Resumen de Role of strigolactones in plant defense: hormonal cross-talk and implication in arbuscular mycorrhizal symbiosis
Phytohormones are the main regulators of the biological functions of plants, playing crucial roles in development, reproduction and stress responses. They include abscisic acid (ABA), cytokinins (CKs), auxins, brassinosteroids (BRs), gibberellins (GAs), salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) (Robert-Seilaniantz et al., 2011; Vanstraelen et al., 2012). In 2008, strigolactones (SLs) were included as a new class of phytohormones, due to its role as inductors of apical dominance in shoot architecture, inhibiting the growth of the axillary buds (Gómez-Roldán et al., 2008; Umehara et al., 2008). Initially, they were described as signalling molecules in the rhizosphere inducing seed germination of root parasitic plants of the Orobanchaceae (Bouwmeester et al., 2003; Cook et al., 1972) and later as inducers of hyphal branching of arbuscular mycorrhizal (AM) fungi, thus promoting the symbiosis between the fungus and the host plant (Akiyama et al., 2005; Bouwmeester et al., 2007). More recently, it has been proposed a role of SLs in the symbiosis Rhizobium-legume (Foo et al., 2011; Pelaez-Vico et al., 2016), thus extending its range of action in the rhizosphere. As phytohormones, it has been shown that SLs are also implicated in other physiological processes in the plant such as leaf senescence, root architecture and reproductive development, among others (Pandey et al., 2016). It is well known that phytohormones regulate vital processes by interacting with each other (Vanstraelen et al., 2012). They can present a synergistic or antagonist relationship, creating a hormonal balance that determines the plant response. For example, it has been shown that the plant defense response against pathogens and pests is regulated mainly by the interaction between SA, JA and ABA, or that shoot branching is mainly determined by the action of auxins, CKs and SLs (Janssen et al., 2014; Robert-Seilaniantz et al., 2011; Ton et al., 2009). This interaction between phytohormones, also known as cross-talk, is key part in the hormonal regulation, and it is the main subject of this PhD Thesis.
One of the greatest challenges in modern agriculture is how to eradicate the pests and diseases that cause huge crop losses (Jung et al., 2012; Xie et al., 2010). The use of pesticides and insecticides as control against harmful organisms is a non-viable solution in the context of sustainable agriculture as required by today's society. In this sense, the improvement of plant defence responses against these organisms using beneficial soil microorganisms, e.g. AM fungi, it is a very promising alternative. In order to implement this strategy, a detailed knowledge of the regulation of the symbiosis, as well as the plant defence responses and the fundamental processes of pathogen/parasite is required, and in this context this Doctoral Thesis is framed. Therefore, the main objective of this Doctoral Thesis is the study of the potential role of SLs in defence and its interaction with other phytohormones regulating plant defence responses during the establishment and development of the AM symbiosis, with particular emphasis on the role of jasmonic acid (JA) and the peptide hormone systemin.
Root parasitic plants of the genera Phelipanche and Orobanche, commonly called broomrapes, cause serious damage to agricultural crops. Current control methods used to eradicate these parasitic weeds are not effective. This is due to most of their life cycle occurs underground and therefore, when the infection is detected is too late since an important damage has already been generated to the crop. Thus, new control strategies are focused on the elimination or reduction of the parasite during the initial stages of the interaction (Fernández-Aparicio et al., 2016; Lopez-Ráez et al., 2009). The infection levels and the damages caused by the parasite are partly regulated by the phytohormones of the host plant. For this reason in Chapter 1, we analysed the transcriptomic regulation of molecular markers associated to different hormonal pathways in roots of tomato plants infected by Phelipanche ramosa during the initial stages of infection. Gene expression analyses showed an induction of markers related to JA, SA and ABA, main regulatory hormones associated to plant defence responses (Robert-Seilaniantz et al., 2011). This suggests a possible induction of the defence mechanisms during the early stages of the tomato-P. ramosa interaction, which are associated to these hormonal pathways (Torres-Vera et al., 2016). Molecular markers for the SL signalling pathway were also analysed, showing an induction of these genes during the early stages of infection. This chapter presents for the first time, a possible role of SLs in the regulation of defence responses, probably through an interaction with the major defence phytohormones. Due to the role of SLs in the rhizosphere as signalling cues and to their possible involvement in defence responses against broomrapes, SLs could be an key element in future strategies to eradicate this agricultural pest (Torres-Vera et al., 2016).
