Different Behaviour of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Stimulating Lateral Root Development in Rice (Oryza sativa L.)

The plant hormone auxin has been shown to be involved in lateral root development and application of auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), increases the number of lateral roots in several plants. We found that the effects of two auxins on lateral root development in the indica rice (Oryza sativa L. cv. IR8) were totally different from each other depending on the application method. When the roots were incubated with an auxin solution, IAA inhibited lateral root development, while IBA was stimulatory. In contrast, when auxin was applied to the shoot, IAA promoted lateral root formation, while IBA did not. The transport of [³H]IAA from shoot to root occurred efficiently (% transported compared to supplied) but that of [³H]IBA did not, which is consistent with the stimulatory effect of IAA on lateral root production when applied to the shoot. The auxin action of IBA has been suggested to be due to its conversion to IAA. However, in rice IAA competitively inhibited the stimulatory effect of IBA on lateral root formation when they were applied to the incubation solution, suggesting that the stimulatory effect of IBA on lateral root development is not through its conversion to IAA.     

Small‐molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin‐deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole‐3‐pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole‐3‐acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin‐containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4‐biphenylboronic acid (BBo) and 4‐phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild‐type Arabidopsis seedlings. Co‐treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (K_i) of BBo and PPBo were 67 and 56 nm , respectively. In addition, PPBo did not interfere with the auxin response of auxin‐marker genes when it was co‐treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.     

Phosphatidylinositol‐specific phospholipase C2 functions in auxin‐modulated root development

Nine phosphatidylinositol‐specific phospholipases C (PLCs) have been identified in the Arabidopsis genome; among the importance of PLC2 in reproductive development is significant. However, the role of PLC2 in vegetative development such as in root growth is elusive. Here, we report that plc2 mutants displayed multiple auxin‐defective phenotypes in root development, including short primary root, impaired root gravitropism, and inhibited root hair growth. The DR5:GUS expression and the endogenous indole‐3‐acetic acid (IAA) content, as well as the responses of a set of auxin‐related genes to exogenous IAA treatment, were all decreased in plc2 seedlings, suggesting the influence of PLC2 on auxin accumulation and signalling. The root elongation of plc2 mutants was less sensitive to the high concentration of exogenous auxins, and the application of 1‐naphthaleneacetic acid or the auxin transport inhibitor N‐1‐naphthylphthalamic acid could rescue the root hair growth of plc2 mutants. In addition, the PIN2 polarity and cycling in plc2 root epidermis cells were altered. These results demonstrate a critical role of PLC2 in auxin‐mediated root development in Arabidopsis, in which PLC2 influences the polar distribution of PIN2.     

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.     

RLF,a cytochrome b5‐like heme/steroid binding domain protein,controls lateral root formation independently of ARF7/19‐mediated auxin signaling in Arabidopsis thaliana

Lateral root (LR) formation is important for the establishment of root architecture in higher plants. Recent studies have revealed that LR formation is regulated by an auxin signaling pathway that depends on auxin response factors ARF7 and ARF19, and auxin/indole‐3‐acetic acid (Aux/IAA) proteins including SOLITARY‐ROOT (SLR)/IAA14. To understand the molecular mechanisms of LR formation, we isolated a recessive mutant rlf (reduced lateral root formation) in Arabidopsis thaliana. The rlf‐1 mutant showed reduction of not only emerged LRs but also LR primordia. Analyses using cell‐cycle markers indicated that the rlf‐1 mutation inhibits the first pericycle cell divisions involved in LR initiation. The rlf‐1 mutation did not affect auxin‐induced root growth inhibition but did affect LR formation over a wide range of auxin concentrations. However, the rlf‐1 mutation had almost no effect on auxin‐inducible expression of LATERAL ORGAN BOUNDARIES‐DOMAIN16/ASYMMETRIC LEAVES2‐LIKE18 (LBD16/ASL18) and LBD29/ASL16 genes, which are downstream targets of ARF7/19 for LR formation. These results indicate that ARF7/19‐mediated auxin signaling is not blocked by the rlf‐1 mutation. We found that the RLF gene encodes At5g09680, a protein with a cytochrome b₅‐like heme/steroid binding domain. RLF is ubiquitously expressed in almost all organs, and the protein localizes in the cytosol. These results, together with analysis of the genetic interaction between the rlf‐1 and arf7/19 mutations, indicate that RLF is a cytosolic protein that positively controls the early cell divisions involved in LR initiation, independent of ARF7/19‐mediated auxin signaling.     

