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As wheat (Triticum aestivum) is an important staple food across the world, preservation of stable yields and increased productivity are major objectives in breeding programs. Drought is a global concern because its adverse impact is expected to be amplified in the future due to the current climate change. Here, we analyzed the effects of edaphic, environmental, and host factors on the wheat root microbiomes collected in soils from six regions in Belgium. Amplicon sequencing analysis of unplanted soil and wheat root endosphere samples indicated that the microbial community variations can be significantly explained by soil pH, microbial biomass, wheat genotype, and soil sodium and iron levels. Under drought stress, the biodiversity in the soil decreased significantly, but increased in the root endosphere community, where specific soil parameters seemingly determine the enrichment of bacterial groups. Indeed, we identified a cluster of drought-enriched bacteria that significantly correlated with soil compositions. Interestingly, integration of a functional analysis further revealed a strong correlation between the same cluster of bacteria and β-glucosidase and osmoprotectant proteins, two functions known to be involved in coping with drought stress. By means of this in silico analysis, we identified amplicon sequence variants (ASVs) that could potentially protect the plant from drought stress and validated them in planta. Yet, ASVs based on 16S rRNA sequencing data did not completely distinguish individual isolates because of their intrinsic short sequences. Our findings support the efforts to maintain stable crop yields under drought conditions through implementation of root microbiome analyses. ispartof: COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL vol:19 pages:4235-4247 ispartof: location:Netherlands status: published

Background When maize (Zea mays L.) is grown in the Northern hemisphere, its development is heavily arrested by chilling temperatures, especially at the juvenile phase. As some endophytes are beneficial for plants under stress conditions, we analyzed the impact of chilling temperatures on the root microbiome and examined whether microbiome-based analysis might help to identify bacterial strains that could promote growth under these temperatures. Results We investigated how the maize root microbiome composition changed by means of 16S rRNA gene amplicon sequencing when maize was grown at chilling temperatures in comparison to ambient temperatures by repeatedly cultivating maize in field soil. We identified 12 abundant and enriched bacterial families that colonize maize roots, consisting of bacteria recruited from the soil, whereas seed-derived endophytes were lowly represented. Chilling temperatures modified the root microbiome composition only slightly, but significantly. An enrichment of several chilling-responsive families was detected, of which the Comamonadaceae and the Pseudomonadaceae were the most abundant in the root endosphere of maize grown under chilling conditions, whereas only three were strongly depleted, among which the Streptomycetaceae. Additionally, a collection of bacterial strains isolated from maize roots was established and a selection was screened for growth-promoting effects on juvenile maize grown under chilling temperatures. Two promising strains that promoted maize growth under chilling conditions were identified that belonged to the root endophytic bacterial families, from which the relative abundance remained unchanged by variations in the growth temperature. Conclusions Our analyses indicate that chilling temperatures affect the bacterial community composition within the maize root endosphere. We further identified two bacterial strains that boost maize growth under chilling conditions. Their identity revealed that analyzing the chilling-responsive families did not help for their identification. As both strains belong to root endosphere enriched families, visualizing and comparing the bacterial diversity in these communities might still help to identify new PGPR strains. Additionally, a strain does not necessarely need to belong to a high abundant family in the root endosphere to provoke a growth-promoting effect in chilling conditions.

Soil microbial communities hold great potential for sustainable and ecologically compatible agriculture. Although numerous plant-beneficial bacterial strains from a wide range of taxonomic groups have been reported, very little evidence is available on the plant-beneficial role of bacteria from the genus Caulobacter. Here, the mode of action of a Caulobacter strain, designated RHG1, which had originally been identified through a microbial screen for plant growth-promoting (PGP) bacteria in maize (Zea mays), is investigated in Arabidopsis thaliana. RHG1 colonized both roots and shoots of Arabidopsis, promoted lateral root formation in the root, and increased leaf number and leaf size in the shoot. The genome of RHG1 was sequenced and was utilized to look for PGP factors. Our data revealed that the bacterial production of nitric oxide, auxins, cytokinins, or 1-aminocyclopropane-1-carboxylate deaminase as PGP factors could be excluded. However, the analysis of brassinosteroid mutants suggests that an unknown PGP mechanism is involved that impinges directly or indirectly on the pathway of this growth hormone.

The bacterium Paenibacillus polymyxa is found naturally in diverse niches. Microbiome analyses have revealed enrichment in the genus Paenibacillus in soils under different adverse conditions, which is often accompanied by improved growth conditions for residing plants. Furthermore, Paenibacillus is a member of the core microbiome of several agriculturally important crops, making its close association with plants an interesting research topic. This review covers the versatile interaction possibilities of P. polymyxa with plants and its applicability in industry and agriculture. Thanks to its array of produced compounds and traits, P. polymyxa is likely an efficient plant growth-promoting bacterium, with the potential of biofertilization, biocontrol and protection against abiotic stresses. By contrast, cases of phytotoxicity of P. polymyxa have been described as well, in which growth conditions seem to play a key role. Because of its adjustable character, we propose this bacterial species as an outstanding model for future studies on host-microbe communications and on the manner how the environment can influence these interactions.

International audience; Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe. In order to germinate, it must perceive various structurally divergent host-exuded signals, including isothiocyanates (ITCs) and strigolactones (SLs). However, the receptors involved are still uncharacterized. Here, we identify five putative SL receptors in P. ramosa and show that PrKAI2d3 is involved in the stimulation of seed germination. We demonstrate the high plasticity of PrKAI2d3, which allows it to interact with different chemicals, including ITCs. The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants. We provide evidence that PrKAI2d3 enzymatic activity confers hypersensitivity to SLs. Additionally, we demonstrate that methylbutenolide-OH binds PrKAI2d3 and stimulates P. ramosa germination with bioactivity comparable to that of ITCs. This study demonstrates that P. ramosa has extended its signal perception system during evolution, a fact that should be considered for the development of specific and efficient biocontrol methods.

