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Symbioses with beneficial microbes are widespread in plants, but these relationships must balance the energy invested by the plants with the nutrients acquired. Symbiosis with arbuscular mycorrhizal (AM) fungi occurs throughout land plants but our understanding of the genes and signals that regulate colonisation levels is limited. Here, we demonstrate that in tomato two CLV3/EMBRYO-SURROUNDING REGION (CLE) peptides,SlCLE10 andSlCLE11, act to suppress AM colonisation of roots. Mutant studies and overexpression via hairy transformation indicateSlCLE11acts locally in the root to limit AM colonisation. Indeed,SlCLE11expression is strongly induced in AM colonised roots butSlCLE11is not required for phosphate suppression of AM colonisation.SlCLE11 may act through as yet uncharacterised signalling pathways, asSlCLE11does not suppress AM colonisation by acting through two previously characterised receptors with roles in regulating AM colonisation,SlFAB (CLAVATA1 orthologue) orSlCLV2.SlCLE10 appears to play a more minor or redundant role, ascle10mutants did not influence AM, although the fact that ectopic overexpression ofSlCLE10did suppress colonisation suggestsSlCLE10may play a role in regulating AM colonisation. Our findings show that CLE peptides regulate AM colonisation in the non-legume species tomato.

The induced dwarf mutant Rht12 was previously shown to have agronomic potential to replace the conventional DELLA mutants Rht-B1b/Rht-D1b in wheat. The Rht12 dwarfing gene is not associated with reduced coleoptile length (unlike the DELLA mutants) and it is dominant, characteristics which are shared with the previously characterized dwarfing genes Rht18 and Rht14. Using the Rht18/Rht14 model, a gibberellin (GA) 2-oxidase gene was identified in the Rht12 region on chromosome 5A. A screen for suppressor mutants in the Rht12 background identified tall overgrowth individuals that were shown to contain loss-of-function mutations in GA2oxidaseA13, demonstrating the role of this gene in the Rht12 dwarf phenotype. It was concluded that Rht12, Rht18, and Rht14 share the same height-reducing mechanism through the increased expression of GA 2-oxidase genes. Some of the overgrowth mutants generated in this study were semi-dwarf and taller than the original Rht12 dwarf, providing breeders with new sources of agronomically useful dwarfism.

The underlying mechanisms that determine whether two species can form a successful graft union (graft compatibility) remain obscure. Two prominent hypotheses are (1) the more closely related species are, the higher the graft success and (2) the vascular anatomy at the graft junction influences graft success. In this paper these two hypotheses are examined in a systematic way using graft combinations selected from a range of (a) phylogenetically close and more distant legume species, (b) species displaying different germination patterns and (c) scions and rootstocks possessing contrasting stem tissues and vascular patterns. Relatedness of species was not a good predictor of graft compatibility, as vascular reconnection can occur between distantly related species and can fail to occur in some more closely related species. Similarly, neither the stem tissues present at the graft junction nor the vascular anatomy correlated with the success of vascular reconnection. Relatedness and stem anatomy therefore do not appear to be the determining factors in successful vascular reconnection after grafting in legumes. These results are discussed in conjunction with other hypotheses such as the role of auxin.

Plant hormones play key roles in plant development and their response to the environment. Hormones also play important roles in the development of two key symbioses formed by land plants, arbuscular mycorrhizal symbioses formed between the majority of land plants and fungi from the phylum Glomeromycota and nodulation, a symbiosis between nitrogen-fixing rhizobia and largely leguminous plants. Different hormones play common and divergent roles in regulating these two symbioses, and this is consistent with shared evolutionary history and signaling pathways as well as the divergent developmental programs that occur in each symbiosis.

Heirloom golden tomato fruit varieties are highly nutritious as they accumulate tetra-cis-lycopene, which has a higher bioavailability and recognised health benefits in treating anti-inflammatory diseases compared to all-trans-lycopene isomers found in red tomatoes. We investigated if photoisomerization of tetra-cis-lycopene occurs in roots of the goldentangerineMicro-Tom variety (tangmic), and how this affects root to shoot biomass, mycorrhizal colonization, abscisic acid accumulation, and responses to drought.tangmicplants grown in soil under glasshouse conditions displayed a reduction in height, number of flowers, fruit yield, and root length compared to wild type (WT). Soil inoculation withRhizophagus irregularisrevealed fewer arbuscules and other fungal structures in the endodermal cells of roots intangmicrelative to WT. The roots oftangmichyperaccumulated acycliccis-carotenes, while only trace levels of xanthophylls and abscisic acid were detected. In response to a water deficit, leaves from thetangmicplants displayed a rapid decline in maximum quantum yield of photosystem II compared to WT, indicating a defective root to shoot signalling response to drought. The lack of xanthophylls biosynthesis intangmicroots reduced abscisic acid levels, thereby likely impairing endomycorrhiza colonisation and drought-induced root to shoot signalling.Research HighlightsPhotoisomerization of prolycopene to lycopene is limited in root plastids.Roots oftangerinereveal an important tissue sink to store micronutrients such as prolycopene.Roots oftangerinelack ABA and show impaired mycorrhizal colonization.Thetangerineplant is drought sensitive and has a smaller biomass as well as reduced yield.

The Heirloom Golden tangerine tomato fruit variety is highly nutritious due to accumulation of tetra-cis-lycopene, that has a higher bioavailability and recognised health benefits in treating anti-inflammatory diseases compared to all-trans-lycopene isomers found in red tomatoes. We investigated if photoisomerization of tetra-cis-lycopene occurs in roots of the MicroTom tangerine (tang

Strigolactones play a potent role in the rhizosphere as a signal to symbiotic microbes including arbuscular mycorrhizal fungi and rhizobial bacteria. This chapter outlines guidelines for application of strigolactones to pea roots to influence symbiotic relationships, and includes careful consideration of type of strigolactones applied, solvent use, frequency of application and nutrient regime to optimize experimental conditions.

