Meristematic Activity during Adventitious Root Primordium Development: Influences of Endogenous Auxin and Applied Gibberellic Acid

Intact brittle willows (Salix fragilis L.) were treated so that developing adventitious root primordia in the stems would be subjected to elevated gibberellic acid or reduced endogenous auxin levels. Observations were made of primordia that were initiated during the experiments and of primordia that were established before the experiments began. The results indicated that as primordia became older and contained more cells, auxin basipetally transported in the stem seemed to be of less importance in determining cell number per primordium. Thus, established primordia depended upon this auxin to a lesser extent than primordia which were being initiated. These observations were explained on the basis of differential contributions during primordium development of cell division in the cambium of the stem and in the primordia themselves. As opposed to the effects of reduced auxin levels, applied gibberellic acid reduced the cell number per primordium most in established primordia. Initiating primordia were least affected by gibberellic acid treatment. Gibberellic acid treatment seemed mainly to reduce intraprimordium cell division, on which continued development of established primordia most depends. Seemingly, at least in brittle willow, applied gibberellic acid blocks the action of auxin in primordium development subsequent to the initiation phase.     

Effects of Dimethyl Sulfoxide and pH on Indoleacetic Acid-Induced Lateral Root Formation in the Radish Seedling Root

Segments (2.5 cm) cut from 3-day-old seedling roots of radish (Raphanus sativus L. `Scarlet Globe') were cultured in medium with or without indoleacetic acid (IAA). Lateral root primordia frequency, determined for the central centimeter of segments, was dependent on IAA concentration and on conditions affecting IAA uptake. Dimethyl sulfoxide treatment, or a relatively low medium pH, greatly enhanced the response to exogenous IAA. It was concluded that a permeation barrier exists between the external medium and the hormone responsive sites within the radish seedling root.     

Ethylene–auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana

Plant root systems display considerable plasticity in response to endogenous and environmental signals. Auxin stimulates pericycle cells within elongating primary roots to enter de novo organogenesis, leading to the establishment of new lateral root meristems. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in root branching are not well characterized. We find that enhanced ethylene synthesis, resulting from the application of low concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), promotes the initiation of lateral root primordia. Treatment with higher doses of ACC strongly inhibits the ability of pericycle cells to initiate new lateral root primordia, but promotes the emergence of existing lateral root primordia: behaviour that is also seen in the eto1 mutation. These effects are correlated with decreased pericycle cell length and increased lateral root primordia cell width. When auxin is applied simultaneously with ACC, ACC is unable to prevent the auxin stimulation of lateral root formation in the root tissues formed prior to ACC exposure. However, in root tissues formed after transfer to ACC, in which elongation is reduced, auxin does not rescue the ethylene inhibition of primordia initiation, but instead increases it by several fold. Mutations that block auxin responses, slr1 and arf7 arf19, render initiation of lateral root primordia insensitive to the promoting effect of low ethylene levels, and mutations that inhibit ethylene-stimulated auxin biosynthesis, wei2 and wei7 , reduce the inhibitory effect of higher ethylene levels, consistent with ethylene regulating root branching through interactions with auxin.     

The Control of Lateral Root Development in Cultured Pea Seedlings. II. Root Fasciation Induced by Auxin Inhibitors

Lateral root development in cultured seedlings of Pisum sativum (cv. Alaska) was modified by the application of auxin transport inhibitors or antagonists. When applied either to replace the root tip or beneath the cotyledonary node, two auxin transport inhibitors, 2,3,5-triiodobenzoic acid (TIBA) and 3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-α]isoindol-8-one (DPX-1840), increased cell division activity opposite the protoxylem poles. This resulted in the formation of masses of cells, which we are calling root primordial masses (RPMs), 2 to 3 days after treatment. RPMs differed from lateral root primordia in that they lacked apical organization. Some roots however developed both RPMs and lateral roots indicating that both structures were similar in terms of the timing and location of cell division in the pericycle and endodermis leading to their initiation. Removal of the auxin transport inhibitors allowed many of the RPMs to organize later into lateral root primordia and to emerge in clusters. When the auxin, indoleacetic acid (IAA) was added to the growth medium along with DPX-1840, 3 ranks of RPMs now in the form of fasciated lateral roots emerged from the primary root. The auxin antagonist, p-chlorophenoxy-isobutyric acid (PCIB), also induced RPM formation. In contrast to DPX-1840 treatment, the addition of IAA during PCIB treatment caused normal lateral root development.     

