Exogenous Application Of Auxin Research Articles

Auxin Signaling in Plant Defense

In their Report “A plant miRNA contributes to antibacterial resistance by repressing auxin signaling” (21 Apr., p. 436), L. Navarro et al. demonstrate a link between auxin signaling in plants and resistance to bacterial pathogens. As part of a plant-induced immune response, bacterial pathogen-associated molecular pattern (PAMP) recognition down-regulates auxin signaling in Arabidopsis by targeting auxin receptor transcripts. These results indicate that decreasing plant auxin signaling can increase resistance to bacterial pathogens; Navarro et al. also showed that exogenous application of auxin enhances susceptibility to the bacterial pathogen.

Genome-wide analysis, evolutionary expansion, and expression of early auxin-responsive SAUR gene family in rice ( Oryza sativa)

Small auxin-up RNAs ( SAURs) are the early auxin-responsive genes represented by a large multigene family in plants. Here, we report the identification of 58 OsSAUR gene family members from rice ( Oryza sativa japonica cv Nipponbare), the model monocot plant, by a reiterative database search and manual reannotation; 2 of these are pseudogenes. The coding sequences of OsSAURs do not possess any intron. Most of the predicted OsSAUR protein sequences harbor a putative nuclear localization signal at their N-terminus. Localized gene duplications appear to be the primary genetic event responsible for SAUR gene family expansion in rice. Interestingly, the duplication of OsSAURs was found to be associated with the chromosomal block duplication as well. The phylogenetic analysis revealed that the SAUR gene family expanded in rice and Arabidopsis due to species-specific expansion of the family in monocots and dicots. The auxin-responsive elements and downstream element are conserved in the upstream and downstream sequences, respectively, of OsSAURs. In addition to the 21 OsSAURs with full-length cDNA sequences and 20 with expressed sequence tags, gene expression analyses of at least 7 OsSAURs by RT-qPCR indicated that the majority of identified OsSAURs most likely are expressed in rice. The transcript abundance of the OsSAURs examined increased within a few minutes of exogenous auxin application with varying kinetics. The present study provides basic genomic information for the rice SAUR gene family and will pave the way for deciphering the precise role of SAURs in plant growth and development.

Exogenous auxin enhances the degradation of a light down-regulated and nuclear-localized OsiIAA1, an Aux/IAA protein from rice, via proteasome

Auxin regulates many aspects of plant growth and development by altering the expression of diverse genes. Among these, the early auxin-responsive genes of Aux/IAA class have been extensively studied in dicots but little information is available on monocots. Earlier, we reported the isolation of OsiIAA1 cDNA, first monocot member of Aux/IAA gene family from rice. Extending this work further, we have isolated the OsiIAA1 gene from rice localized on chromosome 3. The transcriptional start site was mapped to 158 bp upstream to the translational start site. The increased accumulation of OsiIAA1 transcript in auxin-treated rice coleoptiles even in the presence of a protein synthesis inhibitor, cycloheximide, suggested that OsiIAA1 is a primary auxin response gene; the expression of OsiIAA1 gene was also upregulated in the presence of cycloheximide alone. The OsiIAA1 transcript levels were down-regulated in etiolated rice coleoptiles irradiated with far-red, red and blue light, suggesting the existence of a cross-talk between auxin and light signaling. The antibodies raised against His 6–OsiIAA1 recombinant protein could detect the OsiIAA1 protein in the plant extract only in the presence of a proteasome inhibitor, MG132, indicating that OsiIAA1 is rapidly degraded by proteasome complex. The degradation of the protein was enhanced by the application of exogenous auxin. Also, the proteasome inhibitor MG132 stabilized the purified His 6–OsiIAA1 protein to some extent in the cell-free extracts of rice coleoptiles. The OsiIAA1 protein harbors two nuclear localization signals (NLSs), one bipartite and the other resembling SV40 type NLS. Although both the NLSs were able to target the protein to the nucleus, the bipartite NLS was more effective. These studies indicate that nuclear localization of OsiIAA1 could be a prerequisite for its role in auxin signal transduction.

