Improving Sedum plumbizincicola genetic transformation with the SpGRF4-SpGIF1 gene and the self-excision CRE/LoxP system.

MAIN CONCLUSION: S. plumbizincicola genetic transformation was optimized using a self-excision molecular-assisted transformation system by integrating the SpGRF4/SpGIF1 gene with XVE and Cre/loxP. Sedum plumbizincicola, despite being an excellent hyperaccumulator of cadmium and zinc with significant potential for soil pollution phytoremediation on farmland, has nonetheless trailed behind other major model plants in genetic transformation technology. In this study, different explants and SpGRF4-SpGIF1 genes were used to optimize the genetic transformation of S. plumbizincicola. We found that petiole and stem segments had higher genetic transformation efficiency than cluster buds. Overexpression of SpGRF4-SpGIF1 could significantly improve the genetic transformation efficiency and shorten the period of obtaining regenerated buds. However, molecular assistance with overexpression of SpGRF4-SpGIF1 leads to abnormal morphology, resulting in plant tissue enlargement and abnormal growth. Therefore, we combined SpGRF4-SpGIF1 with XVE and Cre/loxP to obtain DNA autocleavage transgenic plants induced by estradiol, thereby ensuring normal growth in transgenic plants. This study optimized the S. plumbizincicola genetic transformation system, improved the efficiency of genetic transformation, and established a self-excision molecular-assisted transformation system. This work also established the basis for studying S. plumbizincicola gene function, and for S. plumbizincicola breeding and germplasm innovation.

Delineation of the "Oropharyngeal Mucosa" and Limiting its Dose in Head and Neck Cancer Patients Spares the Oropharynx Without Compromising Target Coverage.

OBJECTIVES: Radiation-induced oropharyngeal injury is a dose-limiting toxicity in head and neck cancer patients. Delineation of the "oropharyngeal mucosa" and limiting its dose to spare the oropharynx was investigated. METHODS: In this retrospective study, computed tomography imaging from eight patients with previously untreated head and neck cancer was employed. An adaptive contouring brush within the planning software Monaco was used to create an air cavity within the oropharynx, and then the air cavity was expanded uniformly 2 mm to create the "oropharyngeal mucosa". Three plans were independently generated for each patient: Plan1: dose constraint was applied for the oropharynx; Plan2: dose constraints were applied for the oropharynx and the "oropharyngeal mucosa"; Plan3: dose constraint was applied for the "oropharyngeal mucosa". T-tests were used to compare the dosimetry variables. RESULTS: All plans had adequate target coverage and there were no statistical differences among plans. The mean dose, D30%, D45%, D50%, D85%, D90%, D95%, D100%, V25 Gy, V30 Gy, V35 Gy, V40 Gy, and V45 Gy of the oropharynx and "oropharyngeal mucosa" in Plan1 were significantly higher than those in Plan2 and Plan3. There were no significant differences between Plan2 and Plan3. There were no significant differences in the dosimetric parameters of any other organs at risk. CONCLUSION: Delineation of the "oropharyngeal mucosa" and limiting its dose should be an easy and effective method to spare the oropharynx.

Radiation-induced oropharyngeal injury is dose-limiting toxicity in head and neck cancer patients. Delineation of "oropharyngeal mucosa" and limiting its dose should be an easy and effective method to spare the oropharynx.

Phylogenomics and plastomics offer new evolutionary perspectives on Kalanchoideae (Crassulaceae).

