Prevention of auxin-induced epinasty by α-aminooxyacetic acid

Elhylene production and epinastic growth of leaf petioles of tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants sprayed with 0.1 mM naphthyl-1-acetic acid were suppressed when 1 mMα-aminooxyacetic acid (AOA) was simultaneously sprayed on the plants. AOA had no effect on ethylene evolution and epinastic growth resulting from the application of 5 mM 1-aminocyclopropane-1-carboxylic acid, the immediate precursor of ethylene.     

The lateral root initiation index: an integrative measure of primordium formation

Background and Aims

Lateral root initiation is an essential and continuous process in the formation of root systems; therefore, its quantitative analysis is indispensable. In this study a new measure of lateral root initiation is proposed and analysed, namely the lateral root initiation index (I_LRI), which defines how many lateral roots and/or primordia are formed along a parent-root portion corresponding to 100 cortical cells in a file.

Methods

For data collection, a commonly used root clearing procedure was employed, and a new simple root clearing procedure is also proposed. The I_LRI was determined as 100dl, where d is the density of lateral root initiation events (number mm⁻¹) and l is the average fully elongated cortical cell length (mm).

Key Results

Analyses of different Arabidopsis thaliana genotypes and of a crop plant, tomato (Solanum lycopersicum), showed that I_LRI is a more precise parameter than others commonly used as it normalizes root growth for variations in cell length. Lateral root primordium density varied in the A. thaliana accessions Col, Ler, Ws, and C24; however, in all accessions except Ws, I_LRI was similar under the same growth conditions. The nitrogen/carbon ratio in the growth medium did not change the lateral root primordium density but did affect I_LRI. The I_LRI was also modified in a number of auxin-related mutants, revealing new root branching phenotypes in some of these mutants. The rate of lateral root initiation increased with Arabidopsis seedling age; however, I_LRI was not changed in plants between 8 and 14 d post-germination.

Conclusions

The I_LRI allows for a more precise comparison of lateral root initiation under different growth conditions, treatments, genotypes and plant species than other comparable methods.Key words: Arabidopsis thaliana, auxin, lateral root density, lateral root initiation index, mutant phenotype, pericycle, root architecture, root branching, root primordium, Solanum lycopersicum     

Early primordium morphogenesis during lateral root initiation in Arabidopsis thaliana

The first morphogenetic events of lateral root primordium (LRP) formation in the Arabidopsis thaliana (L.) Heynh. pericycle occur soon after cells of the primary root complete elongation. Pericycle cells in direct contact with underlying protoxylem cells participate in LRP formation. Two types of LRP initiation were found, longitudinal uni- and bi-cellular. These occur when a single or two pericycle cells within a file, respectively, become founder cells for the entire longitudinal extent of the LRP. Histochemical and cytological analysis suggests that three is the minimum number of cells required to initiate an LRP. In young primordia comprising less than 32 cells, the average cell-doubling time was 3.7 h, indicating a drastic acceleration of cell cycle progression after lateral root initiation. Early in LRP development, cell growth is limited and therefore cytokinesis leads to a reduction of cell volume, similar to cleavage division cycles during animal and plant embryogenesis. The striking coordination of proliferation between pericycle cells in adjacent files in direct contact with the underlying protoxylem implies that intercellular signaling mechanisms act in the root apical meristem or later in development.     

Calcium is involved in nitric oxide- and auxin-induced lateral root formation in rice

In the present study, the role of nitric oxide (NO) in the regulation of lateral root (LR) formation in rice was examined. Application of sodium nitroprusside (SNP; a NO donor) and indole-3-butyric acid (IBA; a naturally occurring auxin) to rice seedlings induced LR formation. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (cPTIO) blocked the action of SNP and IBA. Endogenous NO was detected by the specific fluorescence probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. SNP- and IBA-induced NO fluorescence was specifically suppressed by cPTIO. Nitrate reductase (NR) inhibitor sodium tungstate completely inhibited IBA-induced LR formation and NO fluorescence. However, nitric oxide synthase inhibitor N ^G-nitro-l-arginine methyl ester hydrochloride slightly reduced IBA-induced LR formation and NO generation. It appears that NO generation that occurs in response to IBA might primarily involve NR activity. Moreover, NO production caused by SNP and IBA was localized in root area corresponding to LR emergence. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin antagonists on LR formation induced by SNP and IBA were also examined. All these inhibitors were effective in reducing the action of SNP and IBA. However, Ca²⁺ chelators and Ca²⁺-channel inhibitors had no effect on SNP- and IBA-induced NO generation. It is concluded that cytosolic levels of Ca²⁺ may regulate SNP and IBA action through calmodulin-dependent mechanism.     

