A conditional mutation in a wheat (Triticum aestivum L.) gene regulating root morphology.

KEY MESSAGE: Characterisation and genetic mapping of a key gene defining root morphology in bread wheat. Root morphology is central to plants for the efficient uptake up of soil water and mineral nutrients. Here we describe a conditional mutant of hexaploid wheat (Triticum aestivum L.) that when grown in soil with high Ca2+ develops a larger rhizosheath accompanied with shorter roots than the wild type. In wheat, rhizosheath size is a reliable surrogate for root hair length and this was verified in the mutant which possessed longer root hairs than the wild type when grown in high Ca2+ soil. We named the mutant Stumpy and showed it to be due to a single semi-dominant mutation. The short root phenotype at high Ca2+ was due to reduced cellular elongation which might also explain the long root hair phenotype. Analysis of root cell walls showed that the polysaccharide composition of Stumpy roots is remodelled when grown at non-permissive (high) Ca2+ concentrations. The mutation mapped to chromosome 7B and sequencing of the 7B chromosomes in both wild type and Stumpy identified a candidate gene underlying the Stumpy mutation. As part of the process to determine whether the candidate gene was causative, we identified wheat lines in a Cadenza TILLING population with large rhizosheaths but accompanied with normal root length. This finding illustrates the potential of manipulating the gene to disconnect root length from root hair length as a means of developing wheat lines with improved efficiency of nutrient and water uptake. The Stumpy mutant will be valuable for understanding the mechanisms that regulate root morphology in wheat.

Photosynthetic Gains in Super-Nodulating Mutants of Medicago truncatula under Elevated Atmospheric CO2 Conditions.

Legumes are generally considered to be more responsive to elevated CO2 (eCO2) conditions due to the benefits provided by symbiotic nitrogen fixation. In response to high carbohydrate demand from nodules, legumes display autoregulation of nodulation (AON) to restrict nodules to the minimum number necessary to sustain nitrogen supply under current photosynthetic levels. AON mutants super-nodulate and typically grow smaller than wild-type plants under ambient CO2. Here, we show that AON super-nodulating mutants have substantially higher biomass under eCO2 conditions, which is sustained through increased photosynthetic investment. We examined photosynthetic and physiological traits across super-nodulating rdn1-1 (Root Determined Nodulation) and sunn4 (Super Numeric Nodules) and non-nodulating nfp1 (Nod Factor Perception) Medicago truncatula mutants. Under eCO2 conditions, super-nodulating plants exhibited increased rates of carboxylation (Vcmax) and electron transport (J) relative to wild-type and non-nodulating counterparts. The substantially higher rate of CO2 assimilation in eCO2-grown sunn4 super-nodulating plants was sustained through increased production of key photosynthetic enzymes, including Rieske FeS. We hypothesize that AON mutants are carbon-limited and can perform better at eCO2 through improved photosynthesis. Nodulating legumes, especially those with higher nitrogen fixation capability, are likely to out-perform non-nodulating plants under future CO2 conditions and will be important tools for understanding carbon and nitrogen partitioning under eCO2 conditions and future crop improvements.

Photolysis of caged cytokinin in single cells of Arabidopsis thaliana.

BACKGROUND: Cytokinins are a class of phytohormone that play a crucial role in the development of plants. They are involved in the regulation of nearly every aspect of plant growth, from germination to senescence. The role of cytokinins in many developmental programs is complex and varies both spatially and temporally. Current techniques used to investigate the functions of cytokinins in plant development lack this spatial and temporal resolution required to observe cell-type specific effects. RESULTS: To this end, we present a method of activating a caged cytokinin in single cells. A caged benzyladenine was synthesized, along with caged adenine as a negative control. In vitro testing confirmed ultraviolet light-mediated uncaging, and subsequent root growth assays demonstrated that uncaging produced a cytokinin phenotype. This uncaging was confined to single cells using multiphoton confocal microscopy. Using an Arabidopsis thaliana cytokinin reporter line expressing TCSn::GFP, the resulting GFP expression was confined to the uncaging region, including in single cells. This study presents a novel cell-targeted method of cytokinin delivery, which has the potential to elucidate a broad range of processes in plant development. CONCLUSIONS: We combined multiphoton confocal microscopy and a caged cytokinin treatment, allowing cell type-specific uncaging of a cytokinin in Arabidopsis roots.

