Relationship Between Downward Oxygen Transport and Intercellular Spaces in Root Cortex in Water Cultured and Moist Cultured Seedlings

In the present investigation, seedlings of rice, pea, sorghum, and maize are raised both in water culture and moist culture. The former culture is to provide the roots with an oxygen deficient condition; while the latter, a direct access to air. The amount of oxygen transported downwards in the seedlings varies not only with the nature of plants but also with the way how they are raised: More oxygen is transported downwards in marsh plant (rice) than in land plants (pea, sorghum, maize); and, in case the same plant is concerned, more in water cultured seedlings than in moist cultured ones. Downward oxygen transport in the various seedlings is intimately correlated with the relative volume of the intercellular spaces in the root: the more the downward transport, the larger the air spaces in the cortex. The fractional volume of the intercellular spaces in a small plant segment can be conveniently estimated by determining the specific gravities of the fresh turgescent segment before and after it is filled with water by vaccum infiltration. The difference between the two consecutive measurements in specific gravity times 100 gives directly the percentage of the volume occupied by air spaces. When large root segments are used, the relative volume can also be determined by weighing before and after vaccum infiltration. To test whether oxygen diffusion in the intercellular spaces of roots could actually account for its downward transport, a model is built of capillary tubings with dimensions and oxygen pressure gradients similar to those found in roots. The amount of oxygen diffused in such a model is measured with a respiratory hydrometer (see Fig. 1) and fits closely that measured in roots. By comparing the amount of oxygen transported downwards in a seedling with that consumed by its excised roots in air, it can be shown that, in case of rice, it could meet (and at times may even exceed) 100% of that consumed by roots in water cultured seedlings, but is less in moist cultured ones. In land plants (pea, sorghum, and maize), however, the downward oxygen supply is far below its requirement, being 80%–100% in water cultured seedlings and 30%–60% in moist cultured ones. The above results, together with those obtained in previous communications, support the view that adaptation of a plant to flooded condition is primarily achieved by its capacity of providing adequate intercellular spaces for downward oxygen diffusion. The capacity depends not only upon the phylogeny of the plant concerned but also upon its ontogenic development.     

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.     

Electron microscopic investigation of water occlusions in intercellular spaces in the inner cortex of lucerne nodules.

It is unclear to what extent oxygen diffusion pathways through the cortex of the nitrogen-fixing zone of indeterminate nodules are liquid filled and whether a blockage of these pathways is involved in varying nodule oxygen permeability to control nitrogenase activity. We examined the proportion of water-filled intercellular spaces of lucerne (Medicago sativa L.) nodules with cryo-scanning electron microscopy. This technique allows for direct observation of water accumulation. Thirty percent of all intercellular spaces in the inner cortex of lucerne nodules were liquid filled. Decreasing the nodule oxygen permeability by detopping of the plant or by increasing the rhizospheric oxygen partial pressure to 80 kPa had no statistically significant effect on the water distribution in the intercellular spaces. Therefore, the hypothesis of a continuous aqueous diffusion barrier in the inner cortex could not be supported. The abundance of glycoproteins in intercellular spaces of the inner cortex was investigated with immunoelectron microscopy. No alteration due to detopping or after increase of the rhizospheric oxygen partial pressure was observed. Therefore, our results do not support the hypothesis of a short-term regulation of oxygen permeability by blockage of diffusion pathways through morphological changes in the cortex region of the nitrogen-fixing zone of lucerne nodules.     

Subcellular localization of glycoprotein epitopes during the development of lupin root nodules

