Multiple Autoregulation of Nodulation (AON) Signals Identified through Split Root Analysis of Medicago truncatula sunn and rdn1 Mutants

Abstract

Nodulation is energetically costly to the host: legumes balance the nitrogen demand with the energy expense by limiting the number of nodules through long-distance signaling. A split root system was used to investigate systemic autoregulation of nodulation (AON) in Medicago truncatula and the role of the AON genes RDN1 and SUNN in the regulatory circuit. Developing nodule primordia did not trigger AON in plants carrying mutations in RDN1 and SUNN genes, while wild type plants had fully induced AON within three days. However, despite lacking an early suppression response, AON mutants suppressed nodulation when roots were inoculated 10 days or more apart, correlated with the maturation of nitrogen fixing nodules. In addition to correlation between nitrogen fixation and suppression of nodulation, suppression by extreme nutrient stress was also observed in all genotypes and may be a component of the observed response due to the conditions of the assay. These results suggest there is more than one systemic regulatory circuit controlling nodulation in M. truncatula. While both signals are present in wild type plants, the second signal can only be observed in plants lacking the early repression (AON mutants). RDN1 and SUNN are not essential for response to the later signal.

Keywords: Medicago truncatula; RDN1; SUNN; autoregulation of nodulation; nitrogen; split-root analysis.

Figure 1

Persistence of the systemic suppression signal in mutant and wild type plants. (a) Wild type plants have consistently fewer nodules on the second root when the second root is inoculated 2–15 days after the first. Percent nodulation was calculated by dividing the mean nodule number of the second root by the mean nodule number of the first root. Values for time points 2–15 are statistically significant from time 0. (b) Wild type values (green dots) compared to the sunn-4 allele values (red boxes). Data points marked with * are not statistically different between wild type and mutant (c) Wild type values (green dots) compared to the rdn1-2 allele values (black boxes). Data points marked with * are not statistically different between wild type and mutant. Percent nodulation was calculated by dividing the mean nodule number of the second root by the mean nodule number of the first root. Statistical comparisons among means are based on factorial ANOVA followed by pairwise mean comparisons. Data are shown as means ± SE. (n = 6 to 47 plants for each time point per genotype).

Figure 2

AON mutants and wild type plants fix equivalent amounts of nitrogen. Roots of A17, rdn1-2 and sunn-4 plants were evaluated 15 days after inoculation with S. medicae. Means ±se are per genotype (a) Mean nodule number (b) Mean number of nitrogen fixing (pink) nodules (c) Mean nodule fresh weight (ten samples of three nodules each from five to six plants per genotype). (d) Hydrogen production (μmol/hr) as a proxy for nitrogen fixation rate (see Experimental Section). Measurements were taken on the same plants at both 10 and 15 days after inoculation with S. medicae in two biological replicates. A17 (n = 9), rdn1-2 (n = 10) and sunn-4 (n = 9). Statistically significant differences from wild type based on Student’s t-test are indicated by * (p < 0.05) and ** (p < 0.01).

Figure 3

Effect of ammonium nitrate versus water treatment of first root on nodulation of second root in a split root system. Split root plants were grown as in [27] and the first root fed with either water or 10 mM NH₄NO₃ for four days before inoculating the second root. Nodules were counted on the second root of A17 (wild type) plants (n = 12) and sunn-4 plants (n = 7) 20 days after inoculation and expressed as percentage of the water controls. Bars indicate standard error of the percentage means calculated by Taylor series expansion.

Figure 4

The effect of prior inoculation of the tester root with different strain of S. meliloti on subsequent nodulation of the responder root in A17, sunn-4, and rdn1-2. The responder root was inoculated with Rm1021 20 days after the tester root inoculation and nodules were counted 21 days later. (a) Rm1021 wild type on both roots, expressed as mean nodulation % of the responder root to the tester. Errors are standard error by Taylor expansion. When inoculating with different strains, data is expressed as mean nodule number ± se on the responder root to mean nodule number ± se for strains used on the tester root in (b) Rm1312 (Nod⁺/Fix⁻), and (c) SL44 (Nod⁻/Fix⁻). Error bars for (b) and (c) are standard error of the mean (n = 5 to 10 plants per genotype per experiment).

Figure 5

AON mutants and wild type plants suppress nodulation after long periods of water only treatment of the first root. Dark grey bars represent nodulation on the second root after four days of water treatment prior to inoculation and is set to 100% (actual mean on top of bar); light grey indicates mean percent reduction in nodulation on the second root after 11 days treatment of the first root prior to inoculation. Bars indicate standard error of the percentage means calculated by Taylor expansion. n = 9–16 plants per genotype per treatment.

Figure 6

Carbon and nitrogen in shoots as a percentage of dry weight determined by ¹³C and ¹⁵N isotope analysis of organic solids. An equal amount of tissue was pooled from three plants for each genotype and condition at the day of inoculation, dried, and analysis performed in triplicate (a). Mean percent nitrogen in leaves from plants in which the first roots was watered with 10 mM NH₄NO₃ for four days from Figure 3 (black bars-data for rdn1-2 not available) with water for four days from Figure 5 (grey bars) and with water for 10 days from Figure 5 (dotted bars) (b). Mean percent carbon from the same material and (c) C/N ratio of plants in (a) and (b).