Changes in growth and structure of pea primary roots (Pisum sativum L. cv. Alaska) as a result of sudden flooding

Pea (Pisum sativum L. cv. Alaska) primary roots were exposed to flooding after growth for 4 or 5 d at 25 degrees C under relatively dry conditions. Flooding after 4 d growth reduced, but did not stop, primary root growth, and cavities caused by degradation of central vascular cells were typically found from 10-60 mm from the tips. Flooding after 5 d stopped primary root growth and caused cell death in the tips, and vascular cavities formed that typically were 20-60 mm from the tips of the roots. Degradation of root tip cells in 5-day-roots was very rapid and began in the elongation zone and later in the apical zone. Root tips discolored, narrowed or curled before growth arrest. The mitotic indices of 5-day-root tips were suppressed by the flooding treatment. A few mitotic figures were observed in roots treated with flooding after 4 d growth. Affected cells had condensed nuclei, but cytoplasms appeared to be normal in the early stages of cell degradation. Later these cells became very vacuolated. The relationship of flooding to root growth, vascular cavity formation, and the morphology of pea primary roots is described with regard to the ability to resist flooding stress.     

Aerenchyma formation in maize roots

Maize (Zea mays L.) is generally considered to be a plant with aerenchyma formation inducible by environmental conditions. In our study, young maize plants, cultivated in various ways in order to minimise the stressing effect of hypoxia, flooding, mechanical impedance or nutrient starvation, were examined for the presence of aerenchyma in their primary roots. The area of aerenchyma in the root cortex was correlated with the root length. Although 12 different maize accessions were used, no plants without aerenchyma were acquired until an ethylene synthesis inhibitor was employed. Using an ACC-synthase inhibitor, it was confirmed that the aerenchyma formation is ethylene-regulated and dependent on irradiance. The presence of TUNEL-positive nuclei and ultrastructural changes in cortical cells suggest a connection between ethylene-dependent aerenchyma formation and programmed cell death. Position of cells with TUNEL-positive nuclei in relation to aerenchyma-channels was described.     

Lysigenous aerenchyma formation involves non-apoptotic programmed cell death in rice (Oryza sativa L.) roots

In waterlogged soil, deficiency of oxygen triggers development of aerenchyma in roots which facilitates gas diffusion between roots and the aerial environment. However, in contrast to other monocots, roots of rice (Oryza sativa L.) constitutively form aerenchyma even in aerobic conditions. The formation of cortical aerenchyma in roots is thought to occur by either lysigeny or schizogeny. Schizogenous aerenchyma is developed without cortical cell death. However, lysigenous gas-spaces are formed as a consequence of senescence of specific cells in primary cortex followed by their death due to autolysis. In the last stage of aerenchyma formation, a ‘spoked wheel’ arrangement is observed in the cortical region of root. Ultrastructural studies show that cell death is constitutive and no characteristic cell structural differentiation takes place in the dying cells with respect to surrounding cells. Cell collapse initiation occurs in the center of the cortical tissues which are characterized by shorter with radically enlarged diameter. Then, cell death proceeds by acidification of cytoplasm followed by rupturing of plasma membrane, loss of cellular contents and cell wall degradation, while cells nuclei remain intact. Dying cells releases a signal through symplast which initiates cell death in neighboring cells. During early stages, middle lamella-degenerating enzymes are synthesized in the rough endoplasmic reticulum which are transported through dictyosome and discharged through plasmalemma beneath the cell wall. In rice several features of root aerenchyma formation are analogous to a gene regulated developmental process called programmed cell death (PCD), for instance, specific cortical cell death, obligate production of aerenchyma under environmental stresses and early changes in nuclear structure which includes clumping of chromatin, fragmentation, disruption of nuclear membrane and apparent engulfment by the vacuole. These processes are followed by crenulation of plasma membrane, formation of electron-lucent regions in the cytoplasm, tonoplast disintegration, organellar swelling and disruption, loss of cytoplasmic contents, and collapse of cell. Many processes in lysing cells are structural features of apoptosis, but certain characteristics of apoptosis i.e., pycnosis of the nucleus, plasma membrane blebbing, and apoptotic bodies formation are still lacking and thus classified as non-apoptotic PCD. This review article, describes most recent observations alike to PCD involved in aerenchyma formation and their systematic distributions in rice roots.     