Developmental regulation of 1‐aminocyclopropane‐1‐carboxylate synthase gene expression during leaf ontogeny in white clover

Reverse‐transcriptase‐dependent polymerase chain reaction (RT‐PCR) was used to identify three distinct DNA sequences (designated TR‐ACS1, TR‐ACS2 and TR‐ACS3), each with identity to 1‐aminocyclopropane‐1‐carboxylate synthase (ACS). Southern analysis confirmed that these sequences represented three distinct genes, and phylogenetic analysis showed that each gene was more closely related to homologues from other plant species than to each other. The expression of these three ACS genes during leaf ontogeny in white clover was examined. Northern analysis revealed that TR‐ACS1 was expressed in the apical structure of the stolon in mature‐green leaf tissue and in leaf tissue at the onset of senescence; RT‐PCR analysis showed that TR‐ACS2 was expressed in the apical structure of the stolon and in newly initiated leaf tissue. Furthermore, the expression of TR‐ACS1 was regulated by the exogenous application of auxin. The third gene, TR‐ACS3, was expressed in senescent leaf tissue, but translation of the nucleotide sequence provided an enzyme with a deleted binding site for pyridoxal‐phosphate and S‐adenosyl methionine (AdoMet), suggesting that this was a pseudogene. Antibodies, raised to the protein product of TR‐ACS1 expressed in Escherichia coli, recognized a protein of 55 kDa in leaf extracts of white clover using Western analysis, with the pattern of protein accumulation corresponding to the pattern of gene expression determined using Northern analysis. The significance of these results is discussed with respect to the developmental expression of ACS genes during leaf ontogeny.

Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation.

The effect of auxin on stamen and pistil development in tobacco flowers was investigated by means of the localized expression of rolB (root loci B), an Agrobacterium oncogene that increases auxin sensitivity in a cell-autonomous fashion. When rolB is driven by the promoter of the meiosis-specific Arabidopsis gene DMC1 (disrupted meiotic cDNA 1), expression occurs earlier in male than in female developing organs, resulting in a delay in anther dehiscence with respect to normal timing of pistil development. As a consequence of this developmental uncoupling, self-pollination is prevented in pDMC1:rolB plants. Histological analysis of pDMC1:GFP plants indicates that in tobacco, this promoter is active not only in meiocytes but also in somatic tissues of the anther. In contrast, simultaneous expression of rolB in anther and pistil somatic tissues, achieved by expressing a construct containing rolB under the control of the promoter of the petunia gene FBP7 (floral binding protein 7), results in a concomitant delay of both anther dehiscence and pistil development without affecting self-pollination of the plants. Analysis of plants harboring the pFBP7:GUS construct shows that in tobacco, this promoter is active not only in the ovules, as described for petunia, but also in pistil and anther somatic tissues involved in the dehiscence program. The delay in anther dehiscence and pistil development could be phenocopied by exogenous application of auxin. Jasmonic acid (JA) could not rescue the delay in anther dehiscence. These results suggest that auxin plays a key role in the timing of anther dehiscence, the dehiscence program is controlled by the somatic tissues of the anther, and auxin also regulates pistil development.

Effect of exogenous indole butyric acid on root formation and peroxidase and indole-3-acetic acid oxidase activities and phenolic contents in date Palm offshoots

Exogenous indole butyric acid had a significant positive effect on the rooting response of date Palm (Phoenix dactylifera L.) offshoots. IBA-treated offshoots rooted earlier and with a much higher frequency while untreated offshoots rooted poorly, later, and tended to produce fewer roots. The effect of exogenous application of auxin was also reflected in the metabolic changes in offshoots during the rooting process. Pox activity showed a minimum on the sixth day and a maximum on the twelfth day after the root-inducing treatment. Changes of IAA oxidase activity showed a parallel time curve while phenolic contents and auxin protector levels showed an inverse trend. These parameters changed slightly one week later in the untreated offshoots.

