Dead end for auxin conjugates in Physcomitrella?

For proper development of plants auxin levels need to be tightly controlled. For this, several routes have evolved and it is plausible that different organisms use these differently. To determine whether members of the family of GH3 proteins, which partially act as auxin conjugate synthetases in Arabidopsis thaliana, have similar roles in the moss Physcomitrella patens, we have investigated the in vitro activity of the two GH3 members in moss. We showed that both proteins can form amino acid conjugates with indole-3-acetic acid (IAA) but also with jasmonic acid (JA). Confirming these findings, single and double knockout-mutants showed lower levels of IAA conjugates than wild type. We discuss the results in light of the possible functions of IAA conjugate formation in lower land plants.Key words: Arabidopsis thaliana, auxin metabolism, jasmonic acid, GH3 genes, moss, Physcomitrella patensAuxins play diverse roles in many aspects of plant growth and development. Their activity is relying on the correct concentration in a given tissue and developmental stage.¹ If higher levels of indole-3-acetic acid (IAA) are present, the hormone can also have an inhibitory effect on growth processes.² Therefore, the tight control of IAA concentrations is absolutely necessary. To this end plants have evolved different mechanisms.³ First, biosynthesis is contributing to increasing IAA concentrations, mostly in young tissues such as meristems. Second, IAA can be transported in a polar way, depending on transport molecules, from cell to cell, away from the site of synthesis, thereby forming an auxin gradient along the plant axis. Third, IAA can be degraded, and fourth, IAA can be reversibly inactivated by conjugation to small molecules such as amino acids or sugars, but also be linked to larger molecules such as peptides or proteins.⁴ The inactive IAA conjugates can be hydrolyzed to yield free (i.e., active) IAA if needed. In higher plants the levels of free IAA constitutes between 5 and 20% depending on the tissue or age of the plant, whereas the conjugated form constitutes the major part.⁴ However, it is not yet clear in which way auxin homeostasis has evolved. The hypothesis that auxin has to be present during the evolution of a body plan has been tested by using different lower land plants which were compared in their mechanism to control auxin homeostasis. In algae, e.g., charophytes, the major metabolic way of controlling IAA is via biosynthesis. In bryophytes, the formation of IAA conjugates has been shown, although the amount was lower than for example in seed plants such as Arabidopsis thaliana.^5,6 Since the molecular biology of auxin homeostasis in Arabidopsis is most advanced, we will use this model plant to compare the knowledge on seed plants with that in the moss Physcomitrella patens. The recent publication of the Physcomitrella genome⁷ gives the possibility to investigate components of the machinery controlling IAA levels in a lower land plant.In general, there seem to be high levels of redundancy involved in the pathways leading to decrease or increase of IAA, respectively. In Figure 1 we compare the current knowledge about genes related to IAA concentrations in Physcomitrella with Arabidopsis. While in Arabidopsis many different biosynthetic routes leading to IAA were identified,⁸ in the Physcomitrella genome homologs of the YUCCA genes have been detected.⁷ The presence of auxin conjugate synthetases has been experimentally verified in the moss (see below) and additional evidence for ester conjugate synthesis comes from sequence homology to UDP-glucosyl transferases.⁷ There is also the possibility of degradation of either IAA or an amino acid conjugate with IAA^9,10 as discussed below.Open in a separate windowFigure 1Comparison of possibilities to regulate auxin homeostasis in Physcomitrella (solid lines) and Arabidopsis (dotted lines). Biosynthesis—AO, aldehyde oxidase; AMI1, amidase; CYP, cytochrome P450; NIT, nitrilase; TAA1, tryptophan aminotransferase; YUCCA, flavin monooxygenase; transport—AUX/LAX, auxin influx facilitator family; PIN, auxin efflux carrier family; PGP, ABC transporter type auxin efflux carrier family; conjugation/hydrolysis—UGT, UDP-glucosyl transferase; GH3, auxin conjugate synthetase family; ILR/IAR, auxin conjugate hydrolase family; Ox-IAA, oxindole-3-acetic acid; Ox-IAAsp, oxindole-3-aspartic acid.So far our work has focussed on the characterization of two members of the so called GH3 family, of which several from Arabidopsis can form conjugates of IAA with a variety of amino acids.¹¹ While 19 members of this family have been described in Arabidopsis, only two are present in Physcomitrella.