Are legumes different? Origins and consequences of evolving nitrogen fixing symbioses.

Nitrogen fixing symbioses between plants and bacteria are ancient and, while not numerous, are formed in diverse lineages of plants ranging from microalgae to angiosperms. One symbiosis stands out as the most widespread one is that between legumes and rhizobia, leading to the formation of nitrogen-fixing nodules. The legume family is one of the largest and most diverse group of plants and legumes have been used by humans since the beginning of agriculture, both as high nitrogen food, as well as pastures and rotation crops. One open question is whether their ability to form a nitrogen-fixing symbiosis has contributed to legumes' success, and whether legumes have any unique characteristics that have made them more diverse and widespread than other groups of plants. This review examines the evolutionary journey that has led to the diversification of legumes, in particular its nitrogen-fixing symbiosis, and asks four questions to investigate which legume traits might have contributed to their success: 1. In what ways do legumes differ from other plant groups that have evolved nitrogen-fixing symbioses? In order to answer this question, the characteristics of the symbioses, and efficiencies of nitrogen fixation are compared between different groups of nitrogen fixing plants. 2. Could certain unique features of legumes be a reason for their success? This section examines the manifestations and possible benefits of a nitrogen-rich 'lifestyle' in legumes. 3. If nitrogen fixation was a reason for such a success, why have some species lost the symbiosis? Formation of symbioses has trade-offs, and while these are less well known for non-legumes, there are known energetic and ecological reasons for loss of symbiotic potential in legumes. 4. What can we learn from the unique traits of legumes for future crop improvements? While exploiting some of the physiological properties of legumes could be used to improve legume breeding, our increasing molecular understanding of the essential regulators of root nodule symbioses raise hope of creating new nitrogen fixing symbioses in other crop species.

Manipulating exudate composition from root apices shapes the microbiome throughout the root system.

Certain soil microorganisms can improve plant growth, and practices that encourage their proliferation around the roots can boost production and reduce reliance on agrochemicals. The beneficial effects of the microbial inoculants currently used in agriculture are inconsistent or short-lived because their persistence in soil and on roots is often poor. A complementary approach could use root exudates to recruit beneficial microbes directly from the soil and encourage inoculant proliferation. However, it is unclear whether the release of common organic metabolites can alter the root microbiome in a consistent manner and if so, how those changes vary throughout the whole root system. In this study, we altered the expression of transporters from the ALUMINUM-ACTIVATED MALATE TRANSPORTER and the MULTIDRUG AND TOXIC COMPOUND EXTRUSION families in rice (Oryza sativa L.) and wheat (Triticum aestivum L.) and tested how the subsequent release of their substrates (simple organic anions, including malate, citrate, and γ-amino butyric acid) from root apices affected the root microbiomes. We demonstrate that these exudate compounds, separately and in combination, significantly altered microbiome composition throughout the root system. However, the root type (seminal or nodal), position along the roots (apex or base), and soil type had a greater influence on microbiome structure than the exudates. These results reveal that the root microbiomes of important cereal species can be manipulated by altering the composition of root exudates, and support ongoing attempts to improve plant production by manipulating the root microbiome.

Differential responses of the sunn4 and rdn1-1 super-nodulation mutants of Medicago truncatula to elevated atmospheric CO2.

