Correction: Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues.

[This corrects the article DOI: 10.1007/s42994-023-00119-3.].

Transcriptomic Analysis of Self-Incompatibility in Alfalfa.

Alfalfa (Medicago sativa L.) is an important forage crop worldwide, but molecular genetics and breeding research in this species are hindered by its self-incompatibility (SI). Although the mechanisms underlying SI have been extensively studied in other plant families, SI in legumes, including alfalfa, remains poorly understood. Here, we determined that self-pollinated pollen tubes could germinate on the stigma of alfalfa, grow through the style, and reach the ovarian cavity, but the ovules collapsed ~48 h after self-pollination. A transcriptomic analysis of dissected pistils 24 h after self-pollination identified 941 differently expressed genes (DEGs), including 784 upregulated and 157 downregulated genes. A gene ontology (GO) analysis showed that the DEGs were highly enriched in functions associated with the regulation of pollen tube growth and pollen germination. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that pentose and glucuronate interconversion, plant hormone signal transduction, the spliceosome, and ribosomes might play important roles in SI. Our co-expression analysis showed that F-box proteins, serine/threonine protein kinases, calcium-dependent protein kinases (CDPKs), bHLHs, bZIPs, and MYB-related family proteins were likely involved in the SI response. Our study provides a catalog of candidate genes for further study to understand SI in alfalfa and related legumes.

Co-expression of GR79 EPSPS and GAT generates high glyphosate-resistant alfalfa with low glyphosate residues.

Weed competition seriously threatens the yield of alfalfa, the most important forage legume worldwide, thus generating herbicide-resistant alfalfa varieties is becoming a necessary cost-effective strategy to assist farmers for weed control. Here, we report the co-expression of plant codon-optimized forms of GR79 EPSPS (pGR79 EPSPS) and N-acetyltransferase (pGAT) genes, in alfalfa, via Agrobacterium-mediated transformation. We established that the pGR79 EPSPS-pGAT co-expression alfalfa lines were able to tolerate up to tenfold higher commercial usage of glyphosate and produced approximately ten times lower glyphosate residues than the conventional cultivar. Our findings generate an elite herbicide-resistant germplasm for alfalfa breeding and provide a promising strategy for developing high-glyphosate-resistant and low-glyphosate-residue forages. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00119-3.

Transcriptional repressor AGL79 positively regulates flowering time in Arabidopsis.

The MADS-box gene family is widely distributed in higher plants and the members of the angiosperm-specific APETALA1/FRUITFULL (AP1/FUL) subfamily plays important roles in the regulation of plant reproductive development. Recent studies revealed that the AP1/FUL subfamily member Dt2, VEGETATIVE1/PsFRUITFULc (VEG1/PsFULc) and MtFRUITFULc (MtFULc) are essential for the stem growth, branching and inflorescence development in legume species soybean (Glycine max), pea (Pisum sativum) and Medicago truncatula. However, the biological function of their homologue in Arabidopsis thaliana, AGAMOUS-LIKE 79 (AGL79), has not been well elucidated. In this study, we investigated the developmental roles of Arabidopsis AGL79 by CRISPR/Cas9-mutagenesis and molecular and physiological analyses. We found that AGL79 mainly acts as a transcriptional repressor and positively regulates Arabidopsis flowering time. We further revealed that AGL79 interacts with SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and represses the expression of TERMINAL FLOWER 1 (TFL1). Our results demonstrated the AGL79-mediated flowering regulation in Arabidopsis and added an additional layer of complexity to the understanding of flowering time regulation in dicot plants.

Biochemical Mechanism of Fresh-Cut Lotus (Nelumbo nucifera Gaertn.) Root with Exogenous Melatonin Treatment by Multiomics Analysis.