Micropropagation of horseradish hairy root by means of adventitious shoot primordia

Adventitious shoot primordia were formed on horseradish hairy root cultured in dark. Plantlet formation frequency from the primordia was higher than that from root fragments. Culture for 26 days provided the adventitious shoot primordia, which had the highest potential for plantlet formation (53% explants at 40 days). Benzyladenine supplementation in the dark caused primordium enlargement, but did not increase the number of primordia formed. After adventitious shoot primordia were encapsulated with calcium alginate, kinetin supplementation (2.0–4.0 M) increased the shoot formation frequency (65–80% explants at 20 days) in the light, but also promoted the undesirable formattion of multiple shoots. Supplementation with naphthaleneacetic acid (0.27–5.4 M) in the calcium alginate beads in light enhanced the root emergence from primordia without inhibition of plantlet formation when the encapsulated beads were put on the agar-medium without naphthaleneacetic acid.     

Experimental Studies on Lateral Root Formation in Radish Seedling Roots: II. Analysis of the Dose-Response to Exogenous Auxin

Application of indoleacetic acid (IAA) and other auxins causes cultured radish (Raphanus sativus L. `Scarlet Globe') seedling root segments to produce an increased frequency (FR, no. cm⁻¹) of lateral roots (LR); in the absence of auxin, segments spontaneously form about 6 LR cm⁻¹. A dose-response study has revealed that the increase in FR follows a biphasic Michaelis-Menten relationship with the medium concentration of the undissociated form of IAA ([IAAH]_m). The fitted curve for phase I has a maximum response level (R_max) of 5.2 LR per centimeter above the spontaneous FR; the [IAAH]_m giving half-maximal response (C_1/2) is 21 nanomolar. For phase II, the values for R_max and C_1/2 are 56 LR per centimeter and 11 micromolar, respectively. The response is variable in the transition concentration region between the two phases; in that region (but not, or much less commonly, at higher or lower [IAAH]_m), LR initiation may resume or continue after the first day. At and above 100 micromolar [IAAH]_m, the roots are hyperstimulated and generally fail to respond. The developmental stage of LR formed in medium with very low [IAAH]_m (10 nanomolar) is enhanced compared to LR formed in medium lacking auxin; the stage is diminished at higher auxin levels, in inverse correlation with FR. Trends in the responses to NAA and IBA were similar, but NAA required only 0.03 times the dose of IAA, while IBA required 6 times the dose of IAA. These findings may be of use in a search for possible auxin receptors involved with LR initiation.     

Root primordia and endogenous auxin in maize roots cultured in vitro

Quantitative analyses of indol-3yl-acetic acid (I aa ) in Zea mays L. (cv. LG 11) root segments cultured in vitro were performed by gas chromatography-mass spectrometry with selected ion monitoring. The root extracts were first purified by highperformance liquid chromatography. Root primordia initiation in intact and decapitated roots showed different patterns: decapitation strongly enhanced primordia initiation in their first 10 mm. During the culture (5 days), I aa content decreased in both intact and decapitated roots. No correlation was found between the level of endogenous auxin and the numher of root primordia initiated from either intact or decapitated maize root segments.     

