The HD-ZIP IV transcription factor GLABRA2 acts as an activator for proanthocyanidin biosynthesis in Medicago truncatula seed coat.

Proanthocyanidins (PAs), a group of flavonoids, are found in leaves, flowers, fruits, and seed coats of many plant species. PAs are primarily composed of epicatechin units in the seed coats of the model legume species, Medicago truncatula. It can be synthesized from two separate pathways, the leucoanthocyanidin reductase (MtLAR) pathway and the anthocyanidin synthase (MtANS) pathway, which produce epicatechin through anthocyanidin reductase (MtANR). These pathways are mainly controlled by the MYB-bHLH-WD40 (MBW) ternary complex. Here, we characterize a class IV homeodomain-leucine zipper (HD-ZIP IV) transcription factor, GLABRA2 (MtGL2), which contributes to PA biosynthesis in the seed coat of M. truncatula. Null mutation of MtGL2 results in dark brown seed coat, which is accompanied by reduced PAs accumulation and increased anthocyanins content. The MtGL2 gene is predominantly expressed in the seed coat during the early stages of seed development. Genetic and molecular analyses indicate that MtGL2 positively regulates PA biosynthesis by directly activating the expression of MtANR. Additionally, our results show that MtGL2 is strongly induced by the MBW activator complexes that are involved in PA biosynthesis. Taken together, our results suggest that MtGL2 acts as a novel positive regulator in PA biosynthesis, expanding the regulatory network and providing insights for genetic engineering of PA production.

GmDFB1, an ARM-repeat superfamily protein, regulates floral organ identity through repressing siRNA- and miRNA-mediated gene silencing in soybean.

The development of flowers in soybean (Glycine max) is essential for determining the yield potential of the plant. Gene silencing pathways are involved in modulating flower development, but their full elucidation is still incomplete. Here, we conducted a forward genetic screen and identified an abnormal flower mutant, deformed floral bud1-1 (Gmdfb1-1), in soybean. We mapped and identified the causal gene, which encodes a member of the armadillo (ARM)-repeat superfamily. Using small RNA sequencing (sRNA-seq), we found an abnormal accumulation of small interfering RNAs (siRNAs) and microRNA (miRNAs) in the Gmdfb1 mutants. We further demonstrated that GmDFB1 interacts with the RNA exosome cofactor SUPER KILLER7 (GmSKI7). Additionally, GmDFB1 interacts with the PIWI domain of ARGONAUTE 1 (GmAGO1) to inhibit the cleavage efficiency on the target genes of sRNAs. The enhanced gene silencing mediated by siRNA and miRNA in the Gmdfb1 mutants leads to the downregulation of their target genes associated with flower development. This study revealed the crucial role of GmDFB1 in regulating floral organ identity in soybean probably by participating in two distinct gene silencing pathways.

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.

The long noncoding RNA LAL contributes to salinity tolerance by modulating LHCB1s' expression in Medicago truncatula.

Long non-coding RNAs (lncRNAs) are abundant in plants, however, their regulatory roles remain unclear in most biological processes, such as response in salinity stress which is harm to plant production. Here we show a lncRNA in Medicago truncatula identified from salt-treated Medicago truncatula is important for salinity tolerance. We name the lncRNA LAL, LncRNA ANTISENSE to M. truncatula LIGHT-HARVESTING CHLOROPHYLL A/B BINDING (MtLHCB) genes. LAL is an antisense to four consecutive MtLHCB genes on chromosome 6. In salt-treated M. truncatula, LAL is suppressed in an early stage but induced later; this pattern is opposite to that of the four MtLHCBs. The lal mutants show enhanced salinity tolerance, while overexpressing LAL disrupts this superior tolerance in the lal background, which indicates its regulatory role in salinity response. The regulatory role of LAL on MtLHCB1.4 is further verified by transient co-expression of LAL and MtLHCB1.4-GFP in tobacco leaves, in which the cleavage of MtLHCB1.4 and production of secondary interfering RNA is identified. This work demonstrates a lncRNA, LAL, functioning as a regulator that fine-tunes salinity tolerance via regulating MtLHCB1s' expression in M. truncatula.

