NCR343 is required to maintain the viability of differentiated bacteroids in nodule cells in Medicago truncatula.

Bacteroid (name for rhizobia inside nodule cells) differentiation is a prerequisite for successful nitrogen-fixing symbiosis. In certain legumes, under the regulation of host proteins, for example, a large group of NCR (nodule cysteine rich) peptides, bacteroids undergo irreversible terminal differentiation. This process causes them to lose the ability to propagate inside nodule cells while boosting their competency for nitrogen fixation. How host cells maintain the viability of differentiated bacteroids while maximizing their nitrogen-reducing activities remains elusive. Here, through mutant screen, map-based cloning, and genetic complementation, we find that NCR343 is required for the viability of differentiated bacteroids. In Medicago truncatula debino1 mutant, differentiated bacteroids decay prematurely, and NCR343 is proved to be the casual gene for debino1. NCR343 is mainly expressed in the nodule fixation zone, where bacteroids are differentiated. In nodule cells, mature NCR343 peptide is secreted into the symbiosomes. RNA-Seq assay shows that many stress-responsive genes are significantly induced in debino1 bacteroids. Additionally, a group of stress response-related rhizobium proteins are identified as putative interacting partners of NCR343. In summary, our findings demonstrate that beyond promoting bacteroid differentiation, NCR peptides are also required in maintaining the viability of differentiated bacteroids.

MYB21 interacts with MYC2 to control the expression of terpene synthase genes in flowers of Freesia hybrida and Arabidopsis thaliana.

Previously, linalool was found to be the most abundant component among the cocktail of volatiles released from flowers of Freesia hybrida. Linalool formation is catalysed by monoterpene synthase TPS1. However, the regulatory network developmentally modulating the expression of the TPS1 gene in Freesia hybrida remains unexplored. In this study, three regulatory genes, FhMYB21L1, FhMYB21L2, and FhMYC2, were screened from 52 candidates. Two MYB transcription factor genes were synchronously expressed with FhTPS1 and could activate its expression significantly when overexpressed, and the binding of FhMYB21L2 to the MYBCORE sites in the FhTPS1 promoter was further confirmed, indicating a direct role in activation. FhMYC2 showed an inverse expression pattern compared with FhTPS1; its expression led to a decreased binding of FhMYB21 to the FhTPS1 promoter to reduce its activation capacity when co-expressed, suggesting a role for an MYB-bHLH complex in the regulation of the FhTPS1 gene. In Arabidopsis, both MYB21 and MYC2 regulators were shown to activate the expression of sesquiterpene synthase genes, and the regulatory roles of AtMYB21 and AtMYC2 in the expression of the linalool synthase gene were also confirmed, implying conserved functions of the MYB-bHLH complex in these two evolutionarily divergent plants. Moreover, the expression ratio between MYB21 and MYC2 orthologues might be a determinant factor in floral linalool emission.

Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia x hybrida.

The development of flower scents was a crucial event in biological evolution, providing olfactory signals by which plants can attract pollinators. In this study, bioinformatics, metabolomics, and biochemical and molecular methodologies were integrated to investigate the candidate genes involved in the biosynthesis of volatile components in two cultivars of Freesia x hybrida, Red River® and Ambiance, which release different categories of compounds. We found that terpene synthase (TPS) genes were the pivotal genes determining spatiotemporal release of volatile compounds in both cultivars. Eight FhTPS genes were isolated and six were found to be functional: FhTPS1 was a single-product enzyme catalyzing the formation of linalool, whereas the other four FhTPS proteins were multi-product enzymes, among which FhTPS4, FhTPS6, and FhTPS7 could recognize geranyl diphosphate and farnesyl diphosphate simultaneously. The FhTPS enzymatic products closely matched the volatile terpenes emitted from flowers, and significant correlations were found between release of volatile terpenes and FhTPS gene expression. Graphical models based on these results are proposed that summarize the biosynthesis of Freesia floral volatile terpenes. The characterization of FhTPS genes paves the way to decipher their roles in the speciation and fitness of Freesia, and this knowledge could also be used to introduce or enhance scent in other plants.

