MALDI-mass spectrometry imaging as a new technique for detecting non-heme iron in peripheral tissues via caudal vein injection of deferoxamine.

As one of the most common iron-chelating agents, deferoxamine (DFO) rapidly chelates iron in the body. Moreover, it does not compete for the iron characteristic of hemoglobin in the blood cells, which is common in the clinical treatment of iron poisoning. Iron is a trace element necessary to maintain organism normal life activities. Iron deficiency can lead to anemia, whereas iron overload can cause elevated levels of cellular oxidative stress and cell damage. As a consequence, detection of the iron content in tissues and blood is of great significance. The traditional techniques for detecting the iron content include inductively coupled plasma-mass spectrometry and atomic absorption spectrometry, which cannot be used for imaging purposes. Laser ablation-ICP-MS and synchrotron radiation micro-X-ray fluorescence can map the concentration and distribution of iron in tissues. However, these methods can only be used to measure the total iron levels in blood or tissues. In recent years, due to the deepening understanding of iron metabolism, diseases related to iron overload have attracted increasing attention. Therefore, we took advantage of the properties of DFO in terms of chelating iron and investigated different sampling times following DFO injection in the tail vein of mice. We used mass spectrometry imaging (MSI) technology to detect the DFO and ferrioxamine content in the blood and different tissues to indirectly characterize the non-heme iron content.

Floral resource partitioning of coexisting bumble bees: Distinguishing species-, colony-, and individual-level effects.

Resource partitioning is considered a key factor in alleviating competitive interactions, enabling coexistence among consumer species. However, most studies have focused on resource partitioning between species, ignoring the potentially critical role of intraspecific variation in resource use. We investigated floral resource partitioning across species, colonies, and individuals in a species-rich bumblebee community in the diversification center of bumblebees. We used a total of 10,598 bumblebees belonging to 13 species across 5 years in the Hengduan Mountains of southwest China. First, we evaluated the influence of a comprehensive set of floral traits, including both those related to attractiveness (flower color and shape) and rewards (pollen, sugar ratio, nectar volume, sugar concentration, and amino acid content) on resource partitioning at the species level in bumblebee-plant networks. Then, we explored intraspecific resource partitioning on the colony and individual levels. Our results suggest that bumblebee species differ substantially in their use of the available floral resources, and that this mainly depends on flower attractiveness (floral color and shape). Interestingly, we also detected floral resource partitioning at the colony level within all commonest bumblebee species evaluated. In general, floral resource partitioning between bumblebee individuals decreased with species- and individual-level variation in body size (intertegular span). These results suggest that bumblebee species may coexist via the flexibility in their preferences for specific floral traits, which filters up to support the co-occurrence of high numbers of species and individuals in this global hotspot of species richness.

The interplay between scale, pollination niche and floral attractiveness on density-dependent plant-pollinator interactions.

Pollinators mediate interspecific and intraspecific plant-plant indirect interactions (competition vs. facilitation) via density-dependent processes, potentially shaping the dynamics of plant communities. However, it is still unclear which ecological drivers regulate density-dependent patterns, including scale, pollination niches (i.e., the main pollinator functional group) and floral attractiveness to pollinators. In this study, we conducted three-year field observations in Hengduan Mountains of southwest China. By gathering data for more than 100 animal-pollinated plant species, we quantified the effect (positive vs. negative) of conspecific and heterospecific flower density on pollination at two scales: plot-level (4 m2) and site-level (100-5000 m2). Then, we investigated how pollination niches and floral attractiveness to pollinators (estimated here as average per-flower visitation rates) modulated density-dependent pollination interactions. Pollinator visitation depended on conspecific and heterospecific flower density, with rare plants subjected to interspecific competition at the plot-level and interspecific facilitation at the site-level. Such interspecific competition at the plot-level was stronger for plants pollinated by diverse insects, while interspecific facilitation at the site-level was stronger for bee-pollinated plants. Moreover, we also found stronger positive conspecific density-dependence for plants with lower floral attractiveness at the site-level, meaning that they become more frequently visited when abundant. Our study indicates that the role of pollination in maintaining rare plants and plant diversity depends on the balance of density-dependent processes in species-rich communities. We show here that such balance is modulated by scale, pollination niches and floral attractiveness to pollinators, indicating the context-dependency of diversity maintenance mechanisms.

