Expression and regulation of theRSI-1 gene during lateral root initiation

The tomato geneRSI-1 was previously identified as a molecular marker for auxin-induced lateral root initiation. We have further characterized the expression mode of theRSI-1 gene in tomato andArabidopsis thaliana. Northern blot analyses revealed that the gene was induced specifically by auxin in tomato roots and hypocotyls. For experiments with transgenic plants, the 5′ flanking region of theRSI-1 gene was linked to a GUS reporter gene, then transformed into tomato andArabidopsis. In these transgenic tomato plants, GUS activity was detected at the sites of initiation for lateral and adventitious roots. Expression of the fusion gene was auxin-dependent and tissue-specific. This was consistent with results from the northern blot analyses. In transgenicArabidopsis, the overall expression pattern of theRSI-GUS gene, including tissue specificity and auxin inducibility, was comparable to that in transgenic tomato seedlings. These results indicate that an identical regulatory mechanism for lateral root initiation might be conserved in both plants. Thus, the expression mode of theRSI-CUS gene inArabidopsis mutants defective in lateral root development should be investigated to provide details of this process.     

Cytokinin inhibits a subset of diageotropica-dependent primary auxin responses in tomato

Many aspects of plant development are regulated by antagonistic interactions between the plant hormones auxin and cytokinin, but the molecular mechanisms of this interaction are not understood. To test whether cytokinin controls plant development through inhibiting an early step in the auxin response pathway, we compared the effects of cytokinin with those of the dgt (diageotropica) mutation, which is known to block rapid auxin reactions of tomato (Lycopersicon esculentum) hypocotyls. Long-term cytokinin treatment of wild-type seedlings phenocopied morphological traits of dgt plants such as stunting of root and shoot growth, reduced elongation of internodes, reduced apical dominance, and reduced leaf size and complexity. Cytokinin treatment also inhibited rapid auxin responses in hypocotyl segments: auxin-stimulated elongation, H(+) secretion, and ethylene synthesis were all inhibited by cytokinin in wild-type hypocotyl segments, and thus mimicked the impaired auxin responsiveness found in dgt hypocotyls. However, cytokinin failed to inhibit auxin-induced LeSAUR gene expression, an auxin response that is affected by the dgt mutation. In addition, cytokinin treatment inhibited the auxin induction of only one of two 1-aminocyclopropane-1-carboxylic acid synthase genes that exhibited impaired auxin inducibility in dgt hypocotyls. Thus, cytokinin inhibited a subset of the auxin responses impaired in dgt hypocotyls, suggesting that cytokinin blocks at least one branch of the DGT-dependent auxin response pathway.     

Multilevel interactions between ethylene and auxin in Arabidopsis roots

Hormones play a central role in the coordination of internal developmental processes with environmental signals. Herein, a combination of physiological, genetic, cellular, and whole-genome expression profiling approaches has been employed to investigate the mechanisms of interaction between two key plant hormones: ethylene and auxin. Quantification of the morphological effects of ethylene and auxin in a variety of mutant backgrounds indicates that auxin biosynthesis, transport, signaling, and response are required for the ethylene-induced growth inhibition in roots but not in hypocotyls of dark-grown seedlings. Analysis of the activation of early auxin and ethylene responses at the cellular level, as well as of global changes in gene expression in the wild type versus auxin and ethylene mutants, suggests a simple mechanistic model for the interaction between these two hormones in roots, according to which ethylene and auxin can reciprocally regulate each other's biosyntheses, influence each other's response pathways, and/or act independently on the same target genes. This model not only implies existence of several levels of interaction but also provides a likely explanation for the strong ethylene response defects observed in auxin mutants.     

Inhibition of auxin-induced ethylene production by salicylic acid in mung bean hypocotyls

Salicylic acid (SA), a common plant phenolic compound, influences diverse physiological and biochemical processes in plants. To gain insight into the mode of interaction between auxin, ethylene, and SA, the effect of SA on auxininduced ethylene production in mung bean hypocotyls was investigated. Auxin markedly induced ethylene production, while SA inhibited the auxin-induced ethylene synthesis in a dose-dependent manner. At 1 mM of SA, auxininduced ethylene production decreased more than 60% in hypocotyls. Results showed that the accumulation of ACC was not affected by SA during the entire period of auxin treatment, indicating that the inhibition of auxin-induced ethylene production by SA was not due to the decrease in ACC synthase activity, the rate-limiting step for ethylene biosynthesis. By contrast, SA effectively reduced not only the basal level of ACC oxidase activity but also the wound-and ethylene-induced ACC oxidase activity, the last step of ethylene production, in a dose-dependent manner. Northern and immuno blot analyses indicate that SA does not exert any inhibitory effect on the ACC oxidase gene expression, whereas it effectively inhibits both the in vivo and in vitro ACC oxidase enzyme activity, thereby abolishing auxin-induced ethylene production in mung bean hypocotyl tissue. It appears that SA inhibits ACC oxidase enzyme activity through the reversible interaction with Fe²⁺, an essential cofactor of this enzyme. These results are consistent with the notion that ethylene production is controlled by an intimate regulatory interaction between auxin and SA in mung bean hypocotyl tissue.     