Characterization of a symbiosis- and auxin-regulated glutathione- S-transferase from Eucalyptus globulus roots

The formation of ectomycorrhizal symbiotic tissues between tree roots and fungal hyphae is accompanied by large morphological and molecular modifications. In the Eucalyptus globulus- Pisolithus microcarpus ectomycorrhiza, it has been shown that many plant genes are up-regulated during the onset of the interaction. Among them, EgHypar showed a high degree of homology with plant auxin-induced glutathione- S-transferases (GST). Its expression was up-regulated not only in the ectomycorrhiza, but also by exogenous application of auxins and hypaphorine, the tryptophane betaine excreted by P. microcarpus during ectomycorrhiza formation. In this study, a recombinant EgHYPAR polypeptide was produced in Escherichia coli. The purified polypeptide showed an in vitro GST activity, which was inhibited by indole-3-acetic acid but not by synthetic auxins or hypaphorine. Antibodies were raised against the recombinant EgHYPAR polypeptide and western blot analyses showed that the accumulation of the EgHYPAR polypeptide was enhanced in ectomycorrhizas. Immunolocalization studies demonstrated a preferential cytosolic localization of EgHYPAR close to the plasma membrane and the cell wall in cells of both un-colonized and colonized roots. The putative role of the EGHYPAR GST in mycorrhiza development will be discussed.

An abscisic acid-sensitive checkpoint in lateral root development of Arabidopsis.

Lateral root (LR) formation displays considerable plasticity in response to developmental and environmental signals. The mechanism whereby plants incorporate diverse regulatory signals into the developmental programme of LRs remains to be elucidated. Current concepts of lateral root regulation focus on the role of auxin. In this study, we show that another plant hormone, abscisic acid (ABA), also plays a critical role in the regulation of this post-embryonic developmental event. In the presence of exogenous ABA, LR development is inhibited. This occurs at a specific developmental stage, i.e. immediately after the emergence of the LR primordium (LRP) from the parent root and prior to the activation of the LR meristem, and is reversible. Interestingly, this inhibition requires 10-fold less ABA than the inhibition of seed germination and is only slightly reduced in characterised abi mutants, suggesting that it may involve novel ABA signalling mechanisms. We also present several lines of evidence to support the conclusion that the ABA-induced lateral root inhibition is mediated by an auxin-independent pathway. First, the inhibition could not be rescued by either exogenous auxin application or elevated auxin synthesis. Secondly, a mutation in the ALF3 gene, which is believed to encode an important component in the auxin-dependent regulatory pathway for the post-emergence LR development, does not affect the sensitivity of LRs to ABA. Thirdly, ABA and the alf3-1 mutation do not act at the same developmental point. To summarise, these results demonstrate a novel ABA-sensitive, auxin-independent checkpoint for lateral root development in Arabidopsis at the post-emergence stage. In addition, we also present data indicating that regulation of this developmental checkpoint may require novel ABA signalling mechanisms and that ABA suppresses auxin response in the LRPs.

GFP accumulation controlled by an auxin-responsive promoter as a non-destructive assay to monitor early auxin response

We have constructed transgenic Arabidopsis lines that contain a gene for green fluorescent protein (GFP) under the control of auxin-responsive domains A and B of the promoter from the pea PS-IAA4/5 gene. The chimeric transgene was named BA-mgfp5-ER. GFP was detected after the application of indole-3-acetic acid at concentrations as low as 100 nM in epidermal cells in the root elongation zone. The induction of the reporter gene was highly specific to auxin and was correlated with the auxin-induced change in epidermal cell shape. No GFP accumulation was observed in the lateral root meristem that was formed as a result of exogenous auxin application. These results suggest that auxin signals were transmitted through several distinct pathways depending on the cell type. The intensity of the GFP signal was strong enough to be observed through the plastic lid of the culture dish using a dissecting microscope, thereby enabling GFP expression to be monitored in an aseptic environment. Thus, the BA-mgfp5-ER transgenic plant can be a powerful tool for screening mutants that are defective in auxin signaling and the expression of early auxin-response genes.

Maize VP1 complements Arabidopsis abi3 and confers a novel ABA/auxin interaction in roots.

