Effects of plant growth regulators on acetylene-reducing associations between Azospirillum brasilense and wheat

Treatment of wheat seedlings with the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-d), induced nodule-like structures or tumours (termed para-nodules) where lateral roots would normally emerge. The formation of these structures promoted increased rates of acetylene reduction at reduced oxygen pressure (0.02–0.04 atm) in seedling inoculated with Azospirillum brasilense, compared to seedlings inoculated without auxin treatment. Fluorescent microscopy, laser scanning confocal microscopy and direct bacterial counts all showed that the 2,4-d treatment stimulated internal colonization of the root system with azospirilla, particularly in the basal region of the nodular structures. Both colonization with azospirilla and acetylene-reducing activity were further stimulated by simultaneous treatment with another synthetic auxin, naphthaleneacetic acid (NAA) and, less reliably, with indoleacetic acid (IAA) and indolebutyric acid (IBA). These auxins produced shortening of many initiated lateral roots, although 20 times the concentration of NAA was required to achieve rounded structures similar to those obtained with 2,4-d. Treatment with NAA, IAA or IBA alone also stimulated colonization with azospirilla and acetylene reduction rates at 0.02 atm oxygen, but less effectively than by treatment with 2,4-d. Such exogenous treatments of wheat seedlings with synthetic growth regulators provide an effective laboratory model for studies on the development of a N₂-fixing system in cereals.     

The indole alkaloids brucine, yohimbine, and hypaphorine are indole-3-acetic acid-specific competitors which do not alter auxin transport

The indole alkaloids brucine and yohimbine, just like hypaphorine, counteract indole-3-acetic acid (IAA) activity in seedling roots, root hairs and shoots, but do not appear to alter auxin transport in roots or in cultured cells. In roots, the interactions between IAA and these three alkaloids appear competitive and specific since these molecules interact with IAA but with neither 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D), two synthetic auxins. The data reported further support the hypothesis that hypaphorine brucine and yohimbine compete with IAA on some auxin-binding proteins likely to be auxin receptors and that 2,4-D and NAA are not always perceived by the same receptor as IAA or the same component of that receptor. At certain steps of plant development and in certain cells, endogenous indole alkaloids could be involved in IAA activity regulation together with other well-described mechanisms such as conjugation or degradation. Hypaphorine with other active indole alkaloids remaining to be identified, might be regarded as a new class of IAA antagonists.     

The role of auxins in differentiation of rice tissues culturein Vitro

Effects of various auxins on callus induction (dedifferentiation) and organ redifferentiation from the callus were studied by using various tissues of rice,Oryza sativa L. cv. Kyoto Asahi. 2,4-D, NAA and IAA were used as auxins for the test of their ability to induce callus. All of these were active. This callus induction by auxin was successful in all tissues used; seed, root, shoot nodule, anther and ovary. In all of the calluses induced by various auxins such as 2,4-D, NAA and IAA and derived from various tissues such as seed, root, shoot nodule, anther and ovary, organ redifferentiation, i.e., formation of shoots and roots was achieved by removing the auxins from the medium used for the callus calture. Cytokinins were not necessary for the organ redifferentiation in these calluses. These results suggest that auxin is the only exogenous factor that determines dedifferentiation and redifferentiation in rice plant tissues culturedin vitro.     

Synthesis and activity of another seleniated auxin: 2,4-dichlorophenylselenoacetic acid

The synthesis of 2,4-dichlorophenylselenoacetic acid (2,4-D-Se) may be completed in three steps starting from 2,4-dichloroaniline. The selenium is inserted in the molecule by reaction of a diazonium salt with potassium selenocyanate. 2,4-D-Se has been tested as an auxin in several bioassays including the regeneration of somatic embryos, adventitious root formation and the associated temporary increase of endogenous auxins at the induction phase, and callus formation, and compared with the natural auxin indoleacetic acid (IAA), the classical synthetic auxin(s) naphthaleneacetic acid (NAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D), and with the synthetic seleniated IAA, 3-(benzo[b]selenienyl) acetic acid, BSAA. These biological assays classified 2,4-D-Se together with BSAA among the most powerful synthetic auxins. The role of selenium is briefly discussed.     

