Rapid cellular responses to auxin and the regulation of growth

Abstract The cellular responses rapidly evoked by auxin are reviewed, and related to a consideration of how growth rate is regulated in excised segments and in whole dicotyledonous plants. Two processes, synthesis of proteins and of cell wall components, are both promoted by auxin and essential for auxin-stimulated growth, whereas other processes show little promotion by auxin or do not appear essential for growth. Current models for the cellular regulation of growth by auxin are briefly discussed, and a new model presented. Auxin is suggested to act by bringing about a transient increase in cytosolic Ca²⁺ levels, which through the stimulation of protein kinases converts a cytoplasmic protein factor to an active state capable of binding auxin. The protein-auxin complex induces mRNA synthesis, which effects the increased synthesis of cell wall components and their incorporation into the wall, resulting in wall loosening and growth. It is proposed that the factor limiting growth in floating excised segments may initially be cell wall pH, but that this is not the case in whole plants and growth is instead mediated by increased protein and matrix cell wall synthesis. Differences are noted between monocotyledonous coleoptiles and dicotyledonous stems in some metabolic processes possibly involved in auxin growth responses, and it is cautioned that observations made on one tissue may not necessarily be applicable to the other. Care should also be taken in applying conclusions drawn from studies on excised tissue to the interpretation of growth regulation in the whole plant.     

Primary or secondary? Versatile nitrilases in plant metabolism

The potential of plant nitrilases to convert indole-3-acetonitrile into the plant growth hormone indole-3-acetic acid has earned them the interim title of "key enzyme in auxin biosynthesis". Although not widely recognized, this view has changed considerably in the last few years. Recent work on plant nitrilases has shown them to be involved in the process of cyanide detoxification, in the catabolism of cyanogenic glycosides and presumably in the catabolism of glucosinolates. All plants possess at least one nitrilase that is homologous to the nitrilase 4 isoform of Arabidopsis thaliana. The general function of these nitrilases lies in the process of cyanide detoxification, in which they convert the intermediate detoxification product beta-cyanoalanine into asparagine, aspartic acid and ammonia. Cyanide is a metabolic by-product in biosynthesis of the plant hormone ethylene, but it may also be released from cyanogenic glycosides, which are present in a large number of plants. In Sorghum bicolor, an additional nitrilase isoform has been identified, which can directly use a catabolic intermediate of the cyanogenic glycoside dhurrin, thus enabling the plant to metabolize its cyanogenic glycoside without releasing cyanide. In the Brassicaceae, a family of nitrilases has evolved, the members of which are able to hydrolyze catabolic products of glucosinolates, the predominant secondary metabolites of these plants. Thus, the general theme of nitrilase function in plants is detoxification and nitrogen recycling, since the valuable nitrogen of the nitrile group is recovered in the useful metabolites asparagine or ammonia. Taken together, a picture emerges in which plant nitrilases have versatile functions in plant metabolism, whereas their importance for auxin biosynthesis seems to be minor.     

Halotropism requires phospholipase Dζ1-mediated modulation of cellular polarity of auxin transport carriers

Endocytosis and relocalization of auxin carriers represent important mechanisms for adaptive plant growth and developmental responses. Both root gravitropism and halotropism have been shown to be dependent on relocalization of auxin transporters. Following their homology to mammalian phospholipase Ds (PLDs), plant PLDζ-type enzymes are likely candidates to regulate auxin carrier endocytosis. We investigated root tropic responses for an Arabidopsis pldζ1-KO mutant and its effect on the dynamics of two auxin transporters during salt stress, that is, PIN2 and AUX1. We found altered root growth and halotropic and gravitropic responses in the absence of PLDζ1 and report a role for PLDζ1 in the polar localization of PIN2. Additionally, irrespective of the genetic background, salt stress induced changes in AUX1 polarity. Utilizing our previous computational model, we found that these novel salt-induced AUX1 changes contribute to halotropic auxin asymmetry. We also report the formation of “osmotic stress-induced membrane structures.” These large membrane structures are formed at the plasma membrane shortly after NaCl or sorbitol treatment and have a prolonged presence in a pldζ1 mutant. Taken together, these results show a crucial role for PLDζ1 in both ionic and osmotic stress-induced auxin carrier dynamics during salt stress.     

