Effect of auxins and plant oligosaccharides on root formation and elongation growth of mung bean hypocotyls

The interaction of auxins – IAA, IBA or NAA – with galactoglucomannan oligosaccharides (GGMOs) on adventitious root formation and elongation growth of mung bean hypocotyl cuttings was studied. GGMOs induced adventitious roots in the absence of auxins; however, their effect was lower compared with IBA or NAA. On the other hand, in the presence of auxins, GGMOs inhibited adventitious root induction. Their effect depended on the concentration of oligosaccharides and the type of auxin used. The highest inhibition effect of GGMOs at a concentration of 10⁻⁸ M in the presence of IBA and NAA was observed. In the presence of IAA their inhibition was non-significant in regard to the concentration. The interaction of auxins with GGMOs resulted in the formation of adventitious roots on a shorter part of hypocotyls compared with the effect of auxins alone. However, roots were induced more extensively along the hypocotyls treated with GGMOs compared with the control. GGMOs inhibited the length of induced adventitious roots in the presence of IAA, while in combination with IBA or NAA they were ineffective. The elongation of hypocotyls induced by IAA or IBA was inhibited by GGMOs, too. However, in the presence of NAA or by endogenous growth they were without any significant effect on elongation growth. These findings suggest that GGMOs in certain concentrations might inhibit rooting and the elongation process dependant on auxin used.     

Regulation of lateral root formation in lettuce (Lactuca sativa) seedling roots: Interacting effects of α-naphthaleneacetic acid and kinetin

Growth substances, α-naphthaleneacetic (NAA) and kinetin, had an important role in the regulation of lateral root (LR) formation in lettuce ( Lactuca sativa L. cv. Grand Rapids) seedling roots. NAA (10^-5 M ) was a potent stimulator of LR initiation and caused a 600% increase in the number of lateral root primordia (LRP) compared to untreated roots. NAA was required for only the first 20 h of the 72 h treatment period for maximum stimulation of LRP initiation. Kinetin (2 × 10^-5 M ) effectively prevented the spontaneous formation of LRP and inhibited the NAA-stimulated production of LRP. Kinetin inhibition was maximal during the first 20 h of NAA treatment and this effect was not overcome by subsequent supply of NAA. Also, lettuce roots were most sensitive to kinetin at 20 h of NAA treatment, when the first signs of cell division were observed in the pericycle.     

Auxins and cytokinins exuded during formation of roots by detached primary leaves and stems of dwarf French bean (Phaseolus vulgaris L.)

Summary Hypocotyls of detached stems standing in culture solution produced adventitious roots sooner than did petioles of detached primary leaves. An auxin, probably indol-3-ylacetic acid, appeared in the solutions before the hypocotyls or petioles produced roots. After attaining a maximum, the amounts of auxin in the solutions decreased as fewer roots were formed. Two cytokinins were found in the culture solutions; one had a similar R_f to zeatin, the other ran more slowly on chromatograms. The amounts of cytokinin in the solutions were associated with root formation. Stems soon died unless their hypocotyls formed roots, but the primary leaves survived without roots forming provided a callus formed on the petiole. Hence adventitious roots, or callus tissues, may have produced cytokinins that replaced those produced by the original roots, found in sap exuded from the stem stumps, and were essential for survival of the stems and leaves.     

Shoot-supplied ammonium targets the root auxin influx carrier AUX1 and inhibits lateral root emergence in Arabidopsis

Deposition of ammonium (NH₄⁺) from the atmosphere is a substantial environmental problem. While toxicity resulting from root exposure to NH₄⁺ is well studied, little is known about how shoot‐supplied ammonium (SSA) affects root growth. In this study, we show that SSA significantly affects lateral root (LR) development. We show that SSA inhibits lateral root primordium (LRP) emergence, but not LRP initiation, resulting in significantly impaired LR number. We show that the inhibition is independent of abscisic acid (ABA) signalling and sucrose uptake in shoots but relates to the auxin response in roots. Expression analyses of an auxin‐responsive reporter, DR5:GUS, and direct assays of auxin transport demonstrated that SSA inhibits root acropetal (rootward) auxin transport while not affecting basipetal (shootward) transport or auxin sensitivity of root cells. Mutant analyses indicated that the auxin influx carrier AUX1, but not the auxin efflux carriers PIN‐FORMED (PIN)1 or PIN2, is required for this inhibition of LRP emergence and the observed auxin response. We found that AUX1 expression was modulated by SSA in vascular tissues rather than LR cap cells in roots. Taken together, our results suggest that SSA inhibits LRP emergence in Arabidopsis by interfering with AUX1‐dependent auxin transport from shoot to root.     

