Insensitivity of the diageotropica tomato mutant to auxin

The sensitivity of excised hypocotyl segments to indoleacetic acid (IAA) in two assays, ethylene production and elongation, was determined in the ethylene-requiring tomato (Lycopersicon esculentum Mill.) mutant, diageotropica (dgt), and its isogenic parent, cv VFN8. Endogenous (uninduced) ethylene synthesis rates were slightly lower in dgt hypocotyls than in VFN8 hypocotyls. Ethylene production was essentially unaffected by IAA in dgt, but was stimulated up to 10-fold by 10 micromolar IAA in VFN8. Elongation of dgt hypocotyls was also insensitive to concentrations of IAA as high as 100 micromolar, as compared to significant elongation of VFN8 hypocotyls in response to 0.1 micromolar IAA. A range of IAA analogs active in VFN8 was also ineffective in stimulating elongation of dgt hypocotyls, suggesting that the differences were not due to rapid metabolism of IAA by dgt tissues. Auxin-induced elongation of VFN8 hypocotyls was unaffected by 2,3,5-triiodobenzoic acid and naphthylphthalamic acid, indicating that polar auxin transport was not a factor in these experiments. Exogenous and auxin-induced ethylene had no effect on the elongation respone of either genotype, nor did exogenous ethylene restore the sensitivity of dgt hypocotyls to IAA. Despite their apparent insensitivity to auxin, dgt hypocotyls elongated dramatically and synthesized ethylene rapidly in response to 1.2 micromolar fusicoccin. These results suggest that the primary effect of the dgt mutation is to reduce the sensitivity of the tissue to auxin. As altered regulation of ethylene synthesis is only one symptom of this fundamental deficiency, dgt should more properly be considered to be the auxin-insensitive tomato mutant.     

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.     

Ineffectiveness of ethylene biosynthetic and action inhibitors in phenotypically reverting theEpinastic mutant of Tomato (Lycopersicon esculentum mill.)

Five-day-old, dark-grown seedlings of theEpinastic (Epi) tomato mutant (Lycopersicon esculentum Mill.) and its parent, cultivar VFN8, were used as a system for assessing the role of ethylene in theEpi phenotype. The distinguishing features ofEpi seedlings are an increase in hypocotyl diameter and reduced hypocotyl length. Treatment of VFN8 seedlings with 0.5 l/liter ethylene closely mimicked theEpi phenotype. The rate of ethylene production by 5-day-old, dark-grownEpi seedlings was double that of VFN8 seedlings. Nevertheless, treatment ofEpi seedlings with inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine or Co²⁺) or ethylene action (silver thiosulfate or norbornadiene) failed to normalize theEpi phenotype.Epi seedlings grown in sealed jars containing ethylene and CO₂ adsorbants also expressed the characteristicEpi phenotype. The results indicate that the physiological lesion resulting from theEpi gene mutation is not simply an overproduction of ethylene.     

Auxin and Ethylene Regulation of Petiole Epinasty in Two Developmental Mutants of Tomato, diageotropica and Epinastic

The epinastic growth responses of petioles to auxin and ethylene were quantified in two developmental mutants of tomato (Lycopersicon esculentum Mill.). In the wild type parent line, cultivar VFN8, the epinastic response of excised petiole sections was approximately log-linear between 0.1 and 100 micromolar indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations, with a greater response to 2,4-D at any concentration. When ethylene synthesis was inhibited by aminoethoxyvinylglycine (AVG), epinasty was no longer induced by auxin, but could be restored by the addition of ethylene gas. In the auxin-insensitive mutant, diageotropica (dgt), no epinastic response to IAA was observed at IAA concentrations that effectively induced epinasty in VFN8. In the absence of added IAA, epinastic growth of dgt petioles in 1.3 microliters per liter exogenous ethylene gas was more than double that of VFN8 petioles. IAA had little additional effect in dgt, but promoted epinasty in VFN8. These results confirm that tomato petiole cells respond directly to ethylene and make it unlikely that the differential growth responsible for epinasty results from lateral auxin redistribution. The second mutant, Epinastic (Epi), exhibits constitutively epinasty, cortical swelling, and root branching symptomatic of possible alternation in auxin or ethylene regulation of growth. Only minor quantitative differences were observed between the epinastic responses to auxin and ethylene of VFN8 and Epi. However, in contrast to VFN8, when ethylene synthesis or action was inhibited in Epi, auxin still induced 40 to 50% of the epinastic response observed in the absence of inhibitors. This indicates that the target cells for epinastic growth in Epi are qualitatively different from those of VFN8, having gained the ability to grow differentially in response to auxin alone. The dgt and Epi mutants provide useful systems in which to study the genetic determination of target cell specificity for hormone action.     