BACKGROUND AND AIMS: Kalanchoideae is one of three subfamilies within Crassulaceae and contains four genera. Despite previous efforts, the phylogeny of Kalanchoideae remains inadequately resolved with persistent issues including low support, unstructured topologies and polytomies. This study aimed to address two central objectives: (1) resolving the pending phylogenetic questions within Kalanchoideae by using organelle-scale 'barcodes' (plastomes) and nuclear data; and (2) investigating interspecific diversity patterns among Kalanchoideae plastomes. METHODS: To explore the plastome evolution in Kalanchoideae, we newly sequenced 38 plastomes representing all four constituent genera (Adromischus, Cotyledon, Kalanchoe and Tylecodon). We performed comparative analyses of plastomic features, including GC and gene contents, gene distributions at the IR (inverted repeat) boundaries, nucleotide divergence, plastomic tRNA (pttRNA) structures and codon aversions. Additionally, phylogenetic inferences were inferred using both the plastomic dataset (79 genes) and nuclear dataset (1054 genes). KEY RESULTS: Significant heterogeneities were observed in plastome lengths among Kalanchoideae, strongly correlated with LSC (large single copy) lengths. Informative diversities existed in the gene content at SSC/IRa (small single copy/inverted repeat a), with unique patterns individually identified in Adromischus leucophyllus and one major Kalanchoe clade. The ycf1 gene was assessed as a shared hypervariable region among all four genera, containing nine lineage-specific indels. Three pttRNAs exhibited unique structures specific to Kalanchoideae and the genera Adromischus and Kalanchoe. Moreover, 24 coding sequences revealed a total of 41 lineage-specific unused codons across all four constituent genera. The phyloplastomic inferences clearly depicted internal branching patterns in Kalanchoideae. Most notably, by both plastid- and nuclear-based phylogenies, our research offers the first evidence that Kalanchoe section Eukalanchoe is not monophyletic. CONCLUSIONS: This study conducted comprehensive analyses on 38 newly reported Kalanchoideae plastomes. Importantly, our results not only reconstructed well-resolved phylogenies within Kalanchoideae, but also identified highly informative unique markers at the subfamily, genus and species levels. These findings significantly enhance our understanding of the evolutionary history of Kalanchoideae.

The AtMINPP Gene, Encoding a Multiple Inositol Polyphosphate Phosphatase, Coordinates a Novel Crosstalk between Phytic Acid Metabolism and Ethylene Signal Transduction in Leaf Senescence.

Plant senescence is a highly coordinated process that is intricately regulated by numerous endogenous and environmental signals. The involvement of phytic acid in various cell signaling and plant processes has been recognized, but the specific roles of phytic acid metabolism in Arabidopsis leaf senescence remain unclear. Here, we demonstrate that in Arabidopsis thaliana the multiple inositol phosphate phosphatase (AtMINPP) gene, encoding an enzyme with phytase activity, plays a crucial role in regulating leaf senescence by coordinating the ethylene signal transduction pathway. Through overexpressing AtMINPP (AtMINPP-OE), we observed early leaf senescence and reduced chlorophyll contents. Conversely, a loss-of-function heterozygous mutant (atminpp/+) exhibited the opposite phenotype. Correspondingly, the expression of senescence-associated genes (SAGs) was significantly upregulated in AtMINPP-OE but markedly decreased in atminpp/+. Yeast one-hybrid and chromatin immunoprecipitation assays indicated that the EIN3 transcription factor directly binds to the promoter of AtMINPP. Genetic analysis further revealed that AtMINPP-OE could accelerate the senescence of ein3-1eil1-3 mutants. These findings elucidate the mechanism by which AtMINPP regulates ethylene-induced leaf senescence in Arabidopsis, providing insights into the genetic manipulation of leaf senescence and plant growth.

Functional identification of anthocyanin glucosyltransferase genes: a Ps3GT catalyzes pelargonidin to pelargonidin 3-O-glucoside painting the vivid red flower color of Paeonia.

