Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

The rib1 mutant of Arabidopsis has alterations in indole-3-butyric acid transport, hypocotyl elongation, and root architecture

Polar transport of the auxin indole-3-butyric acid (IBA) has recently been shown to occur in Arabidopsis (Arabidopis thaliana) seedlings, yet the physiological importance of this process has yet to be fully resolved. Here we describe the first demonstration of altered IBA transport in an Arabidopsis mutant, and show that the resistant to IBA (rib1) mutation results in alterations in growth, development, and response to exogenous auxin consistent with an important physiological role for IBA transport. Both hypocotyl and root IBA basipetal transport are decreased in rib1 and root acropetal IBA transport is increased. While indole-3-acetic acid (IAA) transport levels are not different in rib1 compared to wild type, root acropetal IAA transport is insensitive to the IAA efflux inhibitor naphthylphthalamic acid in rib1, as is the dependent physiological process of lateral root formation. These observed changes in IBA transport are accompanied by altered rib1 phenotypes. Previously, rib1 roots were shown to be less sensitive to growth inhibition by IBA, but to have a wild-type response to IAA in root elongation. rib1 is also less sensitive to IBA in stimulation of lateral root formation and in hypocotyl elongation under most, but not all, light and sucrose conditions. rib1 has wild-type responses to IAA, except under one set of conditions, low light and 1.5% sucrose, in which both hypocotyl elongation and lateral root formation show altered IAA response. Taken together, our results support a model in which endogenous IBA influences wild-type seedling morphology. Modifications in IBA distribution in seedlings affect hypocotyl and root elongation, as well as lateral root formation.     

Overexpression of Maize IAGLU in Arabidopsis thaliana Alters Plant Growth and Sensitivity to IAA but not IBA and 2,4-D

Overexpression of the IAGLU gene from maize (ZmIAAGLU) in Arabidopsis thaliana, under the control of the CaMV 35S promoter, inhibited root but not hypocotyl growth of seedlings in four different transgenic lines. Although hypocotyl growth of seedlings and inflorescence growth of mature plants was not affected, the leaves of mature plants were smaller and more curled as compared to wild-type and empty vector transformed plants. The rosette diameter in transgenic lines with higher ZmIAGLU expression was also smaller compared to the wild type. Free indole-3-acetic acid (IAA) levels in the transgenic plants were comparable to the wild type, even though a decrease in free IAA levels might be expected from overexpression of an IAA-conjugate–forming enzyme. IAA-glucose levels, however, were increased in transgenic lines compared to the wild type, indicating that the ZmIAGLU gene product is active in these plants. In addition, three different 35SZmIAGLU lines showed less inhibition of root growth when cultivated on increasing concentrations of IAA but not indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Feeding IAA to transgenic lines resulted in increased IAA-glucose synthesis, whereas the levels of IAA-aspartate and IAA-glutamine formed were reduced compared to the wild type. Our results show that IAA homeostasis can be altered by heterologous overexpression of a conjugate-forming gene from maize.     

Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology

Summary Mutant lines of Arabidopsis thaliana resistant to the artificial auxin 2,4-dichloro phenoxyacetic acid (2,4-D) were isolated by screening for growth of seedlings in the presence of toxic levels of 2,4-D. Genetic analysis of these resistant lines indicated that 2,4-D resistance is due to a recessive mutation at a locus we have designated Axr-1. Mutant seedlings were resistant to approximately 50-fold higher concentrations of 2,4-D than wild-type and were also resistant to 8-fold higher concentrations of indole-3-acetic acid (IAA) than wild-type. Labelling studies with (¹⁴C)2,4-D suggest that resistance was not due to changes in uptake or metabolism of 2,4-D. In addition to auxin resistance the mutants have a distinct morphological phenotype including alterations of the roots, leaves, and flowers. Genetic evidence indicates that both auxin resistance and the morphological changes are due to the same mutation. Because of the pleiotropic morphological effects of these mutations the Axr-1 gene may code for a function involved in auxin action in all tissues of the plant.     

RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling

The roots curl in naphthylphthalamic acid1 (rcn1) mutant of Arabidopsis (Arabidopsis thaliana) has altered auxin transport, gravitropism, and ethylene response, providing an opportunity to analyze the interplay between ethylene and auxin in control of seedling growth. Roots of rcn1 seedlings were previously shown to have altered auxin transport, growth, and gravitropism, while rcn1 hypocotyl elongation exhibited enhanced ethylene response. We have characterized auxin transport and gravitropism phenotypes of rcn1 hypocotyls and have explored the roles of auxin and ethylene in controlling these phenotypes. As in roots, auxin transport is increased in etiolated rcn1 hypocotyls. Hypocotyl gravity response is accelerated, although overall elongation is reduced, in etiolated rcn1 hypocotyls. Etiolated, but not light grown, rcn1 seedlings also overproduce ethylene, and mutations conferring ethylene insensitivity restore normal hypocotyl elongation to rcn1. Auxin transport is unaffected by treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid in etiolated hypocotyls of wild-type and rcn1 seedlings. Surprisingly, the ethylene insensitive2-1 (ein2-1) and ein2-5 mutations dramatically reduce gravitropic bending in hypocotyls. However, the ethylene resistant1-3 (etr1-3) mutation does not significantly affect hypocotyl gravity response. Furthermore, neither the etr1 nor the ein2 mutation abrogates the accelerated gravitropism observed in rcn1 hypocotyls, indicating that both wild-type gravity response and enhanced gravity response in rcn1 do not require an intact ethylene-signaling pathway. We therefore conclude that the RCN1 protein affects overall hypocotyl elongation via negative regulation of ethylene synthesis in etiolated seedlings, and that RCN1 and EIN2 modulate hypocotyl gravitropism and ethylene responses through independent pathways.     

MASSUGU2 encodes Aux/IAA19, an auxin-regulated protein that functions together with the transcriptional activator NPH4/ARF7 to regulate differential growth responses of hypocotyl and formation of lateral roots in Arabidopsis thaliana

We have isolated a dominant, auxin-insensitive mutant of Arabidopsis thaliana, massugu2 (msg2), that displays neither hypocotyl gravitropism nor phototropism, fails to maintain an apical hook as an etiolated seedling, and is defective in lateral root formation. Yet other aspects of growth and development of msg2 plants are almost normal. These characteristics of msg2 are similar to those of another auxin-insensitive mutant, non-phototropic hypocotyl4 (nph4), which is a loss-of-function mutant of AUXIN RESPONSE FACTOR7 (ARF7) (Harper et al., 2000). Map-based cloning of the MSG2 locus reveals that all four mutant alleles result in amino acid substitutions in the conserved domain II of an Auxin/Indole-3-Acetic Acid protein, IAA19. Interestingly, auxin inducibility of MSG2/IAA19 gene expression is reduced by 65% in nph4/arf7. Moreover, MSG2/IAA19 protein binds to the C-terminal domain of NPH4/ARF7 in a Saccharomyces cerevisiae (yeast) two-hybrid assay and to the whole latter protein in vitro by pull-down assay. These results suggest that MSG2/IAA19 and NPH4/ARF7 may constitute a negative feedback loop to regulate differential growth responses of hypocotyls and lateral root formation.     

Exogenously applied melatonin stimulates root growth and raises endogenous indoleacetic acid in roots of etiolated seedlings of Brassica juncea

Exogenous melatonin was applied to etiolated seedlings of wild leaf mustard (Brassica juncea) and the effect on root growth and endogenous indole-3-acetic acid (IAA) levels determined. The results show that 0.1microM melatonin has a stimulatory effect on root growth, while 100microM is inhibitory. Furthermore, the stimulatory effect was only detectable in young seedlings (2-d old). Older seedlings (4-d old) appear to be less susceptible to both the stimulatory and the inhibitory effect of melatonin. Exogenous application of 0.1microM melatonin also raised the endogenous levels of free IAA in roots, while higher concentrations had no significant effect. The specific mechanism that causes exogenous melatonin to increase the amount of free IAA in roots, paired with a stimulation of root growth, remains to be uncovered.     