Auxin Induced Lateral Root Formation in Chicory

The supply of auxins [2,4-dichlorophenoxy acetic acid (2,4D),indole-3 acetic acid (1AA) and -naphthaleneacetic acid (NAA)]to excised chicory roots induced the formation of lateral rootmeristems mainly located close to the pre-existing apical rootmeristem. Lateral root growth induced in non-excised roots requiredhigher auxin concentrations. Inhibition of root elongation andconcomittant enlargement of the apices was also observed. SupplyingIAA induced the formation of lateral meristems earlier thanNAA, but subsequently favoured root elongation. Conversely,in the presence of 2,4D, reactivation of pericycle cells wasvery intense, but conversion of primordia to laterals was inhibited.Regardless of the auxin used, the responsive area in which lateralmeristems appeared was located a maximum of 4 mm away from theapical meristem. This region remained devoid of any lateralroot formation under control conditions. Pericycle cells oppositethe xylem poles in the diarch stele regained meristematic activityand divided transversally, giving rise to shorter cells. Thesecells subsequently divided periclinally, forming pairs of cellson the same transverse level. The root primordium extruded throughcortical cells and was surrounded by a lacuna formed to thedetriment of cortical cells.Copyright 1998 Annals of BotanyCompany Auxins,Cichorium intybus, chicory, lateral root, root elongation.     

Characteristics of adventitious root formation in cotyledon segments of mango (Mangifera indica L. cv. Zihua): two induction patterns, histological origins and the relationship with polar auxin transport

Cotyledon segments derived from zygote embryos of mango (Mangifera indica L. cv. Zihua) were cultured on agar medium for 28 days. Depending on different pre-treatments with plant growth regulators, two distinct patterns of adventitious roots were observed. A first pattern of adventitious roots was seen at the proximal cut surface, whereas no roots were formed on the opposite, distal cut surface. The rooting ability depended on the segment length and was significantly promoted by pre-treatment of embryos with indol-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for 1 h. A pre-treatment with the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) completely inhibited adventitious root formation on proximal cut surfaces. A second pattern of roots was observed on abaxial surfaces of cotyledon segments when embryos were pre-treated with 2,700 μM 1-naphthalenacetic acid (NAA) for 1 h. Histological observations indicated that both patterns of adventitious roots originated from parenchymal cells, but developmental directions of the root primordia were different. A polar auxin transport assay was used to demonstrate transport of [³H] indole-3-acetic acid (IAA) in cotyledon segments from the distal to the proximal cut surface. In conclusion, we suggest that polar auxin transport plays a role in adventitious root formation at the proximal cut surface, whereas NAA levels (influx by diffusion; carrier mediated efflux) seem to control development of adventitious roots on the abaxial surface of cotyledon segments.     

Cytokinins act directly on lateral root founder cells to inhibit root initiation

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.     

An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis

Local concentration gradients of the plant growth regulator auxin (indole-3-acetic acid [IAA]) are thought to instruct the positioning of organ primordia and stem cell niches and to direct cell division, expansion, and differentiation. High-resolution measurements of endogenous IAA concentrations in support of the gradient hypothesis are required to substantiate this hypothesis. Here, we introduce fluorescence-activated cell sorting of green fluorescent protein–marked cell types combined with highly sensitive mass spectrometry methods as a novel means for analyses of IAA distribution and metabolism at cellular resolution. Our results reveal the presence of IAA concentration gradients within the Arabidopsis thaliana root tip with a distinct maximum in the organizing quiescent center of the root apex. We also demonstrate that the root apex provides an important source of IAA and that cells of all types display a high synthesis capacity, suggesting a substantial contribution of local biosynthesis to auxin homeostasis in the root tip. Our results indicate that local biosynthesis and polar transport combine to produce auxin gradients and maxima in the root tip.     

Root meristems in Medicago truncatula tissue culture arise from vascular-derived procambial-like cells in a process regulated by ethylene

