High-strength and high-toughness ECM films with the potential for peripheral nerve repair.

Extracellular matrix (ECM) scaffolds are widely applied in the field of regeneration as the result of their irreplaceable biological advantages, and the preparation of ECM scaffolds into ECM hydrogels expands the applications to some extent. However, weak mechanical properties of current ECM materials limit the complete exploitation of ECM's biological advantages. To enable ECM materials to be utilized in applications requiring high strength, herein, we created a kind of new ECM material, ECM film, and evaluated its mechanical properties. ECM films exhibited outstanding toughness with no cracks after arbitrarily folding and crumpling, and dramatically high strength levels of 86 ± 17.25 MPa, the maximum of which was 115 MPa. Such spectacular high-strength and high-toughness films, containing only pure ECM without any crosslinking agents and other materials, far exceed current pure natural polymer gel films and even many composite gel films and synthetic polymer gel films. In addition, both PC12 cells and Schwann cells cultured on the surface of ECM films, especially Schwann cells, showed good proliferation, and the neurite outgrowth of the PC12 cells was promoted, indicating the application potential of ECM film in peripheral nerve repair.

The MicroRNA397a-LACCASE17 module regulates lignin biosynthesis in Medicago ruthenica (L.).

Mechanical strength is essential for the upright growth habit, which is one of the most important characteristics of terrestrial plants. Lignin, a phenylpropanoid-derived polymer mainly present in secondary cell walls plays critical role in providing mechanical support. Here, we report that the prostrate-stem cultivar of the legume forage Medicago ruthenica cultivar 'Mengnong No. 1' shows compromised mechanical strength compared with the erect-stem cultivar 'Zhilixing'. The erect-stem cultivar, 'Zhilixing' has significantly higher lignin content, leading to higher mechanical strength than the prostrate-stem cultivar. The low abundance of miRNA397a in the Zhiixing cultivar causes reduced cleavage of MrLAC17 transcript, which results in enhanced expression level of MrLAC17 compared to that in the prostrate-stem cultivar Mengnong No. 1. Complementation of the Arabidopsis lac4 lac17 double mutants with MrLAC17 restored the lignin content to wild-type levels, confirming that MrLAC17 perform an exchangeable role with Arabidopsis laccases. LAC17-mediated lignin polymerization is therefore increased in the 'Zhilixing', causing the erect stem phenotype. Our data reveal the importance of the miR397a in the lignin biosynthesis and suggest a strategy for molecular breeding targeting plant architecture in legume forage.

Glycoside-specific metabolomics combined with precursor isotopic labeling for characterizing plant glycosyltransferases.

Glycosylation by uridine diphosphate-dependent glycosyltransferases (UGTs) in plants contributes to the complexity and diversity of secondary metabolites. UGTs are generally promiscuous in their use of acceptors, making it challenging to reveal the function of UGTs in vivo. Here, we described an approach that combined glycoside-specific metabolomics and precursor isotopic labeling analysis to characterize UGTs in Arabidopsis. We revisited the UGT72E cluster, which has been reported to catalyze the glycosylation of monolignols. Glycoside-specific metabolomics analysis reduced the number of differentially accumulated metabolites in the ugt72e1e2e3 mutant by at least 90% compared with that from traditional untargeted metabolomics analysis. In addition to the two previously reported monolignol glycosides, a total of 62 glycosides showed reduced accumulation in the ugt72e1e2e3 mutant, 22 of which were phenylalanine-derived glycosides, including 5-OH coniferyl alcohol-derived and lignan-derived glycosides, as confirmed by isotopic tracing of [13C6]-phenylalanine precursor. Our method revealed that UGT72Es could use coumarins as substrates, and genetic evidence showed that UGT72Es endowed plants with enhanced tolerance to low iron availability under alkaline conditions. Using the newly developed method, the function of UGT78D2 was also evaluated. These case studies suggest that this method can substantially contribute to the characterization of UGTs and efficiently investigate glycosylation processes, the complexity of which have been highly underestimated.

Dual Mechanisms of Coniferyl Alcohol in Phenylpropanoid Pathway Regulation.

Lignin is a complex phenolic polymer that imparts cell wall strength, facilitates water transport and functions as a physical barrier to pathogens in all vascular plants. Lignin biosynthesis is a carbon-consuming, non-reversible process, which requires tight regulation. Here, we report that a major monomer unit of the lignin polymer can function as a signal molecule to trigger proteolysis of the enzyme L-phenylalanine ammonia-lyase, the entry point into the lignin biosynthetic pathway, and feedback regulate the expression levels of lignin biosynthetic genes. These findings highlight the highly complex regulation of lignin biosynthesis and shed light on the biological importance of monolignols as signaling molecules.