Biochemical and Molecular Characterization of a Flavonoid 3-O-glycosyltransferase Responsible for Anthocyanins and Flavonols Biosynthesis in Freesia hybrida.

The glycosylation of flavonoids increases their solubility and stability in plants. Flowers accumulate anthocyanidin and flavonol glycosides which are synthesized by UDP-sugar flavonoid glycosyltransferases (UFGTs). In our previous study, a cDNA clone (Fh3GT1) encoding UFGT was isolated from Freesia hybrida, which was preliminarily proved to be invovled in cyanidin 3-O-glucoside biosynthesis. Here, a variety of anthocyanin and flavonol glycosides were detected in flowers and other tissues of F. hybrida, implying the versatile roles of Fh3GT1 in flavonoids biosynthesis. To further unravel its multi-functional roles, integrative analysis between gene expression and metabolites was investigated. The results showed expression of Fh3GT1 was positively related to the accumulation of anthocyanins and flavonol glycosides, suggesting its potential roles in the biosynthesis of both flavonoid glycosides. Subsequently, biochemical analysis results revealed that a broad range of flavonoid substrates including flavonoid not naturally occurred in F. hybrida could be recognized by the recombinant Fh3GT1. Both UDP-glucose and UDP-galactose could be used as sugar donors by recombinant Fh3GT1, although UDP-galactose was transferred with relatively low activity. Furthermore, regiospecificity analysis demonstrated that Fh3GT1 was able to glycosylate delphinidin at the 3-, 4-', and 7- positions in a sugar-dependent manner. And the introduction of Fh3GT1 into Arabidopsis UGT78D2 mutant successfully restored the anthocyanins and flavonols phenotypes caused by lost-of-function of the 3GT, indicating that Fh3GT1 functions as a flavonoid 3-O-glucosyltransferase in vivo. In summary, these results demonstrate that Fh3GT1 is a flavonoid 3-O-glycosyltransferase using UDP-glucose as the preferred sugar donor and may involve in flavonoid glycosylation in F. hybrida.

Direct and indirect organogenesis of Clivia miniata and assessment of DNA methylation changes in various regenerated plantlets.

UNLABELLED: Clivia miniata is an important indoor ornamental plant and has been reported to have medicinal value. We developed an efficient in vitro micropropagation protocol from young leaves (indirect organogenesis), young petals (indirect organogenesis) and shoot tips (direct organogenesis) of this plant. Using young leaves and shoot tips as explants, the regeneration frequencies were much higher than those in previous investigation and the regeneration was dependent upon less nutrition. We speculated that the leaf-derived callus can generate amino acids necessary for protein synthesis by itself. We employed the methylation-sensitive amplified polymorphism (MSAP) method to assess cytosine methylation variation in various regenerated plantlets and between organs. The MSAP profiles indicated that the frequency of somaclonal variation in the form of cytosine methylation was highest in petal-derived plantlets followed by secondary leaf-derived, primary leaf-derived and shoot tip-derived plantlets, but the methylation variation in petal-derived plantlets was lower than between petals and leaves of a single plant. The results indicated that the methylation variation in regenerated plantlets was related to the types of explants, regeneration pathways and number of regeneration generations. Two possible factors for the highest somaclonal variation rate in petal-derived plantlets are the callus phase and petal-specific set of epigenetic regulators. The property of meristem integrity can account for the lowest variation rate in shoot tip-derived plantlets. Moreover, the secondary plantlets underwent a longer total period of in vitro culture, which can explain why the methylation variation rate in the secondary plantlets is higher than in the primary ones. KEY MESSAGE: Methylation variation in regenerated plantlets of C. miniata was found to be related to the types of explants, regeneration pathways and number of regeneration generations.