Fucose in the surface deposits of axenic and field grown roots ofZea mays L.

Summary The location of materials containing terminal fucose residues on the surface of axenic and field grown roots of corn has been determined.Binding patterns of FITC-labelled,Lotus purpureus Moench lectin indicate the presence of the fucose residues in the cell walls and mucilage of the peripheral region of the root cap. During development, fucose residues also appear in the outer periclinal walls and overlying mucilage of columnar epidermal cells. Surface material rich in these residues persists between the mature root hairs but is not found on their surface. Fucose-rich mucilage is present on the exposed surface of aerial roots and at the point where they enter the soil. No lectin binding residues are indicated elsewhere in the roots.     

The rhizosphere in Zea: new insight into its structure and development

Some of the nodal roots of field-grown Zea mays L. bear a persistent soil sheath along their entire length underground except for a glistening white soil-free zone which extends approximately 25 mm behind the root cap. These roots are generally unbranched. The histology of the surface and the rhizosphere of the sheathed roots has been examined by correlated light and electron microscopy. All mature peripheral tissues including root hairs, are largely intact and apparently alive where enclosed by the soil sheath. The sheath is permeated by extracellular mucilage which is histochemically distinct from the mucilage at the epidermal surface, but similar to that produced by the root cap. Isolated cells resembling those sloughed from the sides of the root cap persist in the soil sheath along the length of these roots. Fresh whole mounts of the sheath show that these detached cells may be alive and streaming vigorously even at some distance from the root cap. Rhizosphere mucilage is associated with the isolated cells.To whom correspondence should be addressed     

Epidermal derivatives as xylem elements and transfer cells: a study of the host-parasite interface in two species of Triphysaria (Scrophulariaceae)

Summary Haustoria ofTriphysaria pusilla andT. versicolor subsp.faucibarbata from a natural habitat were analysed by light and electron microscopy. The keel-shaped edge of the secondary haustorium generally splits the epidermis and cortex of the host root parallel to the root axis, and penetrates to the host vascular tissue. Anticlinally elongated epidermal cells of the haustorium constitute most of the host/parasite interface. Some of these epidermal cells are divided by oblique cell walls. Some of their oblique daughter cells as well as some undivided epidermal cells differentiate into xylem elements. Single epidermal cells occasionally intrude into the vascular tissue of the host and individual host cells can be invaded. The surface area of the plasmalemma in parasitic parenchymatous interface cells is increased by the differentiation of wall labyrinths characteristic of transfer cells and by the development of membrane-lined cytoplasmic tubules or flattened sacs which become embedded in the partly lignified interface cell-wall. Mycorrhizal fungal hyphae enter the xylem bridge in some haustoria. Implications of these observations for the function of the haustorium are discussed.     

抱茎独行菜MUM4基因的克隆及分析

抱茎独行菜(Lepidium perfoliatum L.)为十字花科具典型粘液繁殖体植物,为探究该植物中种皮粘液质基因（MUCILAGE-MODIFIED4,MUM4,该基因在拟南芥中编码NDP-L-鼠李糖合成酶)的功能,通过生物信息学分析设计引物克隆得到抱茎独行菜MUM4基因,命名为LpMUM4。同源比对分析结果表明,LpMUM4与拟南芥AtMUM4基因具有很高的一致性。qRT-PCR结果表明,该基因在抱茎独行菜各组织中均有表达,在角果和根中的表达量最高,且其表达量随角果的发育表现出渐强的趋势。免疫组织化学定位分析表明,LpMUM4基因于角果发育的早期阶段在内珠被和外珠被都有表达,而在外珠被的表皮和亚表皮中表达量更高,至角果发育的最后阶段,其表达集中于表皮和亚表皮层,这可能与抱茎独行菜的外珠被发育成种皮及粘液质的生成有关。将LpMUM4基因转化拟南芥,该基因的过表达对位于粘液质合成途径中的上游基因AtTTG1具有显著的抑制作用。表型比对观察显示,转基因拟南芥与其野生型植株形态无显著差异,这可能是因为抱茎独行菜种皮的发育和粘液质的形成是一个多基因调控的复杂过程,某一基因的过表达或许不会引起明显的表型变化。     

