ARGONAUTE10 controls cell fate specification and formative cell divisions in the Arabidopsis root.

A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity.

Global dynamics and cytokinin participation of salt gland development trajectory in recretohalophyte Limonium bicolor.

Salt gland is an epidermal Na+ secretory structure that enhances salt resistance in the recretohalophyte sea lavender (Limonium bicolor). To elucidate the salt gland development trajectory and related molecular mechanisms, we performed single-cell RNA sequencing of L. bicolor protoplasts from young leaves at salt gland initiation and differentiation stages. Dimensionality reduction analyses defined 19 transcriptionally distinct cell clusters, which were assigned into four broad populations-promeristem, epidermis, mesophyll, and vascular tissue-verified by in situ hybridization. Cytokinin was further proposed to participate in salt gland development by the expression patterns of related genes and cytological evidence. By comparison analyses of scRNA-seq with exogenous application of 6-benzylaminopurine, we delineated five salt gland development-associated sub-clusters and defined salt gland specific differentiation trajectories from sub-clusters 8, 4, or 6 to sub-cluster 3 and 1. Additionally, we validated the participation of TRIPTYCHON and the interacting protein Lb7G34824 in salt gland development, which regulated the expression of cytokinin metabolism and signaling related genes such as GLABROUS INFLORESCENCE STEMS 2 to maintain cytokinin homeostasis during salt gland development. Our results generated a gene expression map of young leaves at single-cell resolution for the comprehensive investigation of salt gland determinants and cytokinin participation that helps elucidate cell fate determination during epidermis formation and evolution in recretohalophytes.

Age-dependent changes in leaf size in apple are governed by a cytokinin-integrated module.

Plants undergo various age-dependent changes in leaf morphology during the transition from the juvenile to the adult stage. However, the precise molecular mechanisms governing these changes in apple (Malus domestica) remain unknown. Here, we showed that CYTOKININ OXIDASE/DEHYDROGENASE5 (MdCKX5), an age-dependent gene, encodes a functional CKX enzyme and serves as the common downstream target of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor MdSPL14 and WRKY transcription factor MdWRKY24 to control the degradation of cytokinin (CK). As the target of mdm-microRNA156a, MdSPL14 interacts with MdWRKY24 to coordinately repress the transcription of MdCKX5 by forming the age-mediated mdm-miR156a-MdSPL14-MdWRKY24 module, which regulates age-dependent changes in CK during the juvenile-to-adult phase transition. We further demonstrated that MdARR6, a type-A ARABIDOPSIS RESPONSE REGULATOR (ARR), is a negative feedback regulator in the CK signaling pathway. Silencing of MdARR6 in apple resulted in large leaves with smaller epidermal cells and a greater number of epidermal cells. Biochemical analysis showed that the mdm-miR156a-MdSPL14-MdWRKY24 module acts as a transcriptional repressor to directly regulate MdARR6 expression, thus controlling the age-dependent changes in leaf size by reducing CK responses. These findings established a link between the age pathway and CK signaling and revealed the molecular mechanism underlying age-dependent changes during the juvenile-to-adult phase transition; our results also provide targets for the genetic improvement of the vegetative phase transition in apple.

Purine permease (PUP) family gene PUP11 positively regulates the rice seed setting rate by influencing seed development.

KEY MESSAGE: Purine permease PUP11 is essential for rice seed development, regulates the seed setting rate, and influences the cytokinin content, sugar transport, and starch biosynthesis during grain development. The distribution of cytokinins in plant tissues determines plant growth and development and is regulated by several cytokinin transporters, including purine permease (PUP). Thirteen PUP genes have been identified within the rice genome; however, the functions of most of these genes remain poorly understood. We found that pup11 mutants showed extremely low seed setting rates and a unique filled seed distribution. Moreover, seed formation arrest in these mutants was associated with the disappearance of accumulated starch 10 days after flowering. PUP11 has two major transcripts with different expression patterns and subcellular locations, and further studies revealed that they have redundant positive roles in regulating the seed setting rate. We also found that type-A Response Regulator (RR) genes were upregulated in the developing grains of the pup11 mutant compared with those in the wild type. The results also showed that PUP11 altered the expression of several sucrose transporters and significantly upregulated certain starch biosynthesis genes. In summary, our results indicate that PUP11 influences the rice seed setting rate by regulating sucrose transport and starch accumulation during grain filling. This research provides new insights into the relationship between cytokinins and seed development, which may help improve cereal yield.