Strigolactones play a potent role in the rhizosphere as a signal to symbiotic microbes including arbuscular mycorrhizal fungi and rhizobial bacteria. This chapter outlines guidelines for application of strigolactones to pea roots to influence symbiotic relationships, and includes careful consideration of type of strigolactones applied, solvent use, frequency of application and nutrient regime to optimize experimental conditions.

Plants form mutualistic nutrient-acquiring symbioses with microbes, including arbuscular mycorrhizal fungi. The formation of these symbioses is costly, and plants employ a negative feedback loop termed autoregulation of mycorrhizae (AOM) to limit formation of arbuscular mycorrhizae (AM). We provide evidence for the role of one leucine-rich repeat receptor-like kinase (FAB), a hydroxyproline O-arabinosyltransferase enzyme (FIN), and additional evidence for one receptor-like protein (SlCLV2) in the negative regulation of AM formation in tomato. Reciprocal grafting experiments suggest that the FAB gene acts locally in the root, while the SlCLV2 gene may act in both the root and the shoot. External nutrients including phosphate and nitrate can also strongly suppress AM formation. We found that FAB and FIN are required for nitrate suppression of AM but are not required for the powerful suppression of AM colonization by phosphate. This parallels some of the roles of legume homologues in the autoregulation of the more recently evolved symbioses with nitrogen-fixing bacteria leading to nodulation. This deep homology in the symbiotic role of these genes suggests that in addition to the early signalling events that lead to the establishment of AM and nodulation, the autoregulation pathway might also be considered part of the common symbiotic toolkit that enabled plants to form beneficial symbioses.

Plants use a variety of hormonal and peptide signals to control root development, including in adapting root development to cope with nutrient stress. Nitrogen (N) is a major limiting factor in plant growth and in response to N stress plants dramatically modulate root development, including in legumes influencing the formation of N-fixing nodules in response to external N supply. Recently, specific CLE peptides and/or receptors important for their perception, including CLV1 and CLV2, have been found to play important roles in root development in a limited number of species, including in some cases the response to N. In the legume Medicago truncatula, this response also appears to be influenced by RDN1, a member of the hydroxyproline O-arabinosyltransferase (HPAT) family which can modify specific CLE peptides. However, it not known if this signalling pathway plays a central role in root development across species, in particular root responses to N. In this study, we sought to systematically examine the role of homologues of these genes in root development of the legume pea (Pisum sativum. L) and non-legume tomato (Solanum lycopersicum) using a mutant based approach. This included a detailed examination of root development in response to N in these mutant series in tomato. We found no evidence for a role of these genes in pea seedling root development. Furthermore, the CLV1-like FAB gene did not influence tomato root development, including N response. In contrast, both CLV2 and the HPAT FIN appear to positively influence root size in tomato but do not mediate root responses to N. These suggest a relatively species-specific role for these genes in root development, including N regulation of root architecture.

Plants form mutualistic nutrient acquiring symbioses with microbes, including arbuscular mycorrhizal fungi. The formation of these symbioses is costly and plants employ a negative feedback loop termed autoregulation of mycorrhizae (AOM) to limit arbuscular mycorrhizae (AM) formation. We provide evidence for the role of one leucine-rich-repeat receptor like kinase (FAB), a hydroxyproline O-arabinosyltransferase enzyme (FIN) and additional evidence for one receptor like protein (SlCLV2) in the negative regulation of AM formation in tomato. Reciprocal grafting experiments suggest that the FAB gene acts locally in the root, while the SlCLV2 gene may act in both the root and the shoot. External nutrients including phosphate and nitrate can also strongly suppress AM formation. We found that FAB and FIN are required for nitrate suppression of AM but are not required for the powerful suppression of AM colonisation by phosphate. This parallels some of the roles of legume homologs in the autoregulation of the more recently evolved symbioses with nitrogen-fixing bacteria leading to nodulation. This deep homology in the symbiotic role of these genes suggests that in addition to the early signalling events that lead to the establishment of AM and nodulation, the autoregulation pathway might also be considered part of the common symbiotic toolkit that enabled plants to form beneficial symbioses.HighlightWe describe the role of CLV signalling elements in the negative regulation of arbuscular mycorrhizal symbioses of tomato, including influencing nitrate but not phosphate suppression of mycorrhizal colonisation.

Plants use a variety of signals to control root development, including in modifying root development in response to nutrient stress. For example, in response to nitrogen (N) stress, plants dramatically modulate root development, including the formation of N-fixing nodules in legumes. Recently, specific CLE peptides and/or receptors important for their perception, including CLV1 and CLV2, have been found to play roles in root development, including in response to N supply. In the legume Medicago truncatula, this response also appears to be influenced by RDN1, a member of the hydroxyproline-O-arabinosyltransferase (HPAT) family which can modify specific CLE peptides. However, it is not known if this signalling pathway plays a central role in root development across species, and in particular root responses to N. In this study, we systematically examined the role of the CLV signalling pathway genes in root development of the legume pea (Pisum sativum) and non-legume tomato (Solanum lycopersicum) using a mutant-based approach. This included a detailed examination of root development in response to N in tomato mutants disrupted in CLV1- or CLV2-like genes or HPAT family member FIN. We found no evidence for a role of these genes in pea seedling root development. Furthermore, the CLV1-like FAB gene did not influence tomato root development, including the root response to N supply. In contrast, both CLV2 and the HPAT gene FIN appear to positively influence root size in tomato but do not mediate root responses to N. These results suggest the function of these genes may vary somewhat in different species, including the N regulation of root architecture.