Construction of C(CO)-C(CO) Bond via NHC-Catalyzed Radical Cross-Coupling Reaction.

A C(sp2)-C(sp2) bond can be constructed via a photoredox/N-heterocyclic carbene (NHC)-cocatalyzed radical cross-coupling reaction, which provides a complementary strategy to classic electron pair processes. The present protocol represents the first example of an NHC-catalyzed two-component radical cross-coupling reaction involving C(sp2)-centered radical species. The decarboxylative acylation of oxamic acid with acyl fluoride was conducted under mild conditions and allowed the preparation of a variety of useful α-keto amides, including sterically congested ones.

CHIR99021 Maintenance of the Cell Stemness by Regulating Cellular Iron Metabolism.

CHIR99021 is an aminopyrimidine derivative, which can efficiently inhibit the activity of glycogen synthesis kinase 3α (GSK-3α) and GSK-3ß. As an essential component of stem cell culture medium, it plays an important role in maintaining cell stemness. However, the mechanism of its role is not fully understood. In the present study, we first found that removal of CHIR99021 from embryonic stem cell culture medium reduced iron storage in mouse embryonic stem cells (mESCs). CHIR99021-treated Neuro-2a cells led to an upregulation of ferritin expression and an increase in intracellular iron levels, along with GSK3ß inhibition and Wnt/GSK-3ß/ß-catenin pathway activation. In addition, iron treatment activated the classical Wnt pathway by affecting the expression of ß-catenin in the Neuro-2a cells. Our data link the role of iron in the maintenance of cell stemness via the Wnt/GSK-3ß/ß-catenin signaling pathway, and identify intermediate molecules, including Steap1, Bola2, and Kdm6bos, which may mediate the upregulation of ferritin expression by CHIR99021. These findings reveal novel mechanisms of the maintenance of cell stemness and differentiation and provide a theoretical basis for the development of new strategies in stem cell treatment in disease.

Upregulation of protein N-glycosylation plays crucial roles in the response of Camellia sinensis leaves to fluoride.

The tea plant (Camellia sinensis) is one of the three major beverage crops in the world with its leaves consumption as tea. However, it can hyperaccumulate fluoride with about 98% fluoride deposition in the leaves. Our previously studies found that cell wall proteins (CWPs) might play a central role in fluoride accumulation/detoxification in C. sinensis. CWP is known to be glycosylated, however the response of CWP N-glycosylation to fluoride remains unknown in C. sinensis. In this study, a comparative N-glycoproteomic analysis was performed through HILIC enrichment coupled with UPLC-MS/MS based on TMT-labeling approach in C. sinensis leaves. Totally, 237 N-glycoproteins containing 326 unique N-glycosites were identified. 73.4%, 18.6%, 6.3% and 1.7% of these proteins possess 1, 2, 3, and ≥4 modification site, respectively. 93.2% of these proteins were predicted to be localized in the secretory pathway and 78.9% of them were targeted to the cell wall and the plasma membrane. 133 differentially accumulated N-glycosites (DNGSs) on 100 N-glycoproteins (DNGPs) were detected and 85.0% of them exhibited upregulated expression after fluoride treatment. 78.0% DNGPs were extracellular DNGPs, which belonged to CWPs, and 53.0% of them were grouped into protein acting on cell wall polysaccharides, proteases and oxido-reductases, whereas the majority of the remaining DNGPs were mainly related to N-glycoprotein biosynthesis, trafficking and quality control. Our study shed new light on the N-glycoproteome study, and revealed that increased N-glycosylation abundance of CWPs might contribute to fluoride accumulation/detoxification in C. sinensis leave.

