The Role of CLE Peptides in the Suppression of Mycorrhizal Colonization of Tomato.

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 colonization levels is limited, especially in non-legumes. Here, we demonstrate that in tomato, two CLV3/EMBRYO-SURROUNDING REGION (CLE) peptides, SlCLE10 and SlCLE11, act to suppress AM colonization of roots. Mutant studies and overexpression via hairy transformation indicate that SlCLE11 acts locally in the root to limit AM colonization. Indeed, SlCLE11 expression is strongly induced in AM-colonized roots, but SlCLE11 is not required for phosphate suppression of AM colonization. SlCLE11 requires the FIN gene that encodes an enzyme required for CLE peptide arabinosylation to suppress mycorrhizal colonization. However, SlCLE11 suppression of AM does not require two CLE receptors with roles in regulating AM colonization, SlFAB (CLAVATA1 ortholog) or SlCLV2. Indeed, multiple parallel pathways appear to suppress mycorrhizal colonization in tomato, as double mutant studies indicate that SlCLV2 and FIN have an additive influence on mycorrhizal colonization. SlCLE10 appears to play a more minor or redundant role, as cle10 mutants did not influence intraradical AM colonization. However, the fact that cle10 mutants had an elevated number of hyphopodia and that ectopic overexpression of SlCLE10 did suppress mycorrhizal colonization suggests that SlCLE10 may also play a role in suppressing AM colonization. Our findings show that CLE peptides regulate AM colonization in tomato and at least SlCLE11 likely requires arabinosylation for activity.

Cell-layer specific roles for gibberellins in nodulation and root development.

Gibberellins (GA) have a profound influence on the formation of lateral root organs. However, the precise role this hormone plays in the cell-specific events during lateral root formation, rhizobial infection and nodule organogenesis, including interactions with auxin and cytokinin (CK), is not clear. We performed epidermal- and endodermal-specific complementation of the severely GA-deficient na pea (Pisum sativum) mutant with Agrobacterium rhizogenes. Gibberellin mutants were used to examine the spatial expression pattern of CK (TCSn)- and auxin (DR5)-responsive promoters and hormone levels. We found that GA produced in the endodermis promote lateral root and nodule organogenesis and can induce a mobile signal(s) that suppresses rhizobial infection. By contrast, epidermal-derived GA suppress infection but have little influence on root or nodule development. GA suppress the CK-responsive TCSn promoter in the cortex and are required for normal auxin activation during nodule primordia formation. Our findings indicate that GA regulate the checkpoints between infection thread (IT) penetration of the cortex and invasion of nodule primordial cells and promote the subsequent progression of nodule development. It appears that GA limit the progression and branching of IT in the cortex by restricting CK response and activate auxin response to promote nodule primordia development.

Mendel: From genes to genome.

Two hundred years after the birth of Gregor Mendel, it is an appropriate time to reflect on recent developments in the discipline of genetics, particularly advances relating to the prescient friar's model species, the garden pea (Pisum sativum L.). Mendel's study of seven characteristics established the laws of segregation and independent assortment. The genes underlying four of Mendel's loci (A, LE, I, and R) have been characterized at the molecular level for over a decade. However, the three remaining genes, influencing pod color (GP), pod form (V/P), and the position of flowers (FA/FAS), have remained elusive for a variety of reasons, including a lack of detail regarding the loci with which Mendel worked. Here, we discuss potential candidate genes for these characteristics, in light of recent advances in the genetic resources for pea. These advances, including the pea genome sequence and reverse-genetics techniques, have revitalized pea as an excellent model species for physiological-genetic studies. We also discuss the issues that have been raised with Mendel's results, such as the recent controversy regarding the discrete nature of the characters that Mendel chose and the perceived overly-good fit of his segregations to his hypotheses. We also consider the relevance of these controversies to his lasting contribution. Finally, we discuss the use of Mendel's classical results to teach and enthuse future generations of geneticists, not only regarding the core principles of the discipline, but also its history and the role of hypothesis testing.

The role of CLAVATA signalling in the negative regulation of mycorrhizal colonization and nitrogen response of tomato.

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.

Brassinosteroids play multiple roles in nodulation of pea via interactions with ethylene and auxin.

MAIN CONCLUSION: 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.

The role of CLV1, CLV2 and HPAT homologues in the nitrogen-regulation of root development.

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.

What drives interspecies graft union success? Exploring the role of phylogenetic relatedness and stem anatomy.

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.

Auxin transport and stem vascular reconnection - has our thinking become canalized?

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.

Determining the Site of Action of Strigolactones during Nodulation.

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 fewer 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 Nod lipochitooligosaccharides (LCOs) and SL-deficient nodules appear to fix nitrogen at a similar rate to those of wild-type plants. In contrast, an SL receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting that SL deficiency may influence the bacterial partner. We found that this influence of SL deficiency was not due to altered flavonoid exudation or the 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 genes. Importantly, SL synthesis is down-regulated by mutations in genes of the Nod LCO signaling 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 LCOs, 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.

Gibberellins promote nodule organogenesis but inhibit the infection stages of nodulation.