Summary The monoclonal antibodies MAC236 and MAC265, raised against a soluble component of pea nodules, were used to elucidate the presence and subcellular localization of glycoprotein epitopes during the development of lupin (Lupinus albus L. cv. Multolupa) nodules, by means of immunocytochemistry and Western blot analysis. These antibodies recognize a single band of 95 kDa in pea, soybean and bean nodules, whilst two different bands of 240 and 135 kDa cross-react with MAC236 and MAC265 respectively in lupin nodules. This fact may indicate that the recognized epitopes can be present in different subcellular compartments and/or play different roles through the development of functional nodules. The results show that MAC265 is mainly associated with Bradyrhizobium infection and with the development of nodule primordium, in the first stages of nodulation. MAC265 is also detected when glycoprotein transport takes place across the cytoplasm and the cell wall, and also in the intercellular spaces of the middle cortex, attached to cell walls. The amount of MAC265 remains constant through nodule development. In contrast the amount of MAC236 increases with nodule age, parallel to the establishment of nitrogenase activity. This antibody is localized in cytoplasmic globules attached to the inner side of cell walls in the middle cortex, and mainly in the matrix filling the intercellular spaces of the middle and inner cortex. This main site of localization of MAC236 may indicate a role in the functioning of the oxygen diffusion barrier.     

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.     

Gas diffusion pathway in nodules ofCasuarina cunninghamiana

The gas diffusion pathway in nodules was traced by vacuum infiltration with India ink or aniline blue and by electron microscopy. India ink infiltration was observed in the outermost and the innermost cortex in sliced nodules, but not in intact nodules. With aniline blue infiltration, it was observed that intercellular air spaces in the outermost and the innermost cortex were connected to those in nodule roots. No air spaces were in contact with walls of infected cells, although intercellular air spaces existed in some groups of uninfected cells within the infected zone. Infiltration with either India ink or aniline blue could not be observed in the infected zone in essentially all cases. Thus it is suggested that the discontinuity of the intercellular air spaces represents a major resistance to O₂ diffusion in nodules ofCasuarina cunninghamiana.     

Observations on Gas Movement in the Rhizome of Menyanthes trifoliata L., with Comments on the Role of the Endodermis

Patterns of diffusion of oxygen in the rhizome of the aquaticMenyanthes have been investigated after the interruption ofeither stelar or cortical diffusion path-ways. Forty-eight hoursafter ringing, oxygen levels in cortex, stele, and root weresignificantly depressed, although never to danger point. Interruptionof the stele, involving also partial interruption of the cortex,was only significantly effective when made just above the samplingzone. The addition of M/1000 KCN to depress the metabolic uptakeof oxygen, resulted as expected in higher equilibrium levelsof oxygen in all zones. It was shown (a) that the velocity of diffusion of oxygen acrossthe endodermis essentially conformed to that of a process ofaqucous diffusion and (b) that oxygen diffused through the corticalair-space system at about 1/25 of its diffusion velocity inair. The diminished diffusion rate in the cortex is doubtlessdue to the frictional resistance imposed by the small poreswhich connect adjacent air cavities. An analysis has been made of the pertinent anatomical featuresof this rhizome, and the findings as a whole are discussed inrelation to their functional and ecological significance.     

Intercellular location of glycoprotein in soybean nodules: effect of altered rhizosphere oxygen concentration

Abstract. A glycoprotein which occludes intercellular spaces in the inner cortex of legume nodules may be involved in controlling oxygen diffusion into rhizobial-infected cells. Here we investigated this possibility by localizing the glycoprotein using monoclonal antibodies and immunogold labelling in nodulated roots of soybean cv. Clarke inoculated with Bradyrhizobium japonicum strain RCR3442 exposed to atmospheres with either 10, 21 (control) or 40% oxygen for 28d. Infected cells showed evidence of premature senescence when grown in above or below ambient pO₂ particularly at 10% oxygen, although cortical cells appeared to be little altered by oxygen treatment. In the inner cortical cells, more glycoprotein was seen to be occluding intercellular spaces of those nodules subjected to 40% oxygen and less in those nodules exposed to 10% oxygen, when compared to controls. This observation, made at the light microscope level (using silver enhancement) was confirmed under the TEM using immunogold labelling. Therefore, it is suggested that intercellular space glycoprotein is one of the structural components of the diffusion resistance in the cortex of legume nodules.     