Iron promotes neurological function recovery in mice with ischemic stroke through endogenous repair mechanisms.

The endogenous repair mechanisms play an important role in the recovery of nerve function after stroke, such as gliosis, synaptic plasticity, remyelination and nerve regeneration. Iron is the most abundant trace metal element in the brain and plays a crucial role in the maintenance of normal cerebral function. It is an important coenzyme factor in the process of cell metabolism, DNA synthesis, purine catabolism and neurotransmitter synthesis and decomposition. However, it is unclear what role iron plays in the long-term recovery of neurological function after stroke. In this study, we first observed that changes in iron metabolism occurred during neurological function recovery in the mice with distal middle cerebral artery occlusion (dMCAO). Our data showed that plasticity changes due to endogenous repair mechanisms resulted in improvements in cerebral cortex function. These changes involved gliosis, synaptic function reconstruction, remyelination, and activation of neural stem cells. In order to examine the potential role of iron, we synthesized liposomal-encapsulated deferoxamine (DFO) nanoparticles to further explore the effect and the mechanism of iron on the recovery of neurological function in dMCAO mice. Our results showed that liposome-DFO decreased iron deposition and reversed plasticity changes in cerebral cortex function after stroke, which delayed neurological function recovery. This experiment shows that the increasing iron level promotes endogenous repair in ischemic stroke. Our finding reveals the change regularity of iron and emphasizes the beneficial role of iron in the recovery process of neurological function, which provides an important basis for the prevention and/or treatment of ischemia-reperfusion and recovery after stroke.

Investigation of cell wall proteins of C. sinensis leaves by combining cell wall proteomics and N-glycoproteomics.

BACKGROUND: C. sinensis is an important economic crop with fluoride over-accumulation in its leaves, which poses a serious threat to human health due to its leaf consumption as tea. Recently, our study has indicated that cell wall proteins (CWPs) probably play a vital role in fluoride accumulation/detoxification in C. sinensis. However, there has been a lack in CWP identification and characterization up to now. This study is aimed to characterize cell wall proteome of C. sinensis leaves and to develop more CWPs related to stress response. A strategy of combined cell wall proteomics and N-glycoproteomics was employed to investigate CWPs. CWPs were extracted by sequential salt buffers, while N-glycoproteins were enriched by hydrophilic interaction chromatography method using C. sinensis leaves as a material. Afterwards all the proteins were subjected to UPLC-MS/MS analysis. RESULTS: A total of 501 CWPs and 195 CWPs were identified respectively by cell wall proteomics and N-glycoproteomics profiling with 118 CWPs in common. Notably, N-glycoproteomics is a feasible method for CWP identification, and it can enhance CWP coverage. Among identified CWPs, proteins acting on cell wall polysaccharides constitute the largest functional class, most of which might be involved in cell wall structure remodeling. The second largest functional class mainly encompass various proteases related to CWP turnover and maturation. Oxidoreductases represent the third largest functional class, most of which (especially Class III peroxidases) participate in defense response. As expected, identified CWPs are mainly related to plant cell wall formation and defense response. CONCLUSION: This was the first large-scale investigation of CWPs in C. sinensis through cell wall proteomics and N-glycoproteomics. Our results not only provide a database for further research on CWPs, but also an insight into cell wall formation and defense response in C. sinensis.

Genome-wide identification and characterization of phosphate transporter gene family members in tea plants (Camellia sinensis L. O. kuntze) under different selenite levels.

Selenium (Se) is an essential element for human health and an important nutrient for plant growth. Selenite is the main form of Se available to plants in acidic soils. Previous studies have shown that phosphate transporters (PTHs) participate in selenite uptake in plants. Research on the PHT gene family is therefore vital for production of Se-rich products. Here, 23 CsPHT genes were identified in the tea (Camellia sinensis) genome and renamed based on homology with AtPHT genes in Arabidopsis thaliana. The CsPHT genes were divided into four subfamilies: PHT1, PHT3, PHT4, and PHO, containing nine, three, six, and five genes, respectively. Phylogenetic analysis indicated that fewer duplication events occurred in tea plants than in A. thaliana, rice, apple, and poplar. Genes in the same subfamily tended to share similar gene structures, conserved motifs, and potential functions. CsPHT genes were differentially expressed in various tissues and in roots under different Se levels, suggesting key roles in selenite uptake, translocation, and homeostasis. The results illuminate the contributions of CsPHT genes to selenite supply in tea plants, and lay a foundation for follow-up studies on their potential functions in this plant species.