Auxin signaling modulates LATERAL ROOT PRIMORDIUM1 (LRP1) expression during lateral root development in Arabidopsis

Auxin signaling mediated by various auxin/indole‐3‐acetic acid (Aux/IAAs) and AUXIN RESPONSE FACTORs (ARFs) regulate lateral root (LR) development by controlling the expression of downstream genes. LATERAL ROOT PRIMORDIUM1 (LRP1), a member of the SHORT INTERNODES/STYLISH (SHI/STY) family, was identified as an auxin‐inducible gene. The precise developmental role and molecular regulation of LRP1 in root development remain to be understood. Here we show that LRP1 is expressed in all stages of LR development, besides the primary root. The expression of LRP1 is regulated by histone deacetylation in an auxin‐dependent manner. Our genetic interaction studies showed that LRP1 acts downstream of auxin responsive Aux/IAAs‐ARFs modules during LR development. We showed that auxin‐mediated induction of LRP1 is lost in emerging LRs of slr‐1 and arf7arf19 mutants roots. NPA treatment studies showed that LRP1 acts after LR founder cell specification and asymmetric division during LR development. Overexpression of LRP1 (LRP1 OE) showed an increased number of LR primordia (LRP) at stages I, IV and V, resulting in reduced emerged LR density, which suggests that it is involved in LRP development. Interestingly, LRP1‐induced expression of YUC4, which is involved in auxin biosynthesis, contributes to the increased accumulation of endogenous auxin in LRP1 OE roots. LRP1 interacts with SHI, STY1, SRS3, SRS6 and SRS7 proteins of the SHI/STY family, indicating their possible redundant role during root development. Our results suggested that auxin and histone deacetylation affect LRP1 expression and it acts downstream of LR forming auxin response modules to negatively regulate LRP development by modulating auxin homeostasis in Arabidopsis thaliana.     

Sequential induction of auxin efflux and influx carriers regulates lateral root emergence

In Arabidopsis, lateral roots originate from pericycle cells deep within the primary root. New lateral root primordia (LRP) have to emerge through several overlaying tissues. Here, we report that auxin produced in new LRP is transported towards the outer tissues where it triggers cell separation by inducing both the auxin influx carrier LAX3 and cell‐wall enzymes. LAX3 is expressed in just two cell files overlaying new LRP. To understand how this striking pattern of LAX3 expression is regulated, we developed a mathematical model that captures the network regulating its expression and auxin transport within realistic three‐dimensional cell and tissue geometries. Our model revealed that, for the LAX3 spatial expression to be robust to natural variations in root tissue geometry, an efflux carrier is required—later identified to be PIN3. To prevent LAX3 from being transiently expressed in multiple cell files, PIN3 and LAX3 must be induced consecutively, which we later demonstrated to be the case. Our study exemplifies how mathematical models can be used to direct experiments to elucidate complex developmental processes.     

An Auxin-Inducible F-Box Protein CEGENDUO Negatively Regulates Auxin-Mediated Lateral Root Formation in Arabidopsis

Previously, we characterized 92 Arabidopsis genes (AtSFLs) similar to the S-locus F-box genes involved in S-RNase-based self-incompatibility and found that they likely play diverse roles in Arabidopsis. In this study, we investigated the role of one of these genes, CEGENDUO (CEG, AtSFL61), in the lateral root formation. A T-DNA insertion in CEG led to an increased lateral root production, which was complemented by transformation of the wild-type gene. Its downregulation by RNAi also produced more lateral roots in transformed Arabidopsis plants whereas its overexpression generated less lateral roots compared to wild-type, indicating that CEG acts as a negative regulator for the lateral root formation. It was found that CEG was expressed abundantly in vascular tissues of the primary root, but not in newly formed lateral root primordia and the root meristem, and induced by exogenous auxin NAA (α-naphthalene acetic acid). In addition, the ceg mutant was hyposensitive to NAA, IAA (indole-3-acetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), as well as the auxin transport inhibitor TIBA (3,3,5-triiodobenzoic acid), showing that CEG is an auxin-inducible gene. Taken together, our results show that CEG is a novel F-box protein negatively regulating the auxin-mediated lateral root formation in Arabidopsis. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Summary Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N⁶-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.     

Influence of galactoglucomannan oligosaccharides on root culture of <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis>

Galactoglucomannan oligosaccharides (GGMOs) activity in K. humboldtiana root culture has been determined. GGMOs inhibited adventitious root growth and lateral root induction in contrast to IAA, IBA, and NAA stimulating effect in these processes. Similarly, the combination of GGMOs with natural auxins (IAA, IBA) evoked an inhibition of adventitious root growth and lateral root induction that depended on the oligosaccharides concentration and the type of auxin. The growth stimulating effect of the synthetic auxin, NAA, in adventitious roots was negatively affected by GGMOs, but they were without influence on lateral root induction. The presence of oligosaccharides triggered lateral root position on adventitious roots and the anatomy of adventitious roots (diameter, proportion of primary cortex to the central cylinder, number and size of primary cortical cells, intercellular spaces, and the number of starch grains in cells of primary cortex) in dependence on their coactions with auxin.     

