Inhibition of formation of protein-bound hydroxyproline by free hydroxyproline in Avena coleoptiles

Free hydroxyproline inhibits the formation of protein-bound hydroxyproline from proline to a considerably greater extent than it does the incorporation of proline into protein of auxin-treated Avena coleoptiles. This inhibition is greater in the wall than in the cytoplasmic fraction. In the absence of auxin, free hydroxyproline exerts little or no inhibition of hydroxyproline formation. Furthermore free hydroxyproline has no effect on respiration, RNA synthesis or the incorporation of leucine into protein. Hydroxyproline is not a general inhibitor of metabolism or protein synthesis in Avena coleoptiles.

These results suggest that free hydroxyproline may inhibit auxin-induced cell elongation by blocking the formation or utilization of a particular hydroxyproline-rich protein which must be incorporated into the cell wall during auxin-induced wall extension.

     

Inhibition of RNA Synthesis and Auxin-Induced Cell Wall Extensibility and Growth by Actinomycin D

A linear stress strain analyzer was used to determine the effects of inhibitors of RNA and protein synthesis on auxin-induced increases in cell wall extensibility. With etiolated soybean hypocotyl, maize mesocotyl and Avena coleoptile sections and light-grown pea internode sections, inhibition of RNA synthesis resulted in inhibition of auxin-induced extensibility changes and cell expansion. The results with both actinomycin D and cycloheximide support an earlier conclusion that unstable cell constituents, presumably enzymes, are essential for cell wall loosening induced by auxin as well as for cell elongation.     

Kinetics of Adaptation to Osmotic Stress in Lentil (Lens culinaris Med.) Roots

When intact roots of lentil (Lens culinaris Med.) are subjected to severe osmotic stress by treatment with a solution of low water potential, they immediately begin to shrink. Within 10 to 15 minutes, shrinkage ceases, and within 20 minutes, the roots resume growth. The time lag between application of osmoticum and resumption of growth varies from about 10 to 30 minutes over the range of external water potentials of −2 to −12.4 bars. For external water potentials as low as −8.7 bars the new steady rate of growth in the presence of osmoticum is approximately equal to that prevailing before application of osmoticum. For external water potentials between −8.7 and −13 bars growth resumes, but the new rate is less than that prior to addition of osmoticum. Measurements of changes in the internal solute content during adaptation show that the solute content of the root increases but that the magnitude of the increase is, by itself, insufficient to account for the resumption of rapid growth.     

Effects of Actinomycin D and 2,4-Dinitrophenol on the Development of Root Primordia in Azuki Bean Stem Cuttings

In the rooting of disbudded azuki bean stem cuttings, actinomycinD and 2,4- dinitrophenol (DNP) acted as auxin synergists. Althoughcuttings treated with actinomycin D or DNP alone formed almostthe same number of roots as water-treated cuttings, cuttingstreated with actinomycin D or DNP for 24 hr then with auxinfor another 24 hr formed more roots than cuttings treated onlywith auxin during the second 24 hr. Both actinomycin D and DNPincreased the number of root primordia with longitudinally dividedcells, but they acted differently on the first transverse celldivision which led to root primordium formation. ActinomycinD delayed the start of the first transverse cell division, butDNP hastened it. (Received July 7, 1981; Accepted September 21, 1981)     

RNA synthesized during late sporulation is required for germ tube formation inBlastocladiella emersonii

Inhibition of RNA synthesis with actinomycin D as late as 210 min (T₂₁₀) afterBlastocladiella emersonii is induced to sporulate results in complete blockage of germ tube formation in the next generation. In agreement with other reports, actinomycin D added during germination did not block germ tube formation. Protein synthesis during germination is reduced by approximately one-half when actinomycin D is added atT₂₁₀ but remains at virtually control levels when actinomycin D is added during germination. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel analyses of the abundant proteins synthesizedin vivo during the first hour of germination revealed no qualitative differences in the proteins which accumulate when control cells are compared to cells treated with actinomycin D atT₂₁₀. Comparison of proteins synthesized from 20 to 40 min germination vs 40 to 60 min germination demonstrated that actinomycin D alters the temporal pattern of accumulation of some abundant proteins. The RNA synthesized afterT₂₁₀ is associated with polysomes, suggesting that an mRNA fraction made in late sporulation is required for a germ tube. The available data do not exclude the possibility that a regulatory RNA synthesized during late sporulation is required for germ tube formation.     

