Epidermal expression of a sterol biosynthesis gene regulates root growth by a non-cell-autonomous mechanism in Arabidopsis.
ABSTRACT: The epidermis is hypothesized to play a signalling role during plant development. One class of mutants showing defects in signal transduction and radial patterning are those in sterol biosynthesis. The expectation is that living cells require sterols, but it is not clear that all cell types express sterol biosynthesis genes. The HYDRA1 (HYD1) gene of Arabidopsis encodes sterol Δ8-Δ7 isomerase, and although hyd1 seedlings are defective in radial patterning across several tissues, we show that the HYD1 gene is expressed most strongly in the root epidermis. Transgenic activation of HYD1 transcription in the epidermis of hyd1 null mutants reveals a major role in root patterning and growth. HYD1 expression in the vascular tissues and root meristem, though not endodermis or pericycle, also leads to some phenotypic rescue. Phenotypic rescue is associated with rescued patterning of the PIN1 and PIN2 auxin efflux carriers. The importance of the epidermis in controlling root growth and development is proposed to be, in part, due to its role as a site for sterol biosynthesis, and auxin is a candidate for the non-cell-autonomous signal.
Project description:The phytohormone auxin is a vital growth regulator in plants. In the root epidermis auxin steers root organ growth. However, the mechanisms that allow adjacent tissues to integrate growth are largely unknown. Here, the focus is on neighbouring epidermal root tissues to assess the integration of auxin-related growth responses. The pharmacologic, genetic, and live-cell imaging approaches reveal that PIN2 auxin efflux carriers are differentially controlled in tricho- and atrichoblast cells. PIN2 proteins show lower abundance at the plasma membrane of trichoblast cells, despite showing higher rates of intracellular trafficking in these cells. The data suggest that PIN2 proteins display distinct cell-type-dependent trafficking rates to the lytic vacuole for degradation. Based on this insight, it is hypothesized that auxin-dependent processes are distinct in tricho- and atrichoblast cells. Moreover, genetic interference with epidermal patterning supports this assumption and suggests that tricho- and atrichoblasts have distinct importance for auxin-sensitive root growth and gravitropic responses.
Project description:The root apical meristem (RAM) is the plant stem cell niche which provides for the formation and continuous development of the root. Auxin is the main regulator of RAM functioning, and auxin maxima coincide with the sites of RAM initiation and maintenance. Auxin gradients are formed due to local auxin biosynthesis and polar auxin transport. The PIN family of auxin transporters plays a critical role in polar auxin transport, and two mechanisms of auxin maximum formation in the RAM based on PIN-mediated auxin transport have been proposed to date: the reverse fountain and the reflected flow mechanisms.The two mechanisms are combined here in in silico studies of auxin distribution in intact roots and roots cut into two pieces in the proximal meristem region. In parallel, corresponding experiments were performed in vivo using DR5::GFP Arabidopsis plants.The reverse fountain and the reflected flow mechanism naturally cooperate for RAM patterning and maintenance in intact root. Regeneration of the RAM in decapitated roots is provided by the reflected flow mechanism. In the excised root tips local auxin biosynthesis either alone or in cooperation with the reverse fountain enables RAM maintenance.The efficiency of a dual-mechanism model in guiding biological experiments on RAM regeneration and maintenance is demonstrated. The model also allows estimation of the concentrations of auxin and PINs in root cells during development and under various treatments. The dual-mechanism model proposed here can be a powerful tool for the study of several different aspects of auxin function in root.
Project description:3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyses the second step of the mevalonate (MVA) pathway. The HMGS inhibitor (F-244) has been reported to retard growth in wheat, tobacco and Brassica juncea, but the mechanism remains unknown. Although the effects of HMGS on downstream isoprenoid metabolites have been extensively reported, not much is known on how it might affect non-isoprenoid metabolic pathways. Here, the mechanism of F-244-mediated inhibition of primary root growth in Arabidopsis and the relationship between HMGS and non-isoprenoid metabolic pathways were investigated by untargeted SWATH-MS quantitative proteomics, quantitative real-time-PCR and target metabolite analysis. Our results revealed that the inhibition of primary root growth caused by F-244 was a consequence of reduced stigmasterol, auxin and cytokinin levels. Interestingly, proteomic analyses identified a relationship between HMGS and glucosinolate biosynthesis. Inhibition of HMGS activated glucosinolate biosynthesis, resulting from the induction of glucosinolate biosynthesis-related genes, suppression of sterol biosynthesis-related genes and reduction in sterol levels. In contrast, HMGS overexpression inhibited glucosinolate biosynthesis, due to down-regulation of glucosinolate biosynthesis-related genes, up-regulation of sterol biosynthesis-related genes and increase in sterol content. Thus, HMGS might represent a target for the manipulation of glucosinolate biosynthesis, given the regulatory relationship between HMGS in the MVA pathway and glucosinolate biosynthesis.
