Ethylene and auxin-induced cell growth in relation to auxin transport and metabolism and ethylene production in the semi-aquatic plant,Regnellidium diphyllum

Cell elongation in the rachis of the semiaquatic fern Regnellidium diphyllum is induced by the addition of ethylene or indoleacetic acid (IAA). Experiments with whole plants or rachis segments have shown that ethylene-induced growth requires the presence of auxin. Ethylene does not cause a modification in either endogenous auxin levels or in the extent of auxin metabolism but auxin transport is reduced. Rates of ethylene production in Regnellidium are not altered by either mechanical excitation or by the addition of auxin. A two-hormone control of cell expansion is proposed in which an initial, auxin-dependent growth event pre-conditions the cells to a further subsequent (or synchronous) ethylene-dependent growth event.Abbreviation IAA indole-3yl-acetic acid     

The auxin response of actin is altered in the rice mutantYin-Yang

Summary The rice mutantYin-Yang has been selected during a screen for resistance to cytoskeletal drugs and is characterized by alterations in epidermal cell length and a precocious onset of gravitropism. The elongation response of coleoptile segments to auxin does not reveal changes of auxin sensitivity inYin-Yang. However, in contrast to the wild type, cell elongation inYin-Yang is highly sensitive to the actin-polymerisation blocker cytochalasin D. This increased sensitivity to cytochalasin D requires optimal concentrations of auxin to become manifest. The auxin response of actin microfilaments in epidermal cells differs between wild type and mutant. In the wild type, the longitudinal microfilament bundles become loosened in response to auxin. In the mutant, these bundles disintegrate partially and are replaced by a network of short filaments surrounding the nucleus. Several aspects of the mutant phenotype can be mimicked in the wild type by treatment with cytochalasin D. The mutant phenotype is discussed in terms of signal-dependent changes of actin dynamics and the putative role of actin during cell elongation.Abbreviations CD cytochalasin D - EPC ethyl-N-phenylcarbamate     

Impermeant auxin analogues have auxin activity

Protein conjugates of 5-aminonaphthalene-1-acetic acid and of 5-azido-naphthalene-1-acetic acid have been prepared and evaluated for auxin activity in two types of assay. In standard elongation tests with pea (Pisum sativum L.) epicotyl sections the conjugates are inactive. However, if the epicotyls are abraded to perforate the cuticle, auxin activity is observed provided that the conjugates are not too large to traverse the cell wall. In a system lacking a cell wall — tobacco (Nicotiana tabacum L.) protoplasts — conjugates of widely differing size are able to induce membrane hyperpolarization. These results support other recent evidence that auxin receptors are exposed at the exterior face of the plasma membrane and indicate that auxins can produce both rapid and longer-term responses without entering the cell.Abbreviations ABP auxin-binding protein - BSA bovine serum albumin - E_m transmembrane potential difference - KLH keyhole limpet hemocyanin - NAA naphthalene-1-acetic acid This work was partly supported under the Biotechnology Action Programme of the European Economic Communities. We thank Mr. P. Cozens for technical assistance.To whom correspondence should be addressed.     

Morphogenetic Studies on Osmunda cinnamomea L.: THE AUXIN RELATIONSHIPS OF EXPANDING FRONDS

Net production of diffusible auxin by fronds of O. cinnamomeahas been found to extend throughout the period of final expansion,with a maximum which occurs shortly after the rate of elongationhas reached its maximum and begun to decline. Auxin is apparentlyproduced only in the pinnae, and from these it enters the rachiswhere its movement is polar. Diffusible auxin moves throughthe rachis without significant loss except in the region ofextensive elongation where a certain amount disappears. No suchdisappearance was found in ether extraction studies. Moreover,in certain cases, ether-extractable auxin persists for as muchas 48 hours in the rachis after removal of the auxin source,although none can be obtained by diffusion under these conditions.It is concluded that diffusible auxin is more closely relatedto growth phenomena in the frond than is extractable auxin.In many cases, there is an apparent failure of transport inthe leaf base which reduces or eliminates expected auxin yieldsby diffusion at this level. Diffusible auxin is an essentialparticipant in three morphogenetic phenomena in the developingfrond: rachis elongation, crozier uncoiling, and final differentiationof xylem and hypodermal sclerenchyma. In none of these can itbe considered as a specific determining factor since the responseto auxin depends upon the physiological state of the reactingcells. In the intact frond diffusible auxin is present in excessof the minimum required for both rachis elongation and uncoiling.Several aspects of the relationship between auxin distributionand development in the frond are discussed.     

