ROLE OF CELLULASE IN AERENCHYMA DEVELOPMENT IN SUNFLOWER

A hypothesis that ethylene causes aerenchyma development in waterlogged plants through increased cellulase activity was tested with sunflower, Helianthus annuus L. Treatment with commercial cellulase induced aerenchyma development in sunflower stem sections. Some of the cellulase-treated cortical cells enlarged radially and some disintegrated, leading to intercellular space. Cell disintegration started with progressive plasmolysis and severe plasmolysis was associated with or was apparently followed by cell wall breakdown. Localized stem treatment of an intact sunflower with ethylene increased cellulase activity in that part of the stem. Localized stem treatment of an intact sunflower with a water jacket increased cellulase activity in that part of the stem. When the lower part of the sunflower was waterlogged, the cellulase activity in the waterlogged stem increased. Present and earlier results suggest that aerenchyma development is a plant adaptation to waterlogging conditions. The deficiency of oxygen in a waterlogged plant triggers the anaerobic stimulation of ethylene production, which causes an increase in cellulase activity leading to aerenchyma development and enhancing the transport of oxygen to the roots. It is proposed that there is competition between neighboring cortical cells for water after an increase in cell wall plasticity by the action of cellulase. The competition causes progressive plasmolysis and eventual disintegration of weaker cells.     

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.     

Interaction of red to far red light ratio and ethylene in regulating stem elongation of <Emphasis Type="Italic">Helianthus annuus</Emphasis>

Sunflower (Helianthus annuus L.) stems showed increased elongation under two types of vegetative shade: canopy shade (low red to far red [R/FR] ratio) and neighbouring proximity shade (FR enrichment). Hypocotyls also elongated more under narrow-band FR light than under narrow-band R light. Ethylene levels were determined in actively elongating 7-day-old hypocotyls and 17-day-old internodes under three R/FR ratios. Ethylene levels were lower in both sunflower hypocotyls and internodes when the R/FR ratio was reduced. Both FR enrichment of normal R/FR ratio and narrow-band FR light with very low light irradiance resulted in reduction in ethylene levels in 7-day-old hypocotyls. Further, in application experiments, sunflower stems grown under low R/FR ratio were more sensitive to ethephon and less sensitive to aminoethoxyvinylglycine (AVG) than stems grown under high R/FR ratio. Low R/FR ratio appears to initiate reduction in ethylene levels in sunflower seedlings, allowing maximum stem elongation. These results, and findings of other authors, suggest that various plant species may have developed different ways of regulating stem elongation and ethylene levels in response to low R/FR ratio.     

Gravitropism in Higher Plant Shoots : IV. Further Studies on Participation of Ethylene

Ethylene at 1.0 and 10.0 cubic centimeters per cubic meter decreased the rate of gravitropic bending in stems of cocklebur (Xanthium strumarium L.) and tomato (Lycopersicon esculentum Mill), but 0.1 cubic centimeter per cubic meter ethylene had little effect. Treating cocklebur plants with 1.0 millimolar aminoethoxyvinylglycine (AVG) (ethylene synthesis inhibitor) delayed stem bending compared with controls, but adding 0.1 cubic centimeter per cubic meter ethylene in the surrounding atmosphere (or applying 0.1% ethephon solution) partially restored the rate of bending of AVG-treated plants. Ethylene increases in bending stems, and AVG inhibits this. Virtually all newly synthesized ethylene appeared in bottom halves of horizontal stems, where ethylene concentrations were as much as 100 times those in upright stems or in top halves of horizontal stems. This was especially true when horizontal stems were physically restrained from bending. Ethylene might promote cell elongation in bottom tissues of a horizontal stem or indicate other factors there (e.g. a large amount of `functioning' auxin). Or top and bottom tissues may become differentially sensitive to ethylene. Auxin applied to one side of a vertical stem caused extreme bending away from that side; gibberellic acid, kinetin, and abscisic acid were without effect. Acidic ethephon solutions applied to one side of young seedlings of cocklebur, tomato, sunflower (Helianthus annuus L.), and soybean (Glycine max [L.] Merr.) caused bending away from that side, but neutral ethephon solutions did not cause bending. Buffered or unbuffered acid (HCl) caused similar bending. Neutral ethephon solutions produced typical ethylene symptoms (i.e. epinasty, inhibition of stem elongation). HCl or acidic ethephon applied to the top of horizontal stems caused downward bending, but these substances applied to the bottom of such stems inhibited growth and upward bending—an unexpected result.     