Isolation and characterization of cDNA clones corresponding with mRNAs that accumulate during auxin-induced lateral root formation

Lateral root formation in root cultures of Arabidopsis thaliana can be initiated by exogenous addition of auxin. In order to find cDNA clones of which the corresponding mRNAs accumulate during this process, a cDNA library was constructed from root cultures treated with the active auxin 1-naphthaleneacetic acid (1- NAA). Differential screening of this library with cDNA probes derived from mRNA populations isolated from root cultures treated with 1-NAA and the inactive analogue 2-naphthaleneacetic acid (2-NAA) led to the isolation of four cDNA clones, designated AIR1, AIR3, AIR9 and AIR12. Accumulation of the mRNAs starts between 4 and 8 h and continues till at least 24 h after addition of an active auxin. Sequence analysis revealed that AIR1 encodes a protein that is related to a large family of proteins that consist of a proline-rich or glycine- rich N-terminus and a hydrophobic, possibly membrane spanning C- terminus. The putative function of these proteins is coupling of the cell wall to the plasma membrane. Surprisingly, AIR1 lacks the proline-rich or glycine-rich N-terminus which is thought to be important for interaction with the cell wall. AIR3 encodes a subtilisin-like serine protease which is believed to be active outside the plant cell. Although AIR9 and AIR12 do not show any significant homology to sequences in the database, they are also predicted to function outside the cell. Our screening thus indicates that a variety of genes encoding extracellular proteins are activated during auxin-induced lateral root formation.     

Heme oxygenase is involved in nitric oxide- and auxin-induced lateral root formation in rice

Lateral root (LR) development performs the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of nitric oxide (NO), auxin, and hemin (Hm) on LR formation in rice. Treatment with Hm [a highly effective heme oxygenase (HO) inducer], sodium nitroprusside (SNP, an NO donor), or indole-3-butyric acid (IBA, a naturally occurring auxin) induced LR formation and HO activity. LR formation and HO activity induced by SNP and IBA but not Hm was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. As well, Hm, SNP, and IBA could induce OsHO1 mRNA expression. Zn protoporphyrin IX (the specific inhibitor of HO) and hemoglobin (the carbon monoxide/NO scavenger) reduced LR number and HO activity induced by Hm, SNP, and IBA. Our data suggest that HO is required for Hm-, auxin-, and NO-induced LR formation in rice.     

Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments

Branca, C, De Lorenzo, G. and Cervone, F. 1988. Competitive inhibition of the auxin-induced elongation by α-D-oligogalacturonides in pea stem segments. - Physiol. Plant. 72: 499–504.
α-D-galacturonide oligomers (OG) were prepared by partial hydrolysis of sodium polypectate with an homogeneous Aspergillus niger endopolygalacturonase (EC 3.2.1.15). OG, obtained after digestion for 10, 20, 30, 60, 120 min and 24 h, were assayed for their ability to interfere with the IAA-induced elongation of pea ( Pisum sativum L. cv. Alaska) stems. Maximum inhibiting activity was exhibited by oligomers with an approximate degree of polymerization higher than 8. Inhibition by longer OG was much lower, and the products of the 24 h digestion and the unhydrolysed polypectate were ineffective. The addition of OG to pea stems caused a parallel shift to the right of the IAA dose-effect curve. The shift depended on the amount of OG used, showing that oligogalacturonides behave as competitive antagonists of IAA. The presence of OG caused the disappearance of the second maximum of the elongation rate and reduced the first maximum. OG were also tested for their ability to inhibit IAA-induced ethylene evolution of pea stem segments. Maximal inhibition was obtained with OG of the same size as those that interfered with IAA-induced elongation. Inhibition of the auxin action seemed to be specific as OG did not interfere with the activity of gibberellic acid (GA₃) or kinetin. It was concluded that oligogalacturonides strongly interfere with the activity of IAA, although they are by themselves incapable to influence the elongation of pea stem segments directly.     

Coinduction of α-L-arabinofuranosidase and α-D-galactosidase formation in Trichoderma reesei RUT C-30

Abstract Formation of α-L-arabinosidase can be induced in Trichoderma reesei by growing the fungus on L-arabinose or dulcitol, and by adding L-arabinose, L-arabitol, D-galactose, or dulcitol ot non-growing mycelia. The same conditions also stimulated the formation of α-D-galactosidase, but not that of various other enzymes involved in hemicellulose degradation. The optimal inducer concentration with all compounds was 4 mM for both enzymes. Using L-arabinose and D-galactose, the induction efficiency was highest at pH 6.5, whereas induction by arabitol and dulcitol was more efficient at low pH (2.5). The addition of 50 mM glucose did not repress α-L-arabinosidase or α-D-galactosidase formation. These findings suggest coregulation of two hemicellulose side-chain cleaving enzymes in T. reesei .     