BACKGROUND AND AIMS: Nitrogen fixation in legumes requires tight control of carbon and nitrogen balance. Thus, legumes control nodule numbers via an autoregulation mechanism. 'Autoregulation of nodulation' mutants super-nodulate are thought to be carbon-limited due to the high carbon-sink strength of excessive nodules. This study aimed to examine the effect of increasing carbon supply on the performance of super-nodulation mutants. METHODS: We compared the responses of Medicago truncatula super-nodulation mutants (sunn-4 and rdn1-1) and wild type to five CO2 levels (300-850 µmol mol-1). Nodule formation and nitrogen fixation were assessed in soil-grown plants at 18 and 42 d after sowing. KEY RESULTS: Shoot and root biomass, nodule number and biomass, nitrogenase activity and fixed nitrogen per plant of all genotypes increased with increasing CO2 concentration and reached a maximum at 700 µmol mol-1. While the sunn-4 mutant showed strong growth retardation compared with wild-type plants, elevated CO2 increased shoot biomass and total nitrogen content of the rdn1-1 mutant up to 2-fold. This was accompanied by a 4-fold increase in nitrogen fixation capacity in the rdn1-1 mutant. CONCLUSIONS: These results suggest that the super-nodulation phenotype per se did not limit growth. The additional nitrogen fixation capacity of the rdn1-1 mutant may enhance the benefit of elevated CO2 for plant growth and N2 fixation.

The microbiomes on the roots of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) exhibit significant differences in structure between root types and along root axes.

There is increasing interest in understanding how the microbial communities on roots can be manipulated to improve plant productivity. Root systems are not homogeneous organs but are comprised of different root types of various ages and anatomies that perform different functions. Relatively little is known about how this variation influences the distribution and abundance of microorganisms on roots and in the rhizosphere. Such information is important for understanding how root-microbe interactions might affect root function and prevent diseases. This study tested specific hypotheses related to the spatial variation of bacterial and fungal communities on wheat (Triticum aestivum L.) and rice (Oryza sativa L.) roots grown in contrasting soils. We demonstrate that microbial communities differed significantly between soil type, between host species, between root types, and with position along the root axes. The magnitude of variation between different root types and along individual roots was comparable with the variation detected between different plant species. We discuss the general patterns that emerged in this variation and identify bacterial and fungal taxa that were consistently more abundant on specific regions of the root system. We argue that these patterns should be measured more routinely so that localised root-microbe interactions can be better linked with root system design, plant health and performance.

New methods for confocal imaging of infection threads in crop and model legumes.

BACKGROUND: The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. RESULTS: We have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized. CONCLUSIONS: Our methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.

Interaction of Symbiotic Rhizobia and Parasitic Root-Knot Nematodes in Legume Roots: From Molecular Regulation to Field Application.

Legumes form two types of root organs in response to signals from microbes, namely, nodules and root galls. In the field, these interactions occur concurrently and often interact with each other. The outcomes of these interactions vary and can depend on natural variation in rhizobia and nematode populations in the soil as well as abiotic conditions. While rhizobia are symbionts that contribute fixed nitrogen to their hosts, parasitic root-knot nematodes (RKN) cause galls as feeding structures that consume plant resources without a contribution to the plant. Yet, the two interactions share similarities, including rhizosphere signaling, repression of host defense responses, activation of host cell division, and differentiation, nutrient exchange, and alteration of root architecture. Rhizobia activate changes in defense and development through Nod factor signaling, with additional functions of effector proteins and exopolysaccharides. RKN inject large numbers of protein effectors into plant cells that directly suppress immune signaling and manipulate developmental pathways. This review examines the molecular control of legume interactions with rhizobia and RKN to elucidate shared and distinct mechanisms of these root-microbe interactions. Many of the molecular pathways targeted by both organisms overlap, yet recent discoveries have singled out differences in the spatial control of expression of developmental regulators that may have enabled activation of cortical cell division during nodulation in legumes. The interaction of legumes with symbionts and parasites highlights the importance of a comprehensive view of root-microbe interactions for future crop management and breeding strategies.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterisation of Medicago truncatula CLE34 and CLE35 in nitrate and rhizobia regulation of nodulation.

