Nitrogen modulation of legume root architecture signaling pathways involves phytohormones and small regulatory molecules

Nitrogen, particularly nitrate is an important yield determinant for crops. However, current agricultural practice with excessive fertilizer usage has detrimental effects on the environment. Therefore, legumes have been suggested as a sustainable alternative for replenishing soil nitrogen. Legumes can uniquely form nitrogen-fixing nodules through symbiotic interaction with specialized soil bacteria. Legumes possess a highly plastic root system which modulates its architecture according to the nitrogen availability in the soil. Understanding how legumes regulate root development in response to nitrogen availability is an important step to improving root architecture. The nitrogen-mediated root development pathway starts with sensing soil nitrogen level followed by subsequent signal transduction pathways involving phytohormones, microRNAs and regulatory peptides that collectively modulate the growth and shape of the root system. This review focuses on the current understanding of nitrogen-mediated legume root architecture including local and systemic regulations by different N-sources and the modulations by phytohormones and small regulatory molecules.Nadiatul A. Mohd-Radzman was supported by ANU International PhD Scholarship. This work was supported by an Australian Research Council grant to Michael A. Djordjevic and Nijat Imin (DP140103714)

MtCEP1 peptides regulate lateral organ development in the model legume, Medicago truncatula

Plant signalling peptides have been shown to have important roles in plant development. These peptides mediate signal transduction pathways that regulate specific developmental events including meristem development and cellular differentiation. In this thesis, a member of the C- terminally Encoded Peptide (CEP) family of regulatory peptides, MtCEP1, is studied in the model legume, Medicago truncatula, for its regulation of nitrogen-mediated root development, most particularly, lateral root and nodule formation. The MtCEP1 peptide-encoding gene is upregulated by low nitrogen condition, an environmental cue which strongly regulates both lateral root and nodule formation. Therefore, MtCEP1 provides an excellent research avenue as an important regulatory molecule for modulating root architecture in response to exogenous nitrogen levels. In this thesis, I have functionally characterized MtCEP1 for its role in regulating lateral root and nodule development. This is the first characterization of a CEP peptide member in Medicago and outside of Arabidopsis. MtCEP1 negatively regulates lateral root formation and promotes nodulation. There are three characteristic root phenotypes when MtCEP1 was overrepresented to the root (either by overexpressing the peptide-encoding gene or applying the synthetic peptide to the root): (1) reduction of lateral root number, (2) increased in nodulation competency and nodule number, (3) formation of periodic circumferential cell proliferation (CCP) sites. By knocking down MtCEP1 using a multigene RNAi construct to reduce gene redundancy effects, significantly more lateral roots were formed while there was no change in the nodule number. This corroborates the results with MtCEP1 promoter analysis using GUS reporter construct (pMtCEP1:GUS) which showed high expression in lateral root primordia when grown in low nitrogen condition. These results suggest the direct regulation of MtCEP1 in regulating lateral roots. Nonetheless, pMtCEP1:GUS also displayed high expression in young nodule primordia indicating that MtCEP1 could be indirectly regulating nodulation by modulating the root nodulation susceptibility during nitrogen limitation. Analysis of the nodule phenotypes revealed wider zone of susceptibility to nodulation, increased nodule number and nodule morphologies akin to ethylene-insensitive mutant, sickle. Therefore, this thesis further explores MtCEP1 regulation of nodule development, focusing on ethylene-mediated pathway. In Medicago, ethylene regulates nodulation susceptibility and provides positional information for nodulation. In the sickle mutant, a mutant of the EIN2 (ETHYLENE INSENSITIVE 2) in the ethylene signaling pathway, hypernodulation and loss of positional information of the nodules could be observed. By utilizing the sickle mutant, I have demonstrated in this thesis that MtCEP1 requires EIN2-mediated ethylene signaling pathway to increase the nodulation susceptibility. To further understand the regulation of MtCEP1, I have successfully isolated and identified the endogenous forms of MtCEP1 peptides. Nine peptides were identified which corresponds to two MtCEP1 peptide domains. The peptides were also hydroxylated and/or triarabinosylated for their biological activities. This is the first isolation of small signalling peptides in Medicago truncatula and the first characterization of triarabinosylated CEP peptide. Additionally, the differential biological activities imparted by the various MtCEP1 peptides on the root architecture provide a new insight in the complexity of plant signaling peptide regulation. In conclusion, MtCEP1 dynamically regulates lateral organ development through the different endogenous peptide species which provides developmental plasticity for the root in response to nitrogen availability

Root branching plasticity: collective decision-making results from local and global signalling

Cells within tissues can be regarded as autonomous entities that respond to their local environment and to signals from neighbours. Coordination between cells is particularly important in plants, as the architecture of the plant adapts to environmental cues. To explain the architectural plasticity of the root, we propose to view it as a swarm of coupled multi-cellular structures, rhizomers, rather than a large set of autonomous cells. Each rhizomer contains a primed site with the potential to develop a single lateral root. Rhizomers are spaced through oscillatory genetic events that occur at the basal root tip. The decision whether or not to develop a lateral root primordium results from the interplay between local interactions of the rhizomer with its immediate environment, such as local nutrient availability, long-range interactions between the rhizomers and global cues, such as overall nutrient uptake. It can halt lateral root progression through its developmental stages, resulting in the observed complex root architecture

