Oat Roots Research Articles

Growth Inhibition and Disruption of Mitosis by DCPA in Oat (Avena sativa) Roots

One to 5.6 μM DCPA (dimethyl tetrachloroterephthalate) inhibited oat (Avena sativaL. ‘Victory’) root growth within 12 to 18 h. Treated roots were severely stunted and swollen. An analysis of cell division in roots treated with DCPA revealed a disruption of normal mitosis after prophase. Metaphase, anaphase, and telophase division figures were absent 8 to 10 h after treatment with 5.6 μM DCPA. In contrast, a 24-h treatment with 5.6 μM DCPA was necessary to eliminate prophase division figures. The number of aberrant division figures increased concomitantly with the reduction in normal division figures. The predominant type of aberrant division figure was a condensed prophase. When the aberrant division cycle was completed and cells entered interphase, the dispersed chromosomes coalesced to form large, polymorphic nuclei and, occasionally, micronuclei. Approximately 60% of the outer four tiers of cells in roots treated with 5.6 μM DCPA developed abnormal cell walls. These data suggest that DCPA causes root growth inhibition by disrupting several processes involving organized microtubules.

Cytological and ultrastructural studies on root tissues.

The anatomy and fine structure of roots from oat and mung bean seedlings, grown under microgravity conditions for 8 days aboard the Space Shuttle, was examined and compared to that of roots from ground control plants grown under similar conditions. Roots from both sets of oat seedlings exhibited characteristic monocotyledonous tissue organization and normal ultrastructural features, except for cortex cell mitochondria, which exhibited a 'swollen' morphology. Various stages of cell division were observed in the meristematic tissues of oat roots. Ground control and flight-grown mung bean roots also showed normal tissue organization, but root cap cells in the flight-grown roots were collapsed and degraded in appearance, especially at the cap periphery. At the ultrastructural level, these cells exhibited a loss of organelle integrity and a highly-condensed cytoplasm. This latter observation perhaps suggests a differing tissue sensitivity for the two species to growth conditions employed in space flight. The basis for abnormal root cap cell development is not understood, but the loss of these putative gravity-sensing cells holds potential significance for long term plant growth orientation during space flight.

Anion-Sensitive, H+-Pumping ATPase of Oat Roots

To understand the mechanism and molecular properties of the tonoplast-type H(+)-translocating ATPase, we have studied the effect of Cl(-), NO(3) (-), and 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H(+)-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H(+)-pumping ATPase generates a membrane potential (Deltapsi) and a pH gradient (DeltapH) that make up two interconvertible components of the proton electrochemical gradient (Deltamuh(+)). A permeant anion (e.g. Cl(-)), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([(14)C]thiocyanate distribution) and stimulated formation of a pH gradient ([(14)C]methylamine distribution). However, Cl(-)-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl(-) independent of the proton electrochemical gradient. Unlike the plasma membrane H(+)-ATPase, the Cl(-)-stimulated ATPase was inhibited by NO(3) (-) (a permeant anion) and by DIDS. In the absence of Cl(-), NO(3) (-) decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO(3) (-) of Cl(-)-stimulated pH gradient formation and Cl(-)-stimulated ATPase activity was noncompetitive. In the absence of Cl(-), DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl(-)-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H(+)-ATPase by NO(3) (-) or DIDS suggest that the vanadate-insensitive H(+)-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H(+)-ATPase has channels that conduct anions is yet to be established.

Purification of the proton pumping ATPase from plant plasma membranes

The plasma membrane ATPase from oat roots has been purified near homogeneity by a simple procedure. Plasma membranes isolated from sucrose gradients are first extracted with Triton X-100 and KCl and the residue solubilized with lysolecithin. Rate-zonal centrifugation in a vertical rotor with a glycerol gradient results in a preparation of very high specific activity (6 μmoles min −1 mg protein −1 at 30 ° C) and where over 70% of the protein corresponds to a polypeptide of about 100 kilodaltons previously identified as the ATPase. The purified enzyme could be reconstituted in proteoliposomes catalyzing ATP-driven proton transport sensitive to vanadate.

