Legume root nodulation with nitrogen-fixing bacteria requires precise control via root-shoot-root autoregulation of nodulation (AON). Post-translationally modified root-derived CLAVATA3/Embryo Surrounding Region-Related (CLE) peptides signal through shoot acting leucine-rich repeat receptors (CLAVATAs) to regulate nodule number, and this pathway is a target to optimise nodulation. We characterise the AON system in the crop model pea (Pisum sativum L.) and address key gaps in our understanding of AON; the role of parallel signalling pathways, shoot receptor complexes and downstream targets. We use novel mutant combinations, overexpression, grafting, gene expression and careful analysis of infection and nodule organogenesis using GFP-labelled rhizobium. These studies provide evidence that, in pea, both PsCLE12 and PsCLE13 require arabinosylation via PsRDN1. Perception of PsCLE12 and PsCLE13 in the shoot to suppress the mature nodules in the root requires the pea CLAVATA1 orthologue PsNARK and PsCLV2. However, we found little evidence that PsCLE12 and PsCLE13 suppress infection thread development or that they act via PsTML1 and/or PsTML2, root acting suppressors of nodulation, indicating a role for additional CLE signals. Grafting and double mutant studies suggest that PsNARK may act together with PsCLV2, but also independently, to influence nodulation, providing in planta support for shoot receptor complexes that control AON.

Root nodules host nitrogen-fixing bacteria and likely evolved through modifications of the lateral root program. Members of the NOOT-BOP-COCH-LIKE transcriptional coregulator family suppress root identity in nodules and plant hormones play key roles in nodule organogenesis, but the interaction between these pathways is unclear. In this study, we investigate how COCH regulates nodule identity through crosstalk with plant hormones, using the Pisum sativum cochleata (Pscoch) mutant - which forms root-nodule hybrids - in combination with hormone biosensors, double mutants, hormone quantification, and RNA-seq analysis. We found that COCH suppresses cytokinin levels and response during nodule formation. By contrast, PsCOCH promotes auxin accumulation and precise auxin response patterning in nodules. Mutant coch developing nodules have gene expression profiles more similar to that of root primordia, with increased expression of defence and auxin response genes and reduced expression of cytokinin biosynthesis genes compared to wild-type. We found gibberellin is unlikely to act downstream of PsCOCH. Constitutive expression of PsCOCH also produces root-nodule hybrids and we found intriguing links between the autoregulation of nodulation pathway and PsCOCH. We show that PsCOCH is required for spatial tight regulation of auxin and cytokinin during nodule organogenesis and identify key hormone and signalling genes that act downstream of COCH.

Plants regulate root development in response to fluctuating environmental conditions, including establishing symbiotic relationships with arbuscular mycorrhizal fungi and nitrogen-fixing bacteria under nutrient limitation. These processes are orchestrated by plant hormones, particularly gibberellins, and the repressors of gibberellin signalling, DELLA proteins. Gibberellin and DELLAs serve as critical regulators in symbiotic signalling and root organogenesis, integrating hormonal and environmental cues with cellular patterning to direct plant development. This review explores the current understanding of gibberellin and DELLA function in symbiosis and root development, including an analysis of the conservation and divergence of their function in land plant evolution. DELLA proteins play a pivotal role in the common symbiotic signalling pathway, modulating transcriptional responses essential for both arbuscular mycorrhizal and rhizobial symbioses. While gibberellin suppresses early symbiotic signalling and microbial infection by promoting DELLA degradation, gibberellin positively regulates nodule organogenesis and function, demonstrating a cell- and stage-dependent role in symbiotic associations. Indeed, precise spatial and temporal dynamics of gibberellin signalling occurs during nodulation and root development. Key avenues for future research are identified, including understanding how the crosstalk between gibberellin and other key plant hormones fine-tune symbiosis and root development.

