Fusicoccin counteracts inhibitory effects of high temperature on auxin-induced growth and proton extrusion in maize coleoptile segments

Plant growth and development are tightly regulated by both plant growth substances and environmental factors such as temperature. Taking into account the above, it was reasonable to point out that indole-3-acetic acid (IAA), the most abundant type of auxin in plants, could be involved in temperature- dependent growth of plant cells. We have recently shown that growth of maize coleoptile segments in the presence of auxin (IAA) and fusicoccin (FC) shows the maximum value in the range 30–35°C and 35–40°C, respectively. Furthermore, simultaneous measurements of growth and external medium pH indicated that FC at stressful temperatures was not only much more active in the stimulation of growth, but was also more effective in acidifying the external medium than IAA. The aim of this addendum is to determine interrelations between the action of IAA and FC (applied together with IAA) on growth and medium pH of maize coleoptile segments incubated at high temperature (40°C), which was optimal for FC but not for IAA.Key words: auxin, fusicoccin, coleoptile segments, elongation growth, medium pHA well studied aspect of auxin action especially in maize coleoptile, is its effect on cell elongation, proton extrusion and membrane potential.^1–7 It is now generally agreed that indole-3-acetic acid (IAA), as the principal regulator of plant elongation growth, causes (i) acceleration of elongation growth as compared to endogenous growth, (ii) enhancement of proton extrusion as compared to auxin—free medium, and (iii) transient depolarization followed by a slow hyperpolarization of membrane potential. According to the “acid growth theory” of elongation growth,^8–11 auxin induced cell wall acidification provides favorable conditions for cell wall loosening, a requirement for cell elongation. At least in maize coleoptile segments, auxin induced cell wall acidification is mediated by increased activity and/or amount of the PM H⁺-ATPase.^11,12 In the case of fusicoccin, which mimics the effect of auxin in many respects,¹³ it was shown that FC-binding site arises from interaction of the 14-3-3 protein dimmer with the C-terminal autoinhibitory domain of the H⁺-ATPase and that FC stabilizes this complex.^14–18 It should be pointed out that in spite of abundant literature on the mechanism through which IAA or FC control growth of grass coleoptiles, little is know how these substances work at extreme temperatures. Over the past decade, the involvement of 14-3-3 proteins in plant stress responses has often been suggested.¹⁹ For example, work by Chelysheva et al.,²⁰ and Babakov et al.,²¹ demonstrated that under low temperature and high osmolarity conditions, 14-3-3 proteins interact with the C-terminal autoinhibitory domain of the PM H⁺-ATPase activating the proton pump that play a key role in stress responses in higher plants. We have recently shown²² that FC at 40°C induced maximal growth whereas growth observed at the same temperature in the presence of IAA was reduced by 33% compared to the maximal value at 30°C. It was also found²² that at 40°C the kinetics of the pH change differed significantly for both growth substances; the segments treated with IAA at 40°C were virtually not able to acidify the external medium, whereas FC at this temperature caused practically maximal acidification. In this addendum we have shown that application of FC together with IAA conteracted the inhibitory effect of high temperature (40°C) on IAA-induced growth and proton extrusion in maize coleoptile segments (Fig. 1). For example, the total IAA-induced elongation growth of coleoptile segments at 40°C was 1438.1 ± 134.5 µm cm⁻¹ (mean ± SE, n = 11) while elongation of 2747.4 ± 269.7 µm cm⁻¹ (mean ± SE, n = 11) was observed in IAA applied together with FC (Fig. 1A). The data in Figure 1B indicate that coleoptile segments incubated at 40°C (over 2 h), without growth substances (control) characteristically changed the pH of the medium: usually within the first 30–45 min an increase of pH (by ca. 0.5 pH unit) was observed, followed by a slow decrease of pH. When IAA or FC was added (after 2 h of segment''s incubation in control medium), an additional decrease of pH was observed. As can be seen in Figure 1B, FC added at 40°C was much more effective in acidification of the medium, as compared to IAA. For FC, 5h after its addition, the pH of the incubation medium dropped to pH 4.2, whereas for IAA the pH was only 5.4. However, addition of IAA together with FC at 40°C dropped medium pH approximately to the same value as was observed in the presence of FC only.Open in a separate windowFigure 1Effect of high temperature (40°C) on growth (A) and medium pH (B) of maize coleoptile segments incubated in the presence of IAA (10 µM) and FC (1 µM). The growth of a stack of 21 segments, expressed as elongation (µm cm⁻¹), was measured simultaneously with medium pH at 40°C. After preincubation (over 2 h) of the coleoptile segments in control medium, IAA and FC was added (arrow). Values are means of 11 independent experiments. Bars indicate ± SE. In the case of medium pH SE did not exceed 8%.In conclusion, the results presented in this addendum provide further evidence that FC on the receptor level is much more effective than IAA.     