Stress-induced Ethylene Production in the Ethylene-requiring Tomato Mutant Diageotropica

Ethylene synthesis in vegetative tissues is thought to be controlled by indoleacetic acid (IAA). However, ethylene synthesis in the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) was much less sensitive to IAA than in the normal variety (VFN8). Yet, mechanical wounding stimulated ethylene production by the mutant. The dgt tomato provides an opportunity to study the regulation of stress ethylene independent of IAA effects. Waterlogging (i.e. anaerobic stress) stimulated production of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), in the roots. The ACC was transported to the shoot where it was converted to ethylene. The dgt mutant efficiently utilized ACC for ethylene synthesis under aerobic conditions. The results confirm that the genetic lesion in dgt is located at a step prior to the formation of ACC. Furthermore, induction of ethylene synthesis by anaerobic or mechanical stresses in this mutant is independent of IAA action.     

Quantification of indol-3-yl acetic acid in pea and maize seedlings by gas chromatography-mass spectrometry

A procedure is described for the identification and quantification of IAA in plant tissues by GC/MS analysis of the N-heptafluorobutyryl ethyl ester of IAA using [²H₅]IAA as an internal standard. The detection limit is ca 3 pmol IAA/tissue sample. By using this method, IAA levels of 30–90 pmol/g fr. wt were obtained for dark-grown Pisum sativum epicotyls and 71–199 pmol/g fr. wt for dark-grown Zea mays seedlings. When either methanol or ethanol was used as extraction solvent, some esterification of IAA during sample preparation was observed. No evidence for the natural occurrence of methyl or ethyl esters of IAA in Pisum sativum seedlings was found.     

Characterization of an Ethylene Overproducing Mutant of Tomato (Lycopersicon esculentum Mill. Cultivar VFN8)

Ethylene production rates and tissue ethylene concentrations were determined for the single-gene, Epinastic (Epi) tomato (Lycopersicon esculentum Mill.) mutant, and its parent, cv VFN8. The Epi phenotype was characterized by severe leaf epinasty, thickened stems and petioles, and a compact growth habit. In 4-day-old seedlings, ethylene production was significantly higher in Epi than in VFN8. Ethylene production rates also were higher for excised root, hypocotyl, cotyledon, and shoot tissue of 14-day-old Epi seedlings as compared with VFN8. The greatest difference in the ethylene production rate was observed in excised Epi shoot tissue, which was more than 2.5 times higher than in VFN8. Tissue ethylene concentrations of 19−, 25−, and 31-day-old Epi plants were 8, 172, and 307% higher than for VFN8, corresponding to increasing expression of the Epi phenotypic characteristics with age. The highest ethylene concentrations occurred in the shoot apex of both genotypes. Higher ethylene concentrations in Epi resulted from greater 1-aminocyclopropane-1-carboxylic acid content rather than increased ethylene-forming enzyme activity. The elevated ethylene levels in Epi did not result from increased auxin sensitivity. The sensitivity of root growth to inhibition by ethylene did not differ between VFN8 and Epi. Although elevated levels of ethylene in Epi plants apparently exacerbate its epinastic growth characteristics, other evidence indicates that this may not be the fundamental lesion. This mutant may provide a unique system for investigating the regulation of ethylene biosynthesis and the role of target cell types in plant development.     

