Endoplasmic reticulum stress response modulator OsbZIP39 regulates cadmium accumulation via activating the expression of defensin-like gene OsCAL2 in rice.

Accumulation of cadmium (Cd) in rice is not only harmful to the growth of plants but also poses a threat to human health. Exposure to Cd triggers unfolded protein response (UPR) within cells, a process that is still not completely understood. The study demonstrated that the lack of OsbZIP39, an essential endoplasmic reticulum (ER)-resident regulator of the UPR, resulted in decreased Cd intake and reduced Cd levels in the roots, stems, and grains of rice. Upon exposure to Cd stress, GFP-OsbZIP39 translocated from ER to nucleus, initiating UPR. Further investigation revealed that Cd treatment caused changes in sphingolipid levels in the membrane, influencing the localization and activation of OsbZIP39. Yeast one-hybrid and dual-LUC assays were conducted to validate the interaction between activated OsbZIP39 and the promoter of the defensin-like gene OsCAL2, resulting in an increase in its expression. Different variations were identified in the coding region of OsbZIP39, which may explain the varying levels of Cd accumulation observed in the indica and japonica subspecies. Under Cd treatment, OsbZIP39ind exhibited a more significant enhancement in the transcription of OsCAL2 compared to OsbZIP39jap. Our data suggest that OsbZIP39 positively regulates Cd uptake in rice, offering an encouraging objective for the cultivation of low-Cd rice.

PatWRKY71 transcription factor regulates patchoulol biosynthesis and plant defense response.

Patchoulol, a valuable compound belonging to the sesquiterpenoid family, is the primary component of patchouli oil produced by Pogostemon cablin (P. cablin). It has a variety of pharmacological and biological activities and is widely used in the medical and cosmetic industries. However, despite its significance, there is a lack of research on the transcriptional modulation of patchoulol biosynthesis.Salicylic acid (SA), is a vital plant hormone that serves as a critical signal molecule and plays an essential role in plant growth and defense. However, to date, no studies have explored the modulation of patchoulol biosynthesis by SA. In our study, we discovered that the application of SA can enhance the production of patchoulol. Utilizing transcriptome analysis of SA-treated P. cablin, we identified a crucial downstream transcription factor, PatWRKY71. The transcription level of PatWRKY71 was significantly increased with the use of SA. Furthermore, our research has revealed that PatWRKY71 was capable of binding to the promoter of PatPTS, ultimately leading to an increase in its expression. When PatWRKY71 was silenced by a virus, the expression of both PatWRKY71 and PatPTS was reduced, resulting in the down-regulation of patchoulol production. Through our studies, we discovered that heterologous expression of PatWRKY71 leads to an increase in the sensitivity of Arabidopsis to salt and Cd, as well as an outbreak of reactive oxygen species (ROS). Additionally, we uncovered the regulatory role of PatWRKY71 in both patchoulol biosynthesis and plant defense response. This discovery provided a theoretical basis for the improvement of the content of patchoulol and the resistance of P. cablin through genetic engineering.

Genome-Wide Identification and Characterization of Long Non-Coding RNAs in Roots of Rice Seedlings under Nitrogen Deficiency.

Long non-coding RNAs (lncRNAs) regulate gene expression in eukaryotic organisms. Research suggests that lncRNAs may be involved in the regulation of nitrogen use efficiency in plants. In this study, we identified 1628 lncRNAs based on the transcriptomic sequencing of rice roots under low-nitrogen (LN) treatment through the implementation of an integrated bioinformatics pipeline. After 4 h of LN treatment, 50 lncRNAs and 373 mRNAs were significantly upregulated, and 17 lncRNAs and 578 mRNAs were significantly downregulated. After 48 h LN treatment, 43 lncRNAs and 536 mRNAs were significantly upregulated, and 42 lncRNAs and 947 mRNAs were significantly downregulated. Moreover, the interaction network among the identified lncRNAs and mRNAs was investigated and one of the LN-induced lncRNAs (lncRNA24320.6) was further characterized. lncRNA24320.6 was demonstrated to positively regulate the expression of a flavonoid 3'-hydroxylase 5 gene (OsF3'H5). The overexpression of lncRNA24320.6 was shown to improve nitrogen absorption and promote growth in rice seedlings under LN conditions. Our results provide valuable insights into the roles of lncRNAs in the rice response to nitrogen starvation.

