Elongation of Stem Internodes in the Japanese Morning Glory (Pharbitis nil) in Relation to Auxin Destruction

The relationship between auxin destruction and stem internode elongation was investigated in the vines of the Japanese morning glory (Pharbitis nil Choisy). In young plants an age-dependent gradient was demonstrated in which the decreasing rate of elongation of older internodes correlated with an increasing ability of such tissue to destroy indoleacetic acid. Fragments of tissue from old internodes when incubated with indoleacetic acid (IAA), destroyed the hormone immediately and rapidly; in contrast, young, rapidly elongating internode tissue destroyed IAA only after a lag of several hours. In older plants the gradient was more erratic towards the middle of the plant but old and young tissue behaved as in young plants, i.e., old internodes destroyed IAA rapidly whereas young internodes did not. It appears reasonable to conclude that cessation of elongation in maturing internodes is brought about by developing an internal environment in which auxin is rapidly destroyed.     

Light Regulation of beta-Tubulin Gene Expression during Internode Development in Soybean (Glycine max [L.] Merr.)

The relationship between tubulin gene expression and cell elongation was explored in developing internodes of Glycine max (L.) Merr., using light as a variable to alter the rate of elongation. First internodes of etiolated seedlings elongated two to three times more rapidly than did those of seedlings growing under a 12 hour diurnal light/dark cycle. Furthermore, light slowed or completely halted internode elongation in the etiolated seedlings, depending upon the age of the seedlings at the time of the light treatment. Steady state levels of β-tubulin mRNA were determined in Northern blots and by solution hybridization of poly(A)⁺RNA with a probe derived from the coding region of a previously characterized soybean β-tubulin gene. (MJ Guiltinan, DP Ma, RF Barker, MM Bustos, RJ Cyr, R Yadegari, DE Fosket [1987] Plant Mol Biol 10: 171-184). Internodes of light-grown seedlings exhibited levels of β-tubulin mRNA that differed by a factor of three, and varied concomitantly with the elongation rate. Illumination of 10-day-old etiolated seedlings not only stopped first internode elongation, but also brought about a 80% decrease in the steady state level of β-tubulin mRNA over the course of the subsequent 12 hours. This strong down regulation of β-tubulin mRNA occurred without significant changes in the size of the soluble tubulin pool and it was accompanied by a marked increase in chlorophyll a/b binding protein mRNA.     

The involvement of indole-3-acetic acid in the control of stem elongation in dark- and light-grown pea (Pisum sativum) seedlings

We investigated the role of auxin on stem elongation in pea (Pisum sativum L.) grown for 10d in continuous darkness or under low-irradiance blue, red, far red and white light. The third internode of treated seedlings was peeled and the tissues (epidermis and cortex+central cylinder) were separately analyzed for the concentration of free and conjugated indole-3-acetic acid (IAA). Under red, far red and white light internode elongation was linearly related with the free IAA content of all internode tissues, suggesting that phytochrome-dependent inhibition of stem growth may be mediated by a decrease of free IAA levels in pea seedlings. The correlation between IAA and internode elongation, however, did not hold for blue light-grown seedlings. The hypothesis that the growth response under low-irradiance blue light might be correlated with the lack of phytochrome B signalling and changes in gibberellin metabolism is discussed in view of current knowledge on hormonal control of stem growth.     

Growth, in vivo extensibility, and tissue tension in developing pea internodes

The relationship between growth, in vivo extensibility, and tissue tension in the first 3 internodes of 5, 6, and 7 day-old pea plants (Pisum sativum L. cv Alaska), grown under continuous red light was investigated. The upper 15 millimeters of each internode was marked with ink and its elongation growth measured over the next subsequent 8 hours. In vivo extensibility was measured by stretching living tissue at constant force (creep test) in a custom-built extensiometer. Tissue tension was determined by (a) measuring the rate of expansion of the isolated cortical cylinder after adding water and the amount of contraction of the epidermis after peeling, and (b) by use of the `split section test.' A good correlation between rate of elongation growth, in vivo extensibility, and tissue tension was established. The epidermis peeled from the growing third internode of 7 day-old plants and measured immediately showed a plastic extensibility (E<sub>pl</sub> twice that of peels from nongrowing excised sections. This high E<sub>pl</sub>-value was lost on incubation of the sections in distilled water, and was subsequently restored by incubating the sections in auxin (indole-3-acetic acid). We conclude that the in situ growth of the internodes is a function of tissue-tension, which provides the driving force of organ growth, and the extensibility (E<sub>pl</sub> of the outer epidermal wall, which is in the growing plant in a `loosened' state. We furthermore suggest that in the intact plant auxin is causally involved in the wall loosening process in the epidermis.     