Cell specificity in auxin- and ethylene-induced ‘supergrowth’ in Riella helicophylla

Summary In gemmalings of Riella helicophylla, auxin and ethylene stimulate elongation growth, especially of pillar cells. When the two hormones are supplied simultaneously, the effects are additive, i.e. the result is supergrowth. In the cells of the meristem, elongation is enhanced by auxin, but not by ethylene when given alone. However, these cells also respond with supergrowth to a combined treatment with auxin and ethylene. The antiauxin p-chlorophenoxyisobutyric acid suppresses both the ethylene stimulation of cell growth and the additive supergrowth. The results support the concept that auxin pre-conditions the cells to the ethylene-dependent growth event. We suggest that the response elicited by the specific cell types could be related to differences in their level of endogenous auxin.Abbreviations IAA indole-3-acetic acid - PCIB p-chlorophenoxyisobutyric acid     

The outer epidermis of Avena and maize coleoptiles is not a unique target for auxin in elongation growth

A controversy exists as to whether or not the outer epidermis in coleoptiles is a unique target for auxin in elongation growth. The following evidence indicates that the outer epidermis is not the only auxin-responsive cell layer in either Avena sativa L. or Zea mays L. coleoptiles. Coleoptile sections from which the epidermis has been removed by peeling elongate in response to auxin. The magnitude of the response is similar to that of intact sections provided the incubation solution contains both auxin and sucrose. The amount of elongation is independent of the amount of epidermis removed. Sections of oat coleoptiles from which the epidermis has been removed from one side are nearly straight after 22 h in auxin and sucrose, despite extensive growth of the sections. These data indicate that the outer epidermis is not a unique target for auxin in elongation growth, at least in Avena and maize coleoptiles.Abbreviations IAA indole-3-acetic acid - PCIB p-chlorophenoxyiso-butyric This research was supported by grants from the National Aeronautics and Space Administration and from the U.S. Department of Energy. The help of S. Ann Dreyer is gratefully acknowledged.     

Microtubule reorganization,cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia

Summary Microtubule reorganization and cell wall deposition have been monitored during the first 30 hours of regeneration of protoplasts of the filamentous green algaMougeotia, using immunofluorescence microscopy to detect microtubules, and the cell-wall stain Tinopal LPW to detect the orientation of cell wall microfibrils. In the cylindrical cells of the alga, cortical microtubules lie in an ordered array, transverse to the long axis of the cells. In newly formed protoplasts, cortical microtubules exhibit some localized order, but within 1 hour microtubules become disordered. However, within 3 to 4 hours, microtubules are reorganized into a highly ordered, symmetrical array centered on two cortical foci. Cell wall synthesis is first detected during early microtubule reorganization. Oriented cell wall microfibrils, co-aligned with the microtubule array, appear subsequent to microtubule reorganization but before cell elongation begins. Most cells elongate in the period between 20 to 30 hours. Elongation is preceded by the aggregation of microtubules into a band intersecting both foci, and transverse to the incipient axis of elongation. The foci subsequently disappear, the microtubule band widens, and microfibrils are deposited in a band which is co-aligned with the band of microtubules. It is proposed that this band of microfibrils restricts lateral expansion of the cells and promotes elongation. Throughout the entire regeneration process inMougeotia, changes in microtubule organization precede and are paralleled by changes in cell wall organization. Protoplast regeneration inMougeotia is therefore a highly ordered process in which the orientation of the rapidly reorganized array of cortical microtubules establishes the future axis of elongation.     