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     

Thigmomorphogenesis in Solanum lycopersicum: Morphological and biochemical responses in stem after mechanical stimulation

The activation of the phenylpropanoid pathway in plants by environmental stimuli is one of the most universal biochemical stress responses known. In tomato plant, rubbing applied to a young internode inhibit elongation of the rubbed internode and his neighboring one. These morphological changes were correlated with an increase in lignification enzyme activities, phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidases (POD), 24 hours after rubbing of the forth internode. Furthermore, a decrease in indole-3-acetic acid (IAA) content was detected in the rubbed internode and the upper one. Taken together, our results suggest that decrease in rubbed internode length is a consequence of IAA oxidation, increases in enzyme activities (PAL, CAD and POD), and cell wall rigidification associated with induction of lignification process.Key words: Mechanical stimulation, PAL, CAD, POD, IAAIn their environment, plants are constantly submitted to several stimuli such as wind, rain and wounding. The growth response of plants to such stimuli was termed thigmomorphogenesis and was observed in a wide range of plants.^1–3 The most common thigmomorphogenetic response is a retardation of tissue elongation accompanied by an increase in thickness.⁴ The plant response to mechanical perturbation is mainly restricted to the young developing internode, since no influence can be detected when the internode has reached its final length.^5,6 These plant growth modifications, which characterize thigmomorphogenesis, are related to biochemical events associated with lignification process⁷ and ethylene production.^8,9In tomato plant the length of internodes 4 (N4) and 5 (N5) was measured 14 days after rubbing of the fourth internode. Results reported in Figure 1 show that rubbing led to a significant reduction of elongation of the stressed internode (N4) (decrease of N4 length from 4.3 cm in the control plant to 2.9 in the rubbed one). This effect was not limited to the rubbed area but affected also the elongation of the neighboring internodes (N5) that were shorter in rubbed plants than in control ones.Open in a separate windowFigure 1Internode lengths of control and rubbed plants measured 14 day after mechanical stress applied to the fourth internode. Standard errors are indicated by vertical bars.Results reported in Figure 2 show an increase in PAL activity in both internodes N4 and N5, 24 hours after mechanical stress application as compared with corresponding controls. CAD activity was also investigated in N4 and N5, 24 h after rubbing of the fourth internode. Results presented in Figure 3 show that mechanical stress application induces a strong increase of CAD activity in the rubbed internode N4 (5.3 nkatal μg-1 protein) with an approximately two-fold increase when compared to control tomato internodes (2.3 nkatal μg-1 protein). Further, CAD activity in N5 was also increased in the rubbed internode (5.538 nkatal μg-1 protein) as compared with the control one (3.256 nkatal μg-1 protein).Open in a separate windowFigure 2PAL activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Open in a separate windowFigure 3CAD activity of internode 4, and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.Syringaldazine (S-POD) and gaïacol (G-POD) peroxidase activities were measured in tomato N4 and N5. Results reported in Figure 4 show an increase in soluble peroxidase activity with both substrates in the rubbed internode N4 as compared with control plant. Enhancement in peroxidase activities in N4 was more pronounced with gaïacol (80.7 U) as an electron donor than syringaldazine (33.8 U). Similar results were observed in internode 5 as compared with control one (Fig. 4).Open in a separate windowFigure 4(A) Syringaldazine-POD (Syr-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars. (B) Gaiacol-POD (G-POD) activity of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.IAA was quantified in control and rubbed plant internodes 24 h after rubbing of the fourth internode. Results reported in figure 5 show that in control sample and as expected, the content of IAA was found to be higher in the younger internode (N5) as compared to the older one (N4). Rubbing led to a significant decrease in IAA levels in N4 (5.06 nmol g⁻¹ MF⁻¹) as compared with corresponding controls (7.27 nmol g⁻¹ MF⁻¹). Similar results were observed in internode 5, where IAA content was reduced from 16.52 nmol g⁻¹ MF⁻¹ in control internode to 12.35 nmol g⁻¹ MF⁻¹ in the rubbed internode (Fig. 5).Open in a separate windowFigure 5IAA Level of internode 4 and 5 in control and rubbed plants 24 h after rubbing of the fourth internode. Standard errors are indicated by vertical bars.The results reported here establish an evident correlation between growth limitation of the rubbed internode and their degree of lignification, the increase in lignification enzymes activities and auxin degradation after mechanical stress application.Auxin seems to be involved in thigmomorphogenesis.