A comprehensive analysis of the role of brassinosteroids in nodulation, including their interactions with auxin and ethylene revealed that brassinosteroids inhibit infection, promote nodule initiation but do not influence nodule organogenesis or function. Nodulation, the symbiosis between legumes and rhizobial bacteria, is regulated by a suite of hormones including brassinosteroids. Previous studies have found that brassinosteroids promote nodule number by inhibiting ethylene biosynthesis. In this study, we examined the influence of brassinosteroids on the various stages of infection and nodule development. We utilise pea mutants, including brassinosteroid mutants lk, lka and lkb, the ethylene insensitive ein2 mutant and the lk ein2 double mutant, along with transgenic lines expressing the DR5::GUS auxin activity marker to investigate how brassinosteroids interact with ethylene and auxin during nodulation. We show that brassinosteroids inhibit the early stages of nodulation, including auxin accumulation, root hair deformation and infection thread formation, and demonstrate that infection thread formation is regulated by brassinosteroids in an ethylene independent manner. In contrast, brassinosteroids appear to act as promoters of nodule initiation through both an ethylene dependent and independent pathway. Although brassinosteroids positively influence the ultimate number of nodules formed, we found that brassinosteroid-deficiency did not influence nodule structure including the vascular pattern of auxin activity or nitrogen-fixation capacity. These findings suggest that brassinosteroids are negative regulators of infection but positive regulators of nodule initiation. Furthermore, brassinosteroids do not appear to be essential for nodule organogenesis or function. Given the influence of brassinosteroids on discreet stages of nodulation but not nodule function, manipulation of brassinosteroids may be an interesting avenue for future research on the optimisation of nodulation.

Background The presence of a polar auxin transport stream has long been correlated with the differentiation and patterning of vascular cells across vascular plants. As our understanding of auxin transport and vascular development has grown, so too has evidence for the correlation between these processes. However, a clear understanding of the cellular and molecular mechanisms driving this correlation has not been elucidated. Scope This article examines the hypothesis that canalization via polar auxin transport regulates vascular reconnection and patterning in the stem after wounding or grafting. We examine the evidence for the causal nature of the relationship and the suggested role that other hormones may play. Data are presented indicating that in grafted plants the degree of auxin transport may not always correlate with vascular reconnection. Furthermore, data on grafting success using plants with a range of hormone-related mutations indicate that these hormones may not be critical for vascular reconnection. Conclusions In the past, excellent work examining elements of auxin synthesis, transport and response in relation to vascular development has been carried out. However, new experimental approaches are required to test more directly the hypothesis that auxin transport regulates stem vascular reconnection after wounding or grafting. This could include studies on the timing of the re-establishment of auxin transport and vascular reconnection after grafting and the influence of auxin transport mutants and inhibitors on these processes using live imaging.

Semidwarfing genes have improved crop yield by reducing height, improving lodging resistance, and allowing plants to allocate more assimilates to grain growth. In wheat (Triticum aestivum), the Rht18 semidwarfing gene was identified and deployed in durum wheat before it was transferred into bread wheat, where it was shown to have agronomic potential. Rht18, a dominant and gibberellin (GA) responsive mutant, is genetically and functionally distinct from the widely used GA-insensitive semidwarfing genes Rht-B1b and Rht-D1b. In this study, the Rht18 gene was identified by mutagenizing the semidwarf durum cultivar Icaro (Rht18) and generating mutants with a range of tall phenotypes. Isolating and sequencing chromosome 6A of these “overgrowth” mutants showed that they contained independent mutations in the coding region of GA2oxA9. GA2oxA9 is predicted to encode a GA 2-oxidase that metabolizes GA biosynthetic intermediates into inactive products, effectively reducing the amount of bioactive GA (GA1). Functional analysis of the GA2oxA9 protein demonstrated that GA2oxA9 converts the intermediate GA12 to the inactive metabolite GA110. Furthermore, Rht18 showed higher expression of GA2oxA9 and lower GA content compared with its tall parent. These data indicate that the increased expression of GA2oxA9 in Rht18 results in a reduction of both bioactive GA content and plant height. This study describes a height-reducing mechanism that can generate new genetic diversity for semidwarfism in wheat by combining increased expression with mutations of specific amino acid residues in GA2oxA9.

descripción no proporcionada por scopus, EF was supported by Australian Research Council Future Fellowship and Discovery Grants. JALR was supported by grants AGL2015-64990-C2-1R from the Spanish National R&D Plan of the Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (ERDF), and 201640I040 from the Spanish National Research Council (CSIC). JP would like to acknowledge the Australian Research Council for research funding (DE150100408). DR was supported by Danish National Research Foundation.

The key event that initiates nodule organogenesis is the perception of bacterial signal molecules, the Nod factors, triggering a complex of responses in epidermal and cortical cells of the root. The Nod factor signaling pathway interacts with plant hormones, including cytokinins and gibberellins. Activation of cytokinin signaling through the homeodomain-containing transcription factors KNOX is essential for nodule formation. The main regulators of gibberellin signaling, the DELLA proteins are also involved in regulation of nodule formation. However, the interaction between the cytokinin and gibberellin signaling pathways is not fully understood. Here, we show in Pisum sativum L. that the DELLA proteins can activate the expression of KNOX and BELL transcription factors involved in regulation of cytokinin metabolic and response genes. Consistently, pea la cry-s (della1 della2) mutant showed reduced ability to upregulate expression of some cytokinin metabolic genes during nodulation. Our results suggest that DELLA proteins may regulate cytokinin metabolism upon nodulation.

Plant hormones play key roles in nodulation and arbuscular mycorrhizal (AM) associations. These two agriculturally and ecologically important symbioses enable plants to gain access to nutrients, in particular, nitrogen in the case of nodulation and phosphorous in the case of AM. Work over the past few decades has revealed how symbioses with nitrogen-fixing rhizobia, restricted almost exclusively to legumes, evolved in part from ancient AM symbioses formed by more than 80% of land plants. Although overlapping, these symbiotic programs also have important differences, including the de novo development of a new organ found only in nodulation. One emerging area of research is the role of two plant hormone groups, the gibberellins (GAs) and brassinosteroids (BRs), in the development and maintenance of these symbioses. In this review, we compare and contrast the roles of these hormones in the two symbioses, including potential interactions with other hormones. This not only focuses on legumes, most of which can host both symbionts, but also examines the role of these in AM development in non-legumes. GA acts by suppressing DELLA, and this regulatory module acts to negatively influence both rhizobial and mycorrhizal infection but appears to promote nodule organogenesis. While an overall positive role for BRs in nodulation and AM has been suggested by studies using mutants disrupted in BR biosynthesis or response, application studies indicate that BR may play a more complex role in nodulation. Given the nature of these symbioses, with events regulated both spatially and temporally, future studies should examine in more detail how GAs and BRs may influence precise events during these symbioses, including interactions with other hormone groups.

