Cryptochrome photoreceptors cry1 and cry2 antagonistically regulate primary root elongation in Arabidopsis thaliana

Cryptochromes are blue-light receptors controlling multiple aspects of plant growth and development. They are flavoproteins with significant homology to photolyases, but instead of repairing DNA they function by transducing blue light energy into a signal that can be recognized by the cellular signaling machinery. Here we report the effect of cry1 and cry2 blue light receptors on primary root growth in Arabidopsis thaliana seedlings, through analysis of both cryptochrome-mutant and cryptochrome-overexpressing lines. Cry1 mutant seedlings show reduced root elongation in blue light while overexpressing seedlings show significantly increased elongation as compared to wild type controls. By contrast, the cry2 mutation has the opposite effect on root elongation growth as does cry1, demonstrating that cry1 and cry2 act antagonistically in this response pathway. The site of cryptochrome signal perception is within the shoot, and the inhibitor of auxin transport, 1-N-naphthylphthalamic acid, abolishes the differential effect of cryptochromes on root growth, suggesting the blue-light signal is transmitted from the shoot to the root by a mechanism that involves auxin. Primary root elongation in blue light may thereby involve interaction between cryptochrome and auxin signaling pathways.     

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild‐type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss‐of‐function mutations, or in a dominant (gain‐of‐function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.     

<Emphasis Type="Italic">Bacillus methylotrophicus</Emphasis> M4-96 isolated from maize (<Emphasis Type="Italic">Zea mays</Emphasis>) rhizoplane increases growth and auxin content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> via emission of volatiles

Plant growth-promoting rhizobacteria stimulate plant growth and development via different mechanisms. In this study, we characterized the effect of volatiles from Bacillus methylotrophicus M4-96 isolated from the maize rhizosphere on root and shoot development, and auxin homeostasis in Arabidopsis thaliana. Phytostimulation occurred after 4 days of interaction between M4-96 and Arabidopsis grown on opposite sides of divided Petri plates, as revealed by enhanced primary root growth, root branching, leaf formation, and shoot biomass accumulation. Analysis of indole-3-acetic acid content revealed two- and threefold higher accumulation in the shoot and root of bacterized seedlings, respectively, compared to uninoculated plants, which was correlated with increased expression of the auxin response marker DR5::GUS. The auxin transport inhibitor 1-naphthylphthalamic acid inhibited primary root growth and lateral root formation in axenically grown seedlings and antagonized the plant growth-promoting effects of M4-96. Analysis of bacterial volatile compounds revealed the presence of four classes of compounds, including ten ketones, eight alcohols, one aldehyde, and two hydrocarbons. However, the abundance of ketones and alcohols represented 88.73 and 8.05%, respectively, of all airborne signals detected, with acetoin being the main compound produced. Application of acetoin had a different effect from application of volatiles, suggesting that either the entire pool or acetoin acting in concert with another unidentified compound underlies the strong phytostimulatory response. Taken together, our results show that B. methylotrophicus M4-96 generates bioactive volatiles that increase the active auxin pool of plants, stimulate the growth and formation of new organs, and reprogram root morphogenesis.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2 gene defines a glutathione-dependent pathway involved in initiation and maintenance of cell division during postembryonic root development

Activation of cell division in the root apical meristem after germination is essential for postembryonic root development. Arabidopsis plants homozygous for a mutation in the ROOT MERISTEMLESS1 (RML1) gene are unable to establish an active postembryonic meristem in the root apex. This mutation abolishes cell division in the root but not in the shoot. We report the molecular cloning of the RML1 gene, which encodes the first enzyme of glutathione (GSH) biosynthesis, γ-glutamylcysteine synthetase, and which is allelic to CADMIUM SENSITIVE2. The phenotype of the rml1 mutant, which was also evident in the roots of wild-type Arabidopsis and tobacco treated with an inhibitor of GSH biosynthesis, could be relieved by applying GSH to rml1 seedlings. By using a synchronized tobacco cell suspension culture, we showed that the G₁-to-S phase transition requires an adequate level of GSH. These observations suggest the existence of a GSH-dependent developmental pathway essential for initiation and maintenance of cell division during postembryonic root development.     

