Site of Oxygen Absorption for Downward Transport in Seedlings of Rice and Other Plants

Our earlier reports have shown that appreciable portions (ranging from 20% to 70%) of the total amount of oxygen absorbed by the aerial part can be transported downwards to roots in water cultured intact seedlings of rice, barnyard grass, wheat, pea, etc. By interrupting the alternative paths of transport, it has been demonstrated that oxygen moves downwards mainly through gaseous diffusion along the intercellularspaces in the cortex. The aim of the present investigation is to ascertain the site of oxygen absorption for downward transport in the aerial part and to show that such a transport does not necessarily involve active participation of the absorbing organ. The results are summarized below: 1. Provided that a small upper portion of the leaf is left exposed in air, flooding of the aerial part of the rice seedling does not reduce the amount of total oxygen absorption to any appreciable extent (Fig. 1). In agreement with field observation, the unflooded tip is capable of furnishing the submerged part with enough oxygen to keep it alive. 2. Nor does the complete or partial removal of leaves by cutting in seedlings of rice and pea affect downward oxygen transport appreciably, provided that the stem segment or a leaf sheath is left exposed in air. 3. The following common notion has been confirmed by actual measurement: The abnormal excessive elongation of the coleoptile in rice seedling germinated under water, which may easily extend itself above the water surface, is an adaptive device to furnish the seedling with the oxygen required for root development. 4. The "floating" roots developed at the later stage in rice culture have been demonstrated to be a possible site of oxygen absorption for downward transport. 5. When a rice seedling is held up side down, with its roots exposed in air and the shoot submerged, downward oxygen transport still takes place, although to a lesser extent than in its normal position.     

Quantitative Estimations of Downward Oxygen Transport from Aerial to Subterranean Parts in Intact Seedlings of Rice and Other Plants

Quantitative estimations of downward oxygen transport from aerial to subterranean parts in intact seedlings were carried out in the present investigation with the respiratory hydrometer specially designed by us for this purpose. The chief object of the investigation is rice, a crop which is notable for its marshy habitat and whose submerged roots are in particular demand of such transport. Some other common plants (wheat, pea, water cress, etc.), either of marshy or of mesophytic habitat, have also been included in the investigation for comparison. Although rice has long been known for its capability of downward oxygen transport, as has often been revealed by various qualitative demonstrations and indirect estimations; yet, data of direct quantitative measurement of the actual amount transported, so far as we are aware, have been very scanty. The few attempts of bringing about such quantitative measurement in an intact plant are made by enclosing its shoot and root in two adjoining compartments respectively, and gas analysis is made on samples taken from each compartment at intervals. The procedure is so elaborate and tedious that estimations on a large scale could not be readily carried out and the results have often been rendered unreliable by mishandling of the plant and air leakage between the compartments. Proposals to the path and mechanism of downward oxygen transport in higher plants have largely been based upon such scanty quantitative approximations and various qualitative observations, and the conclusions derived therefrom are contraversial and far from being convincing. The presentation in this communication of a simple yet accurate experimental method for the quantitative determination of this kind might be opportune and appropriate. The basic principle of the respiratory hydrometer employed in this investigation has been given previously (Lou et al., 1963). Seedlings raised in water culture are inserted into the vessel of the hydrometer (Fig. 1) with its aerial part in the air space above and roots in the water passage below. As the diffusion rate of oxygen in water is about 1/300,000 that in air, the submerged roots of an intact rice seedling practically have their immediate oxygen supply cut off and have to rely upon the oxygen transported from above. Downward oxygen transport in intact seedlings can be easily estimated through the following procedures and the results thus obtained are summarized below: 1. The difference between two consecutive determinations of the oxygen absorbed by the aerial parts of intact seedlings made before and after their roots are severed gives the amount of oxygen transported downwards to roots. For the marshy plant (rice, water cress), it is about 50% (range: 30%–70%) of the total amount absorbed; whereas for ordinary land plants raised in water culture (wheat, pea), it is 20%–30% of the total. 2. The above results are in good agreement with those obtained by determining the respiratory quotients of intact seedlings first in air (e.g.R.Q. ≌ 1 in case of rice seedling) and then with their roots submerged in water (R.Q. ≌ 0.5). The difference between the two consecutive determinations again gives the fraction of oxygen transported downwards. 3. Either by varying the oxygen supply to the aerial part (from 1/4 to twice the oxygen content in air) or by increasing the oxygen consumption of the root through temperature increase or DNP stimulation, the oxygen concentration gradient along the vertical axis of the plant can be steepened or lessened at will. When such experiment is carried out in rice seedlings, the amount of oxygen transported downwards increases with the gradient.     