¹² The Arabidopsis family clusters in three groups: group I containing the jasmonic acid conjugate synthetase JAR1 and a few others with as yet unkown function, group II the auxin conjugate synthetases, and group III with mostly as yet uncharacterized members.^11,13 Sequence similarity of the GH3 genes from Physcomitrella showed that both cluster within the JAR1 group.¹² Therefore, we analyzed the enzymatic activity of the two Physcomitrella GH3 proteins (PpGH3-1 and PpGH3-2) in vitro¹⁴ and found that both were active on jasmonic acid and a variety of different amino acids, whereas PpGH3-2 was active mostly with IAA. PpGH3-1 showed only weak activity with IAA and only two amino acids. For this reason, it could be assumed that the two Physcomitrella genes evolved by gene duplication, from which the initial activities would be for IAA and jasmonic acid. One of these genes might have evolved into a jasmonate conjugate synthetase (maybe AtJAR1),¹³ thereby loosing its activity on IAA. The second may have given rise to the auxin conjugate synthetase family in Arabidopsis,¹¹ but the conjugate synthetases of Physcomitrella have still activity with both hormones. Interestingly, there is no evidence as yet that jasmonic acid itself has a role during Physcomitrella development, although a possible function of JA-conjugates has not been closely investigated. Since in Arabidopsis the JA conjugate with isoleucine is the active compound to be recognized by the COI1 receptor protein,¹⁵ it could be the case that JA itself has no effect in Physcomitrella. However, in our growth experiments a small growth promoting effect of JA, independently on the presence of GH3 genes was found. Similar observations were made with gibberellins in Physcomitrella.¹⁶Further characterization of single and double KO mutants in each of the PpGH3 genes has led to the hypothesis that GH3 proteins are indeed involved in regulating the auxin homeostasis in Physcomitrella.¹⁴ Both single KO mutants were more sensitive to increasing IAA concentrations in the medium than the wild type. Furthermore, the levels of free IAA were higher and the levels of conjugated IAA concomitantly dropped. A double KO mutant had almost no IAA conjugates when compared to the wild type. However, this mutant was still able to synthesize ester conjugates with IAA. Interestingly, the role of GH3 proteins in auxin conjugation seemed to be only important in the gametophore stage, whereas protonema cultures of GH3 KO mutants did not show any changes in auxin homeostasis. Therefore, we hypothesize that the role of GH3 proteins is dependent on a certain developmental stage of the moss. Additionally, we propose other detoxification mechanisms for example, export or degradation, in protonema.In higher plants the ester conjugate formation of IAA has been shown to be dependent on UDP-glucosyl transferases (AtUGT84B1 for Arabidopsis¹⁷ and ZmIAGLU for maize¹⁸). In the genome of Physcomitrella we could detect candidate sequence(s) for these genes, indicating that Physcomitrella has indeed the potential to synthesise the ester conjugates found in the gametophores in addition to amide conjugates. However, in the Physcomitrella genome, no homolog for an auxin conjugate hydrolase was found. In higher plants, auxin conjugate hydrolysis is thought to contribute to free IAA and depending on the plant species, large gene families with overlapping but distinct substrate preferences for individual amino acid conjugates with IAA are present.^19,20 Since this is not the case for Physcomitrella, one has to ask the question whether the conjugation of auxin is a one-way road for inactivation of excess auxin and whether auxin conjugate hydrolysis has evolved later during plant evolution.In the Selaginella moellendorffii genome (http://genome.jgi-psf.org/Selmo1/Selmo1.home.html), an auxin conjugate hydrolase sequence related to higher plant ones, has been found based on homology searches, but the completion of the genome has to be awaited to draw final conclusions. Likewise, it is not clear, if this effect is specific for Physcomitrella, or found in bryophytes in general. Therefore, additional sequenced bryophyte genomes are needed.²¹Since in Arabidopsis the degradation of the IAA-Aspartate conjugate to Ox-IAA-Asp (see Fig. 1) has been described,^9,10 a similar scenario could be suggested to occur in Physcomitrella with the amino acid conjugates formed. Alternatively, the hydrolysis of IAA conjugates by members of the M20 dipeptidase family can be envisioned. However, this would need the activity of enzymes with very low sequence conservation to auxin conjugate hydrolases. These questions will be addressed in future research by studying the metabolism of IAA and IAA conjugates of Physcomitrella in more detail.     