Design and Efficacy of a Monovalent Bispecific PD-1/CTLA4 Antibody That Enhances CTLA4 Blockade on PD-1+ Activated T Cells.

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1- T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995.

Brain iron deficiency and affected contextual fear memory in mice with conditional Ferroportin1 ablation in the brain.

Fear memory is a pivotal biological function by which organisms can predict possible danger to avoid or reduce harm. However, dysregulation of fear memory processing may lead to pathological fear or anxiety and produce serious clinical symptoms, such as post-traumatic stress disorder (PTSD). Iron deficiency (ID) is reported to inhibit the initiation of fear memory. In our study, we found that ferroportin1 (FPN1), the only known cellular iron export protein in mammals, and ablation in neurons and astrocytes caused iron deficiency in the cortex and hippocampus. However, little is known about its role in the development of fear memory. Moreover, direct evidence of the role of FPN1, or the related molecular mechanisms of such a role, in balancing brain iron homeostasis, especially in neuronal cells, is lacking. Herein, we deleted Fpn1 in mouse neurons, using Nestin-cre transgenic mice, and explored the impact on neuronal iron recycling and brain iron homeostasis in the cortex and hippocampus. We investigated the response of the mice to contextual fear and found that formation of fear memory was impeded after neuronal FPN1 depletion. We also found that FPN1 ablation in neurons and astrocytes caused an atypical expression of iron metabolism-related proteins in these two regions: decreased expression of DMT1, Ft-H, and Ft-L, and increased TfR1 expression. In addition, the decreased FPN1 in brain microvascular endothelial cells (BMVECs) also shed light on the cause of the decreased iron delivery to the brain through the blood-brain barrier (BBB). Our research highlights the major role played by FPN1 in brain iron homeostasis and identifies a potential target for the treatment of PTSD.

Nrf2 knockout altered brain iron deposition and mitigated age-related motor dysfunction in aging mice.

The transcription factor NF-E2-related factor 2 (Nrf2) is a central regulator of cellular antioxidant and detoxification response. The association between Nrf2 activity and iron-related oxidative stress in neurodegenerative diseases has been studied, and Nrf2 was found to transcriptionally regulate the expression of iron transporters and ferroptosis-related factors. However, the role of Nrf2 in age-related motor dysfunction and its link to iron metabolism dysregulation in brain have not been fully elucidated. In this study, with different ages of Nrf2 knockout (KO) and wild type (WT) mice, we investigated the effects of Nrf2 deficiency on brain oxidative stress, iron metabolism and the motor coordination ability of mice. In contrast to the predicted neuroprotective role of Nrf2 in oxidative stress-related diseases, we found that Nrf2 KO remarkably improved the motor coordination of aged mice, which was associated with the reduced ROS level and decreased apoptosis of dopaminergic neurons in substantia nigra (SN) of 18-month-old Nrf2 KO mice. With high-iron and Parkinson's disease (PD) mouse models, we revealed that Nrf2 KO prevented the deposition of brain iron, particularly in SN and striatum, which may subsequently delay motor dysfunction in aged mice. The regulation of Nrf2 KO on brain iron metabolism was likely mediated by decreasing the ferroportin 1 (FPN1) level on brain microvascular endothelial cells, thus hindering the process of iron entry into the brain. Nrf2 may be a potential therapeutic target in age-related motor dysfunction diseases for its role in regulating brain iron homeostasis.

