions between AFM and the developing plastid and is not attributable solely to an increase in endogenous photosensitizers.     

Early and late heat shock proteins in wheats and other cereal species

Coleoptiles and roots of 3-day-old seedlings from five cereal species (Triticum aestivum L., T. durum Desf., Hordeum vulgare L., Secale cereale L., and Triticale) respond to heat shock at 40°C by synthesizing a new set of 13 strong bands (as revealed by one-dimensional sodium dodecyl sulfate gel electrophoresis) as well as some 20°C proteins. Heat shock proteins (HSPs) belong to three different size groups: high molecular mass HSPs in the 103 to 70 kilodalton range, intermediate molecular mass HSPs in the 62 to 32 kilodalton range, and low molecular mass HSPs about 17 to 16 kilodalton in size. At the beginning of the heat shock coleoptiles show a reduced ability to synthesize intermediate molecular mass HSPs but after 4 hours at 40°C they exhibit fully developed HSP patterns identical to that found in roots. Synthesis of early HSPs declines after 7 hours of treatment followed by the appearance of a new set of 12 protein bands (late HSPs) in the ranges 99 to 83, 69 to 35, and 15 to 14 kilodaltons. After 12 hours at 40°C, three other late HSPs of 89, 45, and 38 kilodalton are induced. The induction of late HSPs after 7 hours at 40°C appears to be associated with an enhancement of radioactive methionine incorporation into proteins.     

Does Gibberellic Acid Induce the Transfer of Lipase from Protein Bodies to Lipid Bodies in Barley Aleurone Cells?

We have examined the effect of gibberellic acid (GA₃) on the distribution of the enzyme responsible for mobilizing storage triacylglycerol in aleurone cells of Hordeum vulgare L. cv Himalaya. Using cellular fractionation techniques, we find that, in cells that have not been exposed to hormone, neutral lipase activity is principally associated with a pellet containing the membranes of protein bodies. If the cells are exposed to GA₃ for at least 1 hour, the majority of the lipase activity becomes associated with the lipid body fraction. The nature of the in vivo association between lipid bodies and protein bodies was examined using ultrarapid freezing followed by freeze-fracture electron microscopy. Our analysis indicates that the phospholipid monolayer surrounding the lipid body is directly continuous with the outer leaflet of the bilayer surrounding the protein body. Based on our data, we propose that lipase can be transferred from protein bodies (storage form) to lipid bodies (active form) by lateral diffusion within the plane of the fused phospholipid monolayer, and that the transfer can be controlled by gibberellic acid by an unknown mechanism.     

Temperature Effects on Phosphoenolpyruvate Carboxylase from a CAM and a C(4) Plant : A Comparative Study

The effect of temperature in the range from 10 to 35°C on various characteristics of phosphoenolpyruvate carboxylase from the leaves of a CAM plant, Crassula argentea and a C₄ plant Zea mays shows a number of different effects related to the environment in which these distinct types of metabolic specialization normally operate. The Arrhenius plot of V_max for the two enzyme forms shows that the CAM enzyme has a linear increase with temperature while the C₄ enzyme has an inflection at 27°C implying a conformational or aggregational change in the enzyme or a shift in reaction mechanism to one requiring a lower activation energy. The Arrhenius plot of K_m for the two enzymes reveals the startling fact that at temperatures above 20°C an increasing temperature causes an increase in K_mPEP for the CAM enzyme while the C₄ enzyme displays a decreased K_m as the temperature increases. The inhibitory effect of 5 millimolar malate also shows opposite trends for the two enzymes. For the CAM enzyme the percent inhibition by malate increases from essentially none at 15°C to 70% at 35°C. For the C₄ enzyme the percent inhibition drops from about 60% at 20°C to 2% at 30°C. Similar opposite behavior of the two enzymes is found with the K_i for malate. Pretreatment at high temperatures for periods up to 2 hours was found to result in differences similar to those described above if the treated enzyme were subsequently assayed at 25°C.     

