Analysis the role of arabidopsis <Emphasis Type="Italic">CKRC6/ASA1</Emphasis> in auxin and cytokinin biosynthesis

The crosstalk between auxin and cytokinin (CK) is important for plant growth and development, although the underlying molecular mechanisms remain unclear. Here, we describe the isolation and characterization of a mutant of Arabidopsis Cytokinin-induced Root Curling 6 (CKRC6), an allele of ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) that encodes the á-subunit of AS in tryptophan (Trp) biosynthesis. The ckrc6 mutant exhibits root gravitropic defects and insensitivity to both CK and the ethylene precursor 1-aminocyclopropane-1-carboxylicacid (ACC) in primary root growth. These defects can be rescued by exogenous indole-3-acetic acid (IAA) or tryptophan (Trp) supplementation. Furthermore, our results suggest that the ckrc6 mutant has decreased IAA content, differential expression patterns of auxin biosynthesis genes and CK biosynthesis isopentenyl transferase (IPT) genes in comparison to wild type. Collectively, our study shows that auxin controls CK biosynthesis based on that CK sensitivity is altered in most auxin-resistant mutants and that CKs promote auxin biosynthesis but inhibit auxin transport and response. Our results also suggest that CKRC6/ASA1 may be located at an intersection of auxin, CK and ethylene metabolism and/or signaling.     

Plant-derived auxin plays an accessory role in symptom development upon Rhodococcus fascians infection

The biotrophic phytopathogen Rhodococcus fascians has a profound impact on plant development, mainly through its principal virulence factors, a mix of synergistically acting cytokinins that induce shoot formation. Expression profiling of marker genes for several auxin biosynthesis routes and mutant analysis demonstrated that the bacterial cytokinins stimulate the auxin biosynthesis of plants via specific targeting of the indole-3-pyruvic acid (IPA) pathway, resulting in enhanced auxin signaling in infected tissues. The double mutant tryptophan aminotransferase 1-1 tryptophan aminotransferase related 2-1 (taa1-1 tar2-1) of Arabidopsis (Arabidopsis thaliana), in which the IPA pathway is defective, displayed a decreased responsiveness towards R. fascians infection, although bacterial colonization and virulence gene expression were not impaired. These observations implied that plant-derived auxin was employed to reinforce symptom formation. Furthermore, the increased auxin production and, possibly, the accumulating bacterial cytokinins in infected plants modified the polar auxin transport so that new auxin maxima were repetitively established and distributed, a process that is imperative for symptom onset and maintenance. Based on these findings, we extend our model of the mode of action of bacterial and plant signals during the interaction between R. fascians and Arabidopsis.     

TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development

Plants have evolved a tremendous ability to respond to environmental changes by adapting their growth and development. The interaction between hormonal and developmental signals is a critical mechanism in the generation of this enormous plasticity. A good example is the response to the hormone ethylene that depends on tissue type, developmental stage, and environmental conditions. By characterizing the Arabidopsis wei8 mutant, we have found that a small family of genes mediates tissue-specific responses to ethylene. Biochemical studies revealed that WEI8 encodes a long-anticipated tryptophan aminotransferase, TAA1, in the essential, yet genetically uncharacterized, indole-3-pyruvic acid (IPA) branch of the auxin biosynthetic pathway. Analysis of TAA1 and its paralogues revealed a link between local auxin production, tissue-specific ethylene effects, and organ development. Thus, the IPA route of auxin production is key to generating robust auxin gradients in response to environmental and developmental cues.     

The rice FISH BONE gene encodes a tryptophan aminotransferase,which affects pleiotropic auxin‐related processes

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole‐3‐pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole‐3‐acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin‐related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.     