Localized auxin biosynthesis and postembryonic root development in Arabidopsis

Auxin is a phytohormone essential for plant development. Due to the high redundancy in auxin biosynthesis, the role of auxin biosynthesis in embryogenesis and seedling development, vascular and flower development, shade avoidance and ethylene response were revealed only recently. We previously reported that a vitamin B₆ biosynthesis mutant pdx1 exhibits a short-root phenotype with reduced meristematic zone and short mature cells. By reciprocal grafting, we now have found that the pdx1 short root is caused by a root locally generated signal. The mutant root tips are defective in callus induction and have reduced DR5::GUS activity, but maintain relatively normal auxin response. Genetic analysis indicates that pdx1 mutant could suppress the root hair and root growth phenotypes of the auxin overproduction mutant yucca on medium supplemented with tryptophan (Trp), suggesting that the conversion from Trp to auxin is impaired in pdx1 roots. Here we present data showing that pdx1 mutant is more tolerant to 5-methyl anthranilate, an analogue of the Trp biosynthetic intermediate anthranilate, demonstrating that pdx1 is also defective in the conversion from anthranilate to auxin precursor tryptophan. Our data suggest that locally synthesized auxin may play an important role in the postembryonic root growth.Key words: auxin synthesis, root, PLP, PDX1The plant hormone auxin modulates many aspects of growth and development including cell division and cell expansion, leaf initiation, root development, embryo and fruit development, pattern formation, tropism, apical dominance and vascular tissue differentiation.^1–3 Indole-3-acetic acid (IAA) is the major naturally occurring auxin. IAA can be synthesized in cotyledons, leaves and roots, with young developing leaves having the highest capacity.^4,5Auxin most often acts in tissues or cells remote from its synthetic sites, and thus depends on non-polar phloem transport as well as a highly regulated intercellular polar transport system for its distribution.²The importance of local auxin biosynthesis in plant growth and development has been masked by observations that impaired long-distance auxin transport can result in severe growth or developmental defects.^3,6 Furthermore, a few mutants with reduced free IAA contents display phenotypes similar to those caused by impaired long-distance auxin transport. These phenotypes include defective vascular tissues and flower development, short primary roots and reduced apical dominance, or impaired shade avoidance and ethylene response.^7–15 Since these phenotypes most often could not be rescued by exogenous auxin application, it is difficult to attribute such defects to altered local auxin biosynthesis. By complementing double, triple or quadruple mutants of four Arabidopsis shoot-abundant auxin biosynthesis YUCCA genes with specific YUCCA promoters driven bacterial auxin biosynthesis iaaM gene, Cheng et al. provided unambiguous evidence that auxin biosynthesis is indispensable for embryo, flower and vascular tissue development.^8,13 Importantly, it is clear that auxin synthesized by YUCCAs is not functionally interchangeable among different organs, supporting the notion that auxin synthesized by YUCCAs mainly functions locally or in a short range.^6,8,13The central role of auxin in root meristem patterning and maintenance is well documented,^1,2,16 but the source of such IAA is still unclear. When ¹⁴C-labeled IAA was applied to the five-day-old pea apical bud, the radioactivity could be detected in lateral root primordia but not the apical region of primary roots.¹⁷ Moreover, removal of the shoot only slightly affected elongation of the primary root, and localized application of auxin polar transport inhibitor naphthylphthalamic acid (NPA) at the primary root tip exerted more profound inhibitory effect on root elongation than at any other site.¹⁸ These results suggest that auxin generated near the root tip may play a more important role in primary root growth than that transported from the shoot. In line with this notion, Arabidopsis roots have been shown to harbor multiple auxin biosynthesis sites including root tips and the region upward from the tip.