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.     

Plant growth hormones suppress the development of Harpophora maydis,the cause of late wilt in maize

Late wilt, a severe vascular disease of maize caused by the fungus Harpophora maydis, is characterized by rapid wilting of maize plants before tasseling and until shortly before maturity. The pathogen is currently controlled by resistant maize cultivars, but the disease is constantly spreading to new areas. The plant’s late phenological stage at which the disease appears suggests that plant hormones may be involved in the pathogenesis. This work revealed that plant growth hormones, auxin (Indole-3-acetic acid) and cytokinin (kinetin), suppress H. maydis in culture media and in a detached root assay. Kinetin, and even more auxin, caused significant suppression of fungus spore germination. Gibberellic acid did not alter colony growth rate but had a signal suppressive effect on the pathogens’ spore germination. In comparison, ethylene and jasmonic acid, plant senescing and defense response regulators, had minor effects on colony growth and spore germination rate. Their associate hormone, salicylic acid, had a moderate suppressive effect on spore germination and colony growth rate, and a strong influence when combined with auxin. Despite the anti-fungal auxin success in vitro, field experiments with dimethylamine salt of  2,4-dichlorophenoxyacetic acid (that mimics the influence of auxin) failed to suppress the late wilt. The lines of evidence presented here reveal the suppressive influence of the three growth hormones studied on fungal development and are important to encourage further and more in-depth examinations of this intriguing hormonal complex regulatory and its role in the maize-H. maydis interactions.     

Characterisation of root amino acid exudation in white clover (Trifolium repens L.)

Lateral roots are crucial for the plasticity of root responses to environmental conditions in soil. The bacterivorous microfauna has been shown to increase root branching and to foster auxin producing soil bacteria. However, information on modifications of plant internal auxin content by soil bacteria and bacterivores is missing. Therefore, the effects of a rhizosphere bacterial community and a common soil amoeba (Acanthamoeba castellanii) on root branching and on auxin (indole-3-acetic acid) metabolism in Lepidium sativum and Arabidopsis thaliana were investigated. In a first experimental series, bacteria increased conjugated auxin concentrations in L. sativum shoots, but did not alter free bioactive auxin content nor root branching. In contrast, in presence of soil bacteria plus amoebae free auxin concentrations in shoots and root branching increased, demonstrating that effects of bacteria on auxin metabolism in plants were strongly modified by the bacterivorous amoebae. In a second experiment, A. thaliana reporter plants for auxin (DR5) and cytokinin (ARR5) responded similarly with increased root branching in the presence of amoebae. Surprisingly, in reporter plants cytokinin but not auxin responses were detectable, accompanied by higher soil nitrate concentrations in the presence of amoebae. Likely, increased nitrate concentrations in the rhizosphere led to an accumulation of cytokinin and interactions with free auxin in plants and finally to increased root growth in the presence of amoebae. Altogether, the results show that mutual control mechanisms exist between plant hormone metabolism and microbial signalling, and that effects on hormonal concentrations of plants by free-living bacteria are strongly influenced by bacterial grazers like amoebae.     

The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36-amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin-ethylene interactions.     

ROP3 GTPase Contributes to Polar Auxin Transport and Auxin Responses and Is Important for Embryogenesis and Seedling Growth in Arabidopsis

ROP GTPases are crucial for the establishment of cell polarity and for controlling responses to hormones and environmental signals in plants. In this work, we show that ROP3 plays important roles in embryo development and auxin-dependent plant growth. Loss-of-function and dominant-negative (DN) mutations in ROP3 induced a spectrum of similar defects starting with altered cell division patterning during early embryogenesis to postembryonic auxin-regulated growth and developmental responses. These resulted in distorted embryo development, defective organ formation, retarded root gravitropism, and reduced auxin-dependent hypocotyl elongation. Our results showed that the expression of AUXIN RESPONSE FACTOR5/MONOPTEROS and root master regulators PLETHORA1 (PLT1) and PLT2 was reduced in DN-rop3 mutant embryos, accounting for some of the observed patterning defects. ROP3 mutations also altered polar localization of auxin efflux proteins (PINs) at the plasma membrane (PM), thus disrupting auxin maxima in the root. Notably, ROP3 is induced by auxin and prominently detected in root stele cells, an expression pattern similar to those of several stele-enriched PINs. Our results demonstrate that ROP3 is important for maintaining the polarity of PIN proteins at the PM, which in turn ensures polar auxin transport and distribution, thereby controlling plant patterning and auxin-regulated responses.     

