MtPIN4 plays critical roles in amino acid biosynthesis and metabolism of seed in Medicago truncatula.

The regulation of seed development is critical for determining crop yield. Auxins are vital phytohormones that play roles in various aspects of plant growth and development. However, its role in amino acid biosynthesis and metabolism in seeds is not fully understood. In this study, we identified a mutant with small seeds through forward genetic screening in Medicago truncatula. The mutated gene encodes MtPIN4, an ortholog of PIN1. Using molecular approaches and integrative omics analyses, we discovered that auxin and amino acid content significantly decreased in mtpin4 seeds, highlighting the role of MtPIN4-mediated auxin distribution in amino acid biosynthesis and metabolism. Furthermore, genetic analysis revealed that the three orthologs of PIN1 have specific and overlapping functions in various developmental processes in M. truncatula. Our findings emphasize the significance of MtPIN4 in seed development and offer insights into the molecular mechanisms governing the regulation of seed size in crops. This knowledge could be applied to enhance crop quality by targeted manipulation of seed protein regulatory pathways.

Rewiring of a KNOXI regulatory network mediated by UFO underlies the compound leaf development in Medicago truncatula.

Class I KNOTTED-like homeobox (KNOXI) genes are parts of the regulatory network that control the evolutionary diversification of leaf morphology. Their specific spatiotemporal expression patterns in developing leaves correlate with the degrees of leaf complexity between simple-leafed and compound-leafed species. However, KNOXI genes are not involved in compound leaf formation in several legume species. Here, we identify a pathway for dual repression of MtKNOXI function in Medicago truncatula. PINNATE-LIKE PENTAFOLIATA1 (PINNA1) represses the expression of MtKNOXI, while PINNA1 interacts with MtKNOXI and sequesters it to the cytoplasm. Further investigations reveal that UNUSUAL FLORAL ORGANS (MtUFO) is the direct target of MtKNOXI, and mediates the transition from trifoliate to pinnate-like pentafoliate leaves. These data suggest a new layer of regulation for morphological diversity in compound-leafed species, in which the conserved regulators of floral development, MtUFO, and leaf development, MtKNOXI, are involved in variation of pinnate-like compound leaves in M. truncatula.

Zadek Osteotomy, a Good Treatment Option for Refractory Haglund's Deformity.

INTRODUCTION: Haglund's deformity (a prominence in the posterosuperior aspect of the calcaneum) is a known cause of posterior heel pain. Surgery is reserved for patients after failed conservative treatment. Zadek osteotomy is a dorsal-closing wedge osteotomy that reduces the posterior heel prominence. Zadek osteotomy is becoming a favored procedure, however, there are still relatively few studies focusing on patient-reported outcomes. Our main aim was to assess patient-reported outcomes following the Zadek osteotomy in refractory Haglund's deformity. Our secondary aim was to evaluate the correlation between patient outcomes and changes in their pre and postoperative Fowler-Philip and calcaneal pitch angles. METHODS: We conducted a retrospective review of 19 patients (20 heels) who underwent Zadek osteotomy by a single surgeon at a tertiary hospital over six years. Patient-reported outcomes were collected preoperatively and at 12 months postoperatively using the validated Manchester-Oxford foot questionnaire (MOXFQ) scoring system. We also calculated the difference in their pre and postoperative Fowler-Philip angles and calcaneal pitch using the picture archiving communication system. RESULTS: There was an average improvement of 108 points in the MOXFQ score at 12 months (P<0.05). There was no statistically significant change in calcaneal pitch. However, the Fowler-Phillip angle dropped with an average of 11.4 º (P<0.05). A decrease in the Fowler-Philip angle does improve patient-related outcome measurement scores, however, the relationship is not directly proportional with "r" measured at 0.23. CONCLUSION: Our results show that Zadek osteotomy is a useful procedure to consider in patients with symptomatic refractory Haglund's deformity, with an improvement in patient outcomes at 12 months. However, further studies are needed to give stronger evidence for the efficacy of this procedure and its radiological correlations.

Roles of very long-chain fatty acids in compound leaf patterning in Medicago truncatula.

Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.

Developmental Analysis of Compound Leaf Development in Arachis hypogaea.