Arbuscular mycorrhizal (AM) fungi are known to enhance plant drought tolerance, but the physiological mechanism behind this benefit remains unclear. One explanation is that AM colonization improves root hydraulic conductance (Kr), thereby facilitating more efficient water uptake under soil drying, though this mechanism remains highly debated. Here, we measured Kr in tomato (Solanum lycopersicum L.) and pea (Pisum sativum L.) with and without AM using a noninvasive rehydration technique under soil drying, and this was complemented with the evaporative flux method under hydrated conditions. AM colonization was manipulated either through soil sterilization or by using nonmycorrhizal mutants, ensuring precise control of AM status. In both species, AM colonization had no positive impact on Kr under both well-hydrated and drought conditions. The finding suggests that the improved drought performance often observed in AM-colonized plants is not due to enhanced root water transport capacity. Instead, AM-induced benefits under drought may be mediated by other physiological adjustments.

In this study, we expanded the understanding of cytokinin (CK) perception in legumes by generating and characterizing novel pea cytokinin receptor mutants carrying mutations for the four cytokinin histidine kinase CHK genes in pea, CHK1, CHK2, CHK3 and CHK4. We constructed single, double, triple and quadruple mutants and analyzed their shoot, root, and nodulation phenotypes. We evaluated their contributions to the activation of CK-responsive genes, TCSn promoter activity, and used RNAi knockdowns of CHK1 to explore its role in nodulation. We found key roles for CHK1 in promoting nodulation, CHK3 in delaying leaf senescence and CHK4 in promoting leaf size and axillary shoot branching. Traits such as stem elongation and width as well as shoot and root size were regulated redundantly by the CHK receptors. Overall, this work provides a genetic dissection of cytokinin receptor function in pea, advancing our understanding of hormone signaling in a crop legume and offering genetic insights with potential applications for improving both shoot architecture and symbiotic efficiency.

Root hydraulic conductance (Kr) normalised by root dry mass (including nodules) under hydrated and drought conditions in Pisum sativum wild-type (Frisson, Nod+), non-nodulator (sym19, Nod-), and supernodulator (Psrdn1, Nod++).

Root nodules develop in some legumes that host nitrogen-fixing bacteria and likely evolved through modifications of the ancestral lateral root program with plant hormones playing key regulatory roles. Members of the NOOT-BOP-COCH-LIKE transcriptional co-regulator family suppress root identity in legume nodules, including Pisum sativum coch1 that display root-nodule hybrids. However, how COCH/NOOT interacts with hormones to control nodule organogenesis is unclear. We show that PsCOCH ( COCHLEATA ) is required for spatial tight regulation of auxin and cytokinin during nodule organogenesis and identify key hormone and signalling genes regulated by COCH . COCH suppresses cytokinin levels and response during nodule formation, as cytokinin levels are elevated in Pscoch abnormal nodules and this is mirrored by ectopic cytokinin-responsive TCSn::GUS expression in Pscoch nodule apices, nodule vasculature and in root-like tissue. In contrast, PsCOCH promotes auxin accumulation and precise auxin response patterning in nodules, as Pscoch mutants show significantly reduced auxin levels and severely altered auxin-responsive DR5::GUS expression patterns. RNAseq analysis revealed that Pscoch developing nodules have gene expression profiles more similar to root primordia, with increased expression of defence and auxin response genes (IAA and ARF) and reduced expression of cytokinin biosynthesis genes ( IPT3, CYP735A and LOG2) compared to wild type. We found gibberellin is unlikely to act downstream of PsCOCH , as Pscoch and gibberellin-deficient double mutants still form root-nodule hybrids. Ectopic constitutive expression of PsCOCH also produces root-nodule hybrids and we found intriguing links between autoregulation of nodulation pathway and COCH , suggesting that a complex feedback mechanism acts in COCH control of nodule identity. ### Competing Interest Statement The authors have declared no competing interest. Australian Research Council, https://ror.org/05mmh0f86, CE200100015