Disturbed Local Auxin Homeostasis Enhances Cellular Anisotropy and Reveals Alternative Wiring of Auxin-ethylene Crosstalk in Brachypodium distachyon Seminal Roots

Observations gained from model organisms are essential, yet it remains unclear to which degree they are applicable to distant relatives. For example, in the dicotyledon Arabidopsis thaliana (Arabidopsis), auxin biosynthesis via indole-3-pyruvic acid (IPA) is essential for root development and requires redundant TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and TAA1-RELATED (TAR) genes. A promoter T-DNA insertion in the monocotyledon Brachypodium distachyon (Brachypodium) TAR2-LIKE gene (BdTAR2L) severely down-regulates expression, suggesting reduced tryptophan aminotransferase activity in this mutant, which thus represents a hypomorphic Bdtar2l allele (Bdtar2l^hypo). Counterintuitive however, Bdtar2l^hypo mutants display dramatically elongated seminal roots because of enhanced cell elongation. This phenotype is also observed in another, stronger Bdtar2l allele and can be mimicked by treating wild type with L-kynerunine, a specific TAA1/TAR inhibitor. Surprisingly, L-kynerunine-treated as well as Bdtar2l roots display elevated rather than reduced auxin levels. This does not appear to result from compensation by alternative auxin biosynthesis pathways. Rather, expression of YUCCA genes, which are rate-limiting for conversion of IPA to auxin, is increased in Bdtar2l mutants. Consistent with suppression of Bdtar2l^hypo root phenotypes upon application of the ethylene precursor 1-aminocyclopropane-1-carboxylic-acid (ACC), BdYUCCA genes are down-regulated upon ACC treatment. Moreover, they are up-regulated in a downstream ethylene-signaling component homolog mutant, Bd ethylene insensitive 2-like 1, which also displays a Bdtar2l root phenotype. In summary, Bdtar2l phenotypes contrast with gradually reduced root growth and auxin levels described for Arabidopsis taa1/tar mutants. This could be explained if in Brachypodium, ethylene inhibits the rate-limiting step of auxin biosynthesis in an IPA-dependent manner to confer auxin levels that are sub-optimal for root cell elongation, as suggested by our observations. Thus, our results reveal a delicate homeostasis of local auxin and ethylene activity to control cell elongation in Brachypodium roots and suggest alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis.     

SMALL ACIDIC PROTEIN1 Acts with RUB Modification Components, the COP9 Signalosome, and AXR1 to Regulate Growth and Development of Arabidopsis

Previously, a dysfunction of the SMALL ACIDIC PROTEIN1 (SMAP1) gene was identified as the cause of the anti-auxin resistant1 (aar1) mutant of Arabidopsis (Arabidopsis thaliana). SMAP1 is involved in the response pathway of synthetic auxin, 2,4-dichlorophenoxyacetic acid, and functions upstream of the auxin/indole-3-acetic acid protein degradation step in auxin signaling. However, the exact mechanism by which SMAP1 functions in auxin signaling remains unknown. Here, we demonstrate that SMAP1 is required for normal plant growth and development and the root response to indole-3-acetic acid or methyl jasmonate in the auxin resistant1 (axr1) mutation background. Deletion analysis and green fluorescent protein/glutathione S-transferase pull-down assays showed that SMAP1 physically interacts with the CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME (CSN) via the SMAP1 F/D region. The extremely dwarf phenotype of the aar1-1 csn5a-1 double mutant confirms the functional role of SMAP1 in plant growth and development under limiting CSN functionality. Our findings suggest that SMAP1 is involved in the auxin response and possibly in other cullin-RING ubiquitin ligase-regulated signaling processes via its interaction with components associated with RELATED TO UBIQUITIN modification.     

The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis

The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.     

PIS1, a negative regulator of the action of auxin transport inhibitors in Arabidopsis thaliana

In order to clarify the mechanism underlying the polar auxin transport system, the pis1 mutant in Arabidopsis thaliana that is hypersensitive to N -1-naphthylphthalamic acid (NPA), an auxin transport inhibitor was isolated and characterized. Whereas the pis1 mutant is normally sensitive to phytohormones, auxins, cytokinin and ethylene precursor, this mutant is hypersensitive to NPA over the broad spectrum of its effects such as growth of seedlings, root elongation, root gravitropism, root phototropism and root curling. This result indicates that the pis1 mutant is specifically affected in the polar auxin transport system. This result also defines a genetic factor controlling both gravitropism and phototropism, and strongly indicates the involvement of auxin transport during both tropic responses. NPA, 2,3,5-triiodobenzoic acid (TIBA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) represent different classes of auxin transport inhibitors. The pis1 mutation conferred hypersensitivity to both NPA and TIBA but not to HFCA. These results show the genetic separation of the actions of NPA/TIBA and of HFCA. The PIS1 gene product might be specifically involved in the response pathway of NPA/TIBA, leading to interference with auxin-efflux carriers, and might act as a negative regulator of the action of NPA/TIBA.     

