Abscisic acid coordinates nod factor and cytokinin signaling during the regulation of nodulation in Medicago truncatula

Abstract

Nodulation is tightly regulated in legumes to ensure appropriate levels of nitrogen fixation without excessive depletion of carbon reserves. This balance is maintained by intimately linking nodulation and its regulation with plant hormones. It has previously been shown that ethylene and jasmonic acid (JA) are able to regulate nodulation and Nod factor signal transduction. Here, we characterize the nature of abscisic acid (ABA) regulation of nodulation. We show that application of ABA inhibits nodulation, bacterial infection, and nodulin gene expression in Medicago truncatula. ABA acts in a similar manner as JA and ethylene, regulating Nod factor signaling and affecting the nature of Nod factor-induced calcium spiking. However, this action is independent of the ethylene signal transduction pathway. We show that genetic inhibition of ABA signaling through the use of a dominant-negative allele of ABSCISIC ACID INSENSITIVE1 leads to a hypernodulation phenotype. In addition, we characterize a novel locus of M. truncatula, SENSITIVITY TO ABA, that dictates the sensitivity of the plant to ABA and, as such, impacts the regulation of nodulation. We show that ABA can suppress Nod factor signal transduction in the epidermis and can regulate cytokinin induction of the nodule primordium in the root cortex. Therefore, ABA is capable of coordinately regulating the diverse developmental pathways associated with nodule formation and can intimately dictate the nature of the plants' response to the symbiotic bacteria.

Figure 1.

ABA Inhibits Nodulation and Promotes Lateral Roots in M. truncatula. Plants were grown on differing concentrations of ABA and assayed for lateral root initiation (A), nodule formation (B), and S. meliloti infection events (C). Increasing ABA concentrations promote lateral root formation up to 1 μM ABA but suppress nodulation and S. meliloti infection. Infections were measured following lacZ staining, and both infection foci and foci with infection threads were counted as infection events. Ten to twelve plants were analyzed for each ABA concentration, with different plants being used to assay lateral roots, nodulation, and rhizobial infection. Error bars represent se.

Figure 2.

ABA Suppresses Nod Factor and S. meliloti–Induced Gene Expression. (A) and (B) ABA effect on early nodulin gene expression was assayed using transgenic plants containing ENOD11:GUS (A) or qRT-PCR of RIP1 (B). (A) Pretreatment with ABA (concentrations indicated) inhibits ENOD11 induction following 1 nM Nod factor treatment. The blue color represents GUS expression. (B) Pretreatment with 10 μM ABA abolished S. meliloti induction of RIP1 in both wild-type and ethylene-insensitive skl plants. Error bars represent se.

Figure 3.

ABA Modulates Nod Factor–Induced Calcium Spiking. (A) and (B) Representative calcium traces of M. truncatula root hair cells preinduced with 1 nM Nod factor and secondarily treated with 1 mM ABA. Treatment with ABA inhibits calcium spiking, and this can be recovered following ABA washout in continuous 1 nM Nod factor (A) or by raising the Nod factor concentration to 10 nM (B). Calcium measurements were generated from cameleon transformed plants, and the y axes represent the ratio of cyan fluorescent protein: yellow fluorescent protein (CFP:YFP) in arbitrary units. Error bars represent se. (C) While 1 mM ABA inhibits calcium spiking, lower concentrations of ABA cause a lengthening of the interval between individual calcium spikes. The period between calcium spikes was averaged from 20 min of spiking in 20 cells per treatment, and this period was standardized relative to no ABA treatment.

Figure 4.

ABA and Ethylene Act Independently in the Suppression of Nodulation. (A) and (B) The ethylene-insensitive mutant skl shows ABA induction of lateral roots (A) and ABA suppression of nodulation (B) in a manner similar to that observed in the wild type. However, the lateral root and nodule number is affected by the ethylene-insensitive nature of skl, and the ABA effects are superimposed upon this preexisting state. (C) ENOD11:GUS induction by 1 nM Nod factor in skl (all roots) is suppressed following ABA addition, at the concentrations indicated. (D) Quantification of GUS in wild-type plants carrying ENOD11:GUS and skl plants with ENOD11:GUS. skl shows higher levels of ENOD11 induction by 1 nM Nod factor, which is suppressed by ABA treatment. (E) Representative traces of cells pretreated with 1 nM Nod factor for 30 min prior to calcium imaging. Addition of 1 mM Nod factor leads to inhibition of calcium spiking in all wild-type and skl cells, but sta-1 is partially insensitive to ABA treatment. The calcium imaging was generated following microinjection of Oregon green/Texas red, and the y axes represent the change in florescence of Oregon green versus Texas red in arbitrary units. Error bars represent se.