¹⁰ It was proposed that MIS (Mechanically-induced stress) has opposite effects on auxin levels in the two species studied to date, Phaseolus vulgaris¹⁰ and Bryonia dioica.^11,12 Auxin level as measured by bioassay, increased in Phaseolus vulgaris following rubbing of the stem.¹⁰ It was proposed that a build up of auxin may result from the reduced polar transport of IAA at the rubbed internode, causing a build up of IAA in the stem tissue. Exogenous IAA did not reverse the MIS inhibition of growth in Phaseolus vulgaris and high levels of IAA retarded growth in non-stressed plants.¹⁰ Thus, retardation of extension growth in Phaseolus vulgaris may have been caused by high levels of endogenous auxin and the increase in stem diameter by increased ethylene production.⁴ However, ethylene increases radial growth only if auxin is present.¹³Boyer¹¹ reported a decrease in auxinlike activity in Bryonia dioica following MIS and this was confirmed in the same species by Hofinger et al.¹² who reported a decrease in IAA using gas chromatography-mass spectrometry. Auxin catabolism was accompanied with changes in both soluble and ionically bound cell wall basic peroxidases¹⁴ and the appearance of an additional peroxidase. This can suggest that in Bryonia, auxin catabolism is hastened by mechanical stimulated peroxidase. In addition, Boyer et al.¹⁵ reported that lithium pre-treatment prevents both thigmomorphogenesis and appearance of specific cathodic isoperoxidase in Bryonia plants subjected to MIS. This is give further credence to the possibility that the peroxidase-auxin system is involved in Bryonia thigmomorphogenesis. In addition, ethylene increases peroxidase activity which reduces the auxin content in the tissue to a level low enough not to support normal growth. We have evidence that decrease of auxin level contribute to mechanism leading to tomato internode inhibition subjected to mechanical stress.Growth inhibition has been suggested to be the result of tissues lignification.⁶ As the initial enzyme in the monolignol biosynthesis pathway, PAL has a direct influence on lignin accumulation.¹⁶ The characteristics of lignin differ among cell wall tissues and plant organs.¹⁷ It comprises polyphenolic polymers derived from the oxidative polymerization of different monolignols, including p-coumaryl, coniferyl and sinapyl alcohols via a side pathway of phenylalanine metabolism leading to lignin synthesis.¹⁸ The increase in lignin content in the rubbed tomato internode could be a response mechanism to mechanical damage caused by rubbing.³ It is known that plants create a natural barrier that includes lignin and suberin synthesis, components directly linked to support systems.^19,20The increase in lignin content of rubbed tomato internode³ is paralleled by a rise in CAD activity and whilst such direct proportionality between CAD activity and lignin accumulation does not always agree with the results in the literature, it clearly is responding in ways similar to those of the other enzymes in the pathway.²¹Mechanical stress-induced membrane depolarization would generate different species of free radicals and peroxides, which in turn initiate lipid peroxidation.²² The degradation of cell membranes is suggested to bring about rapid changes in ionic flux, especially release of K⁺ which would result in an enhanced endogenous Ca/K ratio and in leakage of solutes, among them electron donors such as ascorbic acid and phenolic substances. The increased intracellular relative calcium level activated secretion of basic peroxidases²³ into the free space where, in association with the electron donors and may be with the circulating IAA, they eliminate the peroxides, and facilitated binding of basic peroxidases to membrane structures allowing a role as 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidases. The resulting IAA and ACC oxidase-mediated changes in ethylene production²⁴ would further induce (this time through the protein synthesis machinery) an increase in activity of phenylalanine ammonia-lyase and peroxidases. The resulting lignification and cell wall rigidification determines the growth response of tomato internode to the mechanical stress.     