Path of Downward Oxygen Transport from Aerial to Subterranean Parts in Intact Seedlings of Rice and Other Plants

There are two opposite opinions as regards the mechanism and the path of downward oxygen transport in rice and other higher plants. Van Raalte (1940), Yamada (1952), and others maintain that an oxygen pressure gradient of decreasing magnitude from the stem base down to the root tip exists in the intercellular air spaces, which are interconnected throughout the cortex, and the oxygen transported therein is in free molecular form and moves about by diffusion along its own gradient. Recent diffusion experiments in plants by Barber (1962), employing radioactive O15 as indicator, gave direct confirmation of this hypothesis. The opposite view is held by Brown (1947), Soldatenkov (1963) and others. They consider that the passive diffusion of oxygen along its own gradient is inadequate to account for the actual amount transported downwards. The fact that downward oxygen transport in roots comes almost to a standstill, once the aerial part is removed while the cut end of the short stump is still left in air, casts doubts as to the validity of the diffusion hypothesis; and is in favour of their claim that in addition to, or in placement of, diffusion, active participation of living tissues in shoot is necessary to drive enough oxygen to meet the demand of roots. The oxygen in active transport is no longer in free gaseous state but is in dissolved or combined form (as in peroxides) and moves presumably along the vascular bundles in a way which is hitherto unrevealed but is apparently dependent upon the physiological activity of the conducting tissue. In our previous report (Lou et al 1964), we gave data based on quantitative measurement of the amount of oxygen transported downwards from aerial to submerged parts in intact seedlings with the respiratory hydrometer specially designed for the purpose. In seedlings of marshy plants (e.g. rice), it amounts to about 50% of the total oxygen absorbed by the aerial part; in water cultured seedlings of ordinary land plants (e.g. pea), 20%–30%. By deliberately blocking the alternative paths of oxygen transport in seedlings, one at a time, and measuring the downward oxygen transport accordingly in the same way as before, we should be able to decide which one of the two paths is mainly responsible for the transport. The blocking can be conveniently carried out at the upper end of the radical in a pea (or broadbean) seedling by surgical treatment (see Fig.1); either by ringing off the peripheral cortex where most of the air spaces reside; or by piercing through the central cylinder, within which the vascular bundles are confined. The treated radical is then submerged in water and ready for measurement. Without recourse to surgical treatment and mechanical injury, the air space in the cortex can also be blocked by displacing its air content with water through vacuum infiltration. The present investigation has shown that when the intercellular spaces in the cortex of the radical are blocked either by ringing or by infiltration, the aerial part of the treated seedling absorbs much less oxygen than the control as though its radical were completely severed (Table 2); or, in other words, the downward oxygen transport is effectively stopped by such a means. On the other hand, interruption of vascular bundles in the central cylinder only reduces the amount of oxygen in transport to less than one half, which can be accounted for by the combined effect of the reduced root activity due to shortage of food supply and the unavoidable partial disruption of the peripheral cortex. Besides taking actual measurement, downward oxygen transport in intact pea (or broadbean) seedlings can also be detected by simply noticing the growth rate of its radical. As is shown in this investigation, the radical ceases growing in still water, if the oxygen supply from its aerial part is interrupted. As a result of oxygen deficiency, the radical tip deteriorates in a few days. These effects can be easily realized by ringing off the cortex or by infiltrating its air spaces with water. That the peripheral ringing of the radical does no harm to its growth process is revealed by the fact that if air is bubbled through the water culture steadily, normal growth ensues. The above results leave no doubt that in seedlings of rice, pea, and broadbean, downward oxygen transport mainly takes place in the intercellular spaces in the radical cortex, and seems to have no concern with the activities of vascular bundle and cortex. Although there are evidence that rice roots may actively secrete oxygen in the form of peroxides to its immediate neighborhood (the rhizosphere), the actual amount and the distance traversed in such an active transport however, is very much limited and is insignificant as compared with that taking place in the intercellular spaces.     