The effect of waterlogging on the growth and ethylene content of Eucalyptus robusta Sm. (Swamp Mahogany)

Summary When waterlogged over a period of 80 days plants of Eucalyptus robusta Sm. showed symptoms of leaf chlorosis, epinasty and premature abscission, reduction of stem elongation, stem hypertrophy and formation of adventitious shoots; chlorophyll content was reduced and soluble protein content of the upper leaves increased. Waterlogging doubled the rate of release of ethylene from roots and stems within 6 days, but had no effect on the ethylene concentration of leaves.     

Phenotypic plasticity of stem elongation in two ecotypes of Stellaria longipes: the role of ethylene and response to wind

Using two ecotypes of Stellaria longipes an alpine form with low plasticity and a prairie form with high plasticity, we investigated whether ethylene was involved in the response to wind stress and might be important in controlling plasticity of stem elongation. Stem growth inhibition was positively correlated with concentration of ethephon application and elevation in ambient ethylene in alpine ecotypes, whereas stem growth in prairie plants was stimulated by low ethephon concentrations. When treated with high AVG, the effects were reversed: alpine plant growth was promoted and prairie plant growth was inhibited. Prairie plants exhibited a daily rhythm in ethylene evolution which increased and peaked at 1500 h, and which was absent in alpine plants. Ethylene evolution did not change significantly during the first 2 weeks of growth in alpine plants, whereas ethylene in prairie plants increased significantly during periods of rapid stem elongation. Wind treatment inhibited growth in both ecotypes, but only alpine plants showed a recovery of growth to control levels when wind stressed plants were pretreated with STS. In addition, only alpine plants showed an increase in ethylene evolution in response to wind simulation, whereas prairie plant ethylene evolution did not deviate from rhythms observed in unstressed plants. We concluded that ethylene dwarfs stems in alpine S. longipes in response to wind stress. However, low levels of ethylene may stimulate growth in prairie ecotypes and act independently of wind stress intensity. The contrasting ability to synthesize and respond to ethylene can account for part of the difference in plasticity documented between the two ecotypes.     

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.     

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     

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.     

Role of Ethylene in Induction of Flooding Damage in Sunflower

The possibility that symptoms of flooding damage in plants are primarily caused by an accumulation of ethylene was investigated using pot-grown sunflower (Helianthus annuus) plants. When plants were flooded to the basal pairs of leaves, ethylene in roots and stems below the water line began to increase. This coincided with the start of hypocotyl hypertrophy and new root formation in hypocotyls, which continued for 14-16 days. There were highly significant correlations between ethylene concentration and number of roots and hypocotyl diameter. After approximately 4 days of flooding, ethylene concentrations in stems between nodes for the 1st and 3rd basal pairs of leaves started to increase, coinciding with initiation of chlorophyll breakdown and epinasty of the 2nd basal pairs of leaves. Thus, there were correlations between ethylene concentration and chlorophyll breakdown and epinasty. The lower the leaves, the more chlorophyll breakdown among 1st, 2nd, 3rd, and 4th basal pairs of leaves. The longer the flooding, the more severe the flooding damage; and even when returned to normal condition, plants flooded longer than 3 days were not able to recover from flooding damage. A gas chromatographic study revealed that Ethephon was absorbed by roots and decomposed to ethylene in the plant. Damage symptoms caused by soil application of Ethephon, such as reduced stem height, chlorophyll breakdown, epinasty of the 2nd basal pairs of leaves, and hypocotyl hypertrophy, were almost identical with those caused by soil flooding treatment. Microscopic studies revealed that radially enlarged cells and increased intercellular spaces in the cortex were the major contribution to the increased hypocotyl diameter in both flooded and Ethephon-treated plants. It is concluded that the increase in ethylene concentration in flooded plants is largely, although not exclusively, responsible for flooding damage symptoms.     