Growth and development of plant roots are highly dynamic and adaptable to environmental conditions. They are under the control of several plant hormone signaling pathways, and therefore root developmental responses can be used as bioassays to study the action of plant hormones and other small molecules. In this chapter, we present different procedures to measure root traits of the model plant Arabidopsis thaliana. We explain methods for phenotypic analysis of lateral root development, primary root length, root skewing and straightness, and root hair density and length. We describe optimal growth conditions for Arabidopsis seedlings for reproducible root and root hair developmental outputs; and how to acquire images and measure the different traits using image analysis with relatively low-tech equipment. We provide guidelines for a semiautomatic image analysis of primary root length, root skewing, and root straightness in Fiji and a script to automate the calculation of root angle deviation from the vertical and root straightness. By including mutants defective in strigolactone (SL) or KAI2 ligand (KL) synthesis and/or signaling, these methods can be used as bioassays for different SLs or SL-like molecules. In addition, the techniques described here can be used for studying seedling root system architecture, root skewing, and root hair development in any context.

The F-box domain is a conserved structural protein motif that most frequently interacts with the SKP1 protein, the core of the SCFs (SKP1-CULLIN-F-box protein ligase) E3 ubiquitin protein ligases. As part of the SCF complexes, the various F-box proteins recruit substrates for degradation through ubiquitination. In this study, we functionally characterized an F-box gene (MtF-box) identified earlier in a population of Tnt1 retrotransposon-tagged mutants of Medicago truncatula and its Arabidopsis thaliana homolog (AtF-box) using gain- and loss-of-function plants. We highlighted the importance of MtF-box in leaf development of M. truncatula. Protein–protein interaction analyses revealed the 2-isopropylmalate synthase (IPMS) protein as a common interactor partner of MtF-box and AtF-box, being a key enzyme in the biosynthesis pathway of the branched-chain amino acid leucine. For further detailed analysis, we focused on AtF-box and its role during the cell division cycle. Based on this work, we suggest a mechanism for the role of the studied F-box gene in regulation of leucine homeostasis, which is important for growth.

Phelipanche ramosa is an obligate root-parasitic weed threatening major crops in central Europe. For its germination, it has to perceive various structurally diverging host-exuded signals, including isothiocyanates (ITCs) and strigolactones (SLs). However, the receptors involved are still uncharacterized. Here, we identified five putative SL receptors in P. ramosa, of which PrKAI2d3 is involved in seed germination stimulation. We established the high plasticity of PrKAI2d3, allowing interaction with different chemicals, including ITCs. The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants. We provide evidence that the PrKAI2d3 enzymatic activity confers hypersensitivity to SLs. Additionally, we demonstrated that methylbutenolide-OH binds PrKAI2d3 and stimulates P. ramosa germination with a bioactivity comparable to that of ITCs. This study highlights that P. ramosa has extended its signal perception system during evolution, a fact to be considered in the development of specific and efficient biocontrol methods.

Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-like-kinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.

Strigolactones are well-known endogenous plant hormones that play a major role in planta by influencing different physiological processes. Moreover, ex planta, strigolactones are important signaling molecules in root exudates and function as host detection cues to launch mutualistic interactions with arbuscular mycorrhizal fungi in the rhizosphere. However, parasitic plants belonging to the Orobanchaceae family hijacked this communication system to stimulate their seed germination when in close proximity to the roots of a suitable host. As a result, the secretion of strigolactones by the plant can have both favorable and detrimental outcomes. Here, we discuss these dual positive and negative effects of strigolactones and we provide a detailed overview on the role of these molecules in the complex dialogs between plants and different organisms in the rhizosphere.

The phytohormone auxin is a critical signal molecule, regulating fundamental processes in plant growth and development, such as shaping the root and shoot architecture, organ patterning, and nodulation. Auxin regulates plant gene expression mainly through auxin response factors (ARFs), which bind to auxin response elements in the promoter, upstream of auxin-activated genes. Here we examine and assess the function and expression pattern of a gene described as an auxin response factor, containing a DNA-binding pseudobarrel and B3 DNA-binding domains, from Medicago truncatula (MtARF-B3). For the model legume species M. truncatula, stable transgenic plants with MtARF-B3 overexpression, downregulation, and transcriptional reporters were constructed. Phenotypic and morphological evaluation of the obtained transgenic plants confirmed the important role of MtARF-B3 in general plant growth and development, modeling of root architecture, and development of seeds. Detailed histochemical and transcriptional analysis revealed expression of the gene in various stages of somatic embryogenesis, during formation of plant organs and tissues, and symbiotic nodulation. The fact that MtARF-B3 was strongly expressed in stamens and pollen grains in M. truncatula suggests that this gene could play a role in the fertility of this model legume.

In the last decade strigolactones have been recognized as a novel type of plant hormones. They are involved in the control of key developmental processes such as lateral shoot outgrowth and leaf and root development, among others. In addition, strigolactones modulate plant responses to abiotic stresses like phosphate starvation and drought. Here we summarize the current knowledge of the widely conserved functions of strigolactones in the control of plant development and stress responses as well as some of their reported species-specific roles. In addition, we will review their known genetic and functional interactions with other phytohormones. The newly discovered activities of strigolactones as plant hormones raise the possibility of using these compounds and their signalling pathways as tools to optimise species of agronomical importance.