Construction of an Obligate Photoheterotrophic Mutant of the Cyanobacterium Synechocystis 6803 : Inactivation of the psbA Gene Family

psbA in Synechocystis 6803 was found to belong to a small multigene family with three copies. The psbA gene family was inactivated in vitro by insertation of bacterial drug resistance markers. Inactivation of all three genes resulted in a transformant that is unable to grow photosynthetically but can be cultured photoheterotrophically. This mutant lacks oxygen evolving capacity but retains photosystem I activity. Room temperature measurements of chlorophyll a fluorescence induction demonstrated that the transformant exhibits a high fluorescence yield with little or no variable fluorescence. Immunoblot analyses showed complete loss of the psbA gene product (the DI polypeptide) from thylakoid membranes in the transformant. However, the extrinsic 33 kilodalton polypeptide of the water-splitting complex of photosystem II, is still present. The results indicate that assembly of a partial photosystem II complex may occur even in the absence of the intrinsic D1 polypeptide, a protein implicated as a crucial component of the photosystem II reaction center.     

Cyanide-resistant respiration in photosynthetic organs of freshwater aquatic plants

The rate and sensitivity to inhibitors (KCN and salicylhydroxamic acid[SHAM]) of respiratory oxygen uptake has been investigated in photosynthetic organs of several freshwater aquatic plant species: six angiosperms, two bryophytes, and an alga. The oxygen uptake rates on a dry weight basis of angiosperm leaves were generally higher than those of the corresponding stems. Leaves also had a higher chlorophyll content than stems. Respiration of leaves and stems of aquatic angiosperms was generally cyanide-resistant, the percentage of resistance being higher than 50% with very few exceptions. The cyanide resistance of respiration of whole shoots of two aquatic bryophytes and an alga was lower and ranged between 25 and 50%. These results suggested that the photosynthetic tissues of aquatic plants have a considerable alternative pathway capacity. The angiosperm leaves generally showed the largest alternative path capacity. In all cases, the respiration rate of the aquatic plants studied was inhibited by SHAM alone by about 13 to 31%. These results were used for calculating the actual activities of the cytochrome and alternative pathways. These activities were generally higher in the leaves of angiosperms. The basal oxygen uptake rate of Myriophyllum spicatum leaves was not stimulated by sucrose, malate or glycine in the absence of the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP), but was greatly increased by CCCP, either in the presence or in the absence of substrates. These results suggest that respiration was limited by the adenylate system, and not by substrate availability. The increase in the respiratory rate by CCCP was due to a large increase in the activities of both the cytochrome and alternative pathways. The respiration rate of M. spicatum leaves in the presence of substrates was little inhibited by SHAM alone, but the SHAM-resistant rate (that is, the cytochrome path) was greatly stimulated by the further addition of CCCP. Similarly, the cyanide-resistant rate of O₂ uptake was also increased by the uncoupler.     

Different Characteristics of the Two Glutamate Synthases in the Green Leaves of Lycopersicon esculentum

The two glutamate synthases, NAD(P)H- and ferredoxin-dependent, from the green leaves of tomato plants (Lycopersicon esculentum L. cv Hellfrucht frühstamm) differed in their chemical properties and catalytic behavior. Gel filtration of NAD(P)H enzyme gave an apparent molecular size of 158 kilodalton, whereas the ferredoxin enzyme molecular size was 141 kilodalton. Arrhenius plots of the activities of the two enzymes showed that the NAD(P)H enzyme had two activation energies; 109.6 and 70.5 kilojoule per mole; the transition temperature was 22°C. The ferredoxin enzyme however, had only one activation energy; 56.1 kilojoule per mole. The respective catalytic activity pH optima for the NAD(P)H- dependent and the ferredoxin dependent enzymes were around 7.3 and 7.8. In experiments to evaluate the effects of modulators aspartate enhanced the NAD(P)H-linked activity, with a K_a value of 0.25 millimolar, but strongly inhibited that of the ferredoxin-dependent glutamate synthase with a K_i of 0.1 millimolar. 3-Phosphoserine was another inhibitor of the ferredoxin dependent enzyme with a K_i value of 4.9 millimolar. 3-Phosphoglyceric acid was a potent inhibitor of the ferredoxin-dependent form, but hardly affected the NAD(P)H-dependent enzyme. The results are discussed and interpreted to propose different specific functions that these activities may have within the leaf tissue cell.     

