A 49-bp deletion of PmAP2L results in a double flower phenotype in Prunus mume.

The double flower is an important trait with substantial ornamental value. While mutations in PETALOSA TOE-type or AG (AGAMOUS) genes play a crucial role in enhancing petal number in ornamental plants, the complete mechanism underlying the formation of double flowers remains to be fully elucidated. Through the application of bulked segregant analysis (BSA), we identified a novel gene, APETALA2-like (PmAP2L), characterized by a 49-bp deletion in double-flowered Prunus mume. ß-Glucuronidase (GUS) staining and luciferase reporter assays confirmed that the 49-bp deletion in PmAP2L reduced its binding with Pmu-miRNA172a. Phylogenetic analysis and microsynteny analysis suggested that PmAP2L was not a PETALOSA TOE-type gene, and it might be a new gene controlling the formation of double flower in P. mume. Subsequently, overexpression of PmAP2L-D in tobacco led to a significant rise in the number of stamens and the conversion of stamens to petals. Furthermore, silencing of the homologue of RC5G0530900 in rose significantly reduced the number of petals. Using transient gene expression in P. mume flower buds, we determined the functional differences between PmAP2L-D and PmAP2-S in controlling flower development. Meanwhile, DNA-affinity purification sequencing (DAP-seq), yeast hybrid assays and luciferase reporter assays indicated that PmAP2L negatively regulated the floral organ identity genes by forming a repressor complex with PmTPL and PmHDA6/19. Overall, these findings indicate that the variation in PmAP2L is associated with differences in the regulation of genes responsible for floral organ identity, providing new insights into the double-flower trait and double-flower breeding in plants.

Genome-Wide Identification of TCP Gene Family in Dendrobium and Their Expression Patterns in Dendrobium chrysotoxum.

The TCP gene family are plant-specific transcription factors that play important roles in plant growth and development. Dendrobium chrysotoxum, D. nobile, and D. huoshanense are orchids with a high ornamental value, but few studies have investigated the specific functions of TCPs in Dendrobium flower development. In this study, we used these three Dendrobium species to analyze TCPs, examining their physicochemical properties, phylogenetic relationships, gene structures, and expression profiles. A total of 50 TCPs were identified across three Dendrobium species; they were divided into two clades-Class-I (PCF subfamily) and Class-II (CIN and CYC/TB1 subfamilies)-based on their phylogenetic relationships. Our sequence logo analysis showed that almost all Dendrobium TCPs contain a conserved TCP domain, as well as the existence of fewer exons, and the cis-regulatory elements of the TCPs were mostly related to light response. In addition, our transcriptomic data and qRT-PCR results showed that DchTCP2 and DchTCP13 had a significant impact on lateral organs. Moreover, changes in the expression level of DchTCP4 suggested its important role in the phenotypic variation of floral organs. Therefore, this study provides a significant reference for the further exploration of TCP gene functions in the regulation of different floral organs in Dendrobium orchids.

The Transcriptome Profiling of Flavonoids and Bibenzyls Reveals Medicinal Importance of Rare Orchid Arundina graminifolia.

Orchids are very important flowering plants that spend long juvenile phases before flowering. Along with aesthetic importance, they are rich sources of medicinal components. However, their long reproductive cycle is the major hurdle to study the medicinal efficacy. Arundina graminifolia is a rare orchid that grows fast, unlike other orchids, and this characteristic makes it an ideal plant to study the medicinal enrichment of orchids. Therefore, this study presents the identification of important medicinal components in various parts of A. graminifolia. Transcriptome analysis was performed for five stages (FD1-FD5) of flower development and four tissue types (mature flower, silique, root, and leaf) to ascertain genetic regulators of flavonoids and bibenzyls. Most of the genes showed the highest expression in roots as compared with other tissues. Weighted gene coexpression network analysis (WGCNA) was performed to identify the coexpression modules and the candidate genes involving biosynthesis pathways of these chemicals. MEyellow module contained the highly coexpressed genes. Moreover, the concentrations of phenylpropanoid, bibenzyls, and flavone were ascertained through high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Phenylpropanoid and bibenzyl were comparatively high in the leaf, while flavone showed a high concentration in the stem. The selected candidate genes [bibenzyl biosynthesis (BIBSY212), CYP84A1, CYP73A4, 4CLL7, UGT88B1, UGT73C3, anthocyanin synthase (ANS), phenylalanine ammonia-lyase (PAL), flavanone synthase FLS, and CHS8] were validated through quantitative real-time PCR (qRT-PCR). Most of these genes showed high expression in leaf and root as compared with other tissue. Therefore, the presence of bibenzyls and flavonoids in different parts of A. graminifolia and their molecular regulators can provide a quick source to decipher the medicinal efficacy of orchids.

Comprehensive Analysis for GRF Transcription Factors in Sacred Lotus (Nelumbo nucifera).

