Rex N. Paul

Role of peroxidase in the development of water-impermeable seed coats in Sida spinosa L.

The seed coats of S. spinosa (prickly sida, Malvaceae) become impermeable to water during seed development on the mother plant. After the seeds have dehydrated during the final maturation stages, piercing of seed coats is necessary to induce imbibition of water and germination. Onset of impermeability occurs during seed coat browning, well in advance of seed dehydration. I. Marbach and A.M. Mayer (1975, Plant Physiol. 56, 93–96) implicated polyphenol oxidase (PO; EC 1.10.3.1) as catechol oxidase in the formation of insoluble polymers during development of coat impermeability in a wild strain of pea (Pisum elatius) seeds. We found, however, that peroxidase (EC 1.11.1.7), not PO, is instrumental in the development of water-impermeable seed coats in prickly sida. We isolated coats and embryos from seeds harvested at several stages of development. Highest peroxidase activity of coat extracts correlated well with the developmental stages of maximum conversion of soluble phenolics to insoluble lignin polymers. Although seed extracts oxidized dihydroxyphenylalanine, this activity was eliminated by catalase, indicating that the oxidation of phenolics in the coat is catalyzed by peroxidase rather than PO. Histochemical localization of peroxidase was strongest in the palisade layer; both the level and time of appearance of activity was proportional to the spectrophotometric assays of seed-coat extracts. The presence of peroxidase and the absence of PO in the seed coat were also confirmed with immunocytochemistry. Our results support the view that peroxidase is involved in the polymerization of soluble phenolics to insoluble lignin polymers during development of prickly sida seed coats, causing the formation of a water-impermeable barrier prior to seed dehydration. As dehydration proceeds, the chalazal area finally becomes impermeable resulting in the hard mature seeds of prickly sida.

LOCALIZATION OF ARTEMISININ AND ARTEMISITENE IN FOLIAR TISSUES OF GLANDED AND GLANDLESS BIOTYPES OF ARTEMISIA ANNUA L.

The tissue localization of the antimalarial sesquiterpenoid compound artemisinin in annual wormwood (Artemisia annua L.) was determined by differential extraction of a glanded biotype and through the use of a glandless biotype. A 5-s dip in chloroform extracted 97% of the artemisinin from glanded A. annua leaf tissue. In addition, all of the detectable artemisitene, an artemisinin analog, was extracted. This extraction method caused collapse of the subcuticular space of the capitate glands on the leaf surface, whereas no other damage to the leaf surface was observed with SEM. Light microscopy and TEM revealed that this extraction method, despite causing some organelle structural changes, did not disrupt cell membranes. An A. annua biotype without glands contained neither artemisinin nor artemisitene. These results indicate that artemisinin and artemisitene present in foliar tissue are localized entirely in the subcuticular space of capitate glands of A. annua.

Development and Fine Structure of the Glandular Trichomes of Artemisia annua L.

Development of capitate glands on the leaves of annual wormwood (Artemisia annua L.) was monitored with scanning and transmission electron microscopy. Differentiation of foliar cells into gland cells began in the youngest leaf primordia. After differentiation into a 10-celled biseriate structure of two stalk cells, two basal cells, and three pairs of secretory cells, the cuticle of the six secretory cells separated from the cell walls to form a bilobed sac that eventually splits to release its contents. At every developmental stage, the cells of the the gland contained relatively little vacuolar volume. The secretory cells contained extensive endoplasmic reticulum. The plastids of each cell pair were different. At maturity, the apical cells contained proplastids or leucoplasts with only occasional thylakoids. The cell pair below the apical cell pair contained amorphous chloroplasts without starch grains. The basal cell pair contained proplastids or leucoplasts and the stalk cells contained chloroplasts. The stroma to thylakoid ratio in the secretory cell chloroplasts was high. Initially, osmiophilic product was observed most frequently associated with stacked thylakoids, plastid envelopes, and smooth endoplasmic reticulum, although it was associated with all cell membranes. Near the plasma membrane adjacent to cell walls bordering the subcuticular space, the cytoplasm was enriched in smooth endoplasmic reticulum containing osmiophilic material. The apical cell wall of the apical secretory cell pair was reticulated on the inner cytoplasmic side and contained osmiophilic staining on the cuticular side. During early senescence, osmiophilic product was commonly associated with outer mitochondrial membranes.

