Modification by ethylene of the cell growth pattern in different tissues of etiolated lupine hypocotyls

The influence of ethylene on growth in etiolated lupine (Lupinus albus L.) hypocotyls was studied in ethephon-treated plants. Ethephon reduced the length and increased the diameter of hypocotyls. At the end of the hypocotyl growth period (14 days), the fresh weight was reduced by 53%, and the dry weight was reduced by 16%. Thus, ethylene reduced water uptake in the tissues to a greater extent than the incorporation of new materials. Light microscopic measurements showed that the thickness of tissues was stimulated by ethylene, the vascular cylinder and cortex exhibiting greater increases (55 and 45%, respectively) than pith (26%) or epidermis (12%). Ethephon modified the cell growth pattern, stimulating lateral cell expansion and cell wall thickness, while reducing cell elongation. The response to ethylene varied in the different tissues and was higher in cortex and pith cells than in the epidermis cells. The ethylene-induced cell expansion in the cortex varied according to the localization of cells in the tissue: the central and subepidermal layers showed little change, whereas the innermost layers exhibited the greatest increase. Electron microscopy revealed that ethylene increased both the rough endoplasmic reticulum and dictyosomes, suggesting that ethylene stimulated the secretion of cell wall materials. In untreated seedlings, the pattern of cell growth was similar in cells from the epidermis, cortex, and pith. The final cell size varied along the hypocotyl, the cells becoming shorter and broader the closer to the basal zones of the organ.     

Ethylene as a natural agent inducing plumular hook formation in pea seedlings

Summary Removing endogenous ethylene by hypobaric treatment, or displacing it with carbon dioxide inhibits hook development in etiolated pea seedlings. When seedlings are returned to a normal atmosphere, hook formation occurs in darkness. Addition of ethylene accelerates this process. When ethylene induces hook formation, cell division in the hook tissue is rather inhibited by the gas. These data suggest that endogenous ethylene causes formation of the hook by inducing expansion of certain cells.     

Involvement of inorganic elements in tissue reunion in the hypocotyl cortex of Cucumis sativus

Cucumber (Cucumis sativus L.) hypocotyls were transversely cut to half their diameter, and morphological analyses of the tissue-reunion process in the cortex were conducted to elucidate the involvement of root-derived factors. Cell division in the cortex commenced 3 days after cutting, and the cortex was nearly fully united within 7 days. In shoots from which the roots were removed and which were cultured in water, cell division occurred during tissue reunion; however, thick-wall layer formed in the reunion region, and intrusive cell elongation and interdigitation of cortex cells at the cut surface did not occur, even after 7 days. Interdigitation of cells, followed by normal tissue reunion, was observed in shoots from which the roots were removed and which were cultured in squash xylem sap or Murashige and Skoog (MS) medium. The same effect was observed with the simultaneous application of B, Mn, and Zn, which are the major inorganic microelements of MS medium. Our results suggest that application of these inorganic elements, which are taken up from the soil and transferred to the xylem sap, are required for interdigitation of cells during tissue reunion in the cortex of cucumber hypocotyls, possibly because they are required for cell wall function and metabolism.Asahina M and Gocho Y equally contributed to this work.     

Effect of Inhibitors and Stimulators of Ethylene Production on Gall Development in Meloidogyne javanica-Infected Tomato Roots

Excised tomato roots infected with Meloidogyne javanica produced ethylene at 3-6 times the rate of noninfected roots. This increase in ethylene production started 5 days after inoculation. Gall growth and ethylene production in infected roots were accelerated by 1-aminocyclopropane-1-carboxylic acid (ACC), indole acetic acid (IAA), and ethrel known as ethylene production stimulators. When inhibitors of ethylene production, like aminoethoxyvinylglycine (AVG) or aminoxyacetic acid (AOA), or inhibitors of ethylene action like silver thiosulfate (STS), were applied, gall growth and ethylene production were inhibited. Enhanced expansion of parenchymatous cells was observed in sections from nematode-induced galls and ethylene-treated roots. Lignification of xylem elements and fibers in the vascular cylinder was markedly inhibited in the gall, compared with noninfected root tissue. Because ethylene is known to induce cell expansion and to inhibit lignification, it is suggested that this plant hormone plays a major role in the development of M. javanica-induced galls. Ethylene affects gall size by enhancing parenchymatous tissue development and allows expansion of giant cells and the nematode body by reducing tissue lignification.     

