Identification and Evolutionary Analysis of the Auxin Response Factor (ARF) Family Based on Transcriptome Data from Caucasian Clover and Analysis of Expression Responses to Hormones.

Caucasian clover (Trifolium ambiguum M. Bieb.) is an excellent perennial plant in the legume family Fabaceae, with a well-developed rhizome and strong clonal growth. Auxin is one of the most important phytohormones in plants and plays an important role in plant growth and development. Auxin response factor (ARF) can regulate the expression of auxin-responsive genes, thus participating in multiple pathways of auxin transduction signaling in a synergistic manner. No genomic database has been established for Caucasian clover. In this study, 71 TaARF genes were identified through a transcriptomic database of Caucasian clover rhizome development. Phylogenetic analysis grouped the TaARFs into six (1-6) clades. Thirty TaARFs contained a complete ARF structure, including three relatively conserved regions. Physical and chemical property analysis revealed that TaARFs are unstable and hydrophilic proteins. We also analyzed the expression pattern of TaARFs in different tissues (taproot, horizontal rhizome, swelling of taproot, rhizome bud and rhizome bud tip). Quantitative real-time RT-PCR revealed that all TaARFs were responsive to phytohormones (indole-3-acetic acid, gibberellic acid, abscisic acid and methyl jasmonate) in roots, stems and leaves. These results helped elucidate the role of ARFs in responses to different hormone treatments in Caucasian clover.

Interspecies co-expression analysis of lateral root development using inducible systems in rice, Medicago, and Arabidopsis.

Lateral roots are crucial for plant growth and development, making them an important target for research aiming to improve crop yields and food security. However, their endogenous ontogeny and, as it were, stochastic appearance challenge their study. Lateral Root Inducible Systems (LRIS) can be used to overcome these challenges by inducing lateral roots massively and synchronously. The combination of LRISs with transcriptomic approaches significantly advanced our insights in the molecular control of lateral root formation, in particular for Arabidopsis. Despite this success, LRISs have been underutilized for other plant species or for lateral root developmental stages later than the initiation. In this study, we developed and/or adapted LRISs in rice, Medicago, and Arabidopsis to perform RNA-sequencing during time courses that cover different developmental stages of lateral root formation and primordium development. As such, our study provides three extensive datasets of gene expression profiles during lateral root development in three different plant species. The three LRISs are highly effective but timing and spatial distribution of lateral root induction vary among the species. Detailed characterization of the stages in time and space in the respective species enabled an interspecies co-expression analysis to identify conserved players involved in lateral root development, as illustrated for the AUX/IAA and LBD gene families. Overall, our results provide a valuable resource to identify potentially conserved regulatory mechanisms in lateral root development, and as such will contribute to a better understanding of the complex regulatory network underlying lateral root development.

Alleviation of gadolinium stress on Medicago by elevated atmospheric CO2 is mediated by changes in carbohydrates, Anthocyanin, and proline metabolism.

Rare earth elements (REE) like Gadolinium (Gd), are increasingly used in industry and agriculture and this is concomitant with the increasingly leaking of Gd into the environment. Under a certain threshold concentration, REE can promote plant growth, however, beyond this concentration, they exert negative effects on plant growth. Moreover, the effect of Gd on plants growth and metabolism under a futuristic climate with increasingly atmospheric CO2 has not yet been studied. To this end, we investigated the effect of soil contamination with Gd (150 mg/kg soil) on the growth, carbohydrates, proline, and anthocyanin metabolism of Medicago plants grown under ambient (aCO2, 410 ppm) or elevated CO2 (eCO2, 720 ppm) concentration. Gd negatively affected the growth and photosynthesis of plants and imposed oxidative stress i.e., increased H2O2 and lipid peroxidation (MDA) level. As defense lines, the level and metabolism of osmoprotectants (soluble sugars and proline) and antioxidants (phenolics, anthocyanins, and tocopherols) were increased under Gd treatment. High CO2 positively affected the growth and metabolism of Medicago plants. Moreover, eCO2 mitigated the negative impacts of Gd on Medicago growth. It further induced the levels of osmoprotectants and antioxidants. In line with increased proline and anthocyanins, their metabolic enzymes (e.g. OAT, P5CS, PAL, and CS) were also increased. This study advances our understanding of how Gd adversely affects Medicago plant growth and metabolism. It also sheds light on the biochemical mechanisms underlying the Gd stress-reducing impact of eCO2.

