OfBFT genes play an essential role in the proliferate flower formation of Osmanthus fragrans.

Floral organ development is one of the most vital events in flowering plants and is closely related to ornamental properties. The proliferate flower (a new branch or flower occurring in the centre of a flower) in plants is an interesting type, while the specific molecular mechanism remains largely unknown. Osmanthus fragrans 'Tianxiang Taige' has two different flower morphologies: normal flower and proliferate flower. Phenotypic observation suggested that a normal flower was composed of calyx, petal, stamen and pistil (reduced to leaf-like carpel). While in proliferate flower, the leaf-like carpel continued to grow and was replaced by a new branch. Paraffin section indicated that the re-growth of leaf carpels might be the main reason for proliferate flower formation. Transcriptome sequencing of normal and proliferate flower was performed, and the expression levels of related genes were analysed. Among the differentially expressed genes, OfBFT-a and OfBFT-b had differential expression during the proliferate flower formation process. The expression patterns revealed that both OfBFT-a and OfBFT-b were highly accumulated in carpels, and were significantly downregulated during the proliferate flower development process. Subcellular localization indicated that OfBFT-a and OfBFT-b proteins were located in the nucleus. Functional studies in 'Tianxiang Taige' and Arabidopsis showed that OfBFT-a and OfBFT-b had important roles in floral organ development, especially the proliferate flower formation process by downregulating the accumulation of AG and SEP3 homologous genes. These results may shed new light on the study of proliferate flower formation and flower morphology breeding in flowering plants.

DNA methylome analysis reveals novel insights into active hypomethylated regulatory mechanisms of temperature-dependent flower opening in Osmanthus fragrans.

Short-term ambient low temperature (ALT) stimulation is necessary for Osmanthus fragrans to facilitate continued flower opening after floral bud development reaches maturity. DNA methylation, a vital epigenetic modification, regulates various biological processes in response to temperature fluctuations. However, its role in temperature-driven flower opening remains elusive. In this study, we identified the pivotal timeframe during which O. fragrans promptly detected temperature cues. Using whole-genome bisulfite sequencing, we explored global DNA hypomethylation during this phase, with the most significant changes occurring in CHH sequence contexts. Auxin transport inhibitor (TIBA) application revealed that ALT-induced endogenous auxin accumulation promoted peduncle elongation. In our mRNA-seq analysis, we discovered that the differentially expressed genes (DEGs) with hypo-differentially methylated regions (hypo-DMRs) were mainly enriched in auxin and temperature response, RNA processing, and carbohydrate and lipid metabolism. Transcripts of three DNA demethylase genes (OfROS1a, OfDML3, OfDME) showed upregulation. Furthermore, all DNA methylase genes, except OfCMT2b, also displayed increased expression, specifically with two of them, OfCMT3a and OfCMT1, being associated with hypo-DMRs. Promoter assays showed that OfROS1a, with promoters containing low-temperature- and auxin-responsive elements, were activated by ALT and exogenous IAA at low concentrations but inhibited at high concentrations. Overexpression of OfROS1 reduced endogenous auxin levels but enhanced the expression of genes related to auxin response and spliceosome in petunia. Furthermore, OfROS1 promoted sucrose synthesis in petunia corollas. Our data characterized the rapid response of active DNA hypomethylation to ALT and suggested a possible epiregulation of temperature-dependent flower opening in O. fragrans. This study revealed the pivotal role of DNA hypomethylation in O. fragrans during the ALT-responsive phase before flower opening, involving dynamic DNA demethylation, auxin signaling modulation, and a potential feedback loop between hypomethylation and methylation.

Temperature-responsive module of OfAP1 and OfLFY regulates floral transition and floral organ identity in Osmanthus fragrans.

