Single-cell RNA-seq of Lotus japonicus provide insights into identification and function of root cell types of legume

The roots of legume plant play a crucial role in nitrogen fixation. However, the transcriptomes of different cell types of legume root and their functions remain largely unknown. Here, we performed single-cell RNA sequencing and profiled more than 22,000 single cells from root tips of Lotus japonicus, a model species of legume.We identified seven clusters corresponding to seven major cell types, which were validated by in situ hybridization. Further analysis revealed regulatory programs including phytohormone and nodulation associated with specific cell types, and revealed conserved and diverged features for the cell types. Our results represent the first single-cell resolution transcriptome for legume root tips and a valuable resource for studying the developmental and physiological functions of various cell types in legumes.

Multiple Components are Integrated to Determine Leaf Complexity in Lotus japonicus

Transcription factors and phytohormones have been reported to play crucial roles to regulate leaf complexity among plant species. Using the compound-leafed species Lotus japonicus, a model legume plant with five visible leaflets, we characterized four independent mutants with reduced leaf complexity, proliferating floral meristem （pfm）, proliferating floral organ-2 （pfo-2）, fused leaflets1 （ful1） and umbrella leaflets （uml）, which were further identified as loss-of-function mutants of Arabidopsis orthologs LEAFY （LFY ）, UNUSUAL FLORAL ORGANS （UFO）, CUP-SHAPED COTYLEDON 2 （CUC2） and PIN-FORMED 1 （PIN1）, respectively. Comparing the leaf development of wild-type and mutants by a scanning electron microscopy approach, leaflet initiation and/or dissection were found to be affected in these mutants. Expression and phenotype analysis indicated that PFM/LjLFY and PFO/LjUFO determined the basipetal leaflet initiation manner in L. japonicus. Genetic analysis of ful1 and uml mutants and their double mutants revealed that the CUC2-like gene and auxin pathway also participated in leaflet dissection in L. japonicus, and their functions might influence cytokinin biogenesis directly or indirectly. Our results here suggest that multiple genes were interplayed and played conserved functions in controlling leaf complexity during compound leaf development in L. japonicus.

Transcriptional and Post-transcriptional Modulation of SQU and KEW Activities in the Control of Dorsal-Ventral Asymmetric Flower Development in Lotus japonicus

In Papilionoideae legume, Lotusjaponicus, the development of dorsal-ventral （DV） asymmetric flowers is mainly controlled by two TB1/CYCLOIDEA/PCF （TCP） genes, SQUARED STANDARD （SQU） and KEELED WINGS IN LOTUS （KEW）, which determine dorsal and lateral identities, respectively. However, the molecular basis of how these two highly homologous genes orchestrate their diverse functions remains unclear. Here, we analyzed their expression levels, and investigated the transcriptional activities of SQUand KEW. We demonstrated that SQU possesses both activation and repression activities, while KEW acts only as an activator. They form homo- and heterodimers, and then collaboraUvely regulate their expression at the transcription level. Furthermore, we identified two types of post-transcriptional modifications, phosphor- ylation and ATP/GTP binding, both of which could affect their transcriptional activities. Mutations in ATP/ GTP binding motifs of SQU and KEW lead to failure of phosphorylation, and transgenic plants bearing the mutant proteins display defective DV asymmetric flower development, indicating that the two conjugate modifications are essential for their diverse functions. Altogether, SQU and KEW activities are precisely modulated at both transcription and post-transcription levels, which might link DV asymmetric flower development to different physiological status and/or signaling pathways.

Knockdown of LjALD1, AGD2‐like defense response protein 1, influences plant growth and nodulation in Lotus japonicus

The discovery of the enzyme L,L‐diaminopimelate aminotransferase（LL‐DAP‐AT, EC 2.6.1.83） uncovered a unique step in the L‐lysine biosynthesis pathway in plants. In Arabidopsis thaliana, LL‐DAP‐AT has been shown to play a key role in plant‐pathogen interactions by regulation of the salicylic acid（SA） signaling pathway. Here, a full‐length cDNA of LL‐DAP‐AT named as LjALD1 from Lotus japonicus（Regel）Larsen was isolated. The deduced amino acid sequence shares 67% identity with the Arabidopsis aminotransferase AGD2‐LIKE DEFENSE RESPONSE PROTEIN1（AtALD1） and is predicted to contain the same key elements： a conserved aminotransferase domain and a pyridoxal‐5＇‐phosphate cofactor binding site.Quantitative real‐time PCR analysis showed that LjALD1 was expressed in all L. japonicus tissues tested, being strongest in nodules. Expression was induced in roots that had been infected with the symbiotic rhizobium Mesorhizobium loti or treated with SA agonist benzo‐（1, 2, 3）‐thiadiazole‐7‐carbothioic Researchacid. LjALD1 Knockdown exhibited a lower SA content, an increased number of infection threads and nodules, and a slight reduction in nodule size. In addition, compared with wild‐type,root growth was increased and shoot growth was suppressed in LjALD1 RNAi plant lines. These results indicate that LjALD1 may play important roles in plant development and nodulation via SA signaling in L. japonicus.

Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation

The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands;however,such mechanisms regulating nodulation factor(NF)receptor(NFR)-mediated perception of NFs to establish symbiosis remain unclear.In this study,we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1(NIRE1)in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus.We demonstrated that NiRE1 has a dual function in this regulatory process.It associates with both NFR1 and NFR5,facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation.However,following rhizobial inoculation,NFR1 phosphorylates NIRE1ata conserved residue,Tyr-109,inducing a functional switch in NIRE1,which enables NIRE1tomediateK63-linkedpolyubiquitination,thereby stabilizing NFR1/NFR5 in infected root cells.The introduction of phospho-dead NIRE1Y1osF leads to delayed nodule development,underscoring the significance of phosphorylation at Tyr-1o9 in orchestrating symbiotic processes.Conversely,expression of the phospho-mimic NIRE1Y0E results in the formation of spontaneous nodules in L.japonicus,further emphasizing the critical role of the phosphorylation-dependent functional switch in NiRE1.In summary,these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylationdependent functional switch to dynamically and precisely regulate NF receptor protein levels.