Auxin Physiology of the Tomato Mutant diageotropica

The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.     

The Role of Protons in the Auxin-induced Root Growth Inhibition - A Critical Reexamination

According to the acid growth theory of auxin action, it has been proposed that auxin decreases root growth by inhibiting the proton pump, thus causing an alkalinization of the apoplast. This paper critically tests this hypothesis with corn (Zea mays L.) roots. It was found that: i) the pH-growth curve for roots exhibits a broad optimum ranging from pH 4.5 to 9. ii) Any acid-induced growth is of very short duration, iii) The low sensitivity of root growth to external pH is independent of both the pump activity and the buffer capacity of the bathing solution, iv) Neither incubation in acidic buffer nor stimulation of the proton pump reverts the auxin-induced root growth inhibition. It is concluded that the auxin-induced root growth inhibition is not mediated by cell wall alkalinization.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Insensitivity of the diageotropica tomato mutant to auxin

The sensitivity of excised hypocotyl segments to indoleacetic acid (IAA) in two assays, ethylene production and elongation, was determined in the ethylene-requiring tomato (Lycopersicon esculentum Mill.) mutant, diageotropica (dgt), and its isogenic parent, cv VFN8. Endogenous (uninduced) ethylene synthesis rates were slightly lower in dgt hypocotyls than in VFN8 hypocotyls. Ethylene production was essentially unaffected by IAA in dgt, but was stimulated up to 10-fold by 10 micromolar IAA in VFN8. Elongation of dgt hypocotyls was also insensitive to concentrations of IAA as high as 100 micromolar, as compared to significant elongation of VFN8 hypocotyls in response to 0.1 micromolar IAA. A range of IAA analogs active in VFN8 was also ineffective in stimulating elongation of dgt hypocotyls, suggesting that the differences were not due to rapid metabolism of IAA by dgt tissues. Auxin-induced elongation of VFN8 hypocotyls was unaffected by 2,3,5-triiodobenzoic acid and naphthylphthalamic acid, indicating that polar auxin transport was not a factor in these experiments. Exogenous and auxin-induced ethylene had no effect on the elongation respone of either genotype, nor did exogenous ethylene restore the sensitivity of dgt hypocotyls to IAA. Despite their apparent insensitivity to auxin, dgt hypocotyls elongated dramatically and synthesized ethylene rapidly in response to 1.2 micromolar fusicoccin. These results suggest that the primary effect of the dgt mutation is to reduce the sensitivity of the tissue to auxin. As altered regulation of ethylene synthesis is only one symptom of this fundamental deficiency, dgt should more properly be considered to be the auxin-insensitive tomato mutant.     

Tubulin synthesis and auxin-induced root initiation in phaseolus

The auxin-induced formation of roots in the hypocotyls of Phaseolus vulgaris can be prevented by treatment with actinomycin D, colchicine or cytochalasin B if applied within 40 hr of initiation. Shortly after auxin pretreatment, there is an increase in translatable messenger RNA activity. Analysis of the labelled cell-free products indicate, among other changes, a striking increase in a protein co-migrating with tubulin, in the case of RNA isolated from indolebutyric acid (IBA) pretreated hypocotyls. An increase in tubulin content in vivo can also be demonstrated on the basis of SDS-polyacrylamide gel analysis of membrane proteins and functional assays for tubulin polymerization. An increase in the synthesis of tubulin in vivo can also be demonstrated after IBA pretreatment. In addition, the auxin is also able to promote tubulin polymerization when added in vitro. It is suggested that tubulin synthesis and microtubule assembly are early events in auxin-mediated root differentiation.     