Genomic Identification of CCCH-Type Zinc Finger Protein Genes Reveals the Role of HuTZF3 in Tolerance of Heat and Salt Stress of Pitaya (Hylocereus polyrhizus).

Pitaya (Hylocereus polyrhizus) is cultivated in a broad ecological range, due to its tolerance to drought, heat, and poor soil. The zinc finger proteins regulate gene expression at the transcriptional and post-transcriptional levels, by interacting with DNA, RNA, and proteins, to play roles in plant growth and development, and stress response. Here, a total of 81 CCCH-type zinc finger protein genes were identified from the pitaya genome. Transcriptomic analysis showed that nine of them, including HuTZF3, responded to both salt and heat stress. RT-qPCR results showed that HuTZF3 is expressed in all tested organs of pitaya, with a high level in the roots and stems, and confirmed that expression of HuTZF3 is induced by salt and heat stress. Subcellular localization showed that HuTZF3 is targeted in the processing bodies (PBs) and stress granules (SGs). Heterologous expression of HuTZF3 could improve both salt and heat tolerance in Arabidopsis, reduce oxidative stress, and improve the activity of catalase and peroxidase. Therefore, HuTZF3 may be involved in post-transcriptional regulation via localizing to PBs and SGs, contributing to both salt and heat tolerance in pitaya.

Rice miR168a-5p regulates seed length, nitrogen allocation and salt tolerance by targeting OsOFP3, OsNPF2.4 and OsAGO1a, respectively.

Rice microRNA168a (osa-miR168a) plays important roles in mediating flowering time, grain yield and vigor, seeding growth, and immunity by targeting the RNA-induced silencing complex component Argonaute 1 (AGO1). However, the functions of miR168a exerted by targeting other genes require further clarification before it could be used in rice molecular breeding. In this study, we identified a new target gene of osa-miR168a-5p (miR168a-5p) in rice called OsOFP3 (ovate family protein 3) and investigated the roles of miR168a-5p in response to brassinosteroids (BRs), salt stress, and nitrogen allocation. Up- and downregulated miR168a-5p expression respectively decreased and increased the expression of the BR-negative regulator OsOPF3. The results of RNA ligase-mediated rapid amplification of cDNA ends (5'RLM-RACE) revealed cleavage sites in OsOPF3 and OsNPF2.4 mRNAs. The phenotype of miR168a-5p transgenic rice was BR-associated and included the lamina bending response to BR, short seeds, and low 1000-grain weight. MicroRNA 168a-5p also regulated the expression of the nitrate transporter, OsNPF2.4, which affected nitrogen allocation, and regulated OsAGO1a expression in response to salt stress. Taken together, rice miR168a-5p regulates BR-associated pathways, nitrogen transport, and stress by targeting OsOFP3, OsNPF2.4, and OsAGO1a, respectively, resulting in a series of important agronomic traits for rice breeding.

Chronic recurrent multifocal osteomyelitis. A narrative and pictorial review.

Chronic recurrent and multifocal osteomyelitis (CRMO) is a nonsporadic autoinflammatory disorder. Currently, it is diagnosed based on clinical, radiologic, pathological, and longitudinal data. Numerous aspects should be highlighted due to increased knowledge in imaging and immunology. We emphasize the use of whole-body MRI, which is a non-invasive diagnostic strategy. A literature review was carried out on longitudinal studies. Commonly, the mean age at diagnosis is 11 years, ranging between 3 and 17. The most common sites are the long bone metaphysis, particularly femoral and tibial metaphysis. In addition, the pelvis, spine, clavicle, and mandible may be involved. In long bones, the radiologic appearance can show typical structure, mixed lytic and sclerotic, sclerotic or lytic. It is frequently metaphyseal or juxta-physeal, with hyperostosis or periosteal thickening. The involvement of the vertebral skeleton is often multifocal. Therefore, whole-body MRI is essential in identifying subclinical lesions. CRMO is a polymorphic disorder in which whole-body MRI is beneficial to demonstrate subclinical edema. Vertebral collapse requires long-term monitoring.