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.     

Auxin acts independently of DELLA proteins in regulating gibberellin levels

Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA₁, play critical roles in the control of elongation,^1–3 along with environmental and endogenous factors, including other hormones such as the brassinosteroids.^4,5 The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism,⁶ since auxin upregulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and downregulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA₁.⁷ In our recent paper,¹ we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action,^8,9 since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA₁ synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level.¹⁰ However, our recent results,¹ in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined.Key words: auxin, gibberellins, DELLA proteins, interactions, elongation     

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.     

Growing in darkness: The etiolated lupin hypocotyls

Epigeal germination of a dicot, like lupin (Lupinus albus L.), produces a seedling with a characteristic hypocotyl, which grows in darkness showing a steep growth gradient with an elongation zone just below the apex. The role of phytohormones, such as auxin and ethylene, in etiolated hypocotyl growth has been the object of our research for some time. The recent cloning and expression of three genes of influx and efflux carriers for polar auxin transport (LaAUX1, LaPIN1 and LaPIN3) reinforces a previous model proposed to explain the accumulation of auxin in the upper growth zone of the hypocotyl.Key words: auxin carriers, auxin transport gradient, etiolated hypocotyl growth, Lupinus albusMost plants show a typical axial polar and branched (dendritic) morphology to compensate for their immobility by optimally exploiting the resources available in a limited environment.From Julius von Sachs¹ to Tsvi Sachs² many plant physiologists sought to explain how the axis is maintained and what type of signals are interchanged between poles. It was demonstrated that auxins were the determining factors in maintaining the polarity in shoots and roots and a reductionistic approach leads to conclude that such polarity had to be established at the cellular level. A chemiosmotic theory was then proposed, which implied an asymmetric distribution of efflux carriers at the bottom of a cell, linked to pH gradients to maintain different undissociated/dissociated forms of auxin separated between apoplast and symplast spaces.³In recent years, the use of Arabidopsis thaliana as a plant model has given additional support to the hypothesis that polar auxin transport is restricted to certain cells and mediated by influx (AUX1 and LAX1–4 proteins) and efflux carriers (PIN1–8 proteins).^4–6 Currently, we have a good idea of the topology of Arabidopsis carrier distribution, especially in roots.^4,5 Additional (MDR/PGP)⁷ or parallel (TRH1)⁸ components of the transport system are now emerging.However, while accepting the enormous advances and contributions to plant science provided by the use of Arabidopsis thaliana, we remain true (loyal) to the particular model adopted by the Department of Plant Biology, University of Murcia (Spain) in the 1970''s: the hypocotyl of lupin seedlings cultivated in darkness. In such conditions, the organ grows heterotrophically and longer than in light.The cotyledons and meristem at the top supply nutrients and hormones in a basipetal direction.The hypocotyl is a cylindrical column, with a radial symmetry that clearly shows differentiated tissues: epidermis, cortex, vascular cylinder and pith. Its size allows surgical separation of the tissues using suitable glass capillaries.At the beginning lupin was chosen because it had higher IAA-oxidase activity than pea, bean, oat or barley seedlings. At the time, it was thought that growth was mainly controlled through auxin catabolism (a fruitful line involving peroxidases was developed later). However, the etiolated hypocotyl was soon adopted preferentially by our group because of its qualities as a model for studying the relationship between hormone levels (auxin and ethylene) and growth. Our Portuguese colleagues have also used lupin as a model with successful results.⁹Bellow, we detail the landmarks of our research to date. Hypocotyl growth shows a characteristic pattern. Unlike plants grown in the light, in which all the cells along the hypocotyl elongate continuously throughout the growth period,^10,11 there is a steep growth gradient in the dark with an elongation zone just below the apex¹² (see Fig. 1 for details). This cell growth pattern in etiolated hypocotyls was described in lupin and then in Arabidopsis.¹¹ In this pattern, it is important to note that there is compensation along the organ between the cell diameter and the cell wall thickness. Once the cell growth pattern was known, we investigated its relation with the level of two phytohormones, auxin and ethylene, which might participate in the growth regulation. Special attention was paid to the distribution of endogenous IAA and its relation with growth. The results showed good correlation between the auxin levels and the cell size.^13,14 Auxin from the apex appears to be responsible for hypocotyl growth, since decapitation of seedlings strongly reduced growth, which was restored after the application of exogenous IAA to the cut surface.¹⁵ In light of the fact that growth depended on auxin from the apex, we investigated the nature of the auxin transport and demonstrated that this transport is polarized and sensitive to inhibition by specific inhibitors of polar auxin transport (PAT) such as 2,3,5-triiodobenzoic acid and 1-N-naphthylphtalamic acid (NPA).^16,17 Basipetal PAT mainly occurred in the stele,¹⁵ while cells in the epidermis and outer cortex are the limiting factor in auxin-induced shoot growth.^18–20 The finding that during PAT auxin can move laterally from transporting cells in the stele to the outer tissues of the elongation zone¹⁵ could explain the apparent conflict between the localization of PAT and the auxin target cells for elongation. In fact, epidermal cells acted as a sink for lateral auxin movement (LAM).