⁴Many steps of tryptophan synthesis and its conversion to auxin involve transamination reactions, which require the vitamin B₆ pyridoxal 5-phosphate (PLP) as a cofactor. We previously reported that the Arabidopsis mutant pdx1 that is defective in vitamin B₆ biosynthesis displays dramatically reduced primary root growth with smaller meristematic zone and shorter mature cortical cells.¹⁹ In the current investigation, we found that the root tips of pdx1 have reduced cell division capability and reduced DR5::GUS activity, although the induction of this reporter gene by exogenous auxin was not changed. Reciprocal grafting indicates that the short-root phenotype of pdx1 is caused by a root local rather than shoot generated factor(s). Importantly, pdx1 suppresses yucca mutant, an auxin overproducer, in root hair proliferation although it fails to suppress the hypocotyl elongation phenotype.²⁰ Our work thus demonstrated that pdx1 has impaired root local auxin biosynthesis from tryptophan. To test whether the synthesis of tryptophan is also affected in pdx1 mutant, we planted pdx1 together with wild-type seeds on Murashige and Skoog (MS) medium supplemented with 5-mehtyl-anthranilate (5-MA), an analogue of the Trp biosynthetic intermediate anthranilate.²¹ Although pdx1 seedlings grew poorly under the control conditions, the growth of wild-type seedlings was more inhibited than that of the pdx1 seedlings on 10 µM 5-MA media (Fig. 1A–D). Compared with the elongated primary root on MS, wild-type seedlings showed very limited root growth on 5-MA (Fig. 1E). The relatively increased tolerance to 5-MA of pdx1 thus indicates that the pdx1 mutant may be defective in Trp biosynthesis, although amino acid analysis of the bulked seedlings did not find clear changes in Trp levels in the mutants (our unpublished data).Open in a separate windowFigure 1The pdx1 mutant seedlings are relatively less sensitive to toxic 5-methyl anthranilate (5-MA). (A and C) Five-day-old seedlings of the wild type (Col-0) (A) or pdx1 (C) on MS medium. (B and D) Five-day-old seedlings of the wild type (B) or pdx1 (D) on MS medium supplemented with 10 µM 5-MA. (E) Eight-day-old seedlings of the wild type or pdx1 on MS medium without or with 10 µM 5-MA supplement. Sterilized seeds were planted directly on the indicated medium and after two days of cold treatment, the plates were incubated under continuous light at 22–24°C before taking pictures.We reported that PDX1 is required for tolerance to oxidative stresses in Arabidopsis.¹⁹ Interestingly, redox homeostasis appears to play a critical role in Arabidopsis root development. The glutathione-deficient mutant root meristemless1 (rml1) and the vitamin C-deficient mutant vitamin C1 (vtc1) both have similar stunted roots.^22,23 Nonetheless, pdx1 is not rescued by either glutathione or vitamin C¹⁹ suggesting that the pdx1 short-root phenotype may not be resulted from a general reduction of antioxidative capacity. Interestingly, ascorbate oxidase is found to be highly expressed in the maize root quiescent center.²⁴ This enzyme can oxidatively decarboxylate auxin in vitro, suggesting that the quiescent center may be a site for metabolizing auxin to control its homeostasis.²⁵ It is therefore likely that the reduced auxin level in pdx1 root tips could be partially caused by increased auxin catabolism resulted from reduced vitamin B₆ level. We thus conducted experiments to test this possibility. A quiescent center-specific promoter WOX5 driven bacterial auxin biosynthetic gene iaaH²⁶ was introduced into pdx1 mutant. The transgenic seeds were planted on media supplemented with different concentrations of indoleacetamide (IAM), the substrate of iaaH protein. Although promotion of lateral root growth was observed at higher IAM concentrations, which indicates increased tryptophan-independent auxin production from the transgene, no change in root elongation was observed between pdx1 with or without the WOX5::iaaH transgene at any concentration of IAM tested (data not shown), suggesting that the pdx1 short-root phenotype may not be due to increased auxin catabolism.Taken together, in addition to auxin transport; temporally, spatially or developmentally coordinated local auxin biosynthesis defines the plant growth and its response to environmental changes.^8,14,15     

Auxin distribution and lenticel formation in determinate nodule of Lotus japonicus

