Dose responses of cytokinins

Analysis of published data for the most widely used bioassays for cytokinins reveals that the dose responses are usually subsensitive, i.e. that S₉₀/S₁₀> 81. The data are. in general, insufficiently detailed and/or precise for the kinetics (cooperative, multiphasic or more complex) of the subsensitive responses to be determined. Genus-and species-specific differences in response type were found for callus growth, where the responses are ultrasensitive (S₉₀/S₁₀<81) for tabacco and Phaseolus Iunatus but mostly subsensitive for soybean and P. vulgaris.     

Rapid auxin-induced root growth inhibition requires the TIR and AFB auxin receptors

We investigated the relation between auxin-induced gene expression and the rapid auxin-induced growth inhibition in Arabidopsis thaliana roots. The natural auxin indole-3-acetic acid (IAA) induced a strong activation of gene expression as visualized by the DR5rev::GFP reporter gene technique. This effect was specific for active auxins and was abolished in knockout mutants of the F-box auxin receptors. We measured the IAA-induced growth inhibition at high time resolution and show that the F-box auxin receptor mutants failed to display this effect. We conclude that the F-box auxin receptors are needed for the response. In hypocotyls, auxin induces an increase in elongation growth, and this effect has been earlier shown to be independent of the F-box receptors. Based on these findings, we discuss differences in the growth control modes in roots and shoots. We demonstrate that the rapid auxin-induced root growth inhibition, unlike the induction of growth in hypocotyls, requires the presence of the F-box auxin receptors.     

Dose responses of gibberellins

To determine the response type, published data for the most widely used bioassays for gibberellins have been analyzed by means of a computer program for estimating sensitivity parameters, or by interpolation. The dose response data are almost uniformly subsensitive, i. e. more than an 81-fold increase in external gibberellin concentration is required for a change from 10 to 90% of maximal response (S₉₀/S₁₀). Data for the interaction of gibberellins with artificial membranes are, in contrast, markedly ultrasensitive (S₉₀/S₁₀± 10). This difference further strengthens the view that lipid structures do not function as receptors for gibberellins. Most of the subsensitive dose responses for gibberellins can be quite precisely represented by cooperative isotherms. However, available data are insufficiently detailed for an unequivocal choice among cooperative, multiphasic or more complex kinetics.     

A chlorate-hypersensitive, high nitrate/chlorate uptake mutant of Arabidopsis thaliana

N-ethylmaleimide (NEM) Lit 10-100 μ M led to a strong inhibition of the auxin-induced elongation growth of colcoptile segments, while fusicoccin-enhanced growth was not affected. Growth inhibition occurred only if NEM and auxin were allowed to act simultaneously. Preincubation of plant segments with NEM in the absence of auxin caused no inhibition of a subsequent growth stimulation by auxin, whenever NEM was removed before the application of IAA. However, preincubation with NEM plus auxin led to a remaining growth inhibition, which could not be reversed by a second auxin incubation in the absence of NEM. Fusicoccin added to NEM- plus auxin-treated segments was able to restore growth. It is suggested that auxin causes the unmasking of essential SH-groups of a protein to which NEM links covalently. thus inhibiting the growth process. This assumption was further supported by labeling experiments wish [¹⁴C]-NEM using membranes of maize ( Zea mays L. cv. Inraplus) coleoptiles. Two membrane fractions (S₂= 480-1900 g; S₄= 4300-15000 g) revealed a significantly higher [¹⁴C]-NEM labeling in the presence of auxin (2,4-diehlorophe-noxyacctic acid compared to 2,6 dichlorophenoxyacetic acid). This effect disappeared when the membranes were previously washed with EGTA [ethyleneglycolbis-(β-aminoethylether)-N,N,Nr',N'-tetraacetic acid]. The auxin-induced sensitization of coleoptilc segments against thiol-reagents and the auxin-induced expression of SH-groups of proteins of isolated membranes from coleoptiles arc suggested to be events involved in the primary action of auxins.     

Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato

Many aspects of plant development are regulated by antagonistic interactions between the plant hormones auxin and cytokinin, but the molecular mechanisms of this interaction are not understood. To test whether cytokinin controls plant development through inhibiting an early step in the auxin response pathway, we compared the effects of cytokinin with those of the dgt (diageotropica) mutation, which is known to block rapid auxin reactions of tomato (Lycopersicon esculentum) hypocotyls. Long-term cytokinin treatment of wild-type seedlings phenocopied morphological traits of dgt plants such as stunting of root and shoot growth, reduced elongation of internodes, reduced apical dominance, and reduced leaf size and complexity. Cytokinin treatment also inhibited rapid auxin responses in hypocotyl segments: auxin-stimulated elongation, H(+) secretion, and ethylene synthesis were all inhibited by cytokinin in wild-type hypocotyl segments, and thus mimicked the impaired auxin responsiveness found in dgt hypocotyls. However, cytokinin failed to inhibit auxin-induced LeSAUR gene expression, an auxin response that is affected by the dgt mutation. In addition, cytokinin treatment inhibited the auxin induction of only one of two 1-aminocyclopropane-1-carboxylic acid synthase genes that exhibited impaired auxin inducibility in dgt hypocotyls. Thus, cytokinin inhibited a subset of the auxin responses impaired in dgt hypocotyls, suggesting that cytokinin blocks at least one branch of the DGT-dependent auxin response pathway.     

Auxin-regulated changes in protein phosphorylation in pea epicotyls

Auxins regulate various aspects of plant growth and development. However, the mechanism by which these hormones elicit diverse physiological processes is not clear. We present evidence for the role of auxin in protein phosphorylation and the possible involvement of calmodulin in auxin-induced changes. In the presence of auxin, phosphorylation of 23,000, 82,000, 105,000 and 110,000 molecular weight polypeptides markedly decreased whereas phosphorylation of 19,000, 24,000 and 28,000 molecular weight polypeptides increased. These results open up a new experimental approach in understanding the molecular mechanism by which auxins regulate various physiological processes in plants.     

Equilibrium analysis of allosteric interactions shows zero-order effects

The binding of effector to an allosteric protein exhibits a non-Michaelis-Menten behavior, resulting in either ultrasensitive or subsensitive response. In the present work, a modular approach has been developed to determine the response curve for allosteric systems at higher concentration of allosteric enzyme than that of effector (zero-order sensitivity, as observed in enzyme cascades) by equilibrium analysis. The analysis shows that, in an allosteric system, the zero-order effect can make the response ultrasensitive or subsensitive with respect to the enzyme concentration. The response is dependent on the number of binding sites, cooperativity, and the total effector concentration. The framework was further applied to a well studied allosteric protein, the Escherichia coli aspartate transcarbamoylase. The predictions are found to be consistent with the reported experimental data.     

GROWTH AND GRAVITATIONAL RESPONSE IN THE DEVELOPMENT OF LEAF BLADE HYPONASTY

Rapid (4 hr) auxin-induced hyponastic curvature of primary leaves of Phaseolus vulgaris is shown to depend on a positive increase in growth of the lower portion of the blade. The curvature involves laminar growth as well as vein growth and is not due to simple turgor changes. The response is sensitive to gravitational orientation, as inversion and horizontal rotation reduce the auxin-induced curvature. The ethylene-generating compound, 2-chloroethylphosphonic acid, had no hyponastic effect on the leaves when applied to either the upper or lower surface and it inhibited auxin-induced hyponasty. This inhibition was additive to that of inversion. Long-term (24–48 hr) effects of 1 mM auxin depend on the surface of the leaf treated. Application to the upper surface results in epinasty, lower surface application in hyponasty, although the initial response in each case is a hyponastic curvature. A dorsi-ventral auxin transport system and differential auxin sensitivity of upper and lower portions of the leaf blade are postulated to account for these responses.     

Physiological evidence for auxin-induced hydrogen-ion secretion and the epidermal paradox

Summary Peeled Avena coleoptile sections will respond to auxin only if the molarity of the incubation buffer at pH 6.2 is less than 5 mM. This inhibition of auxin-induced growth is not due to toxicity or to a reduction of turgor below the critical value needed for extension but rather appears to be related simply to buffering capacity. These data therefore serve as physiological evidence that H⁺-secretion is an intregal part of auxin-induced cell wall loosening. Other data obtained utilizing peeled plant sections and epidermal strips suggest that the epidermis does not directly control cell extension growth. A model is proposed to explain the curvature response in split-segments tests in terms of a H⁺ gradient across the section. As far as tested this model appears to be an alternative to an older concept which implied that the curvature phenomenon in split sections was mediated by special properties of the epidermal layer. Our results suggest that the curvature response may be more directly attributable to the presence of the cuticle.     