Cytidinediphosphate diacylglycerol synthase—Mediated phosphatidic acid metabolism is crucial for early embryonic development of Arabidopsis

Embryonic development is a key developmental event in plant sexual reproduction; however, regulatory networks of plant early embryonic development, particularly the effects and functional mechanisms of phospholipid molecules are still unknown due to the limitation of sample collection and analysis. We innovatively applied the microspore-derived in vitro embryogenesis of Brassica napus and revealed the dynamics of phospholipid molecules, especially phosphatidic acid (PA, an important second messenger that plays an important role in plant growth, development, and stress responses), at different embryonic developmental stages by using a lipidomics approach. Further analysis of Arabidopsis mutants deficiency of CDS1 and CDS2 (cytidinediphosphate diacylglycerol synthase, key protein in PA metabolism) revealed the delayed embryonic development from the proembryo stage, indicating the crucial effect of CDS and PA metabolism in early embryonic development. Decreased auxin level and disturbed polar localization of auxin efflux carrier PIN1 implicate that CDS-mediated PA metabolism may regulate early embryogenesis through modulating auxin transport and distribution. These results demonstrate the dynamics and importance of phospholipid molecules during embryo development, and provide informative clues to elucidate the regulatory network of embryogenesis.     

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.     

Involvement of nitric oxide and auxin in signal transduction of copper-induced morphological responses in Arabidopsis seedlings

Background and Aims

Plants are able to adapt to the environment dynamically through regulation of their growth and development. Excess copper (Cu²⁺), a toxic heavy metal, induces morphological alterations in plant organs; however, the underlying mechanisms are still unclear. With this in mind, the multiple signalling functions of nitric oxide (NO) in plant cells and its possible regulatory role and relationship with auxin were examined during Cu²⁺-induced morphological responses.

Methods

Endogenous auxin distribution was determined by microscopic observation of X-Gluc-stained DR5::GUS arabidopsis, and the levels of NO, superoxide and peroxynitrite were detected by fluorescence microscopy. As well as wild-type, NO-overproducer (nox1) and -deficient (nia1nia2 and nia1nia2noa1-2) arabidopsis plants were used.

Key Results

Cu²⁺ at a concentration of 50 µm resulted in a large reduction in cotyledon area and hypocotyl and primary root lengths, accompanied by an increase in auxin levels. In cotyledons, a low Cu²⁺ concentration promoted NO accumulation, which was arrested by nitric oxide synthase or nitrate reductase inhibitors. The 5-μm Cu²⁺-induced NO synthesis was not detectable in nia1nia2 or nia1nia2noa1-2 plants. In roots, Cu²⁺ caused a decrease of the NO level which was not associated with superoxide and peroxynitrite formation. Inhibition of auxin transport resulted in an increase in NO levels, while exogenous application of an NO donor reduced DR5::GUS expression. The elongation processes of nox1 were not sensitive to Cu²⁺, but NO-deficient plants showed diverse growth responses.

Conclusions

In plant organs, Cu²⁺ excess results in severe morphological responses during which the endogenous hormonal balance and signal transduction are affected. Auxin and NO negatively regulate each other''s level and NO intensifies the metal-induced cotyledon expansion, but mitigates elongation processes under Cu²⁺ exposure.     

