An Ethylene-Mediated Increase in Sensitivity to Auxin Induces Adventitious Root Formation in Flooded Rumex palustris Sm

The hormonal regulation of adventitious root formation induced by flooding of the root system was investigated in the wetland species Rumex palustris Sm. Adventitious root development at the base of the shoot is an important adaptation to flooded conditions and takes place soon after the onset of flooding. Decreases in either endogenous auxin or ethylene concentrations induced by application of inhibitors of either auxin transport or ethylene biosynthesis reduced the number of adventitious roots formed by flooded plants, suggesting an involvement of these hormones in the rooting process. The rooting response during flooding was preceded by increased endogenous ethylene concentrations in the root system. The endogenous auxin concentration did not change during flooding-induced rooting, but a continuous basipetal transport of auxin from the shoot to the rooting zone appeared to be essential in maintaining stable auxin concentrations. These results suggest that the higher ethylene concentration in soil-flooded plants increases the sensitivity of the root-forming tissues to endogenous indoleacetic acid, thus initiating the formation of adventitious roots.     

Elongation by primary lateral roots and adventitious roots during conditions of hypoxia and high ethylene concentrations

Soil flooding results in unusually low oxygen concentrations and high ethylene concentrations in the roots of plants. This gas composition had a strongly negative effect on root elongation of two Rumex species. The effect of low oxygen concentrations was less severe when roots contained aerenchymatous tissues, such as in R. palustris Sm. R. thyrsiflorus Fingerh., which has little root porosity, was much more affected. Ethylene had an even stronger effect on root elongation than hypoxia, since very small concentrations (0.1 cm³ m^?3) reduced root extension in the two species, and higher concentrations inhibited elongation more severely than did anoxia in the culture medium. Thus, ethylene contributes strongly to the negative effects of flooding on root growth. An exception may be the highly aerenchymatous, adventitious roots of R. palustris. Aerenchyma in these roots provides a low-resistance diffusion pathway for both endogenously produced ethylene and shoot-derived oxygen. This paper shows that extension by roots of R. palustris in flooded soil depends almost completely on this shoot-derived oxygen, and that aerenchyma prevents accumulation of growth-inhibiting levels of ethylene in the root.     

Ethylene accumulation in waterlogged Rumex plants promotes formation of adventitious roots

Accumulation of the gaseous plant hormone ethylene is very importantfor the induction of several responses of plants to flooding.However, little is known about the role of this gas in the formationof flooding-induced adventitious roots. Formation of adventitiousroots in Rumex species is an adaptation of these plants to floodedsoil conditions. The large air-spaces in these roots enablesdiffusion of gases between shoot and roots. Application of ethylene to non-flooded Rumex plants resultedin the formation of adventitious roots. In R. palustris Sm.shoot elongation and epinasty were also observed. The numberof roots in R. thyrsiflorus Fingerh. was much lower than inR. palustris, which corresponds with the inherent differencein root forming capacity between these two species. Ethyleneconcentrations of 1.5–2µI I_{– 1} induced a maximumnumber of roots in both species. Quantification of ethylene escaping from root systems of Rumexplants that were de-submerged after a 24 h submergence periodshowed that average ethylene concentrations in submerged rootsreached 1.8 and 9.1 µl I_–1 in R. palustris and R.thyrsiflorus, respectively. Inhibition of ethylene productionin R. palustris by L--(2-aminoethoxyvinyl)-glycine (AVG) or-aminobutyric acid (AIB) decreased the number of adventitiousroots induced by flooding, indicating that high ethylene concentrationsmay be a prerequisite for the flooding-induced formation ofadventitious roots in Rumex species. Key words: Adventitious roots, epinasty, ethylene, flooding, Rumex, shoot elongation     

Internal oxygen transport inRumex species and its significance for respiration under hypoxic conditions