Regulation of root hair initiation and expansin gene expression in Arabidopsis

The expression of two Arabidopsis expansin genes (AtEXP7 and AtEXP18) is tightly linked to root hair initiation; thus, the regulation of these genes was studied to elucidate how developmental, hormonal, and environmental factors orchestrate root hair formation. Exogenous ethylene and auxin, as well as separation of the root from the medium, stimulated root hair formation and the expression of these expansin genes. The effects of exogenous auxin and root separation on root hair formation required the ethylene signaling pathway. By contrast, blocking the endogenous ethylene pathway, either by genetic mutations or by a chemical inhibitor, did not affect normal root hair formation and expansin gene expression. These results indicate that the normal developmental pathway for root hair formation (i.e., not induced by external stimuli) is independent of the ethylene pathway. Promoter analyses of the expansin genes show that the same promoter elements that determine cell specificity also determine inducibility by ethylene, auxin, and root separation. Our study suggests that two distinctive signaling pathways, one developmental and the other environmental/hormonal, converge to modulate the initiation of the root hair and the expression of its specific expansin gene set.     

Regulation of root growth by auxin and ethylene is influenced by phosphate supply in white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.)

The influence of P-supply on root system architecture (primary root length, number and total length of lateral roots) through the effects of ethylene (ACC) and auxin [1-naphthylacetic acid (NAA)] has been examined in the legume white clover (Trifolium repens L.). Higher concentrations (1 and 10 μM) of ACC and NAA (100 nM) inhibited growth, while lower concentrations (100 nM ACC, 5 nM NAA) either had no effect or stimulated growth in P-sufficient (1 mM Pi) roots. In response to low (10 μM) P, a stimulation of primary root growth, number of lateral roots and mean length of lateral roots was observed, while a super-stimulation of these growth parameters occurred in response to subsequent 100 nM ACC treatment suggesting that the low P treatment increased the sensitivity of the roots to ethylene. Examination of the primary roots of DR5p::GUS transformants suggests that this change in sensitivity induced by low P occurs through the promotion of auxin signalling/transport to the root apex. These results are discussed in terms of the role of ethylene and the significance of changes in sensitivity to the hormone in modulating root system architecture in response to low P-supply.     

Adventitious root initiation in hypocotyl and epicotyl cuttings of eastern white pine (Pinus strobus) seedlings

The present paper reports results of experiments to develop a system for studying adventitious root initiation in cuttings derived from seedlings. Hypocotyl cuttings of 2-week-old eastern white pine (Pinus strobus L.) seedlings were treated for 5 min with 0, 100, 200, 300, 400, 500 or 600 mg l^?1 (0, 0.54, 1.07, 1.61, 2.15, 2.69 or 3.22 mM) 1-naphthaleneacetic acid (NAA) to determine the effect on root initiation. The number of root primordia per cutting was correlated with NAA concentration and the square of NAA concentration. Thus, the number increased from less than one per cutting in the 0 NAA treatment to approximately 40 per cutting at 300 mg l-1 NAA, above which no substantial further increase was observed. The larger number of root primordia formed in response to increasing concentrations of NAA was due to the formation of primordia over a larger proportion of the hypocotyls. Histological analysis of the timing of root primordium formation in hypocotyl cuttings revealed three discernible stages. Progression through these stages was relatively synchronous among NAA-treated hypocotyl cuttings and within a given cutting, but variation was observed in the portion of different cuttings undergoing root formation. Control-treated hypocotyl cuttings formed root primordia at lower frequencies and more slowly than NAA-treated cuttings, with fewer primordia per cutting. Epicotyl cuttings from 11-week-old seedlings also formed adventitious roots, but more slowly than hypocotyl cuttings. NAA treatment of epicotyl cuttings caused more rapid root initiation and also affected the origin of adventitious roots in comparison with nontreated cuttings. NAA-treated epicotyl cuttings formed roots in a manner analogous to that of the hypocotyl cuttings, directly from preformed vascular tissue, while control-treated epicotyl cuttings first formed a wound or callus tissue and subsequently differentiated root primordia within that tissue. This system of inducing adventitious roots in pine stem cuttings lends itself to studying the molecular and biochemical steps that occur during root initiation and development.     

