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Title: Disturbed Local Auxin Homeostasis Enhances Cellular Anisotropy and Reveals Alternative Wiring of Auxin-ethylene Crosstalk in Brachypodium distachyon Seminal Roots}
Article:
Abstract:
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( Bdtar2lhypo). Counterintuitive however, Bdtar2lhypo 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 Bdtar2lhypo 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.
Introduction:
The root system plays a fundamental role for plant growth and survival, not only by providing support, water and nutrients for the shoot, but also by participating in secondary functions, such as hormone biosynthesis or storage of photoassimilates[1],[2]. Root system architecture, that is the number and arrangement of different root types and their branching pattern, is highly plastic and determined by developmental and environmental factors that interact to optimize soil exploration. This is particularly important for the capture of growth limiting macronutrients, including nitrogen and phosphorus, whose edaphic distribution strongly influences post-embryonic root development and, therefore, root system architecture[2]–[4]. However, the root system can only respond to variation in such resources within its inherent developmental limits of growth rate and branching capacity, which are genetically determined. Optimization of root system architecture through breeding is therefore of particular interest in crops to increase root system plasticity with respect to biotic and abiotic stresses[5],[6]. Our knowledge about the molecular genetic control of root growth and branching has been largely obtained from analyses of the dicotyledon plant model system Arabidopsis thaliana( Arabidopsis) through mutagenesis approaches[2],[7]. The genes identified through these efforts have greatly benefitted the isolation of corresponding loci in monocotyledons, such as rice or maize[8]–[11]. Many of them encode proteins with regulatory functions, and among them components of plant hormone signaling pathways are particularly preeminent. For example, interference with the auxin-signaling pathway by mutation typically impairs primary root elongation or root branching, and in extreme cases even abolishes root formation[12]–[14]. The same is true for loss-of-function mutations in genes that encode enzymes involved in tryptophan-dependent auxin biosynthesis. In particular, auxin biosynthesis from tryptophan via indole-3-pyruvic acid( IPA) has been shown to be essential for root formation[15],[16]. Two enzyme classes define this pathway: the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1( TAA1) and TAA1-RELATED( TAR) proteins, which catalyze the conversion of tryptophan to IPA; and the family of YUCCA cytochrome P450s, which catalyze the conversion of IPA to indole-3-acetic acid( IAA), the major active form of auxin[15]–[18]. Whereas the YUCCA genes were originally identified through a gain-of-function approach that led to auxin over-accumulation[19], TAA1/TAR genes were identified through loss-of-function approaches[16],[20],[21]. For instance, one study isolated the taa1 mutant because of its root growth resistance to the application of 1-aminocyclopropane-1-carboxylic-acid( ACC), a rate-limiting precursor for the biosynthesis of another hormone, ethylene[16]. This phenotype arises as a consequence of reduced auxin biosynthesis, which is normally up-regulated by ethylene through induction of TAA1/TAR gene expression. This finding also illustrates the dosage-dependent action of auxin, because although auxin and its perception are essential for root formation and growth, excess auxin application, biosynthesis or signaling are eventually inhibitory[22]–[24]. Indeed, it has been suggested that depending on the species, auxin levels might be supra-optimal for root growth[25]. Phylogenetic analysis has identified bona fide TAA1/TAR homologous genes in monocotyledons, with varying degrees of redundancy. For instance, whereas maize contains five genes of this family, only two are found in both rice and the monocotyledon model system, Brachypodium distachyon( Brachypodium)[26]. So far, only one TAA1/TAR-related mutant has been identified in monocotyledons, the vanishing tassel 2( vt2) mutant of maize[26]. Despite the presence of multiple TAA1/TAR homologs in maize, vt2 null mutants display rather severe shoot phenotypes, such as dwarfism, reduced axillary meristem formation and associated impaired inflorescence development. Free auxin levels are reduced to ca. one third of wild type levels in vt2 mutants, suggesting that VT2 encodes the predominant TAA1/TAR activity in maize. Here we report the isolation and characterization of a Brachypodium mutant in the TAR2-LIKE( BdTAR2L) gene. Unlike vt2, this Bdtar2l mutant displays only mild shoot phenotypes. However, we observed dramatic root phenotypes, which surprisingly appear to result from upwardly disturbed auxin homeostasis.
