Absence Of Auxin Research Articles

Rooting and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris are enhanced by the ectomycorrhizal fungus Hebeloma cylindrosporum

The effect of different strains of the ectomycorrhizal fungus Hebeloma cylindrosporum on rooting in vitro and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris was studied. Two clones of P. pinaster and one of P. sylvestris were unable to root in the absence of auxin, but were induced to root on a medium devoid of auxin by all the fungal strains. Wild‐type and indoleacetic acid (IAA)‐overproducing mutant strains of the fungus stimulated rooting of clones showing a good reactivity to auxin to the same extent. In contrast, with a clone of P. sylvestris that showed low reactivity to auxin, IAA‐overproduction by the fungus was advantageous for the induction of rooting of cuttings. Adventitious roots formed in the presence of a fungal strain were completely surrounded by a loosely packed network of hyphae which formed mycorrhizas as soon as roots grew outside the agar medium. During acclimatization, fungal inoculation improved the survival of rooted cuttings. At the end of acclimatization, fungal mycelia could be easily detected in the culture substrate of cuttings inoculated with dikaryotic strains and most of the pines' short roots were mycorrhizal. Monokaryotic mycelia, which have a lower growth rate and a lower infectivity, displayed poor ability to colonize the substrate and to form mycorrhizas. Two months after the end of acclimatization, fungal inoculation frequently depressed the growth of acclimatized cuttings of the clone J of P. pinaster. No depressive effect was observed with clone 78 and growth stimulation could even be observed with the infective dikaryon D1 which formed numerous mycorrhizas. From these studies, it was concluded that ectomycorrhizal fungi could be a suitable tool for improving rooting in vitro and survival at acclimatization of micropropagated conifer cuttings.

Maturation‐related loss in rooting competence by loblolly pine stem cuttings: The role of auxin transport, metabolism and tissue sensitivity

A comparison of rooting ability of stem cuttings made from hypocotyls and epicotyls from 50‐day‐old seedlings of loblolly pine (Pinus taeda L.) reveals a dramatic decline by epicotyl cuttings, which do not root at all in 20–30 days in the presence or absence of auxin. In contrast, almost all the hypocotyls root during this time, but only in the presence of exogenously applied auxin. The failure of epicotyls to root does not appear to be due to differences in [14C]‐labeled auxin uptake, transport, metabolism, or tissue distribution in the two types of cuttings. At the cellular level, initial responses to auxin, such as differentiation of the cambium into parenchyma, occur in both types of cuttings, but localized rapid cell division and root meristem organization are not observed in epicotyls. Autoradiography revealed that radioactivity from a‐naphthalene acetic acid is bound in the cortex but not concentrated at sites of root meristem organization prior to the organization of the meristem in hypocotys. During the development of the epicotyl. cellular competence to form roots appears to be lost. Although this loss in competence is not associated with a concurrent loss in ability to transport auxin polarly, the latter process appears to play a key role in rooting other than to move auxin to the site of root formation. The phytotropin N‐(1‐naphthyl)phthalamic acid inhibits rooting if applied during the first 3 days after the cutting is made, but does not affect auxin concentration or metabolism at the rooting site.

Stimulation of Root Formation by Thiol Compounds

Root formation in shoot cuttings of soybean (Glycine max L. `Williams'), mungbean (Phaseolus aureas Mdlbg.), English ivy (Hedera helix L.), and apple (Malus ×domestica Borkh. `Jork 9') was stimulated by dithiothreitol and reduced glutathione in the presence and absence of auxin (IAA) shock. In soybean, in the absence of auxin, root formation was stimulated to about the same extent by glutathione alone as with auxin alone. The roots induced by thiol compounds were longer than roots induced by auxin shock and were completely normal in appearance. Roots produced with auxin shock alone were short and exhibited characteristic auxin-induced deformations. With a combination treatment of auxin shock and thiol compounds, roots were more numerous than with either alone, somewhat longer than with auxin alone, and exhibited fewer of the usual deformations characteristic of roots grown in the presence of external auxins. The thiol compounds also were beneficial for rooting Malus shoots propagated from callus in vitro. The thiol compounds were most beneficial with older cuttings where auxin shock was often insufficient to obtain roots. In shoots where rooting was stimulated by thiol agents, shoots grew more rapidly than in those where rooting was induced by auxin shock alone. These findings suggest a use for thiol compounds alone or in combination with auxin shock to induce differentiation of root primordia as well as for stimulation of root growth. Chemical name used: indole-3-acetic acid (IAA).

