The glucosinolate breakdown product indole‐3‐carbinol acts as an auxin antagonist in roots of Arabidopsis thaliana

The glucosinolate breakdown product indole‐3‐carbinol functions in cruciferous vegetables as a protective agent against foraging insects. While the toxic and deterrent effects of glucosinolate breakdown on herbivores and pathogens have been studied extensively, the secondary responses that are induced in the plant by indole‐3‐carbinol remain relatively uninvestigated. Here we examined the hypothesis that indole‐3‐carbinol plays a role in influencing plant growth and development by manipulating auxin signaling. We show that indole‐3‐carbinol rapidly and reversibly inhibits root elongation in a dose‐dependent manner, and that this inhibition is accompanied by a loss of auxin activity in the root meristem. A direct interaction between indole‐3‐carbinol and the auxin perception machinery was suggested, as application of indole‐3‐carbinol rescues auxin‐induced root phenotypes. In vitro and yeast‐based protein interaction studies showed that indole‐3‐carbinol perturbs the auxin‐dependent interaction of Transport Inhibitor Response (TIR1) with auxin/3‐indoleacetic acid (Aux/IAAs) proteins, further supporting the possibility that indole‐3‐carbinol acts as an auxin antagonist. The results indicate that chemicals whose production is induced by herbivory, such as indole‐3‐carbinol, function not only to repel herbivores, but also as signaling molecules that directly compete with auxin to fine tune plant growth and development.     

The inter‐kingdom volatile signal indole promotes root development by interfering with auxin signalling

Recently, emission of volatile organic compounds (VOCs) has emerged as a mode of communication between bacteria and plants. Although some bacterial VOCs that promote plant growth have been identified, their underlying mechanism of action is unknown. Here we demonstrate that indole, which was identified using a screen for Arabidopsis growth promotion by VOCs from soil‐borne bacteria, is a potent plant‐growth modulator. Its prominent role in increasing the plant secondary root network is mediated by interfering with the auxin‐signalling machinery. Using auxin reporter lines and classic auxin physiological and transport assays we show that the indole signal invades the plant body, reaches zones of auxin activity and acts in a polar auxin transport‐dependent bimodal mechanism to trigger differential cellular auxin responses. Our results suggest that indole, beyond its importance as a bacterial signal molecule, can serve as a remote messenger to manipulate plant growth and development.     

Auxin and its transport play a role in plant tolerance to arsenite‐induced oxidative stress in Arabidopsis thaliana

The role of auxin in plant development is well known; however, its possible function in root response to abiotic stress is poorly understood. In this study, we demonstrate a novel role of auxin transport in plant tolerance to oxidative stress caused by arsenite. Plant response to arsenite [As(III)] was evaluated by measuring root growth and markers for stress on seedlings treated with control or As(III)‐containing medium. Auxin transporter mutants aux1, pin1 and pin2 were significantly more sensitive to As(III) than the wild type (WT). Auxin transport inhibitors significantly reduced plant tolerance to As(III) in the WT, while exogenous supply of indole‐3‐acetic acid improved As(III) tolerance of aux1 and not that of WT. Uptake assays using H³‐IAA showed As(III) affected auxin transport in WT roots. As(III) increased the levels of H₂O₂ in WT but not in aux1, suggesting a positive role for auxin transport through AUX1 on plant tolerance to As(III) stress via reactive oxygen species (ROS)‐mediated signalling. Compared to the WT, the mutant aux1 was significantly more sensitive to high‐temperature stress and salinity, also suggesting auxin transport influences a common element shared by plant tolerance to arsenite, salinity and high‐temperature stress.     

Tissue‐specific profiling of the Arabidopsis thaliana auxin metabolome

The plant hormone auxin is believed to influence almost every aspect of plant growth and development. Auxin transport, biosynthesis and degradation combine to form gradients of the hormone that influence a range of key developmental and environmental response processes. There is abundant genetic evidence for the existence of multiple pathways for auxin biosynthesis and degradation. The complexity of these pathways makes it difficult to obtain a clear picture of the relative importance of specific metabolic pathways during development. We have developed a sensitive mass spectrometry‐based method to simultaneously profile the majority of known auxin precursors and conjugates/catabolites in small amounts of Arabidopsis tissue. The method includes a new derivatization technique for quantification of the most labile of the auxin precursors. We validated the method by profiling the auxin metabolome in root and shoot tissues from various Arabidopsis thaliana ecotypes and auxin over‐producing mutant lines. Substantial differences were shown in metabolite patterns between the lines and tissues. We also found differences of several orders of magnitude in the abundance of auxin metabolites, potentially indicating the relative importance of these compounds in the maintenance of auxin levels and activity. The method that we have established will enable researchers to obtain a better understanding of the dynamics of auxin metabolism and activity during plant growth and development.     

CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis

Auxins are growth regulators involved in virtually all aspects of plant development. However, little is known about how plants synthesize these essential compounds. We propose that the level of indole-3-acetic acid is regulated by the flux of indole-3-acetaldoxime through a cytochrome P450, CYP83B1, to the glucosinolate pathway. A T-DNA insertion in the CYP83B1 gene leads to plants with a phenotype that suggests severe auxin overproduction, whereas CYP83B1 overexpression leads to loss of apical dominance typical of auxin deficit. CYP83B1 N-hydroxylates indole-3-acetaldoxime to the corresponding aci-nitro compound, 1-aci-nitro-2-indolyl-ethane, with a K(m) of 3 microM and a turnover number of 53 min(-1). The aci-nitro compound formed reacts non-enzymatically with thiol compounds to produce an N-alkyl-thiohydroximate adduct, the committed precursor of glucosinolates. Thus, indole-3-acetaldoxime is the metabolic branch point between the primary auxin indole-3-acetic acid and indole glucosinolate biosynthesis in Arabidopsis.     

Blue‐light‐induced PIN3 polarization for root negative phototropic response in Arabidopsis

Root negative phototropism is an important response in plants. Although blue light is known to mediate this response, the cellular and molecular mechanisms underlying root negative phototropism remain unclear. Here, we report that the auxin efflux carrier PIN‐FORMED (PIN) 3 is involved in asymmetric auxin distribution and root negative phototropism. Unilateral blue‐light illumination polarized PIN3 to the outer lateral membrane of columella cells at the illuminated root side, and increased auxin activity at the illuminated side of roots, where auxin promotes growth and causes roots bending away from the light source. Furthermore, root negative phototropic response and blue‐light‐induced PIN3 polarization were modulated by a brefeldin A‐sensitive, GNOM‐dependent, trafficking pathway and by phot1‐regulated PINOID (PID)/PROTEIN PHOSPHATASE 2A (PP2A) activity. Our results indicate that blue‐light‐induced PIN3 polarization is needed for asymmetric auxin distribution during root negative phototropic response.     

The rice FISH BONE gene encodes a tryptophan aminotransferase,which affects pleiotropic auxin‐related processes

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole‐3‐pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole‐3‐acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin‐related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.     

Classic myrosinase‐dependent degradation of indole glucosinolate attenuates fumonisin B1‐induced programmed cell death in Arabidopsis

The mycotoxin fumonisin B1 (FB1) causes the accumulation of reactive oxygen species (ROS) which then leads to programmed cell death (PCD) in Arabidopsis. In the process of studying FB1‐induced biosynthesis of glucosinolates, we found that indole glucosinolate (IGS) is involved in attenuating FB1‐induced PCD. Treatment with FB1 elevates the expression of genes related to the biosynthesis of camalexin and IGS. Mutants deficient in aliphatic glucosinolate (AGS) or camalexin biosynthesis display similar lesions to Col‐0 upon FB1 infiltration; however, the cyp79B2 cyp79B3 double mutant, which lacks induction of both IGS and camalexin, displays more severe lesions. Based on the fact that the classic myrosinase β‐thioglucoside glucohydrolase (TGG)‐deficient double mutant tgg1 tgg2, rather than atypical myrosinase‐deficient mutant pen2‐2, is more sensitive to FB1 than Col‐0, and the elevated expression of TGG1, but not of PEN2, correlates with the decrease in IGS, we conclude that TGG‐dependent IGS hydrolysis is involved in FB1‐induced PCD. Indole‐3‐acetonitrile (IAN) and indole‐3‐carbinol (I3C), the common derivatives of IGS, were used in feeding experiments, and this rescued the severe cell death phenotype, which is associated with reduced accumulation of ROS as well as increased activity of antioxidant enzymes and ROS‐scavenging ability. Despite the involvement of indole‐3‐acetic acid (IAA) in restricting FB1‐induced PCD, feeding of IAN and I3C attenuated FB1‐induced PCD in the IAA receptor mutant tir1‐1 just as in Col‐0. Taken together, our results indicate that TGG‐catalyzed breakdown products of IGS decrease the accumulation of ROS by their antioxidant behavior, and attenuate FB1 induced PCD in an IAA‐independent way.     

