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.

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.

Intact Leaves Exhibit a Climacteric-Like Rise in Ethylene Production before Abscission.

The rate of ethylene production by intact, attached leaves of cotton plants (Gossypium hirsutum L.) during aging and senescence was studied using a continuous flow system that allowed air around enclosed leaves to be scrubbed to collect and assay ethylene. Senescence of lower leaves began around 150 d after planting in a controlled environment room. A progressive decline in the ethylene production rate was observed when comparing the 3rd, 6th, and 10th leaves from the base with each other. Ethylene production rates of individual leaves also declined over a 50-d period. However, as leaves began to appear chlorotic, a peak of ethylene production occurred that lasted for about 4 d followed by abscission. This peak involved a 3-fold or greater increase in the rate of ethylene production. The data indicate that intact leaves experience a climacteric-like surge in ethylene production after visible symptoms of senescence appear. This "ethylene climacteric" is apparently the signal that initiates hydrolysis of cell walls in the abscission zone.

Hormonal and Environmental Regulation of the Carrot lea-Class Gene Dc3.

Dc3 is a carrot lea-class gene belonging to a small gene family that encodes Dc3 and Dc3-like RNA sequences. We have examined the responsiveness to water deficit and abscisic acid (ABA) of the promoter/enhancer complex of Dc3 fused to a beta-glucuronidase (GUS) reporter gene in vegetative cells of transgenic tobacco. In 56-d tobacco, GUS expression in leaves increased about 200-fold during a 3-d drying cycle, during which there were small decreases (3 atmospheres or less) in leaf water potential and a 16-fold increase in free ABA. These effects were reversed by rewatering. Changes in GUS activity were closely paralleled by changes in GUS transcript levels during the desiccation/watering cycle, indicating transcriptional regulation of GUS gene expression. The Dc3 promoter responds to exogenous ABA; the effect is time and concentration dependent, with greater than 10-fold induction in 8 h with 10 mum ABA. Histochemical visualization of GUS activity in seedlings induced by water deficit or exogenous ABA revealed Dc3-driven GUS expression in all organs of transgenic tobacco seedlings. We suggest that the Dc3/GUS reporter system is a sensitive analytical tool to study various environmental effects on plant growth and development.

Enhanced Sensitivity to Ethylene in Nitrogen- or Phosphate-Starved Roots of Zea mays L. during Aerenchyma Formation.

Adventitious roots of maize (Zea mays L. cv TX 5855), grown in a well-oxygenated nutrient solution, were induced to form cortical gas spaces (aerenchyma) by temporarily omitting nitrate and ammonium (-N), or phosphate (-P), from the solution. Previously this response was shown (MC Drew, CJ He, PW Morgan [1989] Plant Physiology 91: 266-271) to be associated with a slower rate of ethylene biosynthesis, contrasting with the induction of aerenchyma by hypoxia during which ethylene production is strongly stimulated. In the present paper, we show that aerenchyma formation induced by nutrient starvation was blocked, under noninjurious conditions, by addition of low concentrations of Ag(+), an inhibitor of ethylene action, or of aminoethoxyvinyl glycine, an inhibitor of ethylene biosynthesis. When extending roots were exposed to low concentrations of ethylene in air sparged through the nutrient solution, N or P starvation enhanced the sensitivity to exogenous ethylene at concentrations as low as 0.05 microliters ethylene per liter air, promoting a more rapid and extensive formation of aerenchyma than in unstarved roots. We conclude that temporary deprivation of N or P enhances the sensitivity of ethylene-responsive cells of the root cortex, leading to cell lysis and aerenchyma.

Decreased Ethylene Biosynthesis, and Induction of Aerenchyma, by Nitrogen- or Phosphate-Starvation in Adventitious Roots of Zea mays L.

Plants of Zea mays L. cv TX5855 were grown in a complete, well oxygenated nutrient solution then subjected to nutrient starvation by omitting either nitrate and ammonium or phosphate from the solution. These treatments induced the formation of aerenchyma close to the apex of the adventitious roots that subsequently emerged from the base of the shoot, a response similar to that shown earlier to be induced by hypoxia. Compared with control plants supplied with all nutrients throughout, N- or P-starvation consistently depressed the rates of ethylene release by excised, 25 mm apical segments of adventitious roots. Some enzymes and substrates of the ethylene biosynthetic pathway were examined. The content of 1-amino cyclopropane-1-carboxylic acid (ACC) paralleled the differences in ethylene production rates, being depressed by N or P deficiency, while malonyl-ACC showed a similar trend. Activity of ACC synthase and of ethylene forming enzyme (g(-1) fresh weight) was also greater in control roots than in nutrient starved ones. These results indicate that much of the ethylene biosynthetic pathway is slowed under conditions of N- or P-starvation. Thus, by contrast to the effects of hypoxia, the induction of aerenchyma in roots of Zea mays by nutrient starvation is not related to an enhanced biosynthesis and/or accumulation of ethylene in the root tips.

