Lysigenous aerenchyma formation in Arabidopsis is controlled by LESION SIMULATING DISEASE1

Aerenchyma tissues form gas-conducting tubes that provide roots with oxygen under hypoxic conditions. Although aerenchyma have received considerable attention in Zea mays, the signaling events and genes controlling aerenchyma induction remain elusive. Here, we show that Arabidopsis thaliana hypocotyls form lysigenous aerenchyma in response to hypoxia and that this process involves H(2)O(2) and ethylene signaling. By studying Arabidopsis mutants that are deregulated for excess light acclimation, cell death, and defense responses, we find that the formation of lysigenous aerenchyma depends on the plant defense regulators LESION SIMULATING DISEASE1 (LSD1), ENHANCED DISEASE SUSCEPIBILITY1 (EDS1), and PHYTOALEXIN DEFICIENT4 (PAD4) that operate upstream of ethylene and reactive oxygen species production. The obtained results indicate that programmed cell death of lysigenous aerenchyma in hypocotyls occurs in a similar but independent manner from the foliar programmed cell death. Thus, the induction of aerenchyma is subject to a genetic and tissue-specific program. The data lead us to conclude that the balanced activities of LSD1, EDS1, and PAD4 regulate lysigenous aerenchyma formation in response to hypoxia.     

Ethylene‐dependent aerenchyma formation in adventitious roots is regulated differently in rice and maize

In roots of gramineous plants, lysigenous aerenchyma is created by the death and lysis of cortical cells. Rice (Oryza sativa) constitutively forms aerenchyma under aerobic conditions, and its formation is further induced under oxygen‐deficient conditions. However, maize (Zea mays) develops aerenchyma only under oxygen‐deficient conditions. Ethylene is involved in lysigenous aerenchyma formation. Here, we investigated how ethylene‐dependent aerenchyma formation is differently regulated between rice and maize. For this purpose, in rice, we used the reduced culm number1 (rcn1) mutant, in which ethylene biosynthesis is suppressed. Ethylene is converted from 1‐aminocyclopropane‐1‐carboxylic acid (ACC) by the action of ACC oxidase (ACO). We found that OsACO5 was highly expressed in the wild type, but not in rcn1, under aerobic conditions, suggesting that OsACO5 contributes to aerenchyma formation in aerated rice roots. By contrast, the ACO genes in maize roots were weakly expressed under aerobic conditions, and thus ACC treatment did not effectively induce ethylene production or aerenchyma formation, unlike in rice. Aerenchyma formation in rice roots after the initiation of oxygen‐deficient conditions was faster and greater than that in maize. These results suggest that the difference in aerenchyma formation in rice and maize is due to their different mechanisms for regulating ethylene biosynthesis.     

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.     

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.     

Characterisation of programmed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of maize(Zea mays L.)

Aerenchyma is a tissue type characterised by prominent intercellular spaces which enhance flooding tolerance in some plant species by facilitating gas diffusion between roots and the aerial environment. Aerenchyma in maize roots forms by collapse and death of some of the cortical cells in a process that can be promoted by imposing oxygen shortage or by ethylene treatment. Maize roots grown hydroponically in 3% oxygen, 1 μl l⁻¹ ethylene or 21% oxygen (control) were analysed by a combination of light and electron microscopy. Use of in-situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) suggested internucleosomal cleavage of DNA. However, chromatin condensation detectable by electron microscopy was preceded by cytoplasmic changes including plasma membrane invagination and the formation of vesicles, in contrast to mammalian apoptosis in which chromatin condensation is the first detectable event. Later, cellular condensation, condensation of chromatin and the presence of intact organelles surrounded by membrane resembling apoptotic bodies were observed. All these events were complete before cell wall degradation was apparent. Therefore, aerenchyma formation initiated by hypoxia or ethylene appears to be a form of programmed cell death that shows characteristics in part resembling both apoptosis and cytoplasmic cell death in animal cells. Received: 12 April 2000 / Accepted: 18 June 2000     