Longistylin A from Cajanus cajan (L.) Millsp. disturbs glycerophospholipid metabolism and cytokinin biosynthesis of Nocardia seriolae.

ETHNOPHARMACOLOGICAL RELEVANCE: Nocardiosis is an uncommon infectious disease that bears certain similarities to tuberculosis, with a continuous increase in its incidence and a poor prognosis. In traditional Chinese medicine, the leaves of Cajanus cajan (L.) Millsp. are employed to treat wounds, malaria, coughs, and abdominal pain. AIM OF THE STUDY: In this study, we investigated the effects and mechanisms of longistylin A (LGA), a natural stilbene isolated from C. cajan, as a potential antibiotic against nocardiosis MATERIALS AND METHODS: LGA was isolated from the leaves of C. cajan and assessed using a minimum bactericidal concentration (MBC) determination against Nocardia seriolae. Multi-omics analysis encompassing genes, proteins, and metabolites was conducted to investigate the impact of LGA treatment on N. seriolae. Additionally, quantitative analysis of 40 cytokinins in N. seriolae mycelium was performed to assess the specific effects of LGA treatment on cytokinin levels. Cryo-scanning electron microscopy was utilized to examine morphological changes induced by LGA treatment, particularly in the presence of exogenous trans-zeatin-O-glucoside (tZOG). The therapeutic effect of LGA was investigated by feeding N. seriolae-infected largemouth bass. RESULTS: LGA exhibited significant efficacy against N. seriolae, with MBC value of 2.56µg/mL. Multi-omics analysis revealed that LGA disrupted glycerophospholipid metabolism and hormone biosynthesis by notably reducing the expression of glycerol-3-phosphate dehydrogenase and calmodulin-like protein. Treatment with LGA markedly disrupted 12 distinct cytokinins in N. seriolae mycelium. Additionally, the addition of exogenous tZOG counteracted the inhibitory effects of LGA on filamentous growth, resulting in mycelial elongation and branching. Furthermore, LGA treatment improved the survival rate of largemouth bass infected with N. seriolae. CONCLUSIONS: We found for the first time that LGA from C. cajan exhibited significant efficacy against N. seriolae by interfering with glycerophospholipid metabolism and cytokinin biosynthesis.

CEPs suppress auxin signaling but promote cytokinin signaling to inhibit root growth in Arabidopsis.

C-terminally encoded peptides (CEPs) are peptide hormones that function as mobile signals coordinating crucial developmental programs in plants. Previous studies have revealed that CEPs exert negative regulation on root development through interaction with CEP receptors (CEPRs), CEP DOWNSTREAMs (CEPDs), the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE (AHKs) and the transcriptional repressor Auxin/Indole-3-Acetic Acid (AUX/IAA). However, the precise molecular mechanisms underlying CEPs-mediated regulation of root development via auxin and cytokinin signaling pathways still necessitate further detailed investigation. In this study, we examined prior research and elucidated the underlying molecular mechanisms. The results showed that both synthetic AtCEPs and overexpression of AtCEP5 markedly supressed primary root elongation and lateral root (LR) formation in Arabidopsis. Molecular biology and genetics elucidated how CEPs inhibit root growth by suppressing auxin signaling while promoting cytokinin signaling. In summary, this study elucidated the inhibitory effects of AtCEPs on Arabidopsis root growth and provided insights into their potential molecular mechanisms, thus enhancing our comprehension of CEP-mediated regulation of plant growth and development.

[Identification and expression profile analysis of rice CRF gene family].