Absence of auxin-induced stored growth in Avena coleoptiles and its implication concerning the mechanism of wall extension

Summary We have reinvestigated the ability of Avena coleoptiles to undergo auxin-induced stored growth (stored growth is defined as the ability of a cell to store up a potential for extension during periods of reduced turgor which can be converted into extra extension upon restoration of normal turgor). We could detect little or no stored growth, with either moderate (1–2 bar) or more severe (3–5 bar) reductions in turgor, and with varying periods (10–100 min) of reduced turgor. Earlier reports of a stored growth potential (e.g., Cleland and Bonner, 1956) are shown to be in error, in that the apparent growth potential is probably an artifact of the use of argon or nitrogen as an inhibitor of auxin action. The absence of stored growth reported here is not due to a direct inhibitory effect of the osmoticum itself on auxin action, since coleoptiles can extend in response to auxin even in the presence of mannitol if an external force is applied to the section to replace the normal turgor. These results show that the two components of cell-wall extension, wall loosening and wall extension, usually are inseparable. Two possible explanations are considered; the walls may be extending by the process of chemical creep, or the wall loosening may only occur when the load-bearing bonds are under tension.     

Tubulin synthesis and auxin-induced root initiation in phaseolus

The auxin-induced formation of roots in the hypocotyls of Phaseolus vulgaris can be prevented by treatment with actinomycin D, colchicine or cytochalasin B if applied within 40 hr of initiation. Shortly after auxin pretreatment, there is an increase in translatable messenger RNA activity. Analysis of the labelled cell-free products indicate, among other changes, a striking increase in a protein co-migrating with tubulin, in the case of RNA isolated from indolebutyric acid (IBA) pretreated hypocotyls. An increase in tubulin content in vivo can also be demonstrated on the basis of SDS-polyacrylamide gel analysis of membrane proteins and functional assays for tubulin polymerization. An increase in the synthesis of tubulin in vivo can also be demonstrated after IBA pretreatment. In addition, the auxin is also able to promote tubulin polymerization when added in vitro. It is suggested that tubulin synthesis and microtubule assembly are early events in auxin-mediated root differentiation.     

Timing of the Auxin Response in Coleoptiles and Its Implications Regarding Auxin Action

The timing of the auxin response was followed in oat and corn coleoptile tissue by a sensitive optical method in which the elongation of about a dozen coleoptile segments was recorded automatically. The response possesses a latent period of about 10 min at 23°C, which is extended by low concentrations of KCN or by reducing the temperature, but is not extended by pretreatments with actinomycin D, puromycin, or cycloheximide at concentrations that partially inhibit the elongation response. Analysis of the data indicates that auxin probably does not act on the elongation of these tissues by promoting the synthesis of informational RNA or of enzymatic protein. Not excluded is the possibility that auxin acts at the translational level to induce synthesis of a structural protein, such as cell wall protein or membrane protein. While the data do not provide direct support for this hypothesis, the speed with which cycloheximide inhibits elongation suggests that continual protein synthesis may be important in the mechanism of cell wall expansion.     

Dipyridyl-induced Cell Elongation and Inhibition of Cell Wall Hydroxyproline Biosynthesis

Incubation of soybean hypocotyl sections with 0.1 millimolar 2,2′-dipyridyl in the absence of auxin results in increases in growth rate and in cell wall extensibility lasting for about 3 hours. This is accompanied by greatly decreased biosynthesis of hydroxyproline, which ultimately appears in the wall, and in slightly reduced oxygen uptake, both of which continue for at least 9 hours. Continuous synthesis of hydroxyproline which appears in the cell wall is thus not necessary for short term growth. The decrease in growth and cell wall extensibility that occurs between the 3rd and 9th hours of dipyridyl inhibition cannot be attributed to cross-linking of newly synthesized hydroxyproline, since its synthesis is still inhibited.     