We provide new insights into the autoregulation of nodulation that allow us to better understand how the legume Medicago truncatula restricts further nodulation once enough nodules have been formed., The number of legume root nodules resulting from a symbiosis with rhizobia is tightly controlled by the plant. Certain members of the CLAVATA3/Embryo Surrounding Region (CLE) peptide family, specifically MtCLE12 and MtCLE13 in Medicago truncatula, act in the systemic autoregulation of nodulation (AON) pathway that negatively regulates the number of nodules. Little is known about the molecular pathways that operate downstream of the AON-related CLE peptides. Here, by means of a transcriptome analysis, we show that roots ectopically expressing MtCLE13 deregulate only a limited number of genes, including three down-regulated genes encoding lysin motif receptor-like kinases (LysM-RLKs), among which are the nodulation factor (NF) receptor NF Perception gene (NFP) and two up-regulated genes, MtTML1 and MtTML2, encoding Too Much Love (TML)-related Kelch-repeat containing F-box proteins. The observed deregulation was specific for the ectopic expression of nodulation-related MtCLE genes and depended on the Super Numeric Nodules (SUNN) AON RLK. Moreover, overexpression and silencing of these two MtTML genes demonstrated that they play a role in the negative regulation of nodule numbers. Hence, the identified MtTML genes are the functional counterpart of the Lotus japonicus TML gene shown to be central in the AON pathway. Additionally, we propose that the down-regulation of a subset of LysM-RLK-encoding genes, among which is NFP, might contribute to the restriction of further nodulation once the first nodules have been formed.

Plants associate with an infinite number of microorganisms that interact with their hosts in a mutualistic or parasitic manner. Evidence is accumulating that strigolactones (SLs) play a role in shaping these associations. The best described function of SLs in plant–microbe interactions is in the rhizosphere, where, after being exuded from the root, they activate hyphal branching and enhanced growth and energy metabolism of symbiotic arbuscular mycorrhiza fungi (AMF). Furthermore, an impact of SLs on the quantitative development of root nodule symbiosis with symbiotic nitrogen-fixing bacteria and on the success of fungal and bacterial leaf pathogens is beginning to be revealed. Thus far, the role of SLs has predominantly been studied in binary plant–microbe interactions. It can be predicted that their impact on the bacterial, fungal, and oomycetal communities (microbiomes), which thrive on roots, in the rhizosphere, and on aerial tissues, will be addressed in the near future.

Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most powerful techniques to isolate protein complexes and elucidate protein interaction networks. Here, we describe the development of a TAP-MS strategy for the model legume Medicago truncatula, which is widely studied for its ability to produce valuable natural products and to engage in endosymbiotic interactions. As biological material, transgenic hairy roots, generated through Agrobacterium rhizogenes-mediated transformation of M. truncatula seedlings, were used. As proof of concept, proteins involved in the cell cycle, transcript processing and jasmonate signalling were chosen as bait proteins, resulting in a list of putative interactors, many of which confirm the interologue concept of protein interactions, and which can contribute to biological information about the functioning of these bait proteins in planta. Subsequently, binary protein-protein interactions among baits and preys, and among preys were confirmed by a systematic yeast two-hybrid screen. Together, by establishing a M. truncatula TAP-MS platform, we extended the molecular toolbox of this model species.

Highlight To antagonize the developmental process initiated by Rhodococcus fascians and in response to the bacterial cytokinins, Arabidopsis activates its strigolactone response, partially suppressing shoot branching in the rosette., Leafy gall syndrome is the consequence of modified plant development in response to a mixture of cytokinins secreted by the biotrophic actinomycete Rhodococcus fascians. The similarity of the induced symptoms with the phenotype of plant mutants defective in strigolactone biosynthesis and signalling prompted an evaluation of the involvement of strigolactones in this pathology. All tested strigolactone-related Arabidopsis thaliana mutants were hypersensitive to R. fascians. Moreover, treatment with the synthetic strigolactone mixture GR24 and with the carotenoid cleavage dioxygenase inhibitor D2 illustrated that strigolactones acted as antagonistic compounds that restricted the morphogenic activity of R. fascians. Transcript profiling of the MORE AXILLARY GROWTH1 (MAX1), MAX2, MAX3, MAX4, and BRANCHED1 (BRC1) genes in the wild-type Columbia-0 accession and in different mutant backgrounds revealed that upregulation of strigolactone biosynthesis genes was triggered indirectly by the bacterial cytokinins via host-derived auxin and led to the activation of BRC1 expression, inhibiting the outgrowth of the newly developing shoots, a typical hallmark of leafy gall syndrome. Taken together, these data support the emerging insight that balances are critical for optimal leafy gall development: the long-lasting biotrophic interaction is possible only because the host activates a set of countermeasures—including the strigolactone response—in reaction to bacterial cytokinins to constrain the activity of R. fascians.

Obligate root-parasitic plants belonging to the Orobanchaceae family are deadly pests for major crops all over the world. Because these heterotrophic plants severely damage their hosts even before emerging from the soil, there is an unequivocal need to design early and efficient methods for their control. The germination process of these species has probably undergone numerous selective pressure events in the course of evolution, in that the perception of host-derived molecules is a necessary condition for seeds to germinate. Although most of these molecules belong to the strigolactones, structurally different molecules have been identified. Since strigolactones are also classified as novel plant hormones that regulate several physiological processes other than germination, the use of autotrophic model plant species has allowed the identification of many actors involved in the strigolactone biosynthesis, perception, and signal transduction pathways. Nevertheless, many questions remain to be answered regarding the germination process of parasitic plants. For instance, how did parasitic plants evolve to germinate in response to a wide variety of molecules, while autotrophic plants do not? What particular features are associated with their lack of spontaneous germination? In this review, we attempt to illustrate to what extent conclusions from research into strigolactones could be applied to better understand the biology of parasitic plants.