Browning limits the commercial value of fresh-cut lotus root slices. Melatonin has been reported to play crucial plant roles in growth and development. However, the mechanisms in repressing the browning of fresh-cut lotuses are still unclear. In this study, fresh-cut lotus root slices were treated with melatonin, the physical signs of browning were tested, and then the selected samples (0 d, 6 d, 12 d) were used in multiomics analysis. Fresh-cut lotus root slices with a thickness of 4 mm were soaked in a 40 mmol/L melatonin solution for 10 min; then, the slices were packed in pallets and packages and stored at 10 ± 1 °C. The results show that the 40 mmol/L melatonin selected for repressing the browning of lotus roots significantly delayed the decrease in water, total soluble solid content, and Vitamin C, decreased the growth of microorganisms, enhanced total phenolic content, improved total antioxidant capacity, and decreased ·OH, H2O2, and O2-· contents. Moreover, this treatment enhanced phenylalanine ammonialyase, polyphenol oxidase, superoxide dismutase, and catalase activities and reduced peroxidase activities and soluble quinones. NnSOD (104590242), NnCAT (104609297), and some NnPOD genes showed a similar transcript accumulation pattern with enzyme activity. It can be seen from these results that exogenous melatonin accelerated an enhancement in the antioxidant system and AsA-GSH cycle system by regulating ROS-metabolism-related genes, thereby improving the capacity to withstand browning and the quality of lotus root slices. The microbiome also showed that melatonin suppressed the fertility of spoilage organisms, such as Pseudomonas, Tolumonas, Acinetobacter, Stenotrophomonas, and Proteobacteria. Metabonomics data uncovered that the metabolites of flavonoid biosynthesis, phenylpropanoid biosynthesis, tyrosine metabolism, and phenylalanine metabolism were involved in the process.

The GA 20-Oxidase Encoding Gene MSD1 Controls the Main Stem Elongation in Medicago truncatula.

Plant height is an important agronomic trait that is closely related to biomass yield and crop production. Despite legumes comprise one of the largest monophyletic families that are second only to grasses in terms of economic and nutritional values, due to an ancient genome duplication event, most legume plants have complex genomes, thus the molecular mechanisms that determine plant height are less known in legumes. Here, we report the identification and characterization of MAIN STEM DWARF1 (MSD1), which is required for the plant height in the model legume Medicago truncatula. Loss of function of MSD1 leads to severely reduced main stem height but normal lateral branch elongation in M. truncatula. Histological analysis revealed that the msd1-1 main stem has shorter internodes with reduced cell size and number compared with the wild type, indicating that MSD1 affects cell elongation and cell proliferation. MSD1 encodes a putative GA 20-oxidase that is expressed at significantly higher levels in the main shoot apex than in the lateral shoot apices, suggesting that MSD1 expression is associated with its effect on the main stem elongation. UPLC-MS/MS analysis showed that GA9 and GA4, two identified products of the GA 20-oxidase, were severely reduced in msd1-1, and the dwarf phenotype of msd1-1 could be rescued by supplementation with gibberellic acid GA3, confirming that MSD1 functions as a biologically active GA 20-oxidase. Moreover, we found that disruption of either MtGA20ox7 or MtGA20ox8, homologs of MSD1, has little effects on the elongation of the main stem, while the msd1-1 mtga20ox7-1 mtga20ox8 triple mutants exhibits a severe short main shoot and lateral branches, as well as reduced leaf size, suggesting that MSD1 and its homologs MtGA20ox7 and MtGA20ox8, redundantly regulate M. truncatula shoot elongation and leaf development. Taken together, our findings demonstrate the molecular mechanism of MSD1-mediated regulation of main stem elongation in M. truncatula and provide insights into understanding the functional diversity of GA 20-oxidases in optimizing plant architecture in legumes.

Structure of the unique tetrameric STENOFOLIA homeodomain bound with target promoter DNA.

Homeobox transcription factors are key regulators of morphogenesis and development in both animals and plants. In plants, the WUSCHEL-related homeobox (WOX) family of transcription factors function as central organizers of several developmental programs ranging from embryo patterning to meristematic stem-cell maintenance through transcriptional activation and repression mechanisms. The Medicago truncatula STENOFOLIA (STF) gene is a master regulator of leaf-blade lateral development. Here, the crystal structure of the homeodomain (HD) of STF (STF-HD) in complex with its promoter DNA is reported at 2.1 Šresolution. STF-HD binds DNA as a tetramer, enclosing nearly the entire bound DNA surface. The STF-HD tetramer is partially stabilized by docking of the C-terminal tail of one protomer onto a conserved hydrophobic surface on the head of another protomer in a head-to-tail manner. STF-HD specifically binds TGA motifs, although the promoter sequence also contains TAAT motifs. Helix α3 not only serves a canonical role as a base reader in the major groove, but also provides DNA binding in the minor groove through basic residues located at its C-terminus. The structural and functional data in planta reported here provide new insights into the DNA-binding mechanisms of plant-specific HDs from the WOX family of transcription factors.

MtFDa is essential for flowering control and inflorescence development in Medicago truncatula.