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.

The role of strigolactones during plant interactions with the pathogenic fungus Fusarium oxysporum.

MAIN CONCLUSION: 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.

Common and divergent shoot-root signalling in legume symbioses.

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.

Ethylene Signaling Influences Light-Regulated Development in Pea.

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.

Interactions between ethylene, gibberellins, and brassinosteroids in the development of rhizobial and mycorrhizal symbioses of pea.

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 innaandlkplants 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.

The role of strigolactones in photomorphogenesis of pea is limited to adventitious rooting.

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.

Biosynthesis of the halogenated auxin, 4-chloroindole-3-acetic acid.

Seeds of several agriculturally important legumes are rich sources of the only halogenated plant hormone, 4-chloroindole-3-acetic acid. However, the biosynthesis of this auxin is poorly understood. Here, we show that in pea (Pisum sativum) seeds, 4-chloroindole-3-acetic acid is synthesized via the novel intermediate 4-chloroindole-3-pyruvic acid, which is produced from 4-chlorotryptophan by two aminotransferases, TRYPTOPHAN AMINOTRANSFERASE RELATED1 and TRYPTOPHAN AMINOTRANSFERASE RELATED2. We characterize a tar2 mutant, obtained by Targeting Induced Local Lesions in Genomes, the seeds of which contain dramatically reduced 4-chloroindole-3-acetic acid levels as they mature. We also show that the widespread auxin, indole-3-acetic acid, is synthesized by a parallel pathway in pea.

Plant hormones in arbuscular mycorrhizal symbioses: an emerging role for gibberellins.

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 GA3. 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.

Right time, right place: The dynamic role of hormones in rhizobial infection and nodulation of legumes.

Many legume plants form beneficial associations with rhizobial bacteria that are hosted in new plant root organs, nodules, in which atmospheric nitrogen is fixed. This association requires the precise coordination of two separate programs, infection in the epidermis and nodule organogenesis in the cortex. There is extensive literature indicating key roles for plant hormones during nodulation, but a detailed analysis of the spatial and temporal roles of plant hormones during the different stages of nodulation is required. This review analyses the current literature on hormone regulation of infection and organogenesis to reveal the differential roles and interactions of auxin, cytokinin, brassinosteroids, ethylene, and gibberellins during epidermal infection and cortical nodule initiation, development, and function. With the exception of auxin, all of these hormones suppress infection events. By contrast, there is evidence that all of these hormones promote nodule organogenesis, except ethylene, which suppresses nodule initiation. This differential role for many of the hormones between the epidermal and cortical programs is striking. Future work is required to fully examine hormone interactions and create a robust model that integrates this knowledge into our understanding of nodulation pathways.

Development of the Casparian strip is delayed by blue light in pea stems.

To understand the regulatory mechanisms involved in tissue development by light, the kinetics of regulation of Casparian strip (CS) development in garden pea stems was studied. We found that short-term irradiation with white light delayed the development of the CS and used this delay to assess the quantitative effect of light on CS development. We examined the effect of the duration and fluence rates of white light treatment on CS development and observed a significant relationship between fluence and the delay in CS development indicating that the Bunsen-Roscoe law of reciprocity holds for this response. The effect of white light irradiation was not inhibited in the presence of a photosynthetic inhibitor, DCMU, or a carotenoid biosynthesis inhibitor, Norflurazon, indicating that the delay in CS development by light is a photomorphogenetic response rather than a subsidiary effect mediated by photosynthetic activity. An action spectrum for the response displayed a major peak in the blue-light region, suggesting a dominant role for blue-light receptors. A minor peak in the red-light region also suggested the possible involvement of phytochromes. Although phytochromes are known to contribute to blue-light responses, phytochrome-deficient mutants showed a normal delay of CS development in response to blue light, indicating that the response is not mediated by phytochrome and suggesting a role for one or more specific blue-light receptors.

Relationship between gibberellin, ethylene and nodulation in Pisum sativum.

• Gibberellin (GA) deficiency resulting from the na mutation in pea (Pisum sativum) causes a reduction in nodulation. Nodules that do form are aberrant, having poorly developed meristems and a lack of enlarged cells. Studies using additional GA-biosynthesis double mutants indicate that this results from severe GA deficiency of the roots rather than simply dwarf shoot stature. • Double mutants isolated from crosses between na and three supernodulating pea mutants exhibit a supernodulation phenotype, but the nodule structures are aberrant. This suggests that severely reduced GA concentrations are not entirely inhibitory to nodule initiation, but that higher GA concentrations are required for proper nodule development. • na mutants evolve more than double the amount of ethylene produced by wild-type plants, indicating that low GA concentrations can promote ethylene production. The excess ethylene may contribute to the reduced nodulation of na plants, as application of an ethylene biosynthesis inhibitor increased na nodule numbers. However, these nodules were still aberrant in structure. • Constitutive GA signalling mutants also form significantly fewer nodules than wild-type plants. This suggests that there is an optimum degree of GA signalling required for nodule formation and that the GA signal, and not the concentration of bioactive GA per se, is important for nodulation.