EFFECT OF ETHYLENE ON AERENCHYMA DEVELOPMENT

When applied to a part of stem or basal part of stem and root system, 1 ppm ethylene induced lysigenous aerenchyma development in the stem cortex of Helianthus annuus, Lycopersicon esculentum, and Phaseolus vulgaris. Local application of ethylene to a part of stem significantly increased the activity of cellulase in that part of stem in the above three species. Pretreatment of a part of stem with 100 ppm AgNO₃ counteracted the effects of ethylene which was subsequently applied to the part of stem, completely suppressing aerenchyma development and highly significantly reducing cellulase activity in Helianthus annuus. These results support the earlier proposal that the deficiency of oxygen in waterlogged plants triggers the anaerobic stimulation of ethylene production, which in turn increases the cellulase activity leading to aerenchyma development.     

ANAEROBIC ELEVATION OF ETHYLENE CONCENTRATION IN WATERLOGGED PLANTS

Anaerobic elevation of ethylene concentration in waterlogged and non-waterlogged Helianthus annuus L. and Lycopersicon esculentum Mill. was studied. A balloon method was devised to provide an anaerobic atmosphere around the intact sunflower stem. Anaerobic conditions were also produced by bubbling nitrogen into the floodwater. Ethylene concentration in the stem of waterlogged plants was higher when nitrogen was bubbled through the floodwater than when aerated, the effect being greater for the soil-grown plants than for the sand-cultured plants. Ethylene concentration in the stem of waterlogged plants was highest in the region exposed to anaerobiosis, and less with increasing distance or height on the non-waterlogged part of the stems. Intact sunflower stems increased their ethylene concentration in that part of the stem which was maintained in an oxygen-free atmosphere. The results suggest that enhanced ethylene production in waterlogged plants primarily occurs in the waterlogged part of roots and stems.     

AERENCHYMA DEVELOPMENT IN WATERLOGGED PLANTS

Aerenchyma development in waterlogged Helianthus annuus, Lycopersicon esculentum, and Salix fragilis was studied. More than half of the root cortical tissue sometimes became an air cavity in willow roots which developed in water. There was no cortical aerenchyma in the terminal portion, but more advanced aerenchyma developed towards the base of the sunflower roots formed in water. Waterlogged sunflower and tomato plants developed lysigenous aerenchyma in the cortex of waterlogged stems within two days.     

Induction of Enzymes Associated with Lysigenous Aerenchyma Formation in Roots of Zea mays during Hypoxia or Nitrogen Starvation

Either hypoxia, which stimulates ethylene biosynthesis, or temporary N starvation, which depresses ethylene production, leads to formation of aerenchyma in maize (Zea mays L.) adventitious roots by extensive lysis of cortical cells. We studied the activity of enzymes closely involved in either ethylene formation (1-amino-cyclopropane-1-carboxylic acid synthase [ACC synthase]) or cell-wall dissolution (cellulase). Activity of ACC synthase was stimulated in the apical zone of intact roots by hypoxia, but not by anoxia or N starvation. However, N starvation, as well as hypoxia, did enhance cellulase activity in the apical zone, but not in the older zones of the same roots. Cellulase activity did not increase during hypoxia or N starvation in the presence of aminoethoxyvinylglycine, an inhibitor of ACC synthase, but this inhibition of cellulase induction was reversed during simultaneous exposure to exogenous ethylene. Together these results indicate both the role of ethylene in signaling cell lysis in response to two distinct environmental factors and the significance of hypoxia rather than anoxia in stimulation of ethylene biosynthesis in maize roots.     

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.     

Waterlogging Stress and Ethylene Production in the Dune Slack Plant, Scirpus americanus

The sedge, Scirpus americanus Pers., grows in dune slacks andother freshwater and brackish water wetland communities. Whenwaterlogged in a greenhouse, the concentration of ethylene increased4-fold in stems of S. americanus plants. This increase was associatedwith a decrease in plant height and an increase in aerenchymaas exhibited under waterlogged conditions. Endogenous ethyleneproduction in S. americanus was compared to that in anotherdune slack species, Panicum amarulum, and also to Spartina aherniflorafrom a salt marsh. These species did not respond by increasingendogenous ethylene upon waterlogging. In the field, a 16 cmrainfall significantly increased the endogenous ethylene productionin S. americanus. As the water table subsided the concentrationof accumulated ethylene in stem tissue decreased. Exposure ofS. americanus to exogenous ethylene inhibited stem extensionand increased aerenchyma formation, thus linking ethylene tothe morphological characteristics of waterlogged plants of thisspecies. These experiments support the hypothesis that ethylenemodulates S. americanus morphology in natural waterlogged environmentsand may be of importance in adapting this species to life inthe wetland environment. Key words: Dune slack, waterlogging, ethylene     

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.     