Genetic Dissection of the Relative Roles of Auxin and Gibberellin in the Regulation of Stem Elongation in Intact Light-Grown Peas.

Exogenous gibberellin (GA) and auxin (indoleacetic acid [IAA]) strongly stimulated stem elongation in dwarf GA1-deficient le mutants of light-grown pea (Pisum sativum L.): IAA elicited a sharp increase in growth rate after 20 min followed by a slow decline; the GA response had a longer lag (3 h) and growth increased gradually with time. These responses were additive. The effect of GA was mainly in internodes less than 25% expanded, whereas that of IAA was in the older, elongating internodes. IAA stimulated growth by cell extension; GA stimulated growth by an increase in cell length and cell number. Dwarf lkb GA-response-mutant plants elongated poorly in response to GA (accounted for by an increase in cell number) but were very responsive to IAA. GA produced a substantial elongation in lkb plants only in the presence of IAA. Because lkb plants contain low levels of IAA, growth suppression in dwarf lkb mutants seems to be due to a deficiency in endogenous auxin. GA may enhance the auxin induction of cell elongation but cannot promote elongation in the absence of auxin. The effect of GA may, in part, be mediated by auxin. Auxin and GA control separate processes that together contribute to stem elongation. A deficiency in either leads to a dwarfed phenotype.

Vegetative propagation ofParkia biglobosa(Jacq.) Benth., an undomesticated fruit tree from West Africa

SummaryThe rooting of single node leafy stem cuttings from seedlings of Parkia biglobosa (Jacq.) Benth. was examined with respect to provenance, auxin application, basal wounding, and the nodal position on the shoot. Provenances of P. biglobosa from three West African savanna ecozones: Derived, Guinea and Sudan were used for the study. In the absence of auxins and basal wounding, 40% of the cuttings rooted and 80% survived throughout the observation period of 6–8 weeks. However, the addition of auxins and wounding significantly affected the rooting of the cuttings. 100 ppm of the auxin α-napthalene acetic acid (NAA) was more effective than 100 ppm indole-3-butyric acid (IBA). However, when the concentration of both auxins was increased to 200 ppm, IBA was more effective than NAA. In general, wounding cuttings by making a diagonal cut at the end of the stem enhanced rooting, yet the application of 100 ppm NAA negated the beneficial effect. Bruising the stem was detrimental to rooting, except for cuttings f...

Putrescine Control of Peroxidase Activity in the Inductive Phase of Rooting in Poplar Shoots in vitro, and the Adversary Effect of Spermidine

Poplar shoots raised in vitro rooted by 100 °lo in the presence of an auxin while in its absence they did not root. Putrescine, however, and the inhibitor of spermidine synthase, cyclohexylamine (CHA), which favours putrescine accumulation, were able to promote up to 40 % rooting in the absence of auxin. The inhibitory effect of aminoguanidine (AG), which inhibits diamine oxidase, indicated that the catabolic pathway of putrescine was involved in the rooting process. The inductive phase of rooting in poplar shoots was characterized by an increase of peroxidase activity followed by a rapid decrease. Putrescine and CHA favoured this variation. AG and spermidine had adversary effects.

Rheological analysis of viscoelastic cell wall changes in maize coleoptiles as affected by auxin and osmotic stress

The effects of auxin and osmotic stress on elongation growth of maize (Zea mays L.) coleoptile segments are accompanied by characteristic changes in the extensibility of the growth‐limiting cell walls. At full turgor auxin causes growth by an increase in wall extensibility (wall looseining). Growth can be stopped by an osmotically produced step‐down in turgor of 0.45 MPa. Under these conditions auxin causes the accumulation of a potential for future wall extension which is released after restoration of full turgor. Turgor reduction causes a reversible decrease in wall extensibility (wall stiffening) both in the presence and absence of auxin. These changes in vivo are correlated with corresponding changes in the rheological properties of the cell walls in vitro which can be traced back to specific modifications in the shape of the hysteretic stress‐strain relationship. The longitudinally load‐bearing walls of the coleoptile demonstrate almost perfect viscoelasticity as documented by a nearly closed hysteresis loop. Auxin‐mediated wall loosening causes an increase of loop width and thus affects primarily the amount of hysteresis in the isolated wall. In contrast, turgor reduction by osmotic stress reduces loop length and thus affects primarily the amount of viscoelastic wall extensibility. Pretreatment of segments with anoxia and H2O2 modify the hysteresis loop in agreement with the conclusion that the wall‐stiffening reaction visualized under osmotic stress in vivo is an O2‐dependent process in which O2 can be substituted by H2O2. Cycloheximide specifically inhibits auxin‐mediated wall loosening without affecting wall stiffening, and this is mirrored in specific changes of the hysteresis loop. Corroborating a previous in vivo study (Hohl et al. 1995, Physiol. Plant. 94: 491–498) these results show that cell wall stiffening in vivo can also be demonstrated by Theological measurements with the isolated cell wall and that this process can be separated from cell wall loosening by specific changes in the shape of the hysteresis loop.