Auxin Biosynthesis during Seed Germination in Phaseolus vulgaris

The relative roles of de novo biosynthesis of indoleacetic acid (IAA) and IAA conjugates stored in mature seeds (Phaseolus vulgaris L.) in supplying auxin to germinating bean seedlings were studied. Using ²H oxide and 2,4,5,6,7-[²H]l-tryptophan as tracers of IAA synthesis, we have shown that de novo biosynthesis of IAA, primarily from tryptophan, is an important source of auxin for young bean seedlings. New synthesis of IAA was detected as early as the second day of germination, at which time the seedlings began to accumulate fresh weight intensively and the total content of free IAA began to increase steadily. IAA conjugates that accumulate in large amounts in cotyledons of mature seeds may thus be considered to be only one of the possible sources of IAA required for the growth of bean seedlings.     

DIFFERENT SPECIES,SUCROSE, AND LIGHT IN PLANT SECTION ELONGATION TESTS

Sections from both dark- and light-grown seedlings of 11 species were used to test responses to IAA (indoleacetic acid), sucrose, and an inhibitor prepared from cabbage seedlings. Variability among species was great; however, results indicate that many species, light-grown as well as dark-grown, could prove useful in bioassays and probably should be investigated. Although elongation of segments from high-intensity-light-grown cabbage and cucumber hypocotyls and oat coleoptiles had essentially stopped by the time of cutting, their growth and response to IAA as sections were considerable. Neither oat coleoptile nor pea internode sections can be considered representative because of differences in responses to sucrose, of dark-grown sections to light, and to an inhibitor prepared from cabbage. Sucrose generally did not stimulate and even inhibited response of most hypocotyls to IAA. Sucrose was absorbed by sections, increasing final dry weight while not affecting elongation. Sucrose reduced the rate of respiratory decay in cabbage and sunflower, but IAA did not affect respiration. Changes in length and fresh weight of cucumber hypocotyl sections were comparable.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.     

Inhibitors from Carob (Ceratonia siliqua L.): II. Effect on Growth Induced by Indoleacetic Acid or Gibberellins A(1), A(4), A(5), and A(7)

Two inhibitory fractions (B₁ and C) from extracts of immature fruit of carob were tested for their ability to inhibit the action of indoleacetic acid (IAA) in three bioassays. There was no reduction of IAA-induced reactions in the Avena curvature test, abscission of debladed coleus petioles, or growth of cucumber hypocotyls. The highest ratio of inhibitor to IAA was 10,000 times greater than the ratio necessary to inhibit by 50% the growth caused by an equivalent amount of gibberellin A₃ in pea seedlings. At the highest concentration used, fraction C alone caused curvature of Avena coleoptiles. The inhibitory fractions appeared to enhance the effect of IAA in the cucumber test.     

Effect of drought and ABA on growth, photosynthesis and antioxidant system of Cotinus coggygria seedlings under two different light conditions

We exposed seedlings of Cotinus coggygria var. cinerea to drought and exogenous abscisic acid (ABA) under two different light conditions. Two watering regimes (well-watered and drought), two exogenous ABA applications (no ABA and with ABA) and two light regimes (full sunlight and shade) were employed. Compared with well-watered treatment, drought treatment significantly reduced the relative growth rate, relative water content (RWC), net photosynthesis rate (A) and transpiration (E), but increased chlorophyll a (chla), carbon isotope (δ¹³C), endogenous ABA, malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) contents, and guaiacol peroxidase (POD) and catalase (CAT) activities. There was an apparent alleviation of drought effects by shade, as indicated by the lower relative growth rate, and chlorophyll, MDA and H₂O₂ contents, and increases in indoleacetic acid (IAA) and reduced glutathione (GSH) contents. On the other hand, the exogenous ABA application under shade induced protective effects on drought-stressed seedlings, as visible in RWC, MDA, A, stomatal conductance (g_s), E, δ¹³C, ABA and IAA values. In all, our results suggest that seedlings of C. coggygria are more sensitive to drought under full-light than under shade.     