Electron transfer constituents in plasma membrane fractions of Avena sativa and Saccharomyces cerevisiae

The presence of flavins, cytochromes and NAD(P)H dehydrogenase activities was investigated in plasma membrane preparations obtained from Saccharomyces cerevisiae and from Avena sativa roots. Those preparations contained, respectively, 280 and 216 pmol of non-covalently-bound flavin per mg protein. Besides, both membrane fractions showed levels of NAD(P)H ferricyanide reductase activities which could not be accounted for by contaminating mitochondrial or endoplasmic reticulum dehydrogenases. While the yeast fraction lacked detectable haemoproteins, the plant preparation contained a cytochrome b-like pigment with absorption bands at 428.5, 530 and 560 nm in the room-temperature reduced-minus-oxidized difference spectrum. NADH could only reduce a small part of the cytochrome, which was also detected in an endoplasmic reticulum fraction from oat roots. No significant levels of cythchrome b 5, P-450 and P-420 could be found in the oat preparations of plasma membranes and endoplasmic reticula.

Lysophosphatidylcholine-activated, vanadate-inhibited, Mg 2+-ATPase from radish microsomes

An orthovanadate-inhibited, nitrate-insensitive, phospholipid-requiring Mg 2+-ATPase has been partially purified (approx. 40-fold) from microsomal preparations from 24 h germinated radish seedlings. The specific activity obtained was 10–13 μmol P i · min −1 per mg protein, namely by 4- to 10-fold higher than that reported for the known similar enzyme preparations from corn and oat roots, and by 3- to 10-fold lower than that of the extensively purified plasmalemma enzymes from Neurospora and yeast. The partially purified activity was fairly specific for ATP, other nucleotide triphosphates being hydrolysed at less than 10% the rate with ATP; no activity was present towards ADP, AMP, p-nitrophenyl phosphate and other phosphate esters. The activity was strongly dependent on the presence of phospholipids with a marked preference for lysophosphatidylcholine, and showed an absolute requirement for Mg 2+ or some other divalent cations (CO 2+, Mn 2+, Mg 2+, Ni 2+, Zn 2+ in order of decreasing effectiveness); Ca 2+ could not substitute for Mg 2+ and was strongly inhibitory in its presence. K +, Rb + and Na + and also to a lesser extent NH 4 + and Li + were significantly stimulatory, while the anions NO 3 −, H 2PO 4 −, Cl − and SO 4 2− were ineffective. Orthovanadate, N, N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzensulfonate, tetraiodofluorescein and tetrachlorotetraiodofluorescein were strongly inhibitory. The coincidence of the K m for ATP with that for Mg 2+ suggested that ATP-Mg is the true substrate. Accordingly, the enzyme showed a normal Michaelis-Menten kinetics for ATP-Mg with an apparent K m of approx. 0.5 mM. The similarity of the characteristics of this enzyme with those of the plasmalemma enzymes from lower plants suggests its location at the plasma membrane, while some data ‘in vivo’ and in native sealed vesicle systems indicate its involvement in active proton transport.

The negative surface charge density of plasmalemma vesicles from wheat and oat roots

Plasmalemma vesicles were isolated in a sucrose-containing medium from wheat and oat roots and the net negative surface charge density was determined with 9-aminoacridine fluorescence [Chow, W.S. and Barber, J. (1980) J. Biochem. Biophys. Methods 3, 173-185]. The outer surface of the vesicles (measured in the presence of sucrose) had densities of − 16 to − 20 mC·m −2 and −29 mC·m −2 for wheat and oat roots, respectively. The inner surface - presumed to be the cytoplasmic side and calculated from the values measured in the presence and absence of sucrose - was more negative, and its size depended on the salt status of the roots.

Isolation of avenacins A–1, A–2, B–1, and B–2 from oat roots: structures of their ‘aglycones’, the avenestergenins

Four antifungal avenacins are isolated from oat roots: the structures of their ‘aglycones’, avenestergenins A–1, A–2, B–1, and B–2, are shown to be N-methylanthranilic or benzoic esters of an unusually oxygenated oleanane triterpene.

Structures of the four avenacins, oat root resistance factors to ‘take-all’ disease

The true aglycones of the avenacins are acid sensitive triterpene 12,13-epoxides, not 12-ketones, and one such avenestergenin epoxide is isolated direct from oat roots; this, together with study of the attached trisaccharide, permits complete structures to be proposed for the four avenacins (3a—d); avenestergenins readily undergo an acid-catalysed anhydrodimerisation via acetal formation.