Project description:Elucidating molecular links between cell-fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell-fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem-cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS-box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem-cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS-domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4-dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning.
Project description:BACKGROUND:The control of vascular tissue development in plants is influenced by diverse hormonal signals, but their interactions during this process are not well understood. Wild-type sterol profiles are essential for growth, tissue patterning and signalling processes in plant development, and are required for regulated vascular patterning. METHODOLOGY/PRINCIPAL FINDINGS:Here we investigate the roles of sterols in vascular tissue development, through an analysis of the Arabidopsis mutants hydra1 and fackel/hydra2, which are defective in the enzymes sterol isomerase and sterol C-14 reductase respectively. We show that defective vascular patterning in the shoot is associated with ectopic cell divisions. Expression of the auxin-regulated AtHB8 homeobox gene is disrupted in mutant embryos and seedlings, associated with variably incomplete vascular strand formation and duplication of the longitudinal axis. Misexpression of the auxin reporter proIAA2ratioGUS and mislocalization of PIN proteins occurs in the mutants. Introduction of the ethylene-insensitive ein2 mutation partially rescues defective cell division, localization of PIN proteins, and vascular strand development. CONCLUSIONS:The results support a model in which sterols are required for correct auxin and ethylene crosstalk to regulate PIN localization, auxin distribution and AtHB8 expression, necessary for correct vascular development.
Project description:Patterning in Arabidopsis root development is coordinated via a localized auxin concentration maximum in the root tip, requiring the regulated expression of specific genes. However, little is known about how hormone and gene expression patterning is generated. Using a variety of experimental data, we develop a spatiotemporal hormonal crosstalk model that describes the integrated action of auxin, ethylene and cytokinin signalling, the POLARIS protein, and the functions of PIN and AUX1 auxin transporters. We also conduct novel experiments to confirm our modelling predictions. The model reproduces auxin patterning and trends in wild-type and mutants; reveals that coordinated PIN and AUX1 activities are required to generate correct auxin patterning; correctly predicts shoot to root auxin flux, auxin patterning in the aux1 mutant, the amounts of cytokinin, ethylene and PIN protein, and PIN protein patterning in wild-type and mutant roots. Modelling analysis further reveals how PIN protein patterning is related to the POLARIS protein through ethylene signalling. Modelling prediction of the patterning of POLARIS expression is confirmed experimentally. Our combined modelling and experimental analysis reveals that a hormonal crosstalk network regulates the emergence of patterns and levels of hormones and gene expression in wild-type and mutants.
Project description:Periploca sepium Bunge is a traditional medicinal plant, whose root bark is important for Chinese herbal medicine. Its major bioactive compounds are C21 steroids and periplocin, a kind of cardiac glycoside, which are derived from the steroid synthesis pathway. However, research on P. sepium genome or transcriptomes and their related genes has been lacking for a long time. In this study we estimated this species nuclear genome size at 170 Mb (using flow cytometry). Then, RNA sequencing of four different tissue samples of P. sepium (leaves, roots, adventitious roots, and calli) was done using the sequencing platform Illumina/Solexa Hiseq 2,500. After de novo assembly and quantitative assessment, 90,375 all-transcripts and 71,629 all-unigenes were finally generated. Annotation efforts that used a number of public databases resulted in detailed annotation information for the transcripts. In addition, differentially expressed genes (DEGs) were identified by using digital gene profiling based on the reads per kilobase of transcript per million reads mapped (RPKM) values. Compared with the leaf samples (L), up-regulated genes and down-regulated genes were eventually obtained. To deepen our understanding of these DEGs, we performed two enrichment analyses: gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Here, the analysis focused upon the expression characteristics of those genes involved in the terpene metabolic pathway and the steroid biosynthesis pathway, to better elucidate the molecular mechanism of bioactive steroid synthesis in P. sepium. The bioinformatics analysis enabled us to find many genes that are involved in bioactive steroid biosynthesis. These genes encoded acetyl-CoA acetyltransferase (ACAT), HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (MDD), isopentenylpyrophosphate isomerase (IPPI), farnesyl pyrophosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), cycloartenol synthase (CAS), sterol C-24 methyltransferase (SMT1), sterol-4alpha-methyl oxidase 1 (SMO1), sterol 14alpha-demethylase (CYP51/14-SDM), delta(14)-sterol reductase (FK/14SR), C-8,7 sterol isomerase (HYD1), sterol-4alpha-methyl oxidase 2 (SMO2), delta(7)-sterol-C5(6)-desaturase (STE1/SC5DL), 7-dehydrocholesterol reductase (DWF5/DHCR7), delta (24)-sterol reductase (DWF1/DHCR24), sterol 22-desaturase (CYP710A), progesterone 5beta-reductase (5?-POR), 3-beta-hydroxysteroid dehydrogenase (3?-HSD). This research will be helpful to further understand the mechanism of bioactive steroid biosynthesis in P. sepium, namely C21 steroid and periplocin biosynthesis.
