Auxin-induced cell expansion in relation to cell wall extensibility

Decapitation of 30 mm oat coleoptiles, which are commonly usedfor growth tests, resulted in a decrease in their elastic extensibility(DE) but not in their plastic extensibility (DP). By auxin treatmentunder osmotic stress, old coleoptile (45 mm) cells showed noincrease in subsequent expansion in water, whereas RNA synthesisin these cells was stimulated just as in young ones. Auxin increasedthe DE of young coleoptile cell walls but not that of old ones.Significant increase of DE occurred in only 10 min, and themaximum level of DE was reached in 15 min of the auxin treatment.An antiauxin (2,4,6-trichlorophenoxyacetic acid), mitomycinC and cycloheximide inhibited auxin-induced increases in expansionand DE (or Rex, reversible extensibility) of young coleoptilecells. (Received July 23, 1968; )     

Auxin Stimulates Cl-Uptake by Oat Coleoptiles

The effects of auxin on net ion fluxes near parenchyma of oatcoleoptiles were studied using the non-invasive MIFE systemto measure specific ion fluxes using ion selective microelectrodes.Application of 10 µM1-naphthaleneacetic acid (NAA) for3 h caused doubling of coleoptile segment growth, without changingthe pH of the unbuffered bathing solution from pH 5.4 duringthat time. Short term experiments revealed that auxin led toan immediate three-fold increase of chloride influx to 1200nmol m^-2s^-1, maintained for at least 1 h. In the first minutesafter auxin application, proton fluxes were small (-25 nmolm^-2s^-1, an efflux) and tended to decrease, whereas potassiumand calcium fluxes changed little, fluctuating from -100 to0 nmol m^-2s^-1and from -15 to 0 nmol m^-2s^-1, respectively. Itis suggested that one target of auxin action in plant cellsis the plasma membrane chloride transport system mediating increasedchloride uptake.Copyright 1998 Annals of Botany Company Auxin, chloride transport, ion flux,Avena sativaL., oat.     

Auxin and Ethylene Control of Growth in Seedlings of Zea mays L. and Avena sativa L

The effects of applied ethylene on the growth of coleoptilesand mesocotyls of etiolated monocot seedlings (oat and maize)have been compared with those on the epicotyl of a dicot seedling(the etiolated pea). Significant inhibition of elongation by ethylene (10 µll^–1for 24 h) was found in intact seedlings of all three species,but lateral expansion growth was observed only in the pea internodeand oat mesocotyl tissue. The sensitivity of the growth of seedlingparts to ethylene is in the decreasing order pea internode,oat coleoptile and oat mesocotyl, with maize exhibiting theleast growth response. Although excised segments of mesocotyland coleoptile or pea internode all exhibit enhanced elongationgrowth in IAA solutions (10^–6–2 ? 10^–5 moll^–1), no consistent effects were found in ethylene. Ethyleneproduction in segments was significantly enhanced by applicationof auxin (IAA, 10^–5 mol l^–6 or less) in all tissuesexcept those of the eat mesocotyl. Segments of maize show a slow rate of metabolism of applied[2-¹⁴C]IAA (30 per cent converted to other metabolites within9 h) and a high capacity for polar auxin transport. Ethylene(10 µl l^–1 for 24 h) has little effect on eitherof these processes. The oat has a smaller capacity for polartransport than maize and the rate ef metabolism of auxin isas fast as in the pea (90 per cent metabolized in 6 h). Althoughethylene pretreatment does not change the rate of auxin metabolismin oat, there is a marked reduction in auxin transport. It is proposed that the insensitivity of maize seedlings toethylene is related to the supply and persistence of auxin whichcould protect the seedling against the effects of applied orendogenously produced ethylene. Although the mesocotyl of oatis sensitive to applied ethylene it may be in part protectedagainst ethylene in vivo by the absence of an auxin-enhancedethylene production system. The results are discussed in relationto a model for the auxin and ethylene control of cell growthin the pea.     