¹⁷Open in a separate windowFigure 1Distribution of growth and cell size along the hypocotyl in etiolated lupin seedlings. At 3 d, hypocotyls were marked with ink, delimiting four 5-mm long zones including the apical, middle and basal zones. The hypocotyl growth ceased at day 12 and almost no growth was observed in the basal zone after day 3. From 3 to 6 d the growth was localized between the apical and basal zones, while most growth occurring from 6 to 12 d was localized in apical and middle zones. The cell size represents the cell length and cell diameter (the cell wall excluded) and corresponds to the second cell layer of cortex near the vascular cylinder. Similar results were obtained in cells from epidermis and pith. In each zone the cell length increased and the cell diameter showed little change during hypocotyl ageing. The final size at the end of the growth period varied along the hypocotyl, the cells becoming shorter and broader from the apical to the basal zones. In spite of the fact that cell diameter increased basipetally, no significant variation in hypocotyl diameter was found along the organ during the growth period. A morphometric study revealed that cell wall thickness in the apical cells was twice that in the basal cells at the end of the growth period i.e., the thinner apical cells had thicker cell walls, which may help explain the consistency of hypocotyl diameter along the organ.If PAT provides the auxin for growth and elongating growth is restricted to the apical region in etiolated hypocotyls, the question is: how does auxin accumulate in the elongation region?In a former study, we proposed that variations in auxin transport along actively growing lupin hypocotyl could produce such accumulation.²¹ Recently we extensively studied the variation of PAT along the lupin hypocotyls in seedlings of different ages, finding that certain parameters of PAT, such as transport intensity, polarity (basipetal vs acropetal) and sensitivity to NPA inhibition, showed a good correlation with the distribution of growth along the hypocotyl and its variation with ageing.²² These results suggest that a basipetally decreasing gradient in PAT along the hypocotyl may be responsible for the auxin distribution pattern controlling growth, since the existence of such a PAT gradient might generate the so-called barrier effect, which could produce an auxin gradient along the hypocotyl, the auxin content being higher in the apical elongation zone. To investigate whether these PAT variations can be explained in terms of auxin carrier distribution, we isolated three genes coding for auxin influx (LaAUX1) and efflux (LaPIN1 and LaPIN3) carriers, and studied their expression in different tissues along the hypocotyl at different ages.²³ The expression of LaAUX1 and LaPIN3 occurred both in the stele and in the outer tissues, while the expression of LaPIN1 was restricted to the stele and showed a basipetally decreasing gradient along the hypocotyl. The decisive role ascribed to PIN1 in polar auxin transport due to its localization in the basal end of transporting cells,²⁴ and the existence of such a gradient in the expression of LaPIN1 support the hypothesis of a barrier effect (generated by decreasing auxin transport) previously proposed as being responsible for the auxin gradient which controls the growth pattern in etiolated lupin hypocotyls.The acid-growth theory of auxin action was also tested, observing that the elongation growth of etiolated hypocotyl segments of lupin was stimulated by acid pH and IAA. Both factors stimulated growth in a more than additive way, suggesting a synergistic action between them.²⁵ The recent finding of a soluble auxin receptor (intracellular) reinforces the interest of the above study (which has remained a “sleeping beauty”) because pH affects IAA uptake.There are still several questions that must be answered before we can fully understand the growth pattern exhibited by etiolated lupin hypocotyls. Thus, as regards the cause of the PAT gradient, other factors besides the LaPIN1 gradient must be considered. For example, auxin carriers such as some phosphoglycoproteins (PGP), are also expressed differentially along the Arabidopsis hypocotyl and specific PIN-PGP pairings influence PAT by modulating the rates of cellular auxin movement.⁷ The pathway (symplast or apoplast) and mechanism of LAM remains unknown. Although alternative mechanisms have been proposed,²⁶ a previous study in lupin¹⁵ suggested that LAM is a diffusive process and that the IAA metabolism observed in the outer tissues might generate the radial gradient of auxin necessary for the maintenance of its lateral flow. It is thought that this metabolism of IAA occurs once the hormonal action is completed.^25,27 Although NPA does not inhibit LAM, the involvement of auxin efflux carriers cannot be discarded. In fact, the role of PIN carriers in lateral auxin transport towards and from the stele has been described in the root.²⁸ Other phytohormones besides auxin can modulate hypocotyl growth. Thus, the ethylene production rate, the 1-aminocyclopropane-1-carboxylic acid (ACC) content and the ACC oxidase activity decreased along the hypocotyl during the hypocotyl growth period.²⁹ Sensitivity to exogenous ethylene varied during growth, the young apical region being less sensitive than the older basal region.³⁰ Ethylene modified the cell growth pattern in the different tissues.³¹ The ethylene-induced lupin hypocotyl thickening was irreversible and mainly due to an increase in cell diameter. However, the inhibition of hypocotyl elongation produced by ethylene was reversible and involved irreversible inhibition of cell division and, paradoxically, stimulation of cell elongation to produce cells longer than those of the control.³²Studies in Arabidopsis showed that the hypocotyl growth in both light- and dark-grown plants is a process driven by cross-talk between multiple hormones. Interactions between auxins, ethylene, gibberellins and brassinosteroids have been described.^33,34 We think that the etiolated lupin hypocotyl remains a suitable model for confirming some of these results and for opening up new approaches in phytohormone research.     

