Strigolactones: New players in the nitrogen-phosphorus signalling interplay.

Nitrogen (N) and phosphorus (P) are among the most important macronutrients for plant growth and development, and the most widely used as fertilizers. Understanding how plants sense and respond to N and P deficiency is essential to optimize and reduce the use of chemical fertilizers. Strigolactones (SLs) are phytohormones acting as modulators and sensors of plant responses to P deficiency. In the present work, we assess the potential role of SLs in N starvation and in the N-P signalling interplay. Physiological, transcriptional and metabolic responses were analysed in wild-type and SL-deficient tomato plants grown under different P and N regimes, and in plants treated with a short-term pulse of the synthetic SL analogue 2'-epi-GR24. The results evidence that plants prioritize N over P status by affecting SL biosynthesis. We also show that SLs modulate the expression of key regulatory genes of phosphate and nitrate signalling pathways, including the N-P integrators PHO2 and NIGT1/HHO. The results support a key role for SLs as sensors during early plant responses to both N and phosphate starvation and mediating the N-P signalling interplay, indicating that SLs are involved in more physiological processes than so far proposed.

Exogenous strigolactones impact metabolic profiles and phosphate starvation signalling in roots.

Strigolactones (SLs) are important ex-planta signalling molecules in the rhizosphere, promoting the association with beneficial microorganisms, but also affecting plant interactions with harmful organisms. They are also plant hormones in-planta, acting as modulators of plant responses under nutrient-deficient conditions, mainly phosphate (Pi) starvation. In the present work, we investigate the potential role of SLs as regulators of early Pi starvation signalling in plants. A short-term pulse of the synthetic SL analogue 2'-epi-GR24 promoted SL accumulation and the expression of Pi starvation markers in tomato and wheat under Pi deprivation. 2'-epi-GR24 application also increased SL production and the expression of Pi starvation markers under normal Pi conditions, being its effect dependent on the endogenous SL levels. Remarkably, 2'-epi-GR24 also impacted the root metabolic profile under these conditions, promoting the levels of metabolites associated to plant responses to Pi limitation, thus partially mimicking the pattern observed under Pi deprivation. The results suggest an endogenous role for SLs as Pi starvation signals. In agreement with this idea, SL-deficient plants were less sensitive to this stress. Based on the results, we propose that SLs may act as early modulators of plant responses to P starvation.

A new UHPLC-MS/MS method for the direct determination of strigolactones in root exudates and extracts.

INTRODUCTION: Strigolactones (SLs) are the most representative germination stimulants for seeds of root parasitic plants, and they show activity even at concentrations below 10-10  M. The low amounts of stimulants produced by the host and their rapid degradability make it crucial to develop analytical methods with very low limits of quantification. OBJECTIVE: To develop a sensitive and validated analytical method for the simultaneous quantification of seven SLs [7-oxoorobanchyl acetate (1), solanacol (2), orobanchol (4), strigol (5), fabacyl acetate (6), orobanchyl acetate (7), and 5-deoxystrigol (8)]. METHODS: SLs were analysed using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), with (±)-GR24 (3) employed as internal standard (IS). Validation was based on selectivity, linearity, precision of the peak areas (repeatability and intermediate precision), detection and quantification limits, and stability. RESULTS: A simple, rapid and reliable UHPLC-MS/MS method has been validated for the routine analysis of seven SLs and has been successfully applied to quantify them in exudates and extracts from tomato roots (Solanum lycopersicum). The limits of quantifications range from 0.05 µg/L for 5-deoxystrigol to 0.96 µg/L for solanacol. CONCLUSION: The method provides a useful tool for research in all the fields related to SLs, both for studies related to their function as hormones, and signalling molecules in the rhizosphere, without sample preparation required for extracts and root exudates in less than 11 minutes.

How drought and salinity affect arbuscular mycorrhizal symbiosis and strigolactone biosynthesis?

