Investigating Retinal Circuits and Molecular Localization by Pre-Embedding Immunoelectron Microscopy.

The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.

Floral Response to Heat: A Study of Color and Biochemical Adaptations in Purple Chrysanthemums.

Chrysanthemums are among the world's most popular cut flowers, with their color being a key ornamental feature. The formation of these colors can be influenced by high temperatures. However, the regulatory mechanisms that control the fading of chrysanthemum flower color under high-temperature stress remain unclear. This study investigates the impact of high temperatures on the color and biochemical responses of purple chrysanthemums. Four purple chrysanthemum varieties were exposed to both normal and elevated temperature conditions. High-temperature stress elicited distinct responses among the purple chrysanthemum varieties. 'Zi Feng Che' and 'Chrystal Regal' maintained color stability, whereas 'Zi Hong Tuo Gui' and 'Zi lian' exhibited significant color fading, particularly during early bloom stages. This fading was associated with decreased enzymatic activities, specifically of chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), indicating a critical period of color development under heat stress. Additionally, the color fading of 'Zi Lian' was closely related to the increased activity of the peroxidase (POD) and polyphenol oxidase (PPO). Conversely, a reduction in ß-glucosidase (ßG) activity may contribute significantly to the color steadfastness of 'Zi Feng Che'. The genes Cse_sc027584.1_g010.1 (PPO) and Cse_sc031727.1_g010.1 (POD) might contribute to the degradation of anthocyanins in the petals of 'Zi Hong Tuo Gui' and 'Zi Lian' under high-temperature conditions, while simultaneously maintaining the stability of anthocyanins in 'Zi Feng Che' and 'Chrystal Regal' at the early bloom floral stage. The findings of this research provide new insights into the physiological and biochemical mechanisms by which chrysanthemum flower color responds to high-temperature stress.

Cell-specific localization of ß-synuclein in the mouse retina.

ß-synuclein, a member of the synuclein family, is frequently co-expressed with α-synuclein in the neural system, where it serves to inhibit abnormal aggregation of α-synuclein in neurodegenerative diseases. Beyond its role in pathological conditions, ß-synuclein plays various functions independently of α-synuclein. In our investigation, we discovered a broader expression of ß-synuclein in the mouse retina compared to α-synuclein. This widespread pattern implies its potential significance in the retina. Through detailed examination via light- and electron-microscopic immunocytochemistry, we identified ß-synuclein expression from the inner segment (IS) and outer segment (OS) of photoreceptor cells to the ganglion cell layer (GCL). Our findings unveiled unique features, including ß-synuclein immunoreactive IS and OS of cones, higher expression in cone pedicles than in rod spherules, absence in horizontal cells, limited expression in cone bipolar dendrites and somas, higher expression in cone bipolar terminals, presence in most amacrine cells, and expression in almost majority of somas in GCL with an absence in intrinsically photosensitive retinal ganglion cell (ipRGCs) processes. Notably, all cholinergic amacrine cells express high ß- but not α-synuclein, while dopaminergic amacrine cells express α-synuclein exclusively. These distinctive expression patterns offer valuable insights for further exploration into the functions of ß-synuclein and its potential role in synuclein pathology within the retina.

Global Event-Triggered Funnel Control of Switched Nonlinear Systems via Switching Multiple Lyapunov Functions.

In this article, the global event-triggered (ET) funnel tracking control problem is studied for a class of switched nonlinear systems with structural uncertainties, where the solvability of the control problem for each subsystem is not needed. A switching multiple Lyapunov functions (MLFs) method is established, where MLFs are designed to handle switched inverse dynamics, and a switching barrier Lyapunov function is constructed to address switched sampled errors that may compromise system stability. This is achieved alongside a new switching dynamic event-triggering mechanism (DETM). By combining this method with backstepping, a dwell-time state-dependent switching law and an ET funnel controller of each subsystem are constructed, effectively eliminating the issue of the "explosion of complexity" encountered in traditional backstepping without using dynamic surface control or command filters. Additionally, the designed switching DETM ensures that the tracking error always evolves within a performance funnel in any consecutive triggering interval, excluding Zeno behavior, and guaranteeing positive constant lower bounds for two consecutive triggering intervals and any switching interval, respectively. Finally, an example is provided to show the validity of the theoretical results.