Legumes can establish a symbiosis with rhizobia and form root nodules that function as an apparatus for nitrogen fixation. Nodule development is regulated by several phytohormones including auxin. Although accumulation of auxin is necessary to initiate the nodulation of indeterminate nodules, the functions of auxin on the nodulation of determinate nodules have been less characterized. In this study, the functions of auxin in nodule development in Lotus japonicus have been demonstrated using an auxin responsive promoter and auxin inhibitors. We found that the lenticel formation on the nodule surface was sensitive to the auxin defect. Further analysis indicated that failure in the development of the vascular bundle of the determinate nodule, which was regulated by auxin, was the cause of the disappearance of lenticels.Key words: auxin, lenticel, Lotus japonicus, nodulation, symbiotic nitrogen fixationLegumes (Fabaceae) constitute the third largest plant family with around 700 genera and 20,000 species.¹ Legume plants form root nodules through symbiosis with a soil microbe called rhizobia. This plant-microbe symbiosis in nodules mediates an harmonized exchange of chemical signals between host plants and rhizobia.² Nodules are biologically divided into two different groups, i.e., indeterminate nodules and determinate nodules. Indeterminate nodules, represented by Trifolium repens (white clover) and Medicago truncatula, are initiated from the inner cortex to form a persistent nodule meristem, which allows continuous growth, and leads to the formation of elongated nodules, whereas in determinate legumes, nodules are mostly developed from outer cortical cells and form spherical nodules.³Auxin is one of the most important regulators for nodule development. Since the possible involvement of auxin in nodule formation was first reported by Thimann,⁴ auxin distribution during nodulation has been studied in particular with indeterminate nodules.⁵ However, little is known about auxin involvement in determinate nodule formation. To evaluate auxin functions in the determinate nodulation of legume plants, we performed an auxin-responsive promoter analysis in detail. Using GH3:GUS transformed Lotus japonicus (a kind gift from Dr. Herman P. Spaink, Leiden State University, Netherlands),⁶ we detected auxin signals throughout the nodulation process, e.g., at the basal and front part of the nodule primordia, circumjacent to the infection zone of the young developing nodules (Fig. 1), and at the nodule vascular bundle in mature nodules. We also investigated the effect of several auxin inhibitors, including newly synthesized auxin antagonist PEO-IAA (kindly provided by Dr. Hayashi, Okayama University of Science, Japan),⁷ on the nodulation of L. japonicus, and revealed that auxin was required for forming a nodule vascular bundle and lenticels (Fig. 2).⁸Open in a separate windowFigure 1GH3:GUS expression in determinate nodule at 6 dpi. (A) GUS staining was observed in the central cylinder of the root vascular bundle and in the nodule. (B) Cross section of (A). GUS expression was observed around the infection zone of the nodule. Bars = 100 µm.Open in a separate windowFigure 2The effect of auxin inhibitor on nodule surface. (A) Typical mature nodule of L. japonicus at 21 dpi. Lenticels are pointed out by yellow arrowheads. (B) The treatment of auxin inhibitor (NPA 100 µM) inhibited lenticel formation on the nodule surface. Bars = 500 µm.In indeterminate legumes, auxin is accumulated at the site of rhizobia inoculation.⁹ This is caused by the inhibition of polar auxin transport by accumulation of flavonoids around the infection site, which are known as regulators of auxin transport. When flavonoid biosynthesis is reduced by the gene silencing of chalcone synthase, which catalyzes the first step of flavonoid synthesis, M. truncatula was unable to inhibit polar auxin transport and resulted in reduced nodule number.^10,11 A similar phenotype was observed when the auxin transporter gene was silenced.¹² In addition, treatment of polar auxin transport inhibitors such as NPA and TIBA induce pseudonodule formation,⁹ suggesting that auxin accumulation is required for nodulation of indeterminate legumes. In contrast, the treatment of polar auxin transport inhibitors in determinate nodules did not induce a nodule-like structure, suggesting a different function of auxin between indeterminate and determinate nodules. It is, however, of interest to investigate the involvement of flavonoids in determinate nodule formation, because several genes in the flavonoid biosynthesis pathway are upregulated at 2 dpi (days post inoculation) in L. japonicus.