Control of Internode Length in Pisum sativum (Further Evidence for the Involvement of Indole-3-Acetic Acid)

The effects of altered endogenous indole-3-acetic (IAA) levels on elongation in garden pea (Pisum sativum L.) plants were investigated. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) were applied to elongating internodes of wild-type and mutant lkb plants. The lkb mutant was included because elongating lkb internodes contained 2- to 3-fold less free IAA than those of the wild type. In the wild type, TIBA reduced both the IAA level and internode elongation below the site of application. Both TIBA and HFCA strongly promoted the elongation of lkb internodes and also raised IAA levels above the application site. The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) also markedly increased internode elongation in lkb plants and virtually restored petioles and tendrils to their wild-type length. In contrast, treatment of wild-type plants with TIBA, HFCA, or 2,4-D caused little or no increase in elongation above the application site. The ethylene synthesis inhibitor aminoethoxyvinylglycine also increased stem elongation in lkb plants, and combined application of HFCA and aminoethoxy-vinylglycine restored lkb internodes to the wild-type length. It is concluded that the level of IAA in wild-type internodes is necessary for normal elongation, and that the reduced stature of lkb plants is at least partially attributable to a reduction in free IAA level in this mutant.     

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.     

RECOVERY OF NATIVE AND APPLIED AUXIN FROM THE LIGHT-GROWN ‘ALASKA’ PEA SEEDLING

Scott , Tom K., and Winslow R. Briggs . (Stanford U., Stanford, Calif.) Recovery of native and applied auxin from the light-grown ‘Alaska’ pea seedling. Amer. Jour. Bot. 49(10): 1056–1063. Illus. 1962.—The physiological status of both endogenous and exogenously applied auxin was compared in the epicotyl of the 9-day-old light-grown ‘Alaska’ pea (Pisum sativum L.) by means of agar-diffusion and short-term ether extraction. A detailed analysis of endogenous auxin revealed a linear basipetal decrease in diffusible auxin within the growing region. A decrease in extractable auxin occurred only within the most mature region. The capacity for uptake of indole-3-acetic acid (IAA), applied in lanolin paste, was compared in different regions of the epicotyl. The fifth and most apical internode had the greatest capacity for uptake as measured by extraction. A reduced capacity was found in more basal internodes. The transport rate of applied IAA, under conditions of optimal uptake, was 10–12 mm/hr. An application of IAA for 24 hr resulted in a dramatic increase in auxin content throughout the length of the epicotyl compared to that found in the normal control. There was no apparent gradation in content from apex to base. An increase of diffusible auxin was also found, but only in the fourth and third internodes. That no such increase was detected in the basal 3 internodes suggested that the auxin transport system within this region had special properties related to a transition between shoot and root vascular patterns.     

Control of Auxin-induced Stem Elongathn by the Epidermis

Segments of the 4th and 5th internodes of light-grown pea seedlings were used for the study of control of stem elongation. With 5th internodes, at low turgor as well as at water saturation auxin primarily appeared to cause a change in cell wall properties of the epidermis but it showed little effect on expansion af the inner tissue. This was confirmed by comparison of expansion between peeled and unpeeled segments, split tests and by measurements of stress-relaxation properties of the epidermal cell wall. Segments with the central part re-moved elongated well in response to auxin, but the isolated epidermis showed neither auxin-induced elongation nor cell wall loosening. A fungal β-1,3-glucanase appeared, at least partly, to have a similar effect as that of auxin on elongation, by changing cell wall properties of the epidermal cell wall. Peeled segments of 4th internodes expanded very little and auxin had little effect on their epidermal cell wall properties.     

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.     