Characterization of the host-specific phytotoxin victorin by high-pressure liquid chromatography

Summary The host-specific phytotoxin victorin was purified from culture filtrates of Bipolaris victoriae by solvent extraction, alumina adsorption chromatography and reverse phase high-pressure liquid chromatography (HPLC). Victorin activity was associated with an HPLC fraction containing a peak of absorption at 254 nm; by several criteria this ultra-violet (UV)-absorbing peak was identified as victorin. Victorin purified by HPLC ran as a single ninhydrin-positive spot in several thin-layer chromatography (TLC) systems. Purified victorin inhibited susceptible oat ( Avena sativa ) root growth half-maximally at 0.1 ng/ml, yet did not significantly inhibit resistant oat root growth at 50 g/ml.

Effect of invasion by cereal cyst nematode (Heterodera avenue) on the growth and development of the seminal roots of oats and barley

The effect of juvenile cereal cyst nematodes (Heterodera avenae) on the root growth of oat and barley seedlings was tested in three pot experiments. Invasion by juveniles hindered the extension of the seminal root axes of both cereals; after invasion, axes took 2–3 days to attain normal rates of extension. This response was found in susceptible and resistant cultivars. When oats and barley were exposed to continued invasion over 9 days, barley produced a larger root system than oats and was less stunted by nematode invasion. The density of nematodes per unit of root was less in barley than in oats. The effects on root growth were similar to reported responses of roots to mechanical damage.

Separation of Two Types of Electrogenic H+-Pumping ATPases from Oat Roots

Microsomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (25-45%, w/w) gradient to determine the types and membrane identity of proton-pumping ATPases associated with plant membranes. ATPase activity stimulated by the H(+)/K(+) exchange ionophore nigericin exhibited two peaks of activity on a linear dextran gradient. ATPase activities or ATP-generated membrane potential (inside positive), monitored by SCN(-) distribution, included a vanadate-insensitive and a vanadate-sensitive component. In a previous communication, we reported that ATP-dependent pH gradient formation (acid inside), monitored by quinacrine fluorescence quenching, was also partially inhibited by vanadate (Churchill and Sze 1983 Plant Physiol 71: 610-617). Here we show that the vanadate-insensitive, electrogenic ATPase activity was enriched in the low density vesicles (1-4% dextran or 25-32% sucrose) while the vanadate-sensitive activity was enriched at 4% to 7% dextran or 32% to 37% sucrose. The low-density ATPase was stimulated by Cl(-) and inhibited by NO(-) (3) or 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS). The distribution of Cl(-)-stimulated ATPase activity in a linear dextran gradient correlated with the distribution of H(+) pumping into vesicles as monitored by [(14)C]methylamine accumulation. The vanadate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K(+). These results show that microsomal vesicles of plant tissues have at least two types of electrogenic, proton-pumping ATPases. The vanadate-insensitive and Cl(-)-stimulated, H(+)-pumping ATPase may be enriched in vacuolar-type membranes; the H(+)-pumping ATPase that is stimulated by K(+) and inhibited by vanadate is most likely associated with plasma membrane-type vesicles.

The relationship between Rb+ influx and K+‐stimulated Mg2+‐ATPase activity in oat roots of different K+ status flexible coupling?

The relationship between Rb+ influx and microsomal ATPase activity stimulated by K+ and Mg2++ K+ was investigated for roots of 7‐day‐old seedlings of oat (Avena sativa L., cv. Brighton). Different concentrations of K+ in the roots, K+root were produced by cultivating plants in complete nutrient solutions of different dilutions and dFifferent K+ concentrations at various temperatures. Experiments were performed in both light and darkness. The range of the influx/ATPase ratios was large with a factor of 5 or more between the highest and lowest values. In most cases, the highest ratios were obtained at low K+root and at high temperatures, and the lowest at high K+root and at low temperatures. At high temperatures (20 and 25°C) in the light, the influx/ATPase ratio was constant, independently of K+root, if K+ in the medium was kept constant but the bulk of the nutrient solution diluted. If K+ was varied and the other components of the medium kept constant, the normal relation of decreasing influx/ATPase ratio at increasing K+root was found; thus, Rb+ influx appears regulated by both the internal and external potassium conditions. Also in darkness, at 15°C and with K + in the medium varied, the influx/ATPase ratio was independent of K+root but in the corresponding light experiments, ratio and K+root had the normal, inverse relationship. The difference between light and dark conditions appears to indicate that growth rate is of importance for the relationship between energy input and transport. Our data lead to the concept of “flexible coupling” between transducers) of energy and ion carrier. Without excluding other possibilities, this may be one of the mechanisms for ecological adaptation to variations in the root medium.