From immune checkpoints to therapies: understanding immune checkpoint regulation and the influence of natural products and traditional medicine on immune checkpoint and immunotherapy in lung cancer.

Lung cancer is a disease of global concern, and immunotherapy has brought lung cancer therapy to a new era. Besides promising effects in the clinical use of immune checkpoint inhibitors, immune-related adverse events (irAEs) and low response rates are problems unsolved. Natural products and traditional medicine with an immune-modulating nature have the property to influence immune checkpoint expression and can improve immunotherapy's effect with relatively low toxicity. This review summarizes currently approved immunotherapy and the current mechanisms known to regulate immune checkpoint expression in lung cancer. It lists natural products and traditional medicine capable of influencing immune checkpoints or synergizing with immunotherapy in lung cancer, exploring both their effects and underlying mechanisms. Future research on immune checkpoint modulation and immunotherapy combination applying natural products and traditional medicine will be based on a deeper understanding of their mechanisms regulating immune checkpoints. Continued exploration of natural products and traditional medicine holds the potential to enhance the efficacy and reduce the adverse reactions of immunotherapy.

How to identify juxtaglomerular cell tumor by ultrasound: a case series and review of the literature.

PURPOSE: To study the value of ultrasound in the diagnosis of juxtaglomerular cell tumor (JGCT). METHODS: From January 2005 to July 2020, fifteen patients diagnosed as JGCT by surgical pathology in Peking Union Medical College Hospital were collected. All patients underwent preoperative ultrasound examination. The clinical, laboratory, ultrasound, computed tomography (CT), surgical, and pathological features of the patients were analyzed retrospectively. RESULTS: The 15 patients were 5 males and 10 females with a median age of 29 years (10∼72 years). 14 of them had hypertension and one had normal blood pressure. The tumors were all solitary, with a median diameter of 1.5 cm (0.9-5.9 cm). Among the fifteen patients, eleven were correctly detected by preoperative ultrasound, and four were missed. There was a significant difference in tumor size (2.64 ± 1.48 cm vs. 1.23 ± 0.21 cm) and whether the tumor protruded outward (9/11 vs. 0/4) between the ultrasound-detected group and the ultrasound-missed group (p = 0.010, p = 0.011). Of the 11 tumors detected by ultrasound, four were extremely hypoechoic, two were hypoechoic, three were isoechoic, and two were hyperechoic. Color Doppler showed no blood flow in five tumors with the size range from 0.9 to 2.0 cm, and mild blood flow in six tumors with the size range from 2.8 to 5.9 cm. CONCLUSIONS: JGCT is rare, and has characteristic clinical manifestations. Diagnosis should be suspected in case of secondary hypertension, particularly in young women, if no renal vascular cause was found. Ultrasound, combined with clinical manifestations, was helpful for the diagnosis.

A Dual Triggering Scheme to Adaptive Fuzzy Resilient Control of Switched Nonlinear Systems Against Mixed Attacks.

A dual event-triggered adaptive fuzzy resilient control scheme for a class of switched nonlinear systems with vanishing control gains under mixed attacks is proposed in this article. The scheme proposed achieves dual triggering in the channels of sensor-to-controller and controller-to-actuator by designing two new switching dynamic event-triggering mechanisms (ETMs). An adjustable positive lower bound of interevent times for each ETM is found to preclude Zeno behavior. Meanwhile, mixed attacks, that is, deception attacks on sampled state and controller data and dual random denial-of-service attacks on sampled switching signal data, are handled by constructing event-triggered adaptive fuzzy resilient controllers of subsystems. Compared with the existing works for switched systems with only single triggering, more complex asynchronous switching caused by dual triggering and mixed attacks and subsystem switching is addressed. Further, the obstacle caused by vanishing control gains at some points is eliminated by proposing an event-triggered state-dependent switching law and introducing vanishing control gains into a switching dynamic ETM. Finally, a mass-spring-damper system and a switched RLC circuit system are applied to verify the obtained result.