International audience; In the rhizosphere, strigolactones not only act as crucial signalling molecules in the communication of plants with parasitic weeds and arbuscular mycorrhiza, but they also play a key role in regulating different aspects of the root system. Here we investigated how strigolactones influence the root architecture of Medicago truncatula. We provide evidence that addition of the synthetic strigolactone analogue GR24 has an inhibitory effect on the lateral root density. Moreover, treatment with GR24 of Sinorhizobium meliloti-inoculated M. truncatula plants affects the nodule number both positively and negatively, depending on the concentration. Plants treated with 0.1 microM GR24 had a slightly increased number of nodules, whereas concentrations of 2 and 5 microM strongly reduced it. This effect was independent of the autoregulation of nodulation mechanism that is controlled by SUPER NUMERIC NODULE. Furthermore, we demonstrate that GR24 controls the nodule number through crosstalk with SICKLE-dependent ethylene signalling. Additionally, because the expression of the nodulation marker EARLY NODULATION11 was strongly reduced in GR24-treated plants, we concluded that strigolactones influence nodulation at a very early stage of the symbiotic interaction.

Plant architectural characteristics are under strong genetic regulation. Economically important traits for forage crops such as biomass yield, ground cover and persistence can be improved by selecting for particular aerial architectural characteristics. Here, we present an easily applicable method for the spatiotemporal description of branching patterns in red clover (Trifolium pratense) and perennial ryegrass (Lolium perenne), two of the most important forage crops in Europe. A detailed analysis of genotypes with contrasting branching phenotypes demonstrates that in these species different factors are the main determinants of shoot branching characteristics. In red clover, bud outgrowth and to a lesser extent bud formation explain inter-genotype branching differences. In perennial ryegrass, differences in the capacity to form new buds determined largely the differences between forage and turf types. However, when a set of four forage types was compared in a separate experiment, variation in quantity and pace of bud formation and bud outgrowth explained the differences in aerial architecture. In both crops, branching patterns are likely determined by several processes, and highly branched phenotypes can result from the formation of more buds, an increased probability of bud outgrowth, or a combination of these processes. Furthermore, the presence of more buds is partly caused by more bud outgrowth.

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.

Here we discuss the advantages of the majority of this versatile and diverse group of microorganisms for plant health and growth as demonstrated by their contribution to disease-suppressive soils, their antifungal and antibacterial activities, their ability to produce volatile compounds and their capacity to enhance plant biomass. Although much is still to be discovered about the colonization strategies and molecular interactions between plant roots and these microorganisms, they are destined to become important players in the field of plant growth-promoting rhizobacteria for agriculture.

Highlight Strigolactones monitor lateral root development in a spatiotemporal manner by an interplay with cytokinin., Strigolactones are important rhizosphere signals that act as phytohormones and have multiple functions, including modulation of lateral root (LR) development. Here, we show that treatment with the strigolactone analog GR24 did not affect LR initiation, but negatively influenced LR priming and emergence, the latter especially near the root–shoot junction. The cytokinin module ARABIDOPSIS HISTIDINE KINASE3 (AHK3)/ARABIDOPSIS RESPONSE REGULATOR1 (ARR1)/ARR12 was found to interact with the GR24-dependent reduction in LR development, because mutants in this pathway rendered LR development insensitive to GR24. Additionally, pharmacological analyses, mutant analyses, and gene expression analyses indicated that the affected polar auxin transport stream in mutants of the AHK3/ARR1/ARR12 module could be the underlying cause. Altogether, the data reveal that the GR24 effect on LR development depends on the hormonal landscape that results from the intimate connection with auxins and cytokinins, two main players in LR development.

Jasmonates (JAs) act as conserved elicitors of plant secondary metabolism. JA perception triggers extensive transcriptional reprogramming leading to the concerted activation of entire metabolic pathways. This observation inspired numerous quests for 'master' regulators capable of enhancing the production of specific sets of valuable plant metabolites. Many transcription factors (TFs), often JA-activated themselves, with a role in the JA-modulated regulation of metabolism were discovered. At the same time, it became clear that metabolic reprogramming is subject to complex control mechanisms integrated in robust cellular networks. In this review, we discuss current knowledge of the effect of JA-modulated TFs in the elicitation of secondary metabolism in the model plant Arabidopsis (Arabidopsis thaliana) and a range of medicinal plant species with structurally divergent secondary metabolites. We draw parallels with the regulation of secondary metabolism in fungi and consider the remaining challenges to map and exploit the transcriptional machineries that drive JA-mediated elicitation of plant secondary metabolism.

CLE peptides are involved in the balance between cell division and differentiation throughout plant development, including nodulation. Previously, two CLE genes of Medicago truncatula, MtCLE12 and MtCLE13, had been identified whose expression correlated with nodule primordium formation and meristem establishment. Gain-of-function analysis indicated that both MtCLE12 and MtCLE13 interact with the SUPER NUMERIC NODULES (SUNN)-dependent auto-regulation of nodulation to control nodule numbers. Here we demonstrate that cytokinin, which is essential for nodule organ formation, regulates MtCLE13 expression. In addition, simultaneous knockdown of MtCLE12 and MtCLE13 resulted in an increase in nodule number, implying that both genes play a role in controlling nodule number. Additionally, a weak link may exist with the ethylene-dependent mechanism that locally controls nodule number.

In legumes, the symbiotic nodules are formed as a result of dedifferentiation and reactivation of cortical root cells. A shoot-acting receptor complex, similar to the Arabidopsis (Arabidopsis thaliana) CLAVATA1 (CLV1)/CLV2 receptor, regulating development of the shoot apical meristem, is involved in autoregulation of nodulation (AON), a mechanism that systemically controls nodule number. The targets of CLV1/CLV2 in the shoot apical meristem, the WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family transcription factors, have been proposed to be important regulators of apical meristem maintenance and to be expressed in apical meristem “organizers.” Here, we focus on the role of the WOX5 transcription factor upon nodulation in Medicago truncatula and pea (Pisum sativum) that form indeterminate nodules. Analysis of temporal WOX5 expression during nodulation with quantitative reverse transcription-polymerase chain reaction and promoter-reporter fusion revealed that the WOX5 gene was expressed during nodule organogenesis, suggesting that WOX genes are common regulators of cell proliferation in different systems. Furthermore, in nodules of supernodulating mutants, defective in AON, WOX5 expression was higher than that in wild-type nodules. Hence, a conserved WUS/WOX-CLV regulatory system might control cell proliferation and differentiation not only in the root and shoot apical meristems but also in nodule meristems. In addition, the link between nodule-derived CLE peptides activating AON in different legumes and components of the AON system was investigated. We demonstrate that the identified AON component, NODULATION3 of pea, might act downstream from or beside the CLE peptides during AON.