MAIN CONCLUSION: This paper reviews the importance of AM symbiosis in alleviating plant stress under unfavourable environmental conditions, making emphasis on the role of strigolactones. A better understanding of the mechanisms that regulate this beneficial association will increase its potential use as an innovative and sustainable strategy in modern agriculture. Plants are very dynamic systems with a great capacity for adaptation to a constantly changing environment. This phenotypic plasticity is particularly advantageous in areas damaged or subjected to intensive agriculture. Nowadays, global crop production systems are intensifying the impact on natural resources, such as water availability. Therefore, there is an urgent need to find more sustainable alternatives. One of the plant strategies to improve phenotypic plasticity is to establish mutualistic beneficial associations with soil microorganisms, such as the arbuscular mycorrhizal (AM) fungi. The establishment of AM symbiosis requires a complex network of interconnected signalling pathways, in which phytohormones play a key role. Strigolactones (SLs) are plant hormones acting as modulators of the coordinated development under nutrient shortage. SLs also act as host detection signals for AM fungi, favouring symbiosis establishment. In this review, current knowledge on the effect of water-related stresses, such as drought and salinity, in AM symbiosis and in SL production is discussed. Likewise, how the symbiosis helps the host plant to alleviate stress symptoms is also reviewed. Finally, we highlight how interactions between hormonal signalling pathways modulate all these responses, especially in the cross-talk between SLs and abscisic acid (ABA). Understanding the intricate mechanisms that regulate the establishment of AM symbiosis and the plant responses under unfavourable conditions will contribute to implement the use of AM fungi as bioprotective agents against these stresses.

Phytohormones as integrators of environmental signals in the regulation of mycorrhizal symbioses.

For survival, plants have to efficiently adjust their phenotype to environmental challenges, finely coordinating their responses to balance growth and defence. Such phenotypic plasticity can be modulated by their associated microbiota. The widespread mycorrhizal symbioses modify plant responses to external stimuli, generally improving the resilience of the symbiotic system to environmental stresses. Phytohormones, central regulators of plant development and immunity, are instrumental in orchestrating plant responses to the fluctuating environment, but also in the regulation of mycorrhizal symbioses. Exciting advances in the molecular regulation of phytohormone signalling are providing mechanistic insights into how plants coordinate their responses to environmental cues and mycorrhizal functioning. Here, we summarize how these mechanisms permit the fine-tuning of the symbiosis according to the ever-changing environment.

The tomato CAROTENOID CLEAVAGE DIOXYGENASE8 (SlCCD8) regulates rhizosphere signaling, plant architecture and affects reproductive development through strigolactone biosynthesis.

Strigolactones are plant hormones that regulate both above- and belowground plant architecture. Strigolactones were initially identified as rhizosphere signaling molecules. In the present work, the tomato (Solanum lycopersicum) CAROTENOID CLEAVAGE DIOXYGENASE 8 (SlCCD8) was cloned and its role in rhizosphere signaling and plant physiology assessed by generating knock-down lines. Transgenic SlCCD8 plants were generated by RNAi-mediated silencing. Lines with different levels of strigolactone reduction--confirmed by UPLC-MS/MS--were selected and their phenotypes investigated. Lines exhibiting reduced SlCCD8 levels displayed increased shoot branching, reduced plant height, increased number of nodes and excessive adventitious root development. In addition, these lines exhibited reproductive phenotypes such as smaller flowers, fruits, as well as fewer and smaller seeds per fruit. Furthermore, we show that strigolactone loading to the xylem sap is possibly restricted to orobanchol. Infestation by Phelipanche ramosa was reduced by 90% in lines with a relatively mild reduction in strigolactone biosynthesis and secretion while arbuscular mycorrhizal symbiosis, apical dominance and fruit yield were only mildly affected. This demonstrates that reduction of strigolactone biosynthesis could be a suitable tool in parasitic weed management. Furthermore, our results suggest that strigolactones are involved in even more physiological processes than so far assumed.

Mycorrhiza-induced resistance and priming of plant defenses.