Movement of Endogenous Calcium in the Elongating Zone of Graviresponding Roots of Zea mays

Endogenous calcium (Ca) accumulates along the lower side ofthe elongating zone of horizontally oriented roots of Zea mayscv. Yellow Dent. This accumulation of Ca correlates positivelywith the onset of gravicurvature, and occurs in the cytoplasm,cell walls and mucilage of epidermal cells. Corresponding changesin endogenous Ca do not occur in cortical cells of the elongatingzone of intact roots. These results indicate that the calciumasymmetries associated with root gravicurvature occur in theoutermost layers of the root Calcium, corn, gravitropism (root), Zea mays     

Cellular re-distribution of flavin-containing polyamine oxidase in differentiating root and mesocotyl of <Emphasis Type="Italic">Zea mays</Emphasis> L. seedlings

Plant polyamine oxidases (PAOs; EC 1.5.3.11) are hydrogen peroxide-producing enzymes supposedly involved in cell-wall differentiation processes and defence responses. Maize (Zea mays L.) PAO (MPAO) is a 53 kDa secretory glycoprotein, abundant in primary and secondary cell walls of several tissues. Using biochemical, histochemical, ultrastructural and immunocytochemical techniques, the distribution and sub-cellular compartmentalisation of MPAO in the primary root and mesocotyl of seedlings at different maturation stages or after growth under varying light conditions were analysed. In apical root tissues, MPAO immunoreactivity was mainly detected in the cytoplasmic compartment, while a lower immunoreactivity was observed in the cell walls. In the more mature, basal part of the root, intense immunogold labelling was found in the primary and secondary walls of protoxylem precursors and vessels, while endodermal cells and living metaxylem precursors were immunopositive both in their walls and in their thin cytoplasmic compartments. A re-distribution of MPAO protein from the cytoplasm toward the primary and secondary walls was also recognised when immunoreactivity of basal root tissues from 3-day-old seedlings was compared with that detected in 11-day-old tissues. Accordingly, biochemical analyses revealed MPAO entrapment in the extracellular matrix of mature tissues. In the mesocotyl, an enrichment of MPAO immunolabelling in the cell wall of protoxylem, metaxylem and epidermal tissues, as a function of light exposure, was observed. Taken together, these data support the hypothesised role of PAOs in cell-wall maturation. Moreover, the relevant intraprotoplasmic MPAO localisation observed mainly in differentiating root tissues suggests an additional role in intracellular production of hydrogen peroxide.Alessandra Cona and Sandra Moreno have contributed equally to this paper.     

A new approach for the quantification of root-cap mucilage exudation in the soil

Direct evidence on the functions of root-cap mucilage during plant root growth in soil is limited mainly due to the lack of a method for in situ measurements. In this paper, we offer a method that facilitates the measurement of mucilage exudation when roots are growing in soil. We observed the mucilage exudation directly through a transparent panel located on the side of a root box in which plant roots were growing. We used a CCD camera attached to a microscope to observe and record mucilage exudation. Using image analysis, the activity of mucilage exudation was evaluated based on the area occupied by the mucilage on the root tip. The area of mucilage observed on the root tips after 1-h growth in soil corresponded with the weight of mucilage that was originally observed on the tips before they were transplanted. This relationship suggests that the observed area on root tip relates to total exudation. The area of mucilage exudation on the root tips was high (0.48 mm²) at night and low (0.35 mm²) at midday, suggesting that the activity of mucilage exudation follows diurnal changes. Furthermore, the mucilage exudation positively correlated with the root elongation rate, implying that fast-growing roots exude more mucilage.     