ABCB19‐mediated polar auxin transport modulates Arabidopsis hypocotyl elongation and the endoreplication variant of the cell cycle

Elongation of the Arabidopsis hypocotyl pushes the shoot‐producing meristem out of the soil by rapid expansion of cells already present in the embryo. This elongation process is shown here to be impaired by as much as 35% in mutants lacking ABCB19, an ATP‐binding cassette membrane protein required for polar auxin transport, during a limited time of fast growth in dim white light beginning 2.5 days after germination. The discovery of high ectopic expression of a cyclin B1;1‐based reporter of mitosis throughout abcb19 hypocotyls without an equivalent effect on mitosis prompted investigations of the endoreplication variant of the cell cycle. Flow cytometry performed on nuclei isolated from upper (growing) regions of 3‐day‐old hypocotyls showed ploidy levels to be lower in abcb19 mutants compared with wild type. CCS52A2 messenger RNA encoding a nuclear protein that promotes a shift from mitosis to endoreplication was lower in abcb19 hypocotyls, and fluorescence microscopy showed the CCS52A2 protein to be lower in the nuclei of abcb19 hypocotyls compared with wild type. Providing abcb19 seedlings with nanomolar auxin rescued their low CCS52A2 levels, endocycle defects, aberrant cyclin B1;1 expression, and growth rate defect. The abcb19‐like growth rate of ccs52a2 mutants was not rescued by auxin, placing CCS52A2 after ABCB19‐dependent polar auxin transport in a pathway responsible for a component of ploidy‐related hypocotyl growth. A ccs52A2 mutation did not affect the level or pattern of cyclin B1;1 expression, indicating that CCS52A2 does not mediate the effect of auxin on cyclin B1;1.     

Arabidopsis ROP9 and ROP10 GTPases differentially regulate auxin and ABA responses

Auxin and abscisic acid (ABA) are major plant hormones that act together to modulate numerous aspects of plant growth and development, including seed germination, primary root elongation, and lateral root formation. In this study, we analyzed the loss-of-function mutants of two closely related ROP (Rho of plants) GTPases, ROP9 and ROP10, and found that these ROP GTPases differentially regulate the auxin and ABA responses. rop9 and rop10 mutations enhanced the ABA-induced suppression of seed germination, primary root growth, and lateral root formation and the expression of ABA-responsive genes, whereas rop9 but not rop10 suppressed auxin-induced root phenotypes and auxin-responsive gene expression. These results suggest that both ROP9 and ROP10 function as negative regulators of ABA signaling, and that ROP9, but not ROP10, functions as a positive regulator of auxin signaling. Previously, ROPinteractive CRIB motif-containing protein 1 (RIC1) was reported to participate in auxin and ABA responses, and to have a similar effect as ROP9 and ROP10 on gene expression, root development, and seed germination. Because RIC proteins mediate ROP GTPase signaling, our results suggest that ROP9 and ROP10 GTPases function upstream of RIC1 in auxin- and ABA-regulated root development and seed germination.     

Roles for Auxin, Cytokinin, and Strigolactone in Regulating Shoot Branching

Many processes have been described in the control of shoot branching. Apical dominance is defined as the control exerted by the shoot tip on the outgrowth of axillary buds, whereas correlative inhibition includes the suppression of growth by other growing buds or shoots. The level, signaling, and/or flow of the plant hormone auxin in stems and buds is thought to be involved in these processes. In addition, RAMOSUS (RMS) branching genes in pea (Pisum sativum) control the synthesis and perception of a long-distance inhibitory branching signal produced in the stem and roots, a strigolactone or product. Auxin treatment affects the expression of RMS genes, but it is unclear whether the RMS network can regulate branching independently of auxin. Here, we explore whether apical dominance and correlative inhibition show independent or additive effects in rms mutant plants. Bud outgrowth and branch lengths are enhanced in decapitated and stem-girdled rms mutants compared with intact control plants. This may relate to an RMS-independent induction of axillary bud outgrowth by these treatments. Correlative inhibition was also apparent in rms mutant plants, again indicating an RMS-independent component. Treatments giving reductions in RMS1 and RMS5 gene expression, auxin transport, and auxin level in the main stem were not always sufficient to promote bud outgrowth. We suggest that this may relate to a failure to induce the expression of cytokinin biosynthesis genes, which always correlated with bud outgrowth in our treatments. We present a new model that accounts for apical dominance, correlative inhibition, RMS gene action, and auxin and cytokinin and their interactions in controlling the progression of buds through different control points from dormancy to sustained growth.     

WEG1, which encodes a cell wall hydroxyproline-rich glycoprotein,is essential for parental root elongation controlling lateral root formation in rice

Lateral roots (LRs) determine the overall root system architecture, thus enabling plants to efficiently explore their underground environment for water and nutrients. However, the mechanisms regulating LR development are poorly understood in monocotyledonous plants. We characterized a rice mutant, wavy root elongation growth 1 (weg1), that produced higher number of long and thick LRs (L-type LRs) formed from the curvatures of its wavy parental roots caused by asymmetric cell growth in the elongation zone. Consistent with this phenotype, was the expression of the WEG1 gene, which encodes a putative member of the hydroxyproline-rich glycoprotein family that regulates cell wall extensibility, in the root elongation zone. The asymmetric elongation growth in roots is well known to be regulated by auxin, but we found that the distribution of auxin at the apical region of the mutant and the wild-type roots was symmetric suggesting that the wavy root phenotype in rice is independent of auxin. However, the accumulation of auxin at the convex side of the curvatures, the site of L-type LR formation, suggested that auxin likely induced the formation of L-type LRs. This was supported by the need of a high amount of exogenous auxin to induce the formation of L-type LRs. These results suggest that the MNU-induced weg1 mutated gene regulates the auxin-independent parental root elongation that controls the number of likely auxin-induced L-type LRs, thus reflecting its importance in improving rice root architecture.     