Rumex thyrsiflorus, Rumex crispus andRumex maritimus show a differential flood-tolerance in the river ecosystem in the Netherlands.R. thyrsiflorus occurs at high-elevated habitats and is flood-intolerant, the other two species occur at lower-elevated habitats and are flood-tolerant. We compared their respiratory activity under aerobic and anaerobic conditions in the root environment and quantified the internal gas transport. The results indicate that aerial oxygen can be used for root respiration in both aerobically and anaerobically grown plants. The amount of oxygen used via internal aeration increased with decreasing oxygen concentration in the root environment. Aerobically grown plants ofR. maritimus andR. crispus already showed a high internal aeration, but there was a significant increase in internal oxygen transport in anaerobic plants, where new, aerenchymatous roots had formed. This indicates the functional significance of new root formation for respiration in these species upon hypoxia. After two weeks of anaerobiosis, more than 50% of the total respiration of the roots of young plants ofR. maritumus and 40% of roots of young plants ofR. crispus was due to internal aeration at low oxygen concentrations in the root environment. InR. maritimus both young and old plants performed in this way, inR. crispus only young plants, whileR. thyrsiflorus showed some internal aeration, but this was hardly detectable. These differences can be explained on the basis of a different morphology and concomitant diffusive resistance of both root and shoot system. In experiments with different submergence levels of the shoot, the amount of internal aeration was positively correlated to the total leaf area protruding above the water surface inR. maritimus. This indicates a functional significance of the petiole and leaf elongation response upon total submergence of this species.     

Mediators,Genes and Signaling in Adventitious Rooting

Adventitious roots are a post-embryonic root which arise from the stem and leaves and from non-pericycle tissues in old roots and it is one of the most important ways of vegetative propagation in plants. Many exogenous and endogenous factors regulate the formation of adventitious roots, such as Ca²⁺, sugars, auxin, polyamines, ethylene, nitric oxide, hydrogen peroxide, carbon monoxide, cGMP, MAPKs and peroxidase, etc. These mediators are thought to function as signaling and mediate auxin signal transduction during the formation of adventitious roots. To date, only a few genes have been identified that are associated with the general process of adventitious rooting, such as ARL1, VvPRP1, VvPRP2, HRGPnt3, LRP1 and RML, etc. Auxin has been shown to be intimately involved in the process of adventitious rooting and function as crucial role in adventitious rooting. Great progress has been made in elucidating the auxin-induced genes and auxin signaling pathway, especially in auxin response Aux/IAA and ARF genes family and the auxin receptor TIR1. Although, some of important aspects of adventitious rooting signaling have been revealed, the intricate signaling network remains poorly understood.     

Strigolactones suppress adventitious rooting in Arabidopsis and pea

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.     

Histological analysis of adventitious rooting in Arabidopsis thaliana (L.) Heynh seedlings

ABSTRACT

Adventititous rooting is essential for the post-embryonic growth of the root apparatus in various species. In Arabidopsis thaliana, adventitious rooting has been reported in some mutants, and auxin seems to be the inducer of the process. The objective of the study was to identify the tissues involved in adventitious rooting in the most commonly used ecotypes for molecular and genetic studies (i.e. Columbia, Wassilewskija and Landsberg erecta) both in the presence and absence of exogenous auxin. Seedlings of the three ecotypes were grown under various conditions. When grown under 16 hours light/day for 11 days, all seedlings showed adventitious roots, both with and without auxin, however, both adventitious and lateral rooting were enhanced by exogenous auxin (2 µM naphthaleneacetic acid). Independently of the presence of auxin and of the ecotype, the hypocotyl pericycle produced adventitious roots directly (i.e., according to the same pattern of lateral root formation by the pericycle cells in the primary root). However, in the presence of auxin, roots of indirect origin also, and mainly, formed and their formation was preceded by the exfoliation of the tissues external to the stele. Exfoliation was caused by cell hypertrophy, separation, and disintegration, which mainly involved the endodermis. At the exfoliation site, the pericycle, with a minor contribution of a few endodermal cells, produced the callus from which indirect roots arose. The finding that adventitious rooting occurs in the absence of auxin (all ecotypes) indicates that this process is part of the normal root apparatus in Arabidopsis, with the hypocotyl pericycle as the target tissue of the process. Exogenous auxin alters adventitious rhizogenesis mainly affecting the endodermis response.     