Cell elongation and metabolic turnover of the cell wall as affected by auxin and cell wall degrading enzymes

A cell wall fraction (pectic substances) of oat coleoptile segmentsfed with ¹⁴C-glucose contained more radioactivity under theeffect of auxin than did the control. When labeled segmentswere grown for 6 hr in auxin or glucanase solution the labelin the hemicellulose fraction decreased as growth increased.ß-1,3-Glucanase prepared from the culture of a fungus,Sclerotinia libertiana, induces elongation of segments of thepea stem and the oat coleoptile. Traces of cellulase and pectinmethylesterase contaminating the enzyme preparation are notresponsible for the stimulatory effect. Cellulase seemed tobe rather inhibitory and pectin methylesterase showed only aslight effect on coleoptile elongation. A possible relationshipbetween the metabolic turnover of hemicellulosic polysaccharideand cell wall extension is suggested. (Received February 5, 1968; )     

Distribution of Endogenous Indole-3-acetic Acid and Growth Inhibitor(s) in Phototropically Responding Oat Coleoptiles

The relationship between the flank growth of oat (Avena sativaL. cv. Victory) coleoptiles and the distribution of endogenousindole-3-acetic acid (IAA) and growth inhibitor(s) in the coleoptileswas studied for the second positive phototropic curvature inducedby a continuous unilateral illumination with white light (0.1W.m^–2). The phototropic curvature was caused by growthinhibition at the lighted side and growth promotion at the shadedside. Using electron capture detection gas chromatography, weanalyzed the distribution of endogenous IAA in phototropicallyresponding oat coleoptiles and found that the IAA was evenlydistributed over the lighted and shaded sides during the phototropicresponse; there was also no detectable difference in the amountsof IAA between phototropically stimulated and non-irradiatedcoleoptiles. By contrast, oat coleoptile straight-growth testresults showed that the amount of unknown acidic growth inhibitor(s),different from abscisic acid, increased in the lighted halfof the coleoptiles and decreased in the shaded half, as comparedto the amount in the non-irradiated half. These data suggestthat the phototropic curvature of oat coleoptile is inducedby a difference in lateral flank growth through a lateral gradientof endogenous growth inhibitor(s) rather than of IAA. (Received February 10, 1988; Accepted July 29, 1988)     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

RIBONUCLEIC ACID METABOLISM AND CELL EXPANSION IN OAT COLEOPTILE

RNA metabolism in oat coleoptiles was studied using physiologicalresponses to 5-FU and actinomycin D; autoradiographic detectionof RNA and protein synthesis; and estimation of ribosomal concentrationby analytical ultracentrifugation. 5-FU failed to inhibit growthof either intact coleoptiles or isolated coleoptile segmentsbut completely blocked cell division in roots. Actinomycin Dmarkedly inhibited auxin-induced expansion of coleoptile segments.When supplied to isolated segments from coleoptiles of variouslengths the RNA precursors cytidine, adenine and adenosine allshowed weak incorporation into RNA of nuclei and in some cases,to a lesser extent, RNA of cytoplasm. IAA did not affect thisRNA synthesis but it was considerably reduced by actinomycinD. A proportion of the label incorporated from RNA precursorswas not removable with either RNase, PCA or hot TCA but wasextracted by trypsin. The amount of this spurious incorporationincreased with coleoptile age, as did the ability to incorporatelabelled amino acids. The concentration of both free and boundribosomes does not increase in growing coleoptiles and may evendecline. Free ribosomes decline markedly in fully grown coleoptileswhile the proportion of bound ribosomes increases. It is concludedthat young coleoptiles contain a full complement of ribosomesnecessary for subsequent growth but normal growth is dependenton continued production of an actinomycin D-sensitive messenger-typeRNA. No evidence for auxin mediation of RNA synthesis was found. ¹Present address: Laboratory of Cell Biology, Faculty of Science,Osaka City University, Sugimoto-cho, Sumiyoshi-ku, Osaka, Japan.     