Change in XET activities,cell wall extensibility and hypocotyl elongation of soybean seedlings at low water potential

In dark-grown soybean (Glycine max [L.] Merr.) seedlings, exposing the roots to water-deficient vermiculite (_w=–0.36 MPa) inhibited hypocotyl (stem) elongation. The inhibition was associated with decreased extensibility of the cell walls in the elongation zone. A detailed spatial analysis showed xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity on the basis of unit cell wall dry weight was decreased in the elongation region after seedlings were transplanted to low _w. The decrease in XET activity was at least partially due to an accumulation of cell wall mass. Since cell number was only slightly altered, wall mass had increased per cell and probably led to increased wall thickness and decreased cell wall extensibility. Alternatively, an increase in cell wall mass may represent a mechanism for regulating enzyme activity in cell walls, XET in this case, and therefore cell wall extensibility. Hypocotyl elongation was partially recovered after seedlings were grown in low-_w vermiculate for about 80 h. The partial recovery of hypocotyl elongation was associated with a partial recovery of cell wall extensibility and an enhancement of XET activity in the hypocotyl elongation zone. Our results indicate XTH proteins may play an important role in regulating cell wall extensibility and thus cell elongation in soybean hypocotyls. Our results also showed an imperfect correlation of spatial elongation and XET activity along the hypocotyls. Other potential functions of XTH and their regulation in soybean hypocotyl growth are discussed.     

Rapid-growth responses of corn root segments: Effect of auxin on elongation

The effect of indole-3-acetic acid (IAA) on the elongation rates of 2 mm corn (Zea mays L.) root segments induced by citrate-phosphate buffer (or unbuffered) solutions of pH 4.0 and 7.0 was studied. At pH 7.0, auxin initially reduced the elongation rate in both buffered and unbuffered solutions. Only in buffer at pH 7.0 was auxin at a concentration of 0.1 M found to promote the elongation rate though briefly. THis promoted rate represented only ca. 20% of the rate achieved with only buffer at pH 4.0. Auxin in pH 4.0 buffered and unbuffered solutions only served to reduce the elongation rates of root segments. Some comparative experiments were done using 2 mm corn coleoptile segments. Auxin (pH 6.8) promoted the elongation rate of coleoptile segments to a level equal or greater than the maximal H ion-induced rate. The two responses of root segments to auxin are compared to auxin action in coleoptile growth.     

The positional differentiation of ethylene-responsive cells in rachis abscission zones in leaves of Sambucus nigra and their growth and ultrastructural changes at senescence and separation

Summary In leaves of S. nigra, fragmentation of the rachis follows the autumnal abscission of leaflets and the high levels of ethylene produced by the senescing blades. Fragmentation is accompanied by cell growth and ultrastructural changes in a zone of cells precisely differentiated at the separation zone. Studies with explants from the rachis show that those that contain an abscission zone increase in freshweight by as much as 50% before and during cell separation. Cell growth changes are induced by ethylene but not by auxin, and are restricted to explants that contain the separation zone cells. In ethylene, enlarging cells of the zone show cytoplasmic activation indicated by dilated dictyosomes, enhanced production of Golgi vesicles, elongated profiles of rough endoplasmic reticulum, a crenellated plasmalemma, and the apparent discharge and accumulation of cytoplasmic vesicles within the desmotubules of the branched plasmodesmata. Degradation of the middle lamella and cell wall matrix could be associated with the release of hydrolytic enzymes on the disruption of the vesicles. Although ultrastructural changes of a similar but limited nature occur in all cells of the rachis in response to ethylene, only those that are morphologically delimited as zone cells exhibit the growth and separation that leads to rachis fragmentation. It is proposed that abscission can occur only at the sites of the positional differentiation of these special ethylene-responsive target cells.Abbreviation IAA indole-acetic acid (auxin)     

Transgene-mediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants

Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized. Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light. This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant. Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axrl-3 mutations were also studied. The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings. Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects. All phenotypic effects of iaaM-mediated auxin overproduction (i.e. increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation. The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.     

Total epidermal cell walls of pea stems respond differently to auxin than does the outer epidermal wall alone