Effect of morphactin on stem growth in relation to auxin in precooled rooted tulip bulbs

Effect of morphactin IT 3456, an auxin transport inhibitor, on tulip stem elongation induced by indole-3-acetic acid (IAA) was investigated. Tulip stem growth induced by IAA 0.1 % in lanolin paste applied on the top internode after excision of flower bud and removal of all leaves was greatly inhibited by 0.2 % morphactin IT 3456 applied on the 4th, 3rd, 2nd and 1st internode. The inhibitory effect of the morphactin on tulips stem growth promoted by IAA was restored by additional application of IAA below the morphactin treatment place. Morphactin inhibited also the growth of all internodes induced by flower bud in the absence of leaves. These results suggest a crucial role of auxin in the control growth of all internodes in tulip stem.     

Role of auxin and gibberellin in differentiation of primary Phloem fibers

The hypothesis that auxin and gibberellic acid (GA₃) control the differentiation of primary phloem fibers is confirmed for the stem of Coleus blumei Benth. Indoleacetic acid (IAA) alone sufficed to cause the differentiation of a few primary phloem fibers. In long term experiments auxin induced a considerable number of fibers in mature internodes. GA₃ by itself did not exert any effect on fiber differentiation. Combinatiosn of IAA with GA₃ completely replaced the role of the leaves in primary phloem fiber differentiation qualitatively and quantitatively. Although the combined effect of the two growth hormones diminished considerably with increasing distance from the source of induction, auxin with GA₃ or IAA alone induced fibers in a few internodes below the application site. When various combinations of both hormones were applied, high concentrations of IAA stimulated rapid differentiation of fibers with thick secondary walls, while high levels of GA₃ resulted in long fibers with thin walls. The size of the primary phloem fibers correlated with the dimensions of the differentiating internode, thereby providing evidence that both growth regulators figure in the control of stem extension. High IAA/low GA₃ concentrations have an inhibitory effect on internode elongation, whereas low IAA/high GA₃ concentrations promote maximal stem elongation.     

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.     

Magnitude and Kinetics of Stem Elongation Induced by Exogenous Indole-3-Acetic Acid in Intact Light-Grown Pea Seedlings

Exogenously applied indole-3-acetic acid (IAA) strongly promoted stem elongation over the long term in intact light-grown seedlings of both dwarf (cv Progress No. 9) and tall (cv Alaska) peas (Pisum sativum L.), with the relative promotion being far greater in dwarf plants. In dwarf seedlings, solutions of IAA (between 10-4 and 10-3 M), when continuously applied to the uppermost two internodes via a cotton wick, increased whole-stem growth by at least 6-fold over the first 24 h. The magnitude of growth promotion correlated with the applied IAA concentration from 10-6 to 10-3 M, particularly over the first 6 h of application. IAA applied only to the apical bud or the uppermost internode of the seedling stimulated a biphasic growth response in the uppermost internode and the immediately lower internode, with the response in the latter being greatly delayed. This demonstrates that exogenous IAA effectively promotes growth as it is transported through intact stems. IAA withdrawal and reapplication at various times enabled the separation of the initial growth response (IGR) and prolonged growth response (PGR) induced by auxin. The IGR was inducible by at least 1 order of magnitude lower IAA concentrations than the PGR, suggesting that the process underlying the IGR is more sensitive to auxin induction. In contrast to the magnitude of the IAA effect in dwarf seedlings, applied IAA only doubled the growth in tall seedlings. These results suggest that endogenous IAA is more growth limiting in dwarf plants than in tall plants, and that auxin promotes stem elongation in the intact plant probably by the same mechanism of action as in isolated stem segments. However, since dwarf plants to which IAA was applied failed to reach the growth rate of tall plants, auxin cannot be the only limiting factor for stem growth in peas.     

Promotion of stem elongation by indole-3-butyric acid in intact plants of Pisum sativum L.