Mode of Action, Localization of Production, Chemical Nature, and Activity of Sorgoleone: A Potent PSII Inhibitor in Sorghum spp. Root Exudates1

The root exudates produced by sorghums contain a biologically active constituent known as sorgoleone. Seven sorghum accessions were evaluated for their exudate components. Except for johnsongrass, which yielded 14.8 mg root exudate/g fresh root wt, sorghum accessions consistently yielded approximately 2 mg root exudate/g fresh root wt. Exudates contained four to six major components, with sorgoleone being the major component (> 85%). Three-dimensional structure analysis was performed to further characterize sorgoleones mode of action. These studies indicated that sorgoleone required about half the amount of free energy (493.8 kcal/mol) compared to plastoquinone (895.3 kcal/mol) to dock into the QB-binding site of the photosystem II complex of higher plants. Light, cryo-scanning, and transmission electron microscopy were utilized in an attempt to identify the cellular location of root exudate production. From the ultrastructure analysis, it is clear that exudate is being produced in the root hairs and being deposited between the plasmalemma and cell wall. The exact manufacturing and transport mechanism of the root exudate is still unclear. Studies were also conducted on sorgoleones soil persistence and soil activity. Soil impregnated with sorgoleone had activity against a number of plant species. Recovery rates of sorgoleone-impregnated soil ranged from 85% after 1 h to 45% after 24 h. Growth reduction of 9 14-d-old weed species was observed with foliar applications of sorgoleone. Nomenclature: Sorgoleone (2-hydroxy-5-methoxy-3-[(8′Z,11′Z)-8′,11′,14′-pentadecatriene]-p-hydroquinone); common purslane, Portulaca oleracea L. POROL; common ragweed, Ambrosia artemisiifolia L. AMBEL; cress, Lepidium sativum L. ;ns3 LEPSA; giant foxtail, Setaria faberi Herrm. SETFA; johnsongrass, Sorghum halepense (L.) Pers. SORHA; lambsquarters, Chenopodium album L. CHEAL; large crabgrass, Digitaria sanguinalis (L.) Scop. DIGSA; lettuce, Lactuca sativa L.; nightshade, Solanum spp.; purple photosynthetic bacterium, Rhodopseudomonas viridis; redroot pigweed, Amaranthus retroflexus L. AMARE; sicklepod, Cassia obtusifolia L. CASOB; spinach, Spinacea oleracea; shattercane, Sorghum bicolor (L.) Moensch SORVU; sorghum, S. bicolor (L.) Moensch SORVU; sudex, S. bicolor × Sorghum sudanense; sweet sorghum, S. bicolor ‘Della’; SX-15 and SX-17, S. bicolor × S. sudanense; 8446 and 855-F, S. bicolor (L.) Moensch SORVU; tomato, Lycopersicon esculentum L.; velvetleaf, Abutilon theophrasti Medicus ABUTH. Additional index words: Sorgoleone, root hairs, SORVU, SORHA. Abbreviations: ER, endoplasmic reticulum; HPLC, high-pressure liquid chromatography; PSII, photosystem II; QB, quinone binding; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TLC, thin-layer chromatography; 3D, three dimensional; UV, ultraviolet.