Effect of Auxins (2,4-Dichlorophenoxyacetic acid, Indole-3-Acetic Acid and Naphthaleneacetic Acid) on the Accumulation of {gamma}-Aminobutyric Acid in Excised Rice Root Tips

-Aminobutyric acid (GABA) accumulation occurs in cultured ricecells when ammonium is added to the medium [Kishinami and Ojima(1980) Plant Cell Physiol. 21: 581–589]. Whether thisphenomenon occurs in rice plant tissues was examined with respectto exogenously supplied auxins: 2,4-dichlorophenoxyacetic acid(2,4-D), indole-3-acetic acid (IAA) and naphthalene-acetic acid(NAA). In intact rice plants grown in medium containing ammonium withoutauxin, glutamine first increased, then asparagine graduallyincreased. In both shoots and roots, the asparagine contentbecame the highest among four amino acids after 4 days of cultureperiod. GABA did not increase at all, its level remaining lowin both shoots and roots throughout the culture period. GABA accumulation was observed in excised rice root tips whenthey were incubated in the medium containing ammonium in thepresence of 2,4-D, IAA or NAA. In the absence of auxin, however,excised rice root tips accumulated asparagine and glutamine,but not GABA. Rice root segments obtained from a region in whichroot cells had already developed to maturity did not accumulateGABA but asparagine and glutamine in the presence of both ammoniumand 2,4-D. These results suggest that GABA accumulation occurs in rapidlygrowing and dividing tissue, such as the apical meristem ofrice root in the presence of auxin during ammonium assimilation. (Received June 15, 1987; Accepted March 14, 1988)     

The effects of auxin on lateral root initiation and root gravitropism in a lateral rootless mutant Lrt1 of rice (Oryza sativa L.)

Auxins control growth and development in plants, including lateral rootinitiation and root gravity response. However, how endogenous auxin regulatesthese processes is poorly understood. In this study, the effects of auxins onlateral root initiation and root gravity response in rice were investigatedusing a lateral rootless mutant Lrt1, which fails to formlateral roots and shows a reduced root gravity response. Exogenous applicationof IBA to the Lrt1 mutant restored both lateral rootinitiation and root gravitropism. However, application of IAA, a major form ofnatural auxin, restored only root gravitropic response but not lateral rootinitiation. These results suggest that IBA is more effective than IAA in lateralroot formation and that IBA also plays an important role in root gravitropicresponse in rice. The application of NAA restored lateral root initiation, butdid not completely restore root gravitropism. Root elongation assays ofLrt1 displayed resistance to 2,4-D, NAA, IBA, and IAA.This result suggests that the reduced sensitivity to exogenous auxins may be due tothe altered auxin activity in the root, thereby affecting root morphology inLrt1.     

Auxin structure and activity on tomato morphogenesis in vitro and pea stem elongation

In order to understand better the relationship between auxin structure and activity on morphogenesis and cell elongation, six different auxins were tested on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. The auxins were: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1, 2-benzisoxazole-3-acetic acid (BOA), 1,2-benzisothiazole-3-acetic acid (BIA), 1-naphthalenacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D). All these compounds obey the minimum requirement rules for auxin activity and all were effective on cell elongation. At the dose of 10 M and in the absence of cytokinin, they all, except 2,4-D, induced roots, while in the presence of cytokinin they induced shoots, roots, hairy root-like filaments (HRLF) or callus depending on their concentration. The morphogenetic pattern did not change by varying cytokinin concentration. We conclude that auxin structure plays a minor role in morphogenesis or cell elongation, because it is only responsible for variations in the level of auxin activity.     