Sensitivity to ethylene: the key factor in submergence-induced shoot elongation of Rumex

Rosettes of flooding-resistant Rumex palustris plants show a submergence-induced stimulation of elongation, which is confined to the petioles of young leaves. This response increases the probability of survival. It is induced by ethylene that accumulates in submerged tissues. Flooding-intolerant Rumex acetosella plants do not show this response. We investigated whether differences in shoot elongation between the species, between old and young leaves and between the petiole and leaf blade of a R. palustris plant result from differences in internal ethylene concentration or in sensitivity to the gas. Concentrations of free and conjugated ACC in petioles and leaf blades of R. palustris indicated that ethylene is synthesized throughout the submerged shoot, although production rates varied locally. Nevertheless, no differences in ethylene concentration were found between submerged leaves of various ages. In contrast, dose-response curves showed that only elongation of young petioles of R. palustris was sensitive to ethylene. In R. acetosella, elongation of all leaves was insensitive to ethylene. We conclude that variation in ethylene sensitivity rather than content explains the differences in submergence-induced shoot elongation between the two Rumex species and between leaves of R. palustris.     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

Distinct modes of adventitious rooting in Arabidopsis thaliana

The literature describes different rooting protocols for Arabidopsis thaliana as models to study adventitious rooting, and results are generally perceived as comparable. However, there is a lack of investigations focusing on the distinct features, advantages and limitations of each method in the study of adventitious rooting with both wild-type (WT) ecotypes and their respective mutants. This investigation was undertaken to evaluate the adventitious rooting process in three different experimental systems, all using A. thaliana, analysing the same rooting parameters after transient exposure to auxin (indole-3-acetic acid) and control conditions: excised leaves, de-rooted plants and etiolated seedlings. The founding tissues and sites of origin of roots differed depending on the system used, whereas all rooting patterns were of the direct type (i.e., without callus formation). None of the systems had an absolute requirement for exogenous auxin, although rooting was enhanced by this phytohormone, with the exception of de-rooted plants, which had adventitious rooting strongly inhibited by exogenous auxin. Root elongation was much favoured in isolated leaves. Auxin-overproducing mutants could not be used in the detached leaf system due to precocious senescence; in the de-rooted plant system, these mutants had a WT-like rooting response, whereas the expression of the 'rooty' phenotype was only evident in the etiolated seedling system. Adventitious rooting of etiolated WT seedlings in the presence of exogenous auxin was inhibited by exogenous flavonoids, which act as auxin transport inhibitors; surprisingly, the flavonoid-deficient mutant chs had a lower rooting response compared to WT. Although Arabidopsis is an excellent model system to study adventitious rooting, physiological and developmental responses differed significantly, underlining the importance of avoiding data generalisation on rooting responses derived from different experimental systems with this species.     

Gene note. Isolation and characterization of a cDNA encoding a novel 2-oxoacid-dependent dioxygenase which is up-regulated during adventitious root formation in apple (Malus domestica 'Jork 9') stem discs

A cDNA clone encoding a novel 2-oxoacid-dependent dioxygenase which is up-regulated during the early phases of adventitious root formation in Malus domestica 'Jork 9' has been isolated. The corresponding gene has been designated ARRO-1 (Adventitious Rooting Related Oxygenase) and is represented in the apple genome by a number of distinct gene copies.Keywords: Adventitious rooting, apple, auxin, dioxygenase.      

Changes in growth,porosity, and radial oxygen loss from adventitious roots of selected mono‐ and dicotyledonous wetland species with contrasting types of aerenchyma

Growth in stagnant, oxygen‐deficient nutrient solution increased porosity in adventitious roots of two monocotyledonous (Carex acuta and Juncus effusus) and three dicotyledonous species (Caltha palustris, Ranunculus sceleratus and Rumex palustris) wetland species from 10 to 30% under aerated conditions to 20–45%. The spatial patterns of radial oxygen loss (ROL), determined with root‐sleeving oxygen electrodes, indicated a strong constitutive ‘barrier’ to ROL in the basal root zones of the two monocotyledonous species. In contrast, roots of the dicotyledonous species showed no significant ‘barrier’ to ROL when grown in aerated solution, and only a partial ‘barrier’ when grown in stagnant conditions. This partial ‘barrier’ was strongest in C. palustris, so that ROL from basal zones of roots of R. sceleratus and R. palustris was substantial when compared to the monocotyledonous species. ROL from the basal zones would decrease longitudinal diffusion of oxygen to the root apex, and therefore limit the maximum penetration depth of these roots into anaerobic soil. Further studies of a larger number of dicotyledonous wetland species from a range of substrates are required to elucidate the ecophysiological consequences of developing a partial, rather than a strong, ‘barrier’ to ROL.     

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.     