Effects of CO(2) and O(2) on Photosynthesis and Growth of Autotrophic Tobacco Callus

Gas exchange measurements were made on plants from two natural populations differing in salt tolerance of Andropogon glomeratus, a C₄ nonhalophyte, to examine the effect of salinity on components responsible for differences in photosynthetic capacity. Net CO₂ uptake and stomatal conductance decreased with increasing salinity in both populations, but to a greater extent in the inland (nontolerant) population. The intercellular CO₂ concentrations increased with increasing salinity in the inland population, but decreased in the marsh (tolerant) population. Water use efficiency decreased as salinity increased in the inland population, and remained unchanged in the marsh population. Carboxylation efficiency decreased and CO₂ compensation points increased with increasing salinity in both populations, but to a lesser extent in the marsh population. Carboxylation efficiencies were higher with 2% relative to 21% atmospheric O₂ in salt stressed plants, suggesting that a decrease in the carboxylation:oxygenation ratio of ribulose 1,5-bisphosphate carboxylase/oxygenase was partly responsible for the decrease in photosynthetic capacity. Populational differences in photosynthetic capacity were the result of greater salinity-induced changes in carboxylation efficiency in the inland population, and not due to differences in the stomatal limitation to CO₂ diffusion.     

(2-Chloroethyl)phosphonic Acid Promotes Germination of Immature Spores of Ceratopteris richardii Brongn

Freshly collected spores of strain Hn-n of Ceratopteris richardii Brongn. require storage for several months before attaining maximum germination rate. Treatments using (2-chloroethyl)phosphonic acid increased germination rate in freshly collected spores and decreased germination rate in older spores.     

Chlorophyll-Protein Complexes from the Red-Tide Dinoflagellate, Gonyaulax polyedra Stein : Isolation, Characterization, and the Effect of Growth Irradiance on Chlorophyll Distribution

A comparision of high (330 microeinsteins per meter squared per second) and low (80 microeinsteins per meter squared per second) light grown Gonyaulax polyedra indicated a change in the distribution of chlorophyll a, chlorophyll c₂, and peridinin among detergent-soluble chlorophyll-protein complexes. Thylakoid fractions were prepared by sonication and centrifugation. Chlorophyll-protein complexes were solubilized from the membranes with sodium dodecyl sulfate and resolved by Deriphat electrophoresis. Low light cells yielded five distinct chlorophyll-protein complexes (I to V), while only four (I′ to IV′) were evident in preparations of high light cells. Both high molecular weight complexes I and I′ were dominated by chlorophyll a absorption and associated with minor amounts of chlorophyll c. Both complexes II and II′ were chlorophyll a-chlorophyll c₂-protein complexes devoid of peridinin and unique to dinoflagellates. The chlorophyll a:c₂ molar ratio of both complexes was 1:3, indicating significant chlorophyll c enrichment over thylakoid membrane chlorophyll a:c ratios of 1.8 to 2:1. Low light complex III differed from all other high or low light complexes in that it possessed peridinin and had a chlorophyll a:c₂ ratio of 1:1. Low light complexes IV and V and high light complexes III′ and IV′ were spectrally similar, had high chlorophyll a:c₂ ratios (4:1), and were associated with peridinin. The effects of growth irradiance on the composition of chlorophyll-protein complexes in Gonyaulax polyedra differed from those described for other chlorophyll c-containing plant species.