RhLHY-RhTPPF-Tre6P Inhibits Flowering in Rosa hybrida Under Insufficient Light by Regulating Sugar Distribution.

Light is crucial for flower bud development in plants, serving as both signal and energy source. However, the mechanisms by which daylength and light intensity regulate flowering in modern roses remain unclear. In Rosa hybrida 'Carola', insufficient light delays flowering and reduces the sugar content in terminal buds. RNA sequencing identified the Trehalose-6-phosphate phosphatase F (RhTPPF) gene as a key responder to insufficient light, modulating Tre6P metabolism. Overexpression of RhTPPF in rose calli enhanced sugar accumulation and suppressed the synthesis of RhCO/FT. In tobacco, overexpression of RhTPPF delayed the transition from vegetative growth to flowering, while silencing RhTPPF in roses accelerated flowering. Silencing RhTPPF in roses elevated trehalose-6-phosphate (Tre6P) levels and decreased trehalose. Transcriptome data showed that the expression level of RhTPPF was highly correlated with the circadian rhythm gene LATE ELONGATED HYPOCOTYL (RhLHY). Yeast one-hybrid assays, dual luciferase assays and EMSA revealed that RhLHY directly binds to the RhTPPF promoters. Overexpression of RhLHY suppressed flowering, while silencing RhLHY promoted flowering. Furthermore, altering the expression of RhLHY influenced Tre6P synthesis and the expression of sucrose-related transport genes. These findings suggest a RhLHY-RhTPPF-Tre6P regulatory module that maintains sugar balance and inhibits flower formation under reduced light conditions by modulating sugar distribution.

Oenothera biennis with strong copper toxicity resistance enriches trace copper in seeds under copper pollution soil.

Excess copper (Cu) imparts negative effects on plant growth and productivity in soil. To develop the ability of O. biennis to govern pollution soil containing excessive Cu, we investigated seed germination, seedling growth, and seed yield. Furthermore, Cu content and the expression levels of Cu transport related genes in different tissues were measured under exogenous high concentration Cu. O. biennis seeds were sensitive to excess Cu, with an observed reduction in the germination rate, primary root length, fresh weight, and number of seeds germinated daily. Consecutive Cu stress did not cause fatal damage to evening primrose, yet it slowed down plant growth slightly by reducing the leaf water, chlorophyll, plant yield, and seed oil contents while increasing the soluble sugar, proline, malondialdehyde, and H2O2 contents. The Cu content in different organs of O. biennis was disrupted by excess Cu. In particular, the Cu content in O. biennis seeds and seed oil increased and subsequently decreased with the increase of exogenous Cu, reaching a peak under 600 mg·kg-1 consecutive Cu. Furthermore, the 4-month 900 mg·kg-1 Cu treatment did not induce the excessive accumulation of Cu in peels, seeds, and seed oil, maintaining the Cu content within the range required by the Chinese National Food Safety Standards. The treatment also resulted in an upregulation of Cu-uptake (ObCOPT5, ObZIP4, and ObYSL2) and vigorous efflux (ObHMA1) of transport genes, of which expression levels were significant positive correlation (p < 0.05) with the Cu content. Among all organs, the stem replaced the root as the organ exhibited the greatest ability to absorb and store Cu, and even the Cu transport genes could still function continuously in stem under excess Cu. This work identified a species that can tolerate high Cu content in soil while maintaining a high yield. Furthermore, the results revealed the enrichment of Cu to occur primarily in the O. biennis stem rather than the seeds and peel under excess Cu.

Ferric ion crosslinking-based 3D printing of a graphene oxide hydrogel and its evaluation as a bio-scaffold in tissue engineering.

