Ethylene, a Hormone at the Center-Stage of Nodulation

Abstract

Nodulation is the result of a beneficial interaction between legumes and rhizobia. It is a sophisticated process leading to nutrient exchange between the two types of symbionts. In this association, within a nodule, the rhizobia, using energy provided as photosynthates, fix atmospheric nitrogen and convert it to ammonium which is available to the plant. Nodulation is recognized as an essential process in nitrogen cycling and legume crops are known to enrich agricultural soils in nitrogenous compounds. Furthermore, as they are rich in nitrogen, legumes are considered important as staple foods for humans and fodder for animals. To tightly control this association and keep it mutualistic, the plant uses several means, including hormones. The hormone ethylene has been known as a negative regulator of nodulation for almost four decades. Since then, much progress has been made in the understanding of both the ethylene signaling pathway and the nodulation process. Here I have taken a large view, using recently obtained knowledge, to describe in some detail the major stages of the process. I have not only reviewed the steps most commonly covered (the common signaling transduction pathway, and the epidermal and cortical programs), but I have also looked into steps less understood (the pre-infection step with the plant defense response, the bacterial release and the formation of the symbiosome, and nodule functioning and senescence). After a succinct review of the ethylene signaling pathway, I have used the knowledge obtained from nodulation- and ethylene-related mutants to paint a more complete picture of the role played by the hormone in nodule organogenesis, functioning, and senescence. It transpires that ethylene is at the center of this effective symbiosis. It has not only been involved in most of the steps leading to a mature nodule, but it has also been implicated in host immunity and nodule senescence. It is likely responsible for the activation of other hormonal signaling pathways. I have completed the review by citing three studies which makes one wonder whether knowledge gained on nodulation in the last decades is ready to be transferred to agricultural fields.

Keywords: ethylene signaling; hormones; host immunity; model legumes; nodule organogenesis; nodule senescence; rhizobia; sickle.

FIGURE 1

Plant responses to the presence of rhizobia. The bacterium (purple oval) triggers a defense response (pink box) by producing exopolysaccharides (EPS) and lipopolysaccharides (LPS), flagellin-like molecules (flg22), and type III-effector molecules (T3ss) used to inject Nop proteins in the plant cell. As the plant senses these molecules, especially flg22 with the FLS2 receptor, it mounts a set of defense responses. Among the outcomes are the production of ethylene and the up-regulation of pathogenesis-related (PR) proteins. Simultaneously, the rhizobium secretes Nod factors (NFs) which are perceived by the plant receptors NFR1 and NFR5, which may be recruited to membrane micro-domains by remorins (SYMREM1) and flotillins (FLOT2/4). Perception of NFs initiate the CSTP (green box) composed of eight genes: SYMRK, CASTOR/POLLUX, NUP85 and NUP133, NENA, CCaMK and CYCLOPS. CCAMK decrypts the calcium signal, triggering an epidermal program (orange box) and a cortical program (blue box). Epidermal program: Signaling, via CCaMK, triggers the ubiquitin ligase PUB1, considered a negative regulator of NFR1, and the transcription factor NIN which, with NSP1 and NSP2, and the vapyrin (VPY), affects the formation of the infection thread. For this event to occur, proteins important in the layout of the cytoskeleton, such as NAP1, PIR1, and ARPC1, are likely recruited. NF perception may also directly induce transcription of specific genes, such as the EPS receptor EPR3, the ethylene biosynthetic enzyme ACS, and an ethylene response factor required for nodulation ERN1. Cortical program: CCaMK triggers the cytokinin receptor LHK1 and the downstream transcriptions factors NIN, NSP1 and NSP2. In this program, in contrast to the epidermal program, ERN1 induction appears to be done through NIN and the NSPs. VPY and EFD, an ethylene response factor required for nodule differentiation, are also implicated in the program. The proper decoding of the calcium signal leads to nodule organogenesis. For a nodule to become infected and functioning, all steps must be impeccably orchestrated. Pointed arrows denote stimulation, flat arrows reflect inhibition, and broken arrows indicate speculative action or contradiction in the literature. The numbered stars represent potential location of ethylene signaling or action. The numbers correspond to the order in which these actions are reported in the text. Schematics adapted from Desbrosses and Stougaard (2011). Most of the genes mentioned on these diagrams are those which have been designated for Lotus japonicus; their orthologs for Medicago truncatula are mentioned in the text.