Influence of ethylene and Ag+ on hypocotyl growth in etiolated lupin seedlings. Effects on cell growth and division

Lupin seeds treated with 1-amino-cyclopropane-1-carboxylic acid (ACC) or2-chloroethylphosphonic acid (CEPA) produced hypocotyls showing a typicalethylene growth response (reduced elongation and increased thickness), whichcould be efficiently counteracted by the presence of silver thiosulfate (STS).The fact that ACC and CEPA stimulated the ethylene produced in different zonesalong the hypocotyls suggests that these compounds, which are stored in theseeds during treatment, were transported to and along the hypocotyl. The same istrue in hypocotyls from STS-treated seeds, which indicates that stress ethyleneis induced by metal toxicity. CEPA was more effective than ACC in both producingethylene and influencing growth due to the high capacity of the hypocotyl toconjugate ACC. At the same time that CEPA inhibited hypocotyl elongation, thehypocotyl diameter increased and ethylene production exceeded the maximum valueof the control. The subsequent recovery of hypocotyl elongation coincided with adecrease in ethylene production and involved cell elongation. The final celllength was similar (in ACC-) or higher (in CEPA-treated plants) than in thecontrol, although the hypocotyls were shorter in both cases, while the number ofcells per column was reduced to half that observed in the control. Thisinhibition of cell division caused by ethylene was selective since the number ofcell layers did not change. The variations in cell diameter in the epidermisand, especially, in the cortex and pith were correlated with the variations inhypocotyl diameter produced by ACC, CEPA and STS. The results show that theethylene-induced hypocotyl thickening was irreversible and mainly due to anincrease in cell diameter, while the inhibition of hypocotyl elongation wasreversible and involved irreversible inhibition of cell division and,paradoxically, stimulation of cell elongation to produce cells longer than thoseof the control.     

Gibberellin produced in the cotyledon is required for cell division during tissue reunion in the cortex of cut cucumber and tomato hypocotyls

Cucumber (Cucumis sativus) hypocotyls were cut to one-half of their diameter transversely, and morphological and histochemical analyses of the process of tissue reunion in the cortex were performed. Cell division in the cortex commenced 3 d after cutting, and the cortex was nearly fully united within 7 d. 4',6-Diamidino-2-phenylindole staining and 5-bromo-2'-deoxyuridine labeling experiments indicate that nDNA synthesis occurred during this process. In addition, specific accumulation of pectic substances was observed in the cell wall of attached cells in the reunion region of the cortex. Cell division during tissue reunion was strongly inhibited when the cotyledon was removed. This inhibition was reversed by applying gibberellin (GA, 10(-4) M GA3) to the apical tip of the cotyledon-less plant. Supporting this observation, cell division in the cortex was inhibited by treatment of the cotyledon with 10(-4) M uniconazole-P (an inhibitor of GA biosynthesis), and this inhibition was also reversed by simultaneous application of GA. In contrast to the essential role of cotyledon, normal tissue reunion in cut hypocotyls was still observed when the shoot apex was removed. The requirement of GA for tissue reunion in cut hypocotyls was also evident in the GA-deficient gib-1 mutant of tomato (Lycopersicon esculentum). Our results suggest that GA, possibly produced in cotyledons, is essential for cell division in reuniting cortex of cut hypocotyls.     