Leaves are the primary photosynthetic structures, while photosynthesis is the direct motivation of crop yield formation. As a legume plant, peanut (Arachis hypogaea) is one of the most economically essential crops as well as an important source of edible oil and protein. The leaves of A. hypogaea are in the tetrafoliate form, which is different from the trifoliate leaf pattern of Medicago truncatula, a model legume species. In A. hypogaea, an even-pinnate leaf with a pair of proximal and distal leaflets was developed; however, only a single terminal leaflet and a pair of lateral leaflets were formed in the odd-pinnate leaf in M. truncatula. In this study, the development of compound leaf in A. hypogaea was investigated. Transcriptomic profiles revealed that the common and unique differentially expressed genes were identified in a proximal leaflet and a distal leaflet, which provided a research route to understand the leaf development in A. hypogaea. Then, a naturally occurring mutant line with leaf developmental defects in A. hypogaea was obtained, which displayed a pentafoliate form with an extra terminal leaflet. The characterization of the mutant indicated that cytokinin and class I KNOTTED-LIKE HOMEOBOX were involved in the control of compound leaf pattern in A. hypogaea. These results expand our knowledge and provide insights into the molecular mechanism underlying the formation of different compound leaf patterns among species.

Developmental Analysis of the GATA Factor HANABA TARANU Mutants in Medicago truncatula Reveals Their Roles in Nodule Formation.

Formation of nodules on legume roots results from symbiosis with rhizobial bacteria. Here, we identified two GATA transcription factors, MtHAN1 and MtHAN2, in Medicago truncatula, which are the homologs of HANABA TARANU (HAN) and HANABA TARANU LIKE in Arabidopsis thaliana. Our analysis revealed that MtHAN1 and MtHAN2 are expressed in roots and shoots including the root tip and nodule apex. We further show that MtHAN1 and MtHAN2 localize to the nucleus where they interact and that single and double loss-of-function mutants of MtHAN1 and MtHAN2 did not show any obvious phenotype in flower development, suggesting their role is different than their closest Arabidopsis homologues. Investigation of their symbiotic phenotypes revealed that the mthan1 mthan2 double mutant develop twice as many nodules as wild type, revealing a novel biological role for GATA transcription factors. We found that HAN1/2 transcript levels respond to nitrate treatment like their Arabidopsis counterparts. Global gene transcriptional analysis by RNA sequencing revealed different expression genes enriched for several pathways important for nodule development including flavonoid biosynthesis and phytohormones. In addition, further studies suggest that MtHAN1 and MtHAN2 are required for the expression of several nodule-specific cysteine-rich genes, which they may activate directly, and many peptidase and peptidase inhibitor genes. This work expands our knowledge of the functions of MtHANs in plants by revealing an unexpected role in legume nodulation.

Interaction between the MtDELLA-MtGAF1 Complex and MtARF3 Mediates Transcriptional Control of MtGA3ox1 to Elaborate Leaf Margin Formation in Medicago truncatula.

The molecular mechanisms underlying the diversity of leaf shapes have been of great interest to researchers. Leaf shape depends on the pattern of serrations and the degree of indentation of leaf margins. Multiple transcription factors and hormone signaling pathways are involved in this process. In this study, we characterized the developmental roles of SMALL AND SERRATED LEAF (SSL) by analyzing a recessive mutant in the model legume Medicago truncatula. An ortholog of Arabidopsis thaliana GA3-oxidase 1 (GA3ox1), MtGA3ox1/SSL, is required for GA biosynthesis. Loss of function in MtGA3ox1 results in the small plant and lateral organs. The prominent phenotype of the mtga3ox1 mutant is a more pronounced leaf margin, indicating the critical role of GA level in leaf margin formation. Moreover, 35S:MtDELLA2ΔDELLA and 35S:MtARF3 transgenic plants display leaves with a deeply wavy margin, which resembles those of mtga3ox1. Further investigations show that MtGA3ox1 is under the control of MtDELLA1/2/3-MtGAF1 complex-dependent feedback regulation. Further, MtARF3 behaves as a competitive inhibitor of MtDELLA2/3-MtGAF1 complexes to repress the expression of MtGA3ox1 indirectly. These findings suggest that GA feedback regulatory circuits play a fundamental role in leaf margin formation, in which the posttranslational interaction between transcription factors functions as an additional feature.

MtPIN1 and MtPIN3 Play Dual Roles in Regulation of Shade Avoidance Response under Different Environments in Medicago truncatula.