Suppression of gravitropic response of primary roots by submergence

Primary roots of six plant species were placed horizontally either in humid air or under water, and their growth and gravitropic responses were examined. In air, all the roots showed a normal gravitropic curvature. Under water without aeration, roots of rice (Oryza sativa L.), oat (Avena sativa L.), azuki bean (Vigna angularis Ohwi et Ohashi), and cress (Lepidium sativum L.) curved downward at almost same rate as in air, whereas the curvature of roots of maize (Zea mays L.) and pea (Pisum sativum L.) was strongly suppressed. Submergence did not cause a decrease in growth rate of these roots. When roots of maize and pea were placed horizontally under water without aeration and then rotated in three dimensions on a clinostat in air, they showed a significant curvature, suggesting that the step suppressed by submergence is not graviperception but the subsequent signal transmission or differential growth process. Constant bubbling of air through the water partly restored the gravitropic curvature of maize roots and completely restored that of pea roots. The curvature of pea roots was also partly restored by the addition of an inhibitor of ethylene biosynthesis, aminooxyacetic acid. In air, ethylene suppressed the gravitropic curvature of roots of maize and pea. Furthermore, the level of ethylene in the intercellular space of the roots was increased by submergence. These results suggest that the accumulation of ethylene in the tissue is at least partly involved in suppression of transmission of the gravity signal or of differential growth in maize and pea roots under conditions of submergence.Abbreviations AOA aminooxyacetic acid - 3-D three-dimensional Dedicated to Professor Andreas Sievers on the occasion of his retirementWe thank Professor H. Suge and Drs. H. Takahashi and H. Kataoka, Tohoku University and Dr. T. Suzuki, Yamagata University, for helpful suggestions. The present study was supported in part by a Grant for Basic Research in Space Station Utilization from the Institute of Space and Astronautical Science, Japan.     

Distinctive Features of the Functioning of Lactic Acid and the Alcohol Fermentation Enzyme in Sorghum and Pea Leaves under the Conditions of Oxygen Deficiency

Dynamic changes in the enzymatic activity of lactate dehydrogenase, L-lactate-cytochrome-c-coxidoreductase, and alcohol dehydrogenase were analyzed in the seedlings of sorghum, a plant resistant to oxygen deficiency, and peas, a plant vulnerable to oxygen deficiency, cultivated under the conditions of flooding. The subcellular localization and isoform composition of the enzymes were characterized. The mechanism underlying the adaptive reaction of cell metabolism was devised from analysis of the results obtained. The reaction is supposed to involve lactate dehydrogenase and L-lactate-cytochrome-c-oxidoreductase enzymes that suppress cytoplasm acidification in sorghum and pea roots in the case of flooding.     

Analysis of the distribution of copper amine oxidase in cell walls of legume seedlings