Symbioses between plants and beneficial soil microorganisms like arbuscular-mycorrhizal fungi (AMF) are known to promote plant growth and help plants to cope with biotic and abiotic stresses. Profound physiological changes take place in the host plant upon root colonization by AMF affecting the interactions with a wide range of organisms below- and above-ground. Protective effects of the symbiosis against pathogens, pests, and parasitic plants have been described for many plant species, including agriculturally important crop varieties. Besides mechanisms such as improved plant nutrition and competition, experimental evidence supports a major role of plant defenses in the observed protection. During mycorrhiza establishment, modulation of plant defense responses occurs thus achieving a functional symbiosis. As a consequence of this modulation, a mild, but effective activation of the plant immune responses seems to occur, not only locally but also systemically. This activation leads to a primed state of the plant that allows a more efficient activation of defense mechanisms in response to attack by potential enemies. Here, we give an overview of the impact on interactions between mycorrhizal plants and pathogens, herbivores, and parasitic plants, and we summarize the current knowledge of the underlying mechanisms. We focus on the priming of jasmonate-regulated plant defense mechanisms that play a central role in the induction of resistance by arbuscular mycorrhizas.

Phosphorus availability drives mycorrhiza induced resistance in tomato.

Arbuscular mycorrhizal (AM) symbiosis can provide multiple benefits to the host plant, including improved nutrition and protection against biotic stress. Mycorrhiza induced resistance (MIR) against pathogens and insect herbivores has been reported in different plant systems, but nutrient availability may influence the outcome of the interaction. Phosphorus (P) is a key nutrient for plants and insects, but also a regulatory factor for AM establishment and functioning. However, little is known about how AM symbiosis and P interact to regulate plant resistance to pests. Here, using the tomato-Funneliformis mosseae mycorrhizal system, we analyzed the effect of moderate differences in P fertilization on plant and pest performance, and on MIR against biotic stressors including the fungal pathogen Botrytis cinerea and the insect herbivore Spodoperta exigua. P fertilization impacted plant nutritional value, plant defenses, disease development and caterpillar survival, but these effects were modulated by the mycorrhizal status of the plant. Enhanced resistance of F. mosseae-inoculated plants against B. cinerea and S. exigua depended on P availability, as no protection was observed under the most P-limiting conditions. MIR was not directly explained by changes in the plant nutritional status nor to basal differences in defense-related phytohormones. Analysis of early plant defense responses to the damage associated molecules oligogalacturonides showed primed transcriptional activation of plant defenses occurring at intermediate P levels, but not under severe P limitation. The results show that P influences mycorrhizal priming of plant defenses and the resulting induced-resistance is dependent on P availability, and suggest that mycorrhiza fine-tunes the plant growth vs defense prioritization depending on P availability. Our results highlight how MIR is context dependent, thus unravel molecular mechanism based on plant defence in will contribute to improve the efficacy of mycorrhizal inoculants in crop protection.

Does abscisic acid affect strigolactone biosynthesis?

SUMMARY: *Strigolactones are considered a novel class of plant hormones that, in addition to their endogenous signalling function, are exuded into the rhizosphere acting as a signal to stimulate hyphal branching of arbuscular mycorrhizal (AM) fungi and germination of root parasitic plant seeds. Considering the importance of the strigolactones and their biosynthetic origin (from carotenoids), we investigated the relationship with the plant hormone abscisic acid (ABA). *Strigolactone production and ABA content in the presence of specific inhibitors of oxidative carotenoid cleavage enzymes and in several tomato ABA-deficient mutants were analysed by LC-MS/MS. In addition, the expression of two genes involved in strigolactone biosynthesis was studied. *The carotenoid cleavage dioxygenase (CCD) inhibitor D2 reduced strigolactone but not ABA content of roots. However, in abamineSG-treated plants, an inhibitor of 9-cis-epoxycarotenoid dioxygenase (NCED), and the ABA mutants notabilis, sitiens and flacca, ABA and strigolactones were greatly reduced. The reduction in strigolactone production correlated with the downregulation of LeCCD7 and LeCCD8 genes in all three mutants. *The results show a correlation between ABA levels and strigolactone production, and suggest a role for ABA in the regulation of strigolactone biosynthesis.

