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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Functional characterization of PETIOLULE-LIKE PULVINUS (PLP) gene in abscission zone development in Medicago truncatula and its application to genetic improvement of alfalfa.

Alfalfa (Medicago sativa L.) is one of the most important forage crops throughout the world. Maximizing leaf retention during the haymaking process is critical for achieving superior hay quality and maintaining biomass yield. Leaf abscission process affects leaf retention. Previous studies have largely focused on the molecular mechanisms of floral organ, pedicel and seed abscission but scarcely touched on leaf and petiole abscission. This study focuses on leaf and petiole abscission in the model legume Medicago truncatula and its closely related commercial species alfalfa. By analysing the petiolule-like pulvinus (plp) mutant in M. truncatula at phenotypic level (breakstrength and shaking assays), microscopic level (scanning electron microscopy and cross-sectional analyses) and molecular level (expression level and expression pattern analyses), we discovered that the loss of function of PLP leads to an absence of abscission zone (AZ) formation and PLP plays an important role in leaflet and petiole AZ differentiation. Microarray analysis indicated that PLP affects abscission process through modulating genes involved in hormonal homeostasis, cell wall remodelling and degradation. Detailed analyses led us to propose a functional model of PLP in regulating leaflet and petiole abscission. Furthermore, we cloned the PLP gene (MsPLP) from alfalfa and produced RNAi transgenic alfalfa plants to down-regulate the endogenous MsPLP. Down-regulation of MsPLP results in altered pulvinus structure with increased leaflet breakstrength, thus offering a new approach to decrease leaf loss during alfalfa haymaking process.

From genes to networks: The genetic control of leaf development.

Substantial diversity exists for both the size and shape of the leaf, the main photosynthetic organ of flowering plants. The two major forms of leaf are simple leaves, in which the leaf blade is undivided, and compound leaves, which comprise several leaflets. Leaves form at the shoot apical meristem from a group of undifferentiated cells, which first establish polarity, then grow and differentiate. Each of these processes is controlled by a combination of transcriptional regulators, microRNAs and phytohormones. The present review documents recent advances in our understanding of how these various factors modulate the development of both simple leaves (focusing mainly on the model plant Arabidopsis thaliana) and compound leaves (focusing mainly on the model legume species Medicago truncatula).

Transforming compound leaf patterning by manipulating REVOLUTA in Medicago truncatula.

Leaves are derived from the shoot apical meristem with three distinct axes: dorsoventral, proximodistal and mediolateral. Different regulators are involved in the establishment of leaf polarity. Members of the class III homeodomain-leucine zipper (HD-ZIPIII) gene family are critical players in the determination of leaf adaxial identity mediated by microRNA165/166. However, their roles in compound leaf development are still unclear. By screening of a retrotransposon-tagged mutant population of the model legume plant Medicago truncatula, a mutant line with altered leaflet numbers was isolated and characterized. Mutant leaves partially lost their adaxial identity. Leaflet numbers in the mutant were increased along the proximodistal axis, showing pinnate pentafoliate leaves in most cases, in contrast to the trifoliate leaves of the wild type. Detailed characterization revealed that a lesion in a HD-ZIPIII gene, REVOLUTA (MtREV1), resulted in the defects of the mutant. Overexpression of MtMIR166-insensitive MtREV1 led to adaxialized leaves and ectopic leaflets along the dorsoventral axis. Accompanying the abnormal leaf patterning, the free auxin content was affected. Our results demonstrate that MtREV1 plays a key role in determination of leaf adaxial-abaxial polarity and compound leaf patterning, which is associated with proper auxin homeostasis.

Phospho-Mutant Activity Assays Provide Evidence for the Negative Regulation of Transcriptional Regulator PRE1 by Phosphorylation.

The PACLOBUTRAZOL-RESISTANCE (PRE) gene family encodes a group of atypical helix-loop-helix (HLH) proteins that act as the major hub integrating a wide range of environmental and hormonal signals to regulate plant growth and development. PRE1, as a positive regulator of cell elongation, activates HBI1 DNA binding by sequestering its inhibitor IBH1. Furthermore, PRE1 can be phosphorylated at Ser-46 and Ser-67, but how this phosphorylation regulates the functions of PRE1 remains unclear. Here, we used a phospho-mutant activity assay to reveal that the phosphorylation at Ser-67 negatively regulates the functions of PRE1 on cell elongation. Both of mutations of serine 46, either to phospho-dead alanine or phospho-mimicking glutamic acid, had no significant effects on the functions of PRE1. However, the mutation of serine 67 to glutamic acid (PRE1S67E-Ox), but not alanine (PRE1S67A-Ox), significantly reduced the promoting effects of PRE1 on cell elongation. The mutation of Ser-67 to Glu-67 impaired the interaction of PRE1 with IBH1 and resulted in PRE1 failing to inhibit the interaction between IBH1 and HBI1, losing the ability to induce the expression of the subsequent cell elongation-related genes. Furthermore, we showed that PRE1-Ox and PRE1S67A-Ox both suppressed but PRE1S67E-Ox had no strong effects on the dwarf phenotypes of IBH1-Ox. Our study demonstrated that the PRE1 activity is negatively regulated by the phosphorylation at Ser-67.

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.

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.