Nyctinastic movement in legumes: Developmental mechanisms, factors and biological significance.

In legumes, a common phenomenon known as nyctinastic movement is observed. This movement involves the horizontal expansion of leaves during the day and relative vertical closure at night. Nyctinastic movement is driven by the pulvinus, which consists of flexor and extensor motor cells. The turgor pressure difference between these two cell types generates a driving force for the bending and deformation of the pulvinus. This review focuses on the developmental mechanisms of the pulvinus, the factors affecting nyctinastic movement, and the biological significance of this phenomenon in legumes, thus providing a reference for further research on nyctinastic movement.

The role of Class â ¡ KNOX family in controlling compound leaf patterning in Medicago truncatula.

Compound leaf development requires the coordination of genetic factors, hormones, and other signals. In this study, we explored the functions of Class Ⅱ KNOTTED-like homeobox (KNOXII) genes in the model leguminous plant Medicago truncatula. Phenotypic and genetic analyses suggest that MtKNOX4, 5 are able to repress leaflet formation, while MtKNOX3, 9, 10 are not involved in this developmental process. Further investigations have shown that MtKNOX4 represses the CK signal transduction, which is downstream of MtKNOXⅠ-mediated CK biosynthesis. Additionally, two boundary genes, FUSED COMPOUND LEAF1 (orthologue of Arabidopsis Class M KNOX) and NO APICAL MERISTEM (orthologue of Arabidopsis CUP-SHAPED COTYLEDON), are necessary for MtKNOX4-mediated compound leaf formation. These findings suggest, that among the members of MtKNOXⅡ, MtKNOX4 plays a crucial role in integrating the CK pathway and boundary regulators, providing new insights into the roles of MtKNOXⅡ in regulating the elaboration of compound leaves in M. truncatula.

Late Elongated Hypocotyl Positively Regulates Salt Stress Tolerance in Medicago truncatula.

Abiotic stress, such as drought, osmotic, and salinity stresses, seriously affects plant growth and crop production. Studying stress-resistant genes that enhance plant stress tolerance is an efficient way to facilitate the breeding of crop species with high stress tolerance. In this study, we reported that the core circadian clock component, the LATE ELONGATED HYPOCOTYL (LHY) orthologue MtLHY, plays a positive role in salt stress response in Medicago truncatula. The expression of MtLHY was induced by salt stress, and loss-of-function mutants of MtLHY were shown to be hypersensitive to salt treatment. However, overexpression of MtLHY improved salt stress tolerance through a higher accumulation of flavonoids. Consistently, exogenous flavonol application improved the salt stress tolerance in M. truncatula. Additionally, MtLHY was identified as a transcriptional activator of the flavonol synthase gene, MtFLS. Our findings revealed that MtLHY confers plant salt stress tolerance, at least by modulating the flavonoid biosynthesis pathway, which provides insight into salt stress tolerance that links the circadian clock with flavonoid biosynthesis.

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.

Genome-wide characterization of AINTEGUMENTA-LIKE family in Medicago truncatula reveals the significant roles of AINTEGUMENTAs in leaf growth.

AINTEGUMENTA-LIKE (AIL) transcription factors are widely studied and play crucial roles in plant growth and development. However, the functions of the AIL family in legume species are largely unknown. In this study, 11 MtAIL genes were identified in the model legume Medicago truncatula, of which four of them are MtANTs. In situ analysis showed that MtANT1 was highly expressed in the shoot apical meristem (SAM) and leaf primordium. Characterization of mtant1 mtant2 mtant3 mtant4 quadruple mutants and MtANT1-overexpressing plants revealed that MtANTs were not only necessary but also sufficient for the regulation of leaf size, and indicated that they mainly function in the regulation of cell proliferation during secondary morphogenesis of leaves in M. truncatula. This study systematically analyzed the MtAIL family at the genome-wide level and revealed the functions of MtANTs in leaf growth. Thus, these genes may provide a potential application for promoting the biomass of legume forages.