Biosynthesis of Indoleacetic Acid from Tryptophan-C in Cell-free Extracts of Pea Shoot Tips

A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-¹⁴C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a ¹⁴C-product as IAA was by (a) co-chromatography of authentic IAA and ¹⁴C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of α-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA.     

On ethylene and stem elongation in green pea seedlings

Maximum elongation of excised internodal stem sections of light-grown pea (Pisum sativum L.) seedlings occurred at 10⁻⁵ molar indoleacetic acid (IAA), with submaximal responses occurring at 10⁻⁴ and 10⁻³ molar. Accompanying elongation at concentrations of IAA of 10⁻⁶ to 10⁻³ molar was production of ethylene, with the amount increasing up to 10⁻⁴ molar IAA and then becoming nearly constant. Elongation of light-grown sections was not inhibited by exogenous ethylene up to 10,000 ppm in the presence of 10⁻⁵ molar IAA. Marked (up to 50%) inhibition of elongation of internodal segments in situ was observed after treating whole light-grown seedlings with exogenous ethylene for 20 hours. It is concluded that ethylene is not responsible for the submaximal elongation responses of green pea stem sections at high auxin concentrations, but that IAA per se is accountable.     

Localization of Stem Elongation Control in Cucumis sativus L

Reciprocal grafts, and applications of gibberellin (GA) and indoleacetic acid (IAA) were used to localize the site of control for stem elongation in cucumber (Cucumis sativus L.). Dwarf and tall plants were reciprocally grafted to determine influence of stems and roots on stem elongation. At 21 days there were no significant differences in length between stems grafted to their own roots and those grafted to roots of the other type. GA₃, GA₄₊₇, and IAA were applied to seedlings with and without live apical buds. Seedlings with live apical buds responded to level of added GA, but not to added IAA. GA₄₊₇ was more effective than GA₃. Hypocotyls of tall plants responded more to both GA treatments than did those of the dwarves when both types had live apical buds. When either GA₄₊₇ or IAA was applied to seedlings with dead apical buds, elongation of the hypocotyl responded to level of the growth regulator, but there was no difference in response between the dwarf and tall plants.     

Violaxanthin is an abscisic Acid precursor in water-stressed dark-grown bean leaves

The leaves of dark-grown bean (Phaseolus vulgaris L.) seedlings accumulate considerably lower quantities of xanthophylls and carotenes than do leaves of light-grown seedlings, but they synthesize at least comparable amounts of abscisic acid (ABA) and its metabolites when water stressed. We observed a 1:1 relationship on a molar basis between the reduction in levels of violaxanthin, 9′-cis-neoxanthin, and 9-cis-violaxanthin and the accumulation of ABA, phaseic acid, and dihydrophaseic acid, when leaves from dark-grown plants were stressed for 7 hours. Early in the stress period, reductions in xanthophylls were greater than the accumulation of ABA and its metabolites, suggesting the accumulation of an intermediate which was subsequently converted to ABA. Leaves which were detached, but not stressed, did not accumulate ABA nor were their xanthophyll levels reduced. Leaves from plants that had been sprayed with cycloheximide did not accumulate ABA when stressed, nor were their xanthophyll levels reduced significantly. Incubation of dark-grown stressed leaves in an ¹⁸O₂-containing atmosphere resulted in the synthesis of ABA with levels of ¹⁸O in the carboxyl group that were virtually identical to those observed in light-grown leaves. The results of these experiments indicate that violaxanthin is an ABA precursor in stressed dark-grown leaves, and they are used to suggest several possible pathways from violaxanthin to ABA.     