Stimulation of ethylene production and gas-space (aerenchyma) formation in adventitious roots of Zea mays L. by small partial pressures of oxygen.

Adventitious roots of two to four-weekold intact plants of Zea mays L. (cv. LG11) were shorter but less dense after extending into stagnant, non-aerated nutrient solution than into solution continuously aerated with air. Dissolved oxygen in the non-aerated solutions decreased from 21 kPa to 3-9 kPa within 24 h. When oxygen partial pressures similar to those found in non-aerated solutions (3, 5 and 12 kPa) were applied for 7 d to root systems growing in vigorously bubbled solutions, the volume of gas-space in the cortex (aerenchyma) was increased several fold. This stimulation of aerenchyma was associated with faster ethylene production by 45-mm-long apical root segments. When ethylene production by roots exposed to 5 kPa oxygen was inhibited by aminoethoxyvinylglycine (AVG) dissolved in the nutrient solution, aerenchyma formation was also retarded. The effect of AVG was reversible by concomitant applications of 1-aminocyclopropane-1-carboxylic acid, an immediate precursor of ethylene. Addition of silver nitrate, an inhibitor of ethylene action, to the nutrient solution also prevented the development of aerenchyma in roots given 5 kPa oxygen. Treating roots with only 1 kPa oxygen stimulated ethylene production but failed to promote gas-space formation. These severely oxygen-deficient roots seemed insensitive to the ethylene produced since a supplement of exogeneous ethylene that promoted aerenchyma development in nutrient solution aerated with air (21 kPa oxygen) failed to do so in nutrient solution supplied with 1 kPa oxygen. Both ethylene production and aerenchyma formation were almost completely halted when roots were exposed to nutrient solutions devoid of oxygen. Thus both processes require oxygen and are stimulated by oxygen-deficient surroundings in the 3-to 12-kPa range of oxygen partial pressures when compared with rates observed in air (21 kPa oxygen).

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.

Inhibition by silver ions of gas space (aerenchyma) formation in adventitious roots of Zea mays L. subjected to exogenous ethylene or to oxygen deficiency.

We have studied the role of ethylene in accelerating the lytic formation of gas spaces (aerenchyma) in the cortex of adventitious roots of maize (Zea mays L.) growing in poorly aerated conditions. Such roots had previously been shown to contain increased concentrations of ethylene. Ten day-old maize plants bearing seminal roots and one whorl of emerging adventitious roots were grown in nutrient solution bubbled with air, ethylene in air (0.1 to 5.0 µl l(-1)), or allowed to become oxygen-deficient in nonaerated (but not completely anaerobic) solution. Additions of 0.1 µl l(-1) ethylene or more promoted the formation of aerenchyma, with lysis of up to 47% of the cortical cells. The effects of non-aeration were similar to those of exogenous ethylene. When silver ions, an ethylene antagonist, were present at low, non-toxic concentrations (circa 0.6 µM), aerenchyma formation was prevented in ethylene treated roots and in those exposed to oxygen deficiency. Silver ions also blocked the inhibiting effect of exogenous ethylene on root extension. By contrast, the suppression of aerenchyma formation by silver ions under oxygendeficient conditions was associated with a retardation of root extension, indicating the importance of aerenchyma for root growth in poorly aerated media. Rates of production of ethylene by excised roots were stimulated by a previous non-aeration treatment. The effectiveness of Ag(+) in inhibiting equally the action on cortical cells of exogenous ethylene and of non-aeration, supports the view that gas space (aerenchyma) formation in adventitious roots 'adpted' to oxygendeficient environments is mediated by increased concentrations of endogenous ethylene. The possibility that extra ethylene could arise from increased biosynthesis of a precursor in root tissues with a restricted oxygen supply is discussed.

Ethylene-promoted adventitious rooting and development of cortical air spaces (aerenchyma) in roots may be adaptive responses to flooding in Zea mays L.

The roots and stem base of intact, 10 day old maize (Zea mays L. cv. LG11) plants, grown in nutrient solution, were continuously aerated either with ethylene (5 µl l(-1)) in air or with air alone. Ethylene treatment hastened the emergence of adventitious (nodal) roots from the base of the shoot, but slowed their subsequent extension. Ethylene also promoted the collapse of cells in the cortex of these roots, with lysigenous development of prominent air spaces (aerenchyma). Non-aeration of the nutrient solution caused endogenously produced ethylene to accumulate in the roots, and stimulated both the emergence of adventitious roots and the formation of cortical air spaces in them. With non-aeration the concentration of oxygen did not fall below 1% in the equilibrium gas phase (air=20.8%). Complete deoxygenation of the nutrient solution, produced by passing oxygen-free nitrogen gas, prevented both air space formation and the evolution of ethylene by root segments.These results suggest that adventitious rooting and cortical air space formation in nodal roots in Zea mays may be stimulated by enhanced concentrations of endogenous ethylene arising either from entrapment of the gas by unstirred water layers around the roots and/or by increased biosynthesis. These responses are considered conducive to survival in waterlogged soil.