Transduction of an Ethylene Signal Is Required for Cell Death and Lysis in the Root Cortex of Maize during Aerenchyma Formation Induced by Hypoxia

Ethylene has been implicated in signaling cell death in the lysigenous formation of gas spaces (aerenchyma) in the cortex of adventitious roots of maize (Zea mays) subjected to hypoxia. Various antagonists that are known to modify particular steps in signal transduction in other plant systems were applied at low concentrations to normoxic and hypoxic roots of maize, and the effect on cell death (aerenchyma formation) and the increase in cellulase activity that precedes the appearance of cell degeneration were measured. Both cellulase activity and cell death were inhibited in hypoxic roots in the presence of antagonists of inositol phospholipids, Ca2+- calmodulin, and protein kinases. By contrast, there was a parallel promotion of cellulase activity and cell death in hypoxic and normoxic roots by contact with reagents that activate G-proteins, increase cytosolic Ca2+, or inhibit protein phosphatases. Most of these reagents had no effect on ethylene biosynthesis and did not arrest root extension. These results indicate that the transduction of an ethylene signal leading to an increase in intracellular Ca2+ is necessary for cell death and the resulting aerenchyma development in roots of maize subjected to hypoxia.     

Identification of genes expressed in maize root cortical cells during lysigenous aerenchyma formation using laser microdissection and microarray analyses

? To adapt to waterlogging in soil, some gramineous plants, such as maize (Zea mays), form lysigenous aerenchyma in the root cortex. Ethylene, which is accumulated during waterlogging, promotes aerenchyma formation. However, the molecular mechanism of aerenchyma formation is not understood. ? The aim of this study was to identify aerenchyma formation-associated genes expressed in maize roots as a basis for understanding the molecular mechanism of aerenchyma formation. Maize plants were grown under waterlogged conditions, with or without pretreatment with an ethylene perception inhibitor 1-methylcyclopropene (1-MCP), or under aerobic conditions. Cortical cells were isolated by laser microdissection and their mRNA levels were examined with a microarray. ? The microarray analysis revealed 575 genes in the cortical cells, whose expression was either up-regulated or down-regulated under waterlogged conditions and whose induction or repression was suppressed by pretreatment with 1-MCP. ? The differentially expressed genes included genes related to the generation or scavenging of reactive oxygen species, Ca(2+) signaling, and cell wall loosening and degradation. The results of this study should lead to a better understanding of the mechanism of root lysigenous aerenchyma formation.     

Ethylene Biosynthesis during Aerenchyma Formation in Roots of Maize Subjected to Mechanical Impedance and Hypoxia

Germinated maize (Zea mays L.) seedlings were enclosed in modified triaxial cells in an artificial substrate and exposed to oxygen deficiency stress (4% oxygen, hypoxia) or to mechanical resistance to elongation growth (mechanical impedance) achieved by external pressure on the artificial substrate, or to both hypoxia and impedance simultaneously. Compared with controls, seedlings that received either hypoxia or mechanical impedance exhibited increased rates of ethylene evolution, greater activities of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and cellulase, and more cell death and aerenchyma formation in the root cortex. Effects of hypoxia plus mechanical impedance were strongly synergistic on ethylene evolution and ACC synthase activity; cellulase activity, ACC oxidase activity, or aerenchyma formation did not exhibit this synergism. In addition, the lag between the onset of stress and increases in both ACC synthase activity and ethylene production was shortened by 2 to 3 h when mechanical impedance or impedance plus hypoxia was applied compared with hypoxia alone. The synergistic effects of hypoxia and mechanical impedance and the earlier responses to mechanical impedance than to hypoxia suggest that different mechanisms are involved in the promotive effects of these stresses on maize root ethylene biosynthesis.     