The maize Vp1 gene and abi3 gene of Arabidopsis are believed to be orthologs based on similarities of the mutant phenotypes and amino acid sequence conservation. Here we show that expression of VP1 driven by the 35S promoter can partially complement abi3-6, a deletion mutant allele of abi3. The visible phenotype of seed produced from VP1 expression in the abi3 mutant background is nearly indistinguishable from wild type. VP1 fully restores abscisic acid (ABA) sensitivity of abi3 during seed germination and suppresses the early flowering phenotype of abi3. The temporal regulation of C1-beta-glucuronidase (GUS) and chlorophyll a/b binding protein (cab3)-GUS reporter genes in developing seeds of 35S-VP1 lines were similar to wild type. On the other hand, two qualitative differences are observed between the 35S-VP1 line and wild type. The levels of CRC and C1-GUS expression are markedly lower in the seeds of 35S-VP1 lines than in wild type suggesting incomplete complementation of gene activation functions. Similar to ectopic expression of ABI3 (Parcy et al., 1994), ectopic expression of VP1 in vegetative tissue enhances ABA inhibition of root growth. In addition, 35S-VP1 confers strong ABA inducible expression of the normally seed-specific cruciferin C (CRC) gene in leaves. In contrast, ectopic ABA induction of C1-GUS is restricted to a localized region of the root elongation zone. The ABA-dependent C1-GUS expression expanded to a broader area in the root tissues treated with exogenous application of auxin. Interestingly, auxin-induced lateral root formation is completely suppressed by ABA in 35S-VP1 plants but not in wild type. These results indicate VP1 mediates a novel interaction between ABA and auxin signaling that results in developmental arrest and altered patterns of gene expression.

Foliar and Axial Aspects of Vascular Differentiation: Hypotheses and Evidence

A comparison is made between foliar and axial vascular differentiation. Current thoughts and new evidence are presented on the role of hormones in controlling the differentiation of vascular tissues in organized and tumorous tissues, focusing on the role of auxin and cytokinin in controlling phloem and xylem relationships, vessel size and density, cambium sensitivity, vascular adaptation and xylem evolution in deciduous hardwood trees. The possible role of wounding is also considered. A new hypothesis, namely, the leaf-venation hypothesis, is proposed to explain the hormonal control of vascular differentiation in leaves of dicotyledonous plants. Experimental evidence in support of the hypothesis is presented showing that hydathodes, the watersecreting glands, are the primary sites of auxin synthesis during leaf morphogenesis. Vessel element patterns similar to those found in hydathodes were experimentally induced by exogenous auxin application.

Varietal differences in the susceptibility to pointed fruit malformation in tomatoes: histological studies of the ovaries

Differences were found among three tomato cultivars (cvs.) in their susceptibility to pointed-fruit malformation. Pointed fruits are characterized by a conical, rather than a round shape. This type of fruit malformation is often found in winter-grown plants and is due to an uneven development of the locules. Transverse cross-sections of the fruit revealed that one or more of the locules had not developed, forming a flattened side, while the other side of the fruit was round (normal) in shape, because of the presence of developed placenta(s), with seeds and jelly filling their cavity(ies). Histological examination of the ovaries showed that under high night temperatures all the locules, of all the examined cvs., were normal and contained placentas with attached ovules. Ovaries that developed under lower night temperatures contained deformed locules: in the less severe cases the placenta was retarded, with fewer attached ovules than normal; in the most severe ones the locules were severely deformed and the placentas and ovules were absent. The number of deformed ovules per fruit and the degree of deformation were cultivar- and night temperature-dependent. Artificial pollination of the affected ovaries and exogenous auxin application did not improve the fruit shape. It is, therefore, concluded that the absence of placentas, seeds and jelly from one or more locules brings about the formation of a malformed, pointed (lop-sided) fruit.

AnArabidopsis thalianaMutant with Virus-Inducible Phenotype

The role of host factors in plant viral diseases is not well understood. To study this important aspect of plant–pathogen interaction, we identified anArabidopsis thalianamutant, designatedvid1(virus-inducibledwarf), with altered responses to viral infection. Specifically,vid1resembled the wild-type plants when healthy but developed a severely dwarfed phenotype with a loss of apical dominance following infection by a tobamovirus. Genetic segregation showed that thevid1phenotype is caused by a recessive mutation in a single gene. Since systemic viral infection is thought to interfere with the host plant intercellular transport, we propose that thevid1mutation affects this transport process. Combination of the mutation and viral infection may disrupt transport of developmental regulators, such as hormones, causing formation of thevid1phenotype. Indeed, the effect ofvid1mutation was repressed by exogenous application of a plant hormone auxin. Potentially, thevid1mutant will help characterize the mechanism of virus–plant interaction and formation of plant viral disease symptoms.

Polarity, vascularization and auxin

Polarity, vascularization and auxin

Three 1-aminocyclopropane-1-carboxylate synthase genes regulated by primary and secondary pollination signals in orchid flowers.