Figure 5.

Arabidopsis abi1-1 Induces Insensitivity to ABA and Hypernodulation in M. truncatula. (A) and (B) Transformation of abi1-1 from Arabidopsis leads to a hypernodulation phenotype (B) that was not observed when M. truncatula was transformed with the empty vector (A). The insets indicate nodules from these plants stained with X-gal to reveal bacterial infection inside the nodules. (C) abi1-1 transformation leads to enhanced Nod factor induction of ENOD11:GUS and makes this response insensitive to 10 μM ABA treatment. (D) qRT-PCR shows that the abi1-1–transformed roots are also insensitive to ABA treatment for induction of RD22, a marker of ABA signaling. EV, empty vector. Error bars represent se.

Figure 6.

sta-1 Shows Defects in Plant Growth, Seed Germination, and Stomatal Behavior. (A) sta-1 shows pleiotropic effects for root and shoot growth revealed by a reduced stature of the plant. (B) sta-1 shows delayed seed germination compared with the wild type. The number of germinated seeds was measured using radicle emergence as a marker relative to the time since removing the seeds from 4°C. Two hundred seeds are analyzed per treatment. (C) The fresh weight of detached leaves was used to measure relative water loss that provides an indication of stomatal aperture. sta-1 shows more rapid water loss, indicating an alteration in its stomatal behavior. Error bars represent se.

Figure 7.

sta-1 Is Altered in Its Sensitivity to ABA. (A) sta-1 shows hypersensitivity to ABA for seed germination. In this assay the number of seeds germinated was analyzed at 24 h after removal from 4°C, a time point where the maximum number of seeds have germinated in each line. The data points are presented as the percentage of seeds germinated relative to germination in the absence of ABA. Two hundred seeds are analyzed per treatment. (B) to (E) In contrast with seed germination, sta-1 is insensitive to ABA for lateral root initiation (B) and induction of RD22 (C) as well as stomatal closure ([D] and [E]). In (D) and (E), the plants were pretreated to ensure maximal stomatal opening and then secondarily treated with buffer alone (0) or with buffer containing 25 μM ABA. In wild-type plants, all stomata closed in the ABA treatment, while in sta-1 mutant leaves, all stomata remained open.

Figure 8.

sta-1 Is Insensitive to ABA for Nod Factor–Induced Gene Induction. (A) Nodulation of sta-1 is greatly reduced compared with wild-type plants and suppressed by ABA treatment. (B) qRT-PCR demonstrating that sta-1 shows reduced sensitivity to ABA for the regulation of Nod factor (1 nM for 12 h) induced RIP1 expression. sta-1 shows reduced levels of RIP1 induction compared with wild-type plants, but the suppression of RIP1 by ABA is also greatly reduced in sta-1. (C) sta-1 shows reduced levels of Nod factor (1 nM) induced ENOD11 expression, as measured by qRT PCR, and reduced suppression of this gene induction by ABA. (D) sta-1 shows reduced levels of S. meliloti induction of RIP1 and reduced suppression of this gene induction by ABA. For (B) to (D), 10 plants were used for each treatment and four biological repeats were performed. DAI, days after inoculation; NF, Nod factor. Error bars represent se.

Figure 9.

ABA Can Suppress Cytokinin-Induced Nodulation Responses. (A) Treatment with 0.01 μM cytokinin (BAP) for 10 d induces ENOD40 in M. truncatula, and this is blocked upon treatment with ABA. sta-1 shows increased levels of ABA suppression of ENOD40 induction by cytokinin. (B) Treatment with 0.01 μM BAP induces NIN in M. truncatula, and this is blocked upon treatment with ABA. sta-1 shows increased levels of ABA suppression of NIN induction by cytokinin. (C) Wild-type M. truncatula plants were transformed with abi1-1 or empty vector (EV) and tested for cytokinin (BAP) induction of ENOD40. abi1-1–transformed plants showed much higher levels of ENOD40 induction by cytokinin compared with EV transformed plants. For (A) and (B), 10 plants were used for each treatment and four biological repeats were performed. All error bars represent se.