The MADS-box transcription factor APETELA1 (AP1) is crucially important for reproductive developmental processes. The function of AP1 and the classic LFY-AP1 interaction in woody plants are not widely known. Here, the OfAP1-a gene from the continuously flowering plant Osmanthus fragrans 'Sijigui' was characterized, and its roles in regulating flowering time, petal number robustness and floral organ identity were determined using overexpression in Arabidopsis thaliana and Nicotiana tabacum. The expression of OfAP1-a was significantly induced by low ambient temperature and was upregulated with the floral transition process. Ectopic expression OfAP1-a revealed its classic function in flowering and flower ABC models. The expression of OfAP1-a is inhibited by LEAFY (OfLFY) through direct promoter binding, as confirmed by yeast one-hybrid and dual luciferase assays. Arabidopsis plants overexpressing OfAP1-a exhibited accelerated flowering and altered floral organ identities. Moreover, OfAP1-a-overexpressing plants displayed variable petal numbers. Likewise, the overexpression of OfLFY in Arabidopsis and Nicotiana altered petal number robustness and inflorescence architecture, partially by regulating native AP1 in transformed plants. Furthermore, we performed RNA-seq analysis of transgenic Nicotiana plants. DEGs were identified by transcriptome analysis, and we found that the expression of several floral homeotic genes was altered in both OfAP1-a and OfLFY-overexpressing transgenic lines. Our results suggest that OfAP1-a may play important roles during floral transition and development in response to ambient temperature. OfAP1-a functions as a petal number modulator and may directly activate a subset of flowers to regulate floral organ formation. OfAP1-a and OfLFY mutually regulate the expression of each other and coregulate genes that might be involved in these phenotypes related to flowering. The results provide valuable data for understanding the function of the LFY-AP1 module in the reproductive process and shaping floral structures in woody plants.

Genome-wide identification and analysis of MIKC-type MADS-box genes expression in Chimonanthus salicifolius.

BACKGROUND: MIKC type MADS-box transcription factors are one of the largest gene families and play a pivotal role in flowering time and flower development. Chimonanthus salicifolius belongs to the family Calycanthaceae and has a unique flowering time and flowering morphology compared to other Chimonanthus species, but the research on MIKC type MADS-box gene family of C. salicifolius has not been reported. OBJECTIVE: Identification, comprehensive bioinformatic analysis, the expression pattern of MIKC-type MADS-box gene family from different tissues of C. salicifolius. METHODS: Genome-wide investigation and expression pattern under different tissues of the MIKC-type MADS-box gene family in C. salicifolius, and their phylogenetic relationships, evolutionary characteristics, gene structure, motif distribution, promoter cis-acting element were performed. RESULTS: A total of 29 MIKC-type MADS-box genes were identified from the whole genome sequencing. Interspecies synteny analysis revealed more significant collinearity between C. salicifolius and the magnoliids species compared to eudicots and monocots. MIKC-type MADS-box genes from the same subfamily share similar distribution patterns, gene structure, and expression patterns. Compared with Arabidopsis thaliana, Nymphaea colorata, and Chimonanthus praecox, the FLC genes were absent in C. salicifolius, while the AGL6 subfamily was expanded in C. salicifolius. The selectively expanded promoter (AGL6) and lack of repressor (FLC) genes may explain the earlier flowering in C. salicifolius. The loss of the AP3 homologous gene in C. salicifolius is probably the primary cause of the morphological distinction between C. salicifolius and C. praecox. The csAGL6a gene is specifically expressed in the flowering process and indicates the potential function of promoting flowering. CONCLUSION: This study offers a genome-wide identification and expression profiling of the MIKC-types MADS-box genes in the C. salicifolius, and establishes the foundation for screening flowering development genes and understanding the potential function of the MIKC-types MADS-box genes in the C. salicifolius.

Highly Dispersed Cu Produced by Mechanical Stress-Activated Redox Reaction to Establish Galvanic Corrosion in Fe Implant.