The temporal and spatial expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes were investigated in pollinated orchid (Phalaenopsis spp.) flowers. Pollination signals initiate a cascade of development events in multiple floral organs, including the induction of ethylene biosynthesis, which coordinates several postpollination developmental responses. The initiation and propagation of ethylene biosynthesis is regulated by the coordinated expression of three distinct ACC synthase genes in orchid flowers. One ACC synthase gene (Phal-ACS1) is regulated by ethylene and participates in amplification and interorgan transmission of the pollination signal, as we have previously described in a related orchid genus. Two additional ACC synthase genes (Phal-ACS2 and Phal-ACS3) are expressed primarily in the stigma and ovary of pollinated orchid flowers. Phal-ACS2 mRNA accumulated in the stigma within 1 h after pollination, whereas Phal-ACS1 mRNA was not detected until 6 h after pollination. Similar to the expression of Phal-ACS2, the Phal-ACS3 gene was expressed within 2 h after pollination in the ovary. Exogenous application of auxin, but not ACC, mimicked pollination by stimulating a rapid increase in ACC synthase activity in the stigma and ovary and inducing Phal-ACS2 and Phal-ACS3 mRNA accumulation in the stigma and ovary, respectively. These results provide the basis for an expanded model of interorgan regulation of three ACC synthase genes that respond to both primary (Phal-ACS2 and Phal-ACS3) and secondary (Phal-ACS1) pollination signals.

Reversible protein phosphorylation regulates jasmonic acid-dependent and -independent wound signal transduction pathways in Arabidopsis thaliana.

Plants responses to mechanical injury are complex and include the induced expression of defence-related genes. The phytohormone JA has been reported to mediate some of these responses. To elucidate further the signal transduction processes involved, the action of specific agonists and antagonists of known signalling effectors on the response of Arabidopsis thaliana plantlets to JA and wounding was investigated. The identification and characterization of a reversible protein phosphorylation step in a transduction pathway leading to JA-induced gene transcription is reported. This phosphorylation event involved the opposing activities of a staurosporine-sensitive protein kinase, negatively regulating the pathway, and a protein phosphatase, most probably of type 2 A, which activated JA-responsive gene expression. JA activation via this pathway was blocked in the A. thaliana JA-insensitive mutants jin1, jin4 and coi1, and by exogenous application of cycloheximide or auxins. Wound-induced activation of JA-responsive genes was also regulated by this protein phosphorylation step. An alternative wound signalling pathway, independent of JA, was also identified, leading to the transcriptional activation of a different set of genes. This JA-independent pathway was also regulated by a protein phosphorylation switch, in which the protein kinase positively regulated the pathway while the protein phosphatase negatively regulated it. Moreover, a labile protein apparently repressed the expression of these genes. One of the genes analysed, JR3, had a complex pattern of expression, possibly because it was regulated via both of the wound signalling pathways identified. According to the function of an homologous gene, JR3 may be involved in feedback inhibition of the JA response.

Endogenous plant growth regulators and rooting capacity of different walnut tissues

Rooting ability of three different materials, namely cotyledonar linking areas, cotyledonary portions and embryonic microshoots from walnut seeds was determined. It was shown that while cotyledonar linking areas may be defined as a espontaneous rooting system, the other two tissue types correspond to inducible rooting systems, needing exogenous application of auxins for root induction. Endogenous zeatin and isopentenyl type cytokinins, indole-3-acetic acid (IAA), and abscisic acid (ABA) contents were determined in the three kinds of plant material prior to their culture. Our results show that the most active rooting systems has high levels of cytokinins, particularly dihydrozeatin (diH)Z and its riboside, and low levels of IAA and ABA. This situation could indicate that oxidative metabolism was taking place in cotyledonar linking areas, which is generally associated with the initiation phase of the rooting process. So that, we can assume that the induction phase is already overpassed in these tissues. On the contrary, the other two systems in which high levels of auxins and reduced levels of cytokinins were found, need to undergo the induction phase to be able to show root manifestation. In this case, the exogenous auxins are responsible for triggering this process, by enhancement of oxidative conditions. Therefore, it can be conclude that dynamics of growth regulators levels during the different phases of rooting are more important than specific single levels at fixed time.