Influence of Cycloheximide and Actinomycin D on Initiation and Early Development of Adventitious Roots

From leaf cuttings of the bean Phaseolus vulgaris L. adventitious roots form on the petiole. This root formation is stimulated by treatment with auxin. Simultaneous or subsequent application of cycloheximide irreversibly inhibited dedifferentiation, so that root production was completely prevented. The effects of actinomycin D application depended upon the stage of development of the root primordium. Cells in the first stage of dedifferentiation were extremely sensitive. When actinomycin D was applied later than 6 h after cutting, its inhibiting effect gradually diminished. It is concluded that an actinomycin D-sensitive process occurring early in dedifferentiation is crucial for root initiation. A second, less actinomycin D-sensitive process occurring later in dedifferentiation is required for the further development of the root primordium. During the initiation and development of the root primordium protein synthesis is required.     

Ethylene-forming Systems in Etiolated Pea Seedling and Apple Tissue

Auxin-induced ethylene formation in etiolated pea (Pisum sativum L. var. Alaska) stem segments was inhibited by inhibitors of RNA and protein synthesis. Kinetics of the inhibitions is described for actinomycin D, cordycepin, α-amanitin, and cycloheximide. α-Amanitin was the most potent and fast-acting inhibitor, when added before induction or 6 hours after induction of the ethylene-forming system. The ethylene-forming system of postclimacteric apple (Malus sylvestris L.) tissue, which is already massively induced, was not further stimulated by auxin. Ethylene production in apples was inhibited least by α-amanitin and most by actinomycin D. The relative responses of the ethylene system in apples to RNA inhibitors were different from the ethylene system of pea stems. However, the protein synthesis inhibitor, cycloheximide, appeared to act equally in both tissue systems. The effect of cycloheximide on ethylene production in postclimacteric apple tissue, already producing large quantities of ethylene, suggests a dynamic regulating system for the synthesis and degradation of the ethylene-forming system.     

The Inhibition by Cytokinin of Auxin-Promoted Elongation in Excised Soybean Hypocotyl

The experiments characterize the inhibition by kinetin of auxin-promoted elongation in excised hypocotyl sections of 3-day soybean seedlings (Glycine max cv. Hawkeye 63). It was found that concentrations of kinetin above 4.2 μM did not further inhibit auxin-promoted elongation. Kinetin is as potent an inhibitor of elongation as actinomycin D or cycloheximide. Tissue incubated for 3 or 5 h in the absence of auxin or cytokinin would, upon addition of auxin, exhibit a new growth rate similar to that of tissue grown in auxin for the entire incubation period. Similarly, tissue grown for 3 and 5 h in the presence of auxin would revert to the control rate of elongation upon addition of kinetin. A 10 to 30 min preincubation in kinetin yielded the tissue incapable, for the ensuing 6 h, of increasing its rate of elongation in response to auxin. Zeatin and isopentenyladenine were more potent than kinetin and benzyladenine in the inhibition of elongation. Levels of ethylene produced in the presence of auxin plus cytokinin indicated that it was not involved in this auxin-cytokinin interaction. Kinetin by itself did not promote elongation; nor did it enhance auxin-promoted elongation at low auxin concentrations.     

Auxin-regulated Wall Loosening and Sustained Growth in Elongation

It is proposed that auxin regulates and coordinates both wall loosening and the supply of wall materials in elongation. The tenets of the proposal allowed testable predictions. It was determined that, if the cell walls of Glycine max L. var. Wayne hypocotyl segments are maintained in a loosened state (by excising the segments directly into pH 4 medium), exogenous auxin induced only the second response. It was also predicted and confirmed that elongating systems, e.g. pea epicotyl, with certain early auxin-induced growth kinetics (an initial high non-steady-state rate followed immediately by a drop to a lower steady-state rate) would show a transient second response (in addition to the usual transient first response) when stimulated by pH 4 medium. Finally, it is pointed out that recent results which establish the existence of auxin-induced elongation-associated proteins support the proposition that auxin coordinates wall loosening and the supply of wall materials in elongation.     