Small acidic protein 1 and SCFTIR1 ubiquitin proteasome pathway act in concert to induce 2,4‐dichlorophenoxyacetic acid‐mediated alteration of actin in Arabidopsis roots

2,4‐Dichlorophenoxyacetic acid (2,4‐D), a functional analogue of auxin, is used as an exogenous source of auxin as it evokes physiological responses like the endogenous auxin, indole‐3‐acetic acid (IAA). Previous molecular analyses of the auxin response pathway revealed that IAA and 2,4‐D share a common mode of action to elicit downstream physiological responses. However, recent findings with 2,4‐D‐specific mutants suggested that 2,4‐D and IAA might also use distinct pathways to modulate root growth in Arabidopsis. Using genetic and cellular approaches, we demonstrate that the distinct effects of 2,4‐D and IAA on actin filament organization partly dictate the differential responses of roots to these two auxin analogues. 2,4‐D but not IAA altered the actin structure in long‐term and short‐term assays. Analysis of the 2,4‐D‐specific mutant aar1‐1 revealed that small acidic protein 1 (SMAP1) functions positively to facilitate the 2,4‐D‐induced depolymerization of actin. The ubiquitin proteasome mutants tir1‐1 and axr1‐12, which show enhanced resistance to 2,4‐D compared with IAA for inhibition of root growth, were also found to have less disrupted actin filament networks after 2,4‐D exposure. Consistently, a chemical inhibitor of the ubiquitin proteasome pathway mitigated the disrupting effects of 2,4‐D on the organization of actin filaments. Roots of the double mutant aar1‐1 tir1‐1 also showed enhanced resistance to 2,4‐D‐induced inhibition of root growth and actin degradation compared with their respective parental lines. Collectively, these results suggest that the effects of 2,4‐D on actin filament organization and root growth are mediated through synergistic interactions between SMAP1 and SCF^TIR1 ubiquitin proteasome components.     

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.     

Participation of Auxin Transport in the Early Response of the <i>Arabidopsis</i> Root System to Inoculation with <i>Azospirillum brasilense</i>

The potential of Plant Growth Promoting Rhizobacteria (PGPR) has been demonstrated in the case of plant inoculation with bacteria of the genus Azospirillum which improves yield. A. brasilense produces a wide variety of molecules, including the natural auxin indole-3-acetic acid (IAA), as well as other phytoregulators. However, several studies have suggested that auxin induces changes in plant development during their interaction with the bacteria. The effects of A. brasilense Sp245 on the development of Arabidopsis thaliana root were investigated to help explain the molecular basis of the interaction. The results obtained showed a decrease in primary root length from the first day and remained so throughout the exposure, accompanied by a stimulation of initiation and maturation of lateral root primordia and an increase of lateral roots. An enhanced auxin response was evident in the vascular tissue and lateral root meristems of inoculated plants. However, after five days of bacterization, the response disappeared in the primary root meristems. The role of polar auxin transport (PAT) in auxins relocation involved the PGP1, AXR4-1, and BEN2 proteins, which apparently mediated A. brasilense-induced root branching of Arabidopsis seedlings.     

A role for polar auxin transport in rhizogenesis

Internodal shoot sections of the easy-to-root Forsythia×intermedia cv. Lynwood, and the difficult-to-root Syringa vulgaris cv. Madame Lemoine were used in vitro to investigate the role of polar auxin transport (PAT) in rhizogenesis. Syringa internodes required the distal application of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or naphthaleneacetic acid to induce rooting, while 2,4-dichlorophenoxyacetic acid was ineffective. In contrast, Forsythia internodes rooted equally well when IBA was applied at either end of the internode. Using [³H]IAA showed transport of exogenous auxin was basipetal, and that despite similar transport velocities, the intensity of auxin transport in Syringa was greater than in Forsythia. Basipetal transport of exogenous auxin was blocked using the PAT inhibitors 2,3,5-triiodobenzoic acid (TIBA) and naringenin (Nar); where Forsythia proved more sensitive to TIBA, but less so to Nar, in comparison with Syringa. In both species, percentage rooting and the number of roots formed were greater in 5-mm-long internodes than in shorter internodes. The results demonstrate the importance of PAT for root initiation in Syringa, whereas Forsythia tissue appears to be more sensitive to the direct application of auxin.     

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.     