Cytokinin response factors (CRFs), as unique transcription factors in plants, play crucial roles in regulating development, phytohormone signaling pathway, and stress responses. In this study, we identified nine CRF genes from the rice genome by conducting a BLAST analysis using the protein sequences of twelve Arabidopsis AtCRFs. These genes are located on seven different rice chromosomes. We conducted a comprehensive analysis of the conserved domains, physicochemical properties, secondary structures, and phylogenetic relationships of rice CRF proteins using various online tools and local software. Additionally, we analyzed the exon-intron structures and cis-acting elements of OsCRFs, and found an abundance of elements relevant to phytohormone response and stress response on the promoters of rice CRF genes. Spatial-temporal expression pattern analysis revealed that four of the OsCRFs were barely expressed in all tested samples, while the other five were highly expressed in the leaf, panicle, or seed of rice. Microarray data showed that OsCRF genes are regulated to varying degrees by abscisic acid, auxin, cytokinin, and jasmonic acid. Furthermore, through analyzing the RNA-seq data, we found that OsCRFs are primarily involved in plant response to temperature stress (chilling and heat), with several OsCRFs also implicated in drought response, while hardly any respond to salt stress. This study provides an important basis for the functional characterization of rice CRF family genes.

Profiling of 1-aminocyclopropane-1-carboxylic acid and selected phytohormones in Arabidopsis using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Gaseous phytohormone ethylene levels are directly influenced by the production of its immediate non-volatile precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Owing to the strongly acidic character of the ACC molecule, its quantification has been difficult to perform. Here, we present a simple and straightforward validated method for accurate quantification of not only ACC levels, but also major members of other important phytohormonal classes - auxins, cytokinins, jasmonic acid, abscisic acid and salicylic acid from the same biological sample. RESULTS: The presented technique facilitates the analysis of 15 compounds by liquid chromatography coupled with tandem mass spectrometry. It was optimized and validated for 10 mg of fresh weight plant material. The extraction procedure is composed of a minimal amount of necessary steps. Accuracy and precision were the basis for evaluating the method, together with process efficiency, recovery and matrix effects as validation parameters. The examined compounds comprise important groups of phytohormones, their active forms and some of their metabolites, including six cytokinins, four auxins, two jasmonates, abscisic acid, salicylic acid and 1-aminocyclopropane-1-carboxylic acid. The resulting method was used to examine their contents in selected Arabidopsis thaliana mutant lines. CONCLUSION: This profiling method enables a very straightforward approach for indirect ethylene study and explores how it interacts, based on content levels, with other phytohormonal groups in plants.

A predictive model for ethylene-mediated auxin and cytokinin patterning in the Arabidopsis root.

The interaction between auxin and cytokinin is important in many aspects of plant development. Experimental measurements of both auxin and cytokinin concentration and reporter gene expression clearly show the coexistence of auxin and cytokinin concentration patterning in Arabidopsis root development. However, in the context of crosstalk between auxin, cytokinin and ethylene, little is known about how auxin and cytokinin concentration patterns simultaneously emerge and how they regulate each other in the Arabidopsis root. This work utilizes a wide range of experimental observations to propose a mechanism for simultaneous patterning of auxin and cytokinin concentration. In addition to the regulatory relationships between auxin and cytokinin, the mechanism reveals that ethylene signalling is an important factor in achieving simultaneous auxin and cytokinin patterning, while also predicting other experimental observations. Combining the mechanism with a realistic in silico root model reproduces experimental observations of both auxin and cytokinin patterning. Predictions made by the mechanism can be compared with a variety of experimental observations, including those conducted by our group and other independent experiments reported by other groups. Examples of these predictions include patterning of auxin biosynthesis rate, PIN1 and PIN2 pattern changes in pin3, 4, 7 mutants, cytokinin patterning change in the pls mutant, PLS patterning, as well as various trends in different mutants. This research unravels a plausible mechanism for simultaneous patterning of auxin and cytokinin concentrations in Arabidopsis root development and suggests a key role for ethylene pattern integration.

miR156-PvSPL2 controls culm development by transcriptional repression of switchgrass CYTOKININ OXIDASE/DEHYDROGENASE4.