Multiple signaling pathways control nitrogen-mediated root elongation in maize

Response of root system architecture to nutrient availability is an essential way for plants to adapt to soil environments. Nitrogen can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Low soil N stimulates root elongation in maize. Recent evidence suggests that plant hormones auxin and cytokinin, as well as NO signaling pathway, are involved in the regulation of root elongation by low nitrogen nutrition.Key words: nitrogen, root growth, auxin, cytokinin, NONitrogen acquisition is determined by N demand for plant growth. At low N stress, N demand for maximum plant growth rate is not matched by plant N uptake. To acquire adequate N, plants may increase root length density to explore a larger soil volume and/or increase N uptake activity. High root density is also an important root trait for competition with soil microorganisms.¹ Since nitrate is a highly mobile, non-adsorbing ion, theoretic analysis predicts that its uptake is not limited by transport through soil, and a small root system is sufficient for nitrate acquisition.^2–4 In field conditions, however, genotypes that are efficient in N acquisition generally had a larger root system and higher root length density.^5,6 Under conditions of insufficient N supply, N mass flow to roots may not be adequate to meet the N demand for plant growth. Even in N-sufficient soils, various soil constraints (low water content, etc) may reduce the N mass flow rate. In these cases, large root size and high density will be very important for the utilization of the spatially distributed N, especially newly mineralized N, and the competition for organic N with soil microorganisms.^7,8The development of lateral roots in Arabidopsis in response to nitrate supply has been widely studied.⁹ Less attention has been paid to primary root growth in response to N, possibly because root elongtion is insensitive to increased N supply in Arabidopsis.^10,11 In maize, however, root elongation was sigificantly promoted by suboptimal N supply, and inhibited by overdose supply of N (Fig. 1).^12,13 Until recently less is known about the underlying physiological mechansms. It is well documented that cytokinin is a root-to-shoot signal communicating N availability in addition to nitrate itself.¹⁴ Exogenous cytokinin application suppresses the elongation of primary roots.¹⁵ Recent work in Arabidopsis overexpressing cytokinin synthase (IPT) demonstrate that long-term CK overproduction inhibited primary root elongation by reducing quantitative parameters of primary root meristem.¹⁶ By comparing two maize inbred lines whose root elongation had a differential response to low N stress, it was found that the change of cytokinin content in roots was closely related to low-N induced root elongation.¹³ In the N-sensitive genotype 478, cytokinin (Zeatin + Zeatin riboside) content was significantly lower at low N condition. While in N-insensitive genotype Wu312, cytokinin content was hardly affected at various N supplies. Higher N supply shortened the distance from root apex to the first visible lateral roots, a phenomenen similar to that caused by exogenous cytokinins. Furthermore, exogenous cytokinin 6-benzylaminopurine (6-BA) completely reversed the stimulatory effect of low nitrate on root elongation. All the data suggests that the inhibitory effect of high concentration of nitrate on root elongation is, at least in part, mediated by increased cytokinin level in roots.Open in a separate windowFigure 1Root elongation is inhibited at high nitrate supply.Auxin regulates many cellular responses crucial for plant development. Auxin plays a key role in establishing and elaborating patterns in root meristems.^17,18 Root elongation of Arabidopsis is enhanced by exogenous auxin at low concentrations, but is inhibited at high concentrations.¹⁹ In an earlier report, a high external nitrate supply (8 mM) did cause a 70% decrease in the auxin concentration of the root in soybean.²⁰ In maize, inhibition of root growth by high nitrate was found closely related to the reduction of IAA levels in roots and exogenous NAA and IAA restored primary root growth in high nitrate concentrations.²¹ Interesting, it was found that auxin concentrations in phloem exudates were reduced by a greater nitrate supply, suggesting that shoot-to-root auxin transport may be inhibited by high N supply. Considering the antagonism between auxin and cytokinin.²² it was possible that, by increasing the cytokinin level and decreasing the auxin level, high nitrate supply may have negative influences on root apex activity so that root apical dominance is weakened and, therefore, root elongation is suppressed and lateral roots grow closer to the root apex.Nitric oxide (NO) is emerging as an important messenger molecule associated with many biochemical and physiological processes in plants. The involvement of NO in IAA-induced adventitious root development has also been reported.²³ Given that nitrate is a substrate for NR-catalysed NO production, and root development and growth are closely related to NO, it is expected that NO may play a role in nitrate-dependent root growth. Surprisingly, endogenous levels of NO in the root apices of maize seedlings grown in high nitrate solution were much lower than those in apices grown in low nitrate. The nitrate-induced inhibition of root elongation in maize was markedly reversed by treatments of the roots with a NO donor (SNP) and IAA.²⁴ These data suggest that the arrest of root elongation by high levels of external nitrate concentrations may result from an alteration of endogenous NO levels in root apical cells. NR mediated NO production is unlikely to be involved in the nitrate-dependent NO production and root elongation because NR activity is lower at low N supply. A NO synthase (NOS) inhibitor reduced root elongation in maize plants grown in the low-nitrate medium, suggest that NOS activity may be inhibited in plants grown in high-nitrate solution, thus leading to a reduction of the endogenous NO levels.Taken together, high nitrogen supply increases cytokinin level, but decreases auxin and NO levels in roots of maize. Besides, it was well documented ethylene has a negative effect on root elongation of various plants.^25–27 Exogenous supply of cytokinin increase ethylene production (Stenlid 1982; Bertell et al., 1990). Recently, it was demonstrated in Arabidopsis that auxin transport from the root apex via the lateral root cap is required for ethylene-mediated inhibition of root growth.²⁸ Therefore, a complex multiple siganlling pathways may be involved in N-mediated root elongation (Fig. 2). Further study is required to understand how these pathways interact with each other to reduce root elongation in response to high nitrate supply.Open in a separate windowFigure 2A simplified model explaining nitogen-mediated root elongation in maize.     