Legume nodules are a unique plant organ that contain nitrogen-fixing rhizobial bacteria. For this interaction to be mutually beneficial, plant and bacterial metabolism must be precisely co-ordinated. Plant hormones are known to play essential roles during the establishment of legume-rhizobial symbioses but their role in subsequent nodule metabolism has not been explored in any depth. The plant hormones brassinosteroids, ethylene and gibberellins influence legume infection, nodule number and in some cases nodule function. In this paper, the influence of these hormones on nodule metabolism was examined in a series of well characterised pea mutants with altered hormone biosynthesis or response. A targeted set of metabolites involved in nutrient exchange and nitrogen fixation was examined in nodule tissue of mutant and wild type plants. Gibberellin-deficiency had a major negative impact on the level of several major dicarboxylates supplied to rhizobia by the plant and also led to a significant deficit in the amino acids involved in glutamine-aspartate transamination, consistent with the limited bacteroid development and low fixation rate of gibberellin-deficient na mutant nodules. In contrast, no major effects of brassinosteroid-deficiency or ethylene-insensitivity on the key metabolites in these pathways were found. Therefore, although all three hormones influence infection and nodule number, only gibberellin is important for the establishment of a functional nodule metabolome.

International audience; Gibberellins (GAs) and cytokinins (CKs) are hormones that play antagonistic roles in several developmental processes in plants. However, there has been little exploration of their reciprocal interactions. Recent work in Medicago truncatula has revealed that GA signalling can regulate CK levels and response in roots. Here, we examine the reciprocal interaction, by assessing how CKs and the CRE1 (Cytokinin Response 1) CK receptor may influence endogenous GA levels. Real-Time RT-PCR analyses revealed that the expression of key GA biosynthesis genes is regulated in response to a short-term CK treatment and requires the CRE1 receptor. Similarly, GA quantifications indicated that a short-term CK treatment decreases the GA(1) pool in wild-type plants and that GA levels are increased in the cre1 mutant compared to the wild-type. These data suggest that the M. truncatula CRE1-dependent CK signaling pathway negatively regulates bioactive GA levels.

Plant hormones play key roles in defence against pathogen attack. Recent work has begun to extend this role to encompass not just the traditional disease/stress hormones, such as ethylene, but also growth-promoting hormones. Strigolactones (SLs) are the most recently defined group of plant hormones with important roles in plant–microbe interactions, as well as aspects of plant growth and development, although the knowledge of their role in plant– pathogen interactions is extremely limited. The oomycete Pythium irregulare is a poorly controlled pathogen of many crops. Previous work has indicated an important role for ethylene in defence against this oomycete. We examined the role of ethylene and SLs in response to this pathogen in pea ( Pisum sativum L.) at the molecular and whole-plant levels using a set of well-characterized hormone mutants, including an ethylene-insensitive ein2 mutant and SL-deficient and insensitive mutants.We identified a key role for ethylene signalling in specific cell types that reduces pathogen invasion, extending the work carried out in other species. However, we found no evidence that SL biosynthesis or response influences the interaction of pea with P. irregulare or that synthetic SL influences the growth or hyphal branching of the oomycete in vitro . Future work should seek to extend our understanding of the role of SLs in other plant interactions, including with other fungal, bacterial and viral pathogens, nematodes and insect pests.

Plant responses to light involve a complex network of interactions among multiple plant hormones. In a screen for mutants showing altered photomorphogenesis under red light, we identified a mutant with dramatically enhanced leaf expansion and delayed petal senescence. We show that this mutant exhibits reduced sensitivity to ethylene and carries a nonsense mutation in the single pea (Pisum sativum) ortholog of the ethylene signaling gene ETHYLENE INSENSITIVE2 (EIN2). Consistent with this observation, the ein2 mutation rescues the previously described effects of ethylene overproduction in mature phytochrome-deficient plants. In seedlings, ein2 confers a marked increase in leaf expansion under monochromatic red, far-red, or blue light, and interaction with phytochromeA, phytochromeB, and long1 mutants confirms that ein2 enhances both phytochrome- and cryptochrome-dependent responses in a LONG1-dependent manner. In contrast, minimal effects of ein2 on seedling development in darkness or high-irradiance white light show that ethylene is not limiting for development under these conditions. These results indicate that ethylene signaling constrains leaf expansion during deetiolation in pea and provide further evidence that down-regulation of ethylene production may be an important component mechanism in the broader control of photomorphogenic development by phytochrome and cryptochrome.

Gibberellin plays a dual role in legumes during uptake and accommodation of nitrogen-fixing bacteria; inhibiting infection at the epidermis but promoting nodule organogenesis and ultimate function., Leguminous plant roots can form a symbiosis with soil-dwelling nitrogen-fixing rhizobia, leading to the formation of a new root organ, the nodule. Successful nodulation requires co-ordination of spatially separated events in the root, including infection in the root epidermis and nodule organogenesis deep in the root cortex. We show that the hormone gibberellin plays distinct roles in these epidermal and cortical programmes. We employed a unique set of genetic material in pea that includes severely gibberellin-deficient lines and della-deficient lines that enabled us to characterize all stages of infection and nodule development. We confirmed that gibberellin suppresses infection thread formation and show that it also promotes nodule organogenesis into nitrogen-fixing organs. In both cases, this is achieved through the action of DELLA proteins. This study therefore provides a mechanism to explain how both low and high gibberellin signalling can result in reduced nodule number and reveals a clear role for gibberellin in the maturation of nodules into nitrogen-fixing organs. We also demonstrate that gibberellin acts independently of ethylene in promoting nodule development.