The spatio-temporal biosynthesis of floral flavonols is controlled by differential phylogenetic MYB regulators in Freesia hybrida.

Floral flavonols play specific pivotal roles in pollinator attraction, pollen germination and fertility, in addition to other functions in vegetative organs. For many plants, the process of flavonol biosynthesis in late flower development stages and in mature flower tissues is poorly understood, in contrast to early flower development stages. It is thought that this process may be regulated independently of subgroup 7 R2R3 MYB (SG7 MYB) transcription factors. In this study, two FLS genes were shown to be expressed synchronously with the flower development-specific and tissue-specific biosynthesis of flavonols in Freesia hybrida. FhFLS1 contributed to flavonol biosynthesis in early flower buds, toruses and calyxes, and was regulated by four well-known SG7 MYB proteins, designated as FhMYBFs, with at least partial regulatory redundancy. FhFLS2 accounted for flavonols in late developed flowers and in the petals, stamens and pistils, and was targeted directly by non SG7 MYB protein FhMYB21L2. In parallel, AtMYB21 and AtMYB24 also activated AtFLS1, a gene highly expressed in Arabidopsis anthers and pollen, indicating the conserved regulatory roles of MYB21 against FLS genes in these two evolutionarily divergent angiosperm plants. Our results reveal a novel regulatory and synthetic mechanism underlying flavonol biosynthesis in floral organs and tissues which may be exploited to investigate supplementary roles of flavonols in flowers.

MYB repressors and MBW activation complex collaborate to fine-tune flower coloration in Freesia hybrida.

Floral anthocyanin has multiple ecological and economic values, its biosynthesis largely depends on the conserved MYB-bHLH-WD40 (MBW) activation complex and MYB repressors hierarchically with the MBW complex. In contrast to eudicots, the MBW regulatory network model has not been addressed in monocots because of the lack of a suitable system, as grass plants exhibit monotonous floral pigmentation patterns. Presently, the MBW regulatory network was investigated in a non-grass monocot plant, Freesia hybrida. FhMYB27 and FhMYBx with different functional manners were confirmed to be anthocyanin related R2R3 and R3 MYB repressors, respectively. Particularly, FhMYBx could obstruct the formation of positive MBW complex by titrating bHLH proteins, whereas FhMYB27 mainly defected the activator complex into suppressor via its repression domains in C-terminus. Furthermore, the hierarchical and feedback regulatory loop was verified, indicating the synergistic and sophisticated regulatory network underlying Freesia anthocyanin biosynthesis was quite similar to that reported in eudicot plants.

The Conserved and Particular Roles of the R2R3-MYB Regulator FhPAP1 from Freesia hybrida in Flower Anthocyanin Biosynthesis.

Anthocyanin biosynthesis is mainly controlled by MYB-bHLH-WD40 (MBW) complexes that modulate the expression of anthocyanin biosynthetic genes (ABGs). The MYB regulators involved in anthocyanin biosynthesis arose early during plant evolution and thus might function divergently in different evolutionary lineages. Although the anthocyanin-promoting R2R3-MYB regulators in eudicots have been comprehensively explored, little consensus has been reached about functional discrepancies versus conservation among MYB regulators from different plant lineages. Here, we integrated transcriptome analysis, gene expression profiles, gain-of-function experiments and transient protoplast transfection assays to functionally characterize the monocot Freesia hybrida anthocyanin MYB regulator gene FhPAP1, which showed correlations with late ABGs. FhPAP1 could activate ABGs as well as TT8-clade genes FhTT8L, AtTT8 and NtAN1 when overexpressed in Freesia, Arabidopsis and tobacco, respectively. Consistently, FhPAP1 could interact with FhTT8L and FhTTG1 to form the conserved MBW complex and shared similar target genes with its orthologs from Arabidopsis. Most prominently, FhPAP1 displayed higher transactivation capacity than its homologs in Arabidopsis and tobacco, which was instantiated in its powerful regulation on ABGs. Moreover, we found that FhPAP1 might be the selected gene during the domestication and rapid evolution of the wild Freesia species to generate intensive flower pigmentation. These results showed that while the MBW complex was highly evolutionarily conserved between tested monocot and core eudicot plants, participating MYB regulators showed functional differences in transactivation capacity according to their activation domain and played important roles in the flower coloration domestication and evolution of angiosperms.

Functional Differentiation of Duplicated Flavonoid 3-O-Glycosyltransferases in the Flavonol and Anthocyanin Biosynthesis of Freesia hybrida.