Linking protein kinase CK2 and auxin transport

Studies performed in different organisms have highlighted the importance of protein kinase CK2 in cell growth and cell viability. However, the plant signaling pathways in which CK2 is involved are largely unknown. We have reported that a dominant-negative mutant of CK2 in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. We demonstrated that auxin transport is, indeed, impaired in these mutant plants, and that this correlates with misexpression and mislocalization of PIN efflux transporters and of PINOID. Our data establishes a link between CK2 activity and the regulation of auxin homeostasis in plants, strongly suggesting that CK2 might be required at multiple points of the pathways regulating auxin fluxes.Key words: protein kinase CK2, root development, auxin, PIN, PINOIDThe plant hormone auxin plays critical roles in plant growth and development.¹ The most abundant natural auxin is the indol-3-acetic acid (IAA), which is synthesized in young apical tissues and then transported to the growing zones of the stem and root. The major route for long distance IAA movement is via the vascular tissue, but, additionally, a slower transport via cell-to-cell (called polar transport) is critical to generate auxin gradients within tissues. Formation of correct auxin gradients is thought to be essential for many plant developmental processes.² In recent years, the IAA transporters have been identified, establishing the molecular basis to understand how auxin transport is regulated. In particular, the identification of the family of plasma-resident PIN proteins, the members of which function as IAA efflux carriers, and the knowledge of their polar localization in the plasma membrane (PM), contributed to generate models predicting the direction of IAA fluxes.^3,4The factors that govern PIN targeting to a particular membrane domain are still not understood. It is known that PIN proteins constitutively undergo cycles of exocytosis and endocytosis to and from the PM, using distinct sorting and recycling endosome trafficking pathways.^5–7 Phosphorylation/dephosphorylation by the Ser/Thr kinase PINOID (PID) and the protein phosphatase 2A, respectively, controls PIN proteins apical/basal localization at the PM, via the GNOM-mediated vesicle trafficking system.⁸ Interestingly, PID is a member of the plant AGC kinases, and, as it happens with its mammals AGC counterparts, is activated by a membrane-associated 3-phosphoinositide-dependent kinase (PDK1).⁹ Moreover, a functional similarity between PIN polar localization in response to auxin and glucose receptor (GLUT4) asymmetrical distribution in response to insulin, has been pointed out.¹⁰ In both cases, cargo proteins (GLUT4 and PIN, respectively) are transported from endosomal vesicles to PM and the process is mediated by PDK1-activated AGC kinases.Protein kinase CK2 is a Ser/Thr kinase evolutionary conserved in eukaryotes, which plays key roles in cell survival, cell division and other cellular processes. A loss-of-function mutant of CK2 in Arabidopsis, obtained by overexpression of a CK2α-inactive subunit, confirmed the essential role of this protein kinase for plant viability.¹¹ Moreover, CK2mut plants showed a dramatic decrease of lateral root formation, inhibition of root growth and overproliferation of root hairs. We have further demonstrated that auxin transport is impaired in this plants, which is concomitant with missexpression of most of the PM-resident PIN proteins, and of PID.¹² In addition, PIN proteins accumulated in endosomal vesicles and auxin gradients were disturbed, both in roots and shoots of CK2mut plants. In particular, root columella cells were depleted of auxin, although the maximum at the quiescent center was unchanged. Starch granule staining with lugol revealed that columella cells retained their fate, although their organization and/or cell shape were clearly affected (Fig. 1).Open in a separate windowFigure 1Lugol-stained starch granules in uninduced (−Dex) and Dex-induced (+Dex) CK2mut roots. In the central part of the figure, a sketch of the main morphogenetic characteristics of mutant roots (right plantlet) as compared to wild-type roots (left plantlet) is shown. Note the shorter roots, wavy phenotype, absence of lateral roots and overproliferation of root hairs in mutant plants.Our results strongly suggest that CK2 is a regulator of auxin-dependent responses, most likely by participating in the regulation of auxin transport. Strikingly, depletion of CK2 activity inhibits some auxin-dependent physiological responses whereas it enhances others. For instance, whereas shoot phototropism was completely absent, root gravitropism was enhanced.¹² Figure 2 shows a time-course of DR5rev::GFP-derived signal after changing the gravity vector, in mutant and control Arabidopsis roots. The progressive auxin translocation to the lower side of the root after gravistimulation is more rapid and sustained in mutant than in control roots, which is likely responsible for the enhanced response to gravity found in mutant roots. Based on these results, we postulate that CK2 might act at different points of the auxin-induced regulatory pathway. As far as is known, the core module that regulates auxin transport is constituted by the protein kinase PID and a protein of the NPH3-domain family. NPH3-containing proteins play important roles in phototropic and gravitropic responses, and regulate polarity and endocytosis of PIN proteins.¹³ As has been proposed by other authors, the participation of one AGC kinase and one NPH3-like protein upstream of an ARF factor might be a common theme in response to different stimulus that are signaled by auxin.¹⁴ We propose that one of the functions of CK2 is the regulation of the activity of core proteins (Fig. 3). Mammalian AGC kinases are well known substrates of CK2 and CK2-dependent phosphorylation is critical for a full display of their activity. The PID and the NPH3-containing protein sequences contain numerous acidic-based motifs that are predicted CK2 phosphorylation sites. Moreover, according to Arabidopsis phosphoproteome databases, several members of the NPH3-containing protein family are predicted to be phosphorylated.¹⁵ In addition, we do not discard the possibility that other proteins involved in PIN transport might also be regulated by CK2-dependent phosphorylation. Experiments are in progress in our laboratory to assess the regulatory role of CK2 in auxin transport.Open in a separate windowFigure 2Time course of auxin relocation during root gravitropic response, as visualized by DR5rev::GFP fluorescence. Root pictures were taken at the indicated times after changing the direction of the gravity vector. Translocation of auxin to the lower part of the root is more rapid in Dex-induced CK2mut plants. Arrows indicate asymmetrical DR5::GFP fluorescence.Open in a separate windowFigure 3Proposed model for the role of CK2 in regulating auxin transport. The core module that regulates auxin transport (shown here as a black box) is constituted by the protein kinase PID and a protein of the NPH3-domain family. PID regulates apical-basal targeting of PIN proteins, by phosphorylating conserved Ser residues present in PIN hydrophilic loops.¹⁶ On the other hand, the family of NPH3-containing proteins regulates polarity and endocytosis of PIN proteins.¹³ There is also a functional similarity between the intracellular transport of PIN proteins and that of the glucose receptor (GLUT4),¹⁰ two processes that are signaled by AGC kinases. We propose that CK2 might be a regulator of the activity of the core proteins, by phosphorylating either the AGC kinase and/or the NPH3-containing protein. Mammalian CK2 is a known regulator of the activity of AGC kinases and other proteins participating in signaling pathways, such as in the Wnt/β-catenin signaling pathway.¹⁷     

ARF-GTPase as a Molecular Switch for Polar Auxin Transport Mediated by Vesicle Trafficking in Root Development