Ontogeny and distribution of cell‐wall glycans during aerenchyma formation in roots of Pistia stratiotes (Araceae)

Aerenchyma is widely known to be lysigenous, schizogenous or, more recently, expansigenous. The interpretation and understanding of its function is questionable, given the lack of extensive knowledge on the development and cellular changes of this tissue. The aerenchyma of Pistia stratiotes roots reportedly originates from packet lysigeny. However, our observations suggest schizogenous development. Our objective was to analyse ontogeny of aerenchyma in P. stratiotes roots and evaluate the morphological and chemical changes in the cell wall during the formation of aerenchyma. The aerenchymatous inner cortex of schizogenous origin was observed under light and electron microscopy. Lacunae are formed by the separation, division and stretching of cells, which remain alive until maturity. Analyses using monoclonal anti‐glycan antibodies show that formation of that type of aerenchyma apparently proceeds through the same mechanisms as the genesis of intercellular spaces. However, the greatest changes occur when cells undergo stretching, including the loss of methyl‐esterification and detection of arabinans, which are not directly involved in cell separation. Thus, other factors may account for the formation of schizogenous aerenchyma.     

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     

Rapid changes in cell wall pectic polysaccharides are closely associated with early stages of aerenchyma formation, a spatially localized form of programmed cell death in roots of maize (Zea mays L.) promoted by ethylene

Aerenchyma formation in roots of maize (Zea mays L.) involves programmed death of cortical cells that is promoted by exogenous ethylene (1 µL L⁻¹) or by endogenous ethylene produced in response to external oxygen shortage (3%, v/v). In this study, evidence that degeneration of the cell wall accompanies apoptotic-like changes previously observed in the cytoplasm and nucleus (Gunawardena et al. Planta 212, 205–214, 2001), has been sought by examining de-esterified pectins (revealed by monoclonal antibody JIM 5), and esterified pectins (revealed by monoclonal antibody JIM 7). In controls, de-esterified wall pectins were found at the vertices of triangular junctions between cortical cells (untreated roots). Esterified pectins in control roots were present in the three walls bounding triangular cell-to-cell junctions. After treatment with 3% oxygen or 1 µL L⁻¹ ethylene, this pattern was lost but walls surrounding aerenchyma gas spaces became strongly stained. The results showed that cell wall changes commenced within 0·5 d and evidently were initiated by ethylene in parallel with cytoplasmic and nucleoplasmic events associated with classic intracellular processes of programmed cell death.     

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.     

Lysigenous aerenchyma formation in maize root is confined to cortical cells by regulation of genes related to generation and scavenging of reactive oxygen species

To adapt to waterlogging, maize (Zea mays) forms lysigenous aerenchyma in root cortex as a result of ethylene-promoted programmed cell death (PCD). Respiratory burst oxidase homolog (RBOH) gene encodes a homolog of gp91^phox in NADPH oxidase, and has a role in the generation of reactive oxygen species (ROS). Recently, we found that during aerenchyma formation, RBOH was upregulated in all maize root tissues examined, whereas an ROS scavengingrelated metallothionein (MT) gene was downregulated specifically in cortical cells. Together these changes should lead to high accumulations of ROS in root cortex, thereby inducing PCD for aerenchyma formation. As further evidence of the involvement of ROS in root aerenchyma formation, the PCD was inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Based on these results, we propose a model of cortical cell-specific PCD for root aerenchyma formation.Key words: aerenchyma, ethylene, laser microdissection, maize (Zea mays), metallothionein, programmed cell death, reactive oxygen species, respiratory burst oxidase homologIn both wetland and non-wetland plants, lysigenous aerenchyma is formed in roots by creating gas spaces as a result of death and subsequent lysis of some cortical cells, and allows internal transport of oxygen from shoots to roots under waterlogged soil conditions.^1–3 In rice (Oryza sativa) and some other wetland plant species, lysigenous aerenchyma is constitutively formed under aerobic conditions, and is further enhanced under waterlogged conditions.⁴ On the other hand, in non-wetland plants, including maize (Zea mays), lysigenous aerenchyma does not normally form under well-drained soil conditions, but is induced by waterlogging.⁵ Ethylene is involved in lysigenous aerenchyma formation,^1–3,6,7 but the molecular mechanisms are unclear.We recently identified two reactive oxygen species (ROS)-related genes that were specifically regulated in maize root cortex by waterlogged conditions, but not in the presence of an ethylene perception inhibitor 1-methylcyclopropene (1-MCP).⁵ One was respiratory burst oxidase homolog (RBOH), which has a role in ROS generation and the other was metallothionein (MT), which has a role in ROS scavenging. These results suggest that ROS has a role in ethylene signaling in the PCD that occurs during lysigenous aerenchyma formation.     