The effect of auxin on cell wall mass in the epidermis of third internodes of Pisum sativum L. cv. Alaska grown in dim red light was investigated using epidermal peels, to determine whether epidermal peels reflect the behavior of the outer epidermal cell wall. In contrast to the outer epidermal wall itself, where auxin caused thinning in proportion to growth (M.S. Bret-Harte et al, 1991, Planta 185, 462–471), auxin promoted an increase in wall mass in epidermal peels from treated internode segments in the absence of exogenously supplied sugar. The percentage gain in mass was smaller than the percentage elongation, however, so mass per unit length decreased in peels from auxin-treated segments. Epidermal peels from auxin-treated segments gained more wall mass than control peels even when adhering internal tissue at the basal end of the peel was removed. Epidermal peels also had a gross composition different from that of the outer wall alone (M.S. Bret-Harte and L.D. Talbott, 1993, Planta 190, 369–378). These discrepancies can be explained by the observation that the outer wall makes up only 30% of the mass of the epidermal peel. It appears that the inner walls of the epidermis, and walls of the outer layer of cortical cells that remain attached to the epidermis during peeling, nearly maintain their thickness by biosynthesis while the outer wall loses mass as previously described (Bret-Harte et al. 1991). These results indicate that epidermal peels may not be a good system for examining the biochemical and physiological properties of the outer epidermal cell wall.I would like to thank Dr. Peter M. Ray, of Stanford University, for the use of experimental facilities, helpful discussions, and technical and editorial assistance, Dr. Winslow R. Briggs, of the Carnegie Institute of Washington, for helpful discussions and for the use of experimental facilities, Dr. Paul B. Green, of Stanford University, for financial support, and Dr. Wendy K. Silk, of the Department of Land, Air, and Water Resources, University of California, Davis, for financial support. This work was supported by a National Science Foundation Graduate Fellowship, National Science Foundation grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk in the final writing.     

Physiological Roles of Brassinosteroids in Early Growth of <Emphasis Type="Italic">Arabidopsis:</Emphasis> Brassinosteroids Have a Synergistic Relationship with Gibberellin as well as Auxin in Light-Grown Hypocotyl Elongation

We examined the physiological effects of brassinosteroids (BRs) on early growth of Arabidopsis. Brassinazole (Brz), a BR biosynthesis inhibitor, was used to elucidate the significance of endogenous BRs. It inhibited growth of roots, hypocotyls, and cotyledonous leaf blades dose-dependently and independent of light conditions. This fact suggests that endogenous BRs are necessary for normal growth of individual organs of Arabidopsis in both photomorphogenetic and skotomorphogenetic programs. Exogenous brassinolide (BL) promoted hypocotyl elongation remarkably in light-grown seedlings. Cytological observation disclosed that BL-induced hypocotyl elongation was achieved through cell enlargement rather than cell division. Furthermore, a serial experiment with hormone inhibitors showed that BL induced hypocotyl elongation not through gibberellin and auxin actions. However, a synergistic relationship of BL with gibberellin A₃ (GA₃) and indole-3-acetic acid (IAA) was observed on elongation growth in light-grown hypocotyls, even though gibberellins have been reported to be additive to BR action in other plants. Taken together, our results show that BRs play an important role in the juvenile growth of Arabidopsis; moreover, BRs act on light-grown hypocotyl elongation independent of, but cooperatively with, gibberellins and auxin.     

Lead toxicity effects on indole-3-ylacetic acid-induced cell elongation

In vitro studies of IAA-induced cell elongation in Triticum aestivum have demonstrated that lead causes a large reduction in elongation. Inhibition of elongation can be reduced by increasing the concentration of IAA, or by the addition of calcium. The inhibitory effect appears to be linked with changes in the properties of the cell walls. Experiments are described which show that lead becomes bound strongly to certain chemical substances involved in cell wall architecture.     

The kinetics of bidirectional growth of stem sections from etiolated pea seedlings in response to acid,auxin and fusicoccin

Growth in length and diameter of abraded stem sections from etiolated pea (Pisum sativum L.) seedlings was monitored continuously using a double laser optical level auxanometer system. Acidic solutions (pH 4.0–4.5) induced rapid elongation accompanied by lateral shrinkage (up to 8% of the initial diameter). The shrinkage phase lasted for 30–45 min. Pretreatment with permeant solutes (KCl, NaCl, sucrose or glucose) prevented lateral shrinkage, while pretreatment with the impermeant solute, polyethylene glycol, did not block lateral contraction in response to acid. A slight turgor step-up given during the shrinkage phase inhibited lateral shrinkage and increased the elongation rate. Visual observation confirmed that shrinkage occurred and that the same region of the stem that contracted in diameter also elongated. It is proposed that lateral shrinkage results from a decrease in turgor pressure during acid-stimulated elongation. Elongation induced by auxin and fusicoccin (FC) was also accompanied by a decrease in the diameter; this decrease could be prevented by pretreatment with KCl or glucose. Thus, the early phase of auxin and FC action is acid-like. However, the shrinkage is of shorter duration (14–20 min) and it is less drastic (ca. 2%). In addition, FC caused lateral expansion after a 20-min lag period in stems pretreated with KCl. The results are consistent with an acid-growth mechanism during the early phase (first 20–40 min) of the responses to both auxin and FC. It is suggested that enhanced osmoregulation subsequently inhibits further lateral shrinkage and helps to maintain steady-state growth. FC, unlike auxin, may alter the anisotropic character of the wall.Abbreviations FC fusicoccin - IAA indole-3-acetic acid - LOLA laser optical levar auxanometer - PEG polyethyleneglycol 600     