Copper-containing amine oxidase (CuAO) has been proposed to play a role in H2O2 production in plant cell walls during cell development and in response to pathogen attack. We have compared the localisation of CuAO in pea (Pisum sativum L.), lentil (Lens culinaris M.) and chick pea (Cicer arietinum L.) grown under different light conditions, using both immuno- and histochemical techniques. The enzyme was detected by indirect immunofluorescence in the cell walls of parenchyma tissues of etiolated pea and lentil plants and was particularly abundant at intercellular spaces. Upon de-etiolation, CuAO largely disappeared from cortical cell walls except in the region of intercellular spaces. In the apical internode of light-grown seedlings, CuAO occurred mainly in cortical cell walls and, to some extent, in cell walls of xylem vessels. In both the elongation zone and mature regions of roots, CuAO was restricted to cortical cell walls and some cell junctions close to the meristem. Extensin epitopes co-localised to intercellular spaces of the cortex in de-etiolated pea, indicating that CuAO may have a role in cell wall strengthening at intercellular spaces. In chick pea, the localisation of the enzyme varied between different cultivars that have differing susceptibility to the fungus Ascochyta rabiei. In a susceptible cultivar Calia, immunogold labelling localised CuAO to cell walls of the cortex, as in lentil and pea, while in a resistant cultivar Sultano, it was most abundant in xylem vessels and, in light-grown plants, in the epidermis. These expression patterns are discussed with regard to the possible functions of amine oxidase in cell growth, cell differentiation and pathogen resistance.     

The influence of waterlogging at different temperatures on penetration depth and porosity of roots and on stomatal diffusive resistance of pea and maize seedlings

The 8 days old seedlings of pea (cv. Ilowiecki) and maize (cv. Alma F1) were subjected to differentiated aeration conditions (control — with pore water tension about 15 kPa and flooded treatment) for 12 days at three soil temperatures (7, 15 and 25 °C). The shoots were grown at 25 °C while the soil temperature was differentiated by keeping the cylinders with the soil in thermostated water bath of the appropriate temperature. Lowering the root temperature with respect to the shoot temperature caused under control (oxic) conditions a decrease of the root penetration depth, their mass and porosity as well as a decrease of shoot height, their mass and chlorophyll content; the changes being more pronounced in maize as compared to the pea plants. Flooding the soil diminished the effect of temperature on the investigated parameters; the temperature effect remaining significant only in the case of shoot biomass and root porosity of pea plants. Root porosity of pea plants ranged from 2 to 4 % and that of maize plants — from 4 to 6 % of the root volume. Flooding the soil caused an increase in the root porosity of the pea plants in the entire temperature range and in maize roots at lower temperatures by about 1 % of the root volume. Flooding the soil caused a decrease of root mass and penetration depth as well as a decrease of plant height, biomass and leaf chlorophyll content.     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.     

Water in aerenchyma spaces in roots. A fast diffusion path for solutes

During a study of the diffusivity of sulphorhodamine G in the cortical apoplast of maize roots widely discrepant rates were found between different samples. In roots which had developed large aerenchyma spaces, the diffusion in some regions was very fast, indistinguishable from the rate in water. In other regions the rate was as much as 100 times slower. Examination of frozen intact roots with the cryo-scanning electron microscope showed the presence of liquid filling some of the aerenchyma spaces, while other spaces of the same root contained air. X-ray microanalysis of the liquid (for oxygen) showed that the liquid was water with few detectable ions. Similar liquid was present in small intercellular spaces within the spoke-like radial files of cells between the large spaces, or between remnants of collapsed cell walls at the edges of the large spaces. It is proposed that regions of roots with high diffusivity are those in which some of the aerenchyma spaces are filled with water. In seeking the origin of this liquid, the progress of aerenchyma formation could be followed in the frozen tissues. The first change observed in a group of contiguous cells was a loss of vacuolar solutes and of cell turgor. Next the walls broke apart and collapsed back onto the surrounding turgid cells leaving a volume of ion-poor liquid. The liquid was probably not that found in some aerenchyma spaces of the mature roots, because the final stage of space formation was a loss of the liquid, leaving an air filled cavity surrounded by a composite lining formed from the collapsed walls of the broken cells. It is likely that the liquid in the spaces of mature aerenchyma is exuded from the remaining living cortical cells at times when the root turgor is high. This would be consistent with several recent studies which have shown periodic exudation of water from the surface of turgid roots. The spasmodic occurrence of root cortex tissue with enhanced diffusivity would have important implications for the transport of nutrient ions across the root.Abbreviations CSEM cryo-scanning electron microscope - EDX energy dispersive X-ray microanalysis - SR-G sulphorhodamine G     