As a precursor of graphene, graphene oxide (GO) exhibits excellent mechanical, thermal, and electrical properties, besides appreciable biocompatibility in tissue engineering applications. However, the current GO-3D fabrication technology is still in need of optimization and simplification to ensure fine architecture and reasonable mechanical properties, which would further promote the performance of GO as bio-scaffolds in cell or microorganism attachment and in material transformation. To address this issue, we proposed a GO ink, with appreciable rheological properties and excellent printing performance via high-speed centrifugation and ferric ion-assisted cross-linking. A woodpile structure with controllable micro-pores was produced by micro-extrusion-based 3D printing technology followed by an optimized freeze-drying process. Cellular adhesion and viability were verified by inoculation and culture of HepaRG cells using the fabricated GO 3D structure, thus suggesting ferric ion-assisted cross-linking and controllable pore distribution for improving the performance of the GO construct as a bio-scaffold for in vitro liver tissue models.

Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis.

OBJECTIVES: To investigate the inhibitory effect of cholecystokinin octapeptide (CCK-8) binding to cholecystokinin 2 receptor (CCK2R) on methamphetamine (METH)-induced neuronal apoptosis, and to explore the signal transduction mechanism of ß-arrestin 2 in CCK-8 inhibiting METH-induced neuronal apoptosis. METHODS: SH-SY5Y cell line was cultured, and HEK293-CCK1R and HEK293-CCK2R cell line were constructed by lentivirus transfection. Small interfering RNA (siRNA) was used to knockdown the expression of ß-arrestin 2. Annexin Ⅴ-FITC/PI staining and flow cytometry were used to detect the apoptotic rate of cells, and Western blotting was used to detect the expression of apoptosis-related proteins. RESULTS: The apoptosis of SH-SY5Y cells was induced by 1 mmol/L and 2 mmol/L METH treatment, the number of nuclear fragmentation and pyknotic cells was significantly increased, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were increased. CCK-8 pre-treatment at the dose of 0.1 mmol/L and 1 mmol/L significantly reversed METH-induced apoptosis in SH-SY5Y cells, and inhibited cell nuclear fragmentation, pyknosis and the changes of apoptosis-related proteins induced by METH. In lentivirus transfected HEK293-CCK1R and HEK293-CCK2R cells, the results revealed that CCK-8 had no significant effect on METH-induced changes of apoptosis-related proteins in HEK293-CCK1R cells, but it could inhibit the expression level of apoptosis-related proteins in HEK293-CCK2R cells induced by METH. The inhibitory effect of CCK-8 on METH-induced apoptosis was blocked by the knockdown of ß-arrestin 2 expression in SH-SY5Y cells. CONCLUSIONS: CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the ß-arrestin 2 signal.

Integrative analysis of transcriptomic and proteomic changes related to male sterility in Tagetes erecta.

Male sterile and male fertile two-type lines are important in heterosis utilization and breeding in Tagetes erecta, but the genes and pathways involved in male sterility are poorly understood. To explore these topics, transcriptome data (by RNA-seq) and proteome data (by iTRAQ) were gathered from flower buds of the male sterile line 'MS2-2' and male fertile line 'MF2-2' and integrated for a better understanding of the underlying molecular mechanisms of male sterility in T. erecta. The RNA-seq procedure generated 285,139,740 clean reads and 63359 unigenes and 6640 differentially expressed genes (DEGs) were identified, of which 4136 were downregulated and 2504 were upregulated in 'MS2-2'. DEGs related to flower development, pollen development, pollen wall assembly, endogenous hormones and transcription factors were identified. The iTRAQ analysis identified 3950 proteins in total; 789 were differentially expressed proteins (381 upregulated, 408 downregulated), which were mainly annotated to the Ribosome, Carbon metabolism and Biosynthesis of amino acids pathways. An association analysis revealed strong correlation (r Pearson = 0.6019) between the transcriptomic and proteomic data, and 256 and 34 proteins showed the same and opposite expression patterns with regard to their transcripts, respectively. Pathways such as photosynthesis, fatty acid biosynthesis and phenylpropanoid biosynthesis which influence tapetum and pollen development in male sterile plants, were significantly enriched at the transcript and protein levels. Most genes involved in these pathways were downregulated in 'MS2-2'. The low expression of these genes or functional loss of proteins could be associated with flower development, pollen development and related to changes in fertility in T. erecta. This study provided transcriptomic and proteomic information for T. erecta that could illuminate the mechanism of male sterility.