Bacterial nodulation factors (NFs) are essential signaling molecules for the initiation of a nitrogen-fixing symbiosis in legumes. NFs are perceived by the plant and trigger both local and distant responses, such as curling of root hairs and cortical cell divisions. In addition to their requirement at the start, NFs are produced by bacteria that reside within infection threads. To analyze the role of NFs at later infection stages, several phases of nodulation were studied by detailed light and electron microscopy after coinoculation of adventitious root primordia of Sesbania rostrata with a mixture of Azorhizobium caulinodans mutants ORS571-V44 and ORS571-X15. These mutants are deficient in NF production or surface polysaccharide synthesis, respectively, but they can complement each other, resulting in functional nodules occupied by ORS571-V44. The lack of NFs within the infection threads was confirmed by the absence of expression of an early NF-induced marker, leghemoglobin 6 of S. rostrata. NF production within the infection threads is shown to be necessary for proper infection thread growth and for synchronization of nodule formation with bacterial invasion. However, local production of NFs by bacteria that are taken up by the plant cells at the stage of bacteroid formation is not required for correct symbiosome development.

Most molecular processes during plant development occur with a particular spatio-temporal specificity. Thus far, it has remained technically challenging to capture dynamic protein-protein interactions within a growing organ, where the interplay between cell division and cell expansion is instrumental. Here, we combined high-resolution sampling of the growing maize (Zea mays) leaf with tandem affinity purification followed by mass spectrometry. Our results indicate that the growth-regulating SWI/SNF chromatin remodeling complex associated with ANGUSTIFOLIA3 (AN3) was conserved within growing organs and between dicots and monocots. Moreover, we were able to demonstrate the dynamics of the AN3-interacting proteins within the growing leaf, since copurified GROWTH-REGULATING FACTORs (GRFs) varied throughout the growing leaf. Indeed, GRF1, GRF6, GRF7, GRF12, GRF15, and GRF17 were significantly enriched in the division zone of the growing leaf, while GRF4 and GRF10 levels were comparable between division zone and expansion zone in the growing leaf. These dynamics were also reflected at the mRNA and protein levels, indicating tight developmental regulation of the AN3-associated chromatin remodeling complex. In addition, the phenotypes of maize plants overexpressing miRNA396a-resistant GRF1 support a model proposing that distinct associations of the chromatin remodeling complex with specific GRFs tightly regulate the transition between cell division and cell expansion. Together, our data demonstrate that advancing from static to dynamic protein-protein interaction analysis in a growing organ adds insights in how developmental switches are regulated.

Rhizobia reside as symbiosomes in the infected cells of legume nodules to fix atmospheric nitrogen. The symbiotic relation is strictly controlled, lasts for some time, but eventually leads to nodule senescence. We present a comprehensive transcriptomics study to understand the onset of nodule senescence in the legume Medicago truncatula. Distinct developmental stages with characteristic gene expression were delineated during which the two symbiotic partners were degraded consecutively, marking the switch in nodule tissue status from carbon sink to general nutrient source. Cluster analysis discriminated an early expression group that harbored regulatory genes that might be primary tools to interfere with pod filling-related or stress-induced nodule senescence, ultimately causing prolonged nitrogen fixation. Interestingly, the transcriptomes of nodule and leaf senescence had a high degree of overlap, arguing for the recruitment of similar pathways.

Rhizobia colonize their legume hosts by different modes of entry while initiating symbiotic nitrogen fixation. Most legumes are invaded via growing root hairs by the root hair-curl mechanism, which involves epidermal cell responses. However, invasion of a number of tropical legumes happens through fissures at lateral root bases by cortical, intercellular crack entry. In the semiaquatic Sesbania rostrata , the bacteria entered via root hair curls under nonflooding conditions. Upon flooding, root hair growth was prevented, invasion on accessible root hairs was inhibited, and intercellular invasion was recruited. The plant hormone ethylene was involved in these processes. The occurrence of both invasion pathways on the same host plant enabled a comparison to be made of the structural requirements for the perception of nodulation factors, which were more stringent for the epidermal root hair invasion than for the cortical intercellular invasion at lateral root bases.

De novo shoot organogenesis (i.e., the regeneration of shoots on nonmeristematic tissue) is widely applied in plant biotechnology. However, the capacity to regenerate shoots varies highly among plant species and cultivars, and the factors underlying it are still poorly understood. Here, we evaluated the shoot regeneration capacity of 88 Arabidopsis thaliana accessions and found that the process is blocked at different stages in different accessions. We show that the variation in regeneration capacity between the Arabidopsis accessions Nok-3 and Ga-0 is determined by five quantitative trait loci (QTL): REG-1 to REG-5. Fine mapping by local association analysis identified RECEPTOR-LIKE PROTEIN KINASE1 (RPK1), an abscisic acid-related receptor, as the most likely gene underlying REG-1, which was confirmed by quantitative failure of an RPK1 mutation to complement the high and low REG-1 QTL alleles. The importance of RPK1 in regeneration was further corroborated by mutant and expression analysis. Altogether, our results show that association mapping combined with linkage mapping is a powerful method to discover important genes implicated in a biological process as complex as shoot regeneration.