Discussion:
The root systems of dicotyledons and monocotyledons display some fundamental differences in their organization and ontogeny, as exemplified by the respective model systems, Arabidopsis and Brachypodium[30]. Despite these differences, the principal genes involved in root formation, growth vigor and branching are expected to be homologous in the two systems. This is based on experience in other species such as maize, where several causative mutations that affect root system development are in homologs of auxin signaling components[9],[41]. The effect of manipulating the IPA branch of auxin biosynthesis has been investigated in another monocotyledon crop, rice, through gain- and loss-of-function approaches. For instance, both over-expression and down-regulation of the YUCCA homolog OsYUCCA1 by transgenic means results in strongly reduced root growth[42], whereas a knockout in another YUCCA homolog, CONSTITUTIVELY WILTED 1, displays reduced root branching[43]. Compared to those mutants, the enhanced root elongation phenotype of Bdtar2l mutants is unusual. Our initial interpretation was therefore that auxin levels are supra-optimal for cell elongation in the Brachypodium seminal root, as has been suggested for seminal root growth in rice[25]. However, repeated independent measurements of multiple samples clearly indicated that auxin levels are increased rather than decreased in Bdtar2l mutant roots. This is particularly pronounced in the Bdtar2lhypo allele, and in tendency also observed in the Bdtar2lqnull allele, correlating with quantitatively corresponding BdYUCCA up-regulation. The comparatively severe shoot phenotypes of the Bdtar2lqnull allele, its less pronounced root cell elongation, and the observation that the root meristem gradually breaks down as development progresses indicate that compared to the Bdtar2lhypo allele, IPA levels are eventually limiting in Bdtar2lqnull mutants. This idea is supported by the dose-response curve of wild type to L-kynerunine, where increasing amounts promote cell elongation up to a certain threshold, beyond which root growth is inhibited. Further corroborating this idea, a threshold also exists for the Bdtar2lhypo mutant, which moreover is hypersensitive to L-kynerunine treatment as concentrations that still promote root elongation in wild type are inhibitory in Bdtar2lhypo. A similar dose-response curve is observed for AVG treatment, which inhibits the rate-limiting step in ethylene biosynthesis, but might also impinge on TAA1/TAR activity because of its generic action on enzymes that use PLP as a co-factor[16],[36],[37]. Stimulation of root growth by AVG treatment has also been reported for rice[25], although the reported dosage response is quantitatively different from our assays with Brachypodium. For instance, while in rice 0. 05 µM AVG promoted root growth and 1. 0 µM was already inhibitory, in Brachypodium 5. 0 µM was still stimulating. In part, this could be due to technical issues, for instance the concentration needed in the tissue culture media to reach the same tissue penetration in roots of different thickness or cell permeability. In light of our results, it appears possible that the response of rice to AVG treatment is similar to Brachypodium, i. e. that it could reflect a combined effect of reducing TAA1/TAR as well as ACC synthase activity, thereby boosting auxin levels by removing the inhibitory effect of ethylene on YUCCA expression as long as interference with TAA/TAR1 activity does not lead to limiting IPA levels. The finding that YUCCA expression is rate limiting for auxin biosynthesis in Arabidopsis[17],[33] supports this interpretation, suggesting that this is also likely the case in Brachypodium and/or rice. Corroborating the effects of AVG application and circumventing its ambiguity, the root phenotype of the Bdein2l1hypo mutant confirms the involvement of the ethylene-signaling pathway in auxin homeostasis. However, based on the observed regulatory logic of this hormone crosstalk, a central finding of our study is that the regulation of the IPA branch of auxin biosynthesis through the ethylene pathway observed in Arabidopsis roots might not be conserved in Brachypodium. This idea is based on several convergent observations, for instance that unlike their Arabidopsis counterparts, expression of BdTAR2L as well as BdTAR1L is hardly ethylene-responsive or that BdYUCCAs are repressed upon ACC treatment and up-regulated in Bdein2l1hypo, consistent with the latter's Bdtar2l phenotype. Moreover, unlike Arabidopsis taa1/tar mutants, Bdtar2lhypo mutants are not ACC-resistant. Rather, ACC treatment essentially restores the Bdtar2lhypo phenotype to wild type. Thus, our data thus support a scenario in which the effects of auxin biosynthesis through the IPA branch on root cell elongation are mediated by the ethylene pathway rather than vice versa. Such an inversion of a regulatory relationship could alternatively reflect a shift in the key nodes of the regulatory network linking auxin and ethylene through feedback loops and is a simple way for evolutionary adaptation. Indeed, feedback of ethylene on auxin biosynthesis by repressing YUCCA expression, rather than promoting TAA1/TAR as well as YUCCA expression as in Arabidopsis, is a central feature of the mutant phenotypes described in our paper( Fig. 8). How this feedback is mediated remains unclear for the moment. The recent discovery of an enzymatic link between auxin and ethylene biosynthesis suggests that this crosstalk might very well respond directly to IPA levels[44]. Hypomorphic mutants, such as those employed in our study, might become a crucial tool in future efforts to elaborate such a scenario.
The plant hormone auxin is pivotal for root system development . For instance , its local biosynthesis is essential for root formation and growth in the dicotyledon model Arabidopsis . Thus , increasing interference with auxin biosynthesis results in increasingly shorter roots , partly because of reduced cell elongation . In this study , we isolated a hypomorphic mutant in an auxin biosynthesis pathway enzyme in the monocotyledon model Brachypodium . Counterintuitive , this mutant displays a dramatically longer seminal root , because mature cells are thinner , more elongated and therefore more anisotropic than in wild type . Interestingly , this phenotype can be mimicked in wild type by pharmacological interference with production of a key auxin biosynthesis intermediate , but also by interference with the biosynthesis of another plant hormone , ethylene . The latter controls auxin biosynthesis in Arabidopsis roots . Surprisingly however , auxin levels in the Brachypodium mutant are elevated rather than reduced , because of a simultaneous up-regulation of the second , rate-limiting step of the pathway . Ethylene normally represses this second step , suggesting an inverted regulatory relation between the two hormones as compared to Arabidopsis . Our results point to a complex homeostatic crosstalk between auxin and ethylene in Brachypodium roots , which is fundamentally different from Arabidopsis and might be conserved in other monocotyledons .