Why have chloroplasts developed a unique motility system?

Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins'' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.Key words: actin, Arabidopsis, blue light, kinesin, myosin, organelle movement, phototropinOrganelle movement and positioning are pivotal aspects of the intracellular dynamics in most eukaryotes. Although plants are sessile organisms, their organelles are quickly repositioned in response to fluctuating environmental conditions and certain endogenous signals. By and large, plant organelle movements and positioning are dependent on actin filaments, although microtubules play certain accessory roles in organelle dynamics.^1,2 Actin inhibitors effectively retard the movements of mitochondria,^3–6 peroxisomes,^5,7–11 Golgi stacks,^12,13 endoplasmic reticulum (ER),^14,15 and nuclei.^16–18 These organelles are co-aligned and associated with actin filaments.^{5,7,8,10–12,15,18} Recent progress in this field started to reveal the molecular motility system responsible for the organelle transport in plants.¹⁹Chloroplast movement is among the most fascinating models of organelle movement in plants because it is precisely controlled by ambient light conditions.^20,21 Weak light induces chloroplast accumulation response so that chloroplasts can capture photosynthetic light efficiently (Fig. 1A). Strong light induces chloroplast avoidance response to escape from photodamage (Fig. 1B).²² The blue light-induced chloroplast movement is mediated by the blue light receptor phototropin (phot). In some cryptogam plants, the red light-induced chloroplast movement is regulated by a chimeric phytochrome/phototropin photoreceptor neochrome.^23–25 In a model plant Arabidopsis, phot1 and phot2 function redundantly to regulate the accumulation response,²⁶ whereas phot2 alone is essential for the avoidance response.^27,28 Several additional factors regulating chloroplast movement were identified by analyses of Arabidopsis mutants deficient in chloroplast photorelocation.^29–32 In particular, identification of CHUP1 (chloroplast unusual positioning 1) revealed the connection between chloroplasts and actin filaments at the molecular level.²⁹ CHUP1 is a chloroplast outer membrane protein capable of interacting with F-actin, G-actin and profilin in vitro.^29,33,34 The chup1 mutant plants are defective in both the chloroplast movement and chloroplast anchorage to the plasma membrane,^22,29,33 suggesting that CHUP1 plays an important role in linking chloroplasts to the plasma membrane through the actin filaments. However, how chloroplasts move using the actin filaments and whether chloroplast movement utilizes the actin-based motility system similar to other organelle movements remained to be determined.Open in a separate windowFigure 1Schematic distribution patterns of chloroplasts in a palisade cell under different light conditions, weak (A) and strong (B) lights. Shown as a side view of mid-part of the cell and a top view with three different levels (i.e., top, middle and bottom of the cell). The cell was irradiated from the leaf surface shown as arrows. Weak light induces chloroplast accumulation response (A) and strong light induces the avoidance response (B).Here, we review the recent findings pointing to existence of a novel actin-based mechanisms for chloroplast movement and discuss the differences between the mechanism responsible for movement of chloroplasts and other organelles.     

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.     

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.     

Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Loss of LORELEI function in the pistil delays initiation but does not affect embryo development in Arabidopsis thaliana

Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the pollen tube to the female gametophyte, followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte not only regulates these steps but also controls the subsequent initiation of seed development. Previously, we reported that loss of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the female reproductive tissues causes a delay in initiation of seed development. From these studies, however, it was unclear if embryos derived from fertilization of lre-5 gametophytes continued to lag behind wild-type during seed development. Additionally, it was not determined if the delay in initiation of seed development had any lingering effects during seed germination. Finally, it was not known if loss of LORELEI function affects seedling development given that LORELEI is expressed in eight-day-old seedlings. Here, we showed that despite a delay in initiation, lre-5/lre-5 embryos recover, becoming equivalent to the developing wild-type embryos beginning at 72 hours after pollination. Additionally, lre-5/lre-5 seed germination, and seedling and root development are indistinguishable from wild-type indicating that loss of LORELEI is tolerated, at least under standard growth conditions, in vegetative tissues.Key words: LORELEI, glycosylphosphatidylinositol (GPI)-anchored protein, embryogenesis, DD45, seed germination, primary and lateral root growth, seedling developmentDouble fertilization is unique to flowering plants. Upon female gametophyte reception of a pollen tube, the egg and central cells of the female gametophyte fuse with the two released sperm cells to form zygote and endosperm, respectively and initiate seed development.¹ The female gametophyte controls seed development by (1) repressing premature central cell or egg cell proliferation until double fertilization is completed,^1–3 (2) supplying factors that mediate early stages of embryo and endosperm development^1,4,5 and (3) regulating imprinting of genes required for seed development.^1,6The molecular mechanisms underlying female gametophyte control of early seed development are poorly understood. Although much progress has been made in identifying genes and mechanisms by which the female gametophyte represses premature central cell or egg cell proliferation until double fertilization is completed and regulates imprinting of genes required for seed development,^1,6 only a handful of female gametophyte-expressed genes that affect early embryo^7,8 and endosperm⁹ development after fertilization have been characterized. This is particularly important given that a large number of female gametophyte-expressed genes likely regulate early seed development.⁵We recently reported on a mutant screen for plants with reduced fertility and identification of a mutant that contained a large number of undeveloped ovules and very few viable seeds.¹⁰ TAIL-PCR revealed that this mutant is a new allele of LORELEI(LRE) [At4g26466].^10,11 Four lre alleles have been reported;¹¹ so, this mutant was designated lre-5.¹⁰ The Arabidopsis LORELEI protein contains 165 amino acids and possesses sequence features indicative of a glycosylphosphatidylinositol (GPI)-anchor containing cell surface protein. GPI-anchors serve as an alternative to transmembrane domains for anchoring proteins in cell membranes and GPI-anchored proteins participate in many functions including cell-cell signaling.¹²     

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