Flavonols and anthocyanins are two widely distributed groups of flavonoids that occurred apart during plant evolution and biosynthesized by shared specific enzymes involved in flavonoid metabolism. UDP-glucose, flavonoid 3-O-glycosyltransferase (UF3GT), is one of the common enzymes which could catalyze the glycosylation of both flavonol and anthocyanidin aglycons simultaneously in vitro. However, whether and how UF3GT paralogous genes function diversely at the biochemical and transcriptional levels are largely unknown. Recently, Fh3GT1 was identified to be a member of UF3GTs in Freesia hybrida. However, its expression patterns and enzymatic characteristics could not coincide well with flavonol accumulation. In an attempt to characterize other flavonoids, especially flavonol glycosyltransferase genes in Freesia, three closest candidate UFGT genes-Fh3GT2, Fh3GT3, and Fh3GT4-were mined from the Freesia transcriptomic database and isolated from the flowers of the widely distributed Freesia cultivar, Red River®. Based on bioinformatic analysis and enzymatic assays, Fh3GT2 turned out to be another bona fide glycosyltransferase gene. Biochemical analysis further proved that Fh3GT2 preferentially glucosylated kaempferol while Fh3GT1 controlled the glucosylation of quercetin and anthocyanidins. In addition, transfection assays demonstrated that Fh3GT2 could be mainly activated by the flavonol regulator FhMYBF1 or the anthocyanin regulator FhPAP1, whereas Fh3GT1 could only be activated by FhPAP1. These findings suggested that Fh3GTs might have functionally diverged in flavonoid biosynthesis at both the biochemical and transcriptional levels.

A functional homologue of Arabidopsis TTG1 from Freesia interacts with bHLH proteins to regulate anthocyanin and proanthocyanidin biosynthesis in both Freesia hybrida and Arabidopsis thaliana.

The MBW complex, consisting of MYB, basic helix-loop-helix (bHLH) and WD40 proteins, regulates multiple traits in plants, such as anthocyanin and proanthocyanidin biosynthesis and cell fate determination. The complex has been widely identified in dicot plants, whereas few studies are concentrated on monocot plants which are of crucial importance to decipher its functional diversities among angiosperms during evolution. In present study, a WD40 gene from Freesia hybrida, designated as FhTTG1, was cloned and functionally characterized. Real-time PCR analysis indicated that it was expressed synchronously with the accumulation of both proanthocyanidins and anthocyanins in Freesia flowers. Transient protoplast transfection and biomolecular fluorescence complementation (BiFC) assays demonstrated that FhTTG1 could interact with FhbHLH proteins (FhTT8L and FhGL3L) to constitute the MBW complex. Moreover, the transportation of FhTTG1 to nucleus was found to rely on FhbHLH factors. Outstandingly, FhTTG1 could highly activate the anthocyanin or proanthocyanidin biosynthesis related gene promoters when co-transfected with MYB and bHLH partners, implying that FhTTG1 functioned as a member of MBW complex to control the anthocyanin or proanthocyanidin biosynthesis in Freesia hybrida. Further ectopic expression assays in Arabidopsis ttg1-1 showed the defective phenotypes of ttg1-1 were partially restored. Molecular biological assays validated FhTTG1 might interact with the endogenous bHLH factors to up-regulate genes responsible for anthocyanin and proanthocyanidin biosynthesis and trichome formation, indicating that FhTTG1 might perform exchangeable roles with AtTTG1. These results will not only contribute to the characterization of FhTTG1 in Freesia but also shed light on the establishment of flavonoid regulatory system in monocot plants, especially in Freesia hybrida.

The R2R3-MYB Factor FhMYB5 From Freesia hybrida Contributes to the Regulation of Anthocyanin and Proanthocyanidin Biosynthesis.

The flavonoids are important and nourishing compounds for plants and human. The transcription regulation of anthocyanin and proanthocyanidin (PA) biosynthesis was extensively studied in dicot compared with monocot plants. In this study, we characterized the functionality of an R2R3-MYB gene FhMYB5 from the monocotyledonous flowering plant of Iridaceae, Freesia hybrida. Multiple sequence alignment and phylogenetic analysis implied that FhMYB5 was clustered into grapevine VvMYB5b subclade. Correlation analysis indicated that the spatio-temporal expression patterns of FhMYB5 coincided well with anthocyanin and PA accumulations in Freesia per se. Furthermore, transient transfection assays in Freesia protoplasts revealed that the late flavonoid biosynthetic genes (e.g., DFR and LDOX) were slightly up-regulated by FhMYB5 alone, whereas both early and late biosynthetic genes were significantly activated when FhMYB5 were co-infected with either of the two IIIf clade bHLH genes, FhTT8L and FhGL3L. Moreover, these results were further confirmed by co-transfection of FhMYB5 with either of the bHLH genes aforementioned into protoplasts expressing GUS reporter gene driven by Freesia promoters. In addition, the overexpression of FhMYB5 in tobacco and Arabidopsis could also significantly up-regulate the expression of genes participating in the general flavonoid pathway. In conclusion, FhMYB5 was proved to function in the general flavonoid pathway in Freesia. The results implied a function conservation of flavonoid biosynthesis related MYB regulators in angiosperm plants.

Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids.

Dihydroflavonol-4-reductase (DFR) is a key enzyme in the reduction of dihydroflavonols to leucoanthocyanidins in both anthocyanin biosynthesis and proanthocyanidin accumulation. In many plant species, it is encoded by a gene family, however, how the different copies evolve either to function in different tissues or at different times or to specialize in the use of different but related substrates needs to be further investigated, especially in monocot plants. In this study, a total of eight putative DFR-like genes were firstly cloned from Freesia hybrida. Phylogenetic analysis showed that they were classified into different branches, and FhDFR1, FhDFR2, and FhDFR3 were clustered into DFR subgroup, whereas others fell into the group with cinnamoyl-CoA reductase (CCR) proteins. Then, the functions of the three FhDFR genes were further characterized. Different spatio-temporal transcription patterns and levels were observed, indicating that the duplicated FhDFR genes might function divergently. After introducing them into Arabidopsis dfr (tt3-1) mutant plants, partial complementation of the loss of cyanidin derivative synthesis was observed, implying that FhDFRs could convert dihydroquercetin to leucocyanidin in planta. Biochemical assays also showed that FhDFR1, FhDFR2, and FhDFR3 could utilize dihydromyricetin to generate leucodelphinidin, while FhDFR2 could also catalyze the formation of leucocyanidin from dihydrocyanidin. On the contrary, neither transgenic nor biochemical analysis demonstrated that FhDFR proteins could reduce dihydrokaempferol to leucopelargonidin. These results were consistent with the freesia flower anthocyanin profiles, among which delphinidin derivatives were predominant, with minor quantities of cyanidin derivatives and undetectable pelargonidin derivatives. Thus, it can be deduced that substrate specificities of DFRs were the determinant for the categories of anthocyanins aglycons accumulated in F. hybrida. Furthermore, we also found that the divergence of the expression patterns for FhDFR genes might be controlled at transcriptional level, as the expression of FhDFR1/FhDFR2 and FhDFR3 was controlled by a potential MBW regulatory complex with different activation efficiencies. Therefore, it can be concluded that the DFR-like genes from F. hybrida have diverged during evolution to play partially overlapping roles in the flavonoid biosynthesis, and the results will contribute to the study of evolution of DFR gene families in angiosperms, especially for monocot plants.

Two IIIf Clade-bHLHs from Freesia hybrida Play Divergent Roles in Flavonoid Biosynthesis and Trichome Formation when Ectopically Expressed in Arabidopsis.

The MBW complex, comprised by R2R3-MYB, basic helix-loop-helix (bHLH) and WD40, is a single regulatory protein complex that drives the evolution of multiple traits such as flavonoid biosynthesis and epidermal cell differentiation in plants. In this study, two IIIf Clade-bHLH regulator genes, FhGL3L and FhTT8L, were isolated and functionally characterized from Freesia hybrida. Different spatio-temporal transcription patterns were observed showing diverse correlation with anthocyanin and proanthocyanidin accumulation. When overexpressed in Arabidopsis, FhGL3L could enhance the anthocyanin accumulation through up-regulating endogenous regulators and late structural genes. Unexpectedly, trichome formation was inhibited associating with the down-regulation of AtGL2. Comparably, only the accumulation of anthocyanins and proanthocyanidins was strengthened in FhTT8L transgenic lines. Furthermore, transient expression assays demonstrated that FhGL3L interacted with AtPAP1, AtTT2 and AtGL1, while FhTT8L only showed interaction with AtPAP1 and AtTT2. In addition, similar activation of the AtDFR promoter was found between AtPAP1-FhGL3L/FhTT8L and AtPAP1- AtGL3/AtTT8 combinations. When FhGL3L was fused with a strong activation domain VP16, it could activate the AtGL2 promoter when co-transfected with AtGL1. Therefore, it can be concluded that the functionality of bHLH factors may have diverged, and a sophisticated interaction and hierarchical network might exist in the regulation of flavonoid biosynthesis and trichome formation.