Legumes form a symbiosis with atmospheric nitrogen (N2 )-fixing soil rhizobia, resulting in new root organs called nodules that enable N2 -fixation. Nodulation is a costly process that is tightly regulated by the host through autoregulation of nodulation (AON) and nitrate-dependent regulation of nodulation. Both pathways require legume-specific CLAVATA/ESR-related (CLE) peptides. Nitrogen-induced nodulation-suppressing CLE peptides have not previously been investigated in Medicago truncatula, for which only rhizobia-induced MtCLE12 and MtCLE13 have been characterised. Here, we report on novel peptides MtCLE34 and MtCLE35 in nodulation control. The nodulation-suppressing CLE peptides of five legume species were classified into three clades based on sequence homology and phylogeny. This approached identified MtCLE34 and MtCLE35 and four new CLE peptide orthologues of Pisum sativum. Whereas MtCLE12 and MtCLE13 are induced by rhizobia, MtCLE34 and MtCLE35 respond to both rhizobia and nitrate. MtCLE34 was identified as a pseudogene lacking a functional CLE-domain. MtCLE35 was found to inhibit nodulation in a SUNN- and RDN1-dependent manner via overexpression analysis. Together, our findings indicate that MtCLE12 and MtCLE13 have a specific role in AON, while MtCLE35 regulates nodule numbers in response to both rhizobia and nitrate. MtCLE34 likely had a similar role to MtCLE35, but its function was lost due to a premature nonsense mutation.

Infection of Medicago truncatula by the Root-Knot Nematode Meloidogyne javanica Does Not Require Early Nodulation Genes.

Because of the developmental similarities between root nodules induced by symbiotic rhizobia and root galls formed by parasitic nematodes, we investigated the involvement of nodulation genes in the infection of Medicago truncatula by the root knot nematode (RKN), Meloidogyne javanica. We found that gall formation, including giant cell formation, pericycle and cortical cell division, as well as egg laying, occurred successfully in the non-nodulating mutants nfp1 (nod factor perception1), nin1 (nodule inception1) and nsp2 (nodulation signaling pathway2) and the cytokinin perception mutant cre1 (cytokinin receptor1). Gall and egg formation were significantly reduced in the ethylene insensitive, hypernodulating mutant skl (sickle), and to a lesser extent, in the low nodulation, abscisic acid insensitive mutant latd/nip (lateral root-organ defective/numerous infections and polyphenolics). Despite its supernodulation phenotype, the sunn4 (super numeric nodules4) mutant, which has lost the ability to autoregulate nodule numbers, did not form excessive numbers of galls. Co-inoculation of roots with nematodes and rhizobia significantly reduced nodule numbers compared to rhizobia-only inoculated roots, but only in the hypernodulation mutant skl. Thus, this effect is likely to be influenced by ethylene signaling, but is not likely explained by resource competition between galls and nodules. Co-inoculation with rhizobia also reduced gall numbers compared to nematode-only infected roots, but only in the wild type. Therefore, the protective effect of rhizobia on nematode infection does not clearly depend on nodule number or on Nod factor signaling. Our study demonstrates that early nodulation genes that are essential for successful nodule development are not necessary for nematode-induced gall formation, that gall formation is not under autoregulation of nodulation control, and that ethylene signaling plays a positive role in successful RKN parasitism in M. truncatula.

The Presence of Plant-Associated Bacteria Alters Responses to N-acyl Homoserine Lactone Quorum Sensing Signals that Modulate Nodulation in Medicago Truncatula.

Bacteria use quorum sensing signaling for cell-to-cell communication, which is also important for their interactions with plant hosts. Quorum sensing via N-acyl-homoserine lactones (AHLs) is important for successful symbioses between legumes and nitrogen-fixing rhizobia. Previous studies have shown that plant hosts can recognize and respond to AHLs. Here, we tested whether the response of the model legume Medicago truncatula to AHLs from its symbiont and other bacteria could be modulated by the abundance and composition of plant-associated microbial communities. Temporary antibiotic treatment of the seeds removed the majority of bacterial taxa associated with M. truncatula roots and significantly altered the effect of AHLs on nodule numbers, but lateral root density, biomass, and root length responses were much less affected. The AHL 3-oxo-C14-HSL (homoserine lactone) specifically increased nodule numbers but only after the treatment of seeds with antibiotics. This increase was associated with increased expression of the early nodulation genes RIP1 and ENOD11 at 24 h after infection. A 454 pyrosequencing analysis of the plant-associated bacteria showed that antibiotic treatment had the biggest effect on bacterial community composition. However, we also found distinct effects of 3-oxo-C14-HSL on the abundance of specific bacterial taxa. Our results revealed a complex interaction between plants and their associated microbiome that could modify plant responses to AHLs.