Polar auxin transport mediated by PIN-FORMED (PIN) proteins is critical for plant growth and development. As an environmental cue, shade stimulates hypocotyls, petiole, and stem elongation by inducing auxin synthesis and asymmetric distributions, which is modulated by PIN3,4,7 in Arabidopsis. Here, we characterize the MtPIN1 and MtPIN3, which are the orthologs of PIN3,4,7, in model legume species Medicago truncatula. Under the low Red:Far-Red (R:FR) ratio light, the expression of MtPIN1 and MtPIN3 is induced, and shadeavoidance response is disrupted in mtpin1 mtpin3 double mutant, indicating that MtPIN1 and MtPIN3 have a conserved function in shade response. Surprisingly, under the normal growth condition, mtpin1 mtpin3 displayed the constitutive shade avoidance responses, such as the elongated petiole, smaller leaf, and increased auxin and chlorophyll content. Therefore, MtPIN1 and MtPIN3 play dual roles in regulation of shadeavoidance response under different environments. Furthermore, these data suggest that PIN3,4,7 and its orthologs have evolved conserved and specific functions among species.

Systematic Analysis of Gibberellin Pathway Components in Medicago truncatula Reveals the Potential Application of Gibberellin in Biomass Improvement.

Gibberellins (GAs), a class of phytohormones, act as an essential natural regulator of plant growth and development. Many studies have shown that GA is related to rhizobial infection and nodule organogenesis in legume species. However, thus far, GA metabolism and signaling components are largely unknown in the model legume Medicago truncatula. In this study, a genome-wide analysis of GA metabolism and signaling genes was carried out. In total 29 components, including 8 MtGA20ox genes, 2 MtGA3ox genes, 13 MtGA2ox genes, 3 MtGID1 genes, and 3 MtDELLA genes were identified in M. truncatula genome. Expression profiles revealed that most members of MtGAox, MtGID1, and MtDELLA showed tissue-specific expression patterns. In addition, the GA biosynthesis and deactivation genes displayed a feedback regulation on GA treatment, respectively. Yeast two-hybrid assays showed that all the three MtGID1s interacted with MtDELLA1 and MtDELLA2, suggesting that the MtGID1s are functional GA receptors. More importantly, M. truncatula exhibited increased plant height and biomass by ectopic expression of the MtGA20ox1, suggesting that enhanced GA response has the potential for forage improvement.

Multilayered N-Glycoproteome Profiling Reveals Highly Heterogeneous and Dysregulated Protein N-Glycosylation Related to Alzheimer's Disease.

Protein N-glycosylation is ubiquitous in the brain and is closely related to cognition and memory. Alzheimer's disease (AD) is a multifactorial disorder that lacks a clear pathogenesis and treatment. Aberrant N-glycosylation has been suggested to be involved in AD pathology. However, the systematic variations in protein N-glycosylation and their roles in AD have not been thoroughly investigated due to technical challenges. Here, we applied multilayered N-glycoproteomics to quantify the global protein expression levels, N-glycosylation sites, N-glycans, and site-specific N-glycopeptides in AD (APP/PS1 transgenic) and wild-type mouse brains. The N-glycoproteomic landscape exhibited highly complex site-specific heterogeneity in AD mouse brains. The generally dysregulated N-glycosylation in AD, which involved proteins such as glutamate receptors as well as fucosylated and oligomannose glycans, were explored by quantitative analyses. Furthermore, functional studies revealed the crucial effects of N-glycosylation on proteins and neurons. Our work provides a systematic multilayered N-glycoproteomic strategy for AD and can be applied to diverse biological systems.

HEADLESS Regulates Auxin Response and Compound Leaf Morphogenesis in Medicago truncatula.

WUSCHEL (WUS) is thought to be required for the establishment of the shoot stem cell niche in Arabidopsis thaliana. HEADLESS (HDL), a gene that encodes a WUS-related homeobox family transcription factor, is thought to be the Medicago truncatula ortholog of the WUS gene. HDL plays conserved roles in shoot apical meristem (SAM) and axillary meristem (AM) maintenance. HDL is also involved in compound leaf morphogenesis in M. truncatula; however, its regulatory mechanism has not yet been explored. Here, the significance of HDL in leaf development was investigated. Unlike WUS in A. thaliana, HDL was transcribed not only in the SAM and AM but also in the leaf. Both the patterning of the compound leaves and the shape of the leaf margin in hdl mutant were abnormal. The transcriptional profile of the gene SLM1, which encodes an auxin efflux carrier, was impaired and the plants' auxin response was compromised. Further investigations revealed that HDL positively regulated auxin response likely through the recruitment of MtTPL/MtTPRs into the HDL repressor complex. Its participation in auxin-dependent compound leaf morphogenesis is of interest in the context of the functional conservation and neo-functionalization of the products of WUS orthologs.