The expansion of maize root-cap mucilage during hydration. 1. Kinetics

The conventional view of root-cap mucilage as an expanded blob of mucilage is characteristic only of root tips in contact with free water. In soil, the mucilage is almost always a dry coating over the tip to which soil particles adhere. The kinetics of expansion of root-cap mucilage of Zea mays roots grown in field soil, in soil in pots, and axenically on agar, were determined when the mucilage was exposed to water. On the soil-grown roots the increase in mucilage volume was linear with time, sometimes reaching a constant volume during the 6 h of measurement, but sometimes not. This linear expansion is interpreted as limited by the rate at which the condensed mucilage in the periplasmic and intercellular spaces of the root cap passes to the exterior of the cap, expanding as fast as it arrives outside in the water. The height of the plateau is interpreted as a measure of the amount of mucilage initially present in the interior spaces. Because of the greater availability of water in the axenic roots grown on 1% agar, the mucilage was already outside the root cap, and it expanded more rapidly. It reached a final volume about 10-fold greater than that on the soil-grown roots. The volume increase was curvilinear with time. An analysis of these curves suggested that this swelling on axenic roots was a diffusion of mucilage outwards from the flanks of the root cap, and the diffusivity of the mucilage was estimated as 4 × 10^?8 cm² s^?1. The molecular radius derived from this diffusivity was 34 nm, and the estimated molecular weight was 1.6 × 10⁸ Da.     

A Possible Role for the Thick-walled Epidermal Cells in the Mycorrhizal Hair Roots of Lysinema ciliatum R. Br. and other Epacridaceae

Hair roots ofLysinema ciliatum R. Br. and some other Epacridaceaehave thick-walled cells in the epidermis. These are preferentiallycolonized with mycorrhizal fungi. Individual epidermal cellscontaining hyphal coils separate at the middle lamella and arereleased into the soil. Other colonized cells remain attachedto the roots, usually in groups, surrounded by bare exodermis,where epidermal cells have either collapsed or been sloughedoff. It is suggested that these colonized thick walled cellscan serve to prolong the mycorrhizal association and to infectnew hair roots as these emerge. The thick wall has a very specializedstructure and composition and could have a number of roles,either acting as a substrate or protective coat or in controllingwater status and uptake. Young hair-roots are surrounded bya mucilage sheath that is similar in appearance to that in Ericaceaeand apparently produced by root cap cells, not the epidermis. Lysinema ciliatum R. Br.; ericoid mycorrhiza; hair root; root cap; cortex; epidermis; exodermis     

Plant and bacterial mucilages of the maize rhizosphere: Comparison of their soil binding properties and histochemistry in a model system

Mucilages from the root tips of axenically-grown maize and from a bacterium (Cytophaga sp.) isolated from the rhizosheaths of field-grown roots, were immobilized by drying onto nylon blotting membrane. The mucilage plaques remained in place through repeated rewettings and histochemical treatments. Staining of the plaques showed that both mucilages included acidic groups, and 1,2 diols (the latter notably fewer in bacterial mucilage). Bacterial mucilage plaques stained strongly for protein, plant mucilage was unstained. Plaques of both mucilages bound soil particles strongly if soil was applied to wet mucilage and then dried. Bound soil was not lost with rewetting. Dry weight and densitometer measurements showed that bacterial mucilage bound about 10% more soil than the same surface area of root-cap mucilage. Pretreatment of plaques with periodate oxidation eliminated most soil binding by root-cap mucilage but this was completely reversible by reduction with borohydride. Soil binding to bacterial mucilage was unaffected by periodate but much diminished by borohydride pretreatment (partially restored by subsequent oxidation). Neither pretreatment with cationic dyes nor preincubation in pectinase, pectin methylesterase or protease affected subsequent soil binding by the mucilage plaques. Pretreatment of root-cap mucilage plaques with lectins specific for component sugars also did not alter soil binding. It is concluded that mucilages of both plant and bacterial origin can contribute to the adhesion and cohesion of maize rhizosheaths, but each by a different mechanism. Binding by root-cap mucilage depends on 1,2 diol groups of component sugars, that of bacterial mucilage does not, and is likely to be protein mediated. ei]Section editor: R O D Dixon     

Role of cell-wall biogenesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a growth-limiting protein induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.Abbreviations CHI cycloheximide - COR cordycepin - DCB 2,6-dichlorobenzonitrile - GLP growth-limiting protein(s) - IAA indole-3-acetic acid     