Auxin and Ethylene Regulate Elongation Responses to Neighbor Proximity Signals Independent of Gibberellin and DELLA Proteins in Arabidopsis

Plants modify growth in response to the proximity of neighbors. Among these growth adjustments are shade avoidance responses, such as enhanced elongation of stems and petioles, that help plants to reach the light and outgrow their competitors. Neighbor detection occurs through photoreceptor-mediated detection of light spectral changes (i.e. reduced red:far-red ratio [R:FR] and reduced blue light intensity). We recently showed that physiological regulation of these responses occurs through light-mediated degradation of nuclear, growth-inhibiting DELLA proteins, but this appeared to be only part of the full mechanism. Here, we present how two hormones, auxin and ethylene, coregulate DELLAs but regulate shade avoidance responses through DELLA-independent mechanisms in Arabidopsis (Arabidopsis thaliana). Auxin appears to be required for both seedling and mature plant shoot elongation responses to low blue light and low R:FR, respectively. Auxin action is increased upon exposure to low R:FR and low blue light, and auxin inhibition abolishes the elongation responses to these light cues. Ethylene action is increased during the mature plant response to low R:FR, and this growth response is abolished by ethylene insensitivity. However, ethylene is also a direct volatile neighbor detection signal that induces strong elongation in seedlings, possibly in an auxin-dependent manner. We propose that this novel ethylene and auxin control of shade avoidance interacts with DELLA abundance but also controls independent targets to regulate adaptive growth responses to surrounding vegetation.     

GH3 expression and IAA-amide synthetase activity in pea (Pisum sativum L.) seedlings are regulated by light,plant hormones and auxinic herbicides

The formation of auxin conjugates is one of the important regulatory mechanisms for modulating IAA action. Several auxin-responsive GH3 genes encode IAA-amide synthetases that are involved in the maintenance of hormonal homeostasis by conjugating excess IAA to amino acids. Recently, the data have revealed novel regulatory functions of several GH3 proteins in plant growth, organ development, fruit ripening, light signaling, abiotic stress tolerance and plant defense responses. Indole-3-acetyl-aspartate (IAA-Asp) synthetase catalyzing IAA conjugation to aspartic acid in immature seeds of pea (Pisum sativum L.) was purified and characterized during our previous investigations. In this study, we examined the effect of auxin and other plant hormones (ABA, GA, kinetin, JA, MeJA, SA), different light conditions (red, far-red, blue, white light), and auxinic herbicides (2,4-D, Dicamba, Picloram) on the expression of a putative GH3 gene and IAA-amide synthesizing activity in 10-d-old pea seedlings. Quantitative RT-PCR analysis indicated that the PsGH3-5 gene, weakly expressed in control sample, was visibly induced in response to all plant hormones, different light wavelengths and the auxinic herbicides tested. Protein A immunoprecipitation/gel blot analysis using anti-AtGH3.5 antibodies revealed a similar pattern of changes on the protein levels in response to all treatments. IAA-amide synthetase activity determined with aspartate as a substrate, not detectable in control seedlings, was positively affected by a majority of treatments. Based on these results, we suggest that PsGH3-5 may control the growth and development of pea plants in a way similar to the known GH3 genes from other plant species.     

The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass

Switchgrass (Panicum virgatum L.; family Poaceae) is a warm-season C4 perennial grass. Tillering plays an important role in determining the morphology of aboveground parts and the final biomass yield of switchgrass. Auxin distribution in plants can affect a variety of important growth and developmental processes, including the regulation of shoot and root branching, plant resistance and biological yield. Auxin transport and gradients in plants are mediated by influx and efflux carriers. PvPIN1, a switchgrass PIN1-like gene that is involved in regulating polar transport, is a putative auxin efflux carrier. Neighbor-joining analysis using sequences deposited in NCBI databases showed that the PvPIN1gene belongs to the PIN1 family and is evolutionarily closer to the Oryza sativa japonica group. Tiller emergence and development was significantly promoted in plants subjected toPvPIN1 RNA interference (RNAi), which yielded a phenotype similar to that of wild-type plants treated with the auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid). A transgenic approach that inducedPvPIN1 gene overexpression or suppression altered tiller number and the shoot/root ratio. These data suggest that PvPIN1plays an important role in auxin-dependent adventitious root emergence and tillering.