The Structure of Nitrogen Fixing Root Nodules on the Aquatic Mimosoid Legume Neptunia plena

Nodules of the aquatic mimosoid legume Neptunia plena (L.) Benth.were always found associated with roots but not stems. Theyappeared macroscopically 10 and 20 d after inoculation on plantsgrown hydroponically and in vermiculite, respectively. The developmentof nitrogen-fixing cells occurred in a series of stages notyet reported in legume nodule formation: initial infection wasapparently intercellular and rhizobia spread between cells andthrough intercellular spaces before penetrating individual hostcells by means of infection threads. Subsequently nodule developmentwas broadly similar to that described for indeterminate papilionoidnodules. The infection threads of Neptunia and pea nodules containeda matrix with a common epitope, which was, in Neptunia, extrudedfrom the infection thread at the point of bacterial release. The central tissue contained infected and interstitial cellsand was surrounded by a three-layered cortex and a phellem.Bounding the infected region was a layer two to three cellsthick with large, unoccluded intercellular spaces. Externalto this was a layer, one or more cells thick, in which the cellwalls were interlocked, reducing the number of radially orientedintercellular spaces. The outer layer, several cells thick,contained intercellular spaces many of which were occluded.These features did not vary with growth conditions in a waywhich might influence oxygen diffusion characteristics. However,the phellem of water-cultured nodules was much more aerenchymatousthan that of vermiculite-grown nodules. Aquatic legume, Neptunia plena, nitrogen fixation, oxygen, root nodules, Rhizobium     

Graviresponse in higher plants and its regulation in molecular bases: relevance to growth and development, and auxin polar transport in etiolated pea seedlings]

We review the graviresponse under true and simulated microgravity conditions on a clinostat in higher plants, and its regulation in molecular bases, especially on the aspect of auxin polar transport in etiolated pea (Pisum sativum L. cv. Alaska) seedlings which were the plant materials subjected to STS-95 space experiments. True and simulated microgravity conditions substantially affected growth and development in etiolated pea seedlings, especially the direction of growth of stems and roots, resulting in automorphosis. In etiolated pea seedlings grown in space, epicotyls were the most oriented toward the direction far from the cotyledons, and roots grew toward the aerial space of Plant Growth Chamber. Automorphosis observed in space were well simulated by a clinorotation on a 3-dimensional clinostat and also phenocopied by the application of auxin polar transport inhibitors of 2,3,5-triiodobenzoic acid, N-(1-naphtyl)phthalamic acid and 9-hydroxyfluorene-9-carboxylic acid. Judging from the results described above together with the fact that activities of auxin polar transport in epicotyls of etiolated pea seedlings grown in space substantially were reduced, auxin polar transport seems to be closely related to automorphosis. Strenuous efforts to learn in molecular levels how gravity contributes to the auxin polar transport in etiolated pea epicotyls resulted in successful identification of PsPIN2 and PsAUX1 genes located in plasma membrane which products are considered to be putative efflux and influx carriers of auxin, respectively. Based on the results of expression of PsPIN2 and PsAUX1 genes under various gravistimulations, a possible role of PsPIN2 and PsAUX1 genes for auxin polar transport in etiolated pea seedlings will be discussed.     

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.     

Dimensions and distribution of intercellular spaces in cryo-planed soybean nodules

The ability of legume nodules to regulate their permeability to gas diffusion has been attributed to physiological control over the size and distribution of gas-filed intercellular spaces within the nodule cortex. To examine the size and distribution of intercellular spaces and to determine whether they were filled with gas (high diffusion permeability) or liquid (low diffusion permeability), whole nodules were frozen in liquid nitrogen slush (-210°C), and then either cryo-fractured or cryo-planed before being examined by cold-stage scanning electron microscopy (SEM). The cryo-planed tissue was found to have many advantages over cryo-fractured nodules in providing images which were easier to interpret and quantify. Intercellular spaces throughout the nodule were examined in both tangential and medial planed faces. Since no differences were observed between views in either the size or shape of the open intercellular spaces, it was concluded that the intercellular spaces of nodules were not radially oriented as assumed in many mathematical models of gas diffusion. The inner cortex region in the nodules had the smallest intercellular spaces compared to other zones, and less than 10% of the intercellular spaces were occluded with any type of material in the central zone regions. Vacuum infiltration of nodules with salt solutions and subsequent cryo-planing for SEM examination showed that open and water-filled intercellular spaces could be differentiated. The potential is discussed for using this method to study the mechanism of diffusion barrier regulation in legume nodules.     

