Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula

Abstract

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.

Figure 1.

Ethylene Regulates the Number of Nodules and Infection Events Induced by S. meliloti. Plants were grown on differing concentrations of ACC or AVG and inoculated with Rm1021 (pXLDG4). It is assumed that ethylene levels are highest in plants grown on 10 μM ACC and lowest in plants grown on 1 μM AVG.

Figure 2.

Root Hair Deformation in Response to Nod Factor Reveals a Stronger Response in skl Relative to Wild-Type Plants. Roots were allowed to grow between two thin slabs of medium containing AVG and in some cases Nod factor for 4 days. The root hairs were imaged at set distances along the root from the hypocotyl/root junction. (A) Wild type, 0.1 μM AVG. (B) Wild type, 0.1 μM AVG plus 10 pM Nod factor. (C) skl, 0.1 μM AVG. (D) skl, 0.1 μM AVG plus 10 pM Nod factor.

Figure 3.

The Root Hair Response to Nod Factor Is Regulated by Ethylene. The length of the root hairs was assayed at set distances on the root from the hypocotyl/root junction. (A) Wild type (WT). (B) skl. Open bars represent plants treated with 0.1 μM AVG; closed bars represent plants treated with 0.1 μM AVG and 10 pM Nod factor. Data points represent the average root hair length at each distance along the root for three plants per treatment. Error bars show ±sd.

Figure 4.

Ethylene Modulates Gene Expression Activated by S. meliloti or Nod Factor. (A) Wild-type plants were grown on 0.1 μM AVG or 10 μM ACC, and skl plants were grown in the absence of either AVG or ACC and inoculated with S. meliloti Rm1021 (pXLGD4) at OD₆₀₀ = 0.1. Root tissue was collected at set times after inoculation (hr). RNA gel blots were probed with a region of exon 2 of RIP1 and secondarily with actin to assess loading. (B) The expression levels of RIP1 in (A) were standardized using the actin loading control and quantified as the fold induction of the levels before inoculation with S. meliloti for each treatment. WT, wild type. (C) M. truncatula plants, stably transformed with the ENOD11 promoter driving the expression of GUS, were treated with either 0.1 μM AVG or 10 μM ACC and secondarily treated with 1 nM Nod factor for 6 hr. The photograph shows representative roots from each treatment. Note the nonsymbiotic root cap expression of ENOD11-GUS present in both treatments. The inducible expression of ENOD11-GUS in the epidermis of the root, behind the root tip, is present only in plants treated with AVG.

Figure 5.

Ethylene Regulates the Number of Root Hairs Activated for Calcium Spiking in Response to Nod Factor. (A) Wild-type plants (WT) were grown on either 0.1 μM AVG or 10 μM ACC, and skl plants were grown on 10 μM ACC. Data points represent the percentage of root hairs that were induced for calcium spiking 60 min after the addition of 10 pM Nod factor. (B) Wild-type plants (WT) were grown either on medium alone or on 0.1 μM AVG or 10 μM ACC, and skl plants were grown in the absence of either AVG or ACC. The concentration of Nod factor was increased 10-fold every 30 min, starting at 1 fM and ending at 1 nM. The number of root hairs induced for calcium spiking at each concentration was assessed.

Figure 6.

Ethylene Can Block the Maintenance of Calcium Spiking. The traces show the change in calcium levels of individual cells on the same wild-type or skl plant. Calcium spiking was activated using 10 pM Nod factor and 0.4% ethylene added ∼30 min after the initiation of spiking. (A) to (C) Wild type. (D) to (F) skl. (G) to (I) Wild type, no ethylene. The y axis represents the change in fluorescence after treatment of the raw fluorescence data with the equation Y = X_(n+1) − X_(n). This transformation reduces the level of background fluctuations and amplifies rapid changes in calcium levels, thus aiding in the identification of calcium spikes.

Figure 7.

skl Shows an Increased Period between Calcium Spikes Compared with Wild-Type (WT) Plants. For each cell, the average period was calculated from 30 min of spiking induced by 10 pM Nod factor. The data points represent the average of the average period from multiple cells ±sd.

Figure 8.

A Model Representing the Communication between the Ethylene Perception Pathway and the Nod Factor Perception Pathway. In this model, it is assumed that calcium spiking is part of the Nod factor perception pathway immediately upstream of Nod factor responses. However, the responses downstream of calcium spiking have not been characterized. A protein in the ethylene perception pathway is involved in the inhibition of the Nod factor perception pathway at or upstream of calcium spiking. Additional proteins may be involved in the communication (yellow). Not shown is a possibility discussed in the text whereby ethylene might affect a downstream step of the Nod factor pathway that has feedback to a step upstream of spiking. Mutations in proteins of the ethylene perception pathway at or upstream of the communication will show a loss of ethylene sensitivity for both ethylene-regulated developmental responses and nodulation responses (red). skl must represent one of these proteins. Mutations in proteins downstream of this point will be ethylene insensitive for general ethylene responses but sensitive for nodulation-specific ethylene effects (green). Mutations in proteins involved in communication between the two pathways will be ethylene sensitive for ethylene-regulated developmental responses but insensitive to ethylene for the nodulation-specific ethylene effects (yellow). C, calcium channels; P, calcium pumps.