The FEI2-SOS5 pathway and CELLULOSE SYNTHASE 5 are required for cellulose biosynthesis in the Arabidopsis seed coat and affect pectin mucilage structure

A common adaptation in angiosperms is the deposition of hydrophilic mucilage into the apoplast of seed coat epidermal cells during the course of their differentiation. Upon imbibition, seed mucilage, composed mainly of carbohydrates (i.e. pectins, hemicelluloses and glycans) expands rapidly, encapsulating the seed and aiding in seed dispersal and germination. The FEI1/FEI2 receptor-like kinases and the SOS5 extracellular GPI-anchored protein were previously shown to act on a pathway regulating cellulose biosynthesis during Arabidopsis root elongation. In the highlighted study, we demonstrated that FEI2 and SOS5 regulate the production of the cellulosic rays deposited across the inner adherent-layer of seed mucilage. Mutations in either fei2 or sos5 disrupted the formation of rays, which was associated with an increase in the soluble, outer layer of pectin mucilage and accompanied by a reduction in the inner adherent-layer. Mutations in CELLULOSE SYNTHASE 5 also led to reduced rays and mal-partitioning of the pectic component of seed mucilage, further establishing a structural role for cellulose in seed mucilage. Here, we show that FEI2 expressed from a CaMV 35S promoter complemented both root and seed mucilage defects of the fei1 fei2 double mutant. In contrast, expression of FEI1 from a 35S promoter complemented the root, but not the seed phenotype of the fei1 fei2 double mutant, suggesting that unlike in the root, FEI2 plays a unique and non-redundant role in the regulation of cellulose synthesis in seed mucilage. Altogether, these data suggest a novel role for cellulose in anchoring the pectic component of seed mucilage to the seed surface and indicate that the FEI2 protein has a function distinct from that of FEI1, despite the high sequence similarity of these RLKs.     

Comparison of maize root mucilages isolated from root exudates and root surface extracts by complementary cytological and biochemical investigations

Summary A strategy to obtain fractions enriched in mucilages secreted by root caps or produced by the rhizodermis of axenicallygrown maize seedlings is proposed. It involves a two-step procedure allowing the successive collection of root exudates and surface extracts from the same set of intact, sterile maize plants. Cytological controls were performed at each phase of collection. Whereas root cap mucilage is easily collected in water after one day's extraction, under conditions favouring secretory activity, rhizodermal mucilage remains tightly adherent to the root surface. It can be better extracted using neutral saline buffer assisted by gentle shaking at low temperature. Acidic saline buffer is unsuitable as it induces cell lysis and release of cell wall components.Biochemical analyses confirm that fractions enriched in root cap mucilage contain very high levels of fucose and galactose, high levels of arabinose, xylose and glucose and trace amounts of mannose. Fractions enriched in rhizodermal mucilage contain large amounts of glucose, moderate amounts of arabinose, xylose, mannose and galactose and trace levels of fucose. Isoelectric focusing and SDS-PAGE indicate that there are numerous similarities in the protein composition of materials enriched in root cap mucilages from root exudates or aqueous root surface extracts. However, specific protein bands that could be characteristic of rhizodermal mucilage are obtained using neutral saline buffer extracts. According to these biochemical data, the two-step procedure used in the present study appears to be useful for further biochemical characterization of both types of mucilages.Abbreviations BSA bovine serum albumin - BSTFA N,O-bis (trimethylsilyl)-trifluoroacetamide - DTT dithiothreitol - i. d. internal diameter - MW molecular weight - PATAg periodic acid-thiosemicarbazide-silver proteinate - PVPP polyvinylpolypyrrolidone - RE root exudates - RSE root surface extracts - TMCS trimethylchlorosilane - TMS trimethylsilyl     

SEM-Untersuchungen an myxospermen Diasporen

The epidermal structure of mucilage producing seeds and fruits before and after contact with water is studied. Main emphasis is laid on the elucidation of structural peculiarities of the dessicated mucilaginous substances.