Effects of Local Variations in Soil Moisture on Hydrophobic Deposits and Dye Diffusion in Corn Roots

The effects of local soil water content (SWC) alone on the development of hydrophobic deposits and on diffusivity of root tissues were studied in primary roots of maize seedlings whose top regions were growing at a constant rate in damp soil. Corn primary roots were grown through 30 cm-profiles of soil with pre-set dry (8% SWC), or wet (23% SWC), middle layers, and moist (15% SWC) top and bottom layers. When the root tissues in the soil of variable SWC were maturing (approximately 17% of the total root length), the tips of the roots were in damp soil several centimeters away and growing at the rate characteristic of this environment. Histochemistry and fluorescence microscopy on root sections of similar age showed that in these roots local SWC had no effect on hypodermal and endodermal suberization: both had equally developed Casparian bands and suberized lamellae. However, local SWC did alter the hydrophobic deposits in the walls of the epidermis; they increase in dry soil. To study tissue diffusivity, root portions from dry or wet soil were divided in the transverse direction, and half was dipped in sulphorhodamine G (SRG), sectioned, and observed with fluorescence optics. The other half was studied with a cryo-scanning electron microscope to observe the contents of the cortical intercellular spaces. The rate of SRG diffusion in a portion of root from dry or wet soil was independent of local SWC but was positively correlated with numbers of fluid-filled intercellular spaces. The implications of these observations for the movements of ions through the root cortex are discussed.     

Observations on the Effects of Pressure Differences Between the Bathing Media and Exudates of Excised Maize Roots

Observation of the water fluxes from excised maize roots atvarious levels of suction showed significant differences betweencontrol roots and those which have been subjected to an evacuationpre-treatment. This involved placing excised lengths of rootin bathing medium in a flask which was evacuated (1 cmHg) for10 min; during this time air bubbles emerged from the cut ends. This pre-treatment has been shown to result in an increase inthe density of the root, which is ascribed to the replacementof air in the cortical intercellular spaces by bathing medium.The observed differences in the response to suction in the twogroups of roots is therefore explained as a decrease in theresistance to water flow of the cortex of the treated roots.     

Transport of Abscisic Acid-2-C-14 in Intact Pea Seedlings

When abscisic acid-2-C-14 (AbA-2-C-14), 1 μg in 5 μl 40% ethanol, is applied to the apical bud of light-grown pea seedlings, C-14 is translocated downwards only in very small amounts and does not enter the root. In contrast to this, C-14 from indoleacetic acid-C-14 (IAA-2-C-14) applied in the same manner is translocated to the root where it accumulates. When AbA-2-C-14 is injected to the stem tissue at the apical bud, more labelled material is transported downwards than after application to the surface. Application of AbA-2-C-14 to an expanded leaf results in considerable accumulation of C-14 in the growing apical parts and in the lateral roots.     

Distribution of Intercellular Material in the Apical Part of Pea Seedlings

The sections from the upper part of the third internode, counted from the seed, in etiolated pea seedlings are studied for the distribution of two types of Intercellular spaces: the transparent and the dark. The transparent spaces represent the result of the water logging of passages originally water-lined, while the dark spaces are lined with a lipid-containing substance which may be impregnated with melted paraffin and which forms a ramifying network within the tissue. The intercellular material of the dark spaces has the appearance of a tube, since it concentrically lines the intercellular space, leaving a lumen for air or gas. It is a plastic, isotropic subslance which may be cut transversely and identified in successive sections of the inlernode as belonging to the same continuous material from the intercellular space. The dark and the transparent spaces have a distinct pattern of distribution in the growing internode, and the relative quantity of dark spaces present at different levels of the internode may be measured. The dark spaces predominate in those parts of the stem with a greater potential for growth, while the transparent spaces are located in the regions where growth has ceased or subsided. Since the paraffin is infiltrated instantaneously throughout the network of dark intercellular spaces, it is possible that these may represent a channel for the translocation of substances.     