The MSG1 and AXR1 genes of Arabidopsis are likely to act independently in growth-curvature responses of hypocotyls

Growth-curvature responses of hypocotyls of Arabidopsis thaliana (L.) Heynh. were measured in double mutants between msg1 and axr1, both of which are auxin-resistant and defective in hypocotyl growth curvature induced upon unilateral application of auxin. The msg1 axr1 double mutants showed no auxin-induced growth curvature, that is, they exhibited the msg1 phenotype, though the axr1 defects were partial. Hypocotyls of both the msg1 and axr1 mutants were partially defective in second-positive phototropism, whereas the double mutants lost the response completely. When grown on vertically held agar plates, the axr1 mutant showed normal hypocotyl gravitropism and the mutation did not affect the reduced hypocotyl gravitropism of msg1. Hypocotyls of msg1 and axr1 mutants grew upward like wild-type ones when grown along an agar surface, while they grew more randomly when grown without an agar support, suggesting that axr1 hypocotyls are not completely normal in gravitropism. The extent of defects in growth orientation increased in the order: msg1 axr1 double mutants > msg1 > axr1 > wild type. The hypocotyls of these mutants showed auxin resistance in the order: msg1 axr1 > axr1 > msg1 > wild type. The msg1 mutant had epinastic leaves and axr1 had wrinkled leaves; leaves of the msg1 axr1 double mutants were epinastic and wrinkled. These results suggest that MSG1 and AXR1 act independently in separate pathways of the reactions tested in the present study. In contrast, the phenotype of the msg1 aux1 double mutants shows that AUX1 is not significantly involved in these phenomena. Received: 12 July 1998 / Accepted: 16 August 1998     

Oxidation of NADH by hypocotyl segments from soybean is stimulated by 2,4-D

Sections cut from regions of cell elongation of hypocotyls of dark-grown soybean seedlings oxidized externally supplied NADH as estimated from the decrease in A₃₄₀ measured spectrophotometrically. The oxidation of NADH by 1-cm sections was stimulated 1.5- to 2-fold by 1 μM of the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D-Stimulated oxidation of NADH was resistant to cyanide. Stimulations were also given by the naturally occurring auxin, indole-3-acetic acid (IAA) but not by the growth inactive 2,4-D analog 2,3-dichlorophenoxyacetic acid (2,3-D) and the growth inactive β-naphthaleneacetic acid (β-NAA). Since NADH is a membrane impermeant substrate, the findings confirm studies with inside-out and right-side-out vesicles that show the 2,4-D-stimulated NADH oxidase to be located at the external cell surface. Cut surfaces are not responsible for the activity as shown by experiments with lanolin-sealed sections. The external NADH oxidase measurements do not require special equipment and exhibit characteristics normally associated with enzyme-catalyzed reactions.     

In vitro regeneration of plants from hypocotyl segments of Amaranthus paniculatus

Hypocotyl segments, 5 to 8 mm length from 4 to 7 day old seedlings, callused on B₅ medium supplemented with Kn (0.5 ppm) and NAA (0.1 ppm). Even without transfer, shoots were formed in such cultures. About 20% of the cultures produced multiple shoots. In medium with 1 ppm each of Kn and NAA direct shoots were formed at one end of the hypocotyl segment and callusing was initiated at the other end. The plants obtained in either medium formed roots and could be transferred to soil for further growth.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy acetic acid - GA₃ gibberellic acid - Kn Kinetin - NAA naphthalene acetic acid     

Differential IAA dose response relations of the axr1 and axr2 mutants of Arabidopsis

In comparison to wild type Arabidopsis thaliana, the auxin resistant mutants axr1 and axr2 exhibit reduced inhibition of root elongation in response to auxins. Several auxin-regulated physiological processes are also altered in the mutant plants. When wild-type, axr1 and axr2 seedlings were grown in darkness on media containing indoleacetic acid (IAA), promotion of root growth was observed at low concentrations of IAA (10^?11 to 10^?7M) in 5-day-old axr2 seedlings, but not in axr1 or wild-type seedlings. In axr1 there was little or no measurable root growth response over the same concentration range. In wild type, root growth was inhibited at concentrations greater than 10^?10M and no detectable root growth response was observed at lower concentrations. In addition, production of lateral roots in response to IAA increased in axr2 seedlings and decreased in axr1 seedlings relative to wild type. Promotion of root elongation and initiation of lateral roots in axr2 seedlings in response to auxin indicate that axr2 seedlings are able to perceive and respond to IAA.     