Arabidopsis PROTEIN S‐ACYL TRANSFERASE4 mediates root hair growth

Polar growth of root hairs is critical for plant survival and requires fine‐tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S‐acyl transferases (PATs), mediating the S‐acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site‐directed mutagenesis and genetic analysis with related root‐hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S‐acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild‐type but not an enzyme‐inactive PAT4. Membrane‐associated ROP2 was significantly reduced in pat4, similar to S‐acylation‐deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4‐mediated S‐acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.     

The short-rooted vitamin B<Subscript>6</Subscript>-deficient mutant <Emphasis Type="Italic">pdx1</Emphasis> has impaired local auxin biosynthesis

The phytohormone auxin regulates many aspects of plant growth and development. Auxin often acts distantly from the site of its biosynthesis and this long-distance-transported auxin is well known to play a critical role in eliciting physiological responses including regulating root development. Auxin can be produced in roots, yet the function of locally synthesized auxin in root growth is unclear. The major auxin in plants, indole 3-acetic acid (IAA), is mainly synthesized through tryptophan (Trp)-dependent pathways that require pyridoxal phosphate (an active form of vitamin B₆)-dependent enzymes. We previously reported that the Arabidopsis vitamin B₆ biosynthesis mutant pdx1 has stunted root growth although the underlying cause is unknown. Here we showed that the pdx1 root is deficient in auxin biosynthesis. By reciprocal grafting of pdx1 and the wild type, we demonstrated that the stunted root growth in pdx1 is caused by a locally generated signal(s) in roots. To test whether auxin might be one such signal, the auxin responsive DR5::GUS reporter was introduced into the mutant. The DR5::GUS activity in pdx1 root tips was greatly reduced compared with that in the wild type although the auxin response was unaltered. pdx1 also suppresses the root hair growth defects in the auxin overproduction mutant yucca. These data indicate that pdx1 is impaired in Trp-dependent auxin biosynthesis, which may contribute to the short-root phenotype of pdx1. We suggest that locally synthesized auxin may play a critical role in postembryonic root growth.     

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.     

The Role of Boron in Plant Growth: IV. INTERRELATIONSHIPS BETWEEN BORON AND INDOL-3YL-ACETIC ACID IN THE METABOLISM OF BEAN RADICLES

The hypothesis that boron deficiency is equivalent to a stateof IAA toxicity was explored. Bioassays showed that extractsof substances similar to IAA taken from boron-deficient rootswere significantly more inhibitory to the growth of bean-rootsegments than those from normal roots. Supplied IAA and borondeficiency together restricted root growth to a greater extentthan either deficiency or IAA treatment separately. Roots werefound to recover more quickly from the inhibitory effects ofsupplied IAA if boron was present at high (0.5 ppm) rather thanlow (0.01 ppm) concentrations. Experiments with ¹⁴C-labelled IAA showed that deficient rootsabsorbed ¹⁴C more slowly than boron-fed roots and there wasalso a lower rate of decarboxylation in the deficient tissue.Consideration of the published evidence showed that many ofthe effects of boron deficiency could follow from an upset inIAA metabolism. It is suggested that boron-deficient tissuesuffers from excess auxin either because the element is necessaryfor some growth process, such as cell wall formation or nucleicacid synthesis, which, when impaired, results in the accumulationof auxin, or because the IAA-oxidation system is affected byphenolic inhibitors which boron normally inactivates by complexformation.     

OsCYP2, a chaperone involved in degradation of auxin‐responsive proteins,plays crucial roles in rice lateral root initiation

Auxin plays a pivotal role in many facets of plant development. It acts by inducing the interaction between auxin‐responsive [auxin (AUX)/indole‐3‐acetic acid (IAA)] proteins and the ubiquitin protein ligase SCF^TIR to promote the degradation of the AUX/IAA proteins. Other cofactors and chaperones that participate in auxin signaling remain to be identified. Here, we characterized rice (Oryza sativa) plants with mutations in a cyclophilin gene (OsCYP2). cyp2 mutants showed defects in auxin responses and exhibited a variety of auxin‐related growth defects in the root. In cyp2 mutants, lateral root initiation was blocked after nuclear migration but before the first anticlinal division of the pericycle cell. Yeast two‐hybrid and in vitro pull‐down results revealed an association between OsCYP2 and the co‐chaperone Suppressor of G2 allele of skp1 (OsSGT1). Luciferase complementation imaging assays further supported this interaction. Similar to previous findings in an Arabidopsis thaliana SGT1 mutant (atsgt1b), degradation of AUX/IAA proteins was retarded in cyp2 mutants treated with exogenous 1‐naphthylacetic acid. Our results suggest that OsCYP2 participates in auxin signal transduction by interacting with OsSGT1.