Changes in root development of Arabidopsis promoted by organic matter from oxisols

The presence of ramified and abundant lateral roots is fundamental to plant growth on highly weathered soils. In this work, the effects of humic acid (HA), fulvic acid and hexanic–methanolic (HM) extract, after alkaline extraction from topsoil of seven different oxisols, on the development of roots of Arabidopsis thaliana was evaluated. Furthermore, we used another emergent plant model, that is, micro‐tom (MT) tomatoes with size similar to that of Arabidopsis to test the effects of HA. It was observed that both humic fractions and the HM extract were able to change the root development, improving the number of lateral roots and their development in comparison to control plants. The promotion of root growth by the three organic matter fractions was higher than that observed by 10^?6 mol L^?1 indole acetic acid. The treatment of MT tomato mutant, less sensitive to auxin, with HA did not promote the emergence of lateral roots, being an additional indication of auxin‐like activities of HA. However, some organic matter fractions exhibited, besides promotion of lateral roots number, increase in the length of principal root, which is not a typical auxin effect, indicating that these substances could contain other physiologically active substances.     

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.     

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.     

Shoot‐derived abscisic acid promotes root growth

The phytohormone abscisic acid (ABA) plays a major role in regulating root growth. Most work to date has investigated the influence of root‐sourced ABA on root growth during water stress. Here, we tested whether foliage‐derived ABA could be transported to the roots, and whether this foliage‐derived ABA had an influence on root growth under well‐watered conditions. Using both application studies of deuterium‐labelled ABA and reciprocal grafting between wild‐type and ABA‐biosynthetic mutant plants, we show that both ABA levels in the roots and root growth in representative angiosperms are controlled by ABA synthesized in the leaves rather than sourced from the roots. Foliage‐derived ABA was found to promote root growth relative to shoot growth but to inhibit the development of lateral roots. Increased root auxin (IAA) levels in plants with ABA‐deficient scions suggest that foliage‐derived ABA inhibits root growth through the root growth‐inhibitor IAA. These results highlight the physiological and morphological importance, beyond the control of stomata, of foliage‐derived ABA. The use of foliar ABA as a signal for root growth has important implications for regulating root to shoot growth under normal conditions and suggests that leaf rather than root hydration is the main signal for regulating plant responses to moisture.     

Auxin‐treatment induces recovery of phytoplasma‐infected periwinkle

Aims: To test the effect of auxin‐treatment on plant pathogenic phytoplasmas and phytoplasma‐infected host. Methods and Results: In vitro grown periwinkle shoots infected with different ‘Candidatus Phytoplasma’ species were treated with indole‐3‐acetic acid (IAA) or indole‐3‐butyric acid (IBA). Both auxins induced recovery of phytoplasma‐infected periwinkle shoots, but IBA was more effective. The time period and concentration of the auxin needed to induce recovery was dependent on the ‘Candidatus Phytoplasma’ species and the type of auxin. Two ‘Candidatus Phytoplasma’ species, ‘Ca. P. pruni’ (strain KVI, clover phyllody from Italy) and ‘Ca. P. asteris’ (strain HYDB, hydrangea phyllody), were susceptible to auxin‐treatment and undetected by nested PCR or detected only in the second nested PCR in the host tissue. ‘Ca. P. solani’ (strain SA‐I, grapevine yellows) persisted in the host tissue despite the obvious recovery of the host plant and was always detected in the direct PCR. Conclusions: Both auxins induced recovery of phytoplasma‐infected plants and affected tested ‘Candidatus Phytoplasma’ species in the same manner, implying that the mechanism involved in phytoplasma elimination/survival is common to both, IAA and IBA. Significance and Impact of the Study: The results imply that in the case of some ‘Candidatus Phytoplasma’ species, IBA‐treatment could be used to eliminate phytoplasmas from in vitro grown Catharanthus roseus shoots.     

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.