MAIN CONCLUSION: Glycosylation from an anthocyanidin 3-O-glucosyltransferase Ps3GT (PsUGT78A27) facilitates the accumulation of pelargonidin 3-O-glucoside, which defines the vivid red flower color and occurs only in specific peony tree cultivars. Although tree peony cultivars of Chinese and Japanese both originated from China, vivid red color is only found in flowers of Japanese cultivars but not of Chinese cultivar groups. In this study, a Japanese tree peony cultivar 'Taiyoh' with vivid red petals and a Chinese tree peony cultivar 'Hu Hong' with reddish pink petals were chosen as the experimental materials. Flavonoids profiling indicated that pelargonidin 3-O-glucoside (Pg3G) detected only in Japanese cultivar contributed to vivid red color of tree peony petals, while pelargonidin 3,5-di-O-glucoside (Pg3G5G) found in both of Japanese and Chinese cultivars was responsible for pink flower color. Through the integration of full-length transcriptome sequencing and in vitro enzymatic activity analysis, two anthocyanin glucosyltransferase genes PsUGT78A27 and PsUGT75L45 were isolated from the petals of tree peony, and their encoding products exhibited enzymatic activities of pelargonidin 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively. Further quantitative real-time PCR revealed that PsUGT78A27 displayed high expression in petals of both cultivars and PsUGT75L45 was expressed at high levels in cultivar 'Hu Hong' only. Using a gene gun technique, the GFP fusion proteins of PsUGT78A27 and PsUGT75L45 were visualized to be cytoplasmic and nuclear localization in the epidermal cells of tree peony petals, and the glucosylation function of PsUGT78A27 and PsUGT75L45 to alter petal color of tree peony and herbaceous peony had been directly validated in vivo. These results demonstrated that PsUGT78A27 and PsUGT75L45 are key players for the presence or absence of vivid red flower color in tree peony cultivars. Our findings further elucidated the chemical and molecular mechanism of petal pigmentation of Paeonia and could help breed the Paeonia cultivars possessing novel flower colors.

Exploring Transcriptional Regulation of Hyperaccumulation in Sedum plumbizincicola through Integrated Transcriptome Analysis and CRISPR/Cas9 Technology.

The cadmium hyperaccumulator Sedum plumbizincicola has remarkable abilities for cadmium (Cd) transport, accumulation and detoxification, but the transcriptional regulation mechanisms responsible for its Cd hyperaccumulation remain unknown. To address this knowledge gap, we conducted a comparative transcriptome study between S. plumbizincicola and the non-hyperaccumulating ecotype (NHE) of Sedum alfredii with or without Cd treatment. Our results revealed many differentially expressed genes involved in heavy metal transport and detoxification that were abundantly expressed in S. plumbizincicola. Additionally, we identified a large number of differentially expressed transcription factor genes, highlighting the complexity of transcriptional regulatory networks. We further screened four transcription factor genes that were highly expressed in the roots of S. plumbizincicola as candidate genes for creating CRISPR/Cas9 knockout mutations. Among these, the SpARR11 and SpMYB84 mutant lines exhibited decreased Cd accumulation in their aboveground parts, suggesting that these two transcription factors may play a role in the regulation of the Cd hyperaccumulation in S. plumbizincicola. Although further research will be required to determine the precise targeted genes of these transcription factors, combined transcriptome analysis and CRISPR/Cas9 technology provides unprecedented opportunities for identifying transcription factors related to Cd hyperaccumulation and contributes to the understanding of the transcriptional regulation mechanism of hyperaccumulation in S. plumbizincicola.

Enzymatic basis for stepwise C-glycosylation in the formation of flavonoid di-C-glycosides in sacred lotus (Nelumbo nucifera Gaertn.).