Strigolactones (SLs) influence the ability of legumes to associate with nitrogen-fixing bacteria. In this study we determine the precise stage at which SLs influence nodulation. We show that SLs promote infection thread formation, as a null SL-deficient pea (Pisum sativum) mutant forms significantly less infection threads than wild type plants and this reduction can be overcome by the application of the synthetic SL GR24. We found no evidence that SLs influence physical events in the plant before or after infection thread formation, since SL-deficient plants displayed a similar ability to induce root hair curling in response to rhizobia or lipochito-oligosaccharides (LCOs) and SL-deficient nodules appear to fix nitrogen at a similar rate to wild type plants. In contrast, a SL receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting SL-deficiency may influence the bacterial partner. We found this influence of SL-deficiency was not due to altered flavonoid exudation or ability of root exudates to stimulate bacterial growth. The influence of SL-deficiency on infection thread formation was accompanied by reduced expression of some early nodulation (ENOD) genes. Importantly, SL synthesis is down-regulated by mutations in genes of the Nod LCO signalling pathway and this requires the downstream transcription factor NSP2 but not NIN. This, together with the fact that the expression of certain SL biosynthesis genes can be elevated in response to rhizobia/Nod factors suggests that Nod LCOs may induce SL biosynthesis. SLs appear to influence nodulation independently of ethylene action, as SL-deficient and ethylene insensitive double mutant plants display essentially additive phenotypes and we found no evidence that SLs influence ethylene synthesis or vice versa.

Plant hormones and other mobile signalling elements play key roles in regulating nodulation and N2 fixation in legumes. This includes many hormones associated with regulating general growth and development, such as cytokinin, auxin, gibberellins and strigolactones and plant hormones associated with response to stress, including ethylene. Mobile peptides and microRNAs have also shown to have significant roles in regulating nodule initiation, organogenesis and nutrient response. In this chapter we will discuss the roles of these small signalling molecules in nodulation, highlighting specific examples of their interactions with phosphorous (P) stress. P-induced small peptides and microRNAs have been identified in legumes, but the role of these signals in regulating nodulation response to P stress has not been directly investigated. Similarly, relatively few studies that have specifically examined the role of plant hormones in P response of nodulation and areas for future research are highlighted.

Background and Aims: The number of nodules formed on a legume root system is under the strict genetic control of the autoregulation of nodulation (AON) pathway. Plant hormones are thought to play a role in AON; however, the involvement of two hormones recently described as having a largely positive role in nodulation, strigolactones and brassinosteroids, has not been examined in the AON process. Methods: A genetic approach was used to examine if strigolactones or brassinosteroids interact with the AON system in pea ( Pisum sativum ). Double mutants between shoot-acting ( Psclv2, Psnark ) and root-acting ( Psrdn1 ) mutants of the AON pathway and strigolactone-deficient ( Psccd8 ) or brassinosteroid-deficient ( lk ) mutants were generated and assessed for various aspects of nodulation. Strigolactone production by AON mutant roots was also investigated. Key Results: Supernodulation of the roots was observed in both brassinosteroid- and strigolactone-deficient AON double-mutant plants. This is despite the fact that the shoots of these plants displayed classic strigolactone-deficient (increased shoot branching) or brassinosteroid-deficient (extreme dwarf) phenotypes. No consistent effect of disruption of the AON pathway on strigolactone production was found, but root-acting Psrdn1 mutants did produce significantly more strigolactones. Conclusions: No evidence was found that strigolactones or brassinosteroids act downstream of the AON genes examined. While in pea the AON mutants are epistatic to brassinosteroid and strigolactone synthesis genes, we argue that these hormones are likely to act independently of the AON system, having a role in the promotion of nodule formation.

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.

New roles for the recently identified group of plant hormones, the strigolactones, are currently under active investigation. One of their key roles is to regulate plant symbioses. These compounds act as a rhizosphere signal in arbuscular mycorrhizal symbioses and as a positive regulator of nodulation in legumes. The phosphorous and nitrogen status of the soil has emerged as a powerful regulator of strigolactone production. However, until now, the potential role of strigolactones in regulating mycorrhizal development and nodulation in response to nutrient deficiency has not been proven. In this paper, the role of strigolactone synthesis and response in regulating these symbioses is examined in pea (Pisum sativum L.). Pea is well suited to this study, since there is a range of well-characterized strigolactone biosynthesis and response mutants that is unique amongst legumes. Evidence is provided for a novel endogenous role for strigolactone response within the root during mycorrhizal development, in addition to the action of strigolactones on the fungal partner. The strigolactone response pathway that regulates mycorrhizal development also appears to differ somewhat from the response pathway that regulates nodulation. Finally, studies with strigolactone-deficient pea mutants indicate that, despite strong regulation of strigolactone production by both nitrogen and phosphate, strigolactones are not required to regulate these symbioses in response to nutrient deficiency.

Protein farnesylation is a post-translational modification involving the addition of a 15-carbon farnesyl isoprenoid to the carboxy terminus of select proteins(1-3). Although the roles of this lipid modification are clear in both fungal and animal signalling, many of the mechanistic functions of farnesylation in plant signalling are still unknown. Here, we show that CYP85A2, the cytochrome P450 enzyme that performs the last step in brassinosteroid biosynthesis (conversion of castasterone to brassinolide)(4), must be farnesylated to function in Arabidopsis. Loss of either CYP85A2 or CYP85A2 farnesylation results in reduced brassinolide accumulation and increased plant responsiveness to the hormone abscisic acid (ABA) and overall drought tolerance, explaining previous observations(5). This result not only directly links farnesylation to brassinosteroid biosynthesis but also suggests new strategies to maintain crop yield under challenging climatic conditions.