Culm development in grasses can be controlled by both miR156 and cytokinin. However, the crosstalk between the miR156-SPL module and the cytokinin metabolic pathway remains largely unknown. Here, we found CYTOKININ OXIDASE/DEHYDROGENASE4 (PvCKX4) plays a negative regulatory role in culm development of the bioenergy grass Panicum virgatum (switchgrass). Overexpression of PvCKX4 in switchgrass reduced the internode diameter and length without affecting tiller number. Interestingly, we also found that PvCKX4 was always upregulated in miR156 overexpressing (miR156OE) transgenic switchgrass lines. Additionally, upregulation of either miR156 or PvCKX4 in switchgrass reduced the content of isopentenyl adenine (iP) without affecting trans-zeatin (tZ) accumulation. It is consistent with the evidence that the recombinant PvCKX4 protein exhibited much higher catalytic activity against iP than tZ in vitro. Furthermore, our results showed that miR156-targeted SPL2 bound directly to the promoter of PvCKX4 to repress its expression. Thus, alleviating the SPL2-mediated transcriptional repression of PvCKX4 through miR156 overexpression resulted in a significant increase in cytokinin degradation and impaired culm development in switchgrass. On the contrary, suppressing PvCKX4 in miR156OE transgenic plants restored iP content, internode diameter, and length to wild-type levels. Most strikingly, the double transgenic lines retained the same increased tiller numbers as the miR156OE transgenic line, which yielded more biomass than the wild type. These findings indicate that the miR156-SPL module can control culm development through transcriptional repression of PvCKX4 in switchgrass, which provides a promising target for precise design of shoot architecture to yield more biomass from grasses.

Dissecting the Roles of the Cytokinin Signaling Network: The Case of De Novo Shoot Apical Meristem Formation.

Numerous biotechnological applications require a fast and efficient clonal propagation of whole plants under controlled laboratory conditions. For most plant species, the de novo regeneration of shoots from the cuttings of various plant organs can be obtained on nutrient media supplemented with plant hormones, auxin and cytokinin. While auxin is needed during the early stages of the process that include the establishment of pluripotent primordia and the subsequent acquisition of organogenic competence, cytokinin-supplemented media are required to induce these primordia to differentiate into developing shoots. The perception of cytokinin through the receptor ARABIDOPSIS HISTIDINE KINASE4 (AHK4) is crucial for the activation of the two main regulators of the establishment and maintenance of shoot apical meristems (SAMs): SHOOTMERISTEMLESS (STM) and the WUSCHEL-CLAVATA3 (WUS-CLV3) regulatory circuit. In this review, we summarize the current knowledge of the roles of the cytokinin signaling cascade in the perception and transduction of signals that are crucial for the de novo establishment of SAMs and lead to the desired biotechnological output-adventitious shoot multiplication. We highlight the functional differences between individual members of the multigene families involved in cytokinin signal transduction, and demonstrate how complex genetic regulation can be achieved through functional specialization of individual gene family members.

RETINOBLASTOMA RELATED 1 switches mitosis to meiosis in rice.

The transition from mitosis to meiosis is a critical event in the reproductive development of all sexually reproducing species. However, the mechanisms that regulate this process in plants remain largely unknown. Here, we find that the rice (Oryza sativa L.) protein RETINOBLASTOMA RELATED 1 (RBR1) is essential to the transition from mitosis to meiosis. Loss of RBR1 function results in hyper-proliferative sporogenous-cell-like cells (SCLs) in the anther locules during early stages of reproductive development. These hyper-proliferative SCLs are unable to initiate meiosis, eventually stagnating and degrading at late developmental stages to form pollen-free anthers. These results suggest that RBR1 acts as a gatekeeper of entry into meiosis. Furthermore, cytokinin content is significantly increased in rbr1 mutants, whereas the expression of type-B response factors, particularly LEPTO1, is significantly reduced. Given the known close association of cytokinins with cell proliferation, these findings imply that hyper-proliferative germ cells in the anther locules may be attributed to elevated cytokinin concentrations and disruptions in the cytokinin pathway. Using a genetic strategy, the association between germ cell hyper-proliferation and disturbed cytokinin signaling in rbr1 has been confirmed. In summary, we reveal a unique role of RBR1 in the initiation of meiosis; our results clearly demonstrate that the RBR1 regulatory module is connected to the cytokinin signaling pathway and switches mitosis to meiosis in rice.