Aerenchyma formation and recovery from hypoxia of the flooded root system of nodulated soybean

BACKGROUND AND AIMS: Flooding results in hypoxia of the root system to which N2 fixation of nodulated roots can be especially sensitive. Morphological adaptions, such as aerenchyma formation, can facilitate the diffusion of oxygen to the hypoxic tissues. Using soybean, the aim of the study was to characterize the morphological response of the nodulated root system to flooding and obtain evidence for the recovery of N metabolism. METHODS: Sections from submerged tissues were observed by light microscopy, while sap bleeding from the xylem was analysed for nitrogenous components. KEY RESULTS: Flooding resulted in the rapid formation of adventitious roots and aerenchyma between the stem (immediately above the water line), roots and nodules. In the submerged stem, taproot, lateral roots and adventitious roots, lysigenous aerenchyma arose initially in the cortex and was gradually substituted by secondary aerenchyma arising from cells derived from the pericycle. Nodules developed aerenchyma from cells originating in the phellogen but nodules situated at depths greater than 7-8 cm showed little or no aerenchyma formation. As a result of aerenchyma formation, porosity of the taproot increased substantially between the 4th and 7th days of flooding, coinciding with the recovery of certain nitrogenous products of N metabolism of roots and nodules transported in the xylem. Thus, on the first day of flooding there was a sharp decline in xylem ureides and glutamine (products of N2 fixation), together with a sharp rise in alanine (product of anaerobic metabolism). Between days 7 and 10, recovery of ureides and glutamine to near initial levels was recorded while recovery of alanine was partial. CONCLUSIONS: N metabolism of the nodulated soybean root system can recover at least partially during a prolonged period of flooding, a process associated with aerenchyma formation.     

Developmental programmed cell death in primary roots of Sonoran Desert Cactaceae

Primary roots of two species of Sonoran Desert Cactaceae, Stenocereus gummosus and Pachycereus pringlei, have a determinate pattern of growth: meristematic cells divide only for a limited time and then differentiate. Detecting DNA fragmentation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL), we have shown that programmed cell death (PCD) was not involved in meristem exhaustion. However, we found TUNEL-positive nuclei in the root hair and root cap cells of both species. Programmed cell death in root hair cells has not been previously reported, and the pattern of PCD events in the root cap differed from that described earlier. These data suggest that in the studied Cactaceae, PCD is involved in developmental adaptations related to the formation of a compact root system important for rapid seedling establishment in a desert environment. Participation of PCD in developmental loss of the root cap and in root hair renovation proposed in the current study implicates an evolutionary conserved link between PCD and differentiation processes in plants.     

Comparative spatiotemporal analysis of root aerenchyma formation processes in maize due to sulphate, nitrate or phosphate deprivation

Nitrate (N), phosphate (P) or sulphate (S) deprivation causes aerenchyma formation in maize (Zea mays L.) nodal roots. The exact mechanisms that trigger the formation of aerenchyma under these circumstances are unclear. We have compared aerenchyma distribution across the nodal roots of first whorl (just emerging in 10-day-old seedlings), which were subject to S, N or P deprivation over a period of 10?days in connection with oxygen consumption, ATP concentration, cellulase and polygalacturonase activity in the whole root. The effect of deprivation on aerenchyma formation was examined using light and electron microscopy, along with in situ detection of calcium and of reactive oxygen species (ROS) by fluorescence microscopy. Aerenchyma was not found in the root base regardless of the deprivation. Programmed cell death (PCD) was observed near the root tip, either within the first two days (-N) or a few days later (-S, -P) of the treatment. Roots at day?6 under all three nutrient-deprived conditions showed signs of PCD 1?cm behind the cap, whereas only N-deprived root cells 0.5?cm behind the cap showed severe ultrastructural alterations, due to advanced PCD. The lower ATP concentration and the higher oxygen consumptions observed at day?2 in N-, P- and S-deprived roots compared to the control indicated that PCD may be triggered by perturbations in energy status of the root. The peaks of cellulase activity located between days?3 (-N) and 6 (-P), along with the respective alterations in polygalacturonase activity, indicated a coordination which preceded aerenchyma formation. ROS and calcium seemed to contribute to PCD initiation, with ROS possessing dual roles as signals and eliminators. All the examined parameters presented both common features and characteristic variations among the deprivations.     