Architectural Tradeoffs between Adventitious and Basal Roots for Phosphorus Acquisition

Adventitious rooting contributes to efficient phosphorus acquisition by enhancing topsoil foraging. However, metabolic investment in adventitious roots may retard the development of other root classes such as basal roots, which are also important for phosphorus acquisition. In this study we quantitatively assessed the potential effects of adventitious rooting on basal root growth and whole plant phosphorus acquisition in young bean plants. The geometric simulation model SimRoot was used to dynamically model root systems with varying architecture and C availability growing for 21 days at 3 planting depths in 3 soil types with contrasting nutrient mobility. Simulated root architectures, tradeoffs between adventitious and basal root growth, and phosphorus acquisition were validated with empirical measurements. Phosphorus acquisition and phosphorus acquisition efficiency (defined as mol phosphorus acquired per mol C allocated to roots) were estimated for plants growing in soil in which phosphorus availability was uniform with depth or was greatest in the topsoil, as occurs in most natural soils. Phosphorus acquisition and acquisition efficiency increased with increasing allocation to adventitious roots in stratified soil, due to increased phosphorus depletion of surface soil. In uniform soil, increased adventitious rooting decreased phosphorus acquisition by reducing the growth of lateral roots arising from the tap root and basal roots. The benefit of adventitious roots for phosphorus acquisition was dependent on the specific respiration rate of adventitious roots as well as on whether overall C allocation to root growth was increased, as occurs in plants under phosphorus stress, or was lower, as observed in unstressed plants. In stratified soil, adventitious rooting reduced the growth of tap and basal lateral roots, yet phosphorus acquisition increased by up to 10% when total C allocation to roots was high and adventitious root respiration was similar to that in basal roots. With C allocation to roots decreased by 38%, adventitious roots still increased phosphorus acquisition by 5%. Allocation to adventitious roots enhanced phosphorus acquisition and efficiency as long as the specific respiration of adventitious roots was similar to that of basal roots and less than twice that of tap roots. When adventitious roots were assigned greater specific respiration rates, increased adventitious rooting reduced phosphorus acquisition and efficiency by diverting carbohydrate from other root types. Varying the phosphorus diffusion coefficient to reflect varying mobilities in different soil types had little effect on the value of adventitious rooting for phosphorus acquisition. Adventitious roots benefited plants regardless of basal root growth angle. Seed planting depth only affected phosphorus uptake and efficiency when seed was planted below the high phosphorus surface stratum. Our results confirm the importance of root respiration in nutrient foraging strategies, and demonstrate functional tradeoffs among distinct components of the root system. These results will be useful in developing ideotypes for more nutrient efficient crops.     

N,N′-bis-(2,3-Methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious rooting in Pinus radiata and affect expression of genes induced during adventitious rooting in the presence of exogenous auxin

We have analyzed the effect of N,N′-bis-(2,3-methylenedioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methylenedioxyphenyl)urea (3,4-MDPU), two symmetrically substituted diphenylurea derivatives with no auxin or cytokinin-like activity, on the rooting capacity of Pinus radiata stem cuttings. Results indicate that both diphenylurea derivatives enhance adventitious rooting in the presence of exogenous auxin (indole-3-butyric acid, IBA), even at low auxin concentration, in rooting-competent cuttings, but have no effect on the adventitious rooting of low or null competent-to-root cuttings. Histological analyses show that, in the simultaneous presence of MDPUs and low concentration of exogenous auxin, adventitious root formation is induced in the cell types that retain intrinsic competence to form adventitious roots in response to auxin. The time course of cellular events leading to root formation and the time of root emergence are closely similar to that observed in cuttings treated only with higher auxin concentration. In addition, the mRNA level of a P. radiata SCARECROW-LIKE gene, which is significantly induced in the presence of the optimal concentration (10 μM) of exogenous auxin needed for cuttings to root, is increased in the presence of MDPUs and low concentration of exogenous auxin (1 μM). The expression of a P. radiata SHORT-ROOT gene in rooting-competent cuttings during adventitious rooting is also affected by the presence of MDPUs when combined with auxin. As MDPUs do not affect the expression of either gene in the absence of exogenous auxin, but only in its presence, we suggest that MDPUs could interact, directly or indirectly, with the auxin-signalling pathways in rooting-competent cuttings during adventitious rooting.     

Root Formation in Ethylene-Insensitive Plants

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.     

Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.