Functional conservation and divergence of SEPALLATA-like genes in floral development in Cymbidium sinense.

Cymbidium sinense is one of the most important traditional Chinese Orchids due to its unique and highly ornamental floral organs. Although the ABCDE model for flower development is well-established in model plant species, the precise roles of these genes in C. sinense are not yet fully understood. In this study, four SEPALLATA-like genes were isolated and identified from C. sinense. CsSEP1 and CsSEP3 were grouped into the AGL9 clade, while CsSEP2 and CsSEP4 were included in the AGL2/3/4 clade. The expression pattern of CsSEP genes showed that they were significantly accumulated in reproductive tissues and expressed during flower bud development but only mildly detected or even undetected in vegetative organs. Subcellular localization revealed that CsSEP1 and CsSEP4 were localized to the nucleus, while CsSEP2 and CsSEP3 were located at the nuclear membrane. Promoter sequence analysis predicted that CsSEP genes contained a number of hormone response elements (HREs) and MADS-box binding sites. The early flowering phenotype observed in transgenic Arabidopsis plants expressing four CsSEP genes, along with the expression profiles of endogenous genes, such as SOC1, LFY, AG, FT, SEP3 and TCPs, in both transgenic Arabidopsis and C. sinense protoplasts, suggested that the CsSEP genes played a regulatory role in the flowering transition by influencing downstream genes related to flowering. However, only transgenic plants overexpressing CsSEP3 and CsSEP4 caused abnormal phenotypes of floral organs, while CsSEP1 and CsSEP2 had no effect on floral organs. Protein-protein interaction assays indicated that CsSEPs formed a protein complex with B-class CsAP3-2 and CsSOC1 proteins, affecting downstream genes to regulate floral organs and flowering time. Our findings highlighted both the functional conservation and divergence of SEPALLATA-like genes in C. sinense floral development. These results provided a valuable foundation for future studies of the molecular network underlying floral development in C. sinense.

Distribution and synaptic organization of substance P-like immunoreactive neurons in the mouse retina.

Substance P (SP), a neuroprotective peptidergic neurotransmitter, is known to have immunoreactivity (IR) localized to amacrine and/or ganglion cells in a variety of species' retinas, but it has not yet been studied in the mouse retina. Thus, we investigated the distribution and synaptic organization of SP-IR by confocal and electron microscopy immunocytochemistry in the mouse retina. SP-IR was distributed in the inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Most of the SP-IR somas belonged to amacrine cells (2.5% of all) in the INL and their processes stratified into the S1, S3, and S5 layers of the IPL, with the most intense band in the S5 layer. Some SP-IR somas can also be observed in the GCL, which were identified as displaced amacrine cells (82%, 1269/1550) and ganglion cells (18%, 281/1550) by antibodies against AP2α and RBPMS, respectively. Such SP-IR ganglion cells (1.2% of all RGCs) can be further divided into 3 subgroups expressing SP/α-Synuclein (α-Syn), SP/GAD67, and/or SP/GAD67/α-Syn. Possible physiological and pathological roles of these ganglion cells are discussed. Further, electron microscopy evidence demonstrates that SP-IR amacrine cells receive major inputs from other SP-IR amacrine cell processes (146/242 inputs) and output mostly to SP-negative amacrine cell processes (291/673 outputs), suggesting series inhibition among amacrine cells. These results reveal for the first time an explicit distribution, novel ganglion cell features, and synaptic organization of SP-IR in the mouse retina, which is important for the future use of mouse models to study the roles of SP in healthy and diseased (including Parkinson's disease) retinal states.

Influencing factors of sarcopenia in older adults based on the Newman system model: a case-control study.