A Mutation Altering Auxin Homeostasis and Plant Morphology in Arabidopsis

Many aspects of plant development are associated with changing concentrations of the phytohormone auxin. Several stages of root formation exhibit extreme sensitivities to exogenous auxin and are correlated with shifts in endogenous auxin concentration. In an effort to elucidate mechanisms regulating development of adventitious roots, an ethyl methanesulfonate-mutagenized M2 population of Arabidopsis was screened for mutants altered in this process. A recessive nuclear mutant, rooty (rty), displayed extreme proliferation of roots, inhibition of shoot growth, and other alterations suggesting elevated responses to auxin or ethylene. Wild-type Arabidopsis seedlings grown on auxin-containing media phenocopied rty, whereas rty seedlings were partially rescued on cytokinin-containing media. Analysis by gas chromatography-selected ion monitoring-mass spectrometry showed endogenous indole-3-acetic acid concentrations to be two to 17 times higher in rty than in the wild type. Dose-response assays with exogenous indole-3-acetic acid indicated equal sensitivities to auxin in tissues of the wild type and rty. Combining rty with mutations conferring resistance to auxin (axr1-3) or ethylene (etr1-1) suggested that root proliferation and restricted shoot growth are auxin effects, whereas other phenotypic alterations are due to ethylene. Four mutant alleles from independently mutagenized populations were identified, and the locus was mapped using morphological and restriction fragment length polymorphism markers to 3.9 centimorgans distal to marker m605 on chromosome 2. The wild-type RTY gene product may serve a critical role in regulating auxin concentrations and thereby facilitating normal plant growth and development.     

Disruption of auxin transport is associated with aberrant leaf development in maize

Despite recent progress, the mechanisms governing shoot morphogenesis in higher plants are only partially understood. Classical physiological studies have suggested that gradients of the plant growth regulator auxin may play a role in controlling tissue differentiation in shoots. More recent molecular genetic studies have also identified knotted1 like homeobox (knox) genes as important regulators of shoot development. The maize (Zea mays L.) mutant rough sheath2 (rs2) displays ectopic expression of at least three knox genes and consequently conditions a range of shoot and leaf phenotypes, including aberrant vascular development, ligular displacements, and dwarfism (R. Schneeberger, M. Tsiantis, M. Freeling, J.A. Langdale [1998] Development 125: 2857–2865). In this report, we show that rs2 mutants also display decreased polar auxin transport in the shoot. We also demonstrate that germination of wild-type maize seedlings on agents known to inhibit polar auxin transport mimics aspects of the rs2 mutant phenotype. The phenotype elaborated in inhibitor-treated plants is not correlated with ectopic KNOX protein accumulation.     

Cytokinin receptors are involved in alkamide regulation of root and shoot development in Arabidopsis

Alkamides and N-acilethanolamides are a class of lipid compounds related to animal endocannabinoids of wide distribution in plants. We investigated the structural features required for alkamides to regulate plant development by comparing the root responses of Arabidopsis (Arabidopsis thaliana) seedlings to a range of natural and synthetic compounds. The length of the acyl chain and the amide moiety were found to play a crucial role in their biological activity. From the different compounds tested, N-isobutyl decanamide, a small saturated alkamide, was found to be the most active in regulating primary root growth and lateral root formation. Proliferative-promoting activity of alkamide treatment was evidenced by formation of callus-like structures in primary roots, ectopic blades along petioles of rosette leaves, and disorganized tumorous tissue originating from the leaf lamina. Ectopic organ formation by N-isobutyl decanamide treatment was related to altered expression of the cell division marker CycB1:uidA and an enhanced expression of the cytokinin-inducible marker ARR5:uidA both in roots and in shoots. The involvement of cytokinins in mediating the observed activity of alkamides was tested using Arabidopsis mutants lacking one, two, or three of the putative cytokinin receptors CRE1, AHK2, and AHK3. The triple cytokinin receptor mutant was insensitive to N-isobutyl decanamide treatment, showing absence of callus-like structures in roots, the lack of lateral root proliferation, and absence of ectopic outgrowths in leaves under elevated levels of this alkamide. Taken together our results suggest that alkamides and N-acylethanolamides may belong to a class of endogenous signaling compounds that interact with a cytokinin-signaling pathway to control meristematic activity and differentiation processes during plant development.     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

The boundary-expressed EPIDERMAL PATTERNING FACTOR-LIKE2 gene encoding a signaling peptide promotes cotyledon growth during Arabidopsis thaliana embryogenesis