The formation of stipule requires the coordinated actions of the legume orthologs of Arabidopsis BLADE-ON-PETIOLE and LEAFY.

Stipule morphology is a classical botanical key character used in plant identification. Stipules are considerably diverse in size, function and architecture, such as leaf-like stipules, spines or tendrils. However, the molecular mechanism that regulates stipule identity remains largely unknown. We isolated mutants with abnormal stipules. The mutated gene encodes the NODULE ROOT1 (MtNOOT1), which is the ortholog of BLADE-ON-PETIOLE (BOP) in Medicago truncatula. We also obtained mutants of MtNOOT2, the homolog of MtNOOT1, but they do not show obvious defects in stipules. The mtnoot1 mtnoot2 double mutant shows a higher proportion of transformation from stipules to leaflet-like stipules than the single mutants, suggesting that they redundantly determine stipule identity. Further investigations show that MtNOOTs control stipule initiation together with SINGLE LEAFLET1 (SGL1), which functions in development of lateral leaflets. Increasing SGL1 activity in mtnoot1 mtnoot2 is sufficient for the transformation of stipules to leaves. Moreover, MtNOOTs inhibit SGL1 expression during stipule development, which is probably conserved in legume species. Our study proposes a genetic regulatory model for stipule development, specifically with regard to the MtNOOTs-SGL1 module, which functions in two phases of stipule development, first in the control of stipule initiation and second in stipule patterning.

The Conserved and Specific Roles of the LUX ARRHYTHMO in Circadian Clock and Nodulation.

LUX ARRHYTHMO (LUX) plays a key role in circadian rhythms and flowering. Here, we identified the MtLUX gene which is the putative ortholog of LUX in Medicago truncatula. The roles of MtLUX, in both the nodulation belowground and leaf movement aboveground, were investigated by characterizing a loss-of-function mtlux mutant. MtLUX was required for the control of flowering time under both long-day and short-day conditions. Further investigations showed that the early flowering in the mtlux mutant was correlated with the elevated expression level of the MtFTa1 gene but in a CO-like independent manner. MtLUX played a conserved role in the regulatory interactions with MtLHY, MtTOC1, and MtPRR genes, which is similar to those in other species. Meanwhile, the unexpected functions of MtLUX were revealed in nodule formation and nyctinastic leaf movement, probably through the indirect regulation in MtLHY. Its participation in nodulation is of interest in the context of functional conservation and the neo-functionalization of the products of LUX orthologs.

Successful treatment of an infant with congenital bile acid synthesis disorder type 3 by ursodeoxycholic acid: a case report.

BACKGROUND: Congenital bile acid synthesis disorder type 3 caused by oxysterol 7α-hydroxylase deficiency is an extremely rare genetic liver disease. As it may cause rapid progression to end-stage liver disease, a high cautiousness in diagnosis and early treatment are required. Here we describe the first case of congenital bile acid synthesis disorder type 3 in China that was confirmed by genetic analysis. CASE PRESENTATION: A 5-month-old Chinese male infant suffered skin yellowing since birth. The patient showed significantly increased alanine transaminase, aspartate transaminase, and total and direct bilirubin levels, and enlarged liver at admission. Whole-exome sequencing confirmed homozygous mutation in the CYB7B1 gene that encodes oxysterol 7α-hydroxylase. Ursodeoxycholic acid treatment significantly mitigated the condition of the patient and lowered biochemical indicators. Unfortunately, the patient developed septicemia and gave up treatment. CONCLUSIONS: The patient was successfully treated with ursodeoxycholic acid, which has not been reported previously. Ursodeoxycholic acid replacement therapy is an effective and affordable treatment for congenital bile acid synthesis disorder type 3 caused by oxysterol 7α-hydroxylase deficiency.

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.

Integrated regulation of periclinal cell division by transcriptional module of BZR1-SHR in Arabidopsis roots.