While indole-3-butyric acid (IBA) has been confirmed to be an endogenous form of auxin in peas, and may occur in the shoot tip in a level higher than that of indole-3-acetic acid (IAA), the physiological significance of IBA in plants remains unclear. Recent evidence suggests that endogenous IAA may play an important role in controlling stem elongation in peas. To analyze the potential contribution of IBA to stem growth we determined the effectiveness of exogenous IBA in stimulating stem elongation in intact light-grown pea seedlings. Aqueous IBA, directly applied to the growing internodes via a cotton wick, was found to be nearly as effective as IAA in inducing stem elongation, even though the action of IBA appeared to be slower than that of IAA. Apically applied IBA was able to stimulate elongation of the subtending internodes, indicating that IBA is transported downwards in the stem tissue. The profiles of growth kinetics and distribution suggest that the basipetal transport of IBA in the intact plant stem is slower than that of IAA. Following withdrawal of an application, the residual effect of IBA in growth stimulation was markedly stronger than that of IAA, which may support the notion that IBA conjugates can be a better source of free auxin through hydrolysis than IAA conjugates. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to stem elongation in intact plants.     

Investigations on the Mechanism of the Brassinosteroid Response: I. Indole-3-acetic Acid Metabolism and Transport

A brassinosteroid treatment of light-grown first internode sections of Phaseolus vulgaris results in an increased bending response following unilateral indole-3-acetic acid (IAA) application. Reverse isotope dilution analysis shows that this increased response is not due to an increase in the concentration of applied IAA in the tissue or a change in the amount of IAA conjugated. Treatment with the brassinosteroid also does not affect the rate of IAA transport as measured using the agar block method. These results indicate that even though brassinosteroid potentiates auxin action, it does not have a direct effect on IAA uptake, metabolism, or cell to cell transport.     

Red Light-Regulated Growth : I. Changes in the Abundance of Indoleacetic Acid and a 22-Kilodalton Auxin-Binding Protein in the Maize Mesocotyl

We examined the changes in the levels of indoleacetic acid (IAA), IAA esters, and a 22-kilodalton subunit auxin-binding protein (ABP1) in apical mesocotyl tissue of maize (Zea mays L.) during continuous red light (R) irradiation. These changes were compared with the kinetics of R-induced growth inhibition in the same tissue. Upon the onset of continuous irradiation, growth decreased in a continuous manner following a brief lag period. The decrease in growth continued for 5 hours, then remained constant at 25% of the dark rate. The abundance of ABP1 and the level of free IAA both decreased in the mesocotyl. Only the kinetics of the decrease in IAA within the apical mesocotyl correlated with the initial change in growth, although growth continued to decrease even after IAA content reached its final level, 50% of the dark control. This decrease in IAA within the mesocotyl probably occurs primarily by a change in its transport within the shoot since auxin applied as a pulse moved basipetally in R-irradiated tissue at the same rate but with half the area as dark control tissue. In situ localization of auxin in etiolated maize shoots revealed that R-irradiated shoots contained less auxin in the epidermis than the dark controls. Irradiated mesocotyl grew 50% less than the dark controls even when incubated in an optimal level of auxin. However, irradiated and dark tissue contained essentially the same amount of radioactivity after incubation in [¹⁴C]IAA indicating that the light treatment does not affect the uptake into the tissue through the cut end, although it is possible that a small subset of cells within the mesocotyl is affected. These observations support the hypothesis that R causes a decrease in the level of auxin in epidermal cells of the mesocotyl, consequently constraining the growth of the entire mesocotyl.     

Rapid induction of specific mRNAs by auxin in pea epicotyl tissue

DNA sequences complementary to three indoleacetic acid (IAA)-inducible mRNAs in pea epicotyl tissue were isolated by differential plaque filter hybridization of cDNA libraries constructed in the vector lambda gt10. Clone pIAA6 hybridized to an mRNA encoding the previously identified translational product polypeptide 6 (Mr 22,000), and clone pIAA4/5 hybridized to one or two mRNAs, encoding polypeptides 4 and 5 (Mr 23,000 and 25,000, respectively). The cDNA clones were subsequently used to characterize the hormonally mediated mRNA accumulation. The induction of the mRNAs was rapid, within 15 minutes of exposure to the IAA, and specific to auxins. Anaerobiosis, heat and cold stress did not induce the mRNAs. Other plant hormones, such as gibberellic acid, kinetin, abscisic acid and ethylene were also unable to cause or interfere with the IAA-induced mRNA accumulation. The hormonally regulated mRNAs were induced at least 50 to 100-fold above control levels after two hours of treatment with IAA and the accumulation was (1) independent of protein synthesis, (2) completely abolished by alpha-amanitin, (3) not due to polyadenylylation of pre-existing RNAs, and (4) independent of IAA and fusicoccin-induced H+ secretion. The IAA-induced mRNAs returned to control levels within three hours after removal of IAA, and the hormonally regulated genes were primarily expressed in the third and second internode of the seven-day-old etiolated pea seedling. The data indicate that IAA increases the amount of specific mRNAs rather than alters the translatability of pre-existing mRNAs. Auxin-induced H+ secretion appears not to have a potential role in mediating the induction and perhaps is a consequence of the enhanced biosynthetic activity induced by the hormone. The IAA-mediated mRNA induction is the fastest known for any plant growth regulator and may represent a primary hormonal response to auxin.     