RNA sequencing analysis reveals PgbHLH28 as the key regulator in response to methyl jasmonate-induced saponin accumulation in Platycodon grandiflorus.

Platycodon grandiflorus (Jacq.) A. DC, known for its saponin content, can potentially prevent and treat cerebrovascular diseases and COVID-19. Triterpenoid saponin biosynthesis in plants is enhanced by methyl jasmonate (MeJA) application. However, the underlying molecular mechanisms of MeJA-induced saponin biosynthesis remain unknown in P. grandiflorus. In the current study, exogenous application of 100 µmol/l MeJA was identified to be optimal for promoting saponin accumulation. RNA sequencing analysis demonstrated the PgbHLH28 gene as a key regulatory factor responding to MeJA during saponin accumulation. Overexpression of PgbHLH28 in P. grandiflorus increased saponin content, while silencing of PgbHLH28 significantly inhibited saponin synthesis, suggesting that PgbHLH28 acts as a positive regulator of saponin biosynthesis. Yeast one-hybrid and dual luciferase assays demonstrated that PgbHLH28 directly bound to the promoters of PgHMGR2 and PgDXS2 to activate gene expression. PgHMGR2 and PgDXS2 transformation promoted saponin accumulation, while silencing of these genes inhibited saponin biosynthesis. This study determined that MeJA promoted saponin accumulation in P. grandiflorus by inducing PgbHLH28 gene expression and activating downstream genes (PgHMGR2 and PgDXS2) involved in saponin biosynthesis. In conclusion, a complex regulatory network governing saponin biosynthesis following MeJA treatment was elucidated, offering a theoretical foundation for enhancing saponin content and biosynthesis efficacy in P. grandiflorus.

Genome-Wide Bioinformatics Analysis of SWEET Gene Family and Expression Verification of Candidate PaSWEET Genes in Potentilla anserina.

Sugars act as the main energy sources in many fruit and vegetable crops. The biosynthesis and transportation of sugars are crucial and especially contribute to growth and development. SWEET is an important gene family that plays a vital role in plants' growth, development, and adaptation to various types of stresses (biotic and abiotic). Although SWEET genes have been identified in numerous plant species, there is no information on SWEETs in Potentilla anserina. In the present study, we performed a comprehensive genome-wide bioinformatics analysis and identified a total of 23 candidate PaSWEETs genes in the Potentilla anserina genome, which were randomly distributed on ten different chromosomes. The phylogenetic analysis, chromosomal location, gene structure, specific cis-elements, protein interaction network, and physiological characteristics of these genes were systematically examined. The identified results of the phylogenetic relationship with Arabidopsis thaliana revealed that these PaSWEET genes were divided into four clades (I, II, III, and IV). Moreover, tissue-specific gene expression through quantitative real-time polymerase chain reaction (qRT-PCR) validation exposed that the identified PaSWEETs were differentially expressed in various tissues (roots, stems, leaves, and flowers). Mainly, the relative fold gene expression in swollen and unswollen tubers effectively revealed that PaSWEETs (7, 9, and 12) were highly expressed (300-, 120-, and 100-fold) in swollen tubers. To further elucidate the function of PaSWEETs (7, 9, and 12), their subcellular location was confirmed by inserting them into tobacco leaves, and it was noted that these genes were present on the cell membrane. On the basis of the overall results, it is suggested that PaSWEETs (7, 9, and 12) are the candidate genes involved in swollen tuber formation in P. anserina. In crux, we speculated that our study provides a valuable theoretical base for further in-depth function analysis of the PaSWEET gene family and their role in tuber development and further enhancing the molecular breeding of Potentilla anserina.

Factors influencing the association between depressive symptoms and cardiovascular disease in US population.