Mechanisms of primordium formation during adventitious root development from walnut cotyledon explants

 In walnut (Juglans regia L.), an otherwise difficult-to-root species, explants of cotyledons have been shown to generate complete roots in the absence of exogenous growth regulators. In the present study, this process of root formation was shown to follow a pattern of adventitious, rather than primary or lateral, ontogeny: (i) the arrangement of vascular bundles in the region of root formation was of the petiole type; (ii) a typical root primordium was formed at the side of the procambium within a meristematic ring of actively dividing cells located around each vascular bundle; (iii) the developing root apical meristem was connected in a lateral way with the vascular bundle of the petiole. This adventitious root formation occurred in three main stages of cell division, primordium formation and organization of apical meristem. These stages were characterized by expression of LATERAL ROOT PRIMORDIUM-1 and CHALCONE SYNTHASE genes, which were found to be sequentially expressed during the formation of the primordium. Activation of genes related to root cell differentiation started at the early stage of primordium formation prior to organization of the root apical meristem. The systematic development of adventitious root primordia at a precise site gave indications on the positional and biochemical cues that are necessary for adventitious root formation. Received: 30 July 1999 / Accepted: 16 February 2000     

Regulation of lateral root formation in lettuce (Lactuca sativa) seedling roots: Interacting effects of α-naphthaleneacetic acid and kinetin

Growth substances, α-naphthaleneacetic (NAA) and kinetin, had an important role in the regulation of lateral root (LR) formation in lettuce ( Lactuca sativa L. cv. Grand Rapids) seedling roots. NAA (10^-5 M ) was a potent stimulator of LR initiation and caused a 600% increase in the number of lateral root primordia (LRP) compared to untreated roots. NAA was required for only the first 20 h of the 72 h treatment period for maximum stimulation of LRP initiation. Kinetin (2 × 10^-5 M ) effectively prevented the spontaneous formation of LRP and inhibited the NAA-stimulated production of LRP. Kinetin inhibition was maximal during the first 20 h of NAA treatment and this effect was not overcome by subsequent supply of NAA. Also, lettuce roots were most sensitive to kinetin at 20 h of NAA treatment, when the first signs of cell division were observed in the pericycle.     

Interaction between HY1 and H2O2 in auxin-induced lateral root formation in Arabidopsis

Haem oxygenase-1 (HO-1) and hydrogen peroxide (H₂O₂) are two key downstream signals of auxin, a well-known phytohormone regulating plant growth and development. However, the inter-relationship between HO-1 and H₂O₂ in auxin-mediated lateral root (LR) formation is poorly understood. Herein, we revealed that exogenous auxin, 1-naphthylacetic acid (NAA), could simultaneously stimulate Arabidopsis HO-1 (HY1) gene expression and H₂O₂ generation. Subsequently, LR formation was induced. NAA-induced HY1 expression is dependent on H₂O₂. This conclusion was supported by analyzing the removal of H₂O₂ with ascorbic acid (AsA) and dimethylthiourea (DMTU), both of which could block NAA-induced HY1 expression and LR formation. H₂O₂-induced LR formation was inhibited by an HO-1 inhibitor zinc protoporphyrin IX (Znpp) in wild-type and severely impaired in HY1 mutant hy1-100. Simultaneously, HY1 is required for NAA-mediated H₂O₂ generation, since Znpp inhibition of HY1 blocked the NAA-induced H₂O₂ production and LR formation. Genetic data demonstrated that hy1-100 was significantly impaired in H₂O₂ production and LR formation in response to NAA, compared with wild-type plants. The addition of carbon monoxide-releasing molecule-2 (CORM-2), the carbon monoxide (CO) donor, induced H₂O₂ production and LR formation, both of which were decreased by DMTU. Moreover, H₂O₂ and CORM-2 mimicked the NAA responses in the regulation of cell cycle genes expression, all of which were blocked by Znpp or DMTU, respectively, confirming that both H₂O₂ and CO were important in the early LR initiation. In summary, our pharmacological, genetic and molecular evidence demonstrated a close inter-relationship between HY1 and H₂O₂ existing in auxin-induced LR formation in Arabidopsis.     