Fe has immense potential for biodegradable orthopedic applications, but it degrades slowly in the physiological environment. Inducing galvanic couple by alloying Cu to Fe using ball milling is a promising approach. However, the ductile nature of Cu leads to the cold welding of a large amount of Cu powder during ball milling, which makes it difficult to disperse uniformly in the Fe matrix. Here, a Fe-CuO implant with highly dispersed Cu particles in the matrix was developed by shift-speed ball milling and selective laser melting. Specifically, copper oxide (CuO) particles were selected as precursors and dispersed in Fe powders by ball milling since they were brittle and would not be cold-welded during ball milling. After further milling in higher energy, it was found that CuO particles reacted with Fe and generated Cu particles through a stress-activated redox reaction. Subsequently, the obtained powders were prepared into a Fe-CuO implant using selective laser melting. Microstructure examination revealed that the Cu phases in the implant were dispersed evenly in the Fe matrix, which was considered to establish a large number of galvanic couples and aggravated the galvanic corrosion tendency. Electrochemical tests indicated that the implant had improved performance in degradation behavior in terms of high corrosion current density (22.4 µA/cm2), low corrosion resistance (1319 Ω cm2), and good degradation stability. In addition, it presented antibacterial effects against Escherichia coli and Staphylococcus aureus by diffusion mechanisms with killing rates of 90.7 and 96.7%, respectively, as well as good cytocompatibility.

A WRKY Transcription Factor PmWRKY57 from Prunus mume Improves Cold Tolerance in Arabidopsis thaliana.

Prunus mume, a woody perennial tree, is valued for its ornamental traits and has been cultivated for a long history. Low temperature is the main environmental factor restricting the distribution and affecting the growth of P. mume. In plants, some WRKY transcription factors have been reported to participate in regulating cold tolerance. However, there were few researches about functional characterization of WRKYs involving in P. mume cold response. Here, a cold-induced WRKY gene named as PmWRKY57 was cloned from a P. mume cultivar 'Guhong Zhusha.' PmWRKY57 protein harboring a WRKY domain and a C2H2 zinc finger motif belongs to Group IIc of WRKY family. The PmWRKY57 protein was located to the nucleus and has transcriptional activation activity. PmWRKY57-overexpresing Arabidopsis thaliana lines showed improved cold tolerance, compared to wild-type plants. Under cold treatment, the leaves of transgenic lines contained significantly lower malondialdehyde content, and higher levels of superoxide dismutase activity, peroxidase activity, and proline content than wild-type plants. Furthermore, the expression levels of cold-response genes such as AtCOR6.6, AtCOR47, AtKIN1, and AtRCI2A were up-regulated in leaves of transgenic A. thaliana compared to those in wild-type plants. This study characterized the function of PmWRKY57 in improving cold tolerance of plants.

Accelerated anode and cathode reaction due to direct electron uptake and consumption by manganese dioxide and titanium dioxide composite cathode in degradation of iron composite.

The movement towards the clinical application of iron (Fe) has been hindered by the slow degradation rate in physiological environments. Herein, manganese dioxide (MnO2) particles were compounded with titanium dioxide (TiO2) particles by mechanical ball milling, and then the mixed powders were incorporated into Fe and fabricated into an implant using selective laser melting. On the one hand, MnO2 had a higher work function (5.21 eV) than Fe (4.48 eV), which inclined electrons to transfer from Fe to MnO2 to accelerate the anode reaction. On the other hand, MnO2 catalysed the oxygen reduction reaction (ORR) through a four-step proton-electron-coupled reaction, which caused more oxygen to flow into the sample to improve the cathode performance. Besides, anatase TiO2 with high conductivity was compounded with MnO2 to construct a composite cathode, which facilitated electron transport from the cathode to the electrolyte, further consuming electrons and promoting cathode reaction. Results showed that Fe-MnO2-TiO2 had a high limiting current density of 5.32 mA·cm-2 and a large half-wave potential of -767.4 mV, indicating an enhanced ORR activity. More significantly, Fe-MnO2-TiO2 had a higher average electron transfer number (2.9) than Fe-MnO2 (2.5), demonstrating a faster electronic consumption reaction and higher cathode performance. In addition, the Fe-MnO2-TiO2 also exhibited fast instantaneous and long-term degradation rates (0.33 ± 0.03 and 0.19 ± 0.02 mm/year), suggesting a high anode dissolution rate. In conclusion, introducing the cathode with high work function and ORR activity provides novel pathways for accelerating the degradation rate of Fe-based implants.