Metabolic changes during rooting in stem cuttings of five mangrove species

Vegetative propagation through rooting in stem cuttings in five tree mangroves namely Bruguiera parviflora, Cynometra iripa, Excoecaria agallocha, Heritiera fomes, and Thespesia populnea using IAA, IBA and NAA was reported. Spectacular increase in the root number was noted in the cuttings of H. fomes and C. iripa treated together with IBA (5000 ppm) and NAA (2500 ppm). The highest number of roots was obtained with IBA (2500 ppm) and NAA (500 ppm) in E. agallocha. B. parviflora and T. populnea responded better to IAA and IBA treatment. The species specific variation in the rooting response to exogenous application of auxins was reflected in the metabolic changes during initiation and development of roots in cuttings. Biochemical analysis showed increase of reducing sugar in the above-girdled tissues at initiation as well as subsequent development of roots which was further enhanced by the use of auxins. Decreases in the total sugar, total carbohydrate and polyphenols and increase in total nitrogen were recorded in the girdled tissues and the high C/N ratio at the initial stage helped in initiation of roots in all the species. Interaction of IBA and NAA promoted starch hydrolysis better than IAA and IBA during root development and subsequently reduced the C/N ratio and increased the protein-nitrogen activity during root development which suggest the auxin influenced mobilization of nitrogen to the rooting zone.Abbreviations IAA Indole-3-acetic acid - IBA Indole-butyric acid - NAA A-naphthalene acetic acid     

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.     

Hypaphorine from the ectomycorrhizal fungus Pisolithus tinctorius counteracts activities of indole-3-acetic acid and ethylene but not synthetic auxins in eucalypt seedlings

Very little is known about the molecules regulating the interaction between plants and ectomycorrhizal fungi during root colonization. The role of fungal auxin in ectomycorrhiza has repeatedly been suggested and questioned, suggesting that, if fungal auxin controls some steps of colonized root development, its activity might be tightly controlled in time and in space by plant and/or fungal regulatory mechanisms. We demonstrate that fungal hypaphorine, the betaine of tryptophan, counteracts the activity of indole-3-acetic acid (IAA) on eucalypt tap root elongation but does not affect the activity of the IAA analogs 2,4-D ((2,4-dichlorophenoxy)acetic acid) or NAA (1-naphthaleneacetic acid). These data suggest that IAA and hypaphorine interact during the very early steps of the IAA perception or signal transduction pathway. Furthermore, while seedling treatment with 1-amincocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, results in formation of a hypocotyl apical hook, hypaphorine application as well as root colonization by Pisolithus tinctorius, a hypaphorine-accumulating ectomycorrhizal fungus, stimulated hook opening. Hypaphorine counteraction with ACC is likely a consequence of hypaphorine interaction with IAA. In most plant-microbe interactions studied, the interactions result in increased auxin synthesis or auxin accumulation in plant tissues. The P. tinctorius / eucalypt interaction is intriguing because in this interaction the microbe down-regulates the auxin activity in the host plant. Hypaphorine might be the first specific IAA antagonist identified.     

Auxin binding in roots: A comparison between maize roots and coleoptiles

Auxin binding onto membrane fractions of primary roots of maize seedlings has been demonstrated using naphth-1yl-acetic acid (NAA) and indol-3yl-acetic acid (IAA) as ligands. This binding is compared with the already well characterized interaction between auxins and coleoptile membranes. The results indicate that while kinetic parameters are of the same order for root and coleoptile binding, a number of differences occur with respect to location in cells and relative affinity. The possible significance of the existence of such binding sites in root cells is discussed in relation to auxin action.Abbreviations 4-Cl-PA 4-chlorophenoxyacetic acid - EDTA ethylene diamine tetracetic acid - IAA indol-3yl-acetic acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA naphth-1yl-acetic acid - 2-NAA naphth-2yl-acetic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3 diol - TIBA 2,3,5 triiodobenzoic acid - NPA naphthylphthalamic acid - PCIB 4-chlorophenoxyisobutyric acid - PCPP 4-chlorophenoxyisopropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

The Role of Auxin Metabolism in Root Meristem Regeneration by Pinus lambertiana Embryo Cuttings

Since the existence of root promoting substances that consist of a complex between auxin and another molecule has been suggested, we have examined the role of auxin conversion products in root regeneration by Pinus lambertiana embryo cuttings. Auxin conversion products were detected using radioactive forms of the auxins IAA (indoIe-3-acetic acid), NAA (a-napthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid). 10^?7M NAA was more effective than 10^?6M IAA at promoting rooting, yet it formed conversion products much less rapidly. Also continuous exposure to IAA was necessary for optimum root formation. Based on these and other findings, we conclude that free auxin, and not the conversion products we detected, is essential to root meristem formation.     