Highlight An ethylene-insensitive mutant of pea with enhanced nodulation and arbuscular mycorrhizal development showed that gibberellin and brassinosteroid deficiency affect nodule number via ethylene levels., The regulation of arbuscular mycorrhizal development and nodulation involves complex interactions between the plant and its microbial symbionts. In this study, we use the recently identified ethylene-insensitive ein2 mutant in pea (Pisum sativum L.) to explore the role of ethylene in the development of these symbioses. We show that ethylene acts as a strong negative regulator of nodulation, confirming reports in other legumes. Minor changes in gibberellin1 and indole-3-acetic acid levels in ein2 roots appear insufficient to explain the differences in nodulation. Double mutants produced by crosses between ein2 and the severely gibberellin-deficient na and brassinosteroid-deficient lk mutants showed increased nodule numbers and reduced nodule spacing compared with the na and lk single mutants, but nodule numbers and spacing were typical of ein2 plants, suggesting that the reduced number of nodules in na and lk plants is largely due to the elevated ethylene levels previously reported in these mutants. We show that ethylene can also negatively regulate mycorrhizae development when ethylene levels are elevated above basal levels, consistent with a role for ethylene in reducing symbiotic development under stressful conditions. In contrast to the hormone interactions in nodulation, ein2 does not override the effect of lk or na on the development of arbuscular mycorrhizae, suggesting that brassinosteroids and gibberellins influence this process largely independently of ethylene.

Strigolactones are an ancient group of plant signalling molecules. They play a critical role in the rhizosphere where they facilitate the formation of symbioses with fungi, crucial for the acquisition of plant nutrients in over 80 % of land plant species. Strigolactones have also been exploited by parasitic weeds as a rhizosphere signal indicating the presence of a host species, resulting in devastating losses in some agricultural systems. Recently, they have also been shown to act endogenously as plant hormones controlling shoot branching and have been implicated in a wide range of other physiological processes, including root growth, root-hair elongation, adventitious rooting, secondary growth, photomorphogenesis, seed germination, nodulation, and protonemal development in mosses. Here, we discuss the evidence for the involvement of strigolactones as endogenous regulators of these processes and highlight some examples where the evidence is inconclusive. One major gap in our understanding is the identity of the endogenous strigolactone(s) that are biologically active. A discussion of the interactions between the different plant hormones and the possible role of strigolactones as integrators of the root-to-shoot balance, nutrient acquisition, and thus resource allocation illustrates some important future directions for this area of research.

Strigolactones are recently defined plant hormones with roles in mycorrhizal symbiosis and shoot and root architecture. Their potential role in controlling nodulation, the related symbiosis between legumes and Rhizobium bacteria, was explored using the strigolactone-deficient rms1 mutant in pea (Pisum sativum L.). This work indicates that endogenous strigolactones are positive regulators of nodulation in pea, required for optimal nodule number but not for nodule formation per se. rms1 mutant root exudates and root tissue are almost completely deficient in strigolactones, and rms1 mutant plants have approximately 40% fewer nodules than wild-type plants. Treatment with the synthetic strigolactone GR24 elevated nodule number in wild-type pea plants and also elevated nodule number in rms1 mutant plants to a level similar to that seen in untreated wild-type plants. Grafting studies revealed that nodule number and strigolactone levels in root tissue of rms1 roots were unaffected by grafting to wild-type scions indicating that strigolactones in the root, but not shoot-derived factors, regulate nodule number and provide the first direct evidence that the shoot does not make a major contribution to root strigolactone levels.

The use of shadecloth is common in horticulture to reduce Summer heat load on crops; however, such shading can increase shoot elongation to undesirable levels. A possible solution is to use photoselective shadecloths to overcome the problem of shoot elongation. To examine this question, pea (Pisum sativum L.) plants were grown under no shade, 50% neutral shadecloth, or 50% blue, or 50% red photoselective shadecloths. Under the red and blue shadecloths, the blue fraction was altered, but there was little alteration in other wavelengths relative to neutral shadecloth or to sunlight. Relative to neutral shadecloth, shoot length was reduced significantly under blue shadecloth, and increased significantly under red shadecloth.The effect of the red and blue shadecloths appeared to be mediated by alterations in blue irradiance perceived by the blue light cry1 photoreceptor, as, unlike wild-type (WT) peas, cry1 mutant peas did not respond to the shadecloth treatments. Levels of GA1 were reduced under blue shadecloth in WT pea plants compared with neutral shadecloth, but not in cry1 mutant plants. This study demonstrates that photoselective shadecloths can be used to manipulate plant height and flowering in crops with a strong response to blue irradiance.

Cryptochromes mediate blue light-dependent photomorphogenic responses, such as inhibition of hypocotyl elongation. To investigate the underlying mechanism, we analyzed a genetic suppressor, scc7-D (suppressors of cry1cry2), which suppressed the long-hypocotyl phenotype of the cry1cry2 (cryptochrome1/cryptochrome2) mutant in a light-dependent but wavelength-independent manner. scc7-D is a gain-of-expression allele of the GA2ox8 gene encoding a gibberellin (GA)-inactivating enzyme, GA 2-oxidase. Although scc7-D is hypersensitive to light, transgenic seedlings expressing GA2ox at a level higher than scc7-D showed a constitutive photomorphogenic phenotype, confirming a general role of GA2ox and GA in the suppression of hypocotyl elongation. Prompted by this result, we investigated blue light regulation of mRNA expression of the GA metabolic and catabolic genes. We demonstrated that cryptochromes are required for the blue light regulation of GA2ox1, GA20ox1, and GA3ox1 expression in transient induction, continuous illumination, and photoperiodic conditions. The kinetics of cryptochrome induction of GA2ox1 expression and cryptochrome suppression of GA20ox1 or GA3ox1 expression correlate with the cryptochrome-dependent transient reduction of GA4 in etiolated wild-type seedlings exposed to blue light. Therefore we propose that in deetiolating seedlings, cryptochromes mediate blue light regulation of GA catabolic/metabolic genes, which affect GA levels and hypocotyl elongation. Surprisingly, no significant change in the GA4 content was detected in the whole shoot samples of the wild-type or cry1cry2 seedlings grown in the dark or continuous blue light, suggesting that cryptochromes may also regulate GA responsiveness and/or trigger cell- or tissue-specific changes of the level of bioactive GAs.