International audience; : Strigolactones (SLs) are known not only as plant hormones, but also as rhizosphere signals for establishing symbiotic and parasitic interactions. The design of new specific SL analogs is a challenging goal in understanding the basic plant biology and is also useful to control plant architectures without favoring the development of parasitic plants. Two different molecules (23 (3'-methyl-GR24), 31 (thia-3'-methyl-debranone-like molecule)) already described, and a new one (AR36), for which the synthesis is presented, are biologically compared with the well-known GR24 and the recently identified CISA-1. These different structures emphasize the wide range of parts attached to the D-ring for the bioactivity as a plant hormone. These new compounds possess a common dimethylbutenolide motif but their structure varies in the ABC part of the molecules: 23 has the same ABC part as GR24, while 31 and AR36 carry, respectively, an aromatic ring and an acyclic carbon chain. Detailed information is given for the bioactivity of such derivatives in strigolactone synthesis or in perception mutant plants (pea rms1 and rms4, Arabidopsis max2 and, max4) for different hormonal functions along with their action in the rhizosphere on arbuscular mycorrhizal hyphal growth and parasitic weed germination.

At the base of adventitious root primordia, located on the stem of the tropical legume Sesbania rostrata, nitrogen-fixing nodules are formed upon inoculation with the microsymbiont Azorhizobium caulinodans. This pattern of nodule development presents features of indeterminate and determinate nodules in early and later stages, respectively. A S. rostrata cDNA clone homologous to early nodulin ENOD40 genes was isolated from a cDNA library of developing stem nodules. SrENOD40-1 contained the conserved regions I and II of other ENOD40 genes. By reverse transcriptase PCR, enhanced SrENOD40-1 expression was observed in the adventitious root primordia between 4 and 8 h after inoculation with A. caulinodans. In situ hybridization showed that SrENOD40-1 transcripts, present around the central vascular bundle of the uninfected root primordia, were strongly enhanced upon induction of nodule development. De novo SrENOD40-1 expression was observed in the initiating and growing nodule primordia and around vascular bundles. When cell type specification sets in, the expression became pronounced in cells derived from the meristematic regions. In other parts of the plant, weak SrENOD40-1 expression was associated with vascular bundles and was observed in leaf and stipule primordia.

Upon infection of Sesbania rostrata with Azorhizobium caulinodans, nodules are formed on roots and stems. Stem nodules develop from abundantly distributed dormant root primordia. To acquire more insight into the meristem organization during stem nodule development, the expression patterns of a mitotic B1-type cyclin gene (Sesro; CycB1;1), a cyclin-dependent kinase gene (Cdc2-1Sr), and a histone H4 gene (H4-1Sr) of S. rostrata were followed by in situ hybridization. Cdc2-1Sr transcripts were found in all cells of uninfected and infected root primordia. In uninfected root primordia, Sesro;CycB1;1 transcripts were detected in a few cells of the apical root meristem whereas H4-1Sr transcripts were abundant in this region. Interestingly, after inoculation with A. caulinodans, H4-1Sr transcripts disappeared in the root meristem and a patchy pattern of Sesro;CycB1;1 and H4-1Sr expression appeared in the cortex of the root primordium, reflecting the formation of globular nodule primordia. When bacterial invasion started, a distal nodule meristem was delimited wherein Sesro;CycB1;1 and H4-1Sr expression was concentrated. Approximately 1 week after inoculation, meristem activity ceased, indicated by the loss of Sesro;CycB1;1 and H4-1Sr expression.

LONELY GUY (LOG) genes encode cytokinin riboside 5'-monophosphate phosphoribohydrolases and are directly involved in the activation of cytokinins. To assess whether LOG proteins affect the influence of cytokinin on nodulation, we studied two LOG genes of Medicago truncatula. Expression analysis showed that MtLOG1 and MtLOG2 were upregulated during nodulation in a CRE1-dependent manner. Expression was mainly localized in the dividing cells of the nodule primordium. In addition, RNA interference revealed that MtLOG1 is involved in nodule development and that the gene plays a negative role in lateral root development. Ectopic expression of MtLOG1 resulted in a change in cytokinin homeostasis, triggered cytokinin-inducible genes and produced roots with enlarged vascular tissues and shortened primary roots. In addition, those 35S:LOG1 roots also displayed fewer nodules than the wild-type. This inhibition in nodule formation was local, independent of the SUPER NUMERIC NODULES gene, but coincided with an upregulation of the MtCLE13 gene, encoding a CLAVATA3/EMBRYO SURROUNDING REGION peptide. In conclusion, we demonstrate that in M. truncatula LOG proteins might be implicated in nodule primordium development and lateral root formation.

Plants form symbioses with nitrogen-fixing rhizobia that cause the formation of root nodules. Plant peptides of the CLAVATA3/embryo-surrounding region (CLE), early nodulin 40 (ENOD40), rapid alkalinization factor (RALF), devil1 (DVL1)/rotundifolia4 (ROT4), and nodule-specific cysteine-rich (NCR) families have been implicated in all stages of nodulation. While CLE peptides have roles in controlling the proliferation of nodule cell divisions locally and systemically, ENOD40 peptides act locally in nodule initiation. RALF and DLV1-related peptides play a role in bacterial infection thread formation, while the large family of NCR proteins is nodule specific and controls bacteroid differentiation in mature nodules. Interestingly, parasitic nematodes, which induce feeding structures in host roots that involve similar induction of cell division and differentiation as nodules, are the only organisms known to encode CLE peptides outside the plant kingdom. While rhizobia induce the expression of CLE peptides by the host, parasitic nematodes directly inject CLE peptides into developing feeding cells, mimicking the host peptides.

International audience; Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. * The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. * MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. * This work suggests that regulatory pathways controlling a conserved transcription factor are complex and divergent between M. truncatula and Arabidopsis.