PGI1-mediated vascular oxidative pentose phosphate pathway modulates photosynthesis via long-distance cytokinin signaling.

In Arabidopsis, the plastidial isoform of phosphoglucose isomerase, PGI1, mediates growth and photosynthesis, likely due to its involvement in the vascular production of cytokinins (CK). To examine this hypothesis, we characterized pgi1-2 knockout plants impaired in PGI1 and pgi1-2 plants specifically expressing PGI1 in root tips and vascular tissues. Moreover, to investigate whether the phenotype of pgi1-2 plants is due to impairments in the plastidial oxidative pentose phosphate pathway (OPPP) or the glycolytic pathway, we characterized pgl3-1 plants with reduced OPPP and pfk4pfk5 knockout plants impaired in plastidial glycolysis. Compared with wild-type (WT) leaves, pgi1-2 leaves exhibited weaker expression of photosynthesis- and 2-C-methyl-D-erythritol 4-P (MEP) pathway-related proteins, and stronger expression of oxidative stress protection-related enzymes. Consistently, pgi1-2 leaves accumulated lower levels of chlorophyll, and higher levels of tocopherols, flavonols and anthocyanins than the WT. Vascular- and root tip-specific PGI1 expression countered the reduced photosynthesis, low MEP pathway-derived CK content, dwarf phenotype and the metabolic characteristics of pgi1-2 plants, reverting them to WT-like levels. Moreover, pgl3-1, but not pfk4pfk5 plants phenocopied pgi1-2. Histochemical analyses of plants expressing GUS under the control of promoter regions of genes encoding plastidial OPPP enzymes exhibited strong GUS activity in root tips and vascular tissues. Overall, our findings show that root tip and vascular PGI1-mediated plastidial OPPP activity affects photosynthesis and growth through mechanisms involving long-distance modulation of the leaf proteome by MEP pathway-derived CKs.

Plant hormone profiling of scion and rootstock incision sites and intra- and inter-family graft junctions in Nicotiana benthamiana.

Many previous studies have suggested that various plant hormones play essential roles in the grafting process. In this study, to understand the plant hormones that accumulate in the graft junctions, whether these are supplied from the scion or rootstock, and how these hormones play a role in the grafting process, we performed a hormonome analysis that accumulated in the incision site of the upper plants from the incision as "ungrafted scion" and lower plants from the incision as "ungrafted rootstock" in Nicotiana benthamiana. The results revealed that indole-3-acetic acid (IAA) and gibberellic acid (GA), which regulate cell division; abscisic acid (ABA) and jasmonic acid (JA), which regulate xylem formation; cytokinin (CK), which regulates callus formation, show different accumulation patterns in the incision sites of the ungrafted scion and rootstock. In addition, to try discussing the differences in the degree and speed of each event during the grafting process between intra- and inter-family grafting by determining the concentration and accumulation timing of plant hormones in the graft junctions, we performed hormonome analysis of graft junctions of intra-family grafted plants with N. benthamiana as scion and Solanum lycopersicum as rootstock (Nb/Sl) and inter-family grafted plants with N. benthamiana as scion and Arabidopsis thaliana as rootstock (Nb/At), using the ability of Nicotiana species to graft with many plant species. The results revealed that ABA and CK showed different accumulation timings; IAA, JA, and salicylic acid (SA) showed similar accumulation timings, while different accumulated concentrations in the graft junctions of Nb/Sl and Nb/At. This information is important for understanding the molecular mechanisms of plant hormones in the grafting process and the differences in molecular mechanisms between intra- and inter-family grafting.

Nitric oxide and cytokinin cross-talk and their role in plant hypoxia response.