Leaf explants of Medicago truncatula were used to investigate the origins of auxin-induced root formation. On the application of auxin there is some callus formation (not the massive amount that occurs in response to auxin plus cytokinin) and roots appear shortly after the first visible callus. Histological examination reveals morphologically distinctive sheets of callus cells that emanate from the veins of the leaf explants and, within this cell type, root primordia are produced as well as some vascular tissue cells. What is suggested is that the vein-derived cells (VDCs) are procambial-like and function as pluripotent stem cells with a propensity to form root meristems or vascular tissues in response to added auxin. The development of root primordia from these pluripotent cells was clearly up-regulated by the use of the sickle (skl) mutant, which is a mutant impaired in ethylene signal transduction while the wild type and the sunn mutant, defective in auxin polar transport, produced similar numbers of roots. The skl mutant in generating many more roots concomitantly formed fewer vascular tissues. The root meristems differentiate similarly to normal roots producing a central cylinder of vascular tissue, which connects with the leaf explant veins. The VDCs appear to be derived from the cells of or near the phloem. The leaf observations suggest that a pool of stem cells exist in vascular tissue that, in combination with auxin and perhaps other factors, drive a diversity of plant development outcomes that is species specific. The way auxin interacts with other hormones is a key factor in determining the stem cell fate. The histological data in this study also assist in the interpretation of the molecular analysis of auxin-induced root formation in cultured leaves of M. truncatula.     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. IV. The role of changes in endogenous free and conjugated indole‐3‐acetic acid

We have studied the role of endogenous auxin on adventitious rooting in hypocotyls of derooted sunflower (Helianthus annuus L. var. Dahlgren 131) seedlings. Endogenous free and conjugated indole-3-acetic acid (IAA) were measured in three segments of hypocotyls of equal length (apical, middle, basal) by using gas chromatography-mass spectrometry with [¹³C₆]-IAA as an internal standard. At the time original roots were excised (0 h), the free IAA level in the hypocotyls showed an acropetally decreasing gradient, but conjugated IAA level increased acropetally; i.e. free to total IAA ratio was highest in the basal portion of hypocotyls. The basal portion is the region where most of root primordia were found. Some primordia were seen in this region within 24 h after the roots were excised. The quantity of free IAA in the middle portion of the hypocotyl increased up to 15 h after excision and then decreased. In this middle region there were fewer root primordia, and they could not be seen until 72 h. In the apical portion the amount of free IAA steadily increased and no root primordia were seen by 72 h. Surgical removal of various parts of the hypocotyl tissues caused adventitious root formation in the hypocotyl regions where basipetally transported IAA could accumulate. Reduction in the basipetal flow of auxin by N-1-naphthylphthalamic acid and 2,3,5-tri-iodobenzoic acid resulted in fewer adventitious roots. The fewest root primordia were seen if the major sources of endogenous auxin were removed by decapitation of the cotyledons and apical bud. Exogenous auxins promoted rooting and were able to completely overcome the inhibitory effect of 2,3,5-tri-iodobenzoic acid. Exogenous auxins were only partially able to overcome the inhibitory effect of decapitation. We conclude that in sunflower hypocotyls endogenously produced auxin is necessary for adventitious root formation. The higher concentrations of auxin in the basal portion may be partially responsible for that portion of the hypocotyl producing the greatest number of primordia. In addition to auxins, other factors such as wound ethylene and lowered cytokinin levels caused by excision of the original root system cuttings must also be important.     

NO VEIN Mediates Auxin-Dependent Specification and Patterning in the Arabidopsis Embryo,Shoot, and Root

Local efflux-dependent auxin gradients and maxima mediate organ and tissue development in plants. Auxin efflux is regulated by dynamic expression and subcellular localization of the PIN auxin-efflux proteins, which appears to be established not only through a self-organizing auxin-mediated polarization mechanism, but also through other means, such as cell fate determination and auxin-independent mechanisms. Here, we show that the Arabidopsis thaliana NO VEIN (NOV) gene, encoding a novel, plant-specific nuclear factor, is required for leaf vascular development, cellular patterning and stem cell maintenance in the root meristem, as well as for cotyledon outgrowth and separation. nov mutations affect many aspects of auxin-dependent development without directly affecting auxin perception. NOV is required for provascular PIN1 expression and region-specific expression of PIN7 in leaf primordia, cell type–specific expression of PIN3, PIN4, and PIN7 in the root, and PIN2 polarity in the root cortex. NOV is specifically expressed in developing embryos, leaf primordia, and shoot and root apical meristems. Our data suggest that NOV function underlies cell fate decisions associated with auxin gradients and maxima, thus establishing cell type–specific PIN expression and polarity. We propose that NOV mediates the acquisition of competence to undergo auxin-dependent coordinated cell specification and patterning, thereby eliciting context-dependent auxin-mediated developmental responses.     