Cardiovascular disease (CVD) and depression are common diseases that lead to adverse health outcomes. Depressive Symptoms may be a risk factor for CVD. But few studies focused on the impact of socioeconomic factors, common medical history and dietary intake about this association. This study analyzed National Health and Nutrition Examination Survey (NHANES) 2007-2016. Complex sampling-weighted logistic regression models were used to compare the odds ratios (ORs) of CVD in participants with different depressive symptoms. 11,516 NHANES participants aged ≥ 40 years were included in the final analysis, of whom 1842 had CVD. Compared with participants with no/minimal depression, participants with mild, moderate, and moderately severe/severe depression had OR values of 1.25 (95%  CI 1.01-1.54), 1.98 (95% CI 1.32-2.96), and 2.41 (95% CI 1.63-3.57). The association of depressive symptoms with CVD follow a dose-dependent pattern. The interactions of depressive symptoms with gender (Interaction P = 0.009), diabetes (Interaction P = 0.010), household income level (Interaction P = 0.002), dietary cholesterol intake (Interaction P = 0.017) on CVD were observed. More severe depressive symptoms are associated with increased risk of CVD in US population. The association may be more pronounced in the female population, population with diabetes, low family income level, or high dietary cholesterol intake.

Genome-wide analysis of the WOX gene family and function exploration of RhWOX331 in rose (R. 'The Fairy').

WOXs are a class of plant-specific transcription factors that play key roles in plant growth and stress responses. However, the mechanism by which WOXs influence adventitious root development in Rosa hybrida remains unclear. In this study, RcWOX gene family in rose was identified and phylogenetically analyzed using bioinformatics analysis. A total of 381 RcWOX gene members were localized on seven chromosomes except of nine members. The main cis-acting elements involved in hormonal, light, developmental, and abiotic stress responses were identified in the promoters of RcWOX genes, suggesting their regulation by these signals. Nine RhWOX genes had significant different expression during rooting process of rose. RhWOX331, RhWOX308, RhWOX318 were positive with the formation of rose roots. RhWOX331 was positively involved in the formation of adventitious root primordia, which gene coding a transcription factor localized in the nucleus. The HOX conserved domain in the protein contributed to the self-activating activity of RhWOX331. We obtained genetically modified Arabidopsis to validate the function of RhWOX331. Overexpression of RhWOX331 gene alleviated the inhibition of root length of A. thaliana primary roots by high concentration of IBA and NPA, and significantly increased the number of lateral roots on the primary roots, as well as the height of A. thaliana plants. Additionally, RhWOX331 promoted adventitious root formation in A. thaliana and mitigated hormonal inhibition by exogenous 6-BA, NPA, and GA3. The RhWOX331 promoter contained cis-acting elements such as ABRE, Box 4 and CGTCA-motif et.al. GUS activity analysis showed that the gene acted at the cotyledon attachment site. Taken together, these studies identified a significant expansion of the RcWOX gene family, inferred roles of certain branch members in adventitious root formation, elucidated the function of RhWOX331 in adventitious root initiation, and laid the foundation for further research on the function of WOX gene family in roses.

Genome-Wide Identification of the CYP716 Gene Family in Platycodon grandiflorus (Jacq.) A. DC. and Its Role in the Regulation of Triterpenoid Saponin Biosynthesis.

The main type of saponins occurring in the root of Platycodon grandiflorus (Jacq.) A. DC. are oleanolic acid glycosides. The CYP716 gene family plays a major role in catalyzing the conversion of ß-amyrin into oleanolic acid. However, studies on the CYP716 genes in P. grandiflorus are limited, and its evolutionary history remains poorly understood. In this study, 22 PgCYP716 genes were identified, distributed among seven subfamilies. Cis-acting elements of the PgCYP716 promoters were mainly involved in plant hormone regulation and responses to abiotic stresses. PgCYP716A264, PgCYP716A391, PgCYP716A291, and PgCYP716BWv3 genes were upregulated in the root and during saponin accumulation, as shown by RNA-seq analysis, suggesting that these four genes play an important role in saponin synthesis. The results of subcellular localization indicated that these four genes encoded membrane proteins. Furthermore, the catalytic activity of these four genes was proved in the yeast, which catalyzed the conversion of ß-amyrin into oleanolic acid. We found that the content of ß-amyrin, platycodin D, platycoside E, platycodin D3, and total saponins increased significantly when either of the four genes was over expressed in the transgenic hair root. In addition, the expression of PgSS, PgGPPS2, PgHMGS, and PgSE was also upregulated while these four genes were overexpressed. These data support that these four PgCYP716 enzymes oxidize ß-amyrin to produce oleanolic acid, ultimately promoting saponin accumulation by activating the expression of upstream pathway genes. Our results enhanced the understanding of the functional variation among the PgCYP716 gene family involved in triterpenoid biosynthesis and provided a theoretical foundation for improving saponin content and enriching the saponin biosynthetic pathway in P. grandiflorus.