The Arabidopsis D-type cyclin CYCD2;1 and the inhibitor ICK2/KRP2 modulate auxin-induced lateral root formation

The integration of cell division in root growth and development requires mediation of developmental and physiological signals through regulation of cyclin-dependent kinase activity. Cells within the pericycle form de novo lateral root meristems, and D-type cyclins (CYCD), as regulators of the G₁-to-S phase cell cycle transition, are anticipated to play a role. Here, we show that the D-type cyclin protein CYCD2;1 is nuclear in Arabidopsis thaliana root cells, with the highest concentration in apical and lateral meristems. Loss of CYCD2;1 has a marginal effect on unstimulated lateral root density, but CYCD2;1 is rate-limiting for the response to low levels of exogenous auxin. However, while CYCD2;1 expression requires sucrose, it does not respond to auxin. The protein Inhibitor-Interactor of CDK/Kip Related Protein2 (ICK2/KRP2), which interacts with CYCD2;1, inhibits lateral root formation, and ick2/krp2 mutants show increased lateral root density. ICK2/KRP2 can modulate the nuclear levels of CYCD2;1, and since auxin reduces ICK2/KRP2 protein levels, it affects both activity and cellular distribution of CYCD2;1. Hence, as ICK2/KRP2 levels decrease, the increase in lateral root density depends on CYCD2;1, irrespective of ICK2/CYCD2;1 nuclear localization. We propose that ICK2/KRP2 restrains root ramification by maintaining CYCD2;1 inactive and that this modulates pericycle responses to auxin fluctuations.     

The jasmonate receptor COI1 plays a role in jasmonate-induced lateral root formation and lateral root positioning in Arabidopsis thaliana

Jasmonic acid (JA) regulates a broad range of plant defense and developmental responses. COI1 has been recently found to act as JA receptor. In this report, we show that low micromolar concentrations of JA inhibited primary root (PR) growth and promoted lateral root (LR) formation in Arabidopsis wild-type (WT) seedlings. It was observed that the coi1-1 mutant was less sensitive to JA on pericycle cell activation to induce lateral root primordia (LRP) formation and presented alterations in lateral root positioning and lateral root emergence on bends. To investigate JA-auxin interactions important for remodeling of root system (RS) architecture, we tested the expression of auxin-inducible markers DR5:uidA and BA3:uidA in WT and coi1-1 seedlings in response to indole-3-acetic acid (IAA) and JA and analyzed the RS architecture of a suite of auxin-related mutants under JA treatments. We found that JA did not affect DR5:uidA and BA3:uidA expression in WT and coi1-1 seedlings. Our data also showed that PR growth inhibition in response to JA was likely independent of auxin signaling and that the induction of LRP required ARF7, ARF19, SLR, TIR1, AFB2, AFB3 and AXR1 loci. We conclude that JA regulation of postembryonic root development involves both auxin-dependent and independent mechanisms.     

Cobalt chloride-induced lateral root formation in rice: The role of heme oxygenase

Lateral roots (LRs) perform the essential tasks of providing water, nutrients, and physical support to plants. Therefore, understanding the regulation of LR development is of agronomic importance. Recent findings suggest that heme oxygenase (HO) plays an important role in LR development. In this study, we examined the effect of cobalt chloride (CoCl₂) on LR formation and HO expression in rice. Treatment with CoCl₂ induced LR formation and HO activity. We further observed that CoCl₂ could induce the expression of OsHO1 but not OsHO2. CoCl₂-increased HO activity occurred before LR formation. Zinc protoporphyrin IX (ZnPPIX, the specific inhibitor of HO) and hemoglobin (the carbon monoxide/nitric oxide scavenger) reduced LR formation, HO activity, and OsHO1 expression. Application of biliverdin, a product of HO-catalyzed reaction, to CoCl₂-treated rice seedlings reversed the ZnPPIX-inhibited LR formation and ZnPPIX-decreased HO activity. CoCl₂ had no effect on H₂O₂ content and nitric oxide production. Moreover, application of ascorbate, a H₂O₂ scavenger, failed to affect CoCl₂-promoted LR formation and HO activity. It is concluded that HO is required for CoCl₂-promoted LR formation in rice.     

