The transparent testa4 mutation prevents flavonoid synthesis and alters auxin transport and the response of Arabidopsis roots to gravity and light

We examined whether flavonoids act as endogenous auxin transport regulators during gravity vector and light intensity changes in Arabidopsis thaliana roots. Flavonoid deficient transparent testa4 [tt4(2YY6)] seedlings had elevated root basipetal auxin transport compared with the wild type, consistent with the absence of a negative auxin transport regulator. The tt4(2YY6) roots had delayed gravitropism that was chemically complemented with a flavonoid intermediate. Flavonoid accumulation was found in wild-type columella cells, the site of gravity perception, and in epidermal and cortical cells, the site of differential growth, but flavonoid accumulation was absent in tt4(2YY6) roots. Flavonoid accumulation was higher in gravity-stimulated root tips as compared with vertical controls, with maximum differences coinciding with the timing of gravitropic bending, and was located in epidermal cells. Exogenous indole-3-acetic acid (IAA) also elevated flavonoid accumulation, suggesting that flavonoid changes in response to gravity might be partly as a result of changing IAA distribution. Acropetal IAA transport was also elevated in roots of tt4(2YY6). Flavonoid synthesis was repressed in the dark, as were differences in root acropetal transport in tt4(2YY6). These results are consistent with light- and gravity-induced flavonoid stimulation that alters auxin transport in roots and dependent physiological processes, including gravitropic bending and root development.     

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

 The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsisthaliana (L.) Heynh. seedlings. Moreover, amino acyl-β-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [³H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [¹⁴C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux. Received: 2 December 1999 / Accepted: 16 January 2000     

A chemical complementation approach reveals genes and interactions of flavonoids with other pathways

In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild‐type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome‐wide mRNA accumulation changes in naringenin‐treated and untreated tt5 and wild‐type seedlings, we identified a flavonoid‐responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin‐responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.     

Ethylene modulates flavonoid accumulation and gravitropic responses in roots of Arabidopsis

Plant organs change their growth direction in response to reorientation relative to the gravity vector. We explored the role of ethylene in Arabidopsis (Arabidopsis thaliana) root gravitropism. Treatment of wild-type Columbia seedlings with the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC) reduced root elongation and gravitropic curvature. The ethylene-insensitive mutants ein2-5 and etr1-3 had wild-type root gravity responses, but lacked the growth and gravity inhibition by ACC found in the wild type. We examined the effect of ACC on tt4(2YY6) seedlings, which have a null mutation in the gene encoding chalcone synthase, the first enzyme in flavonoid synthesis. The tt4(2YY6) mutant makes no flavonoids, has elevated indole-3-acetic acid transport, and exhibits a delayed gravity response. Roots of tt4(2YY6), the backcrossed line tt4-2, and two other tt4 alleles had wild-type sensitivity to growth inhibition by ACC, whereas the root gravitropic curvature of these tt4 alleles was much less inhibited by ACC than wild-type roots, suggesting that ACC may reduce gravitropic curvature by altering flavonoid synthesis. ACC treatment induced flavonoid accumulation in root tips, as judged by a dye that becomes fluorescent upon binding flavonoids in wild type, but not in ein2-5 and etr1-3. ACC also prevented a transient peak in flavonoid synthesis in response to gravity. Together, these experiments suggest that elevated ethylene levels negatively regulate root gravitropism, using EIN2- and ETR1-dependent pathways, and that ACC inhibition of gravity response occurs through altering flavonoid synthesis.     