Heterotrophic nitrogen fixation (acetylene reduction) associated to flooded rice: A modified measurement technique in the field

A modifiedin situ technique for measuring heterotrophic nitrogen fixing (acetylene reducing) activity associated to rice is proposed. Ethylene evolution rates measured in opaque cylinders covering the stems of rice plants which have been cut 10 cm over the water level were found independent of the diurnal cycle. Cutting of the leaves resulted in decreased variation between plants and suppression of the acceleration of ethylene evolution rate after 12 h incubation as compared to intact plants. In both systems ethylene evolved was swept by a current of methane and the molar ratio between methane and ethylene was stabilized after 12 h. Methane evolution rates remained stable during 12 h and more than 24 h in whole plants and cut plants respectively. It is suggested that alteration in the active gas transport system after 12 h incubation under 10% acetylene may lead to erroneous evaluation of the actual ethylene production in the root's environment. The average values of ethylene evolution rates by cut plants between 12 and 24 h of incubation may be used for comparative studies of nitrogen fixing activity associated to flooded rice.     

The Never ripe Mutant Provides Evidence That Tumor-Induced Ethylene Controls the Morphogenesis of Agrobacterium tumefaciens-Induced Crown Galls on Tomato Stems

We confirm the hypothesis that Agrobacterium tumefaciens-induced galls produce ethylene that controls vessel differentiation in the host stem of tomato (Lycopersicon esculentum Mill.). Using an ethylene-insensitive mutant, Never ripe (Nr), and its isogenic wild-type parent we show that infection by A. tumefaciens results in high rates of ethylene evolution from the developing crown galls. Ethylene evolution from isolated internodes carrying galls was up to 50-fold greater than from isolated internodes of control plants when measured 21 and 28 d after infection. Tumor-induced ethylene substantially decreased vessel diameter in the host tissues beside the tumor in wild-type stems but had a very limited effect in the Nr stems. Ethylene promoted the typical unorganized callus shape of the gall, which maximized the tumor surface in wild-type stems, whereas the galls on the Nr stems had a smooth surface. The combination of decreased vessel diameter in the host and increased tumor surface ensured water-supply priority to the growing gall over the host shoot. These results indicate that in addition to the well-defined roles of auxin and cytokinin, there is a critical role for ethylene in determining crown-gall morphogenesis.     

Plant growth, nutrient acquisition and mycorrhizal symbioses of a waterlogging tolerant legume (Lotus glaber Mill.) in a saline-sodic soil

Seedlings of Lotus glaberMill., were grown in a native saline-sodic soil in a greenhouse for 50 days and then subjected to waterlogging for an additional period of 40 days. The effect of soil waterlogging was evaluated by measuring plant growth allocation, mineral nutrition and soil chemical properties. Rhizobiumnodules and mycorrhizal colonisation in L. glaberroots were measured before and after waterlogging. Compared to control plants, waterlogged plants had decreased root/shoot ratio, lower number of stems per plant, lower specific root length and less allocation of P and N to roots. Waterlogged plants showed increased N and P concentrations in plant tissues, larger root crown diameter and longer internodes. Available N and P and organic P, pH and amorphous iron increased in waterlogged soil, but total N, EC and exchangeable sodium were not changed. Soil waterlogging decreased root length colonised by arbuscular mycorrhizal (AM) fungi, arbuscular colonisation and number of entry points per unit of root length colonised. Waterlogging also increased vesicle colonisation and Rhizobium nodules on roots. AM fungal spore density was lower at the end of the experiment in non-waterlogged soil but was not reduced under waterlogging. The results indicate that L. glaber can grow, become nodulated by Rhizobium and colonised by mycorrhizas under waterlogged condition. The responses of L. glaber may be related its ability to form aerenchyma.     

Production of ethylene by gravistimulation: a potential problem with the interpretation of data from some experimental techniques

Abstract. Gravistimulation was investigated as a potential and unwanted component in the interpretation of physiological investigations on plants. Using both seedlings and mature sunflower plants, two situations are described where gravistimulation contributes significantly to the outcome of an experiment not initially designed to include this parameter. The number of adventitious roots formed in derooted seedings decreased when the tops of the plants were allowed to bend over under their own weight, and the effect correlated positively with the rate of ethylene production by non-vertical stems. In droughted mature plants, and increase in leaf and stem ethylene caused by water stress was supplemented by additional ethylene produced in the lower halves of stems. Drought had caused these stem tissues to wilt, become gravistimulated, and thus produce more ethylene. Other situations in which gravistimulated ethylene production and its physiological consequences are likely to be complicating factors in experiments are discussed. It is concluded that procedures that unnecessarily place experimental material in non-vertical orientations should be avoided.     

