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Sequential induction of auxin efflux and influx carriers regulates lateral root emergence.

ABSTRACT: In Arabidopsis, lateral roots originate from pericycle cells deep within the primary root. New lateral root primordia (LRP) have to emerge through several overlaying tissues. Here, we report that auxin produced in new LRP is transported towards the outer tissues where it triggers cell separation by inducing both the auxin influx carrier LAX3 and cell-wall enzymes. LAX3 is expressed in just two cell files overlaying new LRP. To understand how this striking pattern of LAX3 expression is regulated, we developed a mathematical model that captures the network regulating its expression and auxin transport within realistic three-dimensional cell and tissue geometries. Our model revealed that, for the LAX3 spatial expression to be robust to natural variations in root tissue geometry, an efflux carrier is required--later identified to be PIN3. To prevent LAX3 from being transiently expressed in multiple cell files, PIN3 and LAX3 must be induced consecutively, which we later demonstrated to be the case. Our study exemplifies how mathematical models can be used to direct experiments to elucidate complex developmental processes.

SUBMITTER: Peret B

PROVIDER: S-EPMC3817398 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Sequential induction of auxin efflux and influx carriers regulates lateral root emergence.

Péret Benjamin B Middleton Alistair M AM French Andrew P AP Larrieu Antoine A Bishopp Anthony A Njo Maria M Wells Darren M DM Porco Silvana S Mellor Nathan N Band Leah R LR Casimiro Ilda I Kleine-Vehn Jürgen J Vanneste Steffen S Sairanen Ilkka I Mallet Romain R Sandberg Göran G Ljung Karin K Beeckman Tom T Benkova Eva E Friml Jiří J Kramer Eric E King John R JR De Smet Ive I Pridmore Tony T Owen Markus M Bennett Malcolm J MJ

Molecular systems biology 20131022

In Arabidopsis, lateral roots originate from pericycle cells deep within the primary root. New lateral root primordia (LRP) have to emerge through several overlaying tissues. Here, we report that auxin produced in new LRP is transported towards the outer tissues where it triggers cell separation by inducing both the auxin influx carrier LAX3 and cell-wall enzymes. LAX3 is expressed in just two cell files overlaying new LRP. To understand how this striking pattern of LAX3 expression is regulated, ...[more]

PMID: 24150423

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Project description:Protein phosphorylation is instrumental to early signaling events. Studying system-wide phosphorylation in relation to processes under investigation requires a quantitative proteomics approach. In Arabidopsis, auxin application can induce pericycle cell divisions and lateral root formation. Initiation of lateral root formation requires transcriptional reprogramming following auxin-mediated degradation of transcriptional repressors. The immediate early signaling events prior this derepression are virtually uncharacterized. To identify the signal molecules responding to auxin application we used the Lateral Root Inducible System which was previously developed to trigger synchronous division of pericycle cells. To identify and quantify the early signaling events following this induction we combined 15N based metabolic labeling and phospho-enrichment and applied a mass spectrometry based approach. In total, 3068 phosphopeptides were identified from auxin treated root tissue. This experiment represents one of the largest quantitative phosphoprotein dataset on Arabidopsis to date. Key proteins responding to auxin treatment included the MDR and PIN2 auxin carriers, auxin response factor2, suppressor of auxin resistance3 and sorting nexin1 (SNX1). Mutational analysis of Serine16 of SNX1 showed that overexpression of the mutated forms of SNX1led to retarded growth and reduction of lateral root formation due to the reduced outgrowth of the primodium, showing proof of principle for our approach. Data Processing: All MS/MS spectra were centroided and merged to a single peak list file using MaxQuant (version 1.0.13.13), which was searched using the Mascot search engine (version 2.2.0, Matrix Science) containing a total 67344 entries (which includes contaminants and an equivalent number of decoy sequences) generated from the publicly available Arabidopsis database (The Arabidopsis Information Resource (TAIR9); June 2009; file name, TAIR9_pep_20090619) with carbamidomethylcysteine as a fixed modification. Oxidized methionine and phosphorylation (serine, threonine, and tyrosine) were searched as variable modifications. Searches were done with tryptic specificity allowing two miscleavages and an initial tolerance on mass measurement of 50 ppm in MS mode and 0.6 Da for MS/MS ions and score cutoff of 20. The resulting .dat files were exported and filtered for a <1% false discovery rate (FDR) at the peptide level using in-house developed software Rockerbox (Version1.1.0) (35). Relative quantification ratios of the identified phosphopeptides and protein were derived by MSQuant (MSQ2.0b4, 2010-02-25) (36). Proteins were quantified with at least two unique non phosphopeptides or single peptide with a score no less than 60. The phospho peptide ratio was normalized by dividing the ratio of its protein of origin. To determine whether phosphorylation ratios for normalized peptides were significantly different a p-value was generated using significance B which is part of the MaxQuant package. The changes were considered significant at a p-value of 0.05 or less. For comparison at the phosphopeptide level the most recent the full dataset with experimental sites was downloaded from PhosPhAt 4.0. Phosphopeptides with ambiguous residues denoted as (s), (t), etc., were converted to S and T, respectively. In addition we used Supplementary Table S1 from Lan et al., (2012) for phosphopeptide comparison. Modifications other than nonambiguous phosphorylation were ignored resulting in a reduction from the original 879 phosphopeptides to 556 peptides. We used pep2pro 'TAIR10 wos' dataset for comparison at the protein level.

Project description:Lateral root (LR) formation is a vital organogenetic process that determines the root architecture in plants. The number of root branches governs the degree of anchorage, efficiency of nutrients acquisition, and water uptake. The molecular pathways involved in LR formation have been extensively studied in Arabidopsis thaliana (At). A plant hormone, Auxin, is a key regulator of root development and promotes LR formation in plants. A plethora of Arabidopsis genes have been identified to regulate LR initiation, patterning, and emergence processes. Recently, the involvement of flowering time control pathways and circadian clock pathways in LR development has come to light, but the connecting link between these processes is still missing. We have established that GIGANTEA (GI), a key component of photoperiodic flowering, can regulate the formation of LRs in Arabidopsis. GI is known to be involved in red light signaling and circadian clock signaling pathways. Here, we report that over-expression of GI enhances LR formation in red light in At. Real-time PCR analysis shows that GI positively regulates the transcription of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) which is an upstream component of auxin signaling. Furthermore, gi-100 mutant downregulates the LR initiation signaling gene, AUXIN RESPONSE FACTOR 7 (ARF7), and its downstream target gene, LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16). Hence, GI acts as a positive regulator of IAA14-ARF7-LBD16 modules during LR initiation. We have also checked the effect of GI on the expression of NAC1 and AIR3 genes which are controlled by TIR1 during LR formation. Our results show that GI induces the NAC1 transcription and its downstream gene, AIR3 expression, which leads to the enhancement of LR initiation. Taken together, our results suggest that GI controls the expression of TIR1 to govern auxin signaling during LR formation in presence of red light and GI can act as a link between circadian clock signaling, flowering time control pathways, light signaling, and lateral root development pathways.

Dataset Information

Sequential induction of auxin efflux and influx carriers regulates lateral root emergence.

Publications

Sequential induction of auxin efflux and influx carriers regulates lateral root emergence.

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