Characteristics of adventitious root formation in cotyledon segments of mango (Mangifera indica L. cv. Zihua): two induction patterns, histological origins and the relationship with polar auxin transport

Cotyledon segments derived from zygote embryos of mango (Mangifera indica L. cv. Zihua) were cultured on agar medium for 28 days. Depending on different pre-treatments with plant growth regulators, two distinct patterns of adventitious roots were observed. A first pattern of adventitious roots was seen at the proximal cut surface, whereas no roots were formed on the opposite, distal cut surface. The rooting ability depended on the segment length and was significantly promoted by pre-treatment of embryos with indol-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for 1 h. A pre-treatment with the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) completely inhibited adventitious root formation on proximal cut surfaces. A second pattern of roots was observed on abaxial surfaces of cotyledon segments when embryos were pre-treated with 2,700 μM 1-naphthalenacetic acid (NAA) for 1 h. Histological observations indicated that both patterns of adventitious roots originated from parenchymal cells, but developmental directions of the root primordia were different. A polar auxin transport assay was used to demonstrate transport of [³H] indole-3-acetic acid (IAA) in cotyledon segments from the distal to the proximal cut surface. In conclusion, we suggest that polar auxin transport plays a role in adventitious root formation at the proximal cut surface, whereas NAA levels (influx by diffusion; carrier mediated efflux) seem to control development of adventitious roots on the abaxial surface of cotyledon segments.     

TAA1-Regulated Local Auxin Biosynthesis in the Root-Apex Transition Zone Mediates the Aluminum-Induced Inhibition of Root Growth in Arabidopsis

The transition zone (TZ) of the root apex is the perception site of Al toxicity. Here, we show that exposure of Arabidopsis thaliana roots to Al induces a localized enhancement of auxin signaling in the root-apex TZ that is dependent on TAA1, which encodes a Trp aminotransferase and regulates auxin biosynthesis. TAA1 is specifically upregulated in the root-apex TZ in response to Al treatment, thus mediating local auxin biosynthesis and inhibition of root growth. The TAA1-regulated local auxin biosynthesis in the root-apex TZ in response to Al stress is dependent on ethylene, as revealed by manipulating ethylene homeostasis via the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid, the inhibitor of ethylene biosynthesis aminoethoxyvinylglycine, or mutant analysis. In response to Al stress, ethylene signaling locally upregulates TAA1 expression and thus auxin responses in the TZ and results in auxin-regulated root growth inhibition through a number of auxin response factors (ARFs). In particular, ARF10 and ARF16 are important in the regulation of cell wall modification–related genes. Our study suggests a mechanism underlying how environmental cues affect root growth plasticity through influencing local auxin biosynthesis and signaling.     

Expression of AMIDASE1 (AMI1) is suppressed during the first two days after germination