PURPOSE: Research on sarcopenia has primarily focused on single fields such as physiology or psychology. However, there is a lack of clear evidence to determine the influence of social factors on sarcopenia. Therefore, our aim was to explore the multidimensional factors that contribute to sarcopenia in older adults within the community. METHODS: In this retrospective case-control study, we applied the diagnostic criteria from The Asian Working Group on Sarcopenia (AWGS) 2019 to categorize study subjects into control and case groups. Our aim was to examine the impact of physical, psychological, and social factors on community-dwelling older adults with sarcopenia across multiple dimensions. We utilized descriptive statistics, as well as simple and multivariate logistic regression analyses, to analyze the data. We compared the odds ratios (OR) of the factors between the two groups and ranked the importance of the influencing factors using the XGBoost algorithm in Python software. RESULTS: Combined with multivariate analysis and XGBoost algorithm results, it can be seen that physical activity is the strongest predictor of sarcopenia [OR] = 0.922(95% CI 0.906-0.948), followed diabetes mellitus [OR] = 3.454(95% CI 1.007-11.854), older age [OR] = 1.112(95% CI 1.023-1.210), divorced or widowed [OR] = 19.148 (95% CI 4.233-86.607), malnutrition [OR] = 18.332(95% CI 5.500-61.099), and depressed [OR] = 7.037(95% CI 2.391-20.710). CONCLUSIONS: Factors associated with the development of sarcopenia in community-dwelling older adults cover a multiplicity of physical, psychological, and social factors, physical activity, diabetes mellitus, age, marital status, nutrition, and depression were important factors that have an impact on sarcopenia. REGISTRATION NUMBER: ChiCTR2200056297.

[Distribution of monocyte subsets and their surface CD31 intensity are associated with disease course and severity of hemorrhagic fever with renal syndrome].

Objective To investigate the relationship between disease courses and severity and monocyte subsets distribution and surface CD31 intensity in patients of hemorrhagic fever with renal syndrome (HFRS). Methods Peripheral blood samples from 29 HFRS patients and 13 normal controls were collected. The dynamic changes of classical monocyte subsets (CD14++CD16-), intermediated monocyte subsets (CD14++CD16+) and non-classical monocyte subsets (CD14+CD16++) and the mean fluorescent intensity (MFI) of CD31 on monocyte subsets were detected by multiple-immunofluorescent staining and flow cytometry. Results In acute phase of HFRS, the ratio of classical monocyte subsets to total monocytes was dramatically decreased compared to convalescent phase and normal control. It was still much lower in convalescent phase compared to normal controls. The ratio of classical monocyte subsets to total monocytes were decreased in HFRS patients compared to that in normal control, whereas there was no difference between severe/critical groups and mild/moderate groups. On the contrary, the ratio of intermediate monocyte subsets to total monocytes in acute phase of HFRS was significantly increased compared to convalescent phase and normal control. The ratio of intermediate monocyte subsets to total monocytes were increased in HFRS patients compared to that in normal control, whereas no difference was found between severe/critical groups and mild/moderate groups. Phases or severity groups had no difference in ratio of non-classical monocyte subsets to total monocytes. Additionally, the ratio of classical monocyte subsets had a tendency to decline and that of intermediate monocyte subsets showed an increase both to total monocytes between the acute and convalescent phases in 11 HFRS patients with paired-samples. Moreover, in acute phase of HFRS, the mean fluorescent intensity (MFI) of CD31 on three monocyte subsets all decreased, specifically classical monocyte subsets showed the highest MFI of CD31 while the normal control reported the highest MFI of CD31 in non-classical monocyte subsets. In convalescent phase, the MFI of CD31 on classical and intermediated monocyte subsets were both lower than that of normal control, while MFI of CD31 was still significantly lower than normal control on non-classical monocyte subsets. Finally, MFI of CD31 on classical and intermediated monocyte subsets in severe/critical group were both lower than those in mild/moderate group, showing no statistical difference in MFI of CD31 on non-classical monocyte subset across groups of different disease severity. Conclusion The ratio of classical and intermediated monocyte subsets to total monocytes are correlated with the course of HFRS, and so are the surface intensity of CD31 on these monocyte subsets with the disease course and severity. The surface intensity of CD31 on non-classical monocyte subsets, however, is correlated only with the course of the disease. Together, the underlying mechanisms for the observed changes in monocyte subsets in HFRS patients should be further investigated.