Selective Inhibition of Auxin-Stimulated NADH Oxidase Activity and Elongation Growth of Soybean Hypocotyls by Thiol Reagents.

The NADH oxidase activity of isolated vesicles of soybean (Glycine max cv Williams 82) plasma membranes and elongation growth of 1-cm-long hypocotyl segments were stimulated by auxins (indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid [2,4-D]). The auxin-induced stimulations of both NADH oxidase and growth were prevented by the thiol reagents N-ethylmaleimide, p-chloromercuribenzoate, 5,5[prime]-dithiobis(2-nitrophenylbenzoic acid), dithiothreitol, and reduced glutathione. These same reagents largely were without effect on or stimulated slightly the basal levels of NADH oxidase and growth when assayed in the absence of auxins. In the presence of dithiothreitol or reduced glutathione, both 2,4-D and indole-3-acetic acid either failed to stimulate or inhibited the NADH oxidase activity. The rapidity of the response at a given concentration of thiol reagent and the degree of inhibition of the 2,4-D-induced NADH oxidase activity were dependent on order of reagent addition. If the thiol reagents were added first, auxin stimulations were prevented. If auxins were added first, the inhibitions by the thiol reagents were delayed or higher concentrations of thiol reagents were required to achieve inhibition. The results demonstrate a fundamental difference between the auxin-stimulated and the constitutive NADH oxidase activities of soybean plasma membranes that suggest an involvement of active-site thiols in the auxin-stimulated but not in the constitutive activity.

Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development

Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype. While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines. The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue. Confirmation that the genomic sequence responsible for the selected phenotype has indeed been isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions. Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated.

Auxin inducibility and developmental expression of axi 1: a gene directing auxin independent growth in tobacco protoplasts

We describe the characterization of axi 1, a tobacco gene isolated by activation T-DNA tagging which apparently plays a role in auxin action. Upon deregulated expression, axi 1 confers on protoplasts the ability to grow in culture not only in the absence of auxin but also in high auxin concentrations where maximal frequencies of cell division are not observed in wild-type protoplasts. In wild-type plants axi 1 is transcribed principally in root tissue. In the tagged plant line, axi 159, axi 1 RNA can be detected in all tissues tested. Freshly isolated wild-type protoplasts require auxin for the accumulation of detectable levels of axi 1 transcript and this precedes maximal levels of cell division. In contrast, axi 1 RNA appears in protoplasts isolated from axi 159 plants in the absence of auxin. axi 1 was localized to 6.2 kb of plant genomic DNA flanking the right T-DNA border sequence. axi 1 is interrupted by nine introns and in tobacco it is a member of a small gene family. Database searching reveals no similarity within the coding region with other genes. Sequences within the fourth intron are similar to those located in the non-coding regions of other plant genes, some of which are known to be auxin inducible. A DNA fragment containing the conserved sequence acts as an auxin responsive element in transient expression assays in wild-type protoplasts and this response is higher in axi 159 protoplasts. This suggests that auxin induced axi 1 expression may be mediated by a region contained within an intron sequence and that the axi 1 product might play a role in this induction.

Auxin inducibility and developmental expression of axi 1: a gene directing auxin independent growth in tobacco protoplasts.