Physiological responses of resistant and susceptible plants to diclofop-methyl and its antagonism by 2,4-D and antioxidants

Diclofop-methyl (DM) sprayed onto 6–8-week-old plants of leafy spurge ( Euphorbia esula L.) caused senescence and abscission of older leaves, while the young leaves and apex remained attached. The phytotoxicity of DM was reversed by the antioxidant, α -tocopherol (vitamin E), in leafy spurge and DM-susceptible oat ( Avena sativa L. cv. Gary). DM and 2,4-dichlorophenoxyacetic acid (2,4-D) increased ethylene evolution in mature leaves of leafy spurge. Vitamin E reduced the DM-induced ethylene by ampproximately 50%, but had no effect on the 2,4-D-induced ethylene. DM did not increase ethylene in DM-resistant pea or tobacco, but 2,4-D induced a 3-fold increase in ethylene evolution over controls in DM-resistant tobacco. 2,4-D amppears to act at a site different from that of DM in the pathway of ethylene formation. Ethylene evolution increased in DM-treated susceptible biotypes of annual ryegrass ( Lolium rigidum L.) and wild oat ( Avena fatua L.), but not in resistant biotypes of these species. DM reduced root and shoot formation and dry weight in hypocotyl segments of etiolated leafy spurge seedlings grown in vitro. Organogenesis and dry weights were increased by the combination of DM+antioxidants. Vitamin E was a more effective antioxidant than ascorbic acid. These results sumpport the hypothesis that DM induces oxidative stress in susceptible plant tissues and that antioxidants reduce the damaging action of the phytotoxic free radicals.     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

Effects of natural and synthetic auxins on the gravitropic growth habit of roots in two auxin-resistant mutants of Arabidopsis, axr1 and axr4: evidence for defects in the auxin influx mechanism of axr4.

The partially agravitropic growth habit of roots of an auxin-resistant mutant of Arabidopsis thaliana, axr4, was restored by the addition of 30-300 nM 1-naphthaleneacetic acid (NAA) to the growth medium. Neither indole 3-acetic acid (IAA) nor 2,4-dichlorophenoxyacetic acid (2,4-D) showed such an effect. Growth of axr4 roots was resistant to IAA and 2,4-D, but not at all to NAA. The differential effects of the three auxins suggest that the defects of axr4 result from a lower auxin influx into its cells. The partially agravitropic growth habit of axr1 roots, which was less severe than that of axr4 roots, was only slightly affected by the three auxins in the growth medium at concentrations up to 300 nM; growth of axr1 roots was resistant to all three of the auxins. These results suggest that the lesion of axrl mutants is different from that of axr4.     

Effects of brassinosteroid, auxin, and cytokinin on ethylene production in Arabidopsis thaliana plants

Inflorescence stalks produced the highest amount of ethylene in response to IAA as compared with other plant parts tested. Leaf age had an effect on IAA-induced ethylene with the youngest leaves showing the greatest stimulation. The highest amount of IAA-induced ethylene was produced in the root or inflorescence tip with regions below this producing less. Inflorescence stalks treated with IAA, 2,4-D, or NAA over a range of concentrations exhibited an increase in ethylene production starting at 1 microM with increasingly greater responses up to 100 microM, followed by a plateau at 500 microM and a significant decline at 1000 microM. Both 2,4-D and NAA elicited a greater response than IAA at all concentrations tested in inflorescence stalks. Inflorescence leaves treated with IAA, 2,4-D, or NAA exhibited the same trend as inflorescence stalks. However, they produced significantly less ethylene. Inflorescence stalks and leaves treated with 100 microM IAA exhibited a dramatic increase in ethylene production 2 h following treatment initiation. Inflorescence stalks showed a further increase 4 h following treatment initiation and no further increase at 6 h. However, there was a slight decline between 6 h and 24 h. Inflorescence leaves exhibited similar rates of IAA-induced ethylene between 2 h and 24 h. Light and high temperature caused a decrease in IAA-induced ethylene in both inflorescence stalks and leaves. Three auxin-insensitive mutants were evaluated for their inflorescence's responsiveness to IAA. aux2 did not produce ethylene in response to 100 microM IAA, while axr1-3 and axr1-12 showed reduced levels of IAA-induced ethylene as compared with Columbia wild type. Inflorescences treated with brassinolide alone had no effect on ethylene production. However, when brassinolide was used in combination with IAA there was a dramatic increase in ethylene production above the induction promoted by IAA alone.     