Auxin, actin and growth of the Arabidopsis thaliana primary root

To understand how auxin regulates root growth, we quantified cell division and elemental elongation, and examined actin organization in the primary root of Arabidopsis thaliana. In treatments for 48 h that inhibited root elongation rate by 50%, we find that auxins and auxin-transport inhibitors can be divided into two classes based on their effects on cell division, elongation and actin organization. Indole acetic acid (IAA), 1-naphthalene acetic acid (NAA) and tri-iodobenzoic acid (TIBA) inhibit root growth primarily through reducing the length of the growth zone rather than the maximal rate of elemental elongation and they do not reduce cell production rate. These three compounds have little effect on the extent of filamentous actin, as imaged in living cells or by chemical fixation and immuno-cytochemistry, but tend to increase actin bundling. In contrast, 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthylphthalamic acid (NPA) inhibit root growth primarily by reducing cell production rate. These compounds remove actin and slow down cytoplasmic streaming, but do not lead to mislocalization of the auxin-efflux proteins, PIN1 or PIN2. The effects of 2,4-D and NPA were mimicked by the actin inhibitor, latrunculin B. The effects of these compounds on actin were also elicited by a 2 h treatment at higher concentration but were not seen in two mutants, eir1-1 and aux1-7, with deficient auxin transport. Our results show that IAA regulates the size of the root elongation zone whereas 2,4-D affects cell production and actin-dependent processes; and, further, that elemental elongation and localization of PINs are appreciably independent of actin.     

LIGHT AND HORMONAL CONTROL OF ROOT FORMATION IN ZEA MAYS CALLUS CULTURES

Callus cultures of Zea mays were used to study the interaction of light with exogenous cytokinin/auxin levels in the initiation, growth and development of roots. Three auxins, indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4 dichlorophenoxyacetic acid (2,4 D) were remarkably different in their effects on callus growth and root initiation. NAA at concentrations of 5 and 25 μM produced the highest combined yields of callus and roots under low light conditions. No significant morphological effects on roots were observed with the three auxins tested nor did low and intermediate light intensities alter root development.
At intermediate light levels the addition of the cytokinin, zeatin, was also able to influence the differentiation of the callus tissue. Increasing the cytokinin/auxin ratio from low to high shifted the development from callus growth to abundant root formation. High light caused the formation of short, thick roots. This effect could be counteracted in part by zeatin which promoted elongation. These observations suggest that both, the cytokinin/auxin ratio and light play an important role in the development of monocotyledonous roots.     

Timing of the response of coleoptiles to the application and withdrawal of various auxins

Summary A study was made of the time courses of growth promotion and the reversal of growth promotion upon the addition and withdrawal of various auxins. Growth promotion by 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) occurs more slowly and is less vigorous than growth promotion by the same concentration of indoleacetic acid (IAA).The time required for the reversal of the stimulation of elongation by auxin is many times greater for 2,4-D-stimulated growth than for IAA- or NAA-stimulated growth (80 min vs. about 10 min). This difference appears to be due to the sluggish exit of 2,4-D since (1) experiments with labeled auxins show that 2,4-D moves out of the tissue more slowly than IAA, and (2) it is possible to shorten the time required for a decline in elongation rate after the removal of 2,4-D to 13 min by adding an auxin antagonist (p-chlorophenoxyisobutyric acid).The rapid reversal of the hormonal stimulation of growth is discussed in relation to possible mechanisms of action of auxin.     