Reduced expression of α-tubulin genes in Arabidopsis thaliana specifically affects root growth and morphology, root hair development and root gravitropism

Different alpha-tubulin cDNA sequences fused in an antisense orientation to a CaMV 35S promoter were introduced into Arabidopsis thaliana plants. Several independent transgenic lines that showed a moderate but clear reduction of alpha-tubulin gene expression (TUA6/AS lines) were obtained and phenotypically characterized. Although no apparent abnormalities were detected in the aerial parts of TUA6/AS plants, root development was severely affected. Cells in TUA6/AS root tips were found to contain aberrant microtubular structures, to expand abnormally and to be unable to undergo regular cell division. These cellular defects caused a dramatic radial expansion of the root tip and inhibited root elongation. In addition, TUA6/AS roots displayed ectopic formation of root hairs, root hair branching and a reduced ability to respond to gravitropic challenges. Our results contribute to an improved understanding of the different roles microtubules play during root development and demonstrate that reverse genetics is a powerful tool to analyze cytoskeletal functions during plant organogenesis.     

The role of the megagametophyte in maintaining loblolly pine (Pinus taeda L.) seedling arginase gene expression in vitro

Following loblolly pine (Pinus taeda L.) seed germination, storage-protein breakdown in the megagametophyte and in the seedling results in a large increase in the seedling's free amino acid pool. A substantial portion of both the storage proteins and the amino acid pool is arginine, a very efficient nitrogen-storage compound. Free arginine is hydrolyzed in the seedling by the enzyme arginase (EC 3.5.3.1), which is under strong developmental control. At present, regulation of arginase in conifers is not well understood. Here we report the utilization of an in vitro culture system to address the separate impacts of the seedling and megagametophyte tissues on arginase enzyme activity, protein levels and patterns of gene expression. We also describe the generation of an anti-arginase antibody prepared from a histidine-tagged loblolly pine arginase fusion protein expressed in Escherichia coli. Our results indicate that arginase gene expression in the seedling is initiated by the seedling itself and then maintained or up-regulated by the megagametophyte. The contribution of storage-protein breakdown and the free amino acid pool, particularly arginine, in this regulation is also addressed.     

Nitric oxide triggers phosphatidic acid accumulation via phospholipase D during auxin-induced adventitious root formation in cucumber

Auxin and nitric oxide (NO) play fundamental roles throughout plant life. NO is a second messenger in auxin signal transduction leading to root developmental processes. The mechanisms triggered by auxin and NO that direct adventitious root (AR) formation are beginning to be unraveled. The goal of this work was to study phospholipid (PL) signaling during the auxin- and NO-induced AR formation in cucumber (Cucumis sativus) explants. Explants were labeled with 32P-inorganic phosphate and treated with the auxins indole-3-acetic acid or 1-naphthylacetic acid, or the NO donor S-nitroso N-acetyl penicillamine, in the presence or absence of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. PLs were separated by thin-layer chromatography and quantified. We report that the signaling PLs phosphatidic acid (PA), phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulated within 1 min after auxin or NO treatment. Both auxin and NO evoked similar and transient time course responses, since signaling PLs returned to control levels after 20 or 30 min of treatment. The results indicate that auxin relies on NO in inducing PA, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate accumulation. Furthermore, we demonstrate that auxin and NO trigger PA formation via phospholipase D (PLD) activity. Explants treated for 10 min with auxin or NO displayed a 200% increase in AR number compared with control explants. In addition, PLD activity was required for the auxin- and NO-induced AR formation. Finally, exogenously applied PA increased up to 300% the number of ARs. Altogether, our data support the idea that PLD-derived PA is an early signaling event during AR formation induced by auxin and NO in cucumber explants.     

Nitric oxide modulates the expression of cell cycle regulatory genes during lateral root formation in tomato

Nitric oxide (NO) is a bioactive molecule involved in diverse physiological functions in plants. It has previously been reported that the NO donor sodium nitroprusside (SNP) applied to germinated tomato seeds was able to induce lateral root (LR) formation in the same way that auxin treatment does. In this paper, it is shown that NO modulates the expression of cell cycle regulatory genes in tomato pericycle cells and leads, in turn, to induced LR formation. The addition of the NO scavenger CPTIO at different time points during auxin-mediated LR development indicates that NO is required for LR primordia formation and not for LR emergence. The SNP-mediated LR promotion could be prevented by the cell cycle inhibitor olomoucine, suggesting that NO is involved in cell cycle regulation. A system was developed in which the formation of LRs was synchronized. It was based on the control of NO availability in roots by treatment with the NO scavenger CPTIO. The expression of the cell cycle regulatory genes encoding CYCA2;1, CYCA3;1, CYCD3;1, CDKA1, and the Kip-Related Protein KRP2 was studied using RT-PCR analysis in roots with synchronized and non-synchronized LR formation. NO mediates the induction of the CYCD3;1 gene and the repression of the CDK inhibitor KRP2 gene at the beginning of LR primordia formation. In addition, auxin-dependent cell cycle gene regulation was dependent on NO.