Polar auxin transport (PAT), which is controlled precisely by both auxin efflux and influx facilitators and mediated by the cell trafficking system, modulates organogenesis, development and root gravitropism. ADP-ribosylation factor (ARF)-GTPase protein is catalyzed to switch to the GTP-bound type by a guanine nucleotide exchange factor (GEF) and promoted for hybridization to the GDP-bound type by a GTPase-activating protein (GAP). Previous studies showed that auxin efflux facilitators such as PIN1 are regulated by GNOM, an ARF-GEF, in Arabidopsis. In the November issue of The Plant Journal, we reported that the auxin influx facilitator AUX1 was regulated by ARF-GAP via the vesicle trafficking system.¹ In this addendum, we report that overexpression of OsAGAP leads to enhanced root gravitropism and propose a new model of PAT regulation: a loop mechanism between ARF-GAP and GEF mediated by vesicle trafficking to regulate PAT at influx and efflux facilitators, thus controlling root development in plants.Key Words: ADP-ribosylation factor (ARF), ARF-GAP, ARF-GEF, auxin, GNOM, polar transport of auxinPolar auxin transport (PAT) is a unique process in plants. It results in alteration of auxin level, which controls organogenesis and development and a series of physiological processes, such as vascular differentiation, apical dominance, and tropic growth.² Genetic and physiological studies identified that PAT depends on efflux facilitators such as PIN family proteins and influx facilitators such as AUX1 in Arabidopsis.Eight PIN family proteins, AtPIN1 to AtPIN8, exist in Arabidopsis. AtPIN1 is located at the basal side of the plasma membrane in vascular tissues but is weak in cortical tissues, which supports the hypothesis of chemical pervasion.³ AtPIN2 is localized at the apical side of epidermal cells and basally in cortical cells.^1,4 GNOM, an ARF GEF, modulates the localization of PIN1 and vesicle trafficking and affects root development.^5,6 The PIN auxin-efflux facilitator network controls root growth and patterning in Arabidopsis.⁴ As well, asymmetric localization of AUX1 occurs in the root cells of Arabidopsis plants,⁷ and overexpression of OsAGAP interferes with localization of AUX1.¹ Our data support that ARF-GAP mediates auxin influx and auxin-dependent root growth and patterning, which involves vesicle trafficking.¹ Here we show that OsAGAP overexpression leads to enhanced gravitropic response in transgenic rice plants. We propose a model whereby ARF GTPase is a molecular switch to control PAT and root growth and development.Overexpression of OsAGAP led to reduced growth in primary or adventitious roots of rice as compared with wild-type rice.¹ Gravitropism assay revealed transgenic rice overxpressing OsAGAP with a faster response to gravity than the wild type during 24-h treatment. However, 1-naphthyl acetic acid (NAA) treatment promoted the gravitropic response of the wild type, with no difference in response between the OsAGAP transgenic plants and the wild type plants (Fig. 1). The phenotype of enhanced gravitropic response in the transgenic plants was similar to that in the mutants atmdr1-100 and atmdr1-100/atpgp1-100 related to Arabidopsis ABC (ATP-binding cassette) transporter and defective in PAT.⁸ The physiological data, as well as data on localization of auxin transport facilitators, support ARF-GAP modulating PAT via regulating the location of the auxin influx facilitator AUX1.¹ So the alteration in gravitropic response in the OsAGAP transgenic plants was explained by a defect in PAT.Open in a separate windowFigure 1Gravitropism of OsAGAP overexpressing transgenic rice roots and response to 1-naphthyl acetic acid (NAA). (A) Gravitropism phenotype of wild type (WT) and OsAGAP overexpressing roots at 6 hr gravi-stimulation (top panel) and 0 hr as a treatment control (bottom panel). (B) Time course of gravitropic response in transgenic roots. (C and D) results correspond to those in (A and B), except for treatment with NAA (5 × 10⁻⁷ M).The polarity of auxin transport is controlled by the asymmetric distribution of auxin transport proteins, efflux facilitators and influx carriers. ARF GTPase is a key member in vesicle trafficking system and modulates cell polarity and PAT in plants. Thus, ARF-GDP or GTP bound with GEF or GAP determines the ARF function on auxin efflux facilitators (such as PIN1) or influx ones (such as AUX1).ARF1, targeting ROP2 and PIN2, affects epidermal cell polarity.⁹ GNOM is involved in the regulation of PIN1 asymmetric localization in cells and its related function in organogenesis and development.⁶ Although VAN3, an ARF-GAP in Arabidopsis, is located in a subpopulation of the trans-Golgi transport network (TGN), which is involved in leaf vascular network formation, it does not affect PAT.¹⁰ OsAGAP possesses an ARF GTPase-activating function in rice.¹¹ Specifically, our evidence supports that ARF-GAP bound with ARF-GTP modulates PAT and gravitropism via AUX1, mediated by vesicle trafficking, including the Golgi stack.¹Therefore, we propose a loop mechanism between ARF-GAP and GEF mediated by the vascular trafficking system in regulating PAT at influx and efflux facilitators, which controls root development and gravitropism in plants (Fig. 2). Here we emphasize that ARF-GEF catalyzes a conversion of ARF-bound GDP to GTP, which is necessary for the efficient delivery of the vesicle to the target membrane.¹² An opposite process of ARF-bound GDP to GTP is promoted by ARF-GTPase-activating protein via binding. A loop status of ARF-GTP and ARF-GDP bound with their appurtenances controls different auxin facilitators and regulates root development and gravitropism.Open in a separate windowFigure 2Model for ARF GTPase as a molecular switch for the polar auxin transport mediated by the vesicle traffic system.     

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.