Implications of long-distance flavonoid movement in Arabidopsis thaliana

Flavonoid synthesis is modulated by developmental and environmental signals that control the amounts and localization of the diverse flavonoids found in plants. Flavonoids are implicated in regulating a number of physiological processes including UV protection, fertilization, auxin transport, plant architecture, gravitropism and pathogenic and symbiotic interactions with other organisms. Recently we showed that flavonoids can move long distances in plants, which may facilitate these molecules reaching positions in the plant where these processes are regulated. The localised application of selective flavonoids to tt4 mutants such as naringenin, dihydrokaempferol and dihydroquercetin showed that they were taken up at the root tip, mid-root or cotyledons and travelled long distances via cell-to-cell movement to distal tissues and converted to quercetin and kaempferol. In contrast, kaempferol and quercetin do not move long distances. They were taken up only at the root tip and did not move from this position. Here we show the movement of endogenous flavonoids by using reciprocal grafting experiments between tt4 and wild-type seedlings. These results demonstrated that to understand the distribution of flavonoids in Arabidopsis, it is necessary to know where the flavonoid biosynthetic enzymes are made and to understand the mechanisms by which certain flavonoids move from their site of synthesis.Key words: flavonoid movement, reciprocal graft, quercetin, kaempferol, Arabidopsis thaliana, fluorescence, aglyconeFlavonoids are plant secondary metabolites made by the phenylpropanoid pathway. The central biosynthetic pathway is known and in Arabidopsis most of the enzymes in flavonoid synthesis are encoded by single copy genes.¹ The isolation of mutants with defects in the genes encoding these flavonoid biosynthetic enzymes has allowed researchers to understand the biochemical complexity of flavonoid synthesis and their biological roles. Flavonoid synthesis is more complex in other species, such as legumes, which produce a greater diversity of flavonoid molecules, and in which gene families encode the key enzymatic branch points of the pathway.^2,3The functions of flavonoids were demonstrated using genetic approaches that blocked flavonoid synthesis in Arabidopsis and other species. In Arabidopsis, flavonoids play important roles in UV protection⁴ and regulate auxin transport and dependent physiological processes, such as gravity responses,^5–7 and lateral root formation.⁸ In petunia, maize and tomato, pollen without flavonoids is infertile and this phenotype is reversed by flavonoid addition.^9–11 However, the enigma of why flavonoid-deficient Arabidopsis seedlings are fertile has not been resolved.¹² Flavonoids appear to interact with Multidrug resistance (MDR)/P-glycoproteins (PGP)/ABC-Type B proteins⁷ that transport auxin, regulate phosphatases and kinases, and may have regulatory roles as scavengers of reactive oxygen species (reviewed in ref. ¹³). These results are consistent with a diversity of important functions for flavonoids in plants that require careful control of flavonoid synthesis and localization.We have explored the possibility that flavonoid accumulation in specific locations is also modulated by movement of early intermediates of the flavonoid pathway. Long-distance movement of secondary metabolites is largely unexplored but potentially has profound developmental effects. Grafting experiments conducted in the early 1900s suggested that alkaloids move from the site of manufacture (the root) to the aerial tissue.¹⁴ More recent grafting experiments showed that root synthesised metabolites, perhaps carotenoids, regulate shoot development,^15,16 flowering inducers travel long distances,¹⁷ and phytohormones are translocated (reviewed in ref. ¹⁸).We recently showed that flavonoids moved long distances in Arabidopsis using several approaches.¹⁹ The roots of Arabidopsis seedlings grown in complete darkness do not accumulate flavonoids⁵ since expression of early genes encoding enzymes of flavonoid biosynthesis are light dependent.²⁰ Yet, flavonoids accumulate in root tips of seedlings with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain (diphenyl boric acid 2-amino ethyl ester [DPBA]), and localised aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating that early intermediates in the flavonoid pathway can move long distances. This was confirmed by time-course fluorescence experiments and HPLC. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application mid-root or to cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol and dihydroquercetin were taken up at the root tip, mid-root or through cotyledons and travelled long distances via cell-to-cell movement to distal tissues followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Uptake of flavonoids at the root tip was inhibited by glybenclamide, a specific inhibitor of ABC type transporters²¹ suggesting a possible role for transporters of this class in the movement of flavonoids.To show that endogenous flavonoids are capable of long distance movement, we performed reciprocal butt grafting between tt4 and wild-type seedlings.²² In these experiments we asked whether flavonoids moved from wild-type tissues to flavonoid-deficient tissues of tt4. DPBA fluorescence detection was used to detect flavonoid movement into tt4 tissues.¹⁹ Seedlings were grafted and grown on filter paper in Petri dishes for 8 d. The seedlings were then transferred to equal parts sand, perlite and vermiculite to avoid the possibility of uptake of pre-existing flavonoids that may be natural soil components. After 14 d, the seedlings were stained with DPBA. When tt4 roots were grafted to tt4 shoots, the samples showed dim greenish autofluorescence in roots and only red chlorophyll fluorescence in the shoot. When either tt4 roots or shoots were grafted onto wild-type shoots or roots, respectively, the tt4 tissues showed bright yellow DPBA fluorescence (Fig. 1). The results of these experiments clearly showed that endogenous flavonoids moved across the graft to the reciprocal tissue. The flavonoid movement is specific to certain tissues, as flavonoids are not transported into the seeds developing on tt4 shoots grafted on wild-type roots, which retain the transparent testa phenotype. Although flavonoids clearly travelled from wild-type root tissue to mutant shoots, they were not capable of complementing the seed colour defect of tt4. In addition, adding naringenin to tt4 plants either to the media, or to the soil, also did not complement the seed colour phenotype in tt4 (data not shown). Recent research by Hsieh and Huang²³ may account for this inability to complement seed colouration, as flavonoids in the Brassicaceae end up in the pollen coat rather than the testa. The testa tissue derives from ovular tissue,²⁴ and thus is maternal in origin.Open in a separate windowFigure 1Grafting shows flavonoid movement occurs across grafts. Reciprocal grafting between wild type and tt4 indicated flavonoid movement from the flavonoid producing tissue to the chalcone synthase-deficient tissue. The order of the graft is indicated by the left legend as aerial tissue over root tissue in the graft. Micrographs are DPBA stained tissue excited with 488 nm wavelength. The tt4/tt4 control graft shows no flavonoids are present, even though a wound has occurred on the leaf which generally exacerbates flavonoid fluorescence. Scale bar = 100 µm. Green fluorescence is from kaempferol and gold from quercetin. The red fluorescence is from chlorophyll.The complexity of the flavonoid biosynthetic pathway and the large number of modified flavonoids that can be made through the complex series of glycosylation reactions suggests that distinct flavonoid molecules may have unique function. To fully understand these molecules, it is necessary to dissect the synthesis pathways for these glycosylated flavonoids. Two unnamed flavonoid glycoside mutants isolated in 1998,²⁵ have profound developmental phenotypes, supporting this hypothesis. These mutations resulted in whorled cauline leaves on inflorescences and double the number of rosette leaves. Our lab is in the process of determining if other phenotypes exist in flavonoid mutants.A critical feature of the observations of flavonoid movement is understanding the biological context of this movement. First, a number of recent studies reported physiological functions of flavonoids in roots, ranging from modulation of auxin transport and root gravitropism,^5–8 to nodulation³ and root branching,⁸ while it is clear that flavonoid synthesis is absent in dark-grown seedlings.⁵ Yet, for flavonoids to function in roots of plants grown in soil, the light signal and/or flavonoid precursors must travel to the roots. Additionally, transient flavonoid accumulation has been reported in roots reoriented relative to the gravity vector.⁶ For flavonoids to transiently accumulate at the root tip (at 2 hours after reorientation) and to return to lower levels (within 2 additional hours), suggests that more than flavonoid synthesis is regulated. Perhaps this transient flavonoid accumulation requires localized enzyme activation and transport mechanisms. As flavonoid transport is inhibited by a compound that blocks ABC transporters, which include the newly identified auxin transporters of the MDR/PGP class, perhaps there are connections between flavonoid and auxin transport that allow this transient accumulation. A more detailed understanding of this role of flavonoid movement in controlling plant development awaits additional experimentation.     