We describe the characterization of axi 1, a tobacco gene isolated by activation T-DNA tagging which apparently plays a role in auxin action. Upon deregulated expression, axi 1 confers on protoplasts the ability to grow in culture not only in the absence of auxin but also in high auxin concentrations where maximal frequencies of cell division are not observed in wild-type protoplasts. In wild-type plants axi 1 is transcribed principally in root tissue. In the tagged plant line, axi 159, axi 1 RNA can be detected in all tissues tested. Freshly isolated wild-type protoplasts require auxin for the accumulation of detectable levels of axi 1 transcript and this precedes maximal levels of cell division. In contrast, axi 1 RNA appears in protoplasts isolated from axi 159 plants in the absence of auxin. axi 1 was localized to 6.2 kb of plant genomic DNA flanking the right T-DNA border sequence. axi 1 is interrupted by nine introns and in tobacco it is a member of a small gene family. Database searching reveals no similarity within the coding region with other genes. Sequences within the fourth intron are similar to those located in the non-coding regions of other plant genes, some of which are known to be auxin inducible. A DNA fragment containing the conserved sequence acts as an auxin responsive element in transient expression assays in wild-type protoplasts and this response is higher in axi 159 protoplasts. This suggests that auxin induced axi 1 expression may be mediated by a region contained within an intron sequence and that the axi 1 product might play a role in this induction.

Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α‐difluoromethylarginine) and DFMO (α‐difluoromethylomithine), aminoguanidine (AG) and methylglyoxal‐bis‐guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence.The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ1‐pyrroline pathway, in the inductive phase of rooting in poplar shoots.

Jasmonate-inducible expression of a potato cathepsin D inhibitor-GUS gene fusion in tobacco cells

A potato gene encoding cathepsin D inhibitor (CDI) is expressed constitutively in tubers and flower buds and it is inducible in leaves upon wounding of the tissue or by treatment with methyl jasmonate (MJA). A fusion gene (CDI:GUS) in which the 2.4 kb long promoter of the CDI gene was translationaly fused with the coding sequence for beta-glucuronidase (GUS) showed MJA-inducible expression in transformed tobacco cells in suspension. The maximum level of induction by MJA was obtained in the absence of auxin and repression of MJA-inducible expression of the fusion gene by auxin was released by aphidicolin, the results suggesting that MJA-inducible expression is repressed during active cell division. JA and MJA showed similar activities in inducing the expression of the fusion gene, while other JA-related compounds such as cucurbic acid, tuberonic acid and dihydrojasmonic acid neither induced expression of the fusion gene nor inhibited the MJA-inducible expression of the fusion gene. Methyl dihydrojasmonate specifically stimulated the MJA-inducible expression of the fusion gene. The MJA-inducible expression of the fusion gene was observed even with a 100 bp long promoter of the CDI gene albeit with significantly decreased level of expression compared to the 2.4 kb long promoter. The 100 bp long CDI promoter did not contain a G-box or hexamer motif that had been implicated in the MJA-responsive expression of several other plant genes. Further mutagenesis of the 100 bp long promoter by deletion or oligonucleotide insertion suggested that although a sequence between -100 and -82 is required for the MJA-responsive expression, the presence of this sequence alone does not confer the MJA-responsive expression.

Organ-Specific and Auxin-Inducible Expression of Two Tobacco par A-Related Genes in Transgenic Plants

We have isolated four genomic DNA clones that contain the transcription initiation site of the parA gene(s) from a tobacco genomic library by hybridization with the 5' segment of the parA cDNA previously isolated. They were classified into two types on the basis of their nucleotide sequences. Southern blot analysis indicated that two types of clones were respectively derived from the two parental species of tobacco, Nicotiana tomentosiformis and Nicotiana sylvestris. The genes corresponding to these clones were designated as parAt and parAs, respectively, and the parA cDNA clone was shown to code for mRNA from parAt on the basis of its nucleotide sequence. The 5' regions about 400 nucleotides upstream from the transcription initiation sites of the parAt and parAs genomic clones were highly homologous to one another, but regions further upstream showed no significant similarity. The coding sequence of the GUS (beta-glucuronidase) reporter gene was linked to the 5'-upstream regions of parAt and parAs, and the sites of expression of these fusion genes were examined in transgenic tobacco plants. In the absence of auxins, both fusion genes were expressed in capsules at a late stage of seed development, mature seeds, a root apex and a root-hair zone whereas no significant expression was seen in other organs. Their expression was enhanced by 2,4-dichlorophenoxyacetic acid in most of the organs of tobacco. The results show that expression of these genes is regulated by both organ-specific and auxin-inducible mechanisms.