Aerenchyma formation in the rice stem and its promotion by H2O2

? Gas spaces (aerenchyma) form as an adaptation to submergence to facilitate gas exchange. In rice (Oryza sativa), aerenchyma develop by cell death and lysis, which are poorly understood at the cellular level. ? Aerenchyma formation was studied in rice stems by light microscopy. It was analyzed in response to submergence, ethylene and hydrogen peroxide (H(2)O(2)) treatment, and in the MT2b::Tos17 mutant. O(2)·(-) was detected with nitroblue tetrazolium and an epinephrine assay. H(2)O(2) was detected with 3,3'-diaminobenzidine. ? Aerenchyma develop constitutively in all internodes of the deep-water rice variety Pin Gaew 56, but are absent from the nodes. Constitutive aerenchyma formation was also observed in two lowland rice varieties, albeit to a lesser degree. A larger number of aerenchyma are present in older internodes, and at the top of each internode, revealing developmental gradients. Submergence or treatment with the ethylene-releasing compound ethephon promoted aerenchyma formation in all genotypes analyzed. Pre-aerenchymal cells contain less starch, no chloroplasts, thinner cell walls and produce elevated levels of O(2)·(-) and H(2)O(2) compared with other parenchymal cells. Ethephon promotes O(2)·(-) formation and H(2)O(2) promotes aerenchyma formation in a dose-dependent manner. Further-more, genetic downregulation of the H(2)O(2) scavenger MT2b enhances aerenchyma formation. ? Aerenchyma formation is mediated by reactive oxygen species.     

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.     

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.     

植物根内通气组织形成的研究进展

植物能否在湿地或淹涝环境中生长,很大程度上取决于植物是否具有健全发达的通气组织。在结合形态学和分子生物学等方面研究的基础上,概述了植物根内通气组织的形成过程,主要涉及生理功能、诱导因子和相关酶等,推测细胞程序性死亡是溶生性通气组织形成的机理,乙烯在整体信号转导网络中起关键性中介作用。     

植物根内通气组织形成机理的研究进展

植物能否在湿地或淹涝环境中生长, 很大程度上取决于植物是否具有健全发达的通气组织。在结合形态学和分子生物学等方面研究的基础上, 概述了植物根内通气组织的形成过程, 主要涉及生理功能、诱导因子和相关酶等, 推测细胞程序性死亡是溶生性通气组织形成的机理, 乙烯在整体信号转导网络中起关键性中介作用。     

Electron microscopy of gas space (aerenchyma) formation in adventitious roots of Zea mays L. subjected to oxygen shortage

This paper examines the ultrastructure of cortical cells in maize root tips during the early stages in lysigenous aerenchyma formation, promoted by oxygen-deficient nutrient solution. The aim was to determine whether changes in fine structure were compatible with oxygen starvation as the primary cause of cell degeneration and death. There was an initial collapse of some cortical cells, indicating loss of turgor, and the cytoplasm became more electron dense. Mitochondria and endoplasmic reticulum appeared normal at this early stage though the tonoplast lost its integrity. Subsequently the cytoplasm became less electron dense than surrounding healthy cells, and underwent further degeneration while the plasmalemma retracted from the cell wall. Cell walls remained unaltered until this stage, but some then became thin and electron transparent. No cells of the stele were found to degenerate. These observations, which do not readily accord with the hypothesis that oxygen starvation was the cause of cell death, are compared with detailed studies of cell degeration in other cell types. An alternative mechanism for the stimulation of cortical cell lysis in poorly oxygenated roots involving the hormone ethylene, is discussed.     