Transcriptional Analysis of Metabolic Pathways and Regulatory Mechanisms of Essential Oil Biosynthesis in the Leaves of Cinnamomum camphora (L.) Presl.

The roots, bark, and leaves of Cinnamomum camphora are rich in essential oils, which mainly comprised monoterpenes and sesquiterpenes. Although the essential oils obtained from C. camphora have been widely used in pharmaceutical, medicinal, perfume, and food industries, the molecular mechanisms underlying terpenoid biosynthesis are poorly understood. To address this lack of knowledge, we performed transcriptome analysis to investigate the key regulatory genes involved in terpenoid biosynthesis in C. camphora. High-oil-yield trees of linalool type and low-oil-yield trees were used to assemble a de novo transcriptome of C. camphora. A total of 121,285 unigenes were assembled, and the total length, average length, N50, and GC content of unigenes were 87,869,987, 724, 1,063, and 41.1%, respectively. Comparison of the transcriptome profiles of linalool-type C. camphora with trees of low oil yield resulted in a total of 3,689 differentially expressed unigenes, among them 31 candidate genes had annotations associated with metabolism of terpenoids and polyketides, including four in the monoterpenoid biosynthesis pathway and three in the terpenoid backbone biosynthesis pathway. Collectively, this genome-wide transcriptome provides a valuable tool for future identification of genes related to essential oil biosynthesis. Additionally, the identification of a cohort of genes in the biosynthetic pathways of terpenoids provides a theoretical basis for metabolic engineering of essential oils in C. camphora.

Preventive Role of Salsalate in Diabetes Is Associated With Reducing Intestinal Inflammation Through Improvement of Gut Dysbiosis in ZDF Rats.

A safe and effective approach is needed to prevent and reduce the incidence of diabetes worldwide. The hypoglycemic efficacy of salicylic acid (salsalate, SAL), which has anti-inflammatory properties, has been empirically demonstrated in studies conducted at the Joslin Diabetes Center and elsewhere. Here, we investigated the potential role of SAL in preventing the onset of diabetes in Zucker diabetic fatty (ZDF) rats and attempted to elucidate its underlying mechanisms. ZDF and Zucker lean (ZL) rats were administered a high-fat diet with or without SAL intervention, and their relative rates of diabetes were compared. Our results showed that all rats in the placebo group developed diabetes, whereas only 10% of the SAL-treated rats presented with impaired glucose tolerance (IGT). None of the latter progressed to diabetes. Relative to the untreated rats, SAL lowered plasma glucagon and insulin while improving insulin sensitivity and ß-cell function. SAL may protect against hyperglycemia by increasing the microbial diversity, ameliorating gut dysbiosis, restoring intestinal epithelial cell connections, inhibiting endotoxin influx into the blood, and attenuating inflammation. Together, these findings suggest that SAL may be a candidate prophylactic therapy against diabetes. The protective role of SAL may be attributed to its ability to reduce intestinal inflammation and improve gut dysbiosis.

Acute Hyperglycemia May Induce Renal Tubular Injury Through Mitophagy Inhibition.

Aim: Acute hyperglycemia is closely related to kidney injury. Oxidative stress activation and notable mitochondria damages were found under acute hyperglycemia treatment in our previous work. In the present study, we explored the dose-effect relationship and the pivotal role of mitophagy in acute hyperglycemia induced tubular injuries. Methods: Forty non-diabetic SD rats were randomly divided and treated with different concentrations of hyperglycemia respectively during the 6-h clamp experiment. Renal morphological and functional alterations were detected. Rat renal tubular epithelial cells were treated with different concentrations of glucose for 6 h. Markers and the regulation pathway of mitophagy were analyzed. Results: Significant tubular injuries but not glomeruli were observed under both light and electron microscope after acute hyperglycemia treatment, which manifested as enlargement of tubular epithelial cells, disarrangement of epithelial cell labyrinths and swelling of mitochondria. Urinary microalbumin, ß2-MG, CysC, NAG, GAL, and NGAL were increased significantly with the increase of blood glucose (P < 0.05). ROS was activated, mitochondrial membrane potential and LC3-II/LC3-I ratio were decreased but P62 and BNIP3L/Nix were increased in hyperglycemia groups (P < 0.05), which were reversed by AMPK activation or mTOR inhibition. Conclusion: Acute hyperglycemia causes obvious tubular morphological and functional injuries in a dose-dependent manner. Acute hyperglycemia could inhibit mitophagy through AMPK/mTOR pathway, which would aggravate mitochondria damage and renal tubular impairment.