Comparative Transcriptome Analysis of CCCH Family in Roles of Flower Opening and Abiotic Stress in Osmanthus fragrans.

CCCH is a zinc finger family with a typical CCCH-type motif which performs a variety of roles in plant growth and development and responses to environmental stressors. However, the information about this family has not been reported for Osmanthus fragrans. In this study, a total of 66 CCCH predicted genes were identified from the O. fragrans genome, the majority of which had multiple CCCH motifs. The 66 OfCCCHs were found to be unevenly distributed on 21 chromosomes and were clustered into nine groups based on their phylogenetic analysis. In each group, the gene structure and domain makeup were comparatively conserved. The expression profiles of the OfCCCH genes were examined in various tissues, the flower-opening processes, and under various abiotic stresses using transcriptome sequencing and qRT-PCR (quantitative real-time PCR). The results demonstrated the widespread expression of OfCCCHs in various tissues, the differential expression of 22 OfCCCHs during flower-opening stages, and the identification of 4, 5, and 13 OfCCCHs after ABA, salt, and drought stress treatment, respectively. Furthermore, characterization of the representative OfCCCHs (OfCCCH8, 23, 27, and 36) revealed that they were all localized in the nucleus and that the majority of them had transcriptional activation in the yeast system. Our research offers the first thorough examination of the OfCCCH family and lays the groundwork for future investigations regarding the functions of CCCH genes in O. fragrans.

Shikimate Kinase Plays Important Roles in Anthocyanin Synthesis in Petunia.

In plants, the shikimate pathway is responsible for the production of aromatic amino acids L-tryptophan, L-phenylalanine, and L-tyrosine. L-Phenylalanine is the upstream substrate of flavonoid and anthocyanin synthesis. Shikimate kinase (SK) catalyzes the phosphorylation of the C3 hydroxyl group of shikimate to produce 3-phosphate shikimate (S3P), the fifth step of the shikimate pathway. However, whether SK participates in flavonoid and anthocyanin synthesis is unknown. This study characterized the single-copy PhSK gene in the petunia (Petunia hybrida) genome. PhSK was localized in chloroplasts. PhSK showed a high transcription level in corollas, especially in the coloring stage of flower buds. Suppression of PhSK changed flower color and shape, reduced the content of anthocyanins, and changed the flavonoid metabolome profile in petunia. Surprisingly, PhSK silencing caused a reduction in the shikimate, a substrate of PhSK. Further qPCR analysis showed that PhSK silencing resulted in a reduction in the mRNA level of PhDHQ/SDH, which encodes the protein catalyzing the third and fourth steps of the shikimate pathway, showing a feedback regulation mechanism of gene expression in the shikimate pathway.

Integrated Transcriptome and Metabolome Analysis of Color Change and Low-Temperature Response during Flowering of Prunus mume.

In China, Prunus mume is a famous flowering tree that has been cultivated for 3000 years. P. mume grows in tropical and subtropical regions, and most varieties lack cold resistance; thus, it is necessary to study the low-temperature response mechanism of P. mume to expand the scope of its cultivation. We used the integrated transcriptomic and metabolomic analysis of a cold-resistant variety of P. mume 'Meiren', to identify key genes and metabolites associated with low temperatures during flowering. The 'Meiren' cultivar responded in a timely manner to temperature by way of a low-temperature signal transduction pathway. After experiencing low temperatures, the petals fade and wilt, resulting in low ornamental value. At the same time, in the cold response pathway, the activities of related transcription factors up- or downregulate genes and metabolites related to low temperature-induced proteins, osmotic regulators, protective enzyme systems, and biosynthesis and metabolism of sugars and acids. Our findings promote research on the adaptation of P. mume to low temperatures during wintering and early flowering for domestication and breeding.