Distribution of alcohol dehydrogenase in roots and shoots of rice (Oryza sativa) and Echinochloa seedlings

Abstract Aerobically germinated seedlings of rice and Echinochloa were found to survive when placed in an anaerobic environment for 4 d, whereas pea and maize seedlings did not. Although root and shoot growth were inhibited in rice and Echinochloa under anaerobiosis, growth resumed when the seedlings were returned to aerobic conditions. Alcohol dehydrogenase (ADH) activity increased more, and protein synthesis was greater, in the shoots than in the roots under anaerobic conditions. These results suggest that, in anaerobiosis-tolerant species, ADH activity and protein synthesis in the shoots represents or results from metabolic adaptations to low oxygen. These results are discussed in terms of plant establishment and growth in a low-oxygen environment.     

The ability of the biological control agent Bacillus subtilis, strain BB, to colonise vegetable brassicas endophytically following seed inoculation

The ability of Bacillus subtilis, strain BB, to colonise cabbage seedlings endophytically was examined following seed inoculation. Strain BB was recovered from different plant parts including leaves (cotyledons), stem (hypocotyl) and roots. While high bacterial populations persisted in the roots and lower stem, they were lower in the upper stem and leaves through time. In addition to cabbage, strain BB colonised endophytically the roots of 5 other vegetable brassicas. Fatty acid methyl ester (FAME) and PCR fingerprinting analysis confirmed the reliability of the detection method. Studies conducted with transmission electron microscope (TEM) showed that BB mainly colonised intercellular spaces of cortical tissues including intercellular spaces close to the conducting elements of roots and stem of cabbage seedlings. Gold labelling was specifically associated with BB and the fibrillar material filling the intercellular spaces where bacterial cells were found.     

Dynamics of CO2 evolution by plants at low pressure

Dynamics of CO₂ evolution at low pressure was studied in barley, maize, pea, wheat and pine seedlings using the gas exchange system with laser photoacoustic spectrometer. The CO₂ evolution from plant surfaces to environment increased with decreasing air pressure. Simultaneously the changes in activities of phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, glyceraldehyde phosphate dehydrogenase, alcohol-dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase in pea and maize leaves were observed. The response depended on plant species used as well as on air pressure and period of its action     

Effect of Water Deficit and Membrane Destruction on Water Diffusion in the Tissues of Maize Seedlings

We investigated diffusion of water in maize seedlings (Zea mays L. cv. Dnepropetrovskaya) following addition of polyethylene glycol (PEG) 6000 (osmotic potential –0.1 and –0.3 MPa) to the root medium by NMR method with pulsed gradient of magnetic field. Diffusion coefficients of different water phases in plant tissues (water of apoplast and vacuoles, water transported through the membranes) have been estimated from multicomponent decays of echo amplitude. Different signs of changes of water diffusion coefficients of fast and slow components of diffusional echo decay in roots and leaves under the influence of PEG-induced water deficits were shown. It has been supposed that under water deficit a sharing of water flows takes places through the different pathways (apoplastic, symplastic and transmembrane). In roots, 1-h water deficit increased the rate of fast diffusing water (water of apoplasm, vacuoles and, perhaps, water contained in intercellular endoplasm system), and decreased the rate of slowly diffusing water (water passing across the membranes). A long-term water deficit increased to a small extent the rate of water transmembrane transfer in root tissue. Leaf response to water stress was in the intensification of rate of transmembrane water transport that could be connected with the expression of water channels, and in the decrease of apoplastic water flow and flow along endoplasm. The possibility of estimation of plant tissue (membrane) integrity on the basis of diffusional data has been demonstrated.     