Ethylene production and ACC-accumulation in Agrobacterium tumefaciens-induced plant tumours and their impact on tumour and host stem structure and function

Ethylene emission from wild-type Agrobacterium tumefaciens (C58)-induced stem tumours of Ricinus communis was continuously measured with two different methods, process gas chromatography and photo-acoustic spectrometry. Ethylene production was as high as 700 pmol g FW^–1 h^–1, namely 140 times greater than emitted by non-tumourized control stems. It was highest in 5-week-old tumours, independent of light, depressed by anoxia and, during water deficit it was stimulated by rewatering. A remarkable concomitant CO-production was discovered. Accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC), the substrate of ACC-oxidase, preceded ethylene emission with a maximum 2 weeks after tumour induction. Simultaneously, the xylem in the tumour-adjacent host stem underwent drastic changes: it increased two to three times in thickness, vessel diameters decreased, the rays remained unlignified and became multiseriate. With increasing emission of ethylene aerenchyma developed in the non-transformed, tumour-surrounding tissue that formerly was stem cortex. Cotyledons reacted with epinastic symptoms indicating induction of senescence. The present results reveal an important role of ethylene, in addition to cytokinin and auxin, for the differentiation and physiology of A. tumefaciens-induced tumours.     

Ethylene and the Regulation of Apple Stem Growth under Stress

Bending stree resulted in an increase in the ethylene concentration in the internal atmosphere of apple stem (Malus domestica Borkh. cv. Winesap). reaching a maximum at about 2 days after bending. The rise in ethylene content was followed by a depression of growth at about 14–21 days. Ethylene content returned to control levels after about 3 weeks. Application of a past naphthaleneacetic acit caused a similar increase in ethylene levels, and the application of ethephon pastes brought about an inhibition of elongation growth. Whereas stress treatment resulted in an inhibition of growth in stem diameter as well as elongation of growth in stem diameter as well as elongation, the ethephon applications resulted in a stimulation of growth in diameter. It is suggested that ethylene may be involved in the growth responses to mechanical stress.     

Oxygen dynamics during submergence in the halophytic stem succulent Halosarcia pergranulata

This study elucidated O2 dynamics in shoots and roots of submerged Halosarcia pergranulata (Salicornioideae), a perennial halophytic stem succulent that grows on floodprone mudflats of salt lakes. Oxygen within shoots and roots was measured using microelectrodes, for plants when waterlogged or completely submerged, with shoots in light or in darkness, in a controlled environment. Net photosynthesis (PN) when underwater, at a range of dissolved CO2 concentrations, was measured by monitoring O2 production rates by excised stems. The bulky nature and apparently low volume of gas-filled spaces of the succulent stems resulted in relatively high radial resistance to gas diffusion. At ambient CO2, quasi-steady state rates of PN by excised succulent stems were estimated to be close to zero; nevertheless, in intact plants, underwater photosynthesis provided O2 to tissues and led to radial O2 loss (ROL) from the roots, at least during the first several hours (the time period measured) after submergence or when light periods followed darkness. The influence of light on tissue O2 dynamics was confirmed in an experiment on a submerged plant in a salt lake in south-western Australia. In the late afternoon, partial pressure of O2 (pO2) in the succulent stem was 23.2 kPa (i.e. approximately 10% above that in the air), while in the roots, it was 6.2-9.8 kPa. Upon sunset, the pO2 in the succulent stems declined within 1 h to below detection, but then showed some fluctuations with the pO2 increasing to at most 2.5 kPa during the night. At night, pO2 in the roots remained higher than in the succulent stems, especially for a root with the basal portion in the floodwater. At sunrise, the pO2 increased in the succulent stems within minutes. In the roots, changes in the pO2 lagged behind those in the succulent stems. In summary, photosynthesis in stems of submerged plants increased the pO2 in the shoots and roots so that tissues experience diurnal changes in the pO2, but O2 from the H2O column also entered submerged plants.     