Auxin-induced assimilate translocation in the bean stem (Phaseolus vulgaris L.)

Decapitation of the fully-elongated fourth internode of Phaseolus vulgaris plants resulted in the disappearance from the internode of soluble acid invertase (EC 3.2.1.26). This loss was prevented by local applications to the internode of indol-3yl-acetic acid (IAA) and, at the point of IAA application, the specific activity of the enzyme increased by up to 3 times its initial value within 48 h of treatment. IAA applications stimulated the acropetal translocation to the internode of ¹⁴C-sucrose applied to the subtending (second) trifoliate leaf 30 h after decapitation and the start of the auxin treatment. Labelled assimilates accumulated in the IAA-treated region of the internode. Following decapitation the concentration of hexose sugars in the internode fell and that of sucrose rose substantially, but these trends were reversed by IAA treatment. However, small local accumulations of sucrose occurred at the point of auxin application where tissue concentrations of IAA were greatest (determined using [1-¹⁴C] IAA).Considerable quantities of starch were present in the ground parenchyma of the internodes at the start of the experiment but, in the absence of IAA, this was remobilised within 48 h of decapitation. IAA prevented starch loss at and below its point of application to the internode, but not from more distal tissues. Cambial proliferation, radial growth and lignification were stimulated in and below IAA-treated regions of the internode. These observations are discussed in relation to the hormonal regulation of assimilate translocation in the phloem.     

Indole-3-acetic acid levels after phytochrome-mediated changes in the stem elongation rate of dark- and light-grownPisum seedlings

The effect of red (R) and far-red (FR) light on stem elongation and indole-3-acetic acid (IAA) levels was examined in dwarf and tall Pisum sativum L. seedlings. Red light reduced the extension-growth rate of etiolated seedlings by 70–90% after 3 h, and this inhibition was reversible by FR. Inhibition occurred throughout the growing zone. After 3 h of R, the level of extractable IAA in whole stem sections from the growing zone of etiolated plants either increased or showed no change. By contrast, extractable IAA from epidermal peels consistently decreased 3 h after R treatments. Decreases of 40% were observed for epidermal peels from the top 1 cm of tall plants receiving 3 h R. Brief R treatments resulted in smaller decreases in epidermal IAA levels and these decreases were not as great when FR followed R. In lightgrown plants, end-of-day FR stimulated growth during the following dark period in a photoreversible manner. The uppermost 1 cm of expanding third internodes was most responsive to the FR. Extractable IAA from epidermal peels from the upper 1 cm of third internodes increased by 30% or more 5 h after FR. When R followed the FR the increases were smaller. Levels of IAA in whole stem sections did not change and were twofold greater than in dark-grown plants. In both dark- and light-grown tall plants, IAA levels were lower in epidermal peels than in whole stem segments. These results provide evidence that IAA is compartmentalized at the tissue level within the growing stem and that phytochrome regulation of stem elongation rates may be partly based on modulating the level of IAA within the epidermis.Abbreviations IAA indole-3-acetic acid - R red light - FR farred light We thank Yu-Xian Zhu for helping to develop methods for IAA analysis, James Reid for supplying the genetic lines of Pisum and Richard Cyr for the use of microscopy equipment. This work was supported by NSF grant DCB-8801880 and by Hatch funds from the College of Agriculture and Life Sciences at Cornell University. The gas chromatograph-mass spectrometer was funded by NSF grant DMB-8505974 and funds from the College of Agriculture and Life Sciences at Cornell University. A preliminary report of some of these experiments has appeared in Plant Growth Substances, 1991 (Behringer et al. 1992 b).