¹³Lenticels regulate gas permeability of nodules.¹⁴ Under low oxygen or water-logged conditions, they develop more extensively, whereas they collapse, or develop very little during insufficient water conditions, or under high oxygen pressure.^14,15 Because lenticel development on the nodule surface is accompanied with the nodule vascular bundle, growth regulators supplied from the vascular system likely facilitate lenticel development.¹⁵ Our data suggests that auxin is necessary to form the nodule vascular bundle, and in fact, auxin itself is one of the candidates of growth substances that control lenticel formation. It is necessary to analyze mutants, which lack in lenticel formation, but can form a nodule vascular bundle, for clarification of further mechanisms of lenticel development.     

Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway

The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.Key words: strigolactone, auxin, ethylene, root, root hair, lateral rootStrigolactones (SLs) are newly identified phytohormones that act as long-distance shoot-branching inhibitors (reviewed in ref. ¹). In Arabidopsis, SLs have been shown to be regulators of root development and architecture, by modulating primary root elongation and lateral root formation.^2,3 In addition, they were shown to have a positive effect on root-hair (RH) elongation.² All of these effects are mediated via the MAX2 F-box.^2,3In addition to SLs, two other plant hormones, auxin and ethylene, have been shown to affect root development, including lateral root formation and RH elongation.^4–6 Since all three phytohormones (SLs, auxin and ethylene) were shown to have a positive effect on RH elongation, we examined the epistatic relations between them by examining RH length.⁷ Our results led to the conclusion that SLs and ethylene are in the same pathway regulating RH elongation, where ethylene may be epistatic to SLs.⁷ Moreover, auxin signaling was shown to be needed to some extent for the RH response to SLs: the auxin-insensitive mutant tir1-1,⁸ was less sensitive to SLs than the wild type under low SL concentrations.⁷On the one hand, ethylene has been shown to induce the auxin response,^9–12 auxin synthesis in the root apex,^11,12 and acropetal and basipetal auxin transport in the root.^4,13 On the other, ethylene has been shown to be epistatic to SLs in the SL-induced RH-elongation response.⁷ Therefore, it might be that at least for RH elongation, SLs are in direct cross talk with ethylene, whereas the cross talk between SL and auxin pathways may converge through that of ethylene.⁷ The reduced response to SLs in tir1-1 may be derived from its reduced ethylene sensitivity;^7,14 this is in line with the notion of the ethylene pathway being a mediator in the cross talk between the SL and auxin pathways.The suggested ethylene-mediated convergence of auxin and SLs may be extended also to lateral root formation, and may involve regulation of auxin transport. In the root, SLs have been suggested to affect auxin efflux,^3,15 whereas ethylene has been shown to have a positive effect on auxin transport.^4,13 Hence, it might be that in the root, the SLs'' effect on auxin flux is mediated, at least in part, via the ethylene pathway. Ethylene''s ability to increase auxin transport in roots was associated with its negative effect on lateral root formation: ethylene was suggested to enhance polar IAA transport, leading to alterations in the quantity of auxin that unloads into the tissues to drive lateral root formation.⁴ Under conditions of sufficient phosphate, SL''s effect was similar to that of ethylene: SLs reduced the appearance of lateral roots; this was explained by their ability to change auxin flux.³ Taken together, one possibility is that the SLs'' ability to affect auxin flux and thereby lateral root formation in the roots is mediated by induction of ethylene synthesis.To conclude, root development may be regulated by a network of auxin, SL and ethylene cross talk.⁷ The possibility that similar networks exist elsewhere in the SLs'' regulation of plant development, including shoot architecture, cannot be excluded.     