Genome-wide analysis of the WRKY genes and their important roles during cold stress in white clover.

Background: White clover (Trifolium repens L) is a high-quality forage grass with a high protein content, but it is vulnerable to cold stress, which can negatively affect its growth and development. WRKY transcription factor is a family of plant transcription factors found mainly in higher plants and plays an important role in plant growth, development, and stress response. Although WRKY transcription factors have been studied extensively in other plants, it has been less studied in white clover. Methods and Results: In the present research, we have performed a genome-wide analysis of the WRKY gene family of white clover, in total, there were 145 members of WRKY transcription factors identified in white clover. The characterization of the TrWRKY genes was detailed, including conserved motif analysis, phylogenetic analysis, and gene duplication analysis, which have provided a better understanding of the structure and evolution of the TrWRKY genes in white clover. Meanwhile, the genetic regulation network (GRN) containing TrWRKY genes was reconstructed, and Gene Ontology (GO) annotation analysis of these function genes showed they contributed to regulation of transcription process, response to wounding, and phosphorylay signal transduction system, all of which were important processes in response to abiotic stress. To determine the TrWRKY genes function under cold stress, the RNA-seq dataset was analyzed; most of TrWRKY genes were highly upregulated in response to cold stress, particularly in the early stages of cold stress. These results were validated by qRT-PCR experiment, implying they are involved in various gene regulation pathways in response to cold stress. Conclusion: The results of this study provide insights that will be useful for further functional analyses of TrWRKY genes in response to biotic or abiotic stresses in white clover. These findings are likely to be useful for further research on the functions of TrWRKY genes and their role in response to cold stress, which is important to understand the molecular mechanism of cold tolerance in white clover and improve its cold tolerance.

Genome-wide analysis of the CDPK gene family and their important roles response to cold stress in white clover.

Calcium-dependent protein kinases (CDPKs) are an important class of calcium-sensitive response proteins that play an important regulatory role in response to abiotic stresses. To date, little is known about the CDPK genes in white clover. White clover is a high-quality forage grass with high protein content, but it is susceptible to cold stress. Therefore, we performed a genome-wide analysis of the CDPK gene family in white clover and identified 50 members of the CDPK genes. Phylogenetic analysis using CDPKs from the model plant Arabidopsis divided the TrCDPK genes into four groups based on their sequence similarities. Motif analysis showed that TrCDPKs within the same group had similar motif compositions. Gene duplication analysis revealed the evolution and expansion of TrCDPK genes in white clover. Meanwhile, a genetic regulatory network (GRN) containing TrCDPK genes was reconstructed, and gene ontology (GO) annotation analysis of these functional genes showed that they contribute to signal transduction, cellular response to stimuli, and biological regulation, all of which are important processes in response to abiotic stresses. To determine the function of TrCDPK genes, we analyzed the RNA-seq dataset and found that most TrCDPK genes were highly up-regulated under cold stress, particularly in the early stages of cold stress. These results were validated by qRT-PCR experiments, implying that TrCDPK genes are involved in various gene regulatory pathways in response to cold stress. Our study may help to further investigate the function of TrCDPK genes and their role in response to cold stress, which is important for understanding the molecular mechanisms of cold tolerance in white clover and improving its cold tolerance.

Genome-Wide Identification and Comparative Analysis of RALF Gene Family in Legume and Non-Legume Species.

Rapid alkalinization factor (RALF) are small secreted peptide hormones that can induce rapid alkalinization in a medium. They act as signaling molecules in plants, playing a critical role in plant development and growth, especially in plant immunity. Although the function of RALF peptides has been comprehensively analyzed, the evolutionary mechanism of RALFs in symbiosis has not been studied. In this study, 41, 24, 17 and 12 RALFs were identified in Arabidopsis, soybean, Lotus and Medicago, respectively. A comparative analysis including the molecular characteristics and conserved motifs suggested that the RALF pre-peptides in soybean represented a higher value of isoelectric point and more conservative motifs/residues composition than other species. All 94 RALFs were divided into two clades according to the phylogenetic analysis. Chromosome distribution and synteny analysis suggested that the expansion of the RALF gene family in Arabidopsis mainly depended on tandem duplication, while segment duplication played a dominant role in legume species. The expression levels of most RALFs in soybean were significantly affected by the treatment of rhizobia. Seven GmRALFs are potentially involved in the release of rhizobia in the cortex cells. Overall, our research provides novel insights into the understanding of the role of the RALF gene family in nodule symbiosis.