Levels of endogenous indole-3-acetic acid and indole-3-acetyl-aspartic acid during adventitious rooting in avocado microcuttings

Quantification of endogenous IAA and lAAsp was carried out during adventitious root formation in avocado microcuttings. Both auxin and conjugate were monitored in control cuttings (rooted in the absence of auxin) as well as in cuttings treated with a rooting promotor (IBA) or an auxin transport inhibitor (TIBA). Additionally, a histological study to follow root differentiation was carried out. In control cuttings IAA levels remained constant throughout the rooting process, however, in IB A-treated cuttings IAA levels increased 2-fold during the first 6 d. Addition of 200 fit* TIBA induced a slight decrease of IAA levels and inhibited root formation. As for lAAsp levels, both control and IBA-treated cuttings showed a big increase before root differentiation occurred and as the process went on, a progressive decrease took place. However, in TIBA-treated cuttings lAAsp levels not only did not increase but diminished progressively during the process. The role of auxin conjugates during the rooting process of avocado is discussed.

Inhibition of Golgi-apparatus function by brefeldin A in maize coleoptiles and its consequences on auxin-mediated growth, cell-wall extensibility and secretion of cell-wall proteins

Brefeldin A (BFA), a fungal metabolite causing dysfunction of the Golgi apparatus in plant and animal cells, was used to investigate the role of secretory processes at the plasma membrane in auxin-mediated elongation growth of maize (Zea mays L.) coleoptiles. In abraded coleoptile segments BFA produced, within less than 30 min, a decrease in the incorporation of [3H]leucine into tightly bound cell-wall proteins, accompanied by an increased incorporation into the intracellular pool of putative cell-wall glycoproteins. Total protein synthesis was not affected. Electron micrographs revealed striking morphological changes in dictyosomes (especially vesiculation of trans-cisternae), accumulation of Golgi vesicles and dilation of the endoplasmic reticulum. These effects are taken as indication that BFA interferes with the secretion of cell-wall components. Elongation growth of coleoptile segments in the presence and absence of auxin was inhibited by 80% in 20 mg·l−1 BFA. If BFA was applied to segments growing in the presence of auxin, maximum inhibition was reached after about 30 min, indicating that the growth response depends on an uninterrupted supply of a cell-wall or plasma-membrane component (“wall-loosening factor”) delivered by the secretory pathway. After its secretion, this factor has a rather short growth-effective life time. The inhibition of auxin-mediated growth by BFA was accompanied by an elimination of auxin-induced cell-wall extensibility and by an inhibition of auxin-induced proton excretion. Fusicoccin-induced proton excretion was similarly affected by BFA. It is concluded that both the wall-loosening process underlying elongation growth as well as proton excretion depend on an intact secretory pathway from the Golgi apparatus to the cell wall; however, a causal relationship between these processes is not warranted by the data.

Four 9-kDa proteins excreted by somatic embryos of grapevine are isoforms of lipid-transfer proteins.

Four 9-kDa small extracellular proteins produced by embryogenic cultures in the absence of auxin have been purified from the extracellular medium of grapevine somatic embryo cultures through cation-exchange chromatography and hydrophobic-interaction chromatography. The partial amino-acid sequences reflect high similarities between the four proteins as well as with the sequences established for carrot, spinach, millet and maize nonspecific lipid-transfer proteins. All these sequences show conservation of three cysteines at positions 4, 14 and 30-32, as well as glycine, valine, tyrosine and lysine residues at positions 5, 7, 17 and 37, respectively. In-vitro lipid-transfer assays reveal that the four proteins catalyze the transfer of phosphatidylcholine from liposomes towards mitochondria with an efficiency similar or higher than that of a purified maize lipid-transfer protein.

Transient changes in length and growth of wheat coleoptile segments following treatments with osmotica and auxin.