The shoot organ boundaries have important roles in plant growth and morphogenesis. It has been reported that a gene encoding a cysteine-rich secreted peptide of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family, EPFL2, is expressed in the boundary domain between the two cotyledon primordia of Arabidopsis thaliana embryo. However, its developmental functions remain unknown. This study aimed to analyze the role of EPFL2 during embryogenesis. We found that cotyledon growth was reduced in its loss-of-function mutants, and this phenotype was associated with the reduction of auxin response peaks at the tips of the primordia. The reduced cotyledon size of the mutant embryo recovered in germinating seedlings, indicating the presence of a factor that acted redundantly with EPFL2 to promote cotyledon growth in late embryogenesis. Our analysis suggests that the boundary domain between the cotyledon primordia acts as a signaling center that organizes auxin response peaks and promotes cotyledon growth.     

Auxin controls the division of root endodermal cells

The root endodermis forms a selective barrier that prevents the free diffusion of solutes into the vasculature; to make this barrier, endodermal cells deposit hydrophobic compounds in their cell walls, forming the Casparian strip. Here, we showed that, in contrast to vascular and epidermal root cells, endodermal root cells do not divide alongside the root apical meristem in Arabidopsis thaliana. Auxin treatment induced division of endodermal cells in wild-type plants, but not in the auxin signaling mutant auxin resistant3-1. Endodermis-specific activation of auxin responses by expression of truncated AUXIN-RESPONSIVE FACTOR5 (ΔARF5) in root endodermal cells under the control of the ENDODERMIS7 promoter (EN7::ΔARF5) also induced endodermal cell division. We used an auxin transport inhibitor to cause accumulation of auxin in endodermal cells, which induced endodermal cell division. In addition, knockout of P-GLYCOPROTEIN1 (PGP1) and PGP19, which mediate centripetal auxin flow, promoted the division of endodermal cells. Together, these findings reveal a tight link between the endodermal auxin response and endodermal cell division, suggesting that auxin is a key regulator controlling the division of root endodermal cells, and that PGP1 and PGP19 are involved in regulating endodermal cell division.

The endodermal auxin response, which is regulated by centripetal auxin flow, determines division of the endodermal cells.     

Two Seven-Transmembrane Domain MILDEW RESISTANCE LOCUS O Proteins Cofunction in Arabidopsis Root Thigmomorphogenesis

Directional root expansion is governed by nutrient gradients, positive gravitropism and hydrotropism, negative phototropism and thigmotropism, as well as endogenous oscillations in the growth trajectory (circumnutation). Null mutations in phylogenetically related Arabidopsis thaliana genes MILDEW RESISTANCE LOCUS O 4 (MLO4) and MLO11, encoding heptahelical, plasma membrane–localized proteins predominantly expressed in the root tip, result in aberrant root thigmomorphogenesis. mlo4 and mlo11 mutant plants show anisotropic, chiral root expansion manifesting as tightly curled root patterns upon contact with solid surfaces. The defect in mlo4 and mlo11 mutants is nonadditive and dependent on light and nutrients. Genetic epistasis experiments demonstrate that the mutant phenotype is independently modulated by the Gβ subunit of the heterotrimeric G-protein complex. Analysis of expressed chimeric MLO4/MLO2 proteins revealed that the C-terminal domain of MLO4 is necessary but not sufficient for MLO4 action in root thigmomorphogenesis. The expression of the auxin efflux carrier fusion, PIN1-green fluorescent protein, the pattern of auxin-induced gene expression, and acropetal as well as basipetal auxin transport are altered at the root tip of mlo4 mutant seedlings. Moreover, addition of auxin transport inhibitors or the loss of EIR1/AGR1/PIN2 function abolishes root curling of mlo4, mlo11, and wild-type seedlings. These results demonstrate that the exaggerated root curling phenotypes of the mlo4 and mlo11 mutants depend on auxin gradients and suggest that MLO4 and MLO11 cofunction as modulators of touch-induced root tropism.     