The timing and extent of cell division are crucial for the correct patterning of multicellular organism. In Arabidopsis, root ground tissue maturation involves the periclinal cell division of the endodermis to generate two cell layers: endodermis and middle cortex. However, the molecular mechanism underlying this pattern formation remains unclear. Here, we report that phytohormone brassinosteroid (BR) and redox signal hydrogen peroxide (H2 O2 ) interdependently promote periclinal division during root ground tissue maturation by regulating the activity of SHORT-ROOT (SHR), a master regulator of root growth and development. BR-activated transcription factor BRASSINAZOLE RESISTANT1 (BZR1) directly binds to the promoter of SHR to induce its expression, and physically interacts with SHR to increase the transcripts of RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs) and elevate the levels of H2 O2 , which feedback enhances the interaction between BZR1 and SHR. Additionally, genetic analysis shows that SHR is required for BZR1-promoted periclinal division, and BZR1 enhances the promoting effects of SHR on periclinal division. Together, our finding reveals that the transcriptional module of BZR1-SHR fine-tunes periclinal division during root ground tissue maturation in response to hormone and redox signals.

LATE MERISTEM IDENTITY1 regulates leaf margin development via the auxin transporter gene SMOOTH LEAF MARGIN1.

Plant leaves have evolved into diverse shapes and LATE MERISTEM IDENTITY1 (LMI1) and its putative paralogous genes encode homeodomain leucine zipper transcription factors that are proposed evolutionary hotspots for the regulation of leaf development in plants. However, the LMI1-mediated regulatory mechanism underlying leaf shape formation is largely unknown. MtLMI1a and MtLMI1b are putative orthologs of LMI1 in the model legume barrelclover (Medicago truncatula). Here, we investigated the role of MtLMI1a and MtLMI1b in leaf margin morphogenesis by characterizing loss-of-function mutants. MtLMI1a and MtLMI1b are expressed along leaf margin in a near-complementary pattern, and they redundantly promote development of leaf margin serrations, as revealed by the relatively smooth leaf margin in their double mutants. Moreover, MtLMI1s directly activate expression of SMOOTH LEAF MARGIN1 (SLM1), which encodes an auxin efflux carrier, thereby regulating auxin distribution along the leaf margin. Further analysis indicates that MtLMI1s genetically interact with NO APICAL MERISTEM (MtNAM) and the ARGONAUTE7 (MtAGO7)-mediated trans-acting short interfering RNA3 (TAS3 ta-siRNA) pathway to develop the final leaf margin shape. The participation of MtLMI1s in auxin-dependent leaf margin formation is interesting in the context of functional conservation. Furthermore, the diverse expression patterns of LMI1s and their putative paralogs within key domains are important drivers for functional specialization, despite their functional equivalency 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.

Brassinosteroid homeostasis is critical for the functionality of the Medicago truncatula pulvinus.

Many plant species open their leaves during the daytime and close them at night as if sleeping. This leaf movement is known as nyctinasty, a unique and intriguing phenomenon that been of great interest to scientists for centuries. Nyctinastic leaf movement occurs widely in leguminous plants, and is generated by a specialized motor organ, the pulvinus. Although a key determinant of pulvinus development, PETIOLULE-LIKE PULVINUS (PLP), has been identified, the molecular genetic basis for pulvinus function is largely unknown. Here, through an analysis of knockout mutants in barrelclover (Medicago truncatula), we showed that neither altering brassinosteroid (BR) content nor blocking BR signal perception affected pulvinus determination. However, BR homeostasis did influence nyctinastic leaf movement. BR activity in the pulvinus is regulated by a BR-inactivating gene PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1), which is directly activated by PLP. A comparative analysis between M. truncatula and the non-pulvinus forming species Arabidopsis and tomato (Solanum lycopersicum) revealed that PLP may act as a factor that associates with unknown regulators in pulvinus determination in M. truncatula. Apart from exposing the involvement of BR in the functionality of the pulvinus, these results have provided insights into whether gene functions among species are general or specialized.