Identification of Single-Nucleotide Polymorphisms (SNPs) Associated with Heat Tolerance at the Reproductive Stage in Synthetic Hexaploid Wheats Using GWAS.

The projected rise in global ambient temperature by 3-5 °C by the end of this century, along with unpredicted heat waves during critical crop growth stages, can drastically reduce grain yield and will pose a great food security challenge. It is therefore important to identify wheat genetic resources able to withstand high temperatures, discover genes underpinning resilience to higher temperatures, and deploy such genetic resources in wheat breeding to develop heat-tolerant cultivars. In this study, 180 accessions of synthetic hexaploid wheats (SHWs) were evaluated under normal and late wheat growing seasons (to expose them to higher temperatures) at three locations (Islamabad, Bahawalpur, and Tando Jam), and data were collected on 11 morphological and yield-related traits. The diversity panel was genotyped with a 50 K SNP array to conduct genome-wide association studies (GWASs) for heat tolerance in SHW. A known heat-tolerance locus, TaHST1, was profiled to identify different haplotypes of this locus in SHWs and their association with grain yield and related traits in SHWs. There was a 36% decrease in grain yield (GY), a 23% decrease in thousand-grain weight (TKW), and an 18% decrease in grains per spike (GpS) across three locations in the population due to the heat stress conditions. GWASs identified 143 quantitative trait nucleotides (QTNs) distributed over all 21 chromosomes in the SHWs. Out of these, 52 QTNs were associated with morphological and yield-related traits under heat stress, while 15 of them were pleiotropically associated with multiple traits. The heat shock protein (HSP) framework of the wheat genome was then aligned with the QTNs identified in this study. Seventeen QTNs were in proximity to HSPs on chr2B, chr3D, chr5A, chr5B, chr6D, and chr7D. It is likely that QTNs on the D genome and those in proximity to HSPs may carry novel alleles for heat-tolerance genes. The analysis of TaHST1 indicated that 15 haplotypes were present in the SHWs for this locus, while hap1 showed the highest frequency of 25% (33 SHWs). These haplotypes were significantly associated with yield-related traits in the SHWs. New alleles associated with yield-related traits in SHWs could be an excellent reservoir for breeding deployment.

Rod bipolar cells receive cone photoreceptor inputs through both invaginating synapses and flat contacts in the mouse and guinea pig retinas.

The light pathways are segregated into rod and cone pathways in which rods synapse with rod bipolar cells (RBCs), while cones contact cone bipolar cells (CBCs). However, previous studies found that cones can make synapse with RBCs (cone-RBC synapses) and rods can contact OFF CBC in primate and rabbit retinas. Recently, such cone-RBC synapses have been reported physiologically and morphologically in the mouse retina. Nevertheless, the precise subcellular evidence to determine whether it is the invaginating synapse or the flat contact remains absent. This is due to a lack of immunochemically verified ultrastructural data. Here, we investigated the precise expression of protein kinase C alpha (PKCα) using pre-embedding immunoelectron microscopy (immuno-EM) with a monoclonal antibody against PKCα, a biomarker for the RBCs. We determined the nanoscale localization of PKCα in the outer plexiform layer of the mouse and guinea pig retinas. Our results demonstrate the existence of both the direct invaginating synapse and the basal/flat contact of the cone-RBCs, providing for the first time immunochemically verified ultrastructural evidence for the cone-RBC synapse in the mouse and guinea pig retinas. These results suggest that the cross talk between cone and rod pathways is much more extensive than previously assumed.

Expression of α-Synuclein in the mouse retina is confined to inhibitory presynaptic elements.