Target Lines for in Planta Gene Stacking in Japonica Rice.

The clustering of transgenes at a chromosome location minimizes the number of segregating loci that needs to be introgressed to field cultivars. Transgenes could be efficiently stacked through site-specific recombination and a recombinase-mediated in planta gene stacking process was described previously in tobacco based on the Mycobacteriophage Bxb1 site-specific integration system. Since this process requires a recombination site in the genome, this work describes the generation of target sites in the Japonica rice genome. Agrobacterium-mediated gene transfer yielded ~4000 random-insertion lines. Seven lines met the criteria of being single copy, not close to a centromere, not inserted within or close to a known gene or repetitive DNA, having precise recombination site sequences on both ends, and able to express the reporter gene. Each target line tested was able to accept the site-specific integration of a new gfp-containing plasmid and in three of those lines, we regenerated fertile plants. These target lines could be used as foundation lines for stacking new traits into Japonica rice.

The putative obtusifoliol 14α-demethylase OsCYP51H3 affects multiple aspects of rice growth and development.

Some members of the CYP51G subfamily has been shown to be obtusifoliol 14α-demethylase, key enzyme of the sterol and brassinosteroid (BR) biosynthesis, which mediate plant development and response to stresses. However, little is known about the functions of CYP51H subfamily in rice. Here, OsCYP51H3, an ortholog of rice OsCYP51G1 was identified. Compared with wild type, the mutants oscyp51H3 and OsCYP51H3-RNAi showed dwarf phenotype, late flowering, erected leaves, lower seed-setting rate, and smaller and shorter seeds. In contrast, the phenotypic changes of OsCYP51H3-OE plants are not obvious. Metabolomic analysis of oscyp51H3 mutant indicated that OsCYP51H3 may also encode an obtusifoliol 14α-demethylase involved in phytosterol and BR biosynthesis, but possibly not that of triterpenes. The RNA-seq results showed that OsCYP51H3 may affect the expression of a lot of genes related to rice development. These findings showed that OsCYP51H3 codes for a putative obtusifoliol 14α-demethylase involved in phytosterol and BR biosynthesis, and mediates rice development.

Diverse function of the PISTILLATA, APETALA 3, and AGAMOUS-like MADS-box genes involved in the floral development in Alpinia hainanensis (Zingiberaceae).

Zingiberaceae is the vital clue and key node in the decreased process of fertile stamens in Zingiberales, helping to understand the evolution of the ginger families. This study focuses on Alpinia hainanensis to investigate the function of B- and C-class MADS-box genes in floral development. The introns size of two B-class genes AhPI and AhAP3, and one C-class gene AhAG are quite variable. By contrast, the positions of the corresponding introns are conserved, resulting in a similar exon size in homologs. The typical region 70 bp-CCAATCA element was not found in the second intron of AhAG compared to AG homologs. The subcellular localization showed that AhAP3 was in both intranuclear and extranuclear. The heterodimer was formed between APETALA3 and PISTILLATA but not between the B- and C-class proteins using Y2H and BiFC. The 35S::AhAG heterologous transformed Arabidopsis had curly and smaller rosette leaves with early flowering. Floral organs had no homeotic conversion, albeit sepals and petals reduced in size. Siliques development was affected and displayed wrinkled and shorter. By contrast, 35S::AhAP3 and 35S::AhPI did not show any modified phenotype in transgenic Arabidopsis thaliana. We first proposed the model for Alpinia flower development. MADS-box transcription factor binding at particular genomic locations and interaction with partners may be crucial for the development of the floral organ.

Simultaneous Determination of Seven Lipophilic and Hydrophilic Components in Salvia miltiorrhiza Bunge by LC-MS/MS Method and Its Application to a Transport Study in a Blood-Brain-Barrier Cell Model.