Clover Root Uptake of Cereal Benzoxazinoids (BXs) Caused Accumulation of BXs and BX Transformation Products Concurrently with Substantial Increments in Clover Flavonoids and Abscisic Acid.

Metabolomic studies on root uptake and transformation of bioactive compounds, like cereal benzoxazinoids (BXs) in non-BX producing plants, are very limited. Therefore, a targeted mass-spectrometry-based metabolomics study was performed to elucidate the root uptake of BXs in white clover (Trifolium repens L.) and the impact of absorbed BXs on intrinsic clover secondary metabolites. Clover plants grew in a medium containing 100 µM of individual BXs (five aglycone and one glycoside BXs) for 3 weeks. Subsequently, plant tissues were analyzed by liquid chromatography-tandem mass spectrometry to quantify the BXs and clover secondary metabolite concentrations. All BXs were taken up by clover roots and translocated to the shoots. Upon uptake of 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), 2-hydroxy-1,4-benzoxazin-3-one (HBOA), and 2-ß-d-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-glc), the parent compounds and a range of transformation products were seen in the roots and shoots. The individual BX concentrations ranged from not detected (nd) to 469 µg/g of dry weight (dw) and from nd to 170 µg/g of dw in the roots and shoots, respectively. The root uptake of BXs altered the composition of intrinsic clover secondary metabolites. In particular, the concentration of flavonoids and the hormone abscisic acid increased substantially in comparison to control plants.

A Heat Shock Transcription Factor TrHSFB2a of White Clover Negatively Regulates Drought, Heat and Salt Stress Tolerance in Transgenic Arabidopsis.

Heat shock transcription factors (HSF) are divided into classes A, B and C. Class A transcription factors are generally recognized as transcriptional activators, while functional characterization of class B and C heat shock transcription factors have not been fully developed in most plant species. We isolated and characterized a novel HSF transcription factor gene, TrHSFB2a (a class B HSF) gene, from the drought stress-sensitive forage crop species, white clover (Trifolium repens). TrHSFB2a was highly homologous to MtHSFB2b, CarHSFB2a, AtHSFB2b and AtHSFB2a. The expression of TrHSFB2a was strongly induced by drought (PEG6000 15% w/v), high temperature (35 °C) and salt stresses (200 mM L-1 NaCl) in white clover, while subcellular localization analysis showed that it is a nuclear protein. Overexpression of the white clover gene TrHSFB2a in Arabidopsis significantly reduced fresh and dry weight, relative water contents (RWC), maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS), while it promoted leaf senescence, relative electrical conductivity (REC) and the contents of malondialdehyde (MDA) compared to a wild type under drought, heat and salt stress conditions of Arabidopsis plants. The silencing of its native homolog (AtHSFB2a) by RNA interference in Arabidopsis thaliana showed opposite trends by significantly increasing fresh and dry weights, RWC, maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS) and reducing REC and MDA contents under drought, heat and salt stress conditions compared to wild type Arabidopsis plants. These phenotypic and physiological indicators suggested that the TrHSFB2a of white clover functions as a negative regulator of heat, salt and drought tolerance. The bioinformatics analysis showed that TrHSFB2a contained the core B3 repression domain (BRD) that has been reported as a repressor activator domain in other plant species that might repress the activation of the heat shock-inducible genes required in the stress tolerance process in plants. The present study explores one of the potential causes of drought and heat sensitivity in white clover that can be overcome to some extent by silencing the TrHSFB2a gene in white clover.

Evolution of the CBL and CIPK gene families in Medicago: genome-wide characterization, pervasive duplication, and expression pattern under salt and drought stress.