Lotus plumule, the embryo of the seed of the sacred lotus (Nelumbo nucifera), contains a high accumulation of secondary metabolites including flavonoids and possesses important pharmaceutical value. Flavonoid C-glycosides, which accumulate exclusively in lotus plumule, have attracted considerable attention in recent decades due to their unique chemical structure and special bioactivities. As well as mono-C-glycosides, lotus plumule also accumulates various kinds of di-C-glycosides by mechanisms which are as yet unclear. In this study we identified two C-glycosyltransferase (CGT) genes by mining sacred lotus genome data and provide in vitro and in planta evidence that these two enzymes (NnCGT1 and NnCGT2, also designated as UGT708N1 and UGT708N2, respectively) exhibit CGT activity. Recombinant UGT708N1 and UGT708N2 can C-glycosylate 2-hydroxyflavanones and 2-hydroxynaringenin C-glucoside, forming flavone mono-C-glycosides and di-C-glycosides, respectively, after dehydration. In addition, the above reactions were successfully catalysed by cell-free extracts from tobacco leaves transiently expressing NnCGT1 or NnCGT2. Finally, enzyme assays using cell-free extracts of lotus plumule suggested that flavone di-C-glycosides (vicenin-1, vicenin-3, schaftoside and isoschaftoside) are biosynthesized through sequentially C-glucosylating and C-arabinosylating/C-xylosylating 2-hydroxynaringenin. Taken together, our results provide novel insights into the biosynthesis of flavonoid di-C-glycosides by proposing a new biosynthetic pathway for flavone C-glycosides in N. nucifera and identifying a novel uridine diphosphate-glycosyltransferase (UGT708N2) that specifically catalyses the second glycsosylation, C-arabinosylating and C-xylosylating 2-hydroxynaringenin C-glucoside.

PAICS is related to glioma grade and can promote glioma growth and migration.

Glioma is a common malignant tumour of the brain. In this study, we aimed to investigate diagnostic biomarkers and its role in glioma. Weighted gene co-expression network analysis (WGCNA) and Cytoscape software were used to screen the marker genes in glioma. RT-qPCR and Western blotting methods were performed to determine the expression of PAICS, ERCC1 and XPA genes in glioma tissues. Expression level of PAICS in different grades of glioma was examined by immunohistochemistry. CCK8 and Colony formation assays were used to detect cell proliferation. Cell adhesion assay was used to detect adhesion ability. Wound healing and transwell tests were used to detect cell migration ability. Flow cytometry was used to detect cell cycle and apoptosis. According to the predicted co-expression network, we identified the hub gene PAICS. Furthermore, we observed that PAICS expression level was up-regulated in glioma tissues compared with normal tissues, and the expression level was correlated with the grade of glioma. Moreover, we found PAICS can promote glioma cells proliferation and migration in vitro. Flow cytometry results showed that si-PAICS cells were stalled at the G1 phase compared with the si-NC cells and knocking down PAICS expression can increase apoptotic rate. PAICS can regulate the mRNA and protein levels of nucleotide excision repair pathway core genes ERCC1 and XPA. l-aspartic acid can affect the expression of PAICS and then inhibit glioma cell proliferation. Our results indicated that PAICS can promote glioma proliferation and migration. PAICS may act as a potential diagnostic marker and a therapeutic target for glioma.

Impact of DRGs-based inpatient service management on the performance of regional inpatient services in Shanghai, China: an interrupted time series study, 2013-2019.

BACKGROUND: The asymmetry of information brings difficulty for government to manage public hospitals. Therefore, Jiading District of Shanghai has been establishing DRGs-based inpatient service management system (ISMS) to effectively compare the output of different hospitals through DRGs, reward desired hospital performance and enhance inpatient service capacity. However, the impact of the implementation of DRGs-based inpatient service management (ISM) policy in Jiading district is still unknow. We therefore conducted this study to evaluate the impact of DRGs-based ISM policy on the performance of inpatient service since its implementation in Jiading District, Shanghai, China in 2017. METHODS: Using an interrupted time series design, we analyzed quarterly data of seven DRGs-based performance measures from the ISMS which covered all five public hospitals in Jiading District from 2013 to 2019. We utilized the segmented linear regression model to assess the change of level and trend of performance indicators before and after ISM policy. Dickey-Fuller test was used to examine the stationary of the data. Durbin-Watson test was performed to check the series autocorrelation of indicators. RESULTS: Significant changes in the following indicators were observed since the implementation of ISM policy. The case-mix index (CMI) level decreased by 0.103 (P < 0.05), the trend increased by 0.008 (P < 0.05). The total weight level decreased by 3719.05 (P < 0.05), and the trend increased by 250.13 (P < 0.05). The time efficiency index (TEI) level increased by 0.12 (P < 0.05), and the trend decreased by 0.01 (P < 0.05). The cost efficiency index (CEI) level increased by 0.31 (P < 0.05), and the trend decreased by 0.02 (P < 0.05). No significant difference was found in the change of DRGs number, inpatient mortality of low-risk group cases (IMLRG) and inpatient mortality of medium-to-low risk group cases (IMMLRG). CONCLUSIONS: Findings highlight the role of ISM policy in improving the capacity and efficiency of regional inpatient service. Three prerequisites, including a good information system, high-quality EMR data, and a management team, are needed for other countries to implement their own ISM policy to help government manage public hospitals and improve the performance of regional inpatient service.