Hormonal and transcriptional profiles highlight common and differential host responses to arbuscular mycorrhizal fungi and the regulation of the oxylipin pathway.

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. The symbiosis significantly affects the host physiology in terms of nutrition and stress resistance. Despite the lack of host range specificity of the interaction, functional diversity between AM fungal species exists. The interaction is finely regulated according to plant and fungal characters, and plant hormones are believed to orchestrate the modifications in the host plant. Using tomato as a model, an integrative analysis of the host response to different mycorrhizal fungi was performed combining multiple hormone determination and transcriptional profiling. Analysis of ethylene-, abscisic acid-, salicylic acid-, and jasmonate-related compounds evidenced common and divergent responses of tomato roots to Glomus mosseae and Glomus intraradices, two fungi differing in their colonization abilities and impact on the host. Both hormonal and transcriptional analyses revealed, among others, regulation of the oxylipin pathway during the AM symbiosis and point to a key regulatory role for jasmonates. In addition, the results suggest that specific responses to particular fungi underlie the differential impact of individual AM fungi on plant physiology, and particularly on its ability to cope with biotic stresses.

Histochemical and Molecular Quantification of Arbuscular Mycorrhiza Symbiosis.

Arbuscular mycorrhizae (AM) are one of the most widespread and studied plant associations with beneficial microorganisms. Indeed, more than 80% of land plants, including most agricultural and horticultural crop species, are able to establish this mutualistic symbiosis with AM fungi. Through this association the fungus helps the plant in the acquisition of water and mineral nutrients, especially under stress conditions. AM symbiosis affects other ecologically and economically important traits such as plant architecture, flowering, and fruit quality but also tolerance against biotic and abiotic stresses. As a consequence, AM fungi have a great potential as biofertilizers and bioprotection agents in sustainable agriculture. However, in order to take advantage of all these benefits, a good and functional symbiosis is required. Here we present methods for reliable quantification of colonization levels which should be useful not only for research but also from the agronomic point of view.

Phosphorus Acquisition Efficiency Related to Root Traits: Is Mycorrhizal Symbiosis a Key Factor to Wheat and Barley Cropping?

Wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) are major crops cultivated around the world, thus playing a crucial role on human diet. Remarkably, the growing human population requires a significant increase in agricultural production in order to feed everybody. In this context, phosphorus (P) management is a key factor as it is component of organic molecules such as nucleic acids, ATP and phospholipids, and it is the most abundant macronutrient in biomass after nitrogen (N), although being one of the scarcest elements in the lithosphere. In general, P fertilization has low efficiency, as only a fraction of the applied P is acquired by roots, leaving a substantial amount to be accumulated in soil as not readily available P. Breeding for P-efficient cultivars is a relatively low cost alternative and can be done through two mechanisms: i) improving P use efficiency (PUE), and/or ii) P acquisition efficiency (PAE). PUE is related to the internal allocation/mobilization of P, and is usually represented by the amount of P accumulated per biomass. PAE relies on roots ability to acquire P from the soil, and is commonly expressed as the relative difference of P acquired under low and high P availability conditions. In this review, plant adaptations related to improved PAE are described, with emphasis on arbuscular mycorrhizal (AM) symbiosis, which is generally accepted to enhance plant P acquisition. A state of the art (1980-2018) of AM growth responses and P uptake in wheat and barley is made to discuss about the commonly accepted growth promoting effect and P increased uptake by AM fungi and the contrasting evidence about the generally accepted lack of positive responses in both plant species. Finally, the mechanisms by which AM symbiosis can affect wheat and barley PAE are discussed, highlighting the importance of considering AM functional diversity on future studies and the necessity to improve PAE definition by considering the carbon trading between all the directly related PAE traits and its return to the host plant.

Strigolactones in Plant Interactions with Beneficial and Detrimental Organisms: The Yin and Yang.