Dehydrozaluzanin C, a natural sesquiterpenolide, causes rapid plasma membrane leakage

Abstract Dehydrozaluzanin C, a natural sesquiterpene lactone, is a weak plant growth inhibitor with an I50 of about 0.5 mM for lettuce root growth. It also causes rapid plasma membrane leakage in cucumber cotyledon discs. Dehydrozaluzanin C is more active at 50 μM than the same concentration of the herbicide acifluorfen. Symptoms include plasmolysis and the disruption of membrane integrity is not light dependent. Reversal of its effects on root growth was obtained with treatment by various amino acids, with histidine and glycine providing ca. 40% reversion. The strong reversal effect obtained with reduced glutathione is due to cross-reactivity with DHZ and the formation of mono- and di-adducts. Photosynthetic, respiratory and mitotic processes, as well as NADH oxidase activity appear to be unaffected by this compound. Our results indicate that dehydrozaluzanin C exerts its effects on plants through two different mechanisms, only one of which is related to the disruption of plasma membrane function.

ANATOMY OF SORGOLEONE-SECRETING ROOT HAIRS OF SORGHUM SPECIES

Johnsongrass (Sorghum halepense [L.] Pers.) and SX‐17 (Sorghum bicolor × Sorghum sudanese) were investigated microscopically to identify specifically the location of root exudate production. Light, cryoscanning electron, and transmission electron microscopy were used to determine the area of exudate secretion. Light micrographs indicated that the exudate is solely produced by the root hairs. Scanning electron microscopy supported this conclusion. Transmission electron microscopy studies of root hairs support the hypothesis that root exudates are manufactured in the cytoplasmically dense root hair cell in association with smooth endoplasmic reticulum and possibly Golgi bodies. Ultrastructure studies indicated that small globules of cytoplasmic exudate are deposited between the cell wall and the plasma membrane, where they coalesce into larger globules that pass through the cell wall to form droplets near the tip of root hairs.

FMC 57020 Effects on Chloroplast Development in Pitted Morningglory (Ipomoea lacunosa) Cotyledons

The effect of FMC 57020 [2(2 chlorophenyl) methyl-4,4dimethyl-3-isoxalidinoneI on chloroplast development was examined in the cotyledons of 5-day-old, etiolated pitted morningglory (Ipomoea lacunosa L. #3 IPOLA) seedlings grown from seeds inbibed for 24 h in water or 0.5 mM FMC 57020. In etiolated tissues, protochlorophyllide content was unaffected by FMC 57020; however, the herbicide eliminated carotenoid accumulation. There was no effect of FMC 57020 on phytoene or phytofluene content, although norflurazon [4chloro -5(methylamino)-2(3-trifluoromethyl) phenyl)3(2H)-pyridazinoneJ increased phytoene content in these tissues. The Shibata shift was greatly retarded in FMC 57020-treated cotyledons, suggesting that phytol levels are also reduced by the herbicide. There were no ultrastructural effects on etioplasts; however, under low white light (150 ME*m2s l PAR), plastids of FMC 57020-treated seedlings did not develop into chloroplasts but rapidly developed ultrastructural symptoms of photobleaching. Starch was not mobilized in herbicide-treated plastids and sugar levels were higher in these plastids than in control plastids. Etiolated hypocotyl growth was inhibited by FMC 57020, whereas norflurazon had no effect upon it. Our results suggest that FMC 57020 blocks both diterpene and tetraterpene synthesis. Additional index words. Carotenoids, bleaching herbicides, terpenoids, IPOLA.