Boron and blue light reduce responsiveness of <Emphasis Type="Italic">Arabidopsis</Emphasis> hypocotyls to exogenous auxins

We reported earlier that boron stimulates hypocotyl growth in several Arabidopsis ecotypes but not in the boron-deficient mutant bor1-1. Others have shown that boron influences the metabolism and transport of the plant hormone auxin. We investigated how boron, in interaction with light, influences Arabidopsis hypocotyl growth responses to the exogenous auxins 1-NAA, 2,4-D and IAA. In either light condition, 1-NAA similarly inhibited hypocotyl growth in bor1-1 and the corresponding WT (Col-0), while in both genotypes, boron did not essentially affect the extent of the inhibition. Whatever the light conditions and in the absence of boron, 2,4-D inhibited hypocotyl elongation in WT, while in BL seedlings, high responsiveness to 2,4-D vanished when boron was added to the culture medium. Hypocotyl of bor1-1 seedlings in all boron concentrations tested and grown in the dark or RL responded to the auxin similar to WT plants. In BL, the mutant hypocotyls retained full sensitivity to 2,4-D at 0.1 mM H₃BO₃ but lost that sensitivity by 2 mM. In both genotypes tested, in the dark or RL, IAA inhibited hypocotyl growth. Conversely, IAA stimulated hypocotyl elongation in both genotypes developed in BL at 0.1 mM H₃BO₃. That stimulation disappeared when the boron supply increased to 2 mM. Our results suggest that specifically in BL, boron reduces hypocotyl responsiveness to auxins 2,4-D or IAA via the functional transporter BOR1. Our results lead to a discussion of how BL and BOR1 influence the mechanisms of auxin transport into and out of the cell.     

Somatic embryogenesis and plant regeneration from embryogenic callus of soybean,Glycine max L.

During induction of somatic embryogenesis from immature embryos of soybean, a smooth-shiny and a rough callus were obtained. The smooth-shiny type developed from callus derived from cotyledonary tissue and cultured on media containing 10 mg/l 2,4-D. The rough type was derived from immature embryos, cotyledons, hypocotyls and hypocotyl segments from germinated seedlings on a medium containing various growth regulators. Plants were obtained from the smooth-shiny type but rough callus did not differentiate into plants. The conditions for formation of both callus types and regeneration of mature soybean plants from the smooth-shiny type were defined.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - ABA abscicic acid - GA3 gibberellic acid - BA benzyladenine - B5 Gamborg et al. - LS Linsmaier & Skoog - MS Murashige & Skoog - P proline     

A small acidic protein 1 (SMAP1) mediates responses of the Arabidopsis root to the synthetic auxin 2,4-dichlorophenoxyacetic acid

2,4-dichlorophenoxyacetic acid (2,4-D), a chemical analogue of indole-3-acetic acid (IAA), is widely used as a growth regulator and exogenous source of auxin. Because 2,4-D evokes physiological and molecular responses similar to those evoked by IAA, it is believed that they share a common response pathway. Here, we show that a mutant, antiauxin resistant1 (aar1), identified in a screen for resistance to the anti-auxin p-chlorophenoxy-isobutyric acid (PCIB), is resistant to 2,4-D, yet nevertheless responds like the wild-type to IAA and 1-napthaleneacetic acid in root elongation and lateral root induction assays. That the aar1 mutation alters 2,4-D responsiveness specifically was confirmed by analysis of GUS expression in the DR5:GUS and HS:AXR3NT-GUS backgrounds, as well as by real-time PCR quantification of IAA11 expression. The two characterized aar1 alleles both harbor multi-gene deletions; however, 2,4-D responsiveness was restored by transformation with one of the genes missing in both alleles, and the 2,4-D-resistant phenotype was reproduced by decreasing the expression of the same gene in the wild-type using an RNAi construct. The gene encodes a small, acidic protein (SMAP1) with unknown function and present in plants, animals and invertebrates but not in fungi or prokaryotes. Taken together, these results suggest that SMAP1 is a regulatory component that mediates responses to 2,4-D, and that responses to 2,4-D and IAA are partially distinct.