Nodulation is enhanced by ectopic expression of MtCEP1 or by peptide application

The effect of overexpressing MtCEP1 on nodule organogenesis was also scored. Although MtCEP1 clearly inhibited lateral root formation, this regulatory peptide promoted root nodule formation. The data showed a 75–310% increase in nodule numbers formed on composite plant roots overexpressing MtCEP1 compared with controls when they were infected by S. meliloti strain 1021 grown at either 20 °C or 25 °C (Fig. 3.6A). There was also a significant increase in nodulation in plants inoculated with the highly effective strain WSM1022, but the magnitude of the increase was less (Fig. 3.6B). WSM1022 induces nodules which develop faster and fix more N than S. meliloti strain 1021 (Terpolilli et al., 2008; Saur et al., 2011). Continuous exposure of plants to MtCEP1 peptide followed by rhizobial inoculation also increased root nodule number.

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Figure 3.6: MtCEP1 overexpression or peptide addition increases nodulation and promotes nodule development at different nitrate concentrations. (A) Comparison of nodulation

between MtCEP1-overexpressing plants and control plants when inoculated with!S. meliloti strain 1021. (B) Partial nitrate tolerance of nodule development induced by S. meliloti WSM1022 on MtCEP1-overexpressing plants. Three-week-old composite plants were inoculated and scored 2 weeks post-inoculation. n ≥30. (C) Acetylene reduction assay. MtCEP1-treated (1 μM) and non-

CEP regulatory peptide affecting root development | 5405

Fig. 6. MtCEP1 overexpression or peptide addition increases nodulation and promotes nodule development at different nitrate concentrations. (A) Comparison of nodulation between MtCEP1-overexpressing plants and control plants when inoculated with S. meliloti strain 1021. (B) Partial nitrate tolerance of nodule development induced by S. meliloti WSM1022 on MtCEP1-overexpressing plants. Three-week-old composite plants were inoculated and scored 2 weeks post-inoculation. n ≥30. (C) Acetylene reduction assay. MtCEP1-treated (1 μM) and non-treated wild-type plants grown in Fåhraeus medium were harvested at 2 weeks post-inoculation by WSM1022. Nitrogenase activity was calculated from peak areas of samples relative to acetylene and ethylene standards to obtain nmol ethylene min–1_{. n ≥3. (D) Effect of MtCEP1 peptide application on nodulation. Plants were grown in 0.8% agar-containing plates}

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treated wild-type plants grown in Fåhraeus medium were harvested at 2 weeks post-inoculation by WSM1022. Nitrogenase activity was calculated from peak areas of samples relative to acetylene and ethylene standards to obtain!nmol ethylene min–1. n ≥3. (D) Effect of MtCEP1 peptide application on nodulation. Plants were grown in 0.8% agar-containing plates supplemented with Fåhraeus medium with or without peptides. Five-day-old seedlings were inoculated with S. meliloti strain WSM1022 and grown for a further 2 weeks. MtCEP1, AFQ(P)TTPGNS(P)GVGH; MtCEP1 G→A, AFQ(P)TTPANS(P)GVGH. (P) is hydroxylproline.! (E) Inoculation of plants with pre-formed CCPs results in root nodule formation at CCP sites at 25 mM KNO₃ at 14 d post-inoculation. Arrowhead, CCP sites; arrows, nodules. (F) Representative nodule sections from MtCEP1 peptide-treated and untreated plants grown at either 0, 5, or 25 mM KNO₃ for 3 weeks. Scale bars=100 μm. Plants were grown at 20 °C. Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001. Error bars indicate the SE.

Nodule number is strictly controlled in legumes by an autoregulatory mechanism (which prevents overnodulation) and by the N status of the root (Carroll et al., 1985). The developmental susceptibility of legume roots is also predominantly restricted to cells occurring in the zone of elongation (Bhuvaneswari et al., 1980; Sargent et al., 1987). Therefore, the developmental susceptibility of legumes is maximal in plants grown under low (<1mM) N or under N starvation, and the suppressive effects of nitrate on root nodule number, development, leghaemoglobin production, and nitrogen fixation, particularly above 3mM, are well documented. Leghaemoglobin production is also an important indicator of symbiotic capacity (Roponen, 1970; Legocki and Verma, 1980; Ott et al., 2005; Madsen et al., 2010). Therefore, the effect of MtCEP1 on symbiotic capacity was measured by the frequency of pink (leghaemoglobin-containing) nodules forming at 5mM and 25mM KNO3. The results showed a

significantly higher numbers of pink nodules on plants overexpressing MtCEP1 or exposed to 1 µM MtCEP1 peptide compared with controls (Appendix Fig. 1.8A, B). At 5mM and 25mM KNO3, the nodule numbers formed on roots overexpressing MtCEP1 were increased

significantly (e.g. 10-fold higher at 25mM KNO3; Fig. 3.6B). A similar trend was observed when

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peptide where Gly8 was replaced with alanine (Fig. 3.6D; Appendix Fig. 1.3D). Across all nitrate regimes, nodules were larger on MtCEP1 peptide-treated roots than on control plants (Fig. 3.6F) or plants treated with the MtCEP1 peptide where Gly8 was replaced with alanine (Fig. 3.6D), and they developed ~2 d faster than the controls. At 3 weeks post-inoculation with S. meliloti, a clear bacteroid-containing zone was apparent in nodule sections on MtCEP1 peptide- treated plants at 0 and 5mM nitrate, and nodules often spanned two xylem poles (Fig. 3.6F). Nodules forming at 25mM nitrate on the MtCEP1-treated plants also possessed leghaemoglobin, whereas controls did not (Fig. 3.6F). The inoculation of rhizobia onto plants grown at 25mM nitrate with pre-formed CCPs (due to overexpressing MtCEP1 or treated with MtCEP1 peptide) resulted in nodules being induced at, but not between, these CCP sites even though they occurred in the mature root zone (Fig. 3.6E). The results showed that for plants exposed to elevated MtCEP1 by overexpression or ectopic application, the susceptibility to infection by S. meliloti and nodulation ability was more tolerant to nitrate, and overall nodulation development was enhanced at the different nitrate levels tested.

To confirm further the enhancement of nodulation by MtCEP1 addition, the nitrogenase activity was measured. The results showed a significant increase in the nitrogenease activity from 35% at 0mM KNO3 to 200% at 5mM KNO3 when 1 µM MtCEP1 peptides were applied (Fig. 3.6C).

Although nitrogenase activity reflects nodule functionality, the overall results show that MtCEP1 overexpression or peptide addition enhances all aspects of root nodule formation. The increased number of nodules formed at 25mM nitrate and their formation at pre-formed CCP sites suggests that these sites show elevated susceptibility for root nodule formation, and this partial nitrate tolerance for nodulation imparted by MtCEP1 could have beneficial agricultural outcomes.

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3.4.6 Identification of genes that are differentially expressed by MtCEP1 overexpression