Protein synthesis and wall extensibility in the Avena coleoptile

Summary The inhibitors cycloheximide and puromycin have been used to examine the relationship between protein synthesis and wall extensibility, as measured with an Instron, in Avena coleoptile segments. Cycloheximide at 4 g/ml almost totally inhibits both auxin-induced cell elongation and protein synthesis with only a slight lag. Wall extensibility is unaffected by the inhibitor if auxin is absent. If added prior to auxin, cycloheximide prevents auxin-induced wall loosening while if added after auxin it causes a substantial decline in the wall extensibility. With puromycin there is a 2–4 hr lag before growth and wall loosening are inhibited. These results support the conclusions that the proteins needed for wall loosening are unstable, and that continued protein synthesis is necessary to maintain the wall loosening process.     

Auxin-induced changes in cell wall extensibility of maize roots

The rheological properties of corn (Zea mays L. cv. Garant) root elongation zones were investigated by means of a computer-controlled extensiometer. Creep closely followed a logarithmic time function, which was used to quantify creep activity. Pretreatment with auxin, which inhibits extension growth in roots, lowered the creep activity and the apparent plastic extensibility. While the time course of the inhibition of apparent plastic extensibility lagged behind the cessation of elongation growth, the drop in creep activity matched the growth inhibition more closely. Creep activity and apparent plastic extensibility were not significantly affected by pH. These data support the view that the auxin-induced cell wall stiffening (e.g. by cross-linking processes), while causal for the growth inhibition, is not brought about by a cell wall alkalinization. Received: 10 December 1996 / Accepted: 19 August 1997     

The positional differentiation of abscission zones during the development of leaves of Sambucus nigra and the response of the cells to auxin and ethylene

Summary Abscission in the leaf rachis of Sambucus nigra L. is preceded by a positional differentiation of zone cells that enlarge and separate in response to ethylene but not to auxin. These cells are absent from youngest leaves, and such leaves do not abscind even in ethylene; other cells of the immature rachii will enlarge in response to auxin. These two classes of target cells are always recognisable by their opposing responses to auxin and ethylene. Prior to separation zone cells exposed to ethylene show considerable activation of the cytoplasm, many polysomes, elongate endoplasmic reticulum and highly dilated dictyosomes with many associated vesicles. Treatment with auxin precludes these changes, and abscission is always retarded: high levels of ethylene must be added to overcome the auxin inhibition. The differentiation of zone cells and their ethylene-stimulated growth and activation are prerequisites for rachis abscission in Sambucus. Such cell development may be of general occurrence prior to organ abscission in plants.Abbreviation IAA indole-3yl-acetic acid     

Changing cell-wall compositions in hypocotyls of dark-grown bean seedlings

Hypocotyls of dark-grown 6-day-old seedlings of Phaseolus vulgaris L. proved to be sufficiently homogeneous to permit studies relating the rate of cell elongation to the composition of the primary cell walls. Whereas the levels of cellulose and uronic acids remained practically constant during and after cell extension, all other components showed major or minor changes. Cell-wall protein, as such, decreased by more than 50%, but indications are that hydroxyproline-rich glycoprotein increased with a decreasing rate of cell elongation, concomitant with a rise in the degree of arabinosylation of wall-bound hydroxyproline. As cell elongation slowed down, non-cellulosic glucose accumulated, presumably in the form of a -(1–4)glucan closely associated with cellulose. These findings confirm the notion that the primary cell wall is a highly dynamic structure.