Flowering plants display a vast diversity of flowering time and inflorescence architecture, which plays an important role in determining seed yield and fruit production. However, the molecular mechanism underlying the flowering control and compound inflorescence development, especially in legumes, remain to be elucidated. Here, we reported the identification of MtFDa, an essential regulator of flowering in the model legume Medicago truncatula. Mutation of MtFDa, led to the late flowering, abnormal secondary inflorescences as well as severe floral organ defects. Biochemical and molecular analyses revealed that MtFDa physically interacts with M. truncaula FLOWERING LOCUS T homolog, MtFTa1, a key regulator of Medicago flowering time, and this interaction facilitates MtFDa's function in activating the expression of MtSOC1a. Moreover, we demonstrated that MtFDa may affect secondary inflorescence development via regulating MtFULc expression in M. truncatula. Our findings help elucidate the mechanism of MtFDa-mediated regulation of flowering time and inflorescence development and provide insights into understanding the genetic regulatory network underlying complex productive development in legumes.

The effects of different temperatures on the storage characteristics of lotus (Nelumbo nucifera G.) root.

Lotus root (Nelumbo nucifera G.) is a high economic value crop in the world. In this study, the storage characteristics (color, sensory, texture, and fatty acids) of lotus root ("Elian No.5″) were evaluated at different harvest periods (September 2018, October 2018, November 2018, December 2018, and January 2019). Moreover, the storage characteristics were evaluated after the short- term and long-term storage of lotus root at 4 °C and 20 °C. The hardness of lotus root significantly decreased at both temperatures (4 °C and 20 °C) during the first 3 days of storage. In contrast, the decrease in hardness delayed at 4 °C (beyond 3 days of storage). Further, genes related to hardness at different storage temperatures were identified using the RNA-seq and qRT-PCR. The results of this study provide a reference for lotus root storage and a basis for the molecular breeding of longterm-storable lotus root.

MtFULc controls inflorescence development by directly repressing MtTFL1 in Medicago truncatula.

Flowering plants display a vast diversity of inflorescence architecture, which plays an important role in determining seed yield and fruit production. Unlike the model eudicot Arabidopsis thaliana that has simple inflorescences, most legume plants have compound types of inflorescences. Recent studies in the model legume species Pisum sativum and Medicago truncatula showed that the MADS-box transcription factors VEGETATIVE1/PsFRUITFULc/MtFRUITFULc (VEG1/PsFULc and MtFULc) are essential for the development of compound inflorescences by specifying the secondary inflorescence meristem identity. In this study, we report the isolation and characterization of two new mtfulc alleles by screening the M. truncatula Tnt1 insertion mutant collection. We found that MtFULc specifies M. truncatula secondary inflorescence meristem identity in a dose-dependent manner. Biochemical analysis and chromatin immunoprecipitation (ChIP) assays revealed that MtFULc acts as a transcriptional repressor to directly repress the expression of MtTFL1 through its promoter and 3' intergenic region. Comprehensive genetic analysis suggest MtFULc coordinates with the primary inflorescence meristem maintainer MtTFL1 and floral meristem regulator MtPIM to control M. truncatula inflorescence development. Our findings help to elucidate the mechanism of MtFULc-mediated regulation of secondary inflorescence meristem identity and provide insights into understanding the genetic regulatory network underlying compound inflorescence development in legumes.

The antagonistic MYB paralogs RH1 and RH2 govern anthocyanin leaf markings in Medicago truncatula.

Patterned leaf coloration in plants generates remarkable diversity in nature, but the underlying mechanisms remain largely unclear. Here, using Medicago truncatula leaf marking as a model, we show that the classic M. truncatula leaf anthocyanin spot trait depends on two R2R3 MYB paralogous regulators, RED HEART1 (RH1) and RH2. RH1 mainly functions as an anthocyanin biosynthesis activator that specifically determines leaf marking formation depending on its C-terminal activation motif. RH1 physically interacts with the M. truncatula bHLH protein MtTT8 and the WDR family member MtWD40-1, and this interaction facilitates RH1 function in leaf anthocyanin marking formation. RH2 has lost transcriptional activation activity, due to a divergent C-terminal domain, but retains the ability to interact with the same partners, MtTT8 and MtWD40-1, as RH1, thereby acting as a competitor in the regulatory complex and exerting opposite effects. Moreover, our results demonstrate that RH1 can activate its own expression and that RH2-mediated competition can repress RH1 expression. Our findings reveal the molecular mechanism of the antagonistic gene paralogs RH1 and RH2 in determining anthocyanin leaf markings in M. truncatula, providing a multidimensional paralogous-antagonistic regulatory paradigm for fine-tuning patterned pigmentation.