Genetic studies on continuous flowering in woody plant Osmanthus fragrans.

Continuous flowering is a key horticultural trait in ornamental plants, whereas the specific molecular regulation mechanism remains largely unknown. In sweet osmanthus (Osmanthus fragrans Lour.), plants based on their flowering characteristics are divided into once-flowering (OF) habit and continuous flowering (CF) habit. Here, we first described the flowering phenology shifts of OF and CF habits in sweet osmanthus through paraffin section and microscope assay. Phenotypic characterization showed that CF plants had constant new shoot growth, floral transition, and blooming for 1 year, which might lead to a continuous flowering trait. We performed the transcriptome sequencing of OF and CF sweet osmanthus and analyzed the transcriptional activity of flowering-related genes. Among the genes, three floral integrators, OfFT, OfTFL1, and OfBFT, had a differential expression during the floral transition process in OF and CF habits. The expression patterns of the three genes in 1 year were revealed. The results suggested that their accumulations corresponded to the new shoots occurring and the floral transition process. Function studies suggested that OfFT acted as a flowering activator, whereas OfBFT was a flowering inhibitor. Yeast one-hybrid assay indicated that OfSPL8 was a common upstream transcription factor of OfFT and OfBFT, suggesting the vital role of OfSPL8 in continuous flowering regulation. These results provide a novel insight into the molecular mechanism of continuous flowering.

Genome-Wide Identification and Expression Analysis of XTH Gene Family during Flower-Opening Stages in Osmanthus fragrans.

Osmanthus fragrans is an aromatic plant which is widely used in landscaping and garden greening in China. However, the process of flower opening is significantly affected by ambient temperature changes. Cell expansion in petals is the primary factor responsible for flower opening. Xyloglucan endoglycolase/hydrolase (XTH) is a cell-wall-loosening protein involved in cell expansion or cell-wall weakening. Through whole-genome analysis, 38 OfXTH genes were identified in O. fragrans which belong to the four main phylogenetic groups. The gene structure, chromosomal location, synteny relationship, and cis-acting elements prediction and expression patterns were analyzed on a genome-wide scale. The expression patterns showed that most OfXTHs were closely associated with the flower-opening period of O. fragrans. At the early flower-opening stage (S1 and S2), transcriptome and qRT-PCR analysis revealed the expression of OfXTH24, 27, 32, 35, and 36 significantly increased under low ambient temperature (19 °C). It is speculated that the five genes might be involved in the regulation of flower opening by responding to ambient temperature changes. Our results provide solid foundation for the functional analysis of OfXTH genes and help to explore the mechanism of flower opening responding to ambient temperature in O. fragrans.

Stress-Induced Dual-Phase Structure to Accelerate Degradation of the Fe Implant.

Fe is considered as a potential candidate for implant materials, but its application is impeded by the low degradation rate. Herein, a dual-phase Fe30Mn6Si alloy was prepared by mechanical alloying (MA). During MA, the motion of dislocations driven by the impact stress promoted the solid solution of Mn in Fe, which transformed α-ferrite into γ-austenite since Mn was an austenite-stabilizing element. Meanwhile, the incorporation of Si decreased the stacking fault energy inside austenite grains, which tangled dislocations into stacking faults and acted as nucleation sites for ε-martensite. Resultantly, Fe30Mn6Si powder had a dual-phase structure composed of 53% γ-austenite and 47% ε-martensite. Afterward, the powders were prepared into implants by selective laser melting. The Fe30Mn6Si alloy had a more negative corrosion potential of -0.76 ± 0.09 V and a higher corrosion current of 30.61 ± 0.41 µA/cm2 than Fe and Fe30Mn. Besides, the long-term weight loss tests also proved that Fe30Mn6Si had the optimal degradation rate (0.25 ± 0.02 mm/year).

Phosphoproteome analysis reveals the involvement of protein dephosphorylation in ethylene-induced corolla senescence in petunia.