Genome-wide characterization of SPL family in Medicago truncatula reveals the novel roles of miR156/SPL module in spiky pod development.

BACKGROUND: SQUAMOSA Promoter Binding Protein-Likes (SPLs) proteins are plant-specific transcription factors that play many crucial roles in plant growth and development. However, there is little information about SPL family in the model legume Medicago truncatula. RESULTS: In this study, a total of 23 MtSPL genes were identified in M. truncatula genome, in which 17 of the MtSPLs contained the putative MtmiR156 binding site at the coding or 3' UTR regions. Tissue-specific expression pattern analysis showed that most MtmiR156-targeted MtSPLs were highly expressed in seed and pod. The observation of MtmiR156B-overexpressing plants reveals that MtmiR156/MtSPL modules are not only involved in the development of leaves and branches, but also in the seed pod development, especially the formation of spine on pod. CONCLUSION: The spines on pods are developed in many plant species, which allow pods to adhere to the animals, and then be transported on the outside. This study sheds light on the new function of SPL family in seed dispersal by controlling the formation of spiky pod, and provides insights on understanding evolutionary divergence of the members of SPL gene family among plant species.

MtBZR1 Plays an Important Role in Nodule Development in Medicago truncatula.

Brassinosteroid (BR) is an essential hormone in plant growth and development. The BR signaling pathway was extensively studied, in which BRASSINAZOLE RESISTANT 1 (BZR1) functions as a key regulator. Here, we carried out a functional study of the homolog of BZR1 in Medicago truncatula R108, whose expression was induced in nodules upon Sinorhizobium meliloti 1021 inoculation. We identified a loss-of-function mutant mtbzr1-1 and generated 35S:MtBZR1 transgenic lines for further analysis at the genetic level. Both the mutant and the overexpression lines of MtBZR1 showed no obvious phenotypic changes under normal growth conditions. After S. meliloti 1021 inoculation, however, the shoot and root dry mass was reduced in mtbzr1-1 compared with the wild type, caused by partially impaired nodule development. The transcriptomic analysis identified 1319 differentially expressed genes in mtbzr1-1 compared with wild type, many of which are involved in nodule development and secondary metabolite biosynthesis. Our results demonstrate the role of MtBZR1 in nodule development in M. truncatula, shedding light on the potential role of BR in legume-rhizobium symbiosis.

AGLF provides C-function in floral organ identity through transcriptional regulation of AGAMOUS in Medicago truncatula.

Floral development is one of the model systems for investigating the mechanisms underlying organogenesis in plants. Floral organ identity is controlled by the well-known ABC model, which has been generalized to many flowering plants. Here, we report a previously uncharacterized MYB-like gene, AGAMOUS-LIKE FLOWER (AGLF), involved in flower development in the model legume Medicago truncatula Loss-of-function of AGLF results in flowers with stamens and carpel transformed into extra whorls of petals and sepals. Compared with the loss-of-function mutant of the class C gene AGAMOUS (MtAG) in M. truncatula, the defects in floral organ identity are similar between aglf and mtag, but the floral indeterminacy is enhanced in the aglf mutant. Knockout of AGLF in the mutants of the class A gene MtAP1 or the class B gene MtPI leads to an addition of a loss-of-C-function phenotype, reflecting a conventional relationship of AGLF with the canonical A and B genes. Furthermore, we demonstrate that AGLF activates MtAG in transcriptional levels in control of floral organ identity. These data shed light on the conserved and diverged molecular mechanisms that control flower development and morphology among plant species.

Genome-Wide Identification of TCP Family Transcription Factors in Medicago truncatula Reveals Significant Roles of miR319-Targeted TCPs in Nodule Development.