Strigolactones (SLs) do not influence spore germination or hyphal growth of Fusarium oxysporum. Mutant studies revealed no role for SLs but a role for ethylene signalling in defence against this pathogen in pea. Strigolactones (SLs) play important roles both inside the plant as a hormone and outside the plant as a rhizosphere signal in interactions with mycorrhizal fungi and parasitic weeds. What is less well understood is any potential role SLs may play in interactions with disease causing microbes such as pathogenic fungi. In this paper we investigate the influence of SLs on the hemibiotrophic pathogen Fusarium oxysporum f.sp. pisi both directly via their effects on fungal growth and inside the plant through the use of a mutant deficient in SL. Given that various stereoisomers of synthetic and naturally occuring SLs can display different biological activities, we used (+)-GR24, (−)-GR24 and the naturally occurring SL, (+)-strigol, as well as a racemic mixture of 5-deoxystrigol. As a positive control, we examined the influence of a plant mutant with altered ethylene signalling, ein2, on disease development. We found no evidence that SLs influence spore germination or hyphal growth of Fusarium oxysporum and that, while ethylene signalling influences pea susceptibility to this pathogen, SLs do not.

Plant hormones play key roles in defence against pathogen attack. Recent work has begun to extend this role to encompass not just the traditional disease/stress hormones, such as ethylene, but also growth‐promoting hormones. Strigolactones (SLs) are the most recently defined group of plant hormones with important roles in plant–microbe interactions, as well as aspects of plant growth and development, although the knowledge of their role in plant–pathogen interactions is extremely limited. The oomycete P ythium irregulare is a poorly controlled pathogen of many crops. Previous work has indicated an important role for ethylene in defence against this oomycete. We examined the role of ethylene and SLs in response to this pathogen in pea (P isum sativum L.) at the molecular and whole‐plant levels using a set of well‐characterized hormone mutants, including an ethylene‐insensitive ein2 mutant and SL‐deficient and insensitive mutants. We identified a key role for ethylene signalling in specific cell types that reduces pathogen invasion, extending the work carried out in other species. However, we found no evidence that SL biosynthesis or response influences the interaction of pea with P . irregulare or that synthetic SL influences the growth or hyphal branching of the oomycete in vitro. Future work should seek to extend our understanding of the role of SLs in other plant interactions, including with other fungal, bacterial and viral pathogens, nematodes and insect pests.

The role of shoot-root signals in the control of nodulation and arbuscular mycorrhizal (AM) development were examined in the divergent legume species pea and blue lupin. These species were chosen as pea can host both symbionts, whereas lupin can nodulate but has lost the ability to form AM. Intergeneric grafts between lupin and pea enabled examination of key long-distance signals in these symbioses. The role of strigolactones, auxin and elements of the autoregulation of nodulation (AON) pathway were investigated. Grafting studies were combined with loss-of-function mutants to monitor symbioses (nodulation, AM) and hormone effects (levels, gene expression and application studies). Lupin shoots suppress AM colonization in pea roots, in part by downregulating strigolactone exudation involving reduced expression of the strigolactone biosynthesis gene PsCCD8. By contrast, lupin shoots enhance pea nodulation, independently of strigolactones, possibly due to a partial incompatibility in AON shoot-root signalling between pea and lupin. This study highlights that nodulation and AM symbioses can be regulated independently and this may be due to long-distance signals, a phenomenon we were able to uncover by working with divergent legumes. We also identify a role for strigolactone exudation in determining the status of non-AM hosts.

In pea (Pisum sativum L.) seedlings, both phytochrome A (phyA) and cryptochrome 1 (cry1) are required for the rapid inhibition of elongation in response to transfer from dark to blue light. The reduction in stem elongation upon blue light exposure is accompanied by a rapid reduction in the level of bioactive gibberellin A1 (GA1) in the shoot, and mutant studies indicate that this process is redundantly regulated by phyA and cry1. The reduction in GA1 levels under blue light may be mediated by phyA and cry1 in part through increased conversion of GA1 to the inactive catabolite GA8. Changes in the transcript level of key GA metabolism genes appear to play an important role in this process. Under blue light, both phyA and cry1 are required to suppress the transcript level of PsGA3ox1, which regulates the conversion of GA20 to GA1. PhyA and cry1 also contribute to upregulation of PsGA2ox2 transcript level under blue light, which encodes a GA 2-oxidase that converts GA1 to inactive GA8. In addition to changes in GA levels, phyA and/or cry1 may also regulate changes in GA responsiveness, an important mechanism for regulating long-term stem elongation in the light.

Shoot branching is inhibited by auxin transported down the stem from the shoot apex. Auxin does not accumulate in inhibited buds and so must act indirectly. We show that mutations in theMAX4gene ofArabidopsisresult in increased and auxin-resistant bud growth. Increased branching inmax4shoots is restored to wild type by grafting to wild-type rootstocks, suggesting thatMAX4is required to produce a mobile branch-inhibiting signal, acting downstream of auxin. A similar role has been proposed for the pea gene,RMS1. Accordingly,MAX4andRMS1were found to encode orthologous, auxin-inducible members of the polyene dioxygenase family.