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.     

Improving of oilseed rape lateral root formation by physiological analogues of auxin

The aim of the present work was to study the effect of auxin physiological analogue TA-12 [1-(2-chloroethoksicarbonylmethyl)-4-naphthalenesulfonic acid calcium salt] on the formation of oilseed rape lateral root and on the mitotic activity of apical meristem cells. Spring oilseed rape (Brassica napus L. ssp. oleifera annua Metzg.) cultivar ‘Mascot’ was chosen as a test object. Anatomical, cytological and histological studies on root development suggest that compound TA-12 induces the activity of parent root pericycle cells, stimulates the formation of lateral roots and enhances the division of apical meristem cells. The auxin transport inhibitor 2,3,5-triiodobenzoic acid suppresses the division of apical meristem cells, while this process is restored by the auxin physiological analogue TA-12 and naphthaleneacetic acid. The compound TA-12, by stimulating primary root growth and lateral root induction, optimised the formation of the oilseed rape root system.     

Quantitative distribution and metabolism of auxin herbicides in roots

The internal concentrations of four auxin herbicides— 2,4-dichlorophenoxyacetic acid, dicamba, picloram, and naphthaleneacetic acid—were measured in the roots of treated pea seedlings. Intact seedlings were immersed in solutions of labeled herbicides at concentrations sufficient to produce toxic symptoms (inhibition of elongation, radial enlargement, and lateral root proliferation). Measurements of volume and herbicide content of segments taken sequentially along the root showed that an acropetal concentration gradient of each herbicide was established within the root immediately following treatment. Although there was a net loss of herbicide in the following 24 hours, the gradient was maintained. Initially, the concentration of herbicide in the root tips exceeded that in the external medium.     

Micropropagation of juvenile and adult Sorbus domestica L.

Successful propagation of seedlings and mature trees of Sorbus domestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75–85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid     

Comparison of the growth promoting activities and toxicities of various auxin analogs on cells derived from wild type and a nonrooting mutant of tobacco

A naphthaleneacetic acid tolerant mutant isolated from a mutagenized culture of tobacco mesophyll protoplasts and impaired in root morphogenesis has been previously characterized by genetic analysis. To understand the biochemical basis for naphthaleneacetic acid resistance, cells derived from this mutant and from wild-type tobacco were compared for their ability to respond to various growth regulators. The growth promoting abilities and cytotoxicities of auxin analogs were different for mutant and wild-type cells. These different activities were not correlated with increased rate of conjugation or breakdown of the auxins by mutant cells. These observations, as well as previous studies on the interaction of the mutant with Agrobacterium, suggest that mutant resistance to auxins is not a result of a specific modification of the process by which auxins induce cell killing, but to a more general alteration of the cellular response to auxin. A screening of auxin-related molecules which induce cell death in wild-type cells but not mutant cells without promoting growth in either was performed. p-Bromophenyleacetic acid was found to display these characteristics.     

The nature of the dual effect of auxin on root formation in Azukia cuttings

In disbudded Azukia stem cuttings, auxin exerted a dual effecton root formation. The first phase of auxin action is identifiedwith the acceleration of cell division, especially longitudinaldivision. In cuttings treated with auxin during the first 24hr, longitudinally divided cells were observed in all 12 rootprimordia, while in water-treated cuttings, such cells wereobserved only in 8 root primordia. The second phase is the promotionof the reaction in which root primordia unable to develop furtherwithout auxin supply develop into roots. Irrespective of thetreatment during the first 24 hr, the auxin-treatment duringthe second 24 hr increased the number of roots protruding fromthe cuttings. Portulal applied during the first 24 hr increased the numberof root primordia which contained longitudinally divided cells.Gibberellin applied during the first 24 hr inhibited both transverseand longitudinal divisions in root primordia. ¹ Supported in part by Grant No. 139011 from the Ministry ofEducation, Japan. ² Present address: Junior College of Toyo University, Hakusan,Bunkyo-ku, Tokyo 112, Japan. (Received June 13, 1978; )