TCP proteins, the plant-specific transcription factors, are involved in the regulation of multiple aspects of plant development among different species, such as leaf development, branching, and flower symmetry. However, thus far, the roles of TCPs in legume, especially in nodulation are still not clear. In this study, a genome-wide analysis of TCP genes was carried out to discover their evolution and function in Medicago truncatula. In total, 21 MtTCPs were identified and classified into class I and class II, and the class II MtTCPs were further divided into two subclasses, CIN and CYC/TB1. The expression profiles of MtTCPs are dramatically different. The universal expression of class I MtTCPs was detected in all organs. However, the MtTCPs in CIN subclass were highly expressed in leaf and most of the members in CYC/TB1 subclass were highly expressed in flower. Such organ-specific expression patterns of MtTCPs suggest their different roles in plant development. In addition, most MtTCPs were down-regulated during the nodule development, except for the putative MtmiR319 targets, MtTCP3, MtTCP4, and MtTCP10A. Overexpression of MtmiR319A significantly reduced the expression level of MtTCP3/4/10A/10B and resulted in the decreased nodule number, indicating the important roles of MtmiR319-targeted MtTCPs in nodulation. Taken together, this study systematically analyzes the MtTCP gene family at a genome-wide level and their possible functions in nodulation, which lay the basis for further explorations of MtmiR319/MtTCPs module in association with nodule development in M. truncatula.

The TERT promoter mutation incidence is modified by germline TERT rs2736098 and rs2736100 polymorphisms in hepatocellular carcinoma.

Telomerase activation via induction of the catalytic component telomerase reverse transcriptase (TERT) plays essential roles in malignant transformation. TERT promoter-activating mutations were recently identified as a novel mechanism to activate telomerase in hepatocellular carcinoma (HCC) and many other malignancies. In addition, single nucleotide polymorphisms (SNPs) in the TERT rs2736098 and rs2736100 are significantly associated with cancer susceptibility. It is currently unclear whether different germline TERT variants modify TERT promoter mutations. Here we analyzed the TERT promoter status and genotyped the TERT SNPs at rs2736098 and rs2736100 in patients with HCC. Thirty percent of HCCs harbored TERT promoter mutations and there was a significant difference in rs2736098 and rs2736100 genotypes between wt and mutant TERT promoter-bearing HCC tumors (P = 0.007 and 0.018, respectively). For rs2736100, the cancer risk genotype CC was significantly associated with a reduced incidence of TERT promoter mutations compared to AA + AC variants [Odds ratio (OR): 0.181, 95% Confidence interval (CI): 0.0543-0.601, P = 0.004]. The rs2736098_CT genotype was significantly associated with the TERT promoter mutation-positive tumors compared to the TT genotype (OR: 5.391, 95% CI: 1.234-23.553, P = 0.025). These differences in genotype distribution did not differ between patients with a wt TERT promoter and controls. The presence of TERT promoter mutations was not associated with clinico-pathological variables. Taken together, the germline TERT genetic background may significantly affect the onset of TERT promoter mutations in HCCs, which provides a better understanding of HCC-related TERT promoter mutations and telomerase regulation in cancer.

Association Between the Telomerase rs2736098_TT Genotype and a Lower Risk of Chronic Hepatitis B and Cirrhosis in Chinese Males.

OBJECTIVES: Chronic hepatitis B (CHB) is caused by infection of hepatitis B virus (HBV) and liver cirrhosis (LC) is its most common complication. The accumulated evidence indicates a genetic context of HBV infection phenotypes. Here we determine a potential association of CHB/LC with the genetic variant of telomerase reverse transcriptase (TERT), a key player in aging including immune-senescence. METHODS: The study included 227 Chinese CHB patients and 315 sex/age-matched healthy controls. TERT rs2736098 and rs2736100 genotyping was performed using pre-designed TaqMan SNP genotyping assay kits. Leukocyte telomere length (LTL) was determined using quantitative PCR. RESULTS: The rs2736098_CT/CC genotypes were significantly associated with risk of CHB compared to the TT one (OR 2.265, 95% CI 1.202-4.269, P=0.015). A similar association was also found in CHB patients with cirrhosis (CT/CC vs TT: OR 2.398, 95% CI 1.168-4.922, P=0.02). Further analyses showed that the rs2736098_TT genotype difference occurred between male controls and patients (P=0.008) and male CT/CC-carriers exhibited highly increased risk of CHB compared to male controls (CT+CC vs TT, OR 3.182, 95% CI 1.350-7.500, P=0.01). There was no difference in the rs2736100 variants between controls and CHB patients. LTL was not different between cases and controls. CONCLUSIONS: The TERT rs2736098_TT genotype is associated with a lower CHB and LC risk in Chinese males, which may have implications in CHB pathogenesis and prevention.