Sacred lotus (Nelumbo nucifera) is an aquatic perennial plant with essential food, ornamental, and pharmacological value. Growth-regulating factor (GRF) is a transcription factor (TF) family that plays an important role in regulating the growth and development of plants. In this study, a comprehensive analysis of the GRF family in N. nucifera was performed, and its role in N. nucifera development was studied. A total of eight GRF genes were identified in the N. nucifera genome. Phylogenetic analysis divided the 38 GRF genes into six clades, while the NuGRFs only contained five clades. The analyses of gene structures, motifs, and cis-acting regulatory elements of the GRF gene family were performed. In addition, the chromosome location and collinearity were analyzed. The expression pattern based on transcriptomic data and real-time reverse transcription-quantitative PCR (qRT-PCR) revealed that the GRF genes were expressed in multiple organs and were abundant in actively growing tissues, and the expression levels decreased as the age of N. nucifera increased. Then, 3D structures of the NuGRF proteins were predicted by homology modeling. Finally, the subcellular localization of GRF1 was ascertained in the tobacco leaf through a vector. Therefore, this study provides a comprehensive overview of the GRF TF family in N. nucifera.

The possible therapeutic role of curcumin and quercetin in the early-stage of COVID-19-Results from a pragmatic randomized clinical trial.

Background: Curcumin (CUR) and quercetin (QUE), two natural polyphenols, possess diverse biological activities including broad-spectrum antiviral, antioxidant, and immunomodulatory effects. Both CUR and QUE have shown inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in in vitro assays. Objective: In the present study we aimed to assess the possible treatment benefits of a combined curcumin and quercetin (CUR-QUE) oral supplement, alongside standard of care (SOC), in the early-stage COVID-19 infection. Methods: This was an exploratory, pragmatic, open-label, randomized controlled clinical trial, conducted at the Department of Pathology, Liaquat University of Medical and Health Sciences, Jamshoro, PK. The study compared the treatment effect of an oral CUR-QUE supplement plus SOC vs. SOC alone, in the early-stage/mild to moderately symptomatic COVID-19 outpatients. Patients were randomized in a 1:1 ratio to CUR-QUE (n = 25) and control (n = 25) treatment groups. The CUR-QUE supplementation consisted of a daily intake of 168 mg curcumin and 260 mg quercetin, as two soft capsules, to be taken twice a day at home for 14 days. Results: After one-week of treatment, most of the patients in the CUR-QUE group showed an expedited clearance of the viral infection i.e., 18 (72.0%) vs. 6 (24.0%) patients in the control group tested negative for SARS-CoV-2 in the nasal-oropharyngeal swab reverse transcription-polymerase chain reaction (RT-PCR) analysis (p = 0.0002). In addition, COVID-19-associated acute symptoms were also speedily resolved in the CUR-QUE treated patients, i.e., 10 (40.0%) vs. 4 (16.0%) patients in the control group (p = 0.061). The CUR-QUE supplementation therapy was well-tolerated by all 25 patients and no treatment-emergent effects or serious adverse events were reported. Conclusion: The results revealed in this exploratory study suggest a possible therapeutic role of curcumin and quercetin in the early-stage of COVID-19. It is proposed that the two agents possibly acting in synergy, interfere the SARS-CoV-2 replication, and thus help a speedy recovery in the early-stage of COVID-19. Further research is highly encouraged. Clinical trial registration: Clinicaltrials.gov, Identifier NCT04603690.

Organ-Specific Gene Expression Reveals the Role of the Cymbidium ensifolium-miR396/Growth-Regulating Factors Module in Flower Development of the Orchid Plant Cymbidium ensifolium.

Orchids are some of the most popular ornamental plants worldwide. Orchid floral morphology has increasingly attracted horticultural and commercial attention. Although multiple genes have been shown to be involved in the formation of the orchid flower, the underlying multi-level regulatory networks are largely unknown. In this study, we analyzed the ontogeny of flower development in Cymbidium ensifolium, a traditional orchid in the tropical and subtropical regions of Asia, by performing deep sequencing of the transcriptome of individual flower organs to discover organ-specific genes potentially involved in their growth. We identified 3,017 differentially-expressed genes (DEGs) during the development of various flower organs, and observed over-representation of GROWTH-REGULATING FACTORS (GRFs) specific to flower column (gynostemium). Eleven C. ensifolium GRFs (CeGRFs) from our transcriptome data clustered into five phylogenetic subgroups. Ten of these GRFs shared a region complementary to C. ensifolium microRNA396 (Ce-miR396), and degradome sequencing confirmed the cleavage of transcripts derived from seven CeGRFs. We cloned Ce-miR396 and used a protoplast-based transient expression system to overexpress it in Cymbidium protoplasts. We observed a significant decrease in the transcripts of several CeGRFs in flowers and leaves, indicating a potential role for miR396-GRF module in organ development through the cleavage of distinct CeGRFs. Temporal and spatial expression analysis indicated that most CeGRF transcripts accumulated in flower buds and column tissues, where Ce-miR396 expression was the lowest. Expression dynamics in wild type and floral-defective mutants further confirmed a strong correlation between Ce-miR396, CeGRFs, and flower organ development and column specification. Moreover, overexpression of Ce-miR396 in Nicotiana tabacum resulted in curved pistils and reduced fertility, implying that the conserved role of Ce-miR396 in floral development. These results provide tools to better understand the biological roles of GRFs in orchid development, and open new avenues for the diversification of orchid floral patterns.