CEP receptor signalling controls root system architecture in Arabidopsis and Medicago.

Root system architecture (RSA) influences the effectiveness of resources acquisition from soils but the genetic networks that control RSA remain largely unclear. We used rhizoboxes, X-ray computed tomography, grafting, auxin transport measurements and hormone quantification to demonstrate that Arabidopsis and Medicago CEP (C-TERMINALLY ENCODED PEPTIDE)-CEP RECEPTOR signalling controls RSA, the gravitropic set-point angle (GSA) of lateral roots (LRs), auxin levels and auxin transport. We showed that soil-grown Arabidopsis and Medicago CEP receptor mutants have a narrower RSA, which results from a steeper LR GSA. Grafting showed that CEPR1 in the shoot controls GSA. CEP receptor mutants exhibited an increase in rootward auxin transport and elevated shoot auxin levels. Consistently, the application of auxin to wild-type shoots induced a steeper GSA and auxin transport inhibitors counteracted the CEP receptor mutant's steep GSA phenotype. Concordantly, CEP peptides increased GSA and inhibited rootward auxin transport in wild-type but not in CEP receptor mutants. The results indicated that CEP-CEP receptor-dependent signalling outputs in Arabidopsis and Medicago control overall RSA, LR GSA, shoot auxin levels and rootward auxin transport. We propose that manipulating CEP signalling strength or CEP receptor downstream targets may provide means to alter RSA.

The Medicago truncatula PIN2 auxin transporter mediates basipetal auxin transport but is not necessary for nodulation.

The development of root nodules leads to an increased auxin response in early nodule primordia, which is mediated by changes in acropetal auxin transport in some legumes. Here, we investigated the role of root basipetal auxin transport during nodulation. Rhizobia inoculation significantly increased basipetal auxin transport in both Medicago truncatula and Lotus japonicus. In M. truncatula, this increase was dependent on functional Nod factor signalling through NFP, NIN, and NSP2, as well as ethylene signalling through SKL. To test whether increased basipetal auxin transport is required for nodulation, we examined a loss-of-function mutant of the M. truncatula PIN2 gene. The Mtpin2 mutant exhibited a reduction in basipetal auxin transport and an agravitropic phenotype. Inoculation of Mtpin2 roots with rhizobia still led to a moderate increase in basipetal auxin transport, but the mutant nodulated normally. No clear differences in auxin response were observed during nodule development. Interestingly, inoculation of wild-type roots increased lateral root numbers, whereas inoculation of Mtpin2 mutants resulted in reduced lateral root numbers compared with uninoculated roots. We conclude that the MtPIN2 auxin transporter is involved in basipetal auxin transport, that its function is not essential for nodulation, but that it plays an important role in the control of lateral root development.

Accumulation of and Response to Auxins in Roots and Nodules of the Actinorhizal Plant Datisca glomerata Compared to the Model Legume Medicago truncatula.

Actinorhizal nodules are structurally different from legume nodules and show a greater similarity to lateral roots. Because of the important role of auxins in lateral root and nodule formation, auxin profiles were examined in roots and nodules of the actinorhizal species Datisca glomerata and the model legume Medicago truncatula. The auxin response in roots and nodules of both species was analyzed in transgenic root systems expressing a beta-glucuronidase gene under control of the synthetic auxin-responsive promoter DR5. The effects of two different auxin on root development were compared for both species. The auxin present in nodules at the highest levels was phenylacetic acid (PAA). No differences were found between the concentrations of active auxins of roots vs. nodules, while levels of the auxin conjugate indole-3-acetic acid-alanine were increased in nodules compared to roots of both species. Because auxins typically act in concert with cytokinins, cytokinins were also quantified. Concentrations of cis-zeatin and some glycosylated cytokinins were dramatically increased in nodules compared to roots of D. glomerata, but not of M. truncatula. The ratio of active auxins to cytokinins remained similar in nodules compared to roots in both species. The auxin response, as shown by the activation of the DR5 promoter, seemed significantly reduced in nodules compared to roots of both species, suggesting the accumulation of auxins in cell types that do not express the signal transduction pathway leading to DR5 activation. Effects on root development were analyzed for the synthetic auxin naphthaleneacetic acid (NAA) and PAA, the dominant auxin in nodules. Both auxins had similar effects, except that the sensitivity of roots to PAA was lower than to NAA. However, while the effects of both auxins on primary root growth were similar for both species, effects on root branching were different: both auxins had the classical positive effect on root branching in M. truncatula, but a negative effect in D. glomerata. Such a negative effect of exogenous auxin on root branching has previously been found for a cucurbit that forms lateral root primordia in the meristem of the parental root; however, root branching in D. glomerata does not follow that pattern.