Identifying the transporters of different flavonoids in plants

We recently identified a new component of flavonoid transport pathways in Arabidopsis. The MATE protein FFT (Flower Flavonoid Transporter) is primarily found in guard cells and seedling roots, and mutation of the transporter results in floral and growth phenotypes. The nature of FFT''s substrate requires further exploration but our data suggest that it is a kaempferol diglucoside. Here we discuss potential partner H⁺-ATPases and possible redundancy among the close homologs within the large Arabidopsis MATE family.Key words: auxin, flavonoid, guard cell, pollen, transporterPlant flavonoids are becoming notorious for their wide and expanding range of possible functions. Beyond UV protection (itself not entirely without debate), further roles have been added in plant development; nodulation and interactions with pathogens; fertilization; and auxin transport. For such a well-described biochemical network, it interesting that few aspects of flavonoid function are clear-cut: perhaps it is the recently established link with auxin, so intimately involved in every aspect of plant development, that consigns them to multiple incompletely-known regulatory pathways. Knowledge is lacking, in particular, about the transport of flavonoids. Such transport is necessary¹ and we now know that selective uptake of flavonoids and movement of flavonoids through the plant occur.^2,3 When naringenin, dihydrokaempferol and dihydroquercetin were added to the Arabidopsis tt4 mutant [lacking the enzyme chalcone synthase (CHS) and thus all flavonoids] at root tip, mid-root or to cotyledons, they were converted to downstream products. Grafting on flavonoid-producing tissues to tt4 could also complement the mutation.³Kitamura⁴ and Buer et al.⁵ speculate that MATE transporters are good candidates to enable flavonoid transport at the membrane, allowing the necessary movement from one membrane system to another. The link between MATE proteins and flavonoid transport is justified by work in tomato⁶ and confirmed by the discovery of TT12.^7,8 Also conforming to this premise is our recent work on FFT (Flower Flavonoid Transporter), a MATE protein probably situated in the tonoplast membrane that has a role in flavonoid transport in specialised guard cells and anthers.⁹     

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.     

Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects.

Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.     

Flavonoid-related regulation of auxin accumulation in<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-induced plant tumors

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.     

Effect of simulated microgravity on auxin polar transport in inflorescence axis of Arabidopsis thaliana.

The morphology, growth and development of higher plants are strongly influenced by environmental stimuli on the earth, which affect the changes in the dynamics of plant hormones in plants. Qualitative and quantitative changes in plant hormones are the most important internal factor to regulate plant growth and development. Among them, auxin (IAA) is of most significant. There are numerous reports concerning the physiological roles of auxin in plant growth and development (Matthysse and Scott 1984). One of the characteristics of auxin is to have the ability of polar transport along the vector of gravity on the earth (Schneider and Wightman 1978), suggesting that the activity of auxin polar transport is also important for the growth and development of plants. It has recently been reported that the normal activity of auxin polar transport in inflorescence axis of Arabidopsis thaliana was required for flower formation (Okada et al. 1991, Ueda et al. 1992). Considering the above evidence together with the fact that gravity affects the morphology, growth and development of higher plants, gravity might affect the qualitative and quantitative changes in plant hormones including the activity of auxin polar transport. In this paper, we report the effect of microgravity condition simulated by a three-dimensional (3-D) or a horizontal clinostat on the activity of auxin polar transport in inflorescence axis of Arabidopsis thaliana.     

Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth

In Arabidopsis thaliana, silencing of hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT), a lignin biosynthetic gene, results in a strong reduction of plant growth. We show that, in HCT-silenced plants, lignin synthesis repression leads to the redirection of the metabolic flux into flavonoids through chalcone synthase activity. Several flavonol glycosides and acylated anthocyanin were shown to accumulate in higher amounts in silenced plants. By contrast, sinapoylmalate levels were barely affected, suggesting that the synthesis of that phenylpropanoid compound might be HCT-independent. The growth phenotype of HCT-silenced plants was shown to be controlled by light and to depend on chalcone synthase expression. Histochemical analysis of silenced stem tissues demonstrated altered tracheary elements. The level of plant growth reduction of HCT-deficient plants was correlated with the inhibition of auxin transport. Suppression of flavonoid accumulation by chalcone synthase repression in HCT-deficient plants restored normal auxin transport and wild-type plant growth. By contrast, the lignin structure of the plants simultaneously repressed for HCT and chalcone synthase remained as severely altered as in HCT-silenced plants, with a large predominance of nonmethoxylated H units. These data demonstrate that the reduced size phenotype of HCT-silenced plants is not due to the alteration of lignin synthesis but to flavonoid accumulation.     

Stems of the <Emphasis Type="Italic">Arabidopsis pin1-1</Emphasis> mutant are not deficient in free indole-3-acetic acid

The pin1-1 mutant of Arabidopsis thaliana has been pivotal for studies on auxin transport and on the role of auxin in plant development. It was reported previously that when whole shoots were analysed, levels of the major auxin, indole-3-acetic acid (IAA) were dramatically reduced in the mutant, compared with the WT (Okada et al. 1991). The cloning of PIN1, however, provided evidence that this gene encodes a facilitator of auxin efflux, raising the question of how the pin1-1 mutation might reduce overall IAA levels as well as IAA transport. We therefore re-examined IAA levels in individual parts of pin1-1 and WT plants, focusing on inflorescence stems. Our data show that there is in fact no systemic IAA deficiency in the mutant. The previously reported difference between mutant and WT may have been due to the inclusion of reproductive structures in the WT harvest: we show here that the inflorescence itself contains high levels of IAA. We reconcile the normal IAA levels of pin1-1 inflorescence stems with their (previously-reported) reduced ability to transport IAA by presenting evidence that the auxin in mutant stems is not imported from their apical portion. Our data also indicate that levels of another auxin, indole-3-butyric acid (IBA), are very low in stems of the genotypes used in this study.     