Formation of Aerenchyma and the Processes of Plant Ventilation in Relation to Soil Flooding and Submergence

Abstract: Enhanced development of gas-spaces beyond that due to the partial cell separation normally found in ground parenchymas and their derivatives creates tissue commonly termed "aerenchyma". Aerenchyma can substantially reduce internal impedance to transport of oxygen, nitrogen and various metabolically generated gases such as carbon dioxide and ethylene, especially between roots and shoots. Such transport lessens the risk of asphyxiation under soil flooding or more complete plant submergence, and promotes radial oxygen loss from roots leading to oxidative detoxification of the rhizo-sphere. Aerenchyma can also increase methane loss from waterlogged sediments via plants to the atmosphere. This review of the formation and functioning of aerenchyma particularly emphasises research findings since 1992 and highlights prospects for the future. Regarding formation, attention is drawn to how little is known of the regulation and processes that create schizogenous aerenchyma with its complex cell arrangements and differential cell to cell adhesion. More progress has been made in understanding lysigenous aerenchyma development. The review highlights recent work on the processes that sense oxygen deficiency and ethylene signals, subsequent transduction processes which initiate cell death, and steps in protoplast and wall degeneration that create the intercellular voids. Similarities between the programmed cell death and its causes in animals and the predictable patterns of cell death that create lysigenous aerenchyma are explored. Recent findings concerning function are addressed in terms of the diffusion aeration of roots, rhizosphere oxygenation and sediment biogeochemistry, photosynthesis and ventilation, pressurised gas-flows and greenhouse gas emissions and aspects of ventilation related to secondary thickening.     

Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10⁻⁵ or 10⁻²m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10⁻²m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10⁻⁵m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.     

Occurrence and Localization of 9.5 Cellulase in Abscising and Nonabscising Tissues

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.     

Dehiscence of Fruit in Oilseed Rape (Brassica nap us L.): II. THE ROLE OF CELL WALL DEGRADING ENZYMES AND ETHYLENE

Pod shatter in oilseed rape is accompanied by the degradationof the cell wall at the site of fruit dehiscence. Cell separationis preceded by an increase in the activity of the hydrolyticenzyme cellulase (ß 1,4-glucanase, E.C. 3.1.2.4 [EC] ),and this rise in enzyme activity is restricted to the dehiscencezone cells. In contrast, the activity of the cell wall degradingenzyme polygalacturonase (E.C. 3.2.1.15 [EC] ) exhibits no correlationeither temporally or spatially with pod dehiscence. An analysisof the ethylene production profile by intact pods during maturationhas revealed the existence of an ethylene climacteric and thisis temporally correlated with the tissue-specific increase incellulase activity. The major site of ethylene production bythe fruit has been identified to be the developing seed. Sincemaintenance of intact pods in ethylene accelerates both thesenescence and dehiscence of the tissue, it is possible thatthis gaseous regulator plays an important role in the processof pod shatter in vivo. Key words: Oilseed rape, Brassica napus, pod shatter, cellulase, polygalacturonase, ethylene     

Ethylene Accumulation in Flooded Plants

Ethylene concentration in sunflower (Helianthus annuus L.) cuttings increased 5-fold within 6 h after submersion in distilled water and then declined. When only the basal half of the cutting was steeped in water, ethylene concentration was slightly over half the concentration of the completely submerged cutting. Ethylene concentration also increased when cuttings were wrapped with moist paper tissue. When wrapped with Saran transparent plastic film, ethylene concentration increased continuously for 12 h. When part of the stem of an intact plant was wrapped with Saran, ethylene also increased in that part of the stem. When wrapping was removed or submersion was discontinued, accumulated ethylene in the cuttings decreased, much faster from unwrapped cuttings than from previously submerged ones. During 3 h submersion, ethylene production rate in submerged cuttings was approximately 10% of that for the controls and over 97% ethylene escaped out of the control cuttings while only 22-52% escaped from the submerged cuttings. Water content increased during submersion and decreased when submersion was discontinued. Water content did not change significantly during wrapping, but decreased when the cuttings were unwrapped. High water content in the submerged cuttings was apparently not related to the high ethylene concentration in the cuttings. Causes of ethylene increase in flooded plants were discussed and it was concluded that one of the first and major causes is the accumulation of ethylene in flooded portions of the plants due to the blockade of ethylene escape by water.     