α-Synuclein (α-Syn) is enriched in presynaptic terminals of the central nervous system including the retina and plays a role in the synaptic vesicle cycle and synaptic transmission. Abnormal aggregation of α-Syn is considered to be the main component of the Lewy bodies that are the pathological hallmarks of Parkinson's disease. Although expression pattern of α-Syn has been described in the retinas, its precise cellular and subcellular locations are poorly understood. We investigated the precise expression of α-Syn using light microscopy (LM) and electron microscopy (EM) with antibodies against α-Syn in the mouse retina. We found that the majority of α-Syn immunoreactivity (IR) is located in GABAergic, glycinergic, and dopaminergic amacrine cells, and their processes often make a direct synapse to other labeled or unlabeled amacrine profiles, bipolar cell terminals, or ganglion cell dendrites. Further, our LM and immuno-EM results confirm the absence of α-Syn in excitatory photoreceptors, bipolar cell bodies, and their ribbon synapses, providing evidence, for the first time, that ribbon synapses do not express α-Syn. Additionally, α-Syn IR is located in the ganglion cells, some of which are intrinsically photosensitive retinal ganglion cells. These results reveal a previously unappreciated inhibitory synapse-specific expression pattern of α-Syn in the retina, suggesting that α-Syn may play a distinct role in the modulation and integration of inhibitory synaptic transmission in the retina.

Dynamic Event-Triggered Adaptive NN Control for Switched Uncertain Nonlinear Systems.

This article is concerned with the problem of dynamic event-triggered adaptive neural network (NN) control for a class of switched strict-feedback uncertainty nonlinear systems. A novel switched command filter-based dynamic event-triggered adaptive NN control approach is set up by exploiting the backstepping and command filter and the common Lyapunov function method. Since adaptive controllers of subsystems are event triggered, then if the switching happens between any two consecutive triggering instants, asynchronous switching will arise between candidate controllers of subsystems and subsystems. Unlike the existing literature, where maximum asynchronous time is restricted, without any strict limitations on maximum asynchronous time being needed in this article, the asynchronous switching problem is directly handled by proposing a novel switching dynamic event-triggered mechanism (DETM) and event-triggered adaptive controllers of subsystems. Moreover, a piecewise constant variable is introduced into the switching DETM, which overcomes the difficulty of switched measurement error being discontinuous. Also, a strictly positive lower bound of interevent times is obtained. Finally, a continuous stirred tank reactor system and a numerical example are presented to demonstrate the effectiveness of the developed approach.

Theta-Burst Stimulation Combined With Virtual-Reality Reconsolidation Intervention for Methamphetamine Use Disorder: Study Protocol for a Randomized-Controlled Trial.

Background: Craving associated with drug-related memory is one of the key factors that induce the relapse of methamphetamine (MA). Disruption or modulation of the reconsolidation of drug-related memory may serve as an option for clinical treatment of MA addiction. This protocol proposes to use virtual reality (VR) to retrieve drug-associated memory and then use transcranial magnetic stimulation (TMS) at the neural circuit that encodes the reward value of drug cues to provide a non-invasive intervention during reconsolidation. We aim to evaluate the effectiveness of TMS treatment after VR retrieval on the reduction of cue reactivity and craving of MA. Methods: This is a randomized, double-blind, sham-controlled, parallel group trial, targeting participants with MA use disorder aged from 18 to 45 years old. Forty-five eligible volunteers in Shanxi Drug Rehabilitation Center will be recruited and be randomly allocated into three parallel groups, receiving either 1) MA-related cues retrieval in VR combined with active TMS (MA VR scene + TBS) or 2) sham TMS (MA VR scene + sham TBS), or 3) neutral cues retrieval in VR combined with active TMS (neutral VR scene + TBS). Two sessions of post-VR-retrieval TBS will be scheduled on two separate days within 1 week. The primary outcome will detect the memory-related activity by the electroencephalography (EEG) reactivity to drug cues in VR scenes. Secondary outcomes are the self-reported MA craving in VR scene, the physiological parameter (cue-induced heart rate) and the scores of psychological questionnaires including anxiety, depression, and mood. All primary and secondary outcomes will be assessed at baseline, 1-week, and 1-month post-intervention. Assessments will be compared between the groups of 1) MA VR scene + TBS, 2) MA VR scene + sham TBS and 3) neutral VR scene + TBS. Discussion: This will be the first study to examine whether the TMS modulation after VR retrieval can reduce self-reported craving and drug-related cue reactivity. It will promote the understanding of the neural circuit mechanism of the reconsolidation-based intervention and provide an effective treatment for MA use disorder patients. Clinical Trial Registration: [Chinese Clinical Trial Registry], identifier [ChiCTR1900026902]. Registered on 26 October 2019.