Plasmodesmata, Tropisms, and Auxin Transport

Attempts were made to disrupt the plasmodesmata between oatcoleoptile cells (Avena saliva L. cv. Victory) by severe plasmolysis.Coleoptiles, allowed to regain turgor after plasmolysis, wereable to execute geotropic and phototropic curvatures and segmentswould grow in response to applied auxin. In coleoptiles similarlytreated, studies with [¹⁴C]IAA have shown that longitudinal,basipetal transport of auxin still takes place and, as in controls,IAA is preferentially redistributed laterally within coleoptilesorientated horizontally. Physical continuity of the symplast of oat coleoptile cellsmay not always be disrupted by severe plasmolysis. Nevertheless,functional continuity appears to be interrupted. Despite this,all the processes involved in the execution of tropistic curvaturesremain intact, including transport of hormones. Plasmodesmatalcontinuity between oat coleoptile cells appears not to be anecessary requirement for auxin transport.     

Action of Inhibitors of RNA and Protein Synthesis on Cell Enlargement

Further studies with inhibitors of protein synthesis are presented to support the conclusion, drawn from work with chloramphenicol, that protein synthesis is a critical limiting factor in auxin-induced cell expansion. The indoleacetic acid-induced elongation of oat coleoptile sections was strongly inhibited by dl-p-fluorophenylalanine, and the inhibition is antagonized by phenylalanine. Puromycin at 10(-4)m very strongly inhibited the indoleacetic acid-induced growth of oat coleoptile and artichoke tuber sections and exerted a less powerful effect on pea stem sections. As found earlier with chloramphenicol, concentrations of puromycin effective in inhibiting the growth of coleoptile sections had quantitatively similar effects on protein synthesis, as measured by the incorporation of C(14)-leucine into protein of the coleoptile tissue. Several analogues of RNA bases were also tested, but while 8-azaguanine very strongly inhibited growth of artichoke tuber disks, 6-azauracil was the only one of this group clearly inhibitory to growth in coleoptile or pea stem sections. Actinomycin D actively inhibited both elongation and the incorporation of C(14)-leucine into protein in oat coleoptile sections. Inhibition of the 2 processes went closely parallel. Actinomycin D also powerfully inhibited growth of artichoke tuber disks. All the compounds effective in inhibiting growth generally inhibited the uptake of leucine as well.The possibility that auxin causes cell enlargement in plants by inducing the synthesis of a messenger RNA and of one or more new but unstable enzymes, is discussed. Possible but less favored alternative explanations are: A) that auxin induces synthesis of a wall protein, or B) that the continued synthesis of some other unstable protein (by a process independent of auxin) may be a prerequisite for cell enlargement.     

Auxin-stimulated RNA Synthesis in Oat Coleoptile Cells

Using oat coleoptile segments the following results were obtained. Ten mg/l auxin (indole-3-acetic acid) increased the incorporation of uracil-2-¹⁴C and orthophosphate-³²P into RNA fraction during a relatively short incubation period. Stimulation of ³²P incorporation due to auxin was found only in the region heavier than ribosomal RNA, probably in the messenger RNA region. The stimulation of uracil-2-¹⁴C incorporation into RNA caused by auxin was not influenced by the presence of 0.3 M mannitol which prevents osmotically the water absorption of cells. It is concluded that auxin primarily stimulates the biosynthesis of RNA, possibly messenger, in oat coleoptile cells.     

The Analysis of Correlative Growth in the Etiolated Oat Seedling in Relation to Carbon Dioxide and Nutrient Supply