Process of aerenchyma formation and reactive oxygen species induced by waterlogging in wheat seminal roots

The development and regulation of aerenchyma in waterlogged conditions were studied in the seminal roots of wheat. Evans blue staining and the first cell death position indicated that the cortical cell death began at the root mid-cortex cells in flooding conditions. Continuous waterlogging treatment caused the spread of cell death from the mid-cortex to the neighboring cells and well-developed aerenchyma was formed after 72 h. Meanwhile, the formation of radial oxygen loss barrier was observed in the exodermis owing to the induction of Casparian bands and lignin deposition. Analysis of aerenchyma along the wheat root revealed that aerenchyma formed at 10 mm from the root tip, significantly increased toward the center of the roots, and decreased toward the basal region of the root. In situ detection of radial oxygen species (ROS) showed that ROS accumulation started in the mid-cortex cells, where cell death began indicating that cell death was probably accompanied by ROS production. Further waterlogging treatments resulted in the accumulation of ROS in the cortical cells, which were the zone for aerenchyma development. Accumulation and distribution of H₂O₂ at the subcellular level were revealed by ultracytochemical localization, which further verified the involvement of ROS in the cortical cell death process (i.e., aerenchyma formation). Furthermore, gene expression analysis indicated that ROS production might be the result of up-regulation of genes encoding for ROS-producing enzymes and the down-regulation of genes encoding for ROS-detoxifying enzymes. These results suggest that aerenchyma development in wheat roots starts in the mid-cortex cells and its formation is regulated by ROS.     

Cortical Aerenchyma formation in hypocotyl and adventitious roots of Luffa cylindrica subjected to soil flooding

BACKGROUND AND AIMS: Aerenchyma formation is thought to be one of the important morphological adaptations to hypoxic stress. Although sponge gourd is an annual vegetable upland crop, in response to flooding the hypocotyl and newly formed adventitious roots create aerenchyma that is neither schizogenous nor lysigenous, but is produced by radial elongation of cortical cells. The aim of this study is to characterize the morphological changes in flooded tissues and the pattern of cortical aerenchyma formation, and to analyse the relative amount of aerenchyma formed. METHODS: Plants were harvested at 16 d after the flooding treatment was initiated. The root system was observed, and sections of fresh materials (hypocotyl, tap root and adventitious root) were viewed with a light or fluorescence microscope. Distributions of porosity along adventitious roots were estimated by a pycnometer method. KEY RESULTS: Under flooded conditions, a considerable part of the root system consisted of new adventitious roots which soon emerged and grew quickly over the soil surface. The outer cortical cells of these roots and those of the hypocotyl elongated radially and contributed to the development of large intercellular spaces. The elongated cortical cells of adventitious roots were clearly T-shaped, and occurred regularly in mesh-like lacunate structures. In these positions, slits were formed in the epidermis. In the roots, the enlargement of the gas space system began close to the apex in the cortical cell layers immediately beneath the epidermis. The porosity along these roots was 11-45 %. In non-flooded plants, adventitious roots were not formed and no aerenchyma developed in the hypocotyl or tap root. CONCLUSIONS: Sponge gourd aerenchyma is produced by the unique radial elongation of cells that make the expansigeny. These morphological changes seem to enhance flooding tolerance by promoting tissue gas exchange, and sponge gourd might thereby adapt to flooding stress.     