BACKGROUND: Calcineurin B-like proteins (CBLs) are ubiquitous Ca2+ sensors that mediate plant responses to various stress and developmental processes by interacting with CBL-interacting protein kinases (CIPKs). CBLs and CIPKs play essential roles in acclimatization of crop plants. However, evolution of these two gene families in the genus Medicago is poorly understood. RESULTS: A total of 68 CBL and 135 CIPK genes have been identified in five genomes from Medicago. Among these genomes, the gene number of CBLs and CIPKs shows no significant difference at the haploid genome level. Phylogenetic and comprehensive characteristic analyses reveal that CBLs and CIPKs are classified into four clades respectively, which is validated by distribution of conserved motifs. The synteny analysis indicates that the whole genome duplication events (WGDs) have contributed to the expansion of both families. Expression analysis demonstrates that two MsCBLs and three MsCIPKs are specifically expressed in roots, mature leaves, developing flowers and nitrogen fixing nodules of Medicago sativa spp. sativa, the widely grown tetraploid species. In particular, the expression of these five genes was highly up-regulated in roots when exposed to salt and drought stress, indicating crucial roles in stress responses. CONCLUSIONS: Our study leads to a comprehensive understanding of evolution of CBL and CIPK gene families in Medicago, but also provides a rich resource to further address the functions of CBL-CIPK complexes in cultivated species and their closely related wild relatives.

Genome-wide analysis of the Glutathione S-Transferase family in wild Medicago ruthenica and drought-tolerant breeding application of MruGSTU39 gene in cultivated alfalfa.

KEY MESSAGE: Transformation of MruGSTU39 in M. ruthenica and alfalfa enhanced growth and survival of transgenic plants by up-regulating GST and glutathione peroxidase activity to detoxify ROS under drought stress. Glutathione S-transferases (GSTs) are ubiquitous supergene family which play crucial roles in detoxification of reactive oxygen species (ROS). Despite studies on GSTs, few studies have focused on them in perennial, wild plant species with high tolerance to environmental stress. Here, we identified 66 MruGST genes from the genome of Medicago ruthenica, a perennial legume species native to temperate grasslands with high tolerance to environmental stress. These genes were divided into eight classes based on their conserved domains, phylogenetic tree and gene structure, with the tau class being the most numerous. Duplication analysis revealed that GST family in M. ruthenica was expanded by segmental and tandem duplication. Several drought-responsive MruGSTs were identified by transcriptomic analyses. Of them, expression of MruGSTU39 was up-regulated much more in a tolerant accession by drought stress. Transformation of MruGSTU39 in M. ruthenica and alfalfa (Medicago sativa) enhanced growth and survival of transgenic seedlings than their wild-type counterparts under drought. We demonstrated that MruGSTU39 can detoxify ROS to reduce its damage to membrane by up-regulating activities of GST and glutathione peroxidase. Our findings provide full-scale knowledge on GST family in the wild legume M. ruthenica with high tolerance to drought, and highlight improvement tolerance of legume forages to drought using genomic information of M. ruthenica.

Salinity Stress Alters the Secondary Metabolic Profile of M. sativa, M. arborea and Their Hybrid (Alborea).

Increased soil salinity, and therefore accumulation of ions, is one of the major abiotic stresses of cultivated plants that negatively affect their growth and yield. Among Medicago species, only Medicago truncatula, which is a model plant, has been extensively studied, while research regarding salinity responses of two important forage legumes of Medicago sativa (M. sativa) and Medicago arborea (M. arborea) has been limited. In the present work, differences between M. arborea, M. sativa and their hybrid Alborea were studied regarding growth parameters and metabolomic responses. The entries were subjected to three different treatments: (1) no NaCl application (control plants), (2) continuous application of 100 mM NaCl (acute stress) and (3) gradual application of NaCl at concentrations of 50-75-150 mM by increasing NaCl concentration every 10 days. According to the results, M. arborea maintained steady growth in all three treatments and appeared to be more resistant to salinity. Furthermore, results clearly demonstrated that M. arborea presented a different metabolic profile from that of M. sativa and their hybrid. In general, it was found that under acute and gradual stress, M. sativa overexpressed saponins in the shoots while M. arborea overexpressed saponins in the roots, which is the part of the plant where most of the saponins are produced and overexpressed. Alborea did not perform well, as more metabolites were downregulated than upregulated when subjected to salinity stress. Finally, saponins and hydroxycinnamic acids were key players of increased salinity tolerance.

Overexpression of the white clover TrSAMDC1 gene enhanced salt and drought resistance in Arabidopsis thaliana.