Allelopathic potential of wild oat (Avena fatua) on spring wheat (Triticum aestivum) growth

Wild oat plants may produce toxic substances that suppress the growth and development of desirable species, thus accounting for severe yield loss in infested fields. The purpose of this study was to determine the allelopathic potential of wild oat (Avena fatua) on the growth of spring wheat (Triticum aestivum var. Fieldwin) in the absence of plant competition. Wild oat and spring wheat plants were grown separately in 250-ml beakers in a sand medium. Root exudates were extracted from wild oat medium at the 1-, 2-, 3-, and 4-leaf stages of wild oat development and added to beakers containing spring wheat in temporally corresponding stages of development. Spring wheat root and leaf dry weights were measured to determine if one or more allelochemical agents were released from wild oat roots. Spring wheat leaf and root dry weights were significantly reduced by exudates from wild oat plants at the 2- and 4-leaf stages of development, respectively. Allelochemicals were isolated from wild oat root exudates at various stages of plant development. Paper chromatography analysis indicated that at least two unknown compounds were present.R f values in benzene-acetic acid-water of the two unknown compounds (0.825 and 0.930) were similar to scopoletin (7-hydroxy-6-methoxycoumarin) and vanillic acid (4-hydroxy-3-methoxybenzoic acid), respectively. Additional tests using diazotizedp-nitraniline, ultraviolet absorption spectra, and gas chromatography analysis also indicated that the unknowns were coumarin-related compounds such as scopoletin and vanillic acid.

Comparison of Atrazine Absorption by Underground Tissues of Several Plant Species

Excised roots of corn (Zea maysL. ‘B73 × Mo 17′) and oat (Avena sativaL. ‘Goodfield’) and potato (Solanum tuberosumL. ‘Kennebec’) tuber slices absorbed14C-atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] in a similar manner during short (1 to 30 min) absorption periods. Within 30 min, atrazine concentrations in tissues and ambient solution were equal. After 24 h of absorption, corn roots contained the most14C, followed in order by velvetleaf (Abutilon theophrastiMedic.) roots, oat roots, and potato tuber slices. Corn and velvetleaf accumulated14C to concentrations above the ambient solution. Efflux of14C was slower for corn than oat, potato, and velvetleaf. Metabolism of atrazine to hydroxyatrazine [2-hydroxy-4-(ethylamino)-6-(isopropylamino)-s-triazine] accounted for accumulation and slow efflux of14C in corn. These experiments show that initial, rapid absorption of atrazine is comparable, but long-term accumulation differs among these tissues.

Anion-Sensitive, H+-Pumping ATPase in Membrane Vesicles from Oat Roots

H(+)-pumping ATPases were detected in microsomal vesicles of oat (Avena sativa L. var Lang) roots using [(14)C]methylamine distribution or quinacrine fluorescent quenching. Methylamine (MeA) accumulation into vesicles and quinacrine quench were specifically dependent on Mg,ATP. Both activities reflected formation of a proton gradient (DeltapH) (acid inside) as carbonyl cyanide m-chlorophenylhydrazone, nigericin (in the presence of K(+)), or gramicidin decreased MeA uptake or increased quinacrine fluorescence. The properties of H(+) pumping as measured by MeA uptake were characterized. The K(m) (app) for ATP was about 0.1 millimolar. Mg,GTP and Mg, pyrophosphate were 19% and 30% as effective as Mg,ATP. MeA uptake was inhibited by N,N'-dicyclohexylcarbodiimide and was mostly insensitive to oligomycin, vanadate, or copper. ATP-dependent MeA was stimulated by anions with decreasing order of potency of Cl(-) > Br(-) > NO(3) (-) > SO(4) (2-), iminodiacetate, benzene sulfonate. Anion stimulation of H(+) pumping was caused in part by the ability of permeant anions to dissipate the electrical potential and in part by a specific requirement of Cl(-) by a H(+) -pumping ATPase. A pH gradient, probably caused by a Donnan potential, could be dissipated by K(+) in the presence or absence of ATP. MeA uptake was enriched in vesicles of relatively low density and showed a parallel distribution with vanadate-insensitive ATPase activity on a continuous dextran gradient. DeltapH as measured by quinacrine quench was partially vanadate-sensitive. These results show that plant membranes have at least two types of H(+) -pumping ATPases. One is vanadate-sensitive and probably enriched in the plasma membrane. One is vanadate-resistant, anion-sensitive and has many properties characteristic of a vacuolar ATPase. These results are consistent with the presence of electrogenic H(+) pumps at the plasma membrane and tonoplast of higher plant cells.

