Genome-Wide Identification and Expression Profiling of Potato (Solanum tuberosum L.) Universal Stress Proteins Reveal Essential Roles in Mechanical Damage and Deoxynivalenol Stress.

Universal stress proteins (USPs) play an important regulatory role in responses to abiotic stress. Most of the research related to USPs so far has been conducted on plant models such as Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa L.), and cotton (Gossypium hirsutum L.). The potato (Solanum tuberosum L.) is one of the four major food crops in the world. The potato is susceptible to mechanical damage and infection by pathogenic fungi during transport and storage. Deoxynivalenol (DON) released by Fusarium can seriously degrade the quality of potatoes. As a result, it is of great significance to study the expression pattern of the potato StUSP gene family under abiotic stress conditions. In this study, a total of 108 USP genes were identified from the genome of the Atlantic potato, divided into four subgroups. Based on their genetic structure, the physical and chemical properties of their proteins and other aspects of their biological characteristics are comprehensively analyzed. Collinear analysis showed that the homologous genes of StUSPs and four other representative species (Solanum lycopersicum, Arabidopsis, Oryza sativa L., and Nicotiana attenuata) were highly conserved. The cis-regulatory elements of the StUSPs promoter are involved in plant hormones, environmental stress, mechanical damage, and light response. RNA-seq analysis showed that there are differences in the expression patterns of members of each subgroup under different abiotic stresses. A Weighted Gene Coexpression Network Analysis (WGCNA) of the central gene showed that the differential coexpression gene is mainly involved in the plant-pathogen response process, plant hormone signal transduction, and the biosynthesis process of secondary metabolites. Through qRT-PCR analysis, it was confirmed that StUSP13, StUSP14, StUSP15, and StUSP41 may be important candidate genes involved in the response to adversity stress in potatoes. The results of this study provide a basis for further research on the functional analysis of StUSPs in the response of potatoes to adversity stress.

Genome-Wide Identification of the NPR1-like Gene Family in Solanum tuberosum and Functional Characterization of StNPR1 in Resistance to Ralstonia solanacearum.

The NPR1 (nonexpressor of pathogenesis-related genes 1) gene is an activator of the systemic acquisition of resistance (SAR) in plants and is one of the central factors in their response to pathogenic bacterial infestation, playing an important role in plant disease resistance. Potato (Solanum tuberosum) is a crucial non-grain crop that has been extensively studied. However, the identification and analysis of the NPR1-like gene within potato have not been understood well. In this study, a total of six NPR1-like proteins were identified in potato, and phylogenetic analysis showed that the six NPR1-like proteins in Solanum tuberosum could be divided into three major groups with NPR1-related proteins from Arabidopsis thaliana and other plants. Analysis of the exon-intron patterns and protein domains of the six NPR1-like genes from potato showed that the exon-intron patterns and protein domains of the NPR1-like genes belonging to the same Arabidopsis thaliana subfamily were similar. By performing quantitative real-time PCR (qRT-PCR) analysis, we found that six NPR1-like proteins have different expression patterns in different potato tissues. In addition, the expression of three StNPR1 genes was significantly downregulated after being infected by Ralstonia solanacearum (RS), while the difference in the expression of StNPR2/3 was insignificant. We also established potato StNPR1 overexpression lines that showed a significantly increased resistance to R. solanacearum and elevated activities of chitinase, ß-1,3-glucanase, and phenylalanine deaminase. Increased peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities, as well as decreased hydrogen peroxide, regulated the dynamic balance of reactive oxygen species (ROS) in the StNPR1 overexpression lines. The transgenic plants activated the expression of the genes associated with the Salicylic acid (SA) defense response but suppressed the expression of the genes associated with Jasmonic acid (JA) signaling. This resulted in resistance to Ralstonia solanacearum.

Iva xanthiifolia leaf extract reduced the diversity of indigenous plant rhizosphere bacteria.