S-adenosylmethionine decarboxylase (SAMDC) mediates the biosynthesis of polyamines (PAs) and plays a positive role in plants' response to adversity stress tolerance. In this study, we isolated a SAMDC gene from white clover, which is located in mitochondria. It was strongly induced when white clover exposed to drought (15% PEG6000), salinity (200 mM NaCl), 20 µM spermidine, 100 µM abscisic acid, and 10 mM H2O2, especially in leaves. The INVSc1 yeast introduced with TrSAMDC1 had tolerance to drought, salt, and oxidative stress. Overexpression of TrSAMDC1 in Arabidopsis showed higher fresh weight and dry weight under drought and salt treatment and without growth inhibition under normal conditions. Leaf senescence induced by drought and saline was further delayed in transgenic plants, regardless of cultivation in 1/2 MS medium and soil. During drought and salt stress, transgenic plants exhibited a significant increase in relative water content, maximum photosynthesis efficiency (Fv/Fm), performance index on the absorption basis (PIABS), activities of antioxidant protective enzymes such as SOD, POD, CAT, and APX, and a significant decrease in accumulation of MDA and H2O2 as compared to the WT. The concentrations of total PAs, putrescine, spermidine, and spermidine in transgenic lines were higher in transgenic plants than in WT under normal and drought conditions. These results suggested that TrSAMDC1 could effectively mitigate abiotic stresses without the expense of production and be a potential candidate gene for improving the drought and salt resistance of crops.

Iron deficient Medicago scutellata grown in nutrient solution at high pH accumulates and secretes large amounts of flavins.

Flavin synthesis and secretion is an integral part of the toolbox of root-borne Fe facilitators used by Strategy I species upon Fe deficiency. The Fe-deficiency responses of the wild legume Medicago scutellata grown in nutrient solution have been studied at two different pH values (5.5 and 7.5). Parameters studied include leaf chlorophyll, nutrient solution pH, concentrations and contents of micronutrients, flavin accumulation in roots, flavin export to the medium, and root ferric chelate reductase and acidification activities. Results show that M. scutellata behaves upon Fe deficiency as a Strategy I species, with a marked capacity for synthesizing flavins (riboflavin and three hydroxylated riboflavin derivatives), which becomes more intense at high pH. Results also show that this species is capable of exporting a large amount of flavins to the external medium, both at pH 5.5 and 7.5. This is the first report of a species having a major flavin secretion at pH 7.5, in contrast with the very low flavin secretion found in other flavin-producing species such as Beta vulgaris and M. truncatula. These results provide further support to the hypothesis that flavin secretion is relevant for Fe acquisition at high pH, and open the possibility to improve the Fe-efficiency responses in legumes of agronomic interest.

Colonization and performance of a pyrene-degrading bacterium Mycolicibacterium sp. Pyr9 on root surfaces of white clover.

Some rhizosphere bacteria could colonize on the root surface of plants, or even form biofilm to promote plant growth, enhance plant resistance to harsh external environments and block the soil contamination. In this study, to explore the effects of pyrene-degrading bacterium on root surface on plant uptake of pyrene, a pyrene-degrading bacterium Mycolicibacterium sp. Pyr9 was isolated from the root surface of Eleusine indica L. Gaertn. in PAH-contaminated fields; after antibiotic labeling, it was colonized onto the root surface of white clover (Trifolium repens L.), and its distribution and performance were monitored under different levels of pyrene contamination. Strain Pyr9 could degrade 98% of pyrene (with an initial concentration of 50 mg L-1) in culture solution within 8 d; it also owns a variety of plant growth promoting characteristics and appreciable tolerance to harsh environments. The transcription of pyrene catabolic genes in Pyr9 enhanced obviously when induced by pyrene. Pyr9 colonized and grew well on the root surface of white clover via root inoculation; some cells could even enter into the root tissues and move to the shoots. Compared with the Pyr9-free treatment, the pyrene contents in the roots and shoots of Pyr9-inoculated white clover decreased by 25%-30% and 33%-42%, respectively. Correspondingly, the pyrene accumulation and translocation factors in white clover decreased as well. These results indicate that Pyr9 would be a good potential to circumvent plant pyrene pollution. This research may provide a theoretical basis and technical support for the safety of agricultural products and human health in PAH-contaminated sites.

EXPath 2.0: An Updated Database for Integrating High-Throughput Gene Expression Data with Biological Pathways.