SpHMA1 is a chloroplast cadmium exporter protecting photochemical reactions in the Cd hyperaccumulator Sedum plumbizincicola.

Sedum plumbizincicola is able to hyperaccumulate cadmium (Cd), a nonessential and highly toxic metal, in the above-ground tissues, but the mechanisms for its Cd hypertolerance are not fully understood. Here, we show that the heavy metal ATPase 1 (SpHMA1) of S. plumbizincicola plays an important role in chloroplast Cd detoxification. Compared with the HMA1 ortholog in the Cd nonhyperaccumulating ecotype of Sedum alfredii, the expression of SpHMA1 in the leaves of S. plumbizincicola was >200 times higher. Heterologous expression of SpHMA1 in Saccharomyces cerevisiae increased Cd sensitivity and Cd transport activity in the yeast cells. The SpHMA1 protein was localized to the chloroplast envelope. SpHMA1 RNA interference transgenic plants and CRISPR/Cas9-induced mutant lines showed significantly increased Cd accumulation in the chloroplasts compared with wild-type plants. Chlorophyll fluorescence imaging analysis revealed that the photosystem II of SpHMA1 knockdown and knockout lines suffered from a much higher degree of Cd toxicity than wild type. Taken together, these results suggest that SpHMA1 functions as a chloroplast Cd exporter and protects photosynthesis by preventing Cd accumulation in the chloroplast in S. plumbizincicola and hyperexpression of SpHMA1 is an important component contributing to Cd hypertolerance in S. plumbizincicola.

Quantification of near-attomole gibberellins in floral organs dissected from a single Arabidopsis thaliana flower.

There remains a methodological bottleneck in the quantification of ultra-trace plant hormones in very tiny plant organs at fresh weights below a milligram. The challenge becomes even more serious in the determination of endogenous gibberellins (GAs), which are a class of compounds that are difficult to separate and detect. Herein, a quantification method using ultra-high-performance liquid chromatography-tandem mass spectrometry was developed, combined with a derivatization technique in which GAs react with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide in ethanol. The method was validated as capable of determining GAs in floral organs (about 80-250 µg) - pistil, stamens, petals, sepals and receptacle - which were dissected from only one flower of Arabidopsis thaliana. Substantially different abundance patterns of GAs were measured from the floral organs at floral stages 13, 14 and 15 along the non-13-hydroxylation pathway and the early 13-hydroxylation pathway in plants. This allows sub-flower-level insights into how GAs affect floral development. The method exhibited excellent limit of detection and limit of quantification down to 5.41 and 18.0 attomole, respectively, and offered a fairly wide linear range from 0.01 to 25 femtomole with linear coefficients above 0.9961. The precision of the method was evaluated with relative standard deviations below 10.6% for intra-day and 11.4% for inter-day assays, and recoveries ranged from 64.0% to 107%.

Heavy metal ATPase 3 (HMA3) confers cadmium hypertolerance on the cadmium/zinc hyperaccumulator Sedum plumbizincicola.