Comparison of betalain compounds in two Beta vulgaris var. cicla and BvCYP76AD27 function identification in betalain biosynthesis.

Beta vulgaris var. cicla is an edible, ornamental and horticultural plant. However, the difference of components and contents of betalain in beets with different leaf color are not well understood. Here, the stress resistance and metabolites of two B. vulgaris var. cicla cultivars were determined. The differences in stress resistance between red leaf-colored chard (RC) and yellow leaf-colored chard (YC) were positively related to betacyanins (BC) and betaxathins (BX) content in the leaves. Furthermore, a total of 3615 distinct metabolites were identified by UPLC-QTOF-MS in two cultivars, including 70 alkaloids and their derivatives, 249 flavonoids, and 264 terpenoids. There were 17 metabolites attributed to betalain biosynthesis pathway, seven of nine BC were up-regulated, and eight BX showed no significant difference in RC compared with YC. The contents of celosianin II and betanin were the highest BC in RC, at approximately 84.38 and 19.97 times that of YC, respectively. The content of portulacaxanthin II was the highest BX in two beets. Additionally, the BvCYP450 genes were identified based on genome, and the members that might be involved in betalain biosynthesis were screened. BvCYP76AD27, a member of the BvCYP76AD subfamily, had a higher expression level in RC than YC under freezing, drought and shading stress. In yeast Saccharomyces cerevisiae, BvCYP76AD5 and BvCYP76AD27 only hydroxylated tyrosine to L-DOPA, which was transformed into portulacaxanthin II by 4,5-DOPA extradiol dioxygenase. The results contribute to illustrating the molecular mechanism of betalain biosynthesis and provide useful information for further investigation of beet chemistry and sufficient utilization of this species.

Genome-Wide Identification and Expression Analysis of the Trehalose-6-phosphate Synthase and Trehalose-6-phosphate Phosphatase Gene Families in Rose (Rosa hybrida cv 'Carola') under Different Light Conditions.

Trehalose, trehalose-6-phosphate synthase (TPS),and trehalose-6-phosphatase (TPP) have been reported to play important roles in plant abiotic stress and growth development. However, their functions in the flowering process of Rosa hybrida have not been characterized. In this study we found that, under a short photoperiod or weak light intensity, the content of trehalose in the shoot apical meristem of Rosa hybrida cv 'Carola' significantly decreased, leading to delayed flowering time. A total of nine RhTPSs and seven RhTPPs genes were identified in the genome. Cis-element analysis suggested that RhTPS and RhTPP genes were involved in plant hormones and environmental stress responses. Transcriptome data analysis reveals significant differences in the expression levels of RhTPSs and RhTPPs family genes in different tissues and indicates that RhTPPF and RhTPPJ are potential key genes involved in rose flower bud development under different light environments. The results of quantitative real-time reverse transcription (qRT-PCR) further indicate that under short photoperiod and weak light intensity all RhTPP members were significantly down-regulated. Additionally, RhTPS1a, RhTPS10, and RhTPS11 were up-regulated under a short photoperiod and showed a negative correlation with flowering time and trehalose content decrease. Under weak light intensity, RhTPS11 was up-regulated and negatively regulated flowering, while RhTPS5, RhTPS6, RhTPS7b, RhTPS9, and RhTPS10 were down-regulated and positively regulated flowering. This work lays the foundation for revealing the functions of RhTPS and RhTPP gene families in the regulation of rose trehalose.