The Maternal Donor of Chrysanthemum Cultivars Revealed by Comparative Analysis of the Chloroplast Genome.

Chrysanthemum (Chrysanthemum morifolium Ramat) is an important floricultural crop and medicinal herb. Modern chrysanthemum cultivars have complex genetic backgrounds because of multiple cycles of hybridization, polyploidization, and prolonged cultivation. Understanding the genetic background and hybrid origin of modern chrysanthemum cultivars can provide pivotal information for chrysanthemum genetic improvement and breeding. By now, the origin of cultivated chrysanthemums remains unclear. In this study, 36 common chrysanthemum cultivars from across the world and multiple wild relatives were studied to identify the maternal donor of modern chrysanthemum. Chloroplast (cp) genomes of chrysanthemum cultivars were assembled and compared with those of the wild relatives. The structure of cp genomes was highly conserved among cultivars and wild relatives. Phylogenetic analyses based on the assembled cp genomes showed that all chrysanthemum cultivars grouped together and shared 64 substitutions that were distinct from those of their wild relatives. These results indicated that a diverged lineage of the genus Chrysanthemum, which was most likely an extinct or un-sampled species/population, provided a maternal source for modern cultivars. These findings provide important insights into the origin of chrysanthemum cultivars, and a source of valuable genetic markers for chrysanthemum breeding programs.

RhWRKY33 Positively Regulates Onset of Floral Senescence by Responding to Wounding- and Ethylene-Signaling in Rose Plants.

Rose plants are one of the most important horticultural crops, whose commercial value mainly depends on long-distance transportation, and wounding and ethylene are the main factors leading to their quality decline and accelerated senescence in the process. However, underlying molecular mechanisms of crosstalk between wounding and ethylene in the regulation of flower senescence remain poorly understood. In relation to this, transcriptome analysis was performed on rose flowers subjected to various treatments, including control, wounding, ethylene, and wounding- and ethylene- (EW) dual treatment. A large number of differentially expressed genes (DEGs) were identified, ranging from 2,442 between the ethylene- and control-treated groups to 4,055 between the EW- and control-treated groups. Using weighted gene co-expression network analysis (WGCNA), we identified a hub gene RhWRKY33 (rchiobhmchr5g0071811), accumulated in the nucleus, where it may function as a transcription factor. Moreover, quantitative reverse transcription PCR (RT-qPCR) results showed that the expression of RhWRKY33 was higher in the wounding-, ethylene, and EW-treated petals than in the control-treated petals. We also functionally characterized the RhWRKY33 gene through virus-induced gene silencing (VIGS). The silencing of RhWRKY33 significantly delayed the senescence process in the different treatments (control, wounding, ethylene, and EW). Meanwhile, we found that the effect of RhWRKY33-silenced petals under ethylene and EW dual-treatment were stronger than those under wounding treatment in delaying the petal senescence process, implying that RhWRKY33 is closely involved with ethylene and wounding mediated petal senescence. Overall, the results indicate that RhWRKY33 positively regulates the onset of floral senescence mediated by both ethylene and wounding signaling, but relies heavily on ethylene signaling.

Complete chloroplast genome sequence of a Chinese traditional cultivar in Chrysanthemum, Chrysanthemum morifolium 'Anhuishiliuye'.

The complete plastid genome of Chrysanthemum morifolium 'Anhuishiliuye', a Chinese traditional cultivar, was determined and analyzed in this work. It had a circular-mapping molecular with the length of 151,059 bp.The LSC and SSC of 82,857 bp and 18,294 bp were separated by two IRs of 24,954 bp. The chloroplast genome of C. morifolium 'Anhuishiliuye' contains 125 genes, including 83 protein-coding genes, 34 ribosomal RNA genes and 8 transfer RNA genes. Phylogenetic analysis showed that C. morifolium 'Anhuishiliuye' clustered together with other Chrysanthemum species. The data provided would be useful for elucidation of phylogenetics and evolution in Chrysanthemum cultivars.