Cadmium (Cd) is highly toxic to most organisms, but some rare plant species can hyperaccumulate Cd in aboveground tissues without suffering from toxicity. The mechanism underlying Cd detoxification by hyperaccumulators is interesting but unclear. Here, the heavy metal ATPase 3 (SpHMA3) gene responsible for Cd detoxification was isolated from the Cd/zinc (Zn) hyperaccumulator Sedum plumbizincicola. RNA interference (RNAi)-mediated silencing and overexpression of SpHMA3 were induced to investigate its physiological functions in S. plumbizincicola and a nonhyperaccumulating ecotype of Sedum alfredii. Heterologous expression of SpHMA3 in Saccharomyces cerevisiae showed Cd-specific transport activity. SpHMA3 was highly expressed in the shoots and the protein was localized to the tonoplast. The SpHMA3-RNAi lines were hypersensitive to Cd but not to Zn, with the growth of shoots and young leaves being severely inhibited by Cd. Overexpressing SpHMA3 in the nonhyperaccumulating ecotype of S. alfredii greatly increased its tolerance to and accumulation of Cd, but not Zn. These results indicate that elevated expression of the tonoplast-localized SpHMA3 in the shoots plays an essential role in Cd detoxification, which contributes to the maintenance of the normal growth of young leaves of S. plumbizincicola in Cd-contaminated soils.

A SAL1 Loss-of-Function Arabidopsis Mutant Exhibits Enhanced Cadmium Tolerance in Association with Alleviation of Endoplasmic Reticulum Stress.

SAL1, as a negative regulator of stress response signaling, has been studied extensively for its role in plant response to environmental stresses. However, the role of SAL1 in cadmium (Cd) stress response and the underlying mechanism is still unclear. Using an Arabidopsis thaliana loss-of-function mutant of SAL1, we assessed Cd resistance and further explored the Cd toxicity mechanism through analysis of the endoplasmic reticulum (ER) stress response. The loss of SAL1 function greatly improved Cd tolerance and significantly attenuated ER stress in Arabidopsis. Exposure to Cd induced an ER stress response in Arabidopsis as evidenced by unconventional splicing of AtbZIP60 and up-regulation of ER stress-responsive genes. Damage caused by Cd was markedly reduced in the ER stress response double mutant bzip28 bzip60 or by application of the ER stress-alleviating chemical agents, tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (4-PBA), in wild-type plants. The Cd-induced ER stress in Arabidopsis was also alleviated by loss of function of SAL1. These results identified SAL1 as a new component mediating Cd toxicity and established the role of the ER stress response in Cd toxicity. Additionally, the attenuated ER stress in the sal1 mutant might also shed new light on the mechanism of diverse abiotic stress resistance in the SAL1 loss-of-function mutants.

An Arabidopsis mutant of inositol pentakisphosphate 2-kinase AtIPK1 displays reduced arsenate tolerance.

Arsenate [As(V)] toxicity is considered to be derived from similarities in the chemical properties of As(V) and phosphate (Pi). An Arabidopsis thaliana mutant of inositol pentakisphosphate 2-kinase (AtIPK1), atipk1-1, has previously exhibited lower level of phytate and higher level of Pi, relative to wild-type (WT). Here, atipk1-1 displayed hypersensitivity to As(V) stress and less As(V) uptake when compared to WT. Overexpression of AtIPK1 controlled by the CaMV 35S promoter partially rescued the As(V)-sensitive phenotype of atipk1-1. When compared to control Pi status, addition of Pi enhanced As(V) tolerance of both WT and atipk1-1 plants, while the arsenic concentration was less reduced in the latter genotype. Despite the higher Pi level in atipk1-1 than did WT plants, the mutant suffered more severe Pi starvation under Pi limitation stress, indicating that Pi homeostasis was altered in the mutant. Gene expression analysis of WT and atipk1-1 plants showed the diverse effect of As(V) stress on Pi starvation-dependent regulation of Pi-responsive genes. Our study suggested that a particular mechanism of As(V) toxicity existed in atipk1-1 mutant, and may offer new insights into the interactions between Pi homeostasis and As(V) detoxification in plants.

High-throughput screening-compatible assays of As(III) S-adenosylmethionine methyltransferase activity.