The regulation of cellular auxin levels is a critical factor in determining plant growth and architecture, as indole-3-acetic acid (IAA) gradients along the plant axis and local IAA maxima are known to initiate numerous plant growth responses. The regulation of auxin homeostasis is mediated in part by transport, conjugation and deconjugation, as well as by de novo biosynthesis. However, the pathways of IAA biosynthesis are yet not entirely characterized at the molecular and biochemical level. It is suggested that several biosynthetic routes for the formation of IAA have evolved. One such pathway proceeds via the intermediate indole-3-acetamide (IAM), which is converted into IAA by the activity of specific IAM hydrolases, such as Arabidopsis AMIDASE1 (AMI1). In this article we present evidence to support the argument that AMI1-dependent IAA synthesis is likely not to be used during the first two days of seedling development.Key words: Arabidopsis thaliana, auxin biosynthesis, AMIDASE1, indole-3-acetic acid, indole-3-acetamide, LEAFY COTYLEDON1, seed developmentAuxins are versatile plant hormones that play diverse roles in regulating many aspects of plant growth and development.¹ To enable auxins to develop their activity, a tight spatiotemporal control of cellular indole-3-acetic acid (IAA) contents is absolutely necessary since it is well-documented that auxin action is dose dependent, and that high IAA levels can have inhibitory effects on plant growth.² To achieve this goal, plants have evolved a set of different mechanisms to control cellular hormone levels. On the one hand, plants possess several pathways that contribute to the de novo synthesis of IAA. This multiplicity of biosynthetic routes presumably facilitates fine-tuning of the IAA production. On the other hand, plants are equipped with a variety of enzymes that are used to conjugate free auxin to either sugars, amino acids or peptides and small proteins, respectively, or on the contrary, that act as IAA-conjugate hydrolases, releasing free IAA from corresponding conjugates. IAA-conjugates serve as a physiologically inactive storage form of IAA from which the active hormone can be quickly released on demand. Alternatively, conjugation of IAA can mark the first step of IAA catabolism. In general, conjugation and deconjugation of free IAA are ways to positively or negatively affect active hormone levels, which adds another level of complexity to the system. Additionally, IAA can be transported from cell to cell in a polar manner, which is dependent on the action of several transport proteins. All together, these means are used to form auxin gradients and local maxima that are essential to initiate plant growth processes, such as root or leaf primordia formation.³     

A dominant negative mutant of protein kinase CK2 exhibits altered auxin responses in Arabidopsis

Protein kinase CK2 is a pleiotropic Ser/Thr kinase, evolutionary conserved in eukaryotes. Studies performed in different organisms, from yeast to humans, have highlighted the importance of CK2 in cell growth and cell-cycle control. However, the signalling pathways in which CK2 is involved have not been fully identified. In plants, the phytohormone auxin is a major regulator of cell growth. Recent discoveries have demonstrated that differential distribution of within auxin plant tissues is essential for developmental processes, and that this distribution is dependent on polar auxin transport. We report here that a dominant-negative mutant of CK2 (CK2mut) in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. However, CK2mut plants exhibit normal responses to exogenous indole-3-acetic acid (IAA) indicating that they are not affected in the perception of the hormone but upstream in the pathway. We demonstrate that mutant plants are not deficient in IAA but are impaired in its transport. Using genetic and pharmacological tools we show that CK2 activity depletion hinders correct formation of auxin gradients and leads to widespread changes in the expression of auxin-related genes. In particular, members of the auxin efflux carrier family (PINs), and the protein kinase PINOID, both key regulators of auxin fluxes, were misexpressed. PIN4 and PIN7 were also found mislocalized, with accumulation in endosomal bodies. We propose that CK2 functions in the regulation of auxin-signalling pathways, particularly in auxin transport.     

The pathway of auxin biosynthesis in plants

The plant hormone auxin, which is predominantly represented by indole-3-acetic acid (IAA), is involved in the regulation of plant growth and development. Although IAA was the first plant hormone identified, the biosynthetic pathway at the genetic level has remained unclear. Two major pathways for IAA biosynthesis have been proposed: the tryptophan (Trp)-independent and Trp-dependent pathways. In Trp-dependent IAA biosynthesis, four pathways have been postulated in plants: (i) the indole-3-acetamide (IAM) pathway; (ii) the indole-3-pyruvic acid (IPA) pathway; (iii) the tryptamine (TAM) pathway; and (iv) the indole-3-acetaldoxime (IAOX) pathway. Although different plant species may have unique strategies and modifications to optimize their metabolic pathways, plants would be expected to share evolutionarily conserved core mechanisms for auxin biosynthesis because IAA is a fundamental substance in the plant life cycle. In this review, the genes now known to be involved in auxin biosynthesis are summarized and the major IAA biosynthetic pathway distributed widely in the plant kingdom is discussed on the basis of biochemical and molecular biological findings and bioinformatics studies. Based on evolutionarily conserved core mechanisms, it is thought that the pathway via IAM or IPA is the major route(s) to IAA in plants.     