Low-temperature accumulation of alcohol dehydrogenase-1 mRNA and protein activity in maize and rice seedlings

Low-temperature stress was shown to cause a rapid increase in steady-state levels of alcohol dehydrogenase-1 message (Adh1) and protein activity (ADH1) in maize (Zea mays) (B37N, A188) and rice (Oryza sativa) (Taipei 309, Calmochi 101) seedlings. Maize roots and rice shoots and roots from 7-day seedlings shifted to low temperature (10°C) contained as much as 15-fold more Adh1 mRNA and 8-fold more ADH1 protein activity than the corresponding tissues from untreated seedlings. Time-course studies showed that these tissues accumulated Adh1 mRNA and ADH1 activity severalfold within 4- to 8-hour, levels plateaued within 20 to 24 hours, and remained elevated at 4 days of cold treatment. Within 24 hours of returning cold-stressed seedlings to ambient temperature, Adh1 mRNA and ADH1 activity decreased to pretreatment levels. Histochemical staining of maize and rice tissue imprints showed that ADH activity was enhanced along the lengths of cold-stressed maize primary roots and rice roots, and along the stems and leaves of rice shoots. Our observations suggest that short-term cold stress induces Adh1 gene expression in certain plant tissues, which, reminiscient of the anaerobic response, may reflect a fundamental shift in energy metabolism to ensure tissue survival during the stress period.     

Morphological and architectural development of root systems in sorghum and maize

Root systems determine the capacity of a plant to access soil water and their architecture can influence adaptation to water-limited conditions. It may be possible to associate that architecture with root attributes of young plants as a basis for rapid phenotypic screening. This requires improved understanding of root system development. This study aimed to characterise the morphological and architectural development of sorghum and maize root systems by (i) clarifying the initiation and origin of roots at germination, and (ii) monitoring and quantifying the development of root systems in young plants. Three experiments were conducted with two maize and four sorghum hybrids. Sorghum produced a sole seminal (primary) root and coleoptile nodal roots emerged at the 4th–5th leaf stage, whereas maize produced 3–7 seminal (primary and scutellum) roots and coleoptile nodal roots emerged at the 2nd leaf stage. Genotypic variation in the flush angle and mean diameter of nodal roots was observed and could be considered a suitable target for large scale screening for root architecture in breeding populations. Because of the relatively late appearance of nodal roots in sorghum, such screening would require a small chamber system to grow plants until at least 6 leaves had fully expanded.     

Effects of diclofop and diclofop-methyl on membrane potentials in roots of intact oat, maize, and pea seedlings

Growth and electrophysiological studies in roots of intact diclofop-methyl susceptible and resistant seedlings were conducted to test the hypothesis that the herbicide acts primarily as a proton ionophore. The ester formulation of diclofop, at 0.2 micromolar, completely inhibited root growth in herbicide-susceptible oat (Avena sativa L.) after a 96 hour treatment, but induced only a delayed transient depolarization of the membrane potential in oat root cortical cells. Root growth in susceptible maize (Zea mays L.) seedlings was dramatically reduced by exposure to 0.8 micromolar diclofop-methyl, while the same diclofop-methyl exposure hyperpolarized the membrane potential within 48 hours after treatment. Furthermore, exposure of maize roots to the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 nanomolar), inhibited growth by only 31%, 96 hours after treatment, while the same CCCP exposure depolarized the resting potential by an average of 32 millivolts. Thus, the protonophore hypothesis cannot account for a differential membrane response to phytotoxic levels of diclofop-methyl in two susceptible species. From the results of others, much of the evidence to support the protonophore hypothesis was obtained using high concentrations of diclofop acid (100 micromolar). At a similar concentration, we also report a rapid (3 minute) diclofop-induced depolarization of the membrane potential in roots of susceptible oat and maize, moderately tolerant barley (Hordeum vulgare L.), and resistant pea (Pisum sativum L.) seedlings. Moreover, 100 micromolar diclofop acid inhibited growth in excised cultured pea roots. In contrast, 100 micromolar diclofop-methyl did not inhibit root growth. Since the membrane response to 100 micromolar diclofop acid does not correspond to differential herbicide sensitivity under field conditions, results obtained with very high levels of diclofop acid are probably physiologically irrelevant. The results of this study suggest that the effect of diclofop-methyl on the membrane potentials of susceptible species is probably unrelated to the primary inhibitory effect of the herbicide on plant growth.