AUXIN-INDUCED HYPONASTY OF THE LEAF BLADE OF PHASEOLUS VULGARIS

The lateral margins of immature primary leaf blades of Phaseolus vulgaris L. cv. ‘Pinto’ curve up and in toward the midrib when auxin is applied to the leaf. The leaves are most sensitive to auxin shortly after they first unfold and leaves which have grown to about 60 % or more of their ultimate area no longer give this hyponastic response. The response is specific for auxins and is inhibited by the anti-auxins, trans-cinnamic acid and para-chlorophenoxyisobutyric acid. Ethylene and ethylene-generating compounds failed to induce hyponasty, suggesting the response is due to a positive growth promotion by auxin. Measurements of the distance between the lateral margins of the leaf at its maximum width were used to provide quantitative estimates of the degree of hyponasty. Between 2 and 4 hr after auxin application a direct proportionality was found between the amount of curvature and the logarithm of the indoleacetic acid concentration over the range of 10⁻⁶ to 10⁻³ m. The relative sensitivity of the leaves to different auxins was qualitatively similar to that observed in many straight-growth bioassays. Similar responses were obtained when auxin was applied by a carborundum wounding procedure. Potential applications of this auxin bioassay for investigations of the role of auxin in the normal plagiotropic growth behavior of leaf lamina and of the role of auxin in the initiation of various plant diseases are suggested.     

Auxin-induced hydrogen-ion secretion in Avena coleoptiles and its implications

Summary The dose response curve for hydrogen-ion-induced extension growth in Avena coleoptile segments has been reinvestigated. The previously published optimum (pH 3.0) is in error by about two orders of magnitude. The correct optimum is around pH 5.0. This discrepancy is thought to be due to the impermeable nature of the cuticle to hydrogen ions. In the present study the cuticular barrier to H⁺ entry was circumvented by using coleoptile segments from which the epidermis with cuticle were physically removed. Using such peeled coleoptile sections, it was also found that auxin can rapidly (20–30 min) initiate H⁺ secretion and that the magnitude of auxin-induced secretion is sufficient to initiate considerable cell-extension growth. Furthermore, it is shown that the secretion response is specific for active auxins, and inhibited by agents which inhibit auxin-induced growth (dinitrophenol, abscisic acid, cycloheximide, valinomycin and others). These results make it very likely that H⁺ secretion is responsible, at least in part, for the initiation of auxin-induced cell wall loosening and extension growth.     

Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein

The soluble bean leaf auxin-binding protein (ABP) has a high affinity for a range of auxins including indole-3-acetic acid (IAA), α-napthaleneacetic acid, phenylacetic acid, 2,4,5-trichlorophenoxyacetic acid, and structurally related auxins. A large number of nonauxin compounds that are nevertheless structurally related to auxins do not displace IAA from bean ABP. Bean ABP has a high affinity for auxin transport inhibitors and antiauxins. The specificity of pea ABP for representative auxins is similar to that found for bean ABP. The bean ABP auxin binding site is similar to the corn endoplasmic reticulum auxin-binding sites in specificity for auxins and sensitivity to thiol reagents and azide. Qualitative similarities between the ligand specificity of bean ABP and the specificity of auxin-induced bean leaf hyponasty provide further evidence, albeit circumstantial, that ABP (ribulose 1,5-bisphosphate carboxylase) can bind auxins in vivo. The high incidence of ABP in bean leaves and the high affinity of this protein for auxins and auxin transport inhibitors suggest possible functions for ABP in auxin transport and/or auxin sequestration.     

Peroxidase genes differentially respond to auxin during the formation of adventitious roots in soybean hypocotyl

In our investigation, auxins (IAA, IBA and NAA) effectively promoted rooting in soybean hypocotyls. The activity of anionic peroxidase (POX) (pI 3.7) and cationic POX (pI 8.5) was significantly suppressed by exogenous auxins on day 2 (the inductive phase). Some particular anionic POXs (pI 4.0 and pI 5.3) significantly increased in IBA-treated tissues as compared with the control when the incubation time was prolonged to day 3 and day 4 (the initiation phase). We sequenced 5′-flanking region of pI 8.5 and pI 5.3 POX genes using the PLACE and PlantCARE databases to identify several potential cis-regulatory elements. The pI 8.5 POX gene promoter contained two sites that were homologous to sequences commonly found in auxin response elements; motifs ARF/AuxRE and CATATGGMSAUR. During the inductive phase, the activity of pI 8.5 POX was significantly suppressed by the exogenously applied auxins. The pI 8.5 POX gene promoter contained both ARF/AuxRE and CATATGGMSAUR motifs that responded to auxins earlier than the pI 5.3 POX gene. Hence, the pI 8.5 POX gene might belong to primary auxin response genes. The pI 5.3 POX gene, which responded to auxins a day or two later, contained only ARF/AuxRE motif. Moreover, unlike pI 8.5 and pI 3.7 POXs that were suppressed by auxins, the pI 5.3 POX was induced or enhanced by the applied auxins, especially IBA. The pI 5.3 POX might generate H₂O₂ which caused the auxin-induced growth at the initiation phase during the formation of adventitious root in soybean hypocotyls.