Arsenic is a naturally existing toxin and carcinogen. As(III) S-adenosylmethionine methyltransferases (AS3MT in mammals and ArsM in microbes) methylate As(III) three times in consecutive steps and play a central role in arsenic metabolism from bacteria to humans. Current assays for arsenic methylation are slow, laborious, and expensive. Here we report the development of two in vitro assays for AS3MT activity that are rapid, sensitive, convenient, and relatively inexpensive and can be adapted for high-throughput assays. The first assay measures As(III) binding by the quenching of the protein fluorescence of a single-tryptophan derivative of an AS3MT ortholog. The second assay utilizes time-resolved fluorescence resonance energy transfer to directly measure the conversion of the AS3MT substrate, S-adenosylmethionine, to S-adenosylhomocysteine catalyzed by AS3MT. These two assays are complementary, one measuring substrate binding and the other catalysis, making them useful tools for functional studies and future development of drugs to prevent arsenic-related diseases.

Engineering arsenic tolerance and hyperaccumulation in plants for phytoremediation by a PvACR3 transgenic approach.

Arsenic (As) pollution is a global problem, and the plant-based cleanup of contaminated soils, called phytoremediation, is therefore of great interest. Recently, transgenic approaches have been designed to develop As phytoremediation technologies. Here, we used a one-gene transgenic approach for As tolerance and accumulation in Arabidopsis thaliana . PvACR3, a key arsenite [As(III)] antiporter in the As hyperaccumulator fern Pteris vittata , was expressed in Arabidopsis , driven by the CaMV 35S promoter. In response to As treatment, PvACR3 transgenic plants showed greatly enhanced tolerance. PvACR3 transgenic seeds could even germinate and grow in the presence of 80 µM As(III) or 1200 µM arsenate [As(V)] treatments that were lethal to wild-type seeds. PvACR3 localizes to the plasma membrane in Arabidopsis and increases arsenite efflux into external medium in short-term experiments. Arsenic determination showed that PvACR3 substantially reduced As concentrations in roots and simultaneously increased shoot As under 150 µM As(V). When cultivated in As(V)-containing soil (10 ppm As), transgenic plants accumulated approximately 7.5-fold more As in above-ground tissues than wild-type plants. This study provides important insights into the behavior of PvACR3 and the physiology of As metabolism in plants. Our work also provides a simple and practical PvACR3 transgenic approach for engineering As-tolerant and -hyperaccumulating plants for phytoremediation.

Phytotoxicities of inorganic arsenic and dimethylarsinic acid to Arabidopsis thaliana and Pteris vittata.

The mechanisms by which Pteris vittata (L.) hyperaccumulates arsenic (As) have not been fully elucidated. To investigate how P. vittata tolerates high concentrations of arsenite, we compared the toxicities of various As compounds to P. vittata and Arabidopsis thaliana (L.). The phytotoxicities of As species were found to be in the order of arsenite > arsenate > dimethylarsinic acid (DMAA) in A. thaliana, and in the order of DMAA > arsenate > arsenite in P. vittata. P. vittata calli displayed a weaker ability to absorb arsenite than arsenate. These results demonstrate that P. vittata possesses mechanisms of As accumulation and detoxification.

Biogenesis of flavor-related linalool is diverged and genetically conserved in tree peony (Paeonia × suffruticosa).