Effect of Ethylene Treatment on Polar IAA Transport, Net IAA Uptake and Specific Binding of N-1-Naphthylphthalamic Acid in Tissues and Microsomes Isolated from Etiolated Pea Epicotyls

The effect of ethylene treatment on polar indole-3-acetic acid (IAA) transport, net IAA uptake in the presence and absence of N-1-naphthylphthalamic acid (NPA) and [³H]NPA binding characteristics was investigated in tissue segments or microsomes isolated from etiolated pea (Pisum sativum L. cv Alaska) epicotyls. Basipetal IAA transport in 5 millimeter segments isolated from ethylene-treated seedlings was inhibited by ethylene in a dose-dependent manner. Threshold, half-maximal and saturating concentrations of ethylene were 0.01, 0.55, 10.0 microliters per liter, respectively. This inhibition became apparent after 6 to 8 hours of ethylene treatment. Transport velocity in both control and ethylene-treated tissues was estimated to be 5 millimeters per hour. Net IAA uptake was stimulated in ethylene-treated tissues and the relative ability of the phytotropin NPA to enhance net IAA uptake was reduced in treated tissues. Specific binding of [³H]NPA to microsomes prepared from both control and ethylene-treated tissues was saturable and consistent with the existence of a single class of binding sites with an apparent affinity (K_d) toward NPA of 8 to 9 nanomolar. The density of these binding sites (per milligram protein) was lower (36% of control) in ethylene-treated tissues. Direct application of ethylene to microsomal preparations isolated from untreated seedlings had no effect on the level of specific [³H]NPA binding.     

Low doses of ultraviolet-B or ultraviolet-C radiation affect phytohormones in young pea plants

Pea (Pisum sativum L., cv. Scinado) seedlings were exposed to low doses of ultraviolet-B (UV-B; 4.4 and 13.3 kJ m⁻² d⁻¹) or UV-C (0.1 and 0.3 kJ m⁻² d⁻¹) radiation for 14 d. Aminocyclopropane carboxylic acid (ACC), indoleacetic acid (IAA) and abscisic acid (ABA) contents were quantified by gas chromatography coupled to mass spectrometry (GC-MS). The accumulation of ACC upon irradiation was dose-dependent. ABA content was reduced and IAA content increased upon UV-C treatment whereas the UV-B doses used did not cause significant changes in ABA and IAA contents.     

In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections

The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which ¹⁴CO₂ is collected following uptake of [1-¹⁴C]IAA by excised tissue sections. After a 30-minute equilibration period, the evolution of ¹⁴CO₂ was found to follow an approximately linear course with respect to both time and tissue weight.

Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the ¹⁴CO₂ evolved per milligram tissue and the percent of [1-¹⁴C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-¹⁴C]IAA-treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation.

     

Polar Transport of Auxin across Gravistimulated Roots of Maize and Its Enhancement by Calcium

The effect of Ca on the polar movement of [³H]indoleacetic acid ([³H] IAA) in gravistimulated roots was examined using 3-day-old seedlings of maize (Zea mays L.). Transport of label was measured by placing an agar donor block containing [³H]IAA on one side of the elongation zone and measuring movement of label across the root into an agar receiver block on the opposite side. In vertically oriented roots, movement of label across the elongation zone into the receiver was slight and was not enhanced by incorporating 10 millimolar CaCl₂ into the receiver block. In horizontally oriented roots, movement of label across the root was readily detectable and movement to a receiver on the bottom was about 3-fold greater than movement in the opposite direction. This polarity was abolished in roots from which the caps were removed prior to gravistimulation. When CaCl₂ was incorporated into the receivers, movement of label across horizontally oriented intact roots was increased about 3-fold in both the downward and upward direction. The ability of Ca to enhance the movement of label from [³H]IAA increased with increasing Ca concentration in the receiver up to 5 to 10 millimolar CaCl₂. With the inclusion of CaCl₂ in the receiver blocks, gravity-induced polar movement of label into receiver blocks from applied [³H]IAA was detectable within 30 minutes, and asymmetric distribution of label within the tissue was detectable within 20 minutes. The results indicate that gravistimulation induces a physiological asymmetry in the auxin transport system of maize roots and that Ca increases the total transport of auxin across the root.