Involvement of nitrate reductase in auxin-induced NO synthesis

It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.¹ Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (N^G-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.^2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.^4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation.Key words: atnoa1, indole-3-butyric acid, nia1, nia2 double mutant, nitric oxideLateral roots are formed from root pericycle cells postembryonically which process is promoted by indole-acetic acid (IAA). It was recognized that IAA share common steps with NO in the signal transduction cascade towards the auxin induced adventitious and lateral root formation.^6–8 Previously it was suggested that besides IAA, indol-3-butyric (IBA) is a true endogenous auxin in Arabidopsis, which acts in adventious and lateral root development.^9,10 Our results showed that IBA induced LR initials emitted intensive NO fluorescence in Arabidopsis. This increased level of NO was present only in the LR initials in contrast to primary root (PR) sections where it remained at the control level.In plants NO can be produced by a number of enzyme systems and non-enzymatic ways. In roots, the most likely candidates of NO synthesis are NR enzymes (cytoplasmic and plasma membrane-bounded isoenzymes, cNR and PM-NR). Recently a new type of enzyme, the PM-bounded nitrite:NO reductase (Ni:NOR) was identified as a possible source of NO in roots.¹¹ Because of the several formation potentials of NO, the identification of its source in plant tissues under different conditions is complicated. Using diverse mutants proved to be a good opportunity to investigate the possible sources of NO. In our experiments wild-type (Col-1), Atnoa1 (nitric oxide synthase associated 1 deficient) and nia1, nia2 (NR deficient) seedlings were applied in order to determine the enzymatic source of NO induced by auxin. In roots of these plants, different NO levels were measured in their control state (i.e., without IBA treatment). The NO content in Atnoa1 roots was similar to that of wild-type, while nia1, nia2 showed lower NO fluorescence than the other groups of plants. This result suggests that NR activity is needed to NO synthesis in roots. Further on, it was demonstrated that IBA induced NO generation in both the wild type and Atnoa1 root primordia, but this induction failed in the NR-deficient mutant. This reveals that the NO accumulation in root primordia induced by auxin requires NR activity. These observations were evidenced also by biochemical manner. On the one part, we applied L-NMMA, which is a specific inhibitor of mammalian NOS, on the other part, the inhibitor of NR enzyme tungstate was used and we monitored NO fluorescence in wild-type roots. The NOS inhibitor displayed no effect on NO levels neither at control state nor during auxin treatment, while tungstate inhibited NO synthesis in lateral roots and primary roots of control plants. The effect of tungstate was similar in auxin-treated roots, since application of this NR enzyme inhibitor decreased NO levels in PRs and LRs (Fig. 1).Open in a separate windowFigure 1NO fluorescence in lateral roots (white columns) and primary roots (grey columns) of control, control + 1 mM tungstate, IBA and IBA + 1 mM tungstate-treated wild-type Arabidopsis thaliana. Vertical bars are standard errors.Some speculations can be made on these results. Although more efforts are needed to make the scene clear, now we can predict that auxin somehow may induce NR isoenzymes, which produce nitrite in root cells. From this point, two further scenarios are possible: as the result of accumulated nitrite, either the NO-producing activity of NR or Ni:NOR activity are promoted, hereby NO is generated from nitrite reduction. NO formed in these two possible ways modulates the expression of certain cell cycle regulatory genes contributing to division of pericycle cells in LR primordia, as was published in tomato.¹²Nowadays research in the “NO-world” of plants is running very actively. Nevertheless, lot of more work is needed to reveal all the unknown faces of this novel multipurpose signaling molecule.     