Floral scent is an important and genetically complex trait in horticultural plants. Tree peony (Paeonia × suffruticosa) originates in the Pan-Himalaya and has nine wild species divided into two subsections, Delavayanae and Vaginatae. Their flowers are beloved worldwide for their sweet floral fragrance, yet the flavor-related volatiles and underlying biosynthetic pathways remain unknown. Here, we characterized the volatile blends of all wild tree peony species and found that the flavor-related volatiles were highly divergent, but linalool was a unique monoterpene in subsect. Delavayanae. Further detection of volatiles in 97 cultivars with various genetic backgrounds showed that linalool was also the characteristic aroma component in Paeonia delavayi hybrid progenies, suggesting that linalool was conserved and dominant within subsect. Delavayanae and its hybrids, instead of species and cultivars from subsect. Vaginatae. Global transcriptome analysis of all wild tree peony species and 60 cultivars revealed five candidate genes that may be involved in key steps of linalool biosynthesis; especially the expressions of three TPS genes, PdTPS1, PdTPS2, and PdTPS4, were significantly positively correlated with linalool emissions across tree peony cultivars. Further biochemical evidence demonstrated that PdTPS1 and PdTPS4 were the pivotal genes determining the species-specific and cultivar-specific emission of linalool. This study revealed a new insight into floral scent divergence in tree peony and would greatly facilitate our understanding of the phylogeny and evolution of Paeonia.

Efficacy and safety of methylphenidate and ginseng in cancer-related fatigue: a network meta-analysis of randomized controlled trials.

Background: Incidence of cancer-related fatigue (CRF), which can persist 5 to 10 years, is nearly 85% in cancer patients. It severely affects the quality of life and is strongly associated with poor prognosis. As clinical trial data on CRF treated with methylphenidate and ginseng, two potential medicines, has been accumulating, an updated meta-analysis was performed to evaluate and compare the efficacy and safety of the two medicines in CRF. Methods: Randomized controlled trials that investigated methylphenidate or ginseng in the treatment of CRF were identified through a literature search. The primary outcome was CRF relief. Standardized mean difference (SMD) was used to analyze the effect. Results: Eight studies on methylphenidate were included and the pooled SMD was 0.18 [95% confidence interval (95% CI): -0.00 to 0.35, P=0.05]. Five studies on ginseng were included and the SMD was 0.32 (95% CI: 0.17-0.46, P<0.0001). Results of network meta-analysis showed that the order was ginseng, methylphenidate, placebo from high efficacy to low and ginseng was significantly better than methylphenidate (SMD =0.23, 95% CI: 0.01-0.45). Incidences of insomnia and nausea caused by ginseng were significantly lower than those caused by methylphenidate (P<0.05). Conclusions: Both methylphenidate and ginseng can significantly ameliorate CRF. Ginseng may be superior to methylphenidate because ginseng may be more effective and might cause less adverse events. Head-to-head trials with fixed protocol are warranted to identify the optimal medical strategy.

The combination of DNA methylation and positive regulation of anthocyanin biosynthesis by MYB and bHLH transcription factors contributes to the petal blotch formation in Xibei tree peony.

Xibei tree peony is a distinctive cultivar group that features red-purple blotches in petals. Interestingly, the pigmentations of blotches and non-blotches are largely independent of one another. The underlying molecular mechanism had attracted lots of attention from investigators, but was still uncertain. Our present work demonstrates the factors that are closely related to blotch formation in Paeonia rockii 'Shu Sheng Peng Mo'. Non-blotch pigmentation is prevented by the silencing of anthocyanin structural genes, among which PrF3H, PrDFR, and PrANS are the three major genes. We characterized two R2R3-MYBs as the key transcription factors that control the early and late anthocyanin biosynthetic pathways. PrMYBa1, which belongs to MYB subgroup 7 (SG7) was found to activate the early biosynthetic gene (EBG) PrF3H by interacting with SG5 member PrMYBa2 to form an 'MM' complex. The SG6 member PrMYBa3 interacts with two SG5 (IIIf) bHLHs to synergistically activate the late biosynthetic genes (LBGs) PrDFR and PrANS, which is essential for anthocyanin accumulation in petal blotches. The comparison of methylation levels of the PrANS and PrF3H promoters between blotch and non-blotch indicated a correlation between hypermethylation and gene silencing. The methylation dynamics of PrANS promoter during flower development revealed a potential early demethylating reaction, which may have contributed to the particular expression of PrANS solely in the blotch area. We suggest that the formation of petal blotch may be highly associated with the cooperation of transcriptional activation and DNA methylation of structural gene promoters.