Project description:In plant roots, auxin is critical for patterning and morphogenesis. It regulates cell elongation and division, the development and maintenance of root apical meristems, and other processes. In Arabidopsis, auxin distribution along the central root axis has several maxima: in the root tip, in the basal meristem and at the shoot/root junction. The distal maximum in the root tip maintains the stem cell niche. Proximal maxima may trigger lateral or adventitious root initiation.We propose a reflected flow mechanism for the formation of the auxin maximum in the root apical meristem. The mechanism is based on auxin's known activation and inhibition of expressed PIN family auxin carriers at low and high auxin levels, respectively. Simulations showed that these regulatory interactions are sufficient for self-organization of the auxin distribution pattern along the central root axis under varying conditions. The mathematical model was extended with rules for discontinuous cell dynamics so that cell divisions were also governed by auxin, and by another morphogen Division Factor which combines the actions of cytokinin and ethylene on cell division in the root. The positional information specified by the gradients of these two morphogens is able to explain root patterning along the central root axis.We present here a plausible mechanism for auxin patterning along the developing root, that may provide for self-organization of the distal auxin maximum when the reverse fountain has not yet been formed or has been disrupted. In addition, the proximal maxima are formed under the reflected flow mechanism in response to periods of increasing auxin flow from the growing shoot. These events may predetermine lateral root initiation in a rhyzotactic pattern. Another outcome of the reflected flow mechanism - the predominance of lateral or adventitious roots in different plant species - may be based on the different efficiencies with which auxin inhibits its own transport in different species, thereby distinguishing two main types of plant root architecture: taproot vs. fibrous.
Project description:Cadmium is one of the most widespread pollutant in both terrestrial and marine environment, and its inhibitory effect on plant growth has been largely demonstrated. However, the molecular mechanisms underlying Cd toxicity in plant and mainly in root, as the first organ sensing soil heavy metals, need to be better investigated. To this aim, in the present work we analyzed the growth and the organization of Arabidopsis thaliana primary root in seedlings exposed to Cd (25 and 50 ?M) for 8 days starting from germination. Root length, root meristem size, and organization were evaluated together with the behavior of some of the major molecular players in root growth and patterning. In particular, by using different GFP transgenic lines, we monitored: (i) the expression pattern of WOX5 and SCR transcription factors involved in the establishment and maintenance of stem cell niche and in the control of meristem size; (ii) the expression pattern of the IAA-inducible pDR5::GFP reporter, PIN 1, 2, 3, 7 auxin carriers and TCSn::GFP cytokinin-sensitive sensor as relevant components of hormone circuit controlling root growth. We report that Cd exposure inhibits primary root growth via affecting RAM stem cell niche and root radial pattern. At the molecular level, an impairment of auxin maximum accumulation at the root tip, related to a down-regulation and mislocalisation of PIN proteins, and an enhancement of TCSn::GFP cytokinin-sensitive sensor signal is also detected under Cd treatment, thus suggesting an alteration in the homeostasis of auxin/cytokinin signaling. Moreover, and for the first time Cd toxicity on root growth and pattern has been related to a misexpression of SCR transcription factors which is known to interplay with auxin/cytokinin cross-talk in the control of RAM maintenance and activity.
Project description:Directional growth of lateral roots is critical for radial expansion and soil coverage. Despite its importance, almost nothing is known about its molecular determinants. Initially, young lateral roots (LRs) grow away from the parental root, maintaining the angle acquired shortly after emergence. A second downwards bending response to gravity terminates the so-called plateau phase and thereby limits radial root expansion. Here, we show that the exit from the plateau phase correlates with an increase in auxin signalling at the tip of the LRs. Moreover, the increase in auxin levels induces the termination of the plateau phase, which requires PIN-FORMED (PIN) auxin efflux carriers. Our data suggests that the developmental increase in auxin triggers the preferential derepression of PIN7 in gravity-sensing columella cells. The subsequent polarization of PIN7 heralds the bending towards gravity and, hence, the exit from the plateau phase. This developmental framework reveals the distinct roles of PIN auxin efflux carriers in controlling the radial growth of root systems.