Salvia miltiorrhiza Bunge (SM) has been extensively used in Alzheimer's disease treatment, the permeability through the blood-brain barrier (BBB) determining its efficacy. However, the transport mechanism of SM components across the BBB remains to be clarified. A simple, precise, and sensitive method using LC-MS/MS was developed for simultaneous quantification of tanshinone I (TS I), dihydrotanshinone I (DTS I), tanshinone IIA (TS IIA), cryptotanshinone (CTS), protocatechuic aldehyde (PAL), protocatechuic acid (PCTA), and caffeic acid (CFA) in transport samples. The analytes were separated on a C18 column by gradient elution. Multiple reaction monitoring mode via electrospray ionization source was used to quantify the analytes in positive mode for TS I, DTS I, TS IIA, CTS, and negative mode for PAL, PCTA, and CFA. The linearity ranges were 0.1-8 ng/mL for TS I and DTS I, 0.2-8 ng/mL for TS IIA, 1-80 ng/mL for CTS, 20-800 ng/mL for PAL and CFA, and 10-4000 ng/mL for PCTA. The developed method was accurate and precise for the compounds. The relative matrix effect was less than 15%, and the analytes were stable for analysis. The established method was successfully applied for transport experiments on a BBB cell model to evaluate the apparent permeability of the seven components.

miR2105 and the kinase OsSAPK10 co-regulate OsbZIP86 to mediate drought-induced ABA biosynthesis in rice.

Mediating induced abscisic acid (ABA) biosynthesis is important for enhancing plant stress tolerance. Here, we found that rice (Oryza sativa L.) osa-miR2105 (miR2105) and the Stress/ABA-activated protein kinase (OsSAPK10) coordinately regulate the rice basic region-leucine zipper transcription factor (bZIP TF; OsbZIP86) at the posttranscriptional and posttranslational levels to control drought-induced ABA biosynthesis via modulation of rice 9-cis-epoxycarotenoid dioxygenase (OsNCED3) expression. OsbZIP86 expression is regulated by miR2105-directed cleavage of the OsbZIP86 mRNA. OsbZIP86 encodes a nuclear TF that binds to the promoter of the ABA biosynthetic gene OsNCED3. OsSAPK10 can phosphorylate and activate OsbZIP86 to enhance the expression of OsNCED3. Under normal growth conditions, altered expression of miR2105 and OsbZIP86 displayed no substantial effect on rice growth. However, under drought conditions, miR2105 knockdown or OsbZIP86 overexpression transgenic rice plants showed higher ABA content, enhanced tolerance to drought, lower rates of water loss, and more stomatal closure of seedlings, compared with wild-type rice Zhonghua 11; in contrast, miR2105 overexpression, OsbZIP86 downregulation, and OsbZIP86 knockout plants displayed opposite phenotypes. Collectively, our results show that the "miR2105-(OsSAPK10)-OsbZIP86-OsNCED3" module regulates the drought-induced ABA biosynthesis without penalty on rice growth under normal conditions, suggesting candidates for improving drought tolerance in rice.

An Integrative Transcriptomic and Metabolomic Analysis of Red Pitaya (Hylocereus polyrhizus) Seedlings in Response to Heat Stress.

Red pitaya (Hylocereus polyrhizus) is a significant functional food that is largely planted in Southeast Asia. Heat stress (HS) induced by high temperatures is likely to restrict the growth and survival of red pitaya. Although pitaya can tolerate temperatures as high as 40 °C, little is known of how it can withstand HS. In this study, the transcriptomic and metabolomic responses of red pitaya seedlings to HS were analyzed. A total of 198 transcripts (122 upregulated and 76 downregulated) were significantly differentially expressed after 24 h and 72 h of exposure to 42 °C compared with a control grown at 28 °C. We also identified 64 differentially accumulated metabolites in pitaya under HS (37 increased and 27 decreased). These differential metabolites, especially amino acids, organic acids, and sugars, are involved in metabolic pathways and the biosynthesis of amino acids. Interaction network analysis of the heat-responsive genes and metabolites suggested that similar pathways and complex response mechanisms are involved in the response of pitaya to HS. Overexpression of one of the upregulated genes (contig10820) in Arabidopsis, which is a homolog of PR-1 and named HuPR-1, significantly increased tolerance to HS. This is the first study showing that HuPR-1 plays a role in the response of pitaya to abiotic stress. These findings provide valuable insights that will aid future studies examining adaptation to HS in pitaya.