Regulation of antibody-mediated complement-dependent cytotoxicity by modulating the intrinsic affinity and binding valency of IgG for target antigen.

Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immunity. Although strategies to improve the CDC activity of antibody therapeutics have primarily focused on enhancing the interaction between the antibody crystallizable fragment (Fc) and the first subcomponent of the C1 complement complex (C1q), the relative importance of intrinsic affinity and binding valency of an antibody to the target antigen is poorly understood. Here we show that antibody binding affinity to a cell surface target antigen evidently affects the extent and efficacy of antibody-mediated complement activation. We further report the fundamental role of antibody binding valency in the capacity to recruit C1q and regulate CDC. More specifically, an array of affinity-modulated variants and functionally monovalent bispecific derivatives of high-affinity anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) therapeutic immunoglobulin Gs (IgGs), previously reported to be deficient in mediating complement activation, were tested for their ability to bind C1q by biolayer interferometry using antigen-loaded biosensors and to exert CDC against a panel of EGFR and HER2 tumor cells of various histological origins. Significantly, affinity-reduced variants or monovalent derivatives, but not their high-affinity bivalent IgG counterparts, induced near-complete cell cytotoxicity in tumor cell lines that had formerly been shown to be resistant to complement-mediated attack. Our findings suggest that monovalent target engagement may contribute to an optimal geometrical positioning of the antibody Fc to engage C1q and deploy the complement pathway.

Accurate position exchange of stamen and stigma by movement in opposite direction resolves the herkogamy dilemma in a protandrous plant, Ajuga decumbens (Labiatae).

Herkogamy is an effective way to reduce sexual interference. However, the separation of stigma and anther potentially leads to a conflict because the pollen may be placed in a location on the pollinator different from the point of stigma contact, which can reduce pollination accuracy. Floral mechanisms aiming to resolve this conflict have seldom been explored. The floral biology of protandrous Ajuga decumbens was studied to uncover how the herkogamy dilemma can be resolved. Flower anthesis was divided into male, middle, female and wilting phases. The positions of stigma and stamen were dissimilar in different flower development stages. We measured the distance of the stamen and stigma to the lower corolla lip at different floral phases, which was the pollinators' approaching way. The pollen viability, stigma receptivity, pollen removal and pollen deposition on stigma were investigated at different phases. During the male phase, the dehisced anthers were lower than the stigma, located at the pollinators' approaching way, and dispersed most pollen with high viability. As the flower developed, the anthers moved upwards, making way for pollen deposition during the female phase. Meanwhile, the stigma becomes receptive by moving into the way and consequently was deposited with sufficient pollen. The position exchange of the stamen and stigma created a dynamic herkogamy at the floral phase with different sexual functions. This floral mechanism effectively avoided sexual interference and maintained pollination accuracy. In Ajuga, the movement herkogamy might be of adaptive significance in response to the changes in the pollination environment.

Genome-wide identification and expression analyses of the LEA protein gene family in tea plant reveal their involvement in seed development and abiotic stress responses.