In Chapter 2, the potential role of SLs in plant defence has been explored in more detail. To this end, the resistance/susceptibility of different SL-deficient tomato plants against different fungal – shoot- and root-borne – pathogens was analysed. It was observed that the SL-deficient plants Slccd8 and Slccd7 were more susceptible to the necrotrophic pathogen Botrytis cinerea than their corresponding wild-types. Slccd8 plants were also more susceptible to the infection by the necrotrophic fungus Alternaria alternata, suggesting a possible involvement of SLs in the defence response against this type of air-borne pathogens. A higher infection rate was also observed in Slccd8 roots by the hemibiotrophic pathogen Fusarium oxysporum f. sp. lycopersici, suggesting a broad action spectrum of SLs in defence responses. On the other hand, the SL-deficient Arabidopsis plants max4-1, blocked at the CCD8 step, were also more susceptible to the infection by B. cinerea, indicating that the SL’ role in defence seems to be conserved across plant species. Hormonal analyses in leaves of Slccd8 plants showed that all the main defence phytohormones (SA, JA and ABA) were reduced in the transgenic line compared to wild-type plants. A further transcriptomic analysis showed a possible predominance of the JA signalling pathway as the main defence pathway where SLs may be involved. Therefore, the role of SLs in defence seems to be an indirect effect, acting synergistically with the major pathways that regulate the defense mechanisms, primarily with the JA pathway (Torres-Vera et al., 2014).
Both SLs and JA play a role in the regulation of AM symbiosis, although apparently to different levels of the plant-AM fungus interaction. SLs act as inducers of hyphal branching of AM fungi during the pre-symbiotic phase, while JA acts regulating hyphal growth and mycorrhizal establishment during the symbiotic phase inside the root (Hause et al., 2007; Pozo et al., 2015), although its specific role is still unclear. Studies with mutants affected in JA levels and signalling showed altered mycorrhizal rates compared to their corresponding wild-types (Herrera-Medina et al., 2008; Isayenkov et al., 2005; Song et al., 2013; Tejeda-Sartorius et al., 2008). This behaviour is also described for SL-deficient plants, which exhibit lower levels of mycorrhizal colonization. SL-deficient plants also present a higher shoot branching and a lower ability to stimulate seed germination of broomrape (Gomez-Roldan et al., 2008; Gutjahr et al., 2012; Kohlen et al., 2012; Koltai et al., 2010; Kretzschmar et al., 2012). In Chapter 3, the possible interaction between SLs and JA in shoot branching regulation was investigated. The total number of branches in different tomato plants altered in JA levels or signalling (spr1, spr2 and jai1) was analysed, only detecting a greater branching in spr2 plants respect to the wild-type. Spr2 plants showed a similar growth pattern to Slccd8 plants, especially with the line L16 which has a deficiency in SLs of about 50% (Kohlen et al., 2012). This result suggested a possible partial SL deficiency in in spr2 plants. However, when analysing the SL levels in these plants, no differences between the mutant and the wild-type were observed. In addition, no differences in the levels of auxin and CKs, key regulators of shoot branching (Teichmann and Muhr, 2015), were detected in spr2 plants. Thus, we did not observe any JA-SLs relationship in the regulation of shoot branching.
Plants affected in prosystemin (PS) levels also present altered JA levels under stress conditions (Fernández I, 2013 Doctoral Thesis). PS is the precursor of systemin, a peptide hormone present in the Solanaceae. Systemin promotes JA biosynthesis and signalling, inducing plant defences against biotic stress as pathogen infection and herbivory in the leaves (Ryan and Pearce, 2003). Fernandez and co-workers found out that PS-deficient (ps-) tomato plants showed lower levels of mycorrhizal colonization than the corresponding wild-type. Conversely, PS-overexpressing (ps+) plants showed higher levels of colonization (Fernandez I, Doctoral Thesis 2013). These authors also showed that ps- and ps+ plants presented lower and higher SL levels, respectively (Fernandez I, Doctoral Thesis 2013). This fact suggests that systemin might be involved in the regulation of mycorrhizal symbiosis by regulating SL levels. In Chapter 4, after exogenous application of systemin into ps- plants grown under low Pi conditions, an increase in SL levels was observed. In addition, the expression of molecular markers for SL biosynthesis was promoted by the treatment with systemin. These results suggest a direct effect of systemin in SL production under conditions of high SL production (Pi deficiency, Lopez-Ráez et al., 2008; Yoneyama et al., 2007), and therefore a role during the pre-symbiotic phase of AM symbiosis.
JA is involved in the regulation of the formation and maintenance of arbuscules, although its role is still unclear (Pozo et al., 2015). The close relationship between systemin and JA suggested the possibility that systemin could be also involved in the symbiotic phase of mycorrhizal colonization. After performing a microscopic analysis of the arbuscular development in ps- and ps+ plants colonized by the AM fungus Rhizophagus irregularis, it was observed that indeed systemin is involved in arbuscular development. However, since none of the JA- and SL- deficient mutants used in this study had committed the arbuscule development, the systemin role here seems to be independent of JA and SLs.
Among other benefits, AM symbiosis promotes induced systemic resistance (ISR), probably through the JA pathway, thus protecting the host plant of both biotic and abiotic stresses (Pieterse et al., 1998; Pozo and Azcón Aguilar, 2007; Segarra et al., 2009; Van Loon et al., 1998). The application of AM fungi as biofertilizers and bioprotection agents into agricultural crops is a promising and sustainable strategy for future agriculture. However, before its application it is necessary to a deeper knowledge about the mechanisms that regulate the establishment and maintenance of AM symbiosis, and the plant defence responses in order to optimize the benefits of this beneficial symbiosis in agriculture. In this Doctoral Thesis, we have advanced in this knowledge and studied the interactions between the different phytohormones that are involved in these processes.