Nitric oxide (NO) and cytokinins (CKs) are known for their crucial contributions to plant development, growth, senescence, and stress response. Despite the importance of both signals in stress responses, their interaction remains largely unexplored. The interplay between NO and CKs emerges as particularly significant not only regarding plant growth and development but also in addressing plant stress response, particularly in the context of extreme weather events leading to yield loss. In this review, we summarize NO and CKs metabolism and signaling. Additionally, we emphasize the crosstalk between NO and CKs, underscoring its potential impact on stress response, with a focus on hypoxia tolerance. Finally, we address the most urgent questions that demand answers and offer recommendations for future research endeavors.

OsRR26, a type-B response regulator, modulates salinity tolerance in rice via phytohormone-mediated ROS accumulation in roots and influencing reproductive development.

MAIN CONCLUSION: OsRR26 is a cytokinin-responsive response regulator that promotes phytohormone-mediated ROS accumulation in rice roots, regulates seedling growth, spikelet fertility, awn development, represses NADPH oxidases, and negatively affects salinity tolerance. Plant two-component systems (TCS) play a pivotal role in phytohormone signaling, stress responses, and circadian rhythm. However, a significant knowledge gap exists regarding TCS in rice. In this study, we utilized a functional genomics approach to elucidate the role of OsRR26, a type-B response regulator in rice. Our results demonstrate that OsRR26 is responsive to cytokinin, ABA, and salinity stress, serving as the ortholog of Arabidopsis ARR11. OsRR26 primarily localizes to the nucleus and plays a crucial role in seedling growth, spikelet fertility, and the suppression of awn development. Exogenous application of cytokinin led to distinct patterns of reactive oxygen species (ROS) accumulation in the roots of both WT and transgenic plants (OsRR26OE and OsRR26KD), indicating the potential involvement of OsRR26 in cytokinin-mediated ROS signaling in roots. The application of exogenous ABA resulted in varied cellular compartmentalization of ROS between the WT and transgenic lines. Stress tolerance assays of these plants revealed that OsRR26 functions as a negative regulator of salinity stress tolerance across different developmental stages in rice. Physiological and biochemical analyses unveiled that the knockdown of OsRR26 enhances salinity tolerance, characterized by improved chlorophyll retention and the accumulation of soluble sugars, K+ content, and amino acids, particularly proline.

Cytokinin signaling promotes root secondary growth and bud formation in Panax ginseng.

Background: Panax ginseng, one of the valuable perennial medicinal plants, stores numerous pharmacological substrates in its storage roots. Given its perennial growth habit, organ regeneration occurs each year, and cambium stem cell activity is necessary for secondary growth and storage root formation. Cytokinin (CK) is a phytohormone involved in the maintenance of meristematic cells for the development of storage organs; however, its physiological role in storage-root secondary growth remains unknown. Methods: Exogenous CK was repeatedly applied to P. ginseng, and morphological and histological changes were observed. RNA-seq analysis was used to elucidate the transcriptional network of CK that regulates P. ginseng growth and development. The HISTIDINE KINASE 3 (PgHK3) and RESPONSE REGULATOR 2 (PgRR2) genes were cloned in P. ginseng and functionally analyzed in Arabidopsis as a two-component system involved in CK signaling. Results: Phenotypic and histological analyses showed that CK increased cambium activity and dormant axillary bud formation in P. ginseng, thus promoting storage-root secondary growth and bud formation. The evolutionarily conserved two-component signaling pathways in P. ginseng were sufficient to restore CK signaling in the Arabidopsis ahk2/3 double mutant and rescue its growth defects. Finally, RNA-seq analysis of CK-treated P. ginseng roots revealed that plant-type cell wall biogenesis-related genes are tightly connected with mitotic cell division, cytokinesis, and auxin signaling to regulate CK-mediated P. ginseng development. Conclusion: Overall, we identified the CK signaling-related two-component systems and their physiological role in P. ginseng. This scientific information has the potential to significantly improve the field-cultivation and biotechnology-based breeding of ginseng.

Corrigendum: Cytokinin and abiotic stress tolerance -What has been accomplished and the way forward?

[This corrects the article DOI: 10.3389/fgene.2022.943025.].

The single-cell transcriptome program of nodule development cellular lineages in Medicago truncatula.

Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

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

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