ATP-Binding Cassette G Transporters SGE1 and MtABCG13 Control Stigma Exsertion.

Stigma exsertion is an important agricultural trait that facilitates the application of heterosis in crop breeding. Although several quantitative trait loci associated with stigma exsertion have been fine-mapped or cloned, the underlying genetic basis, particularly in legumes, remains unclear. In this study, we identified and characterized the exserted stigma mutant stigma exsertion1 (sge1) in the model legume Medicago truncatula The exserted stigma phenotype of sge1 is mainly caused by physical interaction between floral organs, in which normal petal and stamen elongation are inhibited due to flower cuticle defects. SGE1 encodes an ATP-binding cassette G (ABCG) transporter that plays a critical role in regulating floral cutin and wax secretion in M. truncatula SGE1 physically interacts with another half-size transporter, MtABCG13, to form a functional heterodimer. Mutation of MtABCG13 results in flower cuticle defects similar to those in sge1 as well as stigma exsertion, indicating that SGE1 and MtABCG13 are indispensable for flower cuticle secretion and collaboratively control stigma exsertion in M. truncatula Our findings reveal novel functions for ABCG transporters in determining stigma exsertion by affecting the physical interactions of floral organs, providing insight into the molecular mechanism underlying stigma exsertion in leguminous plants with complex zygomorphic flowers.

SMALL LEAF AND BUSHY1 controls organ size and lateral branching by modulating the stability of BIG SEEDS1 in Medicago truncatula.

Organ size is a major agronomic trait that determines grain yield and biomass production in crops. However, the molecular mechanisms controlling organ size, especially in legumes, are poorly understood. Using forward genetic approaches in a Tnt1 insertion mutant population of the model legume Medicago truncatula, we identified SMALL LEAF AND BUSHY1 (SLB1), which is required for the control of organ size and lateral branching. Loss of function of SLB1 led to reduced leaf and flower size but increased lateral branch formation in M. truncatula. SLB1 encodes an F-box protein, an orthologue of Arabidopsis thaliana STERILE APETALA (SAP), that forms part of an SKP1/Cullin/F-box E3 ubiquitin ligase complex. Biochemical and genetic analyses revealed that SLB1 controls M. truncatula organ growth and lateral branching by modulating the stability of BIG SEEDS1 (BS1). Moreover, the overexpression of SLB1 increased seed and leaf size in both M. truncatula and soybean (Glycine max), indicating functional conservation. Our findings revealed a novel mechanism by which SLB1 targets BS1 for degradation to regulate M. truncatula organ size and shoot branching, providing a new genetic tool for increasing seed yield and biomass production in crop and forage legumes.

The MYB Activator WHITE PETAL1 Associates with MtTT8 and MtWD40-1 to Regulate Carotenoid-Derived Flower Pigmentation in Medicago truncatula.

Carotenoids are a group of natural tetraterpenoid pigments with indispensable roles in the plant life cycle and the human diet. Although the carotenoid biosynthetic pathway has been well characterized, the regulatory mechanisms that control carotenoid metabolism, especially in floral organs, remain poorly understood. In this study, we identified an anthocyanin-related R2R3-MYB protein, WHITE PETAL1 (WP1), that plays a critical role in regulating floral carotenoid pigmentation in Medicago truncatula Carotenoid analyses showed that the yellow petals of the wild-type M. truncatula contained high concentrations of carotenoids that largely consisted of esterified lutein and that disruption of WP1 function via Tnt1 insertion led to substantially reduced lutein accumulation. WP1 mainly functions as a transcriptional activator and directly regulates the expression of carotenoid biosynthetic genes including MtLYCe and MtLYCb through its C-terminal acidic activation motif. Further molecular and genetic analyses revealed that WP1 physically interacts with MtTT8 and MtWD40-1 proteins and that this interaction facilitates WP1's function in the transcriptional activation of both carotenoid and anthocyanin biosynthetic genes. Our findings demonstrate the molecular mechanism of WP1-mediated regulation of floral carotenoid pigmentation and suggest that the conserved MYB-basic-helix-loop-helix-WD40 regulatory module functions in carotenoid biosynthesis in M. truncatula, with specificity imposed by the MYB partner.