The rib1 mutant of Arabidopsis has alterations in indole-3-butyric acid transport, hypocotyl elongation, and root architecture

Polar transport of the auxin indole-3-butyric acid (IBA) has recently been shown to occur in Arabidopsis (Arabidopis thaliana) seedlings, yet the physiological importance of this process has yet to be fully resolved. Here we describe the first demonstration of altered IBA transport in an Arabidopsis mutant, and show that the resistant to IBA (rib1) mutation results in alterations in growth, development, and response to exogenous auxin consistent with an important physiological role for IBA transport. Both hypocotyl and root IBA basipetal transport are decreased in rib1 and root acropetal IBA transport is increased. While indole-3-acetic acid (IAA) transport levels are not different in rib1 compared to wild type, root acropetal IAA transport is insensitive to the IAA efflux inhibitor naphthylphthalamic acid in rib1, as is the dependent physiological process of lateral root formation. These observed changes in IBA transport are accompanied by altered rib1 phenotypes. Previously, rib1 roots were shown to be less sensitive to growth inhibition by IBA, but to have a wild-type response to IAA in root elongation. rib1 is also less sensitive to IBA in stimulation of lateral root formation and in hypocotyl elongation under most, but not all, light and sucrose conditions. rib1 has wild-type responses to IAA, except under one set of conditions, low light and 1.5% sucrose, in which both hypocotyl elongation and lateral root formation show altered IAA response. Taken together, our results support a model in which endogenous IBA influences wild-type seedling morphology. Modifications in IBA distribution in seedlings affect hypocotyl and root elongation, as well as lateral root formation.     

The rib1 mutant is resistant to indole-3-butyric acid, an endogenous auxin in Arabidopsis

The presence of indole-3-butyric acid (IBA) as an endogenous auxin in Arabidopsis has been recently demonstrated. However, the in vivo role of IBA remains to be elucidated. We present the characterization of a semi-dominant mutant that is affected in its response to IBA, but shows a wild-type response to indole-3-acetic acid (IAA), the predominant and most studied form of auxin. We have named this mutant rib1 for resistant to IBA. Root elongation assays show that rib1 is specifically resistant to IBA, to the synthetic auxin 2,4-dichlorophenoxyacetic acid, and to auxin transport inhibitors. rib1 does not display increased resistance to IAA, to the synthetic auxin naphthalene acetic acid, or to other classes of plant hormones. rib1 individuals also have other root specific phenotypes including a shortened primary root, an increased number of lateral roots, and a more variable response than wild type to a change in gravitational vector. Adult rib1 plants are morphologically indistinguishable from wild-type plants. These phenotypes suggest that rib1 alters IBA activity in the root, thereby affecting root development and response to environmental stimuli. We propose models in which RIB1 has a function in either IBA transport or response. Our experiments also suggest that IBA does not use the same mechanism to exit cells as does IAA and we propose a model for IBA transport.     

Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation

The pin-formed mutant pin 1-1, one of the Arabidopsis flower mutants, has several structural abnormalities in inflorescence axes, flowers, and leaves. In some cases, pin1-1 forms a flower with abnormal structure (wide petals, no stamens, pistil-like structure with no ovules in the ovary) at the top of inflorescence axes. In other cases, no floral buds are formed on the axes. An independently isolated allelic mutant (pin1-2) shows similar phenotypes. These mutant phenotypes are exactly the same in wild-type plants cultured in the presence of chemical compounds known as auxin polar transport inhibitors: 9-hydroxyfluorene-9-carboxylic acid or N-(1-naphthyl)phthalamic acid. We tested the polar transport activity of indole-3-acetic acid and the endogenous amount of free indole-3-acetic acid in the tissue of inflorescence axes of the pin1 mutants and wild type. The polar transport activity in the pin 1-1 mutant and in the pin1-2 mutant was decreased to 14% and 7% of wild type, respectively. These observations strongly suggest that the normal level of polar transport activity in the inflorescence axes is required in early developmental stages of floral bud formation in Arabidopsis and that the primary function of the pin1 gene is auxin polar transport in the inflorescence axis.