Co-expressed genes tend to have regulatory relationships and participate in similar biological processes. Construction of gene correlation networks from microarray or RNA-seq expression data has been widely applied to study transcriptional regulatory mechanisms and metabolic pathways under specific conditions. Furthermore, since transcription factors (TFs) are critical regulators of gene expression, it is worth investigating TFs on the promoters of co-expressed genes. Although co-expressed genes and their related metabolic pathways can be easily identified from previous resources, such as EXPath and EXPath Tool, this information is not simultaneously available to identify their regulatory TFs. EXPath 2.0 is an updated database for the investigation of regulatory mechanisms in various plant metabolic pathways with 1,881 microarray and 978 RNA-seq samples. There are six significant improvements in EXPath 2.0: (i) the number of species has been extended from three to six to include Arabidopsis, rice, maize, Medicago, soybean and tomato; (ii) gene expression at various developmental stages have been added; (iii) construction of correlation networks according to a group of genes is available; (iv) hierarchical figures of the enriched Gene Ontology (GO) terms are accessible; (v) promoter analysis of genes in a metabolic pathway or correlation network is provided; and (vi) user's gene expression data can be uploaded and analyzed. Thus, EXPath 2.0 is an updated platform for investigating gene expression profiles and metabolic pathways under specific conditions. It facilitates users to access the regulatory mechanisms of plant biological processes. The new version is available at http://EXPath.itps.ncku.edu.tw.

Metabolic regulation of polyamines and γ-aminobutyric acid in relation to spermidine-induced heat tolerance in white clover.

Heat stress decreases crop growth and yield worldwide. Spermidine (Spd) is a small aliphatic amine and acts as a ubiquitous regulator for plant growth, development and stress tolerance. Objectives of this study were to determine effects of exogenous Spd on changes in endogenous polyamine (PA) and γ-aminobutyric acid (GABA) metabolism, oxidative damage, senescence and heat shock protein (HSP) expression in white clover subjected to heat stress. Physiological and molecular methods, including colorimetric assay, high performance liquid chromatography and qRT-PCR, were applied. Results showed that exogenous Spd significantly alleviated heat-induced stress damage. Application of Spd not only increased endogenous putrescine, Spd, spermine and total PA accumulation, but also accelerated PA oxidation and improved glutamic acid decarboxylase activity, leading to GABA accumulation in leaves under heat stress. The Spd-pretreated white clover maintained a significantly higher chlorophyll (Chl) content than untreated plants under heat stress, which could be related to the roles of Spd in up-regulating genes encoding Chl synthesis (PBGD and Mg-CHT) and maintaining reduced Chl degradation (PaO and CHLASE) during heat stress. In addition, Spd up-regulated HSP70, HSP70B and HSP70-5 expression, which might function in stabilizing denatured proteins and helping proteins to folding correctly in white clover under high temperature stress. In summary, exogenous Spd treatment improves the heat tolerance of white clover by altering endogenous PA and GABA content and metabolism, enhancing the antioxidant system and HSP expression and slowing leaf senescence related to an increase in Chl biosynthesis and a decrease in Chl degradation during heat stress.

CEP receptor signalling controls root system architecture in Arabidopsis and Medicago.

Root system architecture (RSA) influences the effectiveness of resources acquisition from soils but the genetic networks that control RSA remain largely unclear. We used rhizoboxes, X-ray computed tomography, grafting, auxin transport measurements and hormone quantification to demonstrate that Arabidopsis and Medicago CEP (C-TERMINALLY ENCODED PEPTIDE)-CEP RECEPTOR signalling controls RSA, the gravitropic set-point angle (GSA) of lateral roots (LRs), auxin levels and auxin transport. We showed that soil-grown Arabidopsis and Medicago CEP receptor mutants have a narrower RSA, which results from a steeper LR GSA. Grafting showed that CEPR1 in the shoot controls GSA. CEP receptor mutants exhibited an increase in rootward auxin transport and elevated shoot auxin levels. Consistently, the application of auxin to wild-type shoots induced a steeper GSA and auxin transport inhibitors counteracted the CEP receptor mutant's steep GSA phenotype. Concordantly, CEP peptides increased GSA and inhibited rootward auxin transport in wild-type but not in CEP receptor mutants. The results indicated that CEP-CEP receptor-dependent signalling outputs in Arabidopsis and Medicago control overall RSA, LR GSA, shoot auxin levels and rootward auxin transport. We propose that manipulating CEP signalling strength or CEP receptor downstream targets may provide means to alter RSA.