Development of paclitaxel loaded pegylated gelatin targeted nanoparticles for improved treatment efficacy in non-small cell lung cancer (NSCLC): an in vitro and in vivo evaluation study.

PURPOSE: To develop and evaluate paclitaxel (PTX) loaded pegylated gelatin targeted nanoparticles for improved efficacy in non-small cell lung cancer (NSCLC) treatment. METHOD: PTX loaded gelatin nanoparticles (PTX-GNP) were prepared by crosslinking with glutaraldehyde aqueous solution. These nanoparticles (NPs) were further incubated with PEG 400 to form PEGylated NPs (PEG-PTX-GNP). The NPs were evaluated for surface morphology, size, zeta potential, encapsulation efficiency, drug loading, in vitro drug release, cytotoxicity in an assay on cancer cell lines L132, in vitro cellular uptake in an assay in L132 and 293T cell lines, in vivo antitumor activity on female Balb/c mice, pulmonary deposition, histopathology, and immunohistochemical properties. RESULTS: The nanoparticles were of spherical shape with smooth surface characteristics. The observed DL was of 20.18 to 32.11%, as particle size was of 90 to 115 nm. Zeta potential and polydispersity index (PDI) were within acceptable ranges. Encapsulation was effective when the NPs had a size of 80.50 nm to 98.12 nm. The PEGylated PTX loaded nanoparticles (PEG-PTX-GNP, GNP4) showed similar PTX release profile to that of the NP4 formulation. PEGylated NPs showed the desired PTX release pattern that is required for cancer treatment. In an in vitro cytotoxicity study, PEG-PTX-GNP showed the maximum antiproliferative activity over the period of 24 hours, followed by PTX-GNP, pure PTX and BPEG-GNP. PEG-PTX-GNP showed the highest internalization within both cell lines, followed by PTX-GNP and pure PTX. The survival rate of animals in PEG-PTX-GNP group was 100%, proving the safety and efficacy of the treatment. PEG-PTX-GNP showed the highest antitumor activity as compared to other formulations. The pulmonary deposition rate was the highest (6.5 to 12.55 µg/g) in PEG-PTX-GNP formulations. Histopathology and immunohistochemical study proved that PEG-PTX-GNP had greater anticancer potential than other tested formulations. CONCLUSION: This study confirms the potential use of paclitaxel loaded PEGylated gelatin targeted nanoparticles for improved efficacy in non-small cell lung cancer (NSCLC) treatment.

CRISPR/Cas9-mediated mutation of OsSWEET14 in rice cv. Zhonghua11 confers resistance to Xanthomonas oryzae pv. oryzae without yield penalty.

BACKGROUND: Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease in Southeast Asia and West Africa. OsSWEET14, encoding a sugar transporter, is known to be a major susceptible gene of bacterial blight targeted by four different transcription activator-like (TAL) effectors from either Asian or African Xoo strains. However, the OsSWEET14 single knockout or promoter mutants in the Kitaake background are moderately resistant or even susceptible to African Xoo strains. Therefore, in this study, we knocked out OsSWEET14 in rice cv. Zhonghua 11 background for disease assessment. RESULTS: In this study, CRISPR/Cas9 was utilized to disrupt the function of OsSWEET14 by modifying its corresponding coding region in the genome of rice cv. Zhonghua 11 (CR-S14). In total, we obtained nine different OsSWEET14-mutant alleles. Besides conferring broad-spectrum resistance to Asian Xoo strains, tested mutant alleles also showed strong resistance to African Xoo strain AXO1947. Moreover, the expression of OsSWEET14 was detected in vascular tissues, including the stem, leaf sheath, leaf blade and root. The disruption of OsSWEET14 led to increased plant height without a reduction in yield. CONCLUSIONS: Disruption of OsSWEET14 in the Zhonghua 11 background is able to confer strong resistance to African Xoo strain AXO1947 and Asian Xoo strain PXO86. CR-S14 has normal reproductive growth and enhanced plant height under normal growth conditions. These results imply that CR-S14 may serve as a better tester line than sweet14 single-knockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance. The genetic background and increased plant height need to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding.