Influence of ACC and Ethephon on cell growth in etiolated lupin hypocotyls. dependence on cell growth state

The possible implication of ethylene on the growth regulation of etiolated lupin hypocotyls was investigated. Excised hypocotyl sections from actively growing seedlings produced ethylene at a rate of 3 nmol h^-1 g^-1 min^-1. The rate of ethylene production was increased about 7 times when sections were treated with 10 mM 1-aminocyclopropane-1-carboxylic acid (ACC). Measurement of endogenous ACC showed that 95 % of total ACC (64.2 nmol g^-1 min^-1) corresponded to conjugated ACC. Treatments to intact seedlings with the ethylene precursor ACC, and the ethylene generating compound, 2-chloroethyl phosphonic acid (ethephon) during the cell elongation phase of the hypocotyl (from 7 to 21 dage), modified the cell growth of the organ. ACC (1 or 5 mM) or low concentrations of ethephon (0.66 mM) produced a transient decrease in the growth rate without modifying the final length of the hypocotyls. Higher concentrations of ethephon reduced the final length; the younger the seedlings were, the greater the reduction. Simultaneously to inhibition of cell elongation, ethephon produced stimulation of the radial expansion of cells in pith and cortex. The growth inhibition period, which lasted for 2 days after the treatments, was followed by another period in which the growth rate of treated plants surpassed that of the control. In both cases differences were observed along the hypocotyls due to the different growth status of the cells. It is suggested that the sensitivity to ethylene and the metabolism of ethylene depend on the growth status of the cells.     

Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6(prc1-1) or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.     

Effects of Calcium Ion Concentration on Cel Wall Synthesis

Cuttings of 6-week-old Norway spruce (Picea abies (L.) Karst.)seedlings were placed in liquid media containing various concentrationsof Ca²⁺. Cytoplasmic concentrations of Ca²⁺ were manipulatedusing the ionophore A 23 187. The effects of Ca²⁺ concentrationson the deposition of total cell wall material as well as onthe deposition of cellulose, lignin, and non-cellulosic polysaccharidesin the hypocotyls were investigated. At low concentrations ofCa²⁺ wall deposition was reduced, mainly as a result of theinhibition of lignin and non-cellulosic polysaccharide deposition.High concentrations of Ca²⁺ stimulated non-cellulosic polysaccharideand lignin deposition, whereas cellulose deposition was almosttotally inhibited. Key words: Conifers, calcium, cell wall, lignin, cellulose     

Ethylene-induced lateral expansion in etiolated pea stems : kinetics, cell wall synthesis, and osmotic potential

Treatment of etiolated pea (Pisum sativum L.) internode tissue with ethylene gas inhibits elongation and induces lateral expansion. Precise kinetics of the induction of this altered mode of growth of excised internode segments were recorded using a double laser optical monitoring device. Inhibition of elongation and promotion of lateral expansion began after about 1 hour of treatment and achieved a maximum by 3 hours. Similar induction kinetics were observed after treating internodes with colchicine and 2,6-dichlorobenzonitrile, an inhibitor of cellulose synthesis. In sealed flask experiments, ethylene had no detectable effect on incorporation of label from [¹⁴C]glucose into any of the classical pectin, hemicellulose, or cellulose wall fractions. Ethylene inhibited fresh weight increase (total cell expansion) of both excised internode segments (in sealed flasks) and intact seedlings. Ethylene treatment resulted in an increase in cell sap osmolality in those tissues (intact and excised) which are inhibited by the gas. A model for ethylene-induced inhibition of elongation and induction of lateral expansion is presented.     