Late embryogenesis abundant (LEA) proteins are widely known to be present in higher plants and are believed to play important functional roles in embryonic development and abiotic stress responses. However, there is a current lack of systematic analyses on the LEA protein gene family in tea plant. In this study, a total of 48 LEA genes were identified using Hidden Markov Model profiles in C. sinensis, and were classified into seven distinct groups based on their conserved domains and phylogenetic relationships. Genes in the CsLEA_2 group were found to be the most abundant. Gene expression analyses revealed that all the identified CsLEA genes were expressed in at least one tissue, and most had higher expression levels in the root or seed relative to other tested tissues. Nearly all the CsLEA genes were found to be involved in seed development, and thirty-nine might play an important role in tea seed maturation concurrent with dehydration. However, only sixteen CsLEA genes were involved in seed desiccation, and furthermore, most were suppressed. Additionally, forty-six CsLEA genes could be induced by at least one of the tested stress treatments, and they were especially sensitive to high temperature stress. Furthermore, it was found that eleven CsLEA genes were involved in tea plant in response to all tested abiotic stresses. Overall, this study provides new insights into the formation of CsLEA gene family members and improves our understanding on the potential roles of these genes in normal development processes and abiotic stress responses in tea plant, particularly during seed development and desiccation. These results are beneficial for future functional studies of CsLEA genes that will help preserve the recalcitrant tea seeds for a long time and genetically improve tea plant.

Transcriptome and Expression Profiling Analysis of Recalcitrant Tea (Camellia sinensis L.) Seeds Sensitive to Dehydration.

The tea plant (Camellia sinensis (L.) O. Kuntze) is an economically important woody perennial nonalcoholic health beverage crop. Tea seeds are categorized as recalcitrant and are sensitive to dehydration treatment. However, the molecular basis of this phenomenon has not been investigated. Thus, we analyzed the genome-wide expression profiles of three dehydration stages using RNA-Seq and digital gene expression (DGE) technologies. We performed de novo assembly and obtained a total of 91,925 nonredundant unigenes, of which 58,472 were extensively annotated. By a hierarchical clustering of differentially expressed genes (DEGs), we found that 8929 DEGs were downregulated and 5875 DEGs were upregulated during dehydration treatment. A series of genes related to ABA biosynthesis and signal transduction, transcription factor, antioxidant enzyme, LEA protein, and proline metabolism that have been reported to function in dehydration process were found to be downregulated. Additionally, the expression profiles of 12 selected genes related to tea seed dehydration treatment were confirmed by qRT-PCR analysis. To our knowledge, this is the first genome-wide study elucidating the possible molecular mechanisms of sensitivity of recalcitrant tea seeds to dehydration. The results obtained in this study contribute to the preservation of tea seeds as genetic resources and can also be used to explore the mechanism of dehydration sensitivity of other recalcitrant seeds.

Transcriptome analysis of differentially expressed genes involved in selenium accumulation in tea plant (Camellia sinensis).

Tea plant (Camellia sinensis) has strong enrichment ability for selenium (Se). Selenite is the main form of Se absorbed and utilized by tea plant. However, the mechanism of selenite absorption and accumulation in tea plant is still unknown. In this study, RNA sequencing (RNA-seq) was used to perform transcriptomic analysis on the molecular mechanism of selenite absorption and accumulation in tea plant. 397.98 million high-quality reads were obtained and assembled into 168,212 unigenes, 89,605 of which were extensively annotated. There were 60,582 and 1,362 differentially expressed genes (DEGs) in roots and leaves, respectively. RNA-seq results were further validated by quantitative RT-PCR. Based on GO terms, the unigenes were mainly involved in cell, binding and metabolic process. KEGG pathway enrichment analysis showed that predominant pathways included ribosome and protein processing in endoplasmic reticulum. Further analysis revealed that sulfur metabolism, glutathione metabolism, selenocompound metabolism and plant hormone signal transduction responded to selenite in tea plant. Additionally, a large number of genes of higher expressions associated with phosphate transporters, sulfur assimilation, antioxidant enzymes, antioxidant substances and responses to ethylene and jasmonic acid were identified. Stress-related plant hormones might play a signaling role in promoting sulfate/selenite uptake and assimilation in tea plant. Moreover, some other Se accumulation mechanisms of tea plant were found. Our study provides a possibility for controlling Se accumulation in tea plant through bio-technologies and will be helpful for breeding new tea cultivars.