Legume root nodulation with nitrogen-fixing bacteria requires precise control via root-shoot-root autoregulation of nodulation (AON). Post-translationally modified root-derived CLAVATA3/Embryo Surrounding Region-Related (CLE) peptides signal through shoot acting leucine-rich repeat receptors (CLAVATAs) to regulate nodule number and this pathway is a target to optimise nodulation. We characterise the AON system in the crop model pea ( Pisum sativum L.) and address key gaps in our understanding of AON; the role of parallel signalling pathways, shoot receptor complexes and downstream targets. We use novel mutant combinations, overexpression, grafting, gene expression and careful analysis of infection and nodule organogenesis using GFP-labelled rhizobium. These studies provide evidence that in pea both Ps CLE12 and Ps CLE13 require arabinosylation via Ps RDN1. Perception of Ps CLE12 and Ps CLE13 in the shoot to suppress the mature nodules in the root requires the pea CLAVATA1 orthologue Ps NARK and Ps CLV2. However, we found little evidence that Ps CLE12 and Ps CLE13 suppress infection thread development or that they act via Ps TML1 and/or Ps TML2, root acting suppressors of nodulation, indicating a role for additional CLE signals. Grafting and double mutant studies indicate that Ps NARK can act together with Ps CLV2, but also independently, to influences nodulation providing in planta evidence for shoot receptor complexes that control AON. Highlight We characterise the specific CLE peptide signalling pathway that the model crop legume pea uses to control the number of nitrogen-fixing nodules formed on the root. ### Competing Interest Statement The authors have declared no competing interest.

Hydraulic, stomatal and anatomical traits significantly influence drought survival and productivity, but the extent to which these traits are coordinated and align with climate of origin (COO) in herbaceous species remains poorly understood. We quantified hydraulic, stomatal and anatomical traits in eight populations of Themeda triandra selected from diverse temperature and precipitation regimes across Australia, grown under common conditions and exposed to drought. Trait-climate-of-origin relationships were quantified using multiple linear mixed models. Leaf xylem embolism resistance (P50) was unrelated to precipitation-of-origin but plants from drier climates exhibited lower maximum stomatal conductance, earlier stomatal closure during drought and higher stomatal safety margins (the difference between water potential at stomatal closure and P50). Plants from warmer climates exhibited greater embolism resistance and higher hydraulic conductance, along with wider vessels and lower vein density, suggesting adaptation to high evaporative demand rather than aridity. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:The CLV3/EMBRYO-SURROUNDING REGION (CLE) peptides control plant development and response to the environment. Key conserved roles include the regulation of shoot apical meristems and the long-distance control of root colonization by nutrient-acquiring microbes, including the widespread symbioses with arbuscular mycorrhizal fungi and nodulation with nitrogen-fixing bacteria in legumes. At least some signalling elements appear to operate across both processes but clear gaps in our understanding remain. In legumes, although CLE peptide signalling has been examined in detail in symbioses, the role of this pathway in shoot apical meristem (SAM) development is poorly understood. SCOPE:In this Research in Context, we review the literature to clarify the conserved and divergent elements of the CLAVATA-CLE peptide signalling pathways that control SAM development, mycorrhizal colonization and nodulation. We used novel pea mutants to determine the role of CLE signalling in regulating SAM development of a model legume, including interactions with temperature. CONCLUSIONS:We found that in pea, both genetic and environmental buffering of the CLE pathway influence SAM development. In pea, the CLAVATA2 (CLV2) CLE receptor-like protein and the unknown gene product encoded by the K301 gene are required to limit SAM size and floral organ production under cool conditions. In contrast, the CLAVATA1 receptor-like kinase promotes SAM proliferation and appears to do so via a CLV2-independent pathway. In contrast, we found no role for the RDN1 enzyme, capable of arabinosylating CLE peptides, in SAM development. Future studies in other legumes are required to examine the role of other CLE peptide signalling elements in SAM control. Studies in non-vascular mycorrhizal hosts could explore if the control of symbioses is also an ancestral role for this signalling pathway.