BACKGROUND: Senescence represents the last stage of flower development. Phosphorylation is the key posttranslational modification that regulates protein functions, and kinases may be more required than phosphatases during plant growth and development. However, little is known about global phosphorylation changes during flower senescence. RESULTS: In this work, we quantitatively investigated the petunia phosphoproteome following ethylene or air treatment. In total, 2170 phosphosites in 1184 protein groups were identified, among which 2059 sites in 1124 proteins were quantified. To our surprise, treatment with ethylene resulted in 697 downregulated and only 117 upregulated phosphosites using a 1.5-fold threshold (FDR < 0.05), which showed that ethylene negatively regulates global phosphorylation levels and that phosphorylation of many proteins was not necessary during flower senescence. Phosphoproteome analysis showed that ethylene regulates ethylene and ABA signalling transduction pathways via phosphorylation levels. One of the major targets of ethylene-induced dephosphorylation is the plant mRNA splicing machinery, and ethylene treatment increases the number of alternative splicing events of precursor RNAs in petunia corollas. CONCLUSIONS: Protein dephosphorylation could play an important role in ethylene-induced senescence, and ethylene treatment increased the number of AS precursor RNAs in petunia corollas.

Suppression of chorismate synthase, which is localized in chloroplasts and peroxisomes, results in abnormal flower development and anthocyanin reduction in petunia.

In plants, the shikimate pathway generally occurs in plastids and leads to the biosynthesis of aromatic amino acids. Chorismate synthase (CS) catalyses the last step of the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, but the role of CS in the metabolism of higher plants has not been reported. In this study, we found that PhCS, which is encoded by a single-copy gene in petunia (Petunia hybrida), contains N-terminal plastidic transit peptides and peroxisomal targeting signals. Green fluorescent protein (GFP) fusion protein assays revealed that PhCS was localized in chloroplasts and, unexpectedly, in peroxisomes. Petunia plants with reduced PhCS activity were generated through virus-induced gene silencing and further characterized. PhCS silencing resulted in reduced CS activity, severe growth retardation, abnormal flower and leaf development and reduced levels of folate and pigments, including chlorophylls, carotenoids and anthocyanins. A widely targeted metabolomics analysis showed that most primary and secondary metabolites were significantly changed in pTRV2-PhCS-treated corollas. Overall, the results revealed a clear connection between primary and specialized metabolism related to the shikimate pathway in petunia.

PaACL silencing accelerates flower senescence and changes the proteome to maintain metabolic homeostasis in Petunia hybrida.

Cytosolic acetyl-CoA is an intermediate of the synthesis of most secondary metabolites and the source of acetyl for protein acetylation. The formation of cytosolic acetyl-CoA from citrate is catalysed by ATP-citrate lyase (ACL). However, the function of ACL in global metabolite synthesis and global protein acetylation is not well known. Here, four genes, PaACLA1, PaACLA2, PaACLB1, and PaACLB2, which encode the ACLA and ACLB subunits of ACL in Petunia axillaris, were identified as the same sequences in Petunia hybrida 'Ultra'. Silencing of PaACLA1-A2 and PaACLB1-B2 led to abnormal leaf and flower development, reduced total anthocyanin content, and accelerated flower senescence in petunia 'Ultra'. Metabolome and acetylome analysis revealed that PaACLB1-B2 silencing increased the content of many downstream metabolites of acetyl-CoA metabolism and the levels of acetylation of many proteins in petunia corollas. Mechanistically, the metabolic stress induced by reduction of acetyl-CoA in PaACL-silenced petunia corollas caused global and specific changes in the transcriptome, the proteome, and the acetylome, with the effect of maintaining metabolic homeostasis. In addition, the global proteome and acetylome were negatively correlated under acetyl-CoA deficiency. Together, our results suggest that ACL acts as an important metabolic regulator that maintains metabolic homeostasis by promoting changes in the transcriptome, proteome. and acetylome.