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

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 axr1 mutants is different from that of axr4. Received 9 June 1999/ Accepted in revised form 16 August 1999     

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     

Differential regulation of Ku gene expression in etiolated mung bean hypocotyls by auxins

Plant Ku genes were identified very recently in Arabidopsis thaliana, and their roles in repair of double-stranded break DNA and maintenance of telomere integrity were scrutinized. In this study, the cDNAs encoding Ku70 (VrKu70) and Ku80 (VrKu80) were isolated from mung bean (Vigna radiata L.) hypocotyls. Both genes were expressed widely among different tissues of mung bean with the highest levels in hypocotyls and leaves. The VrKu gene expression was stimulated by exogenous auxins in a concentration- and time-dependent manner. The stimulation could be abolished by auxin transport inhibitors, N-(1-naphthyl) phthalamic acid and 2,3,5-triiodobenzoic acid implicating that exogenous auxins triggered the effects following their uptake by the cells. Further analysis using specific inhibitors of auxin signaling showed that the stimulation of VrKu expression by 2,4-dichlorophenoxyacetic acid (2,4-D) was suppressed by intracellular Ca(2+) chelators, calmodulin antagonists, and calcium/calmodulin dependent protein kinase inhibitors, suggesting the involvement of calmodulin in the signaling pathway. On the other hand, exogenous indole-3-acetic acid (IAA) and alpha-naphthalene acetic acid (NAA) stimulated VrKu expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. Altogether, it is thus proposed that 2,4-D and IAA (or NAA) regulate the expression of VrKu through two distinct pathways.     

Intercellular colonization and growth promoting effects of Methylobacterium sp. with plant-growth regulators on rice (Oryza sativa L. Cv CO-43)

The Methylobacterium sp. strain NPFM-SB3, isolated from Sesbania rostrata stem nodules possessed nitrogenase activity and nodA genes. Pure culture of NPFM-SB3 strain produced indole-3-acetic acid, cytokinins and on inoculation to rice plants resulted in numerous lateral roots. Inoculation of synthetic auxins 2,4-dichlorophenoxy acetic acid, naphthalene acetic acid or flavonoids naringenin and dihydroxy-4-methoxyisoflavone individually or to bacterial inoculated rice seedlings improved the plant growth and lateral root formation under hydroponic condition. The formation of nodule-like structure and nitrogenase activity which is purely auxin dependent was observed in 2,4-dichlorophenoxy acetic acid treatments to Methylobacterium sp. NPFM-SB3 inoculated rice plants. The rhizobia entered through fissures formed due to lateral root emergence and spread intercellularly in the nodular structures concluded that the effect of 2,4-dichlorophenoxy acetic acid treatment for rice seedlings grown under gnotobiotic conditions is to create a niche in which these bacteria can grow.     

Hormonal Effects on Growth and Morphology of Normal and Hairy Roots of Hyoscyamus muticus

Treatment of normal and Agrobacterium rhizogenes-transformed root cultures of Hyoscyamus muticus with three different auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and naphthaleneacetic acid (NAA), revealed that the response varied considerably among auxins, between transformed and normal roots, and depending on the parameter. In normal roots all three auxins provoked abundant branching, with IBA and NAA being the most effective at 2.5 and 0.5 μm, respectively, whereas IAA was most effective at low concentrations (0.05 and 0.1 μm). In transformed roots exogenously supplied auxins were generally inhibitory or, at best, without effect on growth and branching. Only 0.01 μm IAA significantly enhanced lateral root number, whereas at the higher concentrations IBA, although inhibitory, was the least effective auxin. In both root types IBA had little effect on primary root growth, but normal roots were more sensitive to IAA and NAA. These results suggest a different sensitivity to auxins of normal and transformed roots since there was no significant difference in endogenous free and conjugated IAA content nor in IAA uptake capacity. Ethylene production and biosynthesis were approximately threefold higher in hairy roots, but production could be stimulated up to tenfold that of control levels in normal roots by supplying NAA or 1-aminocyclopropane-1-carboxylic acid (ACC). Treatment with 2.5 μm NAA, but not IAA or IBA, also enhanced ethylene biosynthesis in normal roots but not in transformed ones. ACC and malonyl-1-aminocyclopropane-1-carboxylic acid accumulated to detectable levels only after treatment with an auxin (NAA). Received March 3, 1997; accepted May 28, 1997