The dependence of elongation on the osmotic potential of the medium was investigated, using coleoptile segments (CS) of Triticim aestivum L. (cv. Hartri) and an optoelectronic device. The study aimed at separating the osmoelastic response from the irreversible growth response when an osmoticum (mannitol) was added, and to compare both processes in order to consider the possibility of growth-induced reduction in turgor pressure. The prompt inhibition of elongation registered just after addition of 50 mM mannitol as well as the subsequent resumption of the original elongation rate could be quantitatively explained by the extent and the kinetics of the osmoelastic relaxation. An initial reduction in the irreversible elongation component by mild osmotic stress could not be demonstrated. Above a critical value, the irrevesible growth was insensitive to a further increase in water potential. The minimum turgor pressure required to drive steady growth was not far from zero in both the presence and absence of auxin. The rate (r) of osmotically caused shortening per unit change of water potential [Formula: see text] was determined from the kinetics of CS shortening induced by addition of mannitol at nearly isotonic concentration (300 mM). This parameter relates a fractional change in length to the difference in water potential between inside and outside, and was assumed to depend largely on the hydraulic resistance of the tissue and cuticle. It was found to be independent of IAA. The relatively low value of Γ suggests significant reduction of turgor at high growth rates. In accordance with this conclusion, the extent of osmoelastic shortening after a transfer to 300 mM mannitol (dependent on wall strain) was significantly decreased in the presence of IAA. Addition of 100 μM IAA to CS growing at a constant rate induced pronounced oscillations in the rate of elongation, which may be connected with the change in elastic cell wall strain. Whereas the steady state growth rate before the addition of IAA was the same in the presence and in the absence of 50 mM mannitol, the maximum growth rate found after addition of IAA was substantially reduced in the mannitol variant.

Patch‐clamp analysis establishes a role for an auxin binding protein in the auxin stimulation of plasma membrane current in Zea mays protoplasts

The electrical response of Zea mays protoplasts to different auxins and to antibodies raised against an ER‐located auxin binding protein from maize (Zm‐ERabp1), was investigated using the patch‐clamp technique (whole‐cell configuration). Following a lag‐phase of 30–40 seconds, indole‐3‐acetic acid and 1‐naphthylacetic acid induced an outwardly directed current of positive charge in a concentration‐dependent manner. This current was further increased by the fungal toxin fusicoccin (FC). The current was observed only in the presence of Mg2+‐ATP in the patch‐pipette and was abolished after addition of erythrosin B, an inhibitor of H+‐ATPase, to the protoplasts indicating that the plasma membrane H+‐ATPase is activated by auxins and fusicoccin. Addition of antibodies directed against Zm‐ERabp1 abolished the current induced by auxins, without affecting the response of protoplasts to fusicoccin. Antibodies directed against a peptide representing part of the putative auxin binding domain of Zm‐ERabp1 showed auxin agonist activity, stimulating an outwardly directed membrane current in the absence of auxin. These results suggest that (i) Zm‐ERabp1 or antigenically related proteins represent a site for auxin perception through which the plasma membrane H+‐ATPase is activated, and (ii) that the activation of the H+‐ATPase by such proteins is initiated from outside the plasma membrane.

Isolation and characterization of three families of auxin down-regulated cDNA clones

Five cDNA clones (ADR6, ADR11-1, ADR11-2, ADR12-1 and ADR12-2), representing three families of auxin down-regulated (ADR) genes were isolated and characterized. These were isolated by screening a lambda Zap cDNA library with the partial cDNA clones p6, p11 and p12, isolated earlier (Baulcombe and Key, J Biol Chem 255: 8907-8913, 1980). Hybrid-select translation of ADR6, ADR11-2 and ADR12-2 clones produced polypeptides of 33 kDa 22.5 kDa and a 6 and 7 kDa respectively, when analyzed by SDS-PAGE. ADR6 and ADR12-2 gave one and two spots, respectively, on an IEF-SDS 2D gel. ADR11-2 probably encodes a basic protein as it was only resolved on non-equilibrium pH gradient gel electrophoresis (NEPHGE). Genomic Southern blot analysis of ADR6, ADR11 and ADR12 suggests that each represents a small multigene family. The RNA levels corresponding to ADR6, ADR11 and ADR12 decrease in response to applied auxin by 100-, 15- and 10-fold, respectively (Baulcombe and Key, 1980). Runoff transcription, done in the presence and absence of auxin, showed that the rate of transcription of the genes corresponding to ADR6, ADR11-2 and ADR12-2 was reduced in the presence of auxin, but the decrease was small relative to the decrease in the cytoplasmic levels of these mRNAs, in response to auxin. A comparative analysis of the influence of auxin on in vitro transcription and steady state RNA levels corresponding to these ADR cDNAs suggests that the decrease in rate of transcription due to auxin is not enough to account for the auxin-induced decrease in the steady state levels. Northern analysis showed developmental and organ/tissue-specific response of these ADR genes. Furthermore, the expression of the genes corresponding to ADR6 and ADR12-1 appears to be up-regulated by light, whereas the gene corresponding to ADR11 appears to be down-regulated by light.