Nitric oxide is essential for the development of aerenchyma in wheat roots under hypoxic stress

In response to flooding/waterlogging, plants develop various anatomical changes including the formation of lysigenous aerenchyma for the delivery of oxygen to roots. Under hypoxia, plants produce high levels of nitric oxide (NO) but the role of this molecule in plant‐adaptive response to hypoxia is not known. Here, we investigated whether ethylene‐induced aerenchyma requires hypoxia‐induced NO. Under hypoxic conditions, wheat roots produced NO apparently via nitrate reductase and scavenging of NO led to a marked reduction in aerenchyma formation. Interestingly, we found that hypoxically induced NO is important for induction of the ethylene biosynthetic genes encoding ACC synthase and ACC oxidase. Hypoxia‐induced NO accelerated production of reactive oxygen species, lipid peroxidation, and protein tyrosine nitration. Other events related to cell death such as increased conductivity, increased cellulase activity, DNA fragmentation, and cytoplasmic streaming occurred under hypoxia, and opposing effects were observed by scavenging NO. The NO scavenger cPTIO (2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide potassium salt) and ethylene biosynthetic inhibitor CoCl₂ both led to reduced induction of genes involved in signal transduction such as phospholipase C, G protein alpha subunit, calcium‐dependent protein kinase family genes CDPK, CDPK2, CDPK 4, Ca‐CAMK, inositol 1,4,5‐trisphosphate 5‐phosphatase 1, and protein kinase suggesting that hypoxically induced NO is essential for the development of aerenchyma.     

Aerenchyma (Gas-space) Formation in Adventitious Roots of Rice (Oryza sativa L.) is not Controlled by Ethylene or Small Partial Pressures of Oxygen

Jackson, M. B., Fenning, T. M., and Jenkins, W. 1985 Aerenchyma(gas-space) formation in adventitious roots of rice (Oryza sativaL.) is not controlled by ethylene or small partial pressuresof oxygen.—J. exp. Bot. 36: 1566–1572. The extent of gas-filled voids (aerenchyma) within the cortexof adventitious roots of vegetative rice plants (Oryza sativaL. cv. RB3) was estimated microscopically from transverse sectionswith the aid of a computer-linked digitizer drawing board. Gas-spacewas detectable in 1-d-old tissue and increased in extent withage. After 7 d, approximately 70% of the cortex had degeneratedto form aerenchyma. The extent of the voids in 1-4-d-old tissuewas not increased by stagnant, poorly-aerated external environmentscharacterized by sub-ambient oxygen partial pressures and accumulationsof carbon dioxide and ethylene. Treatment with small oxygenpartial pressures, or with carbon dioxide or ethylene appliedin vigorously stirred nutrient solution also failed to promotethe formation of cortical gas-space. Furthermore, ethylene productionby rice roots was slowed by small oxygen partial pressures typicalof stagnant conditions. Silver nitrate, an inhibitor of ethylene action, did not retardgas-space formation; similarly when endogenous ethylene productionwas inhibited by the application of aminoethoxyvinylglycine(A VG), aerenchyma development continued unabated. Cobalt chloride,another presumed inhibitor of ethylene biosynthesis, did notimpair formation of the gas in rice roots nor did it decreasethe extent of aerenchyma even if A VG was supplied simultaneously.These results contrast with those obtained earlier using rootsof Zea mays L. We conclude that in rice, aerenchyma forms speedily even inwell-aerated environments as an integral part of ordinary rootdevelopment There seems to be little or no requirement for ethyleneas a stimulus in stagnant root-environments where aerenchymais likely to increase the probability of survival. Key words: Rice (Oryza sativa L.), ethylene, flooding, aeration, aerenchyma, environmental stress