Hybrids of Crosses between Oat and Andropogoneae or Paniceae Species

Plant breeders are interested in enlarging the genetic variability within a species of interest and in improving breeding strategies. A wide hybridization program was conducted from 1990 to 1994 in Germany to determine the prospects for sexual gene transfer or haploid production in oat (Avena sativa L.). Five cultivars of oat were pollinated with pearl millet [Pennisetum americanum (L.) Leeke], eastern gamagrass [Tripsacum dactyloides (L.) L.], and maize (Zea mays L.; 2x and 4x) in the greenhouse. Exogenous auxin application, embryo rescue, and early colchicine‐mediated doubling of chromosomes were used to overcome postzygotic barriers. The recovered plants were analyzed cytologically and phenotypically under greenhouse and field conditions. Depending on the pollinator species, the embryo frequencies varied from 0.4% (Z. mays 4x) to 9.8% (pearl millet). In embryo rescue, initial growth occurred frequently, but many plantlets died. Chromosomes of the pollinator species were lost late in embryo and plant development. One to four chromosomes of the pollinator species were found at the tillering stage in root tip cells. Overall, four viable plants were produced, among them hybrids with pearl millet and eastern gamagrass for the first time. The efficiency of haploid production (<0.1%) was too low in all combinations for application in plant breeding programs; however, the transfer of genes or chromosomes appears promising in crosses of oat with maize and pearl millet.

Metabolic changes during rooting in stem cuttings of five mangrove species

Vegetative propagation through rooting in stem cuttings in five tree mangroves namely Bruguiera parviflora, Cynometra iripa, Excoecaria agallocha, Heritiera fomes, and Thespesia populnea using IAA, IBA and NAA was reported. Spectacular increase in the root number was noted in the cuttings of H. fomes and C. iripa treated together with IBA (5000 ppm) and NAA (2500 ppm). The highest number of roots was obtained with IBA (2500 ppm) and NAA (500 ppm) in E. agallocha. B. parviflora and T. populnea responded better to IAA and IBA treatment. The species specific variation in the rooting response to exogenous application of auxins was reflected in the metabolic changes during initiation and development of roots in cuttings. Biochemical analysis showed increase of reducing sugar in the above-girdled tissues at initiation as well as subsequent development of roots which was further enhanced by the use of auxins. Decreases in the total sugar, total carbohydrate and polyphenols and increase in total nitrogen were recorded in the girdled tissues and the high C/N ratio at the initial stage helped in initiation of roots in all the species. Interaction of IBA and NAA promoted starch hydrolysis better than IAA and IBA during root development and subsequently reduced the C/N ratio and increased the protein-nitrogen activity during root development which suggest the auxin influenced mobilization of nitrogen to the rooting zone.

Construction of a trans-dominant inhibitor for members of the TGA family of transcription factors conserved in higher plants.

Activating sequence factor 1 (ASF-1) is a conserved DNA-binding activity that interacts with sequence elements containing TGACG motifs, some of which have been demonstrated to respond to exogenous application of auxin and salicylic acid. Genes encoding transcription factors with similar DNA-binding specificity to ASF-1 have been cloned from diverse plant species and these factors all contain a distinct basic-leucine-zipper (bZIP) motif. Members of this family of DNA-binding proteins, designated as TGA factors, have been shown to interact with similar DNA sequences and at least seven distinct TGA genes are present in the Arabidopsis genome. To study the roles that this family of factors may play in plant development, a trans-dominant inhibitor of ASF-1 was constructed by deleting the basic portion of the bZIP domain in the tobacco factor TGA1a. In vitro co-expression studies demonstrated that this deletion mutant, named TGA1a-D, suppresses the DNA-binding activity of wild-type tobacco TGA1a, and three different members of the Arabidopsis TGA family. In contrast, co-expression of TGA1a-D with another class of bZIP proteins, the G-Box Binding Factor family, showed no suppression of DNA-binding activity. Over-expression of TGA1a-D in transgenic tobacco significantly decreased nuclear ASF-1 relative to several other known factors, indicating that the proteins comprising ASF-1 activity in vivo are likely TGA family members. Thus, TGA1a-D may be a family-specific inhibitor for the TGA family and should facilitate the study of ASF-1 function in vivo.