>To maintain health and productivity,plants must manage a complex set of relationships with both beneficial and harmful microbes. This includes hosting symbiotic microbes to gain access to limited nutrients and defending against invasion by pathogenic microbes. Communication lies at the heart of these interactions via small signalling molecules(Srivastava et al., 2024).

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.

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.

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

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, Sl CLE10 and Sl CLE11, act to suppress AM colonisation of roots. Mutant studies and overexpression via hairy transformation indicate SlCLE11 acts locally in the root to limit AM colonisation. Indeed, SlCLE11 expression is strongly induced in AM colonised roots but SlCLE11 is not required for phosphate suppression of AM colonisation. Sl CLE11 may act through as yet uncharacterised signalling pathways, as SlCLE11 does not suppress AM colonisation by acting through two previously characterised receptors with roles in regulating AM colonisation, Sl FAB (CLAVATA1 orthologue) or Sl CLV2. Sl CLE10 appears to play a more minor or redundant role, as cle10 mutants did not influence AM, although the fact that ectopic overexpression of SlCLE10 did suppress colonisation suggests SlCLE10 may play a role in regulating AM colonisation. Our findings show that CLE peptides regulate AM colonisation in the non-legume species tomato.

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.

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 (tangmic) tomato and how this affects root to shoot biomass, mycorrhizal colonization, abscisic acid accumulation, and responses to drought. tangmic plants 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 with Rhizophagus irregularis revealed fewer arbuscules and other fungal structures in the endodermal cells of roots in tangmic relative to WT. The roots of tangmic hyperaccumulated acyclic cis-carotenes, while only trace levels of xanthophylls and abscisic acid were detected. In response to a water deficit, leaves from the tangmic plants 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 in tangmic roots reduced abscisic acid levels, thereby likely impairing endomycorrhizal colonisation and drought-induced root to shoot signalling.

Chemical dormancy breakers are often used to manipulate floral bud break in sweet cherry production, and their use is increasing due to unpredictable climate effects. The role of plant hormones in regulating the critical transition of floral buds from dormant to opening in deciduous trees is now emerging. By monitoring changes in endogenous hormone levels within floral buds that are undergoing the transition from dormant to the growing state in response to various cues (environmental and/or chemical inducers), we can begin to distinguish the plant hormones that are the drivers of this process. This study sought to identify key hormonal regulators of floral bud break using sweet cherry as a model and modifying timing of bud break through the application of two chemical dormancy breakers, hydrogen cyanamide (HC, Dormex®) and emulsified vegetable oil compound (EVOC, Waiken®), and to determine the effect of these chemicals on fruit growth and quality. Treatments were applied at label rates 35–40 days before estimated bud break. We found that HC-treated tree buds broke earlier, and this was associated with a significant early elevation of the cytokinins dihydrozeatin and dihydrozeatin riboside compared to the control and EVOC-treated tree buds. In contrast, changes in auxin and abscisic acid content did not appear to explain the hastened bud burst induced by hydrogen cyanamide. While HC-treated trees resulted in larger fruit, there was a higher incidence of cracked fruit and the pack-out of A-grade fruit was reduced. The increase in fruit size was attributed to the earlier flowering and hence longer growing period. Harvest assessment of fruit quality showed no treatment effect on most quality parameters, including fruit dry matter content, total soluble solids or malic acid content, but a reduction in fruit compression firmness and stem pull force in EVOC-treated trees was observed. However, all fruit still met the Australian industry fruit quality export market standards. This study offers important insights into bud hormonal activities underpinning the action of these chemical regulators; understanding bud responses is critically important to ensuring consistent and sustainable fruit tree production systems into the future. It also demonstrates that the dormancy-breaking agents HC and EVOC have no detrimental impact on fruit quality at harvest or following storage, however growers need to be aware of the potential for increased fruit cracking when earlier bud break results in a longer growing season which has the potential to increase fruit size. Further studies are required to determine the role of gibberellin in hastening bud break by dormancy breakers.

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.

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.