Irrepressible,truncated Auxin Response Factors: Natural roles and applications in dissecting auxin gene regulation pathways

The molecularly well-characterized auxin signal transduction pathway involves two evolutionarily conserved families interacting through their C-terminal domains III and IV: the Auxin Response Factors (ARFs) and their repressors the Aux/IAAs, to control auxin-responsive genes, among them genes involved in auxin transport.^1,2 We have developed a new genetic tool to study ARF function. Using MONOPTEROS (MP)/ARF5, we have generated a truncated version of MP (MPΔ),³ which has lost the target domains for repression by Aux/IAA proteins. Besides exploring genetic interactions between MP and Aux/IAAs, we used this construct to trace MP’s role in vascular patterning, a previously characterized auxin dependent process.^4,5 Here we summarize examples of naturally occurring truncated ARFs and summarize potential applications of truncated ARFs as analytical tools.     

Arabidopsis FAB1A/B is possibly involved in the recycling of auxin transporters

Fab1/PIKfyve produces Phosphatidylinositol-3,5-bisphosphate (PtdIns (3,5) P₂) from Phosphatidylinositol-3-phosphate (PtdIns 3-P), and is involved not only in vacuole/lysosome homeostasis, but also in transporting various proteins to the vacuole or recycling proteins on the plasma membrane (PM) through the use of endosomes in a variety of eukaryotic cells. We previously demonstrated that Arabidopsis FAB1A/B functions as PtdIns-3,5-kinase in both Arabidopsis and fission yeast and plays a key role in vacuolar acidification and endocytosis. Although the conditional FAB1A/B knockdown mutant revealed an auxin-resistant phenotype to a membrane-impermeable auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), the mutant did not exhibit this phenotype to a membrane-permeable artificial auxin, naphthalene 1-acetic acid (NAA). The difference in the sensitivities to 2,4-D and NAA is similar to those of the auxin-resistant mutants such as aux1. Taken together, these results suggest that impairment of the function of Arabidopsis FAB1A/B might cause a defect in the membrane recycling capabilities of the auxin transporters and inhibit proper auxin transport into the cells in Arabidopsis.Key words: auxin signaling, auxin transporter, recycling of plasma membrane proteinsPhosphatidylinositol-3,5-bisphosphate (PtdIns (3,5) P₂) exists on the external membrane of multi-vesicular bodies (MVBs) at very low levels in eukaryotic cells,^1,2 and plays key roles in endomembrane homeostasis including endocytosis, vacuole/lysosome formation and vacuolar acidification.^1,3 PtdIns (3,5) P₂ deficiency causes an enlarged vacuolar structure in yeast and mammalian cells.^4,5 FAB1 forms a protein complex with its regulatory molecules, and synthesizes PtdIns (3,5) P₂ from PtdIns 3P.^6–9 In Arabidopsis, there are four Fab1/PIKfyve orthologs (FAB1A, FAB1B, FAB1C and FAB1D) in the genome, and the double homozygous mutant of FAB1A and FAB1B exhibited the male gametophyte lethal phenotype.¹⁰ Previously, we reported that conditional loss-of-function and gain-of-function mutants of FAB1A/B impair endomembrane homeostasis and reveal various developmental phenotypes.¹¹ Interestingly, lateral root formation by exogenous auxin, which is known as a typical auxin-responsive phenotype, was largely impaired when FAB1A/B expression was conditionally downregulated or upregulated. From these results, we speculated that the defect in the endocytosis process in fab1a/b mutants might inhibit the precise recycling process of auxin transporters on the PM, thereby inhibiting proper auxin transport into the plant cells.¹¹ In this report, we tested this hypothesis to assess the sensitivity on auxin-dependent lateral root formation to a membrane permeable auxin, NAA, in the fab1a/b knockdown mutant.