Genes controlling legume nodule numbers affect phenotypic plasticity responses to nitrogen in the presence and absence of rhizobia.

We investigated the role of three autoregulation of nodulation (AON) genes in regulating of root and shoot phenotypes when responding to changing nitrogen availability in the model legume, Medicago truncatula. These genes, RDN1-1 (ROOT DETERMINED NODULATION1-1), SUNN (SUPER NUMERIC NODULES), and LSS (LIKE SUNN SUPERNODULAOR), act in a systemic signalling pathway that limits nodule numbers. This pathway is also influenced by nitrogen availability, but it is not well known if AON genes control root and shoot phenotypes other than nodule numbers in response to nitrogen. We conducted a controlled glasshouse experiment to compare root and shoot phenotypes of mutants and wild type plants treated with four nitrate concentrations. All AON mutants showed altered rhizobia-independent phenotypes, including biomass allocation, lateral root length, lateral root density, and root length ratio. In response to nitrogen, uninoculated AON mutants were less plastic than the wild type in controlling root mass ratio, root length ratio, and lateral root length. This suggests that AON genes control nodulation-independent root architecture phenotypes in response to nitrogen. The phenotypic differences between wild type and AON mutants were exacerbated by the presence of nodules, pointing to resource competition as an additional mechanism affecting root and shoot responses to nitrogen.

Functions of Flavonoids in Plant⁻Nematode Interactions.

Most land plants can become infected by plant parasitic nematodes in the field. Plant parasitic nematodes can be free-living or endoparasitic, and they usually infect plant roots. Most damaging are endoparasites, which form feeding sites inside plant roots that damage the root system and redirect nutrients towards the parasite. This process involves developmental changes to the root in parallel with the induction of defense responses. Plant flavonoids are secondary metabolites that have roles in both root development and plant defense responses against a range of microorganisms. Here, we review our current knowledge of the roles of flavonoids in the interactions between plants and plant parasitic nematodes. Flavonoids are induced during nematode infection in plant roots, and more highly so in resistant compared with susceptible plant cultivars, but many of their functions remain unclear. Flavonoids have been shown to alter feeding site development to some extent, but so far have not been found to be essential for root⁻parasite interactions. However, they likely contribute to chemotactic attraction or repulsion of nematodes towards or away from roots and might help in the general plant defense against nematodes. Certain flavonoids have also been associated with functions in nematode reproduction, although the mechanism remains unknown. Much remains to be examined in this area, especially under field conditions.

Flavonoid Functions in Plants and Their Interactions with Other Organisms.

Flavonoids are structurally diverse secondary metabolites in plants, with a multitude of functions. These span from functions in regulating plant development, pigmentation, and UV protection, to an array of roles in defence and signalling between plants and microorganisms. Because of their prevalence in the human diet, many flavonoids constitute important components of medicinal plants and are used in the control of inflammation and cancer prevention. Advances in the elucidation of flavonoid biosynthesis and its regulation have led to an increasing number of studies aimed at engineering the flavonoid pathway for enhancing nutritional value and plant defences against pathogens and herbivores, as well as modifying the feeding value of pastures. Many future opportunities await for the exploitation of this colourful pathway in crops, pastures, and medicinal plants.