STS-95 space experiment for plant growth and development, and auxin polar transport.

The principal objective of the space experiment, BRIC-AUX on STS-95, was the integrated analysis of the growth and development of etiolated pea and maize seedlings in space, and the effect of microgravity conditions in space on auxin polar transport in the segments. Microgravity conditions in space strongly affected the growth and development of etiolated pea and maize seedlings. Etiolated pea and maize seedlings were leaned and curved during space flight, respectively. Finally the growth inhibition of these seedlings was also observed. Roots of some pea seedlings grew toward the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles which were germinated and grown under microgravity conditions in space were significantly low. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and extremely promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.     

Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment.

The principal objectives of the space experiment, BRIC-AUX on STS 95, were the integrated analysis of the growth and development of etiolated pea and maize seedlings in space and a study of the effects of microgravity conditions in space on auxin polar transport in these segments. Microgravity significantly affected the growth and development of etiolated pea and maize seedlings. Epicotyls of etiolated pea seedlings were the most oriented toward about 40 to 60 degrees from the vertical. Mesocotyls of etiolated maize seedlings were curved at random during space flight but coleoptiles were almost straight. Finally the growth inhibition of these seedlings in space was also observed. Roots of some pea seedlings grew toward to the aerial space of Plant Growth Chamber. Extensibilities of cell walls of the third internode of etiolated pea epicotyls and the top region of etiolated maize coleoptiles, which were germinated and grown under microgravity conditions in space, were significantly low as compared with those grown on the ground of the earth. Activities of auxin polar transport in the second internode segments of etiolated pea seedlings and coleoptile segments of etiolated maize seedlings were significantly inhibited and promoted, respectively, under microgravity conditions in space. These results strongly suggest that auxin polar transport as well as the growth and development of plants is controlled under gravity on the earth.     

Hypoxia induced non-apoptotic cellular changes during aerenchyma formation in rice (Oryza sativa L.) roots

The stress of low oxygen concentrations in a waterlogged environment is minimized in some plants that produce aerenchyma, a tissue characterized by prominent intercellular spaces. It is produced by the predictable collapse of root cortex cells, indicating a programmed cell death (PCD) and facilitates gas diffusion between root and the aerial environment. The objective of this study was to characterize the cellular changes take place during aerenchyma formation in root of rice that accompany PCD. Scanning electron microscopy and transmission electron microscopy were used for cellular analysis of roots. Aerenchyma development was observed in both aerobic and flooded conditions. Structural changes in membranes and organelles were examined during development of root cortex cells to compare with previous examples of PCD. There was an initial collapse which started at a specific position in the mid cortex, indicating loss of turgor, and the cytoplasm became more electron dense. These cells were distinct in shape from those located towards the periphery. Mitochondria and endoplasmic reticulum appeared normal at this early stage though the tonoplast lost its integrity. Subsequently it underwent further degeneration while the plasmalemma retracted from the cell wall followed by death of neighboring cells followed a radial path. However, pycnosis of the nucleus, blebbing of plasma membrane and production of apoptotic bodies were not found which in turn indicated nonapoptotic PCD during aerenchyma formation in rice.     

The Pneumathodes of Laguncularia racemosa: Little Known Rootlets of Surprising Structure, and Notes on a New Fluorescent Dye for Lipophilic Substances

Abstract: Two types of negatively geotropic aerial roots may be observed on the root system of Laguncularia racemosa: pneumatophores with secondary growth, and short-lived pneumathodes which remain in the primary anatomical state. The pneumathodes distinguish themselves by the absence of an epidermis; instead, the outer cortex takes the place of the outermost tissue. This tissue forms a three-dimensional network of rod-like cells and gas spaces. The cell walls contain a lipophilic substance which ensures that the intercellular spaces remain gas-filled during submergence. An uniseriate cellular layer separates the outer and inner cortex. This uniseriate cellular layer, which we term a "pore layer", is characterized by cells with suberized and lignified cell walls and occasional pores among the cells. The pores permit the diffusion of oxygen-rich air from the surface of the pneumathode to the aerenchyma of the inner cortex and the escape of carbon dioxide from the interior of the root. The structure of the differentiated pneumathode originates from frequent cell divisions in the part of the apical meristem where the outer cortex emerges. Because of the pressure thereby exerted on the epidermis and hypodermis, these two cell layers tear and become separated from the outer cortex. Their remnants remain visible at the base of the pneumathode and as an appendage of the calyptra. The function and significance of the pneumathodes for L. racemosa are discussed. An extract of Xanthoria parietina was employed as a new fluorescent dye to stain suberine in cell walls. The staining technique is presented in this paper.     