Strigolactones (SLs) are plant hormones that have important roles as modulators of plant development. They were originally described as ex planta signaling molecules in the rhizosphere that induce the germination of parasitic plants, a role that was later linked to encouraging the beneficial symbiosis with arbuscular mycorrhizal (AM) fungi. Recently, the focus has shifted to examining the role of SLs in plant-microbe interactions, and has revealed roles for SLs in the association of legumes with nitrogen-fixing rhizobacteria and in interactions with disease-causing pathogens. Here, we examine the role of SLs in plant interactions with beneficial and detrimental organisms, and propose possible future biotechnological applications.

Strigolactones in the Rhizobium-legume symbiosis: Stimulatory effect on bacterial surface motility and down-regulation of their levels in nodulated plants.

Strigolactones (SLs) are multifunctional molecules acting as modulators of plant responses under nutrient deficient conditions. One of the roles of SLs is to promote beneficial association with arbuscular mycorrhizal (AM) fungi belowground under such stress conditions, mainly phosphorus shortage. Recently, a role of SLs in the Rhizobium-legume symbiosis has been also described. While SLs' function in AM symbiosis is well established, their role in the Rhizobium-legume interaction is still emerging. Recently, SLs have been suggested to stimulate surface motility of rhizobia, opening the possibility that they could also act as molecular cues. The possible effect of SLs in the motility in the alfalfa symbiont Sinorhizobium meliloti was investigated, showing that the synthetic SL analogue GR24 stimulates swarming motility in S. meliloti in a dose-dependent manner. On the other hand, it is known that SL production is regulated by nutrient deficient conditions and by AM symbiosis. Using the model alfalfa-S. meliloti, the impact of phosphorus and nitrogen deficiency, as well as of nodulation on SL production was also assessed. The results showed that phosphorus starvation promoted SL biosynthesis, which was abolished by nitrogen deficiency. In addition, a negative effect of nodulation on SL levels was detected, suggesting a conserved mechanism of SL regulation upon symbiosis establishment.

Differential spatio-temporal expression of carotenoid cleavage dioxygenases regulates apocarotenoid fluxes during AM symbiosis.

Apocarotenoids are a class of compounds that play important roles in nature. In recent years, a prominent role for these compounds in arbuscular mycorrhizal (AM) symbiosis has been shown. They are derived from carotenoids by the action of the carotenoid cleavage dioxygenase (CCD) enzyme family. In the present study, using tomato as a model, the spatio-temporal expression pattern of the CCD genes during AM symbiosis establishment and functioning was investigated. In addition, the levels of the apocarotenoids strigolactones (SLs), C13 α-ionol and C14 mycorradicin (C13/C14) derivatives were analyzed. The results suggest an increase in SLs promoted by the presence of the AM fungus at the early stages of the interaction, which correlated with an induction of the SL biosynthesis gene SlCCD7. At later stages, induction of SlCCD7 and SlCCD1 expression in arbusculated cells promoted the production of C13/C14 apocarotenoid derivatives. We show here that the biosynthesis of apocarotenoids during AM symbiosis is finely regulated throughout the entire process at the gene expression level, and that CCD7 constitutes a key player in this regulation. Once the symbiosis is established, apocarotenoid flux would be turned towards the production of C13/C14 derivatives, thus reducing SL biosynthesis and maintaining a functional symbiosis.

Do strigolactones contribute to plant defence?