     

Composition of Root Mucilage Polysaccharides from Lepidium sativum

Root mucilage polysaccharides were recovered from roots of 3-d-oldcress seedlings by washing with water, followed by ethanol precipitationof the high molecular weight material. The redissolved polysaccharidewas fractionated by combined gel filtration chromatography onBiogel A50 and ion exchange chromatography on DEAE-Sepharose/DEAE-Trisacrylinto four heterogeneous fractions. The fractions could be assignedto two groups based on monosaccharide composition and behaviourduring ion-exchange chromatography. Group One polysaccharidescontained fucose as the major 6-deoxyhexose and were low inuronic acid, not binding to the ion-exchange column. Group Twopolysaccharides contained rhamnose as the major 6-deoxyhexoseand were uronic acid rich. It is suggested that these representroot cap and root epidermal mucilage components respectively. Key words: Root mucilage, recognition, polysaccharides     

Defective Secretion of Mucilage is the Cellular Basis for Agravitropism in Primary Roots of Zea mays cv. Ageotropic

Root caps of primary, secondary, and seminal roots of Z. mayscv. Kys secrete large amounts of mucilage and are in close contactwith the root all along the root apex. These roots are stronglygraviresponsive. Secondary and seminal roots of Z. mays cv.Ageotropic are also strongly graviresponsive. Similarly, theircaps secrete mucilage and closely appress the root all alongthe root apex. However, primary roots of Z. mays cv. Ageotropicare non-responsive to gravity. Their caps secrete negligibleamounts of mucilage and contact the root only at the extremeapex of the root along the calyptrogen. These roots become graviresponsivewhen their tips are coated with mucilage or mucilage-like materials.Peripheral cells of root caps of roots of Z. mays cv. Kys containmany dictyosomes associated with vesicles that migrate to andfuse with the plasmalemma. Root-cap cells of secondary and seminal(i.e. graviresponsive) roots of Z. mays cv. Ageotropic are similarto those of primary roots of Z. mays cv. Kys. However, root-capcells of primary (i.e. non-graviresponsive) roots of Z. mayscv. Ageotropic have distended dictyosomal cisternae filled withan electron-dense, granular material. Large vesicles full ofthis material populate the cells and apparently do not fusewith the plasmalemma. Taken together, these results suggestthat non-graviresponsiveness of primary roots of Z. mays cv.Ageotropic results from the lack of apoplastic continuity betweenthe root and the periphery of the root cap. This is a resultof negligible secretion of mucilage by cells along the edgeof the root cap which, in turn, appears to be due to the malfunctioningof dictyosomes in these cells. Corn, dictyosomes, mucilage, root gravitropism, Zea mays cv. Ageotropic, Zea mays cv. Kys     

Understanding polysaccharide production and properties using seed coat mutants: future perspectives for the exploitation of natural variants

Background

The epidermal cells of the seed coat of certain species accumulate polysaccharides during seed development for cell wall reinforcement or release on imbibition to form mucilage. Seed-coat epidermal cells show natural variation in their structure and mucilage production, which could explain the diverse ecophysiological roles proposed for the latter. Arabidopsis mucilage mutants have proved to be an important tool for the identification of genes involved in the production of seed-coat polysaccharides.

Scope

This review documents genes that have been characterized as playing a role in the differentiation of the epidermal cells of the arabidopsis seed coat, the natural variability in polysaccharide features of these cells and the physiological roles attributed to seed mucilage.

Conclusions

Seed-coat epidermal cells are an excellent model for the study of polysaccharide metabolism and properties. Intra- and interspecies natural variation in the differentiation of these epidermal cells is an under-exploited resource for such studies and promises to play an important part in improving our knowledge of polysaccharide production and ecophysiological function.     