Absorption and translocation of lipidic substances by plants

Young seedlings ofZea mays L. andCucurbita pepo L. were grown in water culture with a layer of oil (linum or olive oil) or liquid paraffin. The seedlings transport oil and parafin into the top and in the young root.Cucurbita seedlings proved to be very sensitive and died within few days.Zea proved to be resistant. InZea this transport of oil was found to be related to the age of the seedlings and the zone of the root in contact with oil. The oil was localised in the cell walls of different tissues of root and top, in the intercellular spaces of cortex and pith and in the xylem vessels.     

Anatomy of the Root-Shoot Junction in Wheat Seedlings with Respect to Internal Oxygen Transport and Root Growth Retardation by External Oxygen Shortage

Anatomical alterations in the root-shoot junction followinghypoxic conditions were studied in young wheat plants (Triticumaestivum L. cv. Hatri) grown in nutrient solution flushed withair or nitrogen gas. The root-shoot junction was characterizedby densely packed tissues with only small intercellular spaces.Seven days of hypoxia did not alter the anatomy of this region,suggesting that it does not constitute an important pathwayfor oxygen diffusion from aerial shoot to the aerenchymatousroots. A likely alternative path for oxygen movement is thegas-filled interspace between coleoptile and shoot base. Rootsemerging from more apical parts of the stem elongated more quicklyin hypoxic conditions than those from more basal parts. Thiswas related to the path length from the main point of entryof atmospheric oxygen into the plant. Additionally, oxygen shortagein the ambient root medium decreased the number of mitoses perroot tip, as determined by the Feulgen method. This effect wasmost severe in the basally inserted roots, that are presumedto be the most oxygen deficient. Triticum aestivum L. cv. Hatri, wheat, hypoxia, root-shoot junction, anatomy, internal oxygen transport, root tip, mitosis     

Aerenchyma and lenticel formation in pine seedlings: A possible avoidance mechanism to anaerobic growth conditions

Seedlings of pond pine ( Pinus serotina Michx.), sand pine [ P. clausa (Engelm.) Sarg.], and loblolly pine ( P. taeda L., wet-site and drought-hardy seed sources) were grown in hydroponic solution culture using a non-circulating, continuously flowing design under anaerobic or aerobic conditions to determine whether flooding tolerance was correlated with enhanced internal root aeration. Transport of atmospheric O₂ from the shoot to the root of anaerobically grown loblolly and pond pine seedlings was demonstrated via rhizosphere oxidation, using both reduced indigo-carmine solution and a polarographic, ensheathing Pt-electrode. Stem and root collar lenticels were the major sites of atmospheric O₂ entry for submerged roots in these seedlings. No O₂ leakage was detected from roots of aerobically grown pine seedlings. Longitudinal and radial pathways for gaseous diffusion via intercellular air spaces in the pericycle and between ray parenchyma cells, respectively, were demonstrated histo-logically in anaerobically grown loblolly and pond pines. Rhizosphere oxidation, and lenticel and aerenchyma development in roots of flood-intolerant sand pine seedlings grown in anaerobic solutions were minimal. Only 15 days of anaerobic growth conditions were necessary to increase internal root porosities of loblolly and pond pine seedlings – although not to the extent found in seedlings treated for 30 or 75 days. Histological results indicated that root tissue in the secondary stage of growth was capable of forming intercellular air spaces, demonstrating a degree of internal plasticity – at least in the more flood-tolerant loblolly and pond pine seedlings.