An AP2/ERF Gene, HuERF1, from Pitaya (Hylocereus undatus) Positively Regulates Salt Tolerance.

Pitaya (Hylocereus undatus) is a high salt-tolerant fruit, and ethylene response factors (ERFs) play important roles in transcription-regulating abiotic tolerance. To clarify the function of HuERF1 in the salt tolerance of pitaya, HuERF1 was heterogeneously expressed in Arabidopsis. HuERF1 had nuclear localization when HuERF1::GFP was expressed in Arabidopsis protoplasts and had transactivation activity when HuERF1 was expressed in yeast. The expression of HuERF1 in pitaya seedlings was significantly induced after exposure to ethylene and high salinity. Overexpression of HuERF1 in Arabidopsis conferred enhanced tolerance to salt stress, reduced the accumulation of superoxide (O2∙) and hydrogen peroxide (H2O2), and improved antioxidant enzyme activities. These results indicate that HuERF1 is involved in ethylene-mediated salt stress tolerance, which may contribute to the salt tolerance of pitaya.

A Rice Autophagy Gene OsATG8b Is Involved in Nitrogen Remobilization and Control of Grain Quality.

Enhancing nitrogen (N) use efficiency is a potential way to reduce excessive nitrogen application and increase yield. Autophagy is a conserved degradation system in the evolution of eukaryotic cells and plays an important role in plant development and stress response. Autophagic cores have two conjugation pathways that attach the product of autophagy-related gene 8 (ATG8) to phosphatidylethanolamine (PE) and ATG5 to ATG12, respectively, which then help with vesicle elongation and enclosure. Rice has six ATG8 genes, which have not been functionally confirmed so far. We identified the rice gene OsATG8b and characterized its role in N remobilization to affect grain quality by generating transgenic plants with its over-expression and knockdown. Our study confirmed the autophagy activity of OsATG8b through the complementation of the yeast autophagy-defective mutant scatg8 and by observation of autophagosome formation in rice. The autophagy activity is higher in OsATG8b-OE lines and lower in OsATG8b-RNAi than that in wild type (ZH11). 15N pulse-chase analysis revealed that OsATG8b-OE plants conferred higher N recycling efficiency to grains, while OsATG8b-RNAi transgenic plants exhibited lower N recycling efficiency and poorer grain quality. The autophagic role of OsATG8b was experimentally confirmed, and it was concluded that OsATG8b-mediated autophagy is involved in N recycling to grains and contributes to the grain quality, indicating that OsATG8b may be a potential gene for molecular breeding and cultivation of rice.

Obtusifoliol 14α-demethylase OsCYP51G1 is involved in phytosterol synthesis and affects pollen and seed development.

As structural components of biological membranes, phytosterols are essential not only for a variety of cellular functions but are also precursors for brassinosteroid (BR) biosynthesis. Plant CYP51 is the oldest and most conserved obtusifoliol 14α-demethylase in eukaryotes and is an essential component of the sterol biosynthesis pathway. However, little is known about rice (Oryza sativa L.) CYP51G1. In this study, we showed that rice OsCYP51G1 shared high homology with obtusifoliol 14α-demethylase and OsCYP51G1 was strongly expressed in most of rice organs. Subcellular localization analysis indicated that OsCYP51G1 was localized to the endoplasmic reticulum. Knockdown and knockout of OsCYP51G1 resulted in delayed flowering, impaired membrane integrity, abnormal pollen, and reduced grain yield, whereas OsCYP51G1 overexpression led to increased grain yield. Knockdown of OsCYP51G1 also reduced the levels of end-products (sitosterol and stigmasterol) and increased those of upstream intermediates (24-methylene-cycloartenol and cycloeucalenol) of the OsCYP51G1-mediated sterol biosynthesis step. In contrast, overexpression of OsCYP51G1 increased the sitosterol and stigmasterol content and reduced that of cycloeucalenol. However, knockdown of OsCYP51G1 by RNAi did not elicit these BR deficiency-related phenotypes, such as dwarfism, erect leaves and small seeds, nor was the leaf lamina angle sensitive to brassinolide treatment. These results revealed that rice OsCYP15G1 encodes an obtusifoliol 14α-demethylase for the phytosterols biosynthesis and possible without affecting the biosynthesis of downstream BRs, which was different from its homolog, OsCYP51G3.