MM31/EIR1 promotes lateral root formation in Arabidopsis

Lateral root formation in Arabidopsis provides a model for the study of auxin function. Tryptophan (Trp) is a precursor of the auxin indoleacetic acid (IAA). To study the physiological function of Trp in auxin-related phenotypes, we examined the effect of Trp on lateral root formation. We found that Trp treatment enhanced lateral root formation and, by screening for mutants in which the effect of Trp on lateral root formation was enhanced, we isolated the mm31 mutant. Based on genetic and physiological analyses, we propose that MM31/EIR1 modulates lateral root formation by regulating the IAA polar transport system, and that auxin transport from the shoot to the root regulates lateral root formation.Key words: lateral root formation, Arabidopsis, EIR1, IAA, auxin     

PP2A mediates lateral root development under NaCl-induced osmotic stress throughout auxin redistribution in Arabidopsis thaliana

Aim

Auxin plays an important role in modulating root system architecture. The effect of salinity on root development has been extensively studied; however, evidence on how salinity affects lateral root development and its underlying molecular mechanism is scarce. Here, we analyzed the role of protein phosphatase PP2A activity in auxin redistribution during Arabidopsis root system adaptation under NaCl-induced osmotic stress.

Method

Arabidopsis Col-0 and DR5::UidA seedlings were grown in MS media containing NaCl alone or in combination with the auxin transport inhibitor naphthylphthalamic acid, the synthetic auxin α-Naphthaleneacetic acid or the phosphatase inhibitor Okadaic acid. After 8 days, primary root length and lateral root number in seedlings were quantified and the auxin distribution was analyzed.

Results

Promotion of primary root shortening and lateral root development induced by osmotic stress correlated with an increase in active auxin content and a >50 % reduction in protein phosphatase type 2A (PP2A) activity. Moreover, the observed effects on seedlings under osmotic stress are more pronounced with the PP2A inhibitor Okadaic acid.

Conclusion

Our data suggest PP2A is a positive regulator of osmotic stress-induced root system architecture modulation, involving auxin redistribution in Arabidopsis thaliana.     

The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroid

Genetic approaches using Arabidopsis thaliana aimed at the identification of mutations affecting events involved in auxin signalling have usually led to the isolation of auxin-resistant mutants. From a selection screen specifically developed to isolate auxin-hypersensitive mutants, one mutant line was selected for its increased sensitivity to auxin (x 2 to 3) for the root elongation response. The genetic analysis of sax1 (hypersensitive to abscisic acid and auxin) indicated that the mutant phenotype segregates as a single recessive Mendelian locus, mapping to the lower arm of chromosome 1. Sax1 seedlings grown in vitro showed a short curled primary root and small, round, dark-green cotyledons. In the greenhouse, adult sax1 plants were characterized by a dwarf phenotype, delayed development and reduced fertility. Further physiological characterization of sax1 seedlings revealed that the most striking trait was a large increase (x 40) in ABA-sensitivity of root elongation and, to a lesser extent, of ABA-induced stomatal closure; in other respects, hypocotyl elongation was resistant to gibberellins and ethylene. These alterations in hormone sensitivity in sax1 plants co-segregated with the dwarf phenotype suggesting that processes involved in cell elongation are modified. Treatment of mutant seedlings with an exogenous brassinosteroid partially rescued a wild-type size, suggesting that brassinosteroid biosynthesis might be affected in sax1 plants. Wild-type sensitivities to ABA, auxin and gibberellins were also restored in sax1 plants by exogenous application of brassinosteroid, illustrating the pivotal importance of the BR-related SAX1 gene.     

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     

OsPIN2, which encodes a member of the auxin efflux carrier proteins,is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice

Auxin flow is important for different root developmental processes such as root formation, emergence, elongation and gravitropism. However, the detailed information about the mechanisms regulating the auxin flow is less well understood in rice. We characterized the auxin transport‐related mutants, Ospin‐formed2‐1 (Ospin2‐1) and Ospin2‐2, which exhibited curly root phenotypes and altered lateral root formation patterns in rice. The OsPIN2 gene encodes a member of the auxin efflux carrier proteins that possibly regulates the basipetal auxin flow from the root tip toward the root elongation zone. According to DR5‐driven GUS expression, there is an asymmetric auxin distribution in the mutants that corresponded with the asymmetric cell elongation pattern in the mutant root tip. Auxin transport inhibitor, N‐1‐naphthylphthalamic acid and Ospin2‐1 Osiaa13 double mutant rescued the curly root phenotype indicating that this phenotype results from a defect in proper auxin distribution. The typical curly root phenotype was not observed when Ospin2‐1 was grown in distilled water as an alternative to tap water, although higher auxin levels were found at the root tip region of the mutant than that of the wild‐type. Therefore, the lateral root formation zone in the mutant was shifted basipetally compared with the wild‐type. These results reflect that an altered auxin flow in the root tip region is responsible for root elongation growth and lateral root formation patterns in rice.