An auxin-inducible gene from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis> L.) is differentially expressed in mature and juvenile-phase shoots and encodes a putative transmembrane protein

We have isolated a gene from loblolly pine, 5NG4, that is highly and specifically induced by auxin in juvenile loblolly pine shoots prior to adventitious root formation, but substantially down-regulated in physiologically mature shoots that are adventitious rooting incompetent. 5NG4 was highly auxin-induced in roots, stems and hypocotyls, organs that can form either lateral or adventitious roots following an auxin treatment, but was not induced to the same level in needles and cotyledons, organs that do not form roots. The deduced amino acid sequence shows homology to the MtN21 nodulin gene from Medicago truncatula. The expression pattern of 5NG4 and its homology to a protein from Medicago involved in a root-related process suggest a possible role for this gene in adventitious root formation. Homology searches also identified similar proteins in Arabidopsis thaliana and Oryza sativa. High conservation across these evolutionarily distant species suggests essential functions in plant growth and development. A 38-member family of genes homologous to 5NG4 was identified in the A. thaliana genome. The physiological significance of this redundancy is most likely associated with functional divergence and/or expression specificity of the different family members. The exact biochemical function of the gene is still unknown, but sequence and structure predictions and 5NG4::GFP fusion protein localizations indicate it is a transmembrane protein with a possible transport function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations ABA Abscisic acid - BA Benzylaminopurine - EST Expressed sequence tag - NAA 1-Naphthaleneacetic acid - GFP Green fluorescent protein - ORF Open reading frame     

New methods to analyse auxin-induced growth I: Classical auxinology goes Arabidopsis

During recent years, genetic approaches have become invaluable tools to analyse signalling chains in plants. The first step to understand auxin signalling is the isolation of signal transduction mutants. In Arabidopsis, screening for auxin resistant plants has identified a number of mutants. However, it is difficult to link the mutated genes to the rapid growth response to auxin. As yet, there is no published method to measure auxin-induced growth at a sufficient temporal resolution. A novel auxanometer described here will close that gap. Hypocotyl segments are immersed in buffer in a flow-through cuvette. A CCD-camera attached to a microscope is used to image markings on the hypocotyl surfaces and to track their movements across the field of view.To illustrate the applicability of this method for analysing mutants we compared the growth responses of tomato wild type with the mutant diageotropica (dgt). We showed that the mutation completely abolished all phases of the rapid growth reaction to applied auxin. This excludes the possibility that the reduced auxin sensitivity reported in the literature is due to a reduced or delayed response. Our technique was modified for use with the tiny Arabidopsis thaliana hypocotyl segments and inflorescence stems. In both systems auxin induced a growth response after a lag phase of 15–20 minutes. The new method will now be used to characterise the physiological effect of the mutations in auxin signalling.     

Altered Gene Expression during Auxin-Induced Root Development from Excised Mung Bean Seedlings

Changes in the pattern of protein synthesis and in the translatable mRNA population have been examined during auxin-induced root development from excised mung bean seedlings. Several proteins, predominantly of low molecular weight and high pI, as shown by two-dimensional polyacrylamide gel electrophoresis, are synthesized specifically by auxin-treated tissue. These auxin-induced proteins appear between 6 and 12 hours of auxin treatment, reach a maximum at 24 hours, and decline at 48 hours. Untreated seedlings (placed in Hoagland solution), known to produce small number of roots at the cut end probably due to endogeneous auxin accumulated at the cut end through basipetal transport, show low level synthesis of auxin-specific proteins. Antiauxin treatment that completely inhibits auxin-induced rooting also prevents the appearance of auxin-induced proteins. The induction of a group of three to four proteins appears to be specific to antiauxin treatment. In vitro translation of mRNA from auxin-treated tissue, but not of mRNA from antiauxin-treated tissue, yields several polypeptides of low molecular weight and high pI. Since the auxin-induced proteins precede root development and are synthesized transitorily, it is likely that they play some regulatory role during the initiation of root development. The result show that auxin-induced root formation involves altered gene expression.