Covalent Design of Cell Membrane Stationary Phase with Enhanced Stability for Fast Screening P-Glycoprotein Inhibitors.

Cell membrane chromatography (CMC) has been widely used for characterizing the interaction between drugs and membrane receptors to screen target components from herbal medicines. However, the column life, stability, and the efficiency cannot meet the needs of high-throughput screening purpose. In this study, a P-glycoprotein immobilized cell membrane stationary phase (P-gp/CMSP) was prepared with a simple and mild two-step aldehyde modification, realizing the covalent bonding between cell membrane and stationary phase. The column life and stability were significantly enhanced compared with the unmodified columns. The P-gp/CMC column was equipped into a comprehensive 2D P-gp/CMC/Capcell-C18/TOFMS system, which actualizes the automated and high-throughput analytical process and rapid identification of complex chemical samples with no data loss. Five compounds with significant retention were screened out and unambiguously identified by the comprehensive 2D analytical system. Baicalin was confirmed as a P-gp inhibitor with ATP depletion inhibition ratio of 83.4%. Moreover, the reversal index of baicalin on DOX significantly increased to 11.13 when its concentration reached 25 µM, revealing that baicalin could effectively reverse the MDR cell model induced by DOX. The integrated system is a practical drug discovery platform and could be applied to other transmembrane protein models.

Arabidopsis Histone Methyltransferase SUVH5 Is a Positive Regulator of Light-Mediated Seed Germination.

Plant lifecycle starts from seed germination, which is regulated by various environmental cues and endogenous hormones. Light promotes seed germination mainly by phytochrome B (PHYB) during the initial phase of imbibition, which involves genome-wide light-responsive transcription changes. Recent studies indicated an involvement of multiple epigenetic factors in the control of seed germination. However, few studies have been reported about the role of a histone methyltransferase in light-mediated seed germination process. Here, we identified SUVH5, a histone H3 lysine 9 methyltransferase, as a positive regulator in light-mediated seed germination in Arabidopsis. Loss of function of SUVH5 leads to decreased PHYB-dependent seed germination. RNA-sequencing analysis displayed that SUVH5 regulates 24.6% of light-responsive transcriptome in imbibed seeds, which mainly related to hormonal signaling pathways and developmental processes. Furthermore, SUVH5 represses the transcription of ABA biosynthesis and signal transduction-related genes, as well as a family of DELAY OF GERMINATION (DOG) genes via dimethylation of histone H3 at lysine 9 (H3K9me2) in imbibed seeds. Taken together, our findings revealed that SUVH5 is a novel positive regulator of light-mediated seed germination in Arabidopsis.

RNA-seq-based selection of reference genes for RT-qPCR analysis of pitaya.

Reverse-transcription quantitative real-time PCR (RT-qPCR) is a primary tool for measuring gene expression levels, and selection of appropriate reference genes is crucial for accurate and reproducible results of gene expression under various experimental conditions. However, no systematic evaluation of reference genes in pitaya (Hylocereus undatus Britt.) has been performed. Here, we examined the expression of five candidate reference genes, namely elongation factor 1-alpha (HuEF1-α), 18S ribosomal RNA (Hu18S rRNA), ubiquitin (HuUBQ), actin (HuACT), and ubiquitin-conjugating enzyme (HuUQT), under different conditions in pitaya. The expression stabilities of these five genes were evaluated using two computation programs: geNorm and NormFinder. The results were further validated by normalizing the expression of the phosphoglycerate kinase (HuPGK) and ethylene-responsive transcription factor (HuERF) genes. Our results indicate that combined use of HuUBQ and HuUQT is the most stable reference under all of the experimental conditions examined. HuEF1-α, HuUBQ, and HuUQT are the top three most stable reference genes under salt stress, drought stress, and heat stress, and across different cultivars. HuEF1-α, HuACT, and HuUQT exhibited the most stable expression patterns across different tissues. Our results will allow researchers to select the most appropriate reference genes for gene expression studies of pitaya under different conditions.