All of the classical plant hormones have been suggested to influence nodulation, including some that interact with the Autoregulation of Nodulation (AON) pathway. Leguminous plants strictly regulate the number of nodules formed through this AON pathway via a root-shoot-root loop that acts to suppress excessive nodulation. A related pathway, the Autoregulation of Mycorrhization (AOM) pathway controls the more ancient, arbuscular mycorrhizal (AM) symbiosis. A comparison of the published responses to the classical hormones in these 2 symbioses shows that most influence the symbioses in the same direction. This may be expected if they affect the symbioses via common components of these symbiotic regulatory pathways. However, some hormones influence these symbioses in opposite directions, suggesting a more complex relationship, and probably one that is not via the common components of these pathways. In a recent paper we showed, using a genetic approach, that strigolactones and brassinosteroids do not act downstream of the AON genes examined and argued that they probably act independently to promote nodule formation. Recently it has been shown that the control of nodulation via the AON pathway involves mobile CLE peptide signals. It is therefore suggested that a more direct avenue to determine if the classical hormones play a direct role in the autoregulatory pathways is to further examine whether CLE peptides and other components of these processes can influence, or be influenced by, the classical hormones. Such studies and other comparisons between the nodulation and mycorrhizal symbioses should allow the role of the classical hormones in these critical symbioses to be rapidly advanced.

The recently discovered group of plant hormones, the strigolactones, have been implicated in regulating photomorphogenesis. We examined this extensively in our strigolactone synthesis and response mutants and could find no evidence to support a major role for strigolactone signaling in classic seedling photomorphogenesis (e.g. elongation and leaf expansion) in pea (Pisum sativum), consistent with two recent independent reports in Arabidopsis. However, we did find a novel effect of strigolactones on adventitious rooting in darkness. Strigolactone-deficient mutants, Psccd8 and Psccd7, produced significantly fewer adventitious roots than comparable wild-type seedlings when grown in the dark, but not when grown in the light. This observation in dark-grown plants did not appear to be due to indirect effects of other factors (e.g. humidity) as the constitutively de-etiolated mutant, lip1, also displayed reduced rooting in the dark. This role for strigolactones did not involve the MAX2 F-Box strigolactone response pathway as Psmax2 f-box mutants did not show a reduction in adventitious rooting in the dark compared with wild-type plants. The auxin-deficient mutant bushy also reduced adventitious rooting in the dark, as did decapitation of wild-type plants. Rooting was restored by the application of indole-3-acetic acid (IAA) to decapitated plants, suggesting a role for auxin in the rooting response. However, auxin measurements showed no accumulation of IAA in the epicotyls of wild-type plants compared with the strigolactone synthesis mutant Psccd8, suggesting that changes in the gross auxin level in the epicotyl are not mediating this response to strigolactone deficiency.

The ramosus (rms) mutation (rms1) of pea (Pisum sativum) causes increased branching through modification of graft-transmissible signal(s) produced in rootstock and shoot. Additional grafting techniques have led us to propose that the novel signal regulated byRms1 moves acropetally in shoots and acts as a branching inhibitor. Epicotyl interstock grafts showed that wild-type (WT) epicotyls grafted between rms1 scions and rootstocks can revert mutant scions to a WT non-branching phenotype. Mutant scions grafted together with mutant and WT rootstocks did not branch despite a contiguous mutant root-shoot system. The primary action ofRms1 is, therefore, unlikely to be to block transport of a branching stimulus from root to shoot. Rather, Rms1may influence a long-distance signal that functions, directly or indirectly, as a branching inhibitor. It can be deduced that this signal moves acropetally in shoots because WT rootstocks inhibit branching in rms1 shoots, and although WT scions do not branch when grafted to mutant rootstocks, they do not inhibit branching in rms1 cotyledonary shoots growing from the same rootstocks. The acropetal direction of transport of theRms1 signal supports previous evidence that therms1 lesion is not in an auxin biosynthesis or transport pathway. The different branching phenotypes of WT andrms1 shoots growing from the same rms1rootstock provides further evidence that the shoot has a major role in the regulation of branching and, moreover, that root-exported cytokinin is not the only graft-transmissible signal regulating branching in intact pea plants.

Background and Aims: Arbuscular mycorrhizal symbioses are important for nutrient acquisition in .80% of terrestrial plants. Recently there have been major breakthroughs in understanding the signals that regulate colonization by the fungus, but the roles of the known plant hormones are still emerging. Here our understanding of the roles of abscisic acid, ethylene, auxin, strigolactones, salicylic acid and jasmonic acid is discussed, and the roles of gibberellins and brassinosteroids examined. Methods: Pea mutants deficient in gibberellins, DELLA proteins and brassinosteroids are used to determine whether fungal colonization is altered by the level of these hormones or signalling compounds. Expression of genes activated during mycorrhizal colonization is also monitored. Key Results: Arbuscular mycorrhizal colonization of pea roots is substantially increased in gibberellin-deficient na-1 mutants compared with wild-type plants. This is reversed by application of GA 3 . Mutant la cry-s , which lacks gibberellin signalling DELLA proteins, shows reduced colonization. These changes were parallelled by changes in the expression of genes associated with mycorrhizal colonization. The brassinosteroid-deficient lkb mutant showed no change in colonization. Conclusions: Biologically active gibberellins suppress arbuscule formation in pea roots, and DELLA proteins are essential for this response, indicating that this role occurs within the root cells.

Arbuscular mycorrhizal symbiosis, formed between more than 80% of land plants and fungi from the phylum Glomeromycota, is an ancient association that is believed to have evolved as plants moved onto land more than 400 mya. Similarly ancient, the plant hormones auxin and strigolactone are thought to have been present in the plant lineage since before the divergence of the bryophytes in the case of auxin and before the colonisation of land in the case of strigolactones. The discovery of auxin in the 1930s predates the discovery of strigolactones as a plant hormone in 2008 by over 70 y. Recent studies in pea suggest that these two signals may interact to regulate mycorrhizal symbiosis. Furthermore, the first quantitative studies are presented that show that low auxin content of the root is correlated with low strigolactone production, an interaction that has implications for how these plant hormones regulate several developmental programs including shoot branching, secondary growth and root development. With recent advances in our understanding of auxin and strigolactone biosynthesis, together with the discovery of the fungal signals that activate the plant host, the stage is set for real breakthroughs in our understanding of the interactions between plant and fungal signals in mycorrhizal symbiosis.