Effects of Flooding, Tilting of Stems, and Ethrel Application on Growth, Stem Anatomy and Ethylene Production of Pinus densiflora Seedlings

Flooding of soil, tilting of seedlings, application of ethrelto stems, and combinations of these treatments, variously alteredthe rate of growth and stem anatomy of 2-year-old Pinus densifloraseedlings. Either flooding or tilting increased stem diametergrowth and induced formation of abnormal xylem. Whereas floodingdecreased the rate of dry weight increment of roots and needlesand increased growth of bark tissues, tilting of stems did not.However, tilting decreased the rate of height growth, stimulatedtracheid production, and induced formation of well-developedcompression wood with rounded, thick-walled tracheids, witha high lignin content but without an S3 layer in the tracheidwall. Ethylene appeared to have an important regulatory rolein stimulating growth of bark tissues as shown by thicker barkin flooded seedlings or those treated with ethrel. Ethyleneappeared to have a less important role in regulating formationof compression wood. Flooding increased the ethylene contentsof stems and induced formation of rounded, thick-walled tracheids.However, these tracheids lacked such features of well-developedcompression wood tracheids as a thick S2 layer, high lignincontent, and absence of an S3 layer. Furthermore, applicationof ethrel to vertical stems greatly increased their ethylenecontents but did not induce formation of well-developed compressionwood. Furthermore, ethrel application blocked development ofcertain characteristics of compression wood when applied totilted seedlings. For example an S3 wall layer was absent intracheids of tilted seedlings but present in tracheids of tilted,ethrel-treated seedlings. Also lignification of tracheids wasincreased on the under side of tilted stems, but reduced intilted, ethrel-treated seedlings, further de-emphasizing a directrole of ethylene in the formation of compression wood. Ethreltreatment induced formation of longitudinal resin ducts in thexylem whereas flooding or tilting of stems did not. Key words: Pinus densiflora, xylogenesis, reaction wood, compression wood, lignification, ethrel, ethylene     

Knockout of the AtCESA2 gene affects microtubule orientation and causes abnormal cell expansion in Arabidopsis

Complete cellulose synthesis is required to form functional cell walls and to facilitate proper cell expansion during plant growth. AtCESA2 is a member of the cellulose synthase A family in Arabidopsis (Arabidopsis thaliana) that participates in cell wall formation. By analysis of transgenic seedlings, we demonstrated that AtCESA2 was expressed in all organs, except root hairs. The atcesa2 mutant was devoid of AtCESA2 expression, leading to the stunted growth of hypocotyls in seedlings and greatly reduced seed production in mature plants. These observations were attributed to alterations in cell size as a result of reduced cellulose synthesis in the mutant. The orientation of microtubules was also altered in the atcesa2 mutant, which was clearly observed in hypocotyls and petioles. Complementary expression of AtCESA2 in atcesa2 could rescue the mutant phenotypes. Together, we conclude that disruption of cellulose synthesis results in altered orientation of microtubules and eventually leads to abnormal plant growth. We also demonstrated that the zinc finger-like domain of AtCESA2 could homodimerize, possibly contributing to rosette assemblies of cellulose synthase A within plasma membranes.     

Thigmomorphogenesis: Changes in Cell Division and Elongation in the Internodes of Mechanically-perturbed or Ethrel-treated Bean Plants

Examination of first internodes of young Phaseolus vulgarisL. plants which have been subjected to mechanical perturbationshows decreased elongation and increased radial growth. Thedecreased elongation can be attributed to both reduced cellelongation of epidermal and cortical cells and a reduced numberof cells in the vascular and pith tissues. The increased radialenlargement is due to increased cortical cell expansion andincreased secondary xylem production resulting from increasedcambial activity. All of these responses are observable withina few hours of a single mechanical perturbation. Treatment ofplants with ethrel mimics all of these effects of mechanicalperturbation. Phaseolus vulgarisL, Kidney bean, thigmomorphogenesis, mechanical perturbation, ethrel, (2-chloroethyl phosphonic acid), cell division, internode elongation     