Hypoxic stress triggers a programmed cell death pathway to induce vascular cavity formation in Pisum sativum roots

Flooding at warm temperatures induces hypoxic stress in Pisum sativum seedling roots. In response, some undifferentiated cells in the primary root vascular cylinder start degenerating and form a longitudinal vascular cavity. Changes in cellular morphology and cell wall ultrastructure detected previously in the late stages of cavity formation suggest possible involvement of programmed cell death (PCD). In this study, cytological events occurring in the early stages of cavity formation were investigated. Systematic DNA fragmentation, a feature of many PCD pathways, was detected in the cavity‐forming roots after 3 h of flooding in situ by terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling assay and in isolated total DNA by gel electrophoresis. High molecular weight DNA fragments of about 20–30 kb were detected by pulse‐field gel electrophoresis, but no low‐molecular weight internucleosomal DNA fragments were detected by conventional gel electrophoresis. Release of mitochondrial cytochrome c protein into the cytosol, an integral part of mitochondria‐dependent PCD pathways, was detected in the cavity‐forming roots within 2 h of flooding by fluorescence microscopy of immunolabeled cytochrome c in situ and in isolated mitochondrial and cytosolic protein fractions by western blotting. DNA fragmentation and cytochrome c release remained confined to the undifferentiated cells in center of the root vascular cylinders, even after 24 h of flooding, while outer vascular cylinder cells and cortical cells maintained cellular integrity and normal activity. These findings confirm that hypoxia‐induced vascular cavity formation in P. sativum roots involves PCD, and provides a chronological model of cytological events involved in this rare and understudied PCD system.     

Formation and function of secondary aerenchyma in hypocotyl, roots and nodules of soybean (Glycine max) under flooded conditions

Flooding is a major problem in many areas of the world and soybean is susceptible to the stress. Understanding the morphological mechanisms of flooding tolerance is important for developing flood-tolerant genotypes. We investigated secondary aerenchyma formation and function in soybean (Glycine max) seedlings grown under flooded conditions. Secondary aerenchyma, a white and spongy tissue, was formed in the hypocotyl, tap root, adventitious roots and root nodules after 3 weeks of flooding. Under irrigated conditions aerenchyma development was either absent or rare and phellem was formed in the hypocotyl, tap root, adventitious roots and root nodules. Secondary meristem partially appeared at the outer parts of the interfascicular cambium and girdled the stele, and then cells differentiated to construct secondary aerenchyma in the flooded hypocotyl. These morphological changes proceeded for 4 days after the initiation of the flooding. After 14 days of treatment, porosity exceeded 30% in flooded hypocotyl with well-developed secondary aerenchyma, while it was below 10% in hypocotyl of irrigated plants that had no aerenchyma. When Vaseline was applied to the hypocotyl of plants from a flooded treatment to prevent the entry of atmospheric oxygen into secondary aerenchyma, plant growth, especially that of roots, was sharply inhibited. Thus secondary aerenchyma might be an adaptive response to flooding.     

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.     

Demonstration of osmotically dependent promotion of aerenchyma formation at different levels in the primary roots of rice using a 'sandwich' method and X-ray computed tomography

Background and Aims

The effect of environmental factors on the regulation of aerenchyma formation in rice roots has been discussed for a long time, because aerenchyma is constitutively formed under aerated conditions. To elucidate this problem, a unique method has been developed that enables sensitive detection of differences in the development of aerenchyma under two different environmental conditions. The method is tested to determine whether aerenchyma development in rice roots is affected by osmotic stress.

Methods

To examine aerenchyma formation both with and without mannitol treatment in the same root, germinating rice (Oryza sativa) caryopses were sandwiched between two agar slabs, one of which contained 270 mm of mannitol. The roots were grown touching both slabs and were thereby exposed unilaterally to osmotic stress. As a non-invasive approach, refraction contrast X-ray computed tomography (CT) using a third-generation synchrotron facility, SPring-8 (Super photon ring 8 GeV, Japan Synchrotron Radiation Research Institute), was used to visualize the three-dimensional (3-D) intact structure of aerenchyma and its formation in situ in rice roots. The effects of unilateral mannitol treatment on the development of aerenchyma were quantitatively examined using conventional light microscopy.