Distribution of expansins in graviresponding maize roots

To test if expansins, wall loosening proteins that disrupt binding between microfibrils and cell wall matrix, participate in the differential elongation of graviresponding roots, Zea mays L. cv. Merit roots were gravistimulated and used for immunolocalization with anti-expansin. Western blots showed cross-reaction with two proteins of maize, one of the same mass as cucumber expansin (29 kDa), the second slightly larger (32 kDa). Maize roots contained mainly the larger protein, but both were found in coleoptiles. The expansin distribution in cucumber roots and hypocotyls was similar to the distribution in maize. Roots showed stronger expansin signals on the expanding convex side than the concave flank as early as 30 min after gravistimulation. Treatment with brefeldin A, a vesicle transport inhibitor, or the auxin transport inhibitor, naphthylphthalamic acid, showed delayed graviresponse and the appearance of differential staining. Our results indicate that expansins may be transported and secreted to cell walls via vesicles and function in wall expansion.     

ALTERED RESPONSE TO GRAVITY is a peripheral membrane protein that modulates gravity-induced cytoplasmic alkalinization and lateral auxin transport in plant statocytes

ARG1 (ALTERED RESPONSE TO GRAVITY) is required for normal root and hypocotyl gravitropism. Here, we show that targeting ARG1 to the gravity-perceiving cells of roots or hypocotyls is sufficient to rescue the gravitropic defects in the corresponding organs of arg1-2 null mutants. The cytosolic alkalinization of root cap columella cells that normally occurs very rapidly upon gravistimulation is lacking in arg1-2 mutants. Additionally, vertically grown arg1-2 roots appear to accumulate a greater amount of auxin in an expanded domain of the root cap compared with the wild type, and no detectable lateral auxin gradient develops across mutant root caps in response to gravistimulation. We also demonstrate that ARG1 is a peripheral membrane protein that may share some subcellular compartments in the vesicular trafficking pathway with PIN auxin efflux carriers. These data support our hypothesis that ARG1 is involved early in gravitropic signal transduction within the gravity-perceiving cells, where it influences pH changes and auxin distribution. We propose that ARG1 affects the localization and/or activity of PIN or other proteins involved in lateral auxin transport.     

Cytokinin receptors are required for normal development of auxin-transporting vascular tissues in the hypocotyl but not in adventitious roots

Plants alter the architecture of their root systems to adapt to the environment by modulating post-embryonic (lateral and adventitious) root formation and growth. To understand better the genetic basis of this regulation, we screened ethylmethane sulfonate-mutagenized lines of Arabidopsis thaliana for adventitious rooting mutants. One mutant showed retardation of the primary root growth, no production of lateral roots and enhanced formation of adventitious roots. Mapping and genetic complementation revealed that this mutant named wooden leg-3 (wol-3) was an allele of ARABIDOPSIS HISTIDINE KINASE 4 (AHK4), a locus known to encode a cytokinin receptor. Although the vascular system of the primary root and hypocotyl in the wol-3 mutant was aborted, that of the adventitious roots was normally developed. In the hypocotyl of the wol-3 mutant, auxin signals accumulated around the aborted vascular system. The application of auxin to primary roots induced lateral root formation in the wol-3 mutant. Transport of radiolabeled auxin from the top of the hypocotyl to the primary root was inhibited in wol-3. Although only a single amino acid alteration had occurred in AHK4, the root morphology in the wol-3 mutant was quite similar to that in the ahk2 ahk3 ahk4 triple mutant, which is a loss-of-function mutant of the three cytokinin receptors. This implies that the functional disturbance of AHK4 affects the function of the other receptors. Our results suggest that cytokinin receptors are necessary for the formation of auxin-transporting vascular tissues in the hypocotyl, but not in adventitious roots.     

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.     

Rapid auxin-induced root growth inhibition requires the TIR and AFB auxin receptors

We investigated the relation between auxin-induced gene expression and the rapid auxin-induced growth inhibition in Arabidopsis thaliana roots. The natural auxin indole-3-acetic acid (IAA) induced a strong activation of gene expression as visualized by the DR5rev::GFP reporter gene technique. This effect was specific for active auxins and was abolished in knockout mutants of the F-box auxin receptors. We measured the IAA-induced growth inhibition at high time resolution and show that the F-box auxin receptor mutants failed to display this effect. We conclude that the F-box auxin receptors are needed for the response. In hypocotyls, auxin induces an increase in elongation growth, and this effect has been earlier shown to be independent of the F-box receptors. Based on these findings, we discuss differences in the growth control modes in roots and shoots. We demonstrate that the rapid auxin-induced root growth inhibition, unlike the induction of growth in hypocotyls, requires the presence of the F-box auxin receptors.     

Transgenic white clover. Studies with the auxin-responsive promoter,GH3, in root gravitropism and lateral root development

We report an improved method for white clover (Trifolium repens) transformation usingAgrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. Thebar gene and phosphinothricin selection was shown to be a more effective selection system thannptII (kanamycin selection) oraadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.