The role of cell wall synthesis in sustained auxin-induced growth

The dependence of auxin-induced growth on continued cell wall synthesis was investigated in stem segments of etiolated pea ( Pisum sativum L. cv. Alaska) seedlings using the cell wall synthesis inhibitors monensin and 2,6-dichlorobenzonitrile (DCB). Monensin (5 μ M ) potently inhibited indole-3-acetic acid (IAA)-induced growth, particularly during the second hour of treatment, whereas growth in fusicoccin (FC) was inhibited much less effectively. Incorporation of [¹⁴C]-glucose into both matrix and cellulose fractions of the wall showed a sharp increase beginning after about 60 min, this rise being promoted by both IAA and FC. Monensin inhibited this rise in incorporation of label and completely removed the promotion of this by IAA, although some promotion by FC remained. Monensin inhibited incorporation into cellulose in a manner similar to that into matrix, but the use of the apparently specific cellulose synthesis inhibitor DCB showed that cellulose synthesis could be strongly inhibited without effect on growth, at least in the short term. The results support the view that sustained auxin-induced growth depends upon the incorporation of new matrix cell wall components into the wall.     

Comparison of the effects of white light and the growth retardant paclobutrazol on the ethylene production in bean hypocotyls

At a concentration of 17 µmol·L^–1, paclobutrazol (PP), a triazole plant growth retardant, effectively reduced the elongation and increased the thickness of hypocotyls in 6-day-old Phaseolus vulgaris L. cv. Juliska seedlings, both in the light and in the dark. PP treatment did not increase the cell number in transverse sections of hypocotyls. The diameter of hypocotyls was uniform from the zone of intensive elongation along the whole hypocotyl in etiolated plants, but those grown in the light exhibited an additional lateral expansion at the base. Ethylene evolution was not reduced by PP in etiolated hypocotyls, and did not differ significantly in the elongating apical and fully grown basal zones. PP reduced the ethylene release by the growing zones in green hypocotyls, but not in the basal parts, which resulted in an increasing ethylene gradient towards the hypocotyl base. The level of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, was much higher in retardant-treated hypocotyls than in the controls, which was due in part to the reduced malonylation. The swelling of the hypocotyl bases could be eliminated by inhibitors of ethylene biosynthesis or action, or could be induced by 10 µmol·L^–1ACC in control plants in the light. None of these treatments had a significant effect on the lateral expansion of hypocotyls in etiolated seedlings. PP treatment induced a similar effect to that of white light in etiolated seedlings, and amplified the effect of light in green plants with respect to the ACC distribution, and consequently, the ethylene production in the hypocotyls of 6-day-old bean seedlings. It can be concluded that the lateral expansion of hypocotyl bases in PP-treated green plants is controlled by ethylene.     

Ethylene effects on peroxidases and cell growth patterns in Picea abies hypocotyl cuttings

The effect of 2-chloroethylphosphonic acid (ethrel) on cell growth patterns and per-oxidase activity (EC 1.11.1.7) and location in young Norway spruce cuttings ( Picea abies [L.] Karst.) was investigated. The peroxidase activity in a fraction containing soluble and membrane bound enzymes show a diurnal variation, with decreased activity during the light period and a corresponding increase during the following dark period. The decrease during the day could to some extent be counteracted by treatment with ethrel. It appears that ethrel affects only peroxidases in the isolated membrane fraction, since peroxidases bound to the cell wall were not affected by ethrel. In vitro experiments indicated that the hydrophobicity of soluble peroxidases was increased by treatment with ethylene. Cytochemical localization of peroxidase activity in differentiating tracheids revealed a clear ethrel-induced increase in the tonoplast. It appears that ethylene affects soluble peroxidases in vivo in such a way that they are directed to a more hydrophobic environment, like the tonoplast. Treatment with ethrel also changed the appearance of the rough endoplasmic reticulum (ER) and Golgi apparatus. Dilated ER cisternae were observed on electron micrographs, as a result of treatment with ethrel. The number of vesicles produced by the Golgi apparatus and also the amount of vesicles fusing with the plasma membrane in secondary-wall-forming tracheids increased considerably. The results clearly indicate that the stimulatory effect of ethylene in spruce seedlings on lignification and cell wall formation, is due to a general stimulation on both synthesis, transport and secretion of cell wall material and not on a stimulation of peroxidase activity as reported for other species.     