Relationship between ethylene production and sensitivity to ethylene in roots of several species

Ethylene concentrations in the soil atmosphere can greatly exceed levels known to markedly influence plant growth. The ethylene is microbial in origin and the rate of production under anaerobic conditions is correlated with organic matter content. Under field conditions the highest concentrations occur when soil temperatures and moisture levels are high, resulting in development of anaerobic zones. Crop species differ widely in the sensitivity of their roots to ethylene, this sensitivity being broadly correlated with known intolerance of waterlogged (anaerobic) environments.     

Radial Oxygen Losses from Intact Rice Roots as Affected by Distance from the Apex, Respiration and Waterlogging

Radial oxygen losses (ROL) from the roots of intact rice plants were assayed by the cylindrical Pt electrode technique. At 23°C losses from roots grown in waterlogged soil proved to be about double those from non-waterlogged plants. Cooling which lowers respiratory activity led to increased ROL and it was estimated that at 23°C respiratory activity had been reducing oxygen loss by 8 to 10 10^–8g O₂ cm^–2 root surface min^–1 (c. 50 %) in the non waterlogged, and by 4.5 to 5.5 10^–8g O₂ cm^–2 min^–1 (2C–30 %) in the waterlogged roots. Lacunae formation occurred nearer to the apex and was eventually more extensive in the waterlogged roots while the presence of more intact and presumably functional tissue in the non-waterlogged roots coincides with the greater respiratory effect noted. Estimated flux rates at 23°C (respiration inactive) were respectively 15–17 × 10^–8g O₂ cm^–2 min^–1 (non-waterlogged) and 20–23 × 10^–8g O₂ cm^–2 min^–1 (waterlogged). A major part of this difference can probably be accounted for directly by the differences in root porosity, and Meakiness' superimposed upon lower porosity in the non-waterlogged plants may account for the remainder. ROL was also examined in relation to distance from the apex. With respiratory activity lowered by cooling, two patterns of oxygen loss were detected. Pattern I was a property of younger roots of length between 5–9 cm, while pattern 2 was found in longer roots 11–16 cm bearing numerous emergent laterals. In both, ROL fell rapidly towards the base and at 4–5 cm approached zero in pattern 1 and near zero to about 16% of the maximum in pattern 2. The rapid drop in oxygen loss in both patterns which indicates a concomitant decrease in root wall permeability was associated with the appearance of cortical lacunae at 2–3 cm from the apex. In pattern 2 a rise in ROL began at 5–6 cm from the apex. The presence of lateral root initials in both the pericycle and unbroken segments of cortex was associated with maintained permeability in this pattern as well as with the basal increase in ROL. With a 3-electrode system placed around the apical 3 cm regions of waterlogged roots, it was found that ROL was substantially affected by respiratory activity at 0.5 cm, a little less so at 1.7 cm, but much less or not at all at 3 cm from the apex. The drop in respiratory effect parallelled the formation of cortical lacunae.     

Polarity of the stem and ontogenetic changes in sunflow (Helianthus annuus L.) leaves in relation to endogenou cytokinins and ethylene

The content of endogenous cytokinin-like substances and the release of ethylene were determined in leaves of different insertion of sunflower plants during their ontogeny. The content of cytokinin-like substances was highest in the leaves on the middle part of the stem (that is in leaves just before full expansion), with a decrease occurring both towards the base and the apex of the stem, when followed at four growth phases (vegetative plants, plants with inflorescence diameter up to 0.5 cm, plants with inflorescence diameter up to 3 cm, and plants in flower). Changes in the content of cytokinin-like substances during the ontogeny of the leaf also corresponded to this pattern. Data obtained with the leaf at the third node from the basis of the stem showed that the level of cytokinin-like substances first sharply increased, and then after reaching maximal value (at the time when leaf blade area reached approximately 70 % of the final value) slowly and continuously decreased. The highest amount of ethylene released from the leaves was recorded in basal leaves and then also in apical leaves, whereas the leaves with the largest blade area situated at the central part of the stem released the lowest amount of ethylene. This pattern was repeatedly found at all four selected growth phases of sunflower plants.