Key Results

Structural continuity of aerenchyma was clearly visualized in 3-D in the primary root of rice and in situ using X-ray CT. Light microscopy and X-ray CT showed that the development of aerenchyma was promoted on the mannitol-treated side of the root. Detailed light microscopic analysis of cross-sections cut along the root axis from the tip to the basal region demonstrated that aerenchyma developed significantly closer to the root tip on the mannitol-treated side of the root.

Conclusions

Continuity of the aerenchyma along the rice root axis was morphologically demonstrated using X-ray CT. By using this ‘sandwich’ method it was shown that mannitol promoted aerenchyma formation in the primary roots of rice.     

Roots of willow (Salix viminalis L.) show marked tolerance to oxygen shortage in flooded soils and in solution culture

Responses to soil flooding and oxygen shortage were studied in field, glasshouse and controlled environment conditions. Established stools ofSalix viminalis L., were compared at five field sites in close proximity but with contrasting water table levels and flooding intensities during the preceding winter. There was no marked effect of site on shoot extension rate, time to half maximum length or final length attained. When rooted cuttings were waterlogged for 4 weeks in a glasshouse, soil redox potentials quickly decreased to below zero. Shoot extension was slowed after a delay of 20 d, while, in the upper 100 mm of soil, formation and outgrowth of unbranched adventitious roots with enhanced aerenchyma development was promoted after 7 d. At depths of 100–200 mm and 200–300 mm, extension by existing root axes was halted by soil flooding, while adventitious roots from above failed to penetrate these deeper zones. After 4 weeks waterlogging, all arrested root tips recommenced elongation when the soil was drained; their extension rates exceeding those of roots that were well-drained throughout. Growth in fresh mass was also stimulated. The additional aerenchyma found in adventitious roots in the upper 100 mm of soil may have been ethylene regulated since gas space development was inhibited by silver nitrate, an ethylene action inhibitor. The effectiveness of aerenchyma was tested by blocking the entry of atmospheric oxygen into plants with lanolin applied to lenticels of woody shoots of plants grown in solution culture. Root extension was halved, while shoot growth remained unaffected. H Lambers Section editor     

DEVELOPMENT OF THE WOODY PORTION OF THE ROOT SYSTEM OF BETULA PAPYRIFERA

Our ontogenetic analysis of paper birch root systems shows that the fate of a root tip is related to its relative primary xylem diam (PXD). Lateral root tips with an initial PXD less than about 25 % of the parent root PXD are ephemeral. Some tips having a PXD of more than about 25 % of the parent root PXD become the permanent portions of the root system, enlarging over time as they elongate. In seedling root systems, the primary root and first-formed laterals are initially about the same size, and their PXD's all enlarge with increasing distance from the stem as the tips elongate to form the initial horizontal woody framework. Permanent lateral root branches with a large relative PXD develop after root tip injury, when the root tip is forced to grow in a curve, or from other unaccountable causes. Our observations show the importance of using relative diameters when classifying roots and when applying the concept of heterorhizy to paper birch root systems. Evidence points to the existence of some form of apical dominance in roots.     

Progressive Cortical Senescence and Formation of Lysigenous Gas Space (Aerenchyma) Distinguished by Nuclear Staining in Adventitious Roots of Zea mays

The pattern of loss of nuclear integrity in the epidermis andcortex of maize adventitious roots was examined during (1) non-pathogeniccortical senescence associated with root ageing, and (2) lysigenousaerenchyma formation, to determine whether these phenomena arerelated. Nuclear integrity was estimated by counting the percentageof cells with nuclei detectable by acridine orange fluorescence. In roots of both soil-grown (90 d) and solution-grown (19 d)plants, nuclei were lost progressively, from the epidermis andfrom successively deeper cortical cell layers, with increasingdistance behind the root tips; this occurred irrespective ofthe degree of aeration in solution culture, and independentlyof aerenchyma formation. Aerenchyma developed in soil-grownplants and in sub-ambient oxygen concentrations (<5 kPa partialpressure) in solution culture. It started to form in the middlecortex and coincided with a marked loss of nuclear stainingin the inner cortex, especially in the innermost cortical celllayer next to the endodermis, but not in the remaining cellsof the middle cortex. Two distinct patterns of nuclear deletionfrom the cortex were thus demonstrated; they occurred independentlybut simultaneously in some conditions. These findings are discussed in relation to mechanisms of celldeath, and the metabolic status of root cortical cells participatingin ion transport to the xylem. Zea mays L., maize, roots, aerenchyma, cell death, nuclei     

