Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro-and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

Most legumes can form a unique type of lateral organ on their roots: root nodules. These structures host symbiotic nitrogen-fixing bacteria called rhizobia. Several different types of nodules can be found in nature, but the two best-studied types are called indeterminate and determinate nodules. These two types differ with respect to the presence or absence of a persistent nodule meristem, which consistently correlates with the cortical cell layers giving rise to the nodule primordia. Similar to other plant developmental processes, auxin signalling overlaps with the site of organ initiation and meristem activity. Here, we review how auxin contributes to early nodule development. We focus on changes in auxin transport, signalling, and metabolism during nodule initiation, describing both experimental evidence and computer modelling. We discuss how indeterminate and determinate nodules may differ in their mechanisms for generating localized auxin response maxima and highlight outstanding questions for future research.

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 presence of nitrogen inhibits legume nodule formation, but the mechanism of this inhibition is poorly understood. We found that 2.5 mM nitrate and above significantly inhibited nodule initiation but not root hair curling in Medicago trunatula. We analyzed protein abundance in M. truncatula roots after treatment with either 0 or 2.5 mM nitrate in the presence or absence of its symbiont Sinorhizobium meliloti after 1, 2 and 5 days following inoculation. Two-dimensional gel electrophoresis combined with mass spectrometry was used to identify 106 differentially accumulated proteins responding to nitrate addition, inoculation or time point. While flavonoid-related proteins were less abundant in the presence of nitrate, addition of Nod gene-inducing flavonoids to the Sinorhizobium culture did not rescue nodulation. Accumulation of auxin in response to rhizobia, which is also controlled by flavonoids, still occurred in the presence of nitrate, but did not localize to a nodule initiation site. Several of the changes included defense- and redox-related proteins, and visualization of reactive oxygen species indicated that their induction in root hairs following Sinorhizobium inoculation was inhibited by nitrate. In summary, the presence of nitrate appears to inhibit nodulation via multiple pathways, including changes to flavonoid metabolism, defense responses and redox changes.

Legumes enter into a symbiotic relationship with nitrogen-fixing rhizobia, leading to nodule development. Two main types of nodules have been widely studied, indeterminate and determinate, which differ in the location of the first cell division in the root cortex, and persistency of the nodule meristem. Here, we compared the control of auxin transport, content, and response during the early stages of indeterminate and determinate nodule development in the model legumes Medicago truncatula and Lotus japonicus, respectively, to investigate whether differences in auxin transport control could explain the differences in the location of cortical cell divisions. While auxin responses were activated in dividing cortical cells during nodulation of both nodule types, auxin (indole-3-acetic acid) content at the nodule initiation site was transiently increased in M. truncatula, but transiently reduced in L. japonicus. Root acropetal auxin transport was reduced in M. truncatula at the very start of nodule initiation, in contrast to a prolonged increase in acropetal auxin transport in L. japonicus. The auxin transport inhibitors 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid (NPA) only induced pseudonodules in legume species forming indeterminate nodules, but failed to elicit such structures in a range of species forming determinate nodules. The development of these pseudonodules in M. truncatula exhibited increased auxin responses in a small primordium formed from the pericycle, endodermis, and inner cortex, similar to rhizobia-induced nodule primordia. In contrast, a diffuse cortical auxin response and no associated cortical cell divisions were found in L. japonicus. Collectively, we hypothesize that a step of acropetal auxin transport inhibition is unique to the process of indeterminate nodule development, leading to auxin responses in pericycle, endodermis, and inner cortex cells, while increased auxin responses in outer cortex cells likely require a different mechanism during the formation of determinate nodules.

Legumes form two types of root organs in response to signals from microbes: 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 defence responses, activation of host cell division and differentiation, nutrient exchange and alteration of root architecture. Rhizobia activate changes in defence 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.

Most field-grown plants are surrounded by microbes, especially from the soil. Some of these, including bacteria, fungi and nematodes, specifically manipulate the growth and development of their plant hosts, primarily for the formation of structures housing the microbes in roots. These developmental processes require the correct localization of the phytohormone auxin, which is involved in the control of cell division, cell enlargement, organ development and defense, and is thus a likely target for microbes that infect and invade plants. Some microbes have the ability to directly synthesize auxin. Others produce specific signals that indirectly alter the accumulation of auxin in the plant by altering auxin transport. This review highlights root–microbe interactions in which auxin transport is known to be targeted by symbionts and parasites to manipulate the development of their host root system. We include case studies for parasitic root–nematode interactions, mycorrhizal symbioses as well as nitrogen fixing symbioses in actinorhizal and legume hosts. The mechanisms to achieve auxin transport control that have been studied in model organisms include the induction of plant flavonoids that indirectly alter auxin transport and the direct targeting of auxin transporters by nematode effectors. In most cases, detailed mechanisms of auxin transport control remain unknown.

Root phenotyping describes methods for measuring root properties, or traits. While root phenotyping can be challenging, it is advancing quickly. In order for the field to move forward, it is essential to understand the current state and challenges of root phenotyping, as well as the pressing needs of the root biology community. In this letter, we present and discuss the results of a survey that was created and disseminated by members of the Graduate Student and Postdoc Ambassador Program at the 11th symposium of the International Society of Root Research. This survey aimed to (1) provide an overview of the objectives, biological models and methodological approaches used in root phenotyping studies, and (2) identify the main limitations currently faced by plant scientists with regard to root phenotyping. Our survey highlighted that (1) monocotyledonous crops dominate the root phenotyping landscape, (2) root phenotyping is mainly used to quantify morphological and architectural root traits, (3) 2D root scanning/imaging is the most widely used root phenotyping technique, (4) time-consuming tasks are an important barrier to root phenotyping, (5) there is a need for standardised, high-throughput methods to sample and phenotype roots, particularly under field conditions, and to improve our understanding of trait-function relationships.