Strigolactones are multifunctional molecules involved in several processes outside and within the plant. As signalling molecules in the rhizosphere, they favour the establishment of arbuscular mycorrhizal symbiosis, but they also act as host detection cues for root parasitic plants. As phytohormones, they are involved in the regulation of plant architecture, adventitious rooting, secondary growth and reproductive development, and novel roles are emerging continuously. In the present study, the possible involvement of strigolactones in plant defence responses was investigated. For this purpose, the resistance/susceptibility of the strigolactone-deficient tomato mutant Slccd8 against the foliar fungal pathogens Botrytis cinerea and Alternaria alternata was assessed. Slccd8 was more susceptible to both pathogens, pointing to a new role for strigolactones in plant defence. A reduction in the content of the defence-related hormones jasmonic acid, salicylic acid and abscisic acid was detected by high-performance liquid chromatography coupled to tandem mass spectrometry in the Slccd8 mutant, suggesting that hormone homeostasis is altered in the mutant. Moreover, the expression level of the jasmonate-dependent gene PinII, involved in the resistance of tomato to B. cinerea, was lower than in the corresponding wild-type. We propose here that strigolactones play a role in the regulation of plant defences through their interaction with other defence-related hormones, especially with the jasmonic acid signalling pathway.

Tomato strigolactones: a more detailed look.

Strigolactones are plant signaling molecules that induce germination of parasitic plant seeds, initiate host plant - arbuscular mycorrhizal fungus symbiosis and act as plant hormones controlling shoot branching and root architecture. To date four unique strigolactones (e.g., orobanchol, didehydroorobanchol isomers 1 and 2 and the aromatic strigolactone solanacol) have been reported in the root exudates and extracts of tomato (Solanum lycopersicum). Here we report on the presence of several additional strigolactones in tomato root exudates and extracts, orobanchyl acetate, two 7-hydroxyorobanchol isomers, 7-oxoorobanchol and two additional didehydroorobanchol isomers and discuss their possible biological relevance.

Arbuscular mycorrhizal symbiosis decreases strigolactone production in tomato.

Strigolactones are a new class of plant hormones emerging as important signals in the control of plant architecture. In addition, they are key elements in plant communication with several rhizosphere organisms. Strigolactones are exuded into the soil, where they act as host detection signals for arbuscular mycorrhizal (AM) fungi, but also as germination stimulants for root parasitic plant seeds. Under phosphate limiting conditions, plants up-regulate the secretion of strigolactones into the rhizosphere to promote the formation of AM symbiosis. Using tomato as a model plant, we have recently shown that AM symbiosis induces changes in transcriptional and hormonal profiles. Using the same model system, here we analytically demonstrate, using liquid chromatography-tandem mass spectrometry, that strigolactone production is also significantly reduced upon AM symbiosis. Considering the dual role of the strigolactones in the rhizosphere as signals for AM fungi and parasitic plants, we discuss the potential implications of these changes in the plant interaction with both organisms.

AM symbiosis alters phenolic acid content in tomato roots.

Arbuscular mycorrhizal (AM) fungi colonize the roots of most plants to establish a mutualistic symbiosis leading to important benefits for plant health. We have recently shown that AM symbiosis alters both transcriptional and hormonal profiles in tomato roots, many of these changes related to plant defence. Here, we analytically demonstrate that the levels of other important defence-related compounds as phenolic acids are also altered in the symbiosis. Both caffeic and chlorogenic acid levels significantly decreased in tomato roots upon mycorrhization, while ferulic acid increased. Moreover, in the case of caffeic acid a differential reduction was observed depending on the colonizing AM fungus. The results confirm that AM associations imply the regulation of plant defence responses, and that the host changes may vary depending on the AM fungus involved. The potential implications of altered phenolic acid levels on plant control over mycorrhizal colonization and in the plant resistance to pathogens is discussed.

Strigolactones: ecological significance and use as a target for parasitic plant control.

Parasitic weeds cause severe damage to important agricultural crops. Although some promising control methods against these parasitic plants have been developed, new strategies continue to be relevant in integrated approaches. The life cycle for root parasitic weeds is intimately associated with their host and is a suitable target for such new control strategies, particularly when directed at the early stages of the host-parasite interaction. Here, the authors focus on knowledge of the germination stimulants-strigolactones-for the root parasitic plants Striga and Orobanche spp. and discuss their biosynthetic origin, ecological significance and physiological and biochemical regulation. In addition, the existing and possible new control strategies that are based on this knowledge, and that could lead to more efficient control methods against these root parasitic weeds, are reviewed.