The rib1 mutant is resistant to indole-3-butyric acid, an endogenous auxin in Arabidopsis

The presence of indole-3-butyric acid (IBA) as an endogenous auxin in Arabidopsis has been recently demonstrated. However, the in vivo role of IBA remains to be elucidated. We present the characterization of a semi-dominant mutant that is affected in its response to IBA, but shows a wild-type response to indole-3-acetic acid (IAA), the predominant and most studied form of auxin. We have named this mutant rib1 for resistant to IBA. Root elongation assays show that rib1 is specifically resistant to IBA, to the synthetic auxin 2,4-dichlorophenoxyacetic acid, and to auxin transport inhibitors. rib1 does not display increased resistance to IAA, to the synthetic auxin naphthalene acetic acid, or to other classes of plant hormones. rib1 individuals also have other root specific phenotypes including a shortened primary root, an increased number of lateral roots, and a more variable response than wild type to a change in gravitational vector. Adult rib1 plants are morphologically indistinguishable from wild-type plants. These phenotypes suggest that rib1 alters IBA activity in the root, thereby affecting root development and response to environmental stimuli. We propose models in which RIB1 has a function in either IBA transport or response. Our experiments also suggest that IBA does not use the same mechanism to exit cells as does IAA and we propose a model for IBA transport.     

Arabidopsis AtGSTF2 is regulated by ethylene and auxin, and encodes a glutathione S-transferase that interacts with flavonoids

Expression of the Arabidopsis glutathione S-transferase (GST) gene AtGSTF2 is induced by several stimuli, but the function of this GST remains unknown. We demonstrate that AtGSTF2 expression is also induced by glutathione, paraquat, copper, and naphthalene acetic acid (NAA) via a mechanism independent of ethylene perception, as determined by analysis of the ethylene-insensitive etr1 mutant. Deletion analyses identified two promoter regions important for regulation of AtGSTF2 expression in response to several of these inducers. Previous studies have suggested that AtGSTF2 interacts with indole-3-acetic acid (IAA) and the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA). We show that recombinant AtGSTF2 directly binds IAA, NPA, and the artificial auxin NAA. As NPA may act as an endogenous flavonoid regulator of auxin transport, competition between NPA and flavonoids for binding to AtGSTF2 was examined. Both quercetin and kaempferol competed with NPA for AtGSTF2 binding, indicating that all three compounds bind AtGSTF2 at the same site. In transgenic Arabidopsis seedlings, AtGSTF2::GUS expression occurred at the root-shoot transition zone and was induced in this region, as well as at the root distal elongation zone, after treatment with IAA. In wild-type seedlings, AtGSTF2 is localized near the plasma membrane of cells in the root-shoot transition zone. However, both AtGSTF2::GUS expression and localization of AtGSTF2 protein were disrupted in flavonoid-deficient tt4 seedlings. Our results indicate that AtGSTF2 is involved not only in stress responses but also in development under normal growth conditions.     

The role of flavonoids in root-rhizosphere signalling: opportunities and challenges for improving plant-microbe interactions

The flavonoid pathway produces a diverse array of plant compounds with functions in UV protection, as antioxidants, pigments, auxin transport regulators, defence compounds against pathogens and during signalling in symbiosis. This review highlights some of the known function of flavonoids in the rhizosphere, in particular for the interaction of roots with microorganisms. Depending on their structure, flavonoids have been shown to stimulate or inhibit rhizobial nod gene expression, cause chemoattraction of rhizobia towards the root, inhibit root pathogens, stimulate mycorrhizal spore germination and hyphal branching, mediate allelopathic interactions between plants, affect quorum sensing, and chelate soil nutrients. Therefore, the manipulation of the flavonoid pathway to synthesize specifically certain products has been suggested as an avenue to improve root-rhizosphere interactions. Possible strategies to alter flavonoid exudation to the rhizosphere are discussed. Possible challenges in that endeavour include limited knowledge of the mechanisms that regulate flavonoid transport and exudation, unforeseen effects of altering parts of the flavonoid synthesis pathway on fluxes elsewhere in the pathway, spatial heterogeneity of flavonoid exudation along the root, as well as alteration of flavonoid products by microorganisms in the soil. In addition, the overlapping functions of many flavonoids as stimulators of functions in one organism and inhibitors of another suggests caution in attempts to manipulate flavonoid rhizosphere signals.