Potassium uptake in atrazine-treated roots of sensitive and resistan plants

The action of atrazine and its biodegradation products on the membrane transport of potassium in roots was evaluated in both sensitive and resistant plants. Excised roots of maize and oat showed inhibition of potassium uptake efficiency in the presence of 1.4 × 10 −4 M atrazine and 1.4 × 10 −4 M deethylated atrazine. Other biodegradation products such as 2-chloro-4-amino-6-ethylamino-1,3,5-triazine,2-chloro-4,6-,bisamino-1,3,5-triazine, and 2-chloro-4-amino-1,3,5-triazine showed no inhibitory effect on the K + uptake capacity. Two maize hybrids showing different uptake efficiency were inhibited differently by atrazine. We suggest that atrazine and deethylated atrazine inhibited the K + transport interacting directly with the plant cell membranes without discerning between resistant and sensitive plants.

Limiting oxygen flux densities for oat root extension

Seedlings of winter oatAvena sativa L. (cv. Pennal) were grown in columns of soil packed in ‘Perspex’ tubes in a growth cabinet with constant temperatures (10 or 7°C) and an illumination flux density equivalent to autumn daylight. The soil was waterlogged one week after seedlings emerged. Roots grew along the walls of sloping ‘Perspex’ tubes and measurements were made of root tip position, oxygen flux to a bare platinum electrode, the oxygen diffusion rate and the concentration of dissolved oxygen. We measured also nitrate and ethylene in the soil solution and root and shoot dimensions, and we examined root structure. Root extension was slower in waterlogged soil than in freely-drained soil at oxygen flux densities less than 56 ng cm−2 min−1, at oxygen diffusion rates less than 121 ng cm−2 min−1 and at concentrations of dissolved oxygen at or below 15% v/v at 10°C. With some simplifying assumptions the limiting oxygen flux was that just needed to maintain unrestricted respiration of root tip tissue.

Field Properties and Ion Uptake of Wheat and Oat: Are They Expressed in the Modulatory Influences of Ions on Membrane ATPases?

The CaATPase activities of microsomal fractions (10,000 g x 15 min 30,000 g X 60 min) of roots from wheat (Triticum aestivum L. cv. Svenno) and oat (Avena sativa L. cv. Brighton) are inhibited by HCO3 and Cr; and there is no effect by K' on the CaATPase. If the ATPase is activated by Mg2 or by Mn2 there is little or no effect by HCOj or CL. In wheat preparations, there is again no effect of K' on the Mgor MnATPase(s); but in preparations from oat roots both the MgATPase and the MnATPase are strongly stimulated by K'. A relation between MgATPase and potassium transport is normally accepted, and the difference between wheat and oat could reflect different strength of coupling between energy (ATPase) and carrier for potassium; with the strong coupling in oat as one of the biochemical adaptations that makes oat functionable as a crop plant on much more acidic soils than wheat. Regulation of anion transport should be a general problem; and alternative hypotheses are presented for how CaATPase may be influenced if a connection with anion transport is assumed. It is pointed out that unity in principles may well be compatible with variation in the specific biochemical solutions of transport problems; and that the biochemical solution may, in turn, be one of the factors that determine the ecological possibilities of an organism.

Sensitivity of isolated oat root cap cells and protoplasts to victorin

Isolated root cap cells can be used to study the effects of victorin on large populations of intact oat cells. The isolation of oat root cap protoplasts allowed a comparison of victorin-induced death of cells, with or without cell walls. Isolated root cap cells and protoplasts were incubated at 35°C in 0·6 M sorbitol, with or without victorin. Cell or protoplast viability was judged using fluorescein diacetate. Fifty percent of the cells and protoplasts from a susceptible oat cultivar were killed after 10 h in 20 units ml−1 victorin; 50–150 units ml−1 victorin caused 100% death within 6 h. Cells and protoplasts from resistant oat cultivars were not killed by up to 200 units ml−1 victorin. There was no difference in the rate of death of cells with or without cell walls; therefore, the cell wall is not the site of victorin's lethal effects. The root cap cell bioassay was also used to compare the sensitivity of other oat cell types. Root epidermal cells died at the same rate as root cap cells, but root hair and leaf mesophyll cells were at least 10-fold more sensitive to victorin than root cap cells. These results emphasize the need for caution in interpreting pathotoxin studies which employ intact plant tissues that contain a variety of cell types.