BACKGROUND: Iva xanthiifolia, native to North America, is now widely distributed in northeastern China and has become a vicious invasive plant. This article aims to probe the role of leaf extract in the invasion of I. xanthiifolia. METHODS: We collected the rhizosphere soil of Amaranthus tricolor and Setaria viridis in the invasive zone, the noninvasive zone and the noninvasive zone treated with extract from I. xanthiifolia leaf, and obtained I. xanthiifolia rhizosphere soil in the invasive zone. All wild plants were identified by Xu Yongqing. I. xanthiifolia (collection number: RQSB04100), A. tricolor (collection number: 831,030) and S. viridis (collection number: CF-0002-034) are all included in Chinese Virtual Herbarium ( https://www.cvh.ac.cn/index.php ). The soil bacterial diversity was analyzed based on the Illumina HiSeq sequencing platform. Subsequently, taxonomic analysis and Faprotax functional prediction were performed. RESULTS: The results showed that the leaf extract significantly reduced the diversity of indigenous plant rhizosphere bacteria. A. tricolor and S. viridis rhizobacterial phylum and genus abundances were significantly reduced under the influence of I. xanthiifolia or its leaf extract. The results of functional prediction showed that bacterial abundance changes induced by leaf extracts could potentially hinder nutrient cycling in native plants and increased bacterial abundance in the A. tricolor rhizosphere related to aromatic compound degradation. In addition, the greatest number of sensitive Operational Taxonomic Units (OTUs) appeared in the rhizosphere when S. viridis was in response to the invasion of I. xanthiifolia. It can be seen that A. tricolor and S. viridis have different mechanisms in response to the invasion of I. xanthiifolia. CONCLUSION: I. xanthiifolia leaves material has potential role in invasion by altering indigenous plant rhizosphere bacteria.

Combined Transcriptome and Metabolome Analysis Reveals Adaptive Defense Responses to DON Induction in Potato.

Phytophthora infestans poses a serious threat to potato production, storage, and processing. Understanding plant immunity triggered by fungal elicitors is important for the effective control of plant diseases. However, the role of the potato stress response to Fusarium toxin deoxynivalenol (DON)-induced stress is still not fully understood. In this study, the metabolites of DON-treated potato tubers were studied for four time intervals using UPLC-MS/MS. We identified 676 metabolites, and differential accumulation metabolite analysis showed that alkaloids, phenolic acids, and flavonoids were the major differential metabolites that directly determined defense response. Transcriptome data showed that differentially expressed genes (DEGs) were significantly enriched in phenylpropane and flavonoid metabolic pathways. Weighted gene co-expression network analysis (WGCNA) identified many hub genes, some of which modulate plant immune responses. This study is important for understanding the metabolic changes, transcriptional regulation, and physiological responses of active and signaling substances during DON induction, and it will help to design defense strategies against Phytophthora infestans in potato.

Mechanism and Prospect of Gastrodin in Osteoporosis, Bone Regeneration, and Osseointegration.

Gastrodin, a traditional Chinese medicine ingredient, is widely used to treat vascular and neurological diseases. However, recently, an increasing number of studies have shown that gastrodin has anti-osteoporosis effects, and its mechanisms of action include its antioxidant effect, anti-inflammatory effect, and anti-apoptotic effect. In addition, gastrodin has many unique advantages in promoting bone healing in tissue engineering, such as inducing high hydrophilicity in the material surface, its anti-inflammatory effect, and pro-vascular regeneration. Therefore, this paper summarized the effects and mechanisms of gastrodin on osteoporosis and bone regeneration in the current research. Here we propose an assumption that the use of gastrodin in the surface loading of oral implants may greatly promote the osseointegration of implants and increase the success rate of implants. In addition, we speculated on the potential mechanisms of gastrodin against osteoporosis, by affecting actin filament polymerization, renin-angiotensin system (RAS) and ferroptosis, and proposed that the potential combination of gastrodin with Mg2+, angiotensin type 2 receptor blockers or artemisinin may greatly inhibit osteoporosis. The purpose of this review is to provide a reference for more in-depth research and application of gastrodin in the treatment of osteoporosis and implant osseointegration in the future.

Co-Delivery of Dihydroartemisinin and Indocyanine Green by Metal-Organic Framework-Based Vehicles for Combination Treatment of Hepatic Carcinoma.

Dihydroartemisinin (DHA), a widely used antimalarial agent, has clinical potential for the treatment of hepatic carcinoma. Although chemotherapy is indispensable for tumor therapy, it is generally limited by poor solubility, low efficiency, rapid clearance, and side effects. As an emerging treatment method, photothermal therapy (PTT) has many outstanding properties, but suffers from poor photostability of photosensitizer and incomplete ablation. Multimodal therapies could combine the advantages of different therapy methods to improve antitumor efficiency. Hence, we designed a nano-delivery system (ICG&DHA@ZIF-8) using zeolitic imidazolate framework-8 (ZIF-8) with a