Salt Stress-induced Programmed Cell Death in Rice Root Tip Cells

Salt stressed rice root tips were used to investigate the changes of reactive oxygen species （ROS） and antioxidant enzymes at the early stages of programmed cell death （PCD）. The results indicated that 500 mmol/L NaCI treatment could lead to specific features of PCD in root tips, such as DNA ladder, nuclear condense and deformation, and transferase mediated dUTP nick end labeling positive reaction, which were initiated at 4 h of treatment and pro- gressed thereafter. Cytochrome c release from mitochondria into cytoplasm was also observed, which occurred at 2 h and was earlier than the above nuclear events. In the very early phase of PCD, an immediate burst in hydrogen peroxide and superoxide anion production rate was accompanied by two-phase changes of superoxide dismutases and ascorbate peroxidase. A short period of increase in the activity was followed by prolonged impairment. Thus, we conclude that salt can induce PCD in rice root tip cells, and propose that in the early phase of rice root tip cell PCD, salt stress-induced oxidative burst increased the antioxidant enzyme activity, which, in turn, scavenged the ROS and abrogated PCD. Also, when the stress is prolonged, the antioxidant system is damaged and accumulated ROS induces the PCD process, which leads to cytochrome c release and nuclear change.     

Physiological adaptation of waxapple to waterlogging

Waxapple (Syzygium samarangense Men. et. Perry) plants receiving up to 40d of continuous flooding treatment showed no symptoms of physiological disorder, but the treatment resulted in early flowering. In this report, several physiological parameters of flooded plants are compared with those of nonflooded plants. Both control plants and 9-d-flooding-treated plants exhibited aerenchyma formation in the cortex tissue beginning 5 cm from the root tip. After 7d flooding treatment, the oxygen consumption rate of the root section was only 20% of that of the controls. Following flooding treatment, the roots showed an increase in alcohol dehydrogenase activity as well as an increase in three isozymes. However, malate dehydrogenase activity was decreased, and no significant change of NADP-malic enzyme activity was observed. There were no significant differences in levels of ethylene, 1-aminocyclopropane-1-carboxylic acid and 1-(malonylamino) cyclopropane-1-carboxylic acid in petiole and roots of flooded and non-flooded plants during the stage examined. It is inferred that the presence of aerenchyma in the root cortex allows a higher level of internal gas exchange, and thus, makes waxapple surprisingly flood tolerant. However, reduced root oxygen consumption rate may have limited root respiration rate and vegetative growth.     

Aerenchyma formation and radial O2 loss along adventitious roots of wheat with only the apical root portion exposed to O2 deficiency

This study investigated aerenchyma formation and function in adventitious roots of wheat (Triticum aestivum L.) when only a part of the root system was exposed to O₂ deficiency. Two experimental systems were used: (1) plants in soil waterlogged at 200 mm below the surface; or (2) a nutrient solution system with only the apical region of a single root exposed to deoxygenated stagnant agar solution with the remainder of the root system in aerated nutrient solution. Porosity increased two‐ to three‐fold along the entire length of the adventitious roots that grew into the water‐saturated zone 200 mm below the soil surface, and also increased in roots that grew in the aerobic soil above the water‐saturated zone. Likewise, adventitious roots with only the tips growing into deoxygenated stagnant agar solution developed aerenchyma along the entire main axis. Measurements of radial O₂ loss (ROL), taken using root‐sleeving O₂ electrodes, showed this aerenchyma was functional in conducting O_2. The ROL measured near tips of intact roots in deoxygenated stagnant agar solution, while the basal part of the root remained in aerated solution, was sustained when the atmosphere around the shoot was replaced by N₂. This illustrates the importance of O₂ diffusion into the basal regions of roots within an aerobic zone, and the subsequent longitudinal movement of O₂ within the aerenchyma, to supply O₂ to the tip growing in an O₂ deficient zone.