AGAMOUS AND TERMINAL FLOWER controls floral organ identity and inflorescence development in Medicago truncatula.

Angiosperms integrate a multitude of endogenous and environmental signals to control floral development, thereby ensuring reproductive success. Here, we report the identification of AGAMOUS AND TERMINAL FLOWER (AGTFL), a novel regulator of floral development in Medicago truncatula. Mutation of AGTFL led to the transformation of carpels and stamens into numerous sepals and petals and altered primary inflorescence identity. AGTFL encodes a nucleus-localized protein containing a putative Myb/SANT-like DNA-binding domain and a PKc kinase domain. Molecular and genetic analyses revealed that AGTFL regulates the transcription of MtAGs and MtTFL1 to control floral organ identity and inflorescence development.

HEADLESS, a WUSCHEL homolog, uncovers novel aspects of shoot meristem regulation and leaf blade development in Medicago truncatula.

The formation and maintenance of the shoot apical meristem (SAM) are critical for plant development. However, the underlying molecular mechanism of regulating meristematic cell activity is poorly understood in the model legume Medicago truncatula. Using forward genetic approaches, we identified HEADLESS (HDL), a homolog of Arabidopsis WUSCHEL, required for SAM maintenance and leaf development in M. truncatula. Disruption of HDL led to disorganized specification and arrest of the SAM and axillary meristems, resulting in the hdl mutant being locked in the vegetative phase without apparent stem elongation. hdl mutant leaves are shorter in the proximal-distal axis due to reduced leaf length elongation, which resulted in a higher blade width/length ratio and altered leaf shape, uncovering novel phenotypes undescribed in the Arabidopsis wus mutant. HDL functions as a transcriptional repressor by recruiting MtTPL through its conserved WUS-box and EAR-like motif. Further genetic analysis revealed that HDL and STENOFOLIA (STF), a key regulator of M. truncatula lamina outgrowth, act independently in leaf development although HDL could recruit MtTPL in the same manner as STF does. Our results indicate that HDL has conserved and novel functions in regulating shoot meristems and leaf shape in M. truncatula, providing new avenues for understanding meristem biology and plant development.

Functional Specialization of Duplicated AGAMOUS Homologs in Regulating Floral Organ Development of Medicago truncatula.

The C function gene AGAMOUS (AG) encodes for a MADS-box transcription factor required for floral organ identity and floral meristem (FM) determinacy in angiosperms. Unlike Arabidopsis, most legume plants possess two AG homologs arose by an ancient genome duplication event. Recently, two euAGAMOUS genes, MtAGa and MtAGb, were characterized and shown to fulfill the C function activity in the model legume Medicago truncatula. Here, we reported the isolation and characterization of a new mtaga allele by screening the Medicago Tnt1 insertion mutant collection. We found that MtAGa was not only required for controlling the stamen and carpel identity but also affected pod and seed development. Genetic analysis indicated that MtAGa and MtAGb redundantly control Medicago floral organ identity, but have minimal distinct functions in regulating stamen and carpel development in a dose-dependent manner. Interestingly, the stamens and carpels are mostly converted to numerous vexillum-like petals in the double mutant of mtaga mtagb, which is distinguished from Arabidopsis ag. Further qRT-PCR analysis in different mtag mutants revealed that MtAGa and MtAGb can repress the expression of putative A and B function genes as well as MtWUS, but promote putative D function genes expression in M. truncatula. In addition, we found that the abnormal dorsal petal phenotype observed in the mtaga mtagb double mutant is associated with the upregulation of CYCLOIDEA (CYC)-like TCP genes. Taken together, our data suggest that the redundant MtAGa and MtAGb genes of M. truncatula employ a conserved mechanism of action similar to Arabidopsis in determining floral organ identity and FM determinacy but may have evolved distinct function in regulating floral symmetry by coordinating with specific floral dorsoventral identity factors.

[Genome editing technology and its application in forage legumes].

Genome editing is a novel targeted genome modification biotechnology, which could successfully mutate specific loci as well as generate gene replacement and insertion in various organisms. So far, genome editing technology has been widely applied in investigating gene function and developing valuable traits in both model plants and major crops. In this review, we briefly survey the historical development of genome editing technology, summarize recent progress using the CRISPR/Cas9 system for plant genome editing and explore the potential of the CRISPR/Cas technology in improving forage legumes.