Laboratory assessment of the allelopathic effects of fine leaf fescues

Laboratory screening studies were conducted to evaluate the allelopathic potential of fine leaf fescues. Of the seven accessions selected from prior field evaluations for weed-suppressive ability, all inhibited root growth of large crabgrass and curly cress in laboratory assays. Grown in agar as a growth medium and in the presence of living fescue seedlings for 14 or 21 days, test species were sensitive depending on the fescue cultivars. Growth inhibition increased when fescue was grown for increasing periods of time in agar. Seedling fescues produced significant quantities of bioactive root exudates, which were released into the agar medium. Bioactive root exudates were extracted from living fescue roots by using methylene chloride. Shoot tissue was extracted in water and the aqueous extract was partitioned against hexane, ethyl acetate, and methylene chloride. Extracts were tested for inhibitory activity on seedling growth as measured by inhibition of curly cress germination and radicle elongation. Root exudates were more toxic (70% inhibition) than shoot extracts (up 40% inhibition), when formulated at 0.25 mg/ml concentration. Light microscopy and transmission electron microscopy were utilized in an attempt to identify the cellular location of production of secondary products contained in bioactive root exudates. Ultrastructural analysis indicated that the exudate is produced in actively dividing tips of fibrous root cells. The mode of release of these exudates into the environment remains unknown.

Ultrastructural effects of AAL-toxin TA from the fungus Alternaria alternata on black nightshade (Solanum nigrum L.) leaf discs and correlation with biochemical measures of toxicity

Ultrastructural effects of AAL-toxin TA from Alternaria alternata on black nightshade (Solanum, nigrum L.) leaf discs and correlation with biochemical measures of toxicity. In black nightshade (Solanum nigrum L.) leaf discs floating in solutions of AAL-toxin TA (0.01-200 microM) under continuous light at 25 degrees C, electrolyte leakage, chlorophyll loss, autolysis, and photobleaching were observed within 24 h. Electrolyte leakage, measured by the conductivity increase in the culture medium, began after 12 h with 200 microM AAL-toxin T(A), but was observed after 24 h with 0.01 to 50 microM AAL-toxin T(A), when it ranged from 25%) to 63% of total releasable electrolytes, respectively. After 48 h incubation, leakage ranged from 39% to 79% of total for 0.01 to 200 microM AAL-toxin T(A), respectively, while chlorophyll loss ranged from 5% to 32% of total, respectively. Ultrastructural examination of black night-shade leaf discs floating in 10 microM AAL-toxin TA under continuous light at 25 degrees C revealed cytological damage beginning at 30 h, consistent with the time electrolyte leakage and chlorophyll reduction were observed. After 30 h incubation chloroplast starch grains were enlarged in control leaf discs, but not in AAL-toxin T(A)-treated discs, and the thylakoids of treated tissue contained structural abnormalities. After 36-48 h incubation with 10 microM AAL-toxin T(A), all tissues were destroyed with only cell walls, starch grains, and thylakoid fragments remaining. Toxicity was light-dependent, because leaf discs incubated with AAL-toxin T(A) in darkness for up to 72 h showed little phytotoxic damage. Within 6 h of exposure to > or =0.5 microM toxin, phytosphingosine and sphinganine in black nightshade leaf discs increased markedly, and continued to increase up to 24 h exposure. Thus, phy siological and ultrastructural changes occurred in parallel with disruption of sphingolipid synthesis, consistent with the hypothesis that AAL-toxin T(A) causes phytotoxicity by interrupting sphingolipid biosynthesis, thereby damaging cellular membranes.

9,10‐Anthraquinone Reduces the Photosynthetic Efficiency of Oscillatoria perornata and Modifies Cellular Inclusions

The natural compound 9,10‐anthraquinone was found to inhibit the growth of the musty odor‐producing cyanobacterium Oscillatoria perornata at a low concentration (1 &mgr;M) in previous laboratory studies. In this study, the mode of action of 9,10‐anthraquinone was investigated by observing ultrastructural changes in O. perornata and by monitoring chlorophyll fluorescence as an indicator of photosynthetic efficiency. Results indicate that 9,10‐anthraquinone inhibits photosynthetic electron transport, probably at PSII, and thereby affects growth. Moreover, 9,10‐anthraquinone treatment caused thylakoid disorganization and reduced the number of ribosomes in O. perornata. The thylakoid disorganization is identical to reported modification in a cyanobacterium treated with simazine, a PSII inhibitor.