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.
Regulatory peptides, legume, Medicago truncatula, root development, lateral roots, nodules, plant development, ethylene, nitrogen fixation
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