An R2R3-MYB Transcription Factor RmMYB108 Responds to Chilling Stress of Rosa multiflora and Conferred Cold Tolerance of Arabidopsis.

A sudden cooling in the early spring or late autumn negatively impacts the plant growth and development. Although a number of studies have characterized the role of the transcription factors (TFs) of plant R2R3-myeloblastosis (R2R3-MYB) in response to biotic and abiotic stress, plant growth, and primary and specific metabolisms, much less is known about their role in Rosa multiflora under chilling stress. In the present study, RmMYB108, which encodes a nuclear-localized R2R3-MYB TF with a self-activation activity, was identified based on the earlier published RNA-seq data of R. multiflora plants exposed to short-term low-temperature stress and also on the results of prediction of the gene function referring Arabidopsis. The RmMYB108 gene was induced by stress due to chilling, salt, and drought and was expressed in higher levels in the roots than in the leaves. The heterologous expression of RmMYB108 in Arabidopsis thaliana significantly enhanced the tolerance of transgenic plants to freezing, water deficit, and high salinity, enabling higher survival and growth rates, earlier flowering and silique formation, and better seed quantity and quality compared with the wild-type (WT) plants. When exposed to a continuous low-temperature stress at 4°C, transgenic Arabidopsis lines-overexpressing RmMYB108 showed higher activities of superoxide dismutase and peroxidase, lower relative conductivity, and lower malondialdehyde content than the WT. Moreover, the initial fluorescence (F o) and maximum photosynthetic efficiency of photosystem II (F v/F m) changed more dramatically in the WT than in transgenic plants. Furthermore, the expression levels of cold-related genes involved in the ICE1 (Inducer of CBF expression 1)-CBFs (C-repeat binding factors)-CORs (Cold regulated genes) cascade were higher in the overexpression lines than in the WT. These results suggest that RmMYB108 was positively involved in the tolerance responses when R. multiflora was exposed to challenges against cold, freeze, salt, or drought and improved the cold tolerance of transgenic Arabidopsis by reducing plant damage and promoting plant growth.

Cool-Warm Temperature Stratification and Simulated Bird Digestion Optimize Removal of Dormancy in Rosa rugosa Seeds.

Rosa rugosa Thunb. has been explored multi-function in medicinal, edible, cosmetic, ornamental and ecological etc. However, R. rugosa natural populations have recently declined substantially in China, besides of global climate change, this species also has the defect of limiting the reproduction of itself such as the hard-to-release seed dormancy. In this study, only 30% of R. rugosa seeds were viable, and the others were incompletely developed or diseased seeds. Without stratification, morphologically complete viable seeds imbibed water but those seeds could not germinate even after seed husk removal under suitable condition to exhibit a physiological dormancy. After cold (4°C) and warm (18 ± 2°C) stratification, macromolecular substances containing carbon or nitrogen accumulated, and respiration, antioxidant enzyme activity, and gibberellin (GA3) /abscisic acid (ABA) and auxin (IAA)/ABA ratios increased significantly in seeds. Water absorption also increased as endocarps softened. Thus, physiological dormancy of seed was broken. Although warm and cold stratification increased separation between endocarp and embryo, the endocarp binding force was removed insufficiently, because only 10.20% of seeds germinated. Therefore, stratified seeds were treated with simulated bird digestion. Then, folds and cracks in loosened endocarps increased permeability, and water absorption rate increased to 64.43% compare to 21.14% in cold and warm stratification treatment. With simulated digestion, 24.20% of radicles broke through the endocarp with plumules and cambiums to develop into seedlings. Thus, the seed dormancy type of R. rugosa is physiological as seeds imbibed water and possessed fully developed embryos with a low growth potential in combination with a mechanical constraint from the endocarp. Cold stratification helped remove physiological dormancy, and additional warm stratification accelerated the process. The optimal stratification treatment was 4°C for 45 days followed by 18 ± 2°C for 15 days. After warm and cold stratification, simulated bird digestion broke the mechanical constraint from the seed covering layers. Based on this research, production of R. rugosa seedlings can be greatly increased to help protect the species from further declines.