Cell surface inCalluna vulgaris L. hair roots

Summary Calluna vulgaris possesses small roots called hair roots, which in natural conditions are colonized by symbiotic mycorrhizal fungi. A specialized cell surface-consisting of the cell wall and the overlaying mucilage-has been hypothesized to be important for the establishment of ericoid mycorrhizae. In this work the cell surface of hair roots of plants growing in sterile conditions has been characterized by using in situ techniques, integrated when possible, by biochemical analysis. The mucilage is abundant around the apex, while it becomes thinner and thinner on the differentiated parts. Sugar residues such as mannose, glucose and galactose are regularly distributed along the whole root length, while N-acetylglucosamine residues are limited to the differentiated part of the hair root. Cellobiohydrolase-gold complex, used to reveal -1, 4-glucans, regularly labels mucilage and cell walls of apical and differentiated regions. Polygalacturonic acids revealed by monoclonal antibodies are found at the surface of the cap cells and on the cell walls of the inner tissues in the differentiated zones, but never at the surface of the epidermal cells.The labeling continuity between mucilage and cell walls demonstrates that some molecules such as -1, 4-glucans are common to the two compartments, but probably have a different status of aggregation. On the contrary, other molecules, such as N-acetylglucosamine or polygalacturonic acid display a precise pattern of localization following root differentiation.Abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - Con A concanavalin A - RCA₁₂₀ Ricinus communis agglutinin - UEA Ulex europaeus agglutinin - CBH I cellobiohydrolase I - TEM transmission electron microscopy - MeNH₂ methylamine - PATAg periodic acid-thiocarbohydrazide-silver proteinate reaction - PAS periodic acid-Schiff reaction     

Purification effects show seed and root mucilage's ability to respond to changing rhizosphere conditions

Mucilage, a polysaccharide-containing hydrogel, is hypothesized to play a key role in the rhizosphere as a self-organized system because it may vary its supramolecular structure with changes in the surrounding solution. However, there is currently limited research on how these changes are reflected in the physical properties of real mucilage. This study examines the role of solutes in maize root, wheat root, chia seed, and flax seed mucilage in relation to their physical properties. Two purification methods, dialysis and ethanol precipitation, were applied to determine the purification yield, cation content, pH, electrical conductivity, surface tension, viscosity, transverse ¹H relaxation time, and contact angle after drying of mucilage before and after purification. The two seed mucilage types contain more polar polymers that are connected to larger assemblies via multivalent cation crosslinks, resulting in a denser network. This is reflected in higher viscosity and water retention ability compared to root mucilage. Seed mucilage also contains fewer surfactants, making them better wettable after drying compared to the two root mucilage types. The root mucilage types, on the other hand, contain smaller polymers or polymer assemblies and become less wettable after drying. However, wettability not only depends on the amount of surfactants but also on their mobility, as well as the strength and mesh size of the network structure. The changes in physical properties and cation composition observed after ethanol precipitation and dialysis suggest that the polymer network of seed mucilage is more stable and specialized in protecting the seeds from unfavorable environmental conditions. In contrast, root mucilage is characterized by fewer cationic interactions and its network relies more on hydrophobic interactions. This allows root mucilage to be more flexible in responding to changing environmental conditions, facilitating nutrient and water exchange between root surfaces and the rhizosphere soil.     

Cell wall peroxidase activity,hydrogen peroxide level and NaCl-inhibited root growth of rice seedlings

The changes in cell-wall peroxidase (POD) activity and H₂O₂ level in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mM progressively reduced root growth and increased ionically bound cell-wall POD activity. NaCl had no effect on covalently bound cell-wall POD activities. The reduction of root growth by NaCl is closely correlated with the increase in H₂O₂ level. Exogenous H₂O₂ was found to inhibit root growth of rice seedlings. Since ammonium and proline accumulation are associated with root growth inhibition caused by NaCl, we determined the effects of NH₄Cl or proline on root growth, cell-wall POD activity and H₂O₂level in roots. External application of NH₄Cl or proline markedly inhibited root growth, increased cell-wall POD activity and increased H₂O₂ level in roots of rice seedlings in the absence of NaCl. An increase in cell-wall POD activity and H₂O₂ level preceded inhibition of root growth caused by NaCl, NH₄Cl or proline. NaCl or proline treatment also increased NADH-POD and diamine oxidase (DAO) activities in roots of rice seedlings, suggesting that NADH-POD and DAO contribute to the H₂O₂ generation in the cell wall of NaCl- or proline-treated roots. NH₄Cl treatment increased NADH-POD activity but had no effect on DAO activity, suggesting that NADH-POD but not DAO is responsible for H₂O₂ generation in cell wall of NH₄Cl-treated roots.