D6PK AGCVIII Kinases Are Required for Auxin Transport and Phototropic Hypocotyl Bending in Arabidopsis

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.     

Effect of indoleacetic Acid and hydroxyproline on isoenzymes of peroxidase in wheat coleoptiles

Indoleacetic acid at 0.017 millimolar inhibited the formation of three peroxidase isoenzymes in both soluble and wall-bound enzyme fractions of wheat coleoptile (Triticum vulgare) tissue. Hydroxyproline at 1 millimolar prevented the indoleacetic acid-induced inhibition. Indoleacetic acid oxidase activity in the soluble fraction was decreased by indoleacetic acid and was restored by hydroxyproline. Most of the indoleacetic acid oxidase activity was located in the electrophoretic zones occupied by two of the peroxidase isoenzymes influenced by indoleacetic acid and hydroxyproline. At least part of the effect of hydroxyproline on auxin-induced elongation of coleoptile tissue may be through control of auxin levels by indoleacetic acid oxidase.     

Mechano-Chemical Aspects of Organ Formation in Arabidopsis thaliana: The Relationship between Auxin and Pectin

How instructive signals are translated into robust and predictable changes in growth is a central question in developmental biology. Recently, much interest has centered on the feedback between chemical instructions and mechanical changes for pattern formation in development. In plants, the patterned arrangement of aerial organs, or phyllotaxis, is instructed by the phytohormone auxin; however, it still remains to be seen how auxin is linked, at the apex, to the biochemical and mechanical changes of the cell wall required for organ outgrowth. Here, using Atomic Force Microscopy, we demonstrate that auxin reduces tissue rigidity prior to organ outgrowth in the shoot apex of Arabidopsis thaliana, and that the de-methyl-esterification of pectin is necessary for this reduction. We further show that development of functional organs produced by pectin-mediated ectopic wall softening requires auxin signaling. Lastly, we demonstrate that coordinated localization of the auxin transport protein, PIN1, is disrupted in a naked-apex produced by increasing cell wall rigidity. Our data indicates that a feedback loop between the instructive chemical auxin and cell wall mechanics may play a crucial role in phyllotactic patterning.     

SLOW MOTION Is Required for Within-Plant Auxin Homeostasis and Normal Timing of Lateral Organ Initiation at the Shoot Meristem in Arabidopsis

The regular arrangement of leaves and flowers around a plant''s stem is a fascinating expression of biological pattern formation. Based on current models, the spacing of lateral shoot organs is determined by transient local auxin maxima generated by polar auxin transport, with existing primordia draining auxin from their vicinity to restrict organ formation close by. It is unclear whether this mechanism encodes not only spatial information but also temporal information about the plastochron (i.e., the interval between the formation of successive primordia). Here, we identify the Arabidopsis thaliana F-box protein SLOW MOTION (SLOMO) as being required for a normal plastochron. SLOMO interacts genetically with components of polar auxin transport, and mutant shoot apices contain less free auxin. However, this reduced auxin level at the shoot apex is not due to increased polar auxin transport down the stem, suggesting that it results from reduced synthesis. Independently reducing the free auxin level in plants causes a similar lengthening of the plastochron as seen in slomo mutants, suggesting that the reduced auxin level in slomo mutant shoot apices delays the establishment of the next auxin maximum. SLOMO acts independently of other plastochron regulators, such as ALTERED MERISTEM PROGRAM1 or KLUH/CYP78A5. We propose that SLOMO contributes to auxin homeostasis in the shoot meristem, thus ensuring a normal rate of the formation of auxin maxima and organ initiation.     

Auxin Physiology of the Tomato Mutant diageotropica

The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.