To overcome the reduced extension growth of the coleoptile whichoccurs when oats are grown in air enriched with 5 per cent.CO¹, plants have been provided with nutrients via the roots.2 per cent, sucrose, glucose or mannitol so applied furtherpromoted the mesocotyl and further depressed the coleoptile.Root growth was also depressed. To induce promotion of coleoptile growth by externally appliedsucrose, seedlings were heated in darkness at 40° C. for3 hours so restricting selectively the growth of the mesocotyl.Promotion of the coleoptile, however, was not observed. Application of mixed Na and K nitrates occasioned an immediategrowth promotion of doleoptile and leaves in both the presenceand absence of CO², and also a.much less pronounced promotionof the mesocotyl in CO²; there was no effect in air. This enhancedgrowth of the coleoptile and leaves was coupled with a correspondinglygreater dry weight and also with an increased outflow of reservesfrom the endosperm into the plumule. Thus, while externally applied sugars seemed not to reach thecoleoptile, those made available from the endosperm as a resultof improved nitrogen supply were rapidly translocated to it.Simultaneous provision to the roots of nitrate and sucrose didnot improve the absorption and translocation of sugar. An analysis of covariance has been computed using the mesocotyland coleoptile length data together with the outflow from theendosperm and the conclusions so derived are discussed in relationto the problem of growth integration in etiolated oat seedlings.     

Coleoptile Senescence in Rice (Oryza sativa L.)

We investigated the cellular events associated with cell deathin the coleoptile of rice plants (Oryza sativa L.). Seeds germinatedunder submergence produced coleoptiles that were more elongatedthan those grown under aerobic conditions. Transfer of seedlingsto aerobic conditions was associated with coleoptile opening(i.e. splitting) due to death of specific cells in the sideof the organ. Another type of cell death occurred in the formationof lysigenous aerenchyma. Senescence of the coleoptile was alsonoted, during which discolouration of the chlorophyll and tissuebrowning were apparent. DNA fragmentation was observed by deoxynucleotidyltransferase-mediateddUTP nick end labelling (TUNEL) assay, and further confirmedby the appearance of oligonucleosomal DNA ladders in senescentcoleoptile cells. Two nucleases (Nuc-a and Nuc-b) were detectedby in-gel-assay from proteins isolated from coleoptiles. Nuc-a,commonly observed in three cell death phases required eitherCa²⁺or Mg²⁺, whereas Nuc-b which appeared during senescencerequired both Ca²⁺and Mg²⁺. Both nucleases were strongly inhibitedby Zn²⁺. Copyright 2000 Annals of Botany Company Aerenchyma, rice, cell death, coleoptile, fragmentation, nuclease, Oryza sativa, senescence, split, submergence, TUNEL     

Polar auxin transport and auxin-induced elongation in the absence of cytoplasmic streaming

Summary When cytoplasmie streaming in oat and maize coleoptile cells is completely inhibited by cytochalasin B (CB), polar transport of auxin (indole-3-acetic acid) continues at a slightly reduced rate. Therefore, cytoplasmic streaming is not required for polar transport. Auxin induces elongation in CB-inhibited coleoptile and pea stem segments, but elongation rate is reduced about 40% by CB. Therefore, stimulation of cytoplasmic streaming cannot be the means by which auxin promotes cell elongation, but streaming may be beneficial to elongation growth although not essential to it. A more severe inhibition of elongation develops after several hours in CB. With coleoptiles this could be due to inhibition of sugar uptake; in pea tissue it may be due to permeability changes and cytoplasmic degeneration. CB does not disorganize or disorient microfilament bundles when it inhibits streaming in maize, but appears instead to cause hypercondensation of microfilament material.     

Inhibiting Effect of Auxin on Glucosamine Incorporation into Cell Walls of Rice Coleoptiles

Auxin induced growth and decreased the hexosamine content ofthe cell walls of rice coleoptile sections. Indole-3-aceticacid (IAA) at 10^–5 M inhibited the incorporation of ¹⁴C-glucosamineinto the cell walls. IAA did not affect the ¹⁴C-incorporationinto the cytoplasm, while inhibitors of glycoprotein synthesis,unicamycin and monensin, suppressed the incorporation into boththe cytoplasm and the cell walls. The radioactivity due to labeledglucosamine in the cell walls increased during the chase, butthis increase was inhibited by IAA. Among the cell wall fractions,the increase in radioactivity and its inhibition by IAA wereconspicuous in the hemicellulose I fraction. The inhibitoryeffect of IAA on glucosamine incorporation into the cell wallswas observed even in the presence of 0.15 M mannitol solutionwhich completely suppressed the IAA-induced growth. These resultssuggest that auxin induces growth at least partly by inhibitingthe transport of asparagine-linked glycoproteins from the cytoplasmto the cell walls. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka 558, Japan (Received July 23, 1986; Accepted December 22, 1986)     