穿叶眼子菜茎叶结构及其通气组织发育解剖学研究

通气组织(aerenchyma)是植物薄壁组织内一些气室或腔隙的集合,对于水生及湿地植物体内的气体运输至关重要。该实验以沉水植物穿叶眼子菜为材料,利用石蜡切片技术,通过对茎的纵切面及横切面结构进行观察,从时间和空间上分析其茎、叶通气组织的发生过程。结果表明:(1)穿叶眼子菜的茎结构包括表皮、皮层及维管柱,通气组织发达,存在于内皮层与表皮之间;茎通气组织由距茎尖约0.6mm处开始形成,并成熟于约2.4mm处。(2)穿叶眼子菜的叶由表皮、皮层薄壁细胞及维管柱组成,其通气组织形成于靠近茎尖的第2~3片新生叶且仅形成于主叶脉。(3)穿叶眼子菜的茎和叶通气组织的发育过程相似,起初为排列致密的细胞团,然后由皮层细胞的分裂产生小的细胞间隙,随后的腔隙膨大过程涉及细胞的生长分裂及细胞降解,最终形成发达的通气组织。(4)穿叶眼子菜的通气组织发育过程可划分为实心期、形成期、膨大期、成熟期四个时期;不同时期茎通气组织的发达程度差异很大,实心期、形成期、膨大期和成熟期的孔隙度分别为0.54%、10.90%、27.61%和57.58%;但节处通气组织不发达,成熟期的节处孔隙度仅为3.62%。     

A flooding-induced xyloglucan endo-transglycosylase homolog in maize is responsive to ethylene and associated with aerenchyma.

Development of aerenchyma (soft cortical tissue with large intercellular air spaces) in flooded plants results from cell-wall hydrolysis and eventual cell lysis and is promoted by endogenous ethylene. Despite its adaptive significance, the molecular mechanisms behind aerenchyma development remain unknown. We recently isolated a flooding-induced maize (Zea mays L.) gene (wusl1005[gfu]; abbreviated as 1005) encoding a homolog of xyloglucan endo-transglycosylase (XET), a putative cell-wall-loosening enzyme active during germination, expansion, and fruit softening. XET and related enzymes may also be involved in cell-wall metabolism during flooding-induced aerenchyma development. Under flooding, 1005 mRNA accumulated in root and mesocotyl locations that subsequently exhibited aerenchyma development and reached maximum levels within 12 h of treatment. Aerenchyma development was observed in the same locations by 48 h of treatment. Treatment with the ethylene synthesis inhibitor (aminooxy) acetic acid (AOA), which prevented cortical air space formation under flooding, almost completely inhibited 1005 mRNA accumulation in both organs. AOA treatment had little effect on the accumulation of mRNA encoded by adh1, indicating that it did not cause general suppression of flooding-responsive genes. Additionally, ethylene treatment under aerobic conditions resulted in aerenchyma development as well as induction of 1005 in both organs. These results indicate that 1005 is responsive to ethylene. Treatment with anoxia, which suppresses ethylene accumulation and aerenchyma development, also resulted in 1005 induction. However, in contrast to flooding, AOA treatment under anoxia did not affect 1005 mRNA accumulation, indicating that 1005 is induced via different mechanisms under flooding (hypoxia) and anoxia.     

Polyamine Content and Action in Roots of Zea mays L. in Relation to Aerenchyma Development

Roots of Zea mays L. developed more aerenchyma (cortical gas-filledspace) when partially deficient in oxygen (3 kPa) than whensupplied with air (20·8 kPa oxygen) in association withfaster production of ethylene (ethene). The possibility wastested that the additional ethylene production resulted fromdecreases in spermidine (spd) and spermine (spm) which share,with ethylene, a common precursor, S-adenosylmethionine. However,no decreases in spd and spm were seen in root tissue up to 4d-old. Removing oxygen completely also had little effect onspd and spm, but strongly suppressed both ethylene productionand aerenchyma formation. Partial oxygen shortage (3 kPa) increased the concentrationof putrescine (put), the precursor of spd and spm. This increasewas not a response to the extra ethylene formed by such rootssince ethylene treatment did no reproduce the effect. Applicationof inhibitors of put biosynthesis, difluoromethylarginine anddifluoromethylornithine, led to increased aerenchyma formation.Exogenous put inhibited the development of aerenchyma whilestimulating rather than inhibiting ethylene production, whentested in either air or 3 kPa oxygen. Thus, put appears to limitaerenchyma formation by suppressing ethylene action rather thanits production.Copyright 1993, 1999 Academic Press Ethylene, ethene, roots, aerenchyma, polyamines, oxygen shortage, anaerobiosis, environmental stress, Zea mays