The symbiotic interaction between legumes and rhizobia results in root nodules with nitrogen-fixing bacteroids. Throughout the lifespan of the nodules, the exchange of C sources and N compounds between the host plant and the bacteria is tightly balanced. Sucrose plays a major role in the provision of C skeletons and energy to the bacteroids. Transcription of MtATB2, encoding a bZIP transcription factor, is shown to be regulated by sucrose and is enhanced during nodule senescence. Transcripts occur in the nodule apex and in the vascular tissue of nodules and roots. Ectopic expression of the gene diminished nodule formation and affected root growth. Presumably, MtATB2 controls processes that are under sucrose homeostasis and are important for nodule and root growth.

Summary Legumes acquired the ability to engage in a symbiotic interaction with soil-borne bacteria and establish a nitrogen-fixing symbiosis in a novel root organ, the nodule. Most legume crops and the model legumes Medicago truncatula and Lotus japonicus are infected intracellularly in root hairs via infection threads that lead the bacteria towards a nodule primordium in the root cortex. This infection process, however, does not reflect the great diversity of infection strategies that are used by leguminous plants. An alternative, intercellular invasion occurs in the semiaquatic legume Sesbania rostrata. Bacteria colonize epidermal fissures at lateral root bases and trigger cortical cell death for infection pocket formation and subsequent intercellular and intracellular infection thread progression towards the primordium. This infection mode evolved as an adaptation to waterlogged conditions that inhibit intracellular invasion. In this review, we discuss the molecular basis for this adaptation and how insights into this process contribute to general knowledge of the rhizobial infection process.

Under nitrogen-limiting conditions, legumes interact with symbiotic rhizobia to produce nitrogen-fixing root nodules. We have previously shown that glutathione and homoglutathione [(h)GSH] deficiencies impaired Medicago truncatula symbiosis efficiency, showing the importance of the low M r thiols during the nodulation process in the model legume M. truncatula. In this study, the plant transcriptomic response to Sinorhizobium meliloti infection under (h)GSH depletion was investigated using cDNA-amplified fragment length polymorphism analysis. Among 6,149 expression tags monitored, 181 genes displayed significant differential expression between inoculated control and inoculated (h)GSH depleted roots. Quantitative reverse transcription polymerase chain reaction analysis confirmed the changes in mRNA levels. This transcriptomic analysis shows a down-regulation of genes involved in meristem formation and a modulation of the expression of stress-related genes in (h)GSH-depleted plants. Promoter-β-glucuronidase histochemical analysis showed that the putative MtPIP2 aquaporin might be up-regulated during nodule meristem formation and that this up-regulation is inhibited under (h)GSH depletion. (h)GSH depletion enhances the expression of salicylic acid (SA)-regulated genes after S. meliloti infection and the expression of SA-regulated genes after exogenous SA treatment. Modification of water transport and SA signaling pathway observed under (h)GSH deficiency contribute to explain how (h)GSH depletion alters the proper development of the symbiotic interaction.

The Fabaceae comprise the third largest family of higher plants, and numerous legume species are spread worldwide in a variety of climates from the artic circle to the tropics. Many legumes obtain their nitrogen sources via a symbiotic interaction with nitrogen-fixing rhizobia and are therefore important natural fertilizers and pioneer plants on eroded soils. Moreover, some legumes have protein-rich seeds, making them essential for food and feed. Medicago truncatula and Lotus japonicus are the model species for legume research as well as for nodulation, the process that results in the symbiotic organs, the nodules, in which the rhizobia reside for nitrogen fixation. Via biotechnological tools such as genome analysis, bioinformatics, mutagenesis, transcriptomics, proteomics and metabolomics, researchers are investigating the molecular and biological principles of the nodulation process. These studies will ultimately lead to the improvement of existing symbioses and eventually to the extension of the process to non-nodulating crops. In this chapter, an overview is given of the current knowledge of the nodulation process in the model legumes.

Protein ubiquitination is a posttranslational regulatory process essential for plant growth and interaction with the environment. E3 ligases, to which the seven in absentia (SINA) proteins belong, determine the specificity by selecting the target proteins for ubiquitination. SINA proteins are found in animals as well as in plants, and a small gene family with highly related members has been identified in the genome of rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), Medicago truncatula, and poplar (Populus trichocarpa). To acquire insight into the function of SINA proteins in nodulation, a dominant negative form of the Arabidopsis SINAT5 was ectopically expressed in the model legume M. truncatula. After rhizobial inoculation of the 35S:SINAT5DN transgenic plants, fewer nodules were formed than in control plants, and most nodules remained small and white, a sign of impaired symbiosis. Defects in rhizobial infection and symbiosome formation were observed by extensive microscopic analysis. Besides the nodulation phenotype, transgenic plants were affected in shoot growth, leaf size, and lateral root number. This work illustrates a function for SINA E3 ligases in a broad spectrum of plant developmental processes, including nodulation.

The symbiosis between legumes and rhizobia is essential for the nitrogen input into the life cycle on our planet. New root organs, the nodules, are established, which house N 2 -fixing bacteria internalized into the host cell cytoplasm as horizontally acquired organelles, the symbiosomes. The interaction is initiated by bacterial invasion via epidermal root hair curling and cell division in the cortex, both triggered by bacterial nodulation factors. Of the several genes involved in nodule initiation that have been identified, one encodes the leucine-rich repeat-type receptor kinase SymRK. In SymRK mutants of Lotus japonicus or its orthologs in Medicago sp. and Pisum sativum , nodule initiation is arrested at the level of the root hair interaction. Because of the epidermal block, the role of SymRK at later stages of nodule development remained enigmatic. To analyze the role of SymRK downstream of the epidermis, the water-tolerant legume Sesbania rostrata was used that has developed a nodulation strategy to circumvent root hair responses for bacterial invasion. Evidence is provided that SymRK plays an essential role during endosymbiotic uptake in plant cells.