Diverse peptide hormones affecting root growth identified in the Medicago truncatula secreted peptidome

Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyze the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP (C-TERMINALLY ENCODED PEPTIDE), two CLE (CLV3/ENDOSPERM SURROUNDING REGION RELATED) and six XAP (XYLEM SAP ASSOCIATED PEPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N- and C-terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer than expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesized to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N-terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species.This work was supported by ARC grants to MAD: DP150104050 and LP150100826. NP was partly supported by an Endeavor Fellowship. NAMR was supported by an ANU Ph.D. scholarship supported by DP120101893. AI was supported by an Australian Post-graduate Award and an AW Howard Memorial Award. LC was supported by the Bruce-Veness Chandler and the John A. Lamberton research scholarship

CEP receptor signalling controls root system architecture in Arabidopsis and Medicago

© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust 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

Compartmentalisation: A strategy for optimising symbiosis and tradeoff management.

Funder: European Research Executive AgencyFunder: Gatsby Charitable Foundation; doi: http://dx.doi.org/10.13039/501100000324Funder: Bill and Melinda Gates Foundation; doi: http://dx.doi.org/10.13039/100000865Plant root architecture is developmentally plastic in response to fluctuating nutrient levels in the soil. Part of this developmental plasticity is the formation of dedicated root cells and organs to host mutualistic symbionts. Structures like nitrogen-fixing nodules serve as alternative nutrient acquisition strategies during starvation conditions. Some root systems can also form myconodules-globular root structures that can host mycorrhizal fungi. The myconodule association is different from the wide-spread arbuscular mycorrhization. This range of symbiotic associations provides different degrees of compartmentalisation, from the cellular to organ scale, which allows the plant host to regulate the entry and extent of symbiotic interactions. In this review, we discuss the degrees of symbiont compartmentalisation by the plant host as a developmental strategy and speculate how spatial confinement mitigates risks associated with root symbiosis.Gatsby Foundation, Bill and Melinda Gates Foundation, European Comissio

Compartmentalisation: A strategy for optimising symbiosis and tradeoff management.

Plant root architecture is developmentally plastic in response to fluctuating nutrient levels in the soil. Part of this developmental plasticity is the formation of dedicated root cells and organs to host mutualistic symbionts. Structures like nitrogen-fixing nodules serve as alternative nutrient acquisition strategies during starvation conditions. Some root systems can also form myconodules-globular root structures that can host mycorrhizal fungi. The myconodule association is different from the wide-spread arbuscular mycorrhization. This range of symbiotic associations provides different degrees of compartmentalisation, from the cellular to organ scale, which allows the plant host to regulate the entry and extent of symbiotic interactions. In this review, we discuss the degrees of symbiont compartmentalisation by the plant host as a developmental strategy and speculate how spatial confinement mitigates risks associated with root symbiosis.Gatsby Foundation, Bill and Melinda Gates Foundation, European Comissio

Small-peptide signals that control root nodule number, development, and symbiosis

Many legumes have the capacity to enter into a symbiotic association with soil bacteria generically called 'rhizobia' that results in the formation of new lateral organs on roots called nodules within which the rhizobia fix atmospheric nitrogen (N). Up to 200 million tonnes of N per annum is fixed by this association. Therefore, this symbiosis plays an integral role in the N cycle and is exploited in agriculture to support the sustainable fixation of N for cropping and animal production in developing and developed nations. Root nodulation is an expendable developmental process and competency for nodulation is coupled to low-N conditions. Both nodule initiation and development is suppressed under high-N conditions. Although root nodule formation enables sufficient N to be fixed for legumes to grow under N-deficient conditions, the carbon cost is high and nodule number is tightly regulated by local and systemic mechanisms. How legumes co-ordinate nodule formation with the other main organs of nutrient acquisition, lateral roots, is not fully understood. Independent mechanisms appear to regulate lateral roots and nodules under low-and high-N regimes. Recently, several signalling peptides have been implicated in the local and systemic regulation of nodule and lateral root formation. Other peptide classes control the symbiotic interaction of rhizobia with the host. This review focuses on the roles played by signalling peptides during the early stages of root nodule formation, in the control of nodule number, and in the establishment of symbiosis. Here, we highlight the latest findings and the gaps in our understanding of these processes

Nitrogen modulation of legume root architecture signalling pathways involves phytohormones and small regulatory molecules

Nitrogen, particularly nitrate is an important yield determinant for crops. However, current agricultural practice with excessive fertilizer usage has detrimental effects on the environment. Therefore, legumes have been suggested as a sustainable alternative for replenishing soil nitrogen. Legumes can uniquely form nitrogen-fixing nodules through symbiotic interaction with specialized soil bacteria. Legumes possess a highly plastic root system which modulates its architecture according to the nitrogen availability in the soil. Understanding how legumes regulate root development in response to nitrogen availability is an important step to improving root architecture. The nitrogen-mediated root development pathway starts with sensing soil nitrogen level followed by subsequent signal transduction pathways involving phytohormones, microRNAs and regulatory peptides that collectively modulate the growth and shape of the root system. This review focuses on the current understanding of nitrogen-mediated legume root architecture including local and systemic regulations by different N-sources and the modulations by phytohormones and small regulatory molecules