Flux Studies and Compartmentation Analysis of Gibberellin A1 in Oat Coleoptiles

Both uptake and loss of GA₁ consist of two distinct transportcomponents. The distribution of GA₁ within oat coleoptile cellsmay thus be in at least two pools, which might arise from compartmentation,localization, or binding of GA₁. There is a net uptake of GA₁,supplied at physiological levels (1.16 x 10^–7 M), intothe slowly exchanging pool and an immobile metabolite of GA₁(probably GA₈) is produced. This is treated as a third poolwithin the cells.     

The spontaneous growth response of maize coleoptile segments and the change in tissue 8

Decapitated segments from maize (Zea mays L.) coleoptiles orientedvertically in an upright position show a strong spontaneousgrowth response (SGR) 3 h after decapitation. The latent periodof the SGR is markedly reduced when these segments are orientedin an inverted position. Coleoptile segments with intact tipsexhibit a weak and transient SGR in the vertical upright orientation.However, in the inverted orientation, these segments show atypical SGR. The data are inconsistent with the current hypothesisthat the SGR is caused by a time-dependent increase in tissuesensitivity to auxin. The parallel increase in membrane potentialdifference and growth rate during the time-course of the SGRindicates a possible role for PM H⁺-ATPase in the establishmentof the SGR in maize coleoptile segments. Key words: Auxin, spontaneous growth response, membrane potential, plasma membrane H⁺-ATPase, Zea mays L.     

Correlation of cytokinins and abscisic acid with monocarpic senescence in soybeans

The involvement of cytokinins and abscisic acid (ABA) in themonocarpic senescence (foliar yellowing following fruit development)of soybeans was examined. Foliar sprays of cytokinin (10^–4M zeatin or 10^–5 M benzyladenine), begun when the plantsfirst set fruit and repeated every other day, significantlydelayed, but did not prevent, monocarpic senescence. Foliarsprays of 10^–4 M ABA, applied in the same manner, significantlyhastened senescence of fruiting soybeans but apparently hadno effect on depodded plants. Leaf and stem material from pre-senescentand senescent plants was extracted, chromatographed, and bioassayedfor cytokinin-lilce activity (Amaranthus betacyanin productionassay) and ABA-like activity (oat coleoptile straight growthassay for inhibitors). ABA-like activity increased, and cytokinin-likeactivity decreased in shoot tissue before the plants began tosenesce. Cytokinin-like activity in the fruit also declinedduring this period. These results implicate a decrease in cytokininsand an increase in ABA-like inhibitors in the control of monocarpicsenescence of soybeans, but neither alone is causal. ¹ Supported in part by Research Grant 416-15-79 from the USDACooperative State Research Service under PL 89–106. ² Present address: Biology Dept., College of St. Benedict'sSt. Joseph, Minn. 56374, U.S.A. (Received February 4, 1978; )     

Effect of cycloheximide and cordycepin on auxin-induced elongation and {beta}-glucan degradation of noncellulosic polysaccharides of Avena coleoptile cell wall

The effect of cycloheximide (10^–5 M) and cordycepin (10^–4M) used as protein and RNA synthesis inhibitors, respectively,on auxin action in noncellulosic ß-glucan degradationof Avena coleoptile cell wall was investigated. Both depressedauxin-induced ßglucan degradation of the cell wallas well as auxin-induced elongation and cell wall loosening,suggesting that the process of ß-glucan degradationof the cell wall is closely associated with cell wall looseningand that auxin enhances the activity of an enzyme for ß-glucandegradation through de novo synthesis of RNA and protein butnot through activation of the enzyme in situ. Kinetic studywith the inhibitors showed that RNA metabolism involved in ß-glucandegradation was stimulated by auxin treatment of only 15 minwhile a longer lag phase (about 1 hr) existed for the synthesisof the enzyme. (Received December 16, 1978; )