FIGURE 2

Rhizobial release and bacteroid differentiation within an infected plant cell of Medicago truncatula. Once the rhizobia are released in the infection droplets, they divide and elongate. They later on differentiate into bacteroids; many bacterial genes, such as those necessary for division, are then turned off, whereas genes the products of which are necessary for bacteroid metabolism (nitrogenase, transporters, etc…) are turned on. As all these events are important steps in the symbiosis, they are controlled tightly and many genes and their products are implicated in their control. All proteins within a black box are known to be involved with the NF perception. Those in a green box are known to be part of the common symbiotic transduction pathway. Proteins in a blue box are thought to play a role in both the epidermal and cortical nodulation programs. Proteins in a brown box are not known to play a role early in the nodulation process; they may be specific for controlling these specific steps. NFs are continuously produced by rhizobia within the infection thread and when the rhizobia are released, the NFs are perceived by NFP and LYK3, the Nod factor receptors. These receptors are likely to be recruited in a micro-domain by SYMREM1 expressed in the plasmalemma of the infected cell. SNAREs and NF-YA1 are essential in bacterial release, whereas NAC074, VAMP721, and RSD are required for bacterial elongation. The bacteria differentiate once they have perceived the antimicrobial NCRs, which are found in the bacteroid after they crossed both symbiosome and bacterial membranes. DNF1 is necessary for the entry of NCRs in the symbiosome. Bacterial differentiation is also under the control of DNF2. Finally, MtNAC920 working in concert with CP2 trigger bacteroid senescence. The numbered stars represent potential location of ethylene signaling or action and the numbers correspond to the order in which these actions are reported in the text.

FIGURE 3

Ethylene biosynthesis and transduction pathway. Methionine is the precursor of ethylene (pink triangle). It is converted into S-adenosyl-methionine (SAM) by SAM synthetase (SAMS); SAM is converted into 1-aminocyclopropane-1-carboxylate (ACC) by ACC synthase (ACS). ACC is later converted into ethylene by ACC oxidase (ACO). Both auxin and cytokinin are known to promote ethylene production; the former up-regulates ACS expression whereas the latter prevents ACS degradation. Ethylene is known to modulate its own levels by stimulating (+) or inhibiting (-) ACO activity. ACC deaminase is an enzyme found in certain types of bacteria; it converts ACC into ammonia and α-ketobutyrate and both may be used as nutrients by the bacteria. When no ethylene is sensed in a cell (circle 1), ethylene receptors (such as ETR1) located on the ER membrane promote CTR1 activity; in doing so, they allow CTR1 to phosphorylate EIN2 which inactivates it. Upon ethylene perception (circle 2), ETR1 is dephosphorylated and CTR1 is switched off. ETR1 can now bind efficiently to EIN2 which is activated. Its C-end terminus (small yellow star) is cleaved and moves to the nucleus where it stabilizes the transcription factors EIN3 and EIL1, which are then able as dimers to trigger the expression of ethylene-responsive genes, inducing many developmental and biochemical events. When ethylene is not perceived, EIN3 and EIL1 are targeted to proteasomes via the action of EBF1/2. MAP kinases 3 and 6 (MAPK3/6) are known to activate ACS enzymes and to phosphorylate EIN3 in Arabidopsis, protecting it from degradation. Jasmonic acid is known to work synergistically with ethylene by promoting the degradation of JAZ2, a protein known to repress EIN3/EIL1. Finally, EIN3/EIL1 inhibits salicylic acid synthesis as they bind to the promoter of SID2, one of its biosynthetic genes, thus inactivating it.