Upon submergence, Azorhizobium caulinodans infects the semiaquatic legume Sesbania rostrata via the intercellular crack entry process, resulting in lateral root-based nodules. A gene encoding a gibberellin (GA) 20-oxidase, SrGA20ox1, involved in GA biosynthesis, was transiently up-regulated during lateral root base nodulation. Two SrGA20ox1 expression patterns were identified, one related to intercellular infection and a second observed in nodule meristem descendants. The infection-related expression pattern depended on bacterially produced nodulation (Nod) factors. Pharmacological studies demonstrated that GAs were involved in infection pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation, and that they were also needed for nodule primordium development. Moreover, GAs inhibited the root hair curling process. These results show that GAs are Nod factor downstream signals for nodulation in hydroponic growth.

A novel marker for the early stages of nodulation of Sesbania rostrata was found to encode a putative member of the Kunitz family of protease inhibitors (SrPI1). Its expression was enhanced during nodulation, and was not up-regulated by wounding or upon infection with wide host-range pathogens. In situ expression patterns resembled those previously described for functions that may be implicated in delimiting infected nodule tissues from the rest of the plant. Thus, SrPI1 may be a component of a multi-layered barrier that restrains the invading rhizobia.

Covering: up to the end of 2013 The genus Medicago, a member of the legume (Fabaceae) family, comprises 87 species of flowering plants, including the forage crop M. sativa (alfalfa) and the model legume M. truncatula (barrel medic). Medicago species synthesize a variety of bioactive natural products that are used to engage into symbiotic interactions but also serve to deter pathogens and herbivores. For humans, these bioactive natural products often possess promising pharmaceutical properties. In this review, we focus on the two most interesting and well characterized secondary metabolite classes found in Medicago species, the triterpene saponins and the flavonoids, with a detailed overview of their biosynthesis, regulation, and profiling methods. Furthermore, their biological role within the plant as well as their potential utility for human health or other applications is discussed. Finally, we give an overview of the advances made in metabolic engineering in Medicago species and how the development of novel molecular and omics toolkits can influence a better understanding of this genus in terms of specialized metabolism and chemistry. Throughout, we critically analyze the current bottlenecks and speculate on future directions and opportunities for research and exploitation of Medicago metabolism.

Free full text to read and download available at PMC, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC22898/. Copyright © 1998, The National Academy of Sciences, Leguminous plants in symbiosis with rhizobia form either indeterminate nodules with a persistent meristem or determinate nodules with a transient meristematic region. Sesbania rostrata was thought to possess determinate stem and root nodules. However, the nature of nodule development is hybrid, and the early stages resemble those of indeterminate nodules. Here we show that, depending on the environmental conditions, mature root nodules can be of the indeterminate type. In situ hybridizations with molecular markers for plant cell division, as well as the patterns of bacterial nod and nif gene expression, confirmed the indeterminate nature of 30-day-old functional root nodules. Experimental data provide evidence that the switch in nodule type is mediated by the plant hormone ethylene., This work was supported by a grant from the European Union Training and Mobility Network (CHRX-CT94-0656). W.D. is indebted to the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie for a predoctoral fellowship. M.H. is a Research Director of the Fund for Scientific Research (Flanders).

Stem nodules on the tropical legume Sesbania rostrata develop after intercellular infection of adventitious root primordia by the microsymbiont Azorhizobium caulinodans strain ORS571. Development of these nodules is dependent on Nod factors and shows hybrid features with a brief indeterminate phase followed by maturation, resulting in the formation of determinate nodules.

Sesbania rostrata is a tropical legume that can be nodulated on the stem as well as on the root. Dormant adventitious root primordia present along the stem can develop into adventitious roots upon immersion in water or into nitrogen fixing stem nodules upon inoculation with the microsymbiont Azorhizohium caulinodans. In a quest for new genes of which the expression is changed during stem nodule development, RNA populations from non-infected root primordia were compared with RNA populations from infected root primordia. This resulted in the isolation of several interesting genes.

The symbiotic interaction between the tropical leguminous plant Sesbania rostrata and the bacterium Azorhizobium caulinodans strain ORS571 is in many aspects a unique one (De Bruijn, 1989). In contrast to most legumes that form nitrogen-fixing nodules only on the roots, S. rostrata can be nodulated on the roots and the stem. The aerial nodules are formed at predetermined sites that are dormant root primordia located in vertical rows along the stem. These primordia develop into roots when immersed in water and/or into nodules upon infection with A. caulinodans. Root and stem nodule organogenesis are very similar and are intermediate between the indeterminate and determinate types of nodule development (Ndoye et al., 1994). Nodulation of S. rostrata stems is relatively unaffected by combined nitrogen and can be very extensive resulting in an extremely high fixation rate per plant. The closest relative of A. caulinodans is the diazotroph Xanthobacter, and A. caulinodans is unique amongst rhizobia in being capable of both symbiotic and free-living nitrogen fixation, the latter with assimilation of the formed ammonium for growth.

The Azorhizobium caulinodans strain ORS571 nodulation genes nodSUIJ were located downstream from nodABC. Complementation data and transcriptional analysis suggest that nodABCSUIJ form a single operon. Mutants with Tn5 insertions in the genes nodS, nodU, and nodJ were delayed in nodulation of Sesbania rostrata roots and stems. The NodS amino acid sequences of ORS571, Bradyrhizobium japonicum, and Rhizobium sp. strain NGR234, contain a consensus with similarity to S-adenosylmethionine (SAM)-utilizing methyltransferases. A naringenin-inducible nodS-dependent protein of approximately 25 kDa could be cross-linked to radiolabelled SAM. By applying L-[methyl-3H]-methionine in vivo, Nod factors of ORS571, known to be N-methylated, could be labelled in wild type and nodU mutants but not in nodS mutants. Therefore, we propose that NodS is a SAM-utilizing methyltransferase involved in Nod factor synthesis.