Gene expression profiles in different stem internodes reveal the genetic regulation of primary and secondary stem development in <Emphasis Type="Italic">Betula platyphylla</Emphasis>

Secondary growth of stems is an important process for the radial increase of trees. To gain an insight into the molecular mechanisms underlying stem development from primary to secondary growth and to provide information for molecular research and breeding in Betula platyphylla (birch), the gene expression profiles of material from the first, third, and fifth internodes (IN) of 3-month-old seedlings were analyzed. Compared with the first IN, 177 genes were up-regulated and 157 genes down-regulated in the third IN; in the fifth IN, 180 genes were up-regulated and 275 genes were down-regulated. The expressions of 24 genes were up-regulated and 6 genes were down-regulated in the fifth IN relative to the third IN. The differentially expressed genes were annotated as having roles in cambium, xylem, and phloem development and formation; including cell wall expansion, cellulose biosynthesis, lignin biosynthesis and deposition, xylem extension, cell wall modification, and growth hormone responses. The expressions of genes related to cell wall expansion and cellulose biosynthesis in the primary cell wall were down-regulated in the third and fifth IN relative to the first IN. Genes involved in lignin biosynthesis, xylem extension, and cellulose synthesis in the secondary cell wall were up-regulated in the third and fifth IN relative to the first IN. These results described the patterns of gene expression during stem development in birch and provided candidate genes for further functional characterization.     

Does growth correlate with turgor-induced elastic strain in stems? A re-evaluation of de Vries' classical experiments

The correlation between growth and turgor-induced elastic expansion was studied in hypocotyls of sunflower (Helianthus annuus) seedlings under various growth conditions. Turgor-induced elastic cell wall strain was greater in hypocotyls of faster growing seedlings, i.e. in etiolated versus light-grown ones. It also was higher in rapidly growing young seedlings as compared with nongrowing mature ones. However, analysis of the spatial distribution of elastic strain and growth demonstrated that their correspondence was only apparent. Profiles of elastic strain declined steadily from the top of the hypocotyls toward the basis, whereas the profiles of relative elemental growth rate along the hypocotyls showed maxima within the growing zones. In contrast to earlier hypotheses, we conclude that turgor-induced elastic cell wall strain and growth do not correlate precisely in growing hypocotyls.     

ACAULIS5 controls Arabidopsis xylem specification through the prevention of premature cell death

Cell size and secondary cell wall patterning are crucial for the proper functioning of xylem vessel elements in the vascular tissues of plants. Through detailed anatomical characterization of Arabidopsis thaliana hypocotyls, we observed that mutations in the putative spermine biosynthetic gene ACL5 severely affected xylem specification: the xylem vessel elements of the acl5 mutant were small and mainly of the spiral type, and the normally predominant pitted vessels as well as the xylem fibers were completely missing. The cell-specific expression of ACL5 in the early developing vessel elements, as detected by in situ hybridization and reporter gene analyses, suggested that the observed xylem vessel defects were caused directly by the acl5 mutation. Exogenous spermine prolonged xylem element differentiation and stimulated cell expansion and cell wall elaboration in xylogenic cell cultures of Zinnia elegans, suggesting that ACL5 prevents premature death of the developing vessel elements to allow complete expansion and secondary cell wall patterning. This was further supported by our observations that the vessel elements of acl5 seemed to initiate the cell death program too early and that the xylem defects associated with acl5 could be largely phenocopied by induction of premature, diphtheria toxin-mediated cell death in the ACL5-expressing vessel elements. We therefore provide, for the first time, mechanistic evidence for the function of ACL5 in xylem specification through its action on the duration of xylem element differentiation.