Overexpression of LiDXS and LiDXR From Lily (Lilium 'Siberia') Enhances the Terpenoid Content in Tobacco Flowers.

Lilium, the famous and significant cut flower, emits a variety of volatile organic compounds, which mainly contain monoterpenes, such as myrcene, (E)-ß-ocimene, and linalool. To understand the molecular mechanism of monoterpene synthesis in Lilium, we cloned two potential genes in the methylerythritol 4-phosphate pathway, namely LiDXS and LiDXR, from the strong-flavored oriental Lilium 'Siberia' using a homology-based PCR strategy. The expression levels of LiDXS and LiDXR were consistent with the emission and accumulation of monoterpenes in different floral organs and during the floral development, indicating that these two genes may play key roles in monoterpene synthesis. Subcellular localization demonstrated that LiDXS and LiDXR are expressed in the chloroplasts. Ectopic expression in transgenic tobacco suggested that the flowers of LiDXS and LiDXR transgenic lines accumulated substantially more diterpene, sclareol, compared to the plants transformed with empty vector. Surprisingly, increased content of the monoterpene, linalool and sesquiterpene, caryophyllene, were detected in the LiDXR transgenic lines, whereas the emission of caryophyllene, increased in one of the LiDXS transgenic tobacco lines, indicating that these two genes play significant roles in the synthesis of floral volatiles in the transgenic plants. These results demonstrate that LiDXR can contribute to monoterpene biosynthesis in Lilium 'Siberia'; however, the role of LiDXS in the biosynthesis of monoterpenes needs further study.

Comprehensive Cloning of Prunus mume Dormancy Associated MADS-Box Genes and Their Response in Flower Bud Development and Dormancy.

Dormancy Associated MADS-box genes are SVP/MADs-box members and supposed to play crucial roles in plant dormancy of perennial species. In Prunus mume, PmDAM6 has been previously identified to induce plant dormancy. In the current study, six PmDAMs were cloned in P. mume and functionally analyzed in yeast and tobacco to detect the roles of the genes paralogous to PmDAM6. The expression patterns together with sequence similarities indicate that PmDAMs are divided into two sub-clades within SVP group. Moreover, PmDAMs are verified to take part in the development of different plant organs, specifically the flower buds, in some intricate patterns. Furthermore, the PmDAM proteins are found to have special functions by forming corresponding protein complex during the development of flower bud and induction of dormancy. In particular, when PmDAM1 dominating in flower bud in the warm months, the protein complexes are consisted of PmDAM1 itself or with PmDAM2. With the decrease temperatures in the following months, PmDAM6 was found to be highly expressed and gradually changed the complex structure to PmDAM6-protein complex due to strong binding tendencies with PmDAM1 and PmDAM3. Finally, the homodimers of PmDAM6 prevailed to induce the dormancy. The results obtained in the current study highlight the functions of PmDAMs in the tissue development and dormancy, which provide available suggestions for further explorations of protein-complex functions in association with bud growth and dormancy.

Peripheral markers of Alzheimer's disease: surveillance of white blood cells.

Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. This is a mechanism of innate immunity, which may cause an increase in the number of monocytes and neutrophils circulating in the blood. Literature indicated that chronic inflammation might be a factor in developing neurological problems, including Alzheimer's, Parkinson's and other similar illnesses. Our main objective is to identify peripheral markers of Alzheimer's disease and for that purpose; we are looking at the profile of white blood cells focusing on monocytes, neutrophils, lymphocytes and basophils. Twenty-seven patients of Alzheimer's disease (AD), diagnosed by magnetic resonance imaging and neuropsychological tests were observed for their blood profile. Key observations during this study were that the levels of monocytes in the blood of the diagnosed AD patients were high irrespective of their age and sex. For those patients whose monocytes were in normal range their neutrophil levels were significantly high. Whereas blood levels of lymphocytes and basophils were found to be constantly low. Escalated levels of monocytes and neutrophils are hallmarks of chronic inflammation and may be precursor to Alzheimer's disease. A low lymphocyte count specifies that the body's resistance to fight infection is substantially reduced, whereas low basophil levels indicates their over utilization due to chronic allergic inflammatory condition. Future studies involved closer look at the cytokines produced by these white blood cells especially TNF IL-1, and IL-12, which are products of monocytes. Likewise, blood glucose and creatinine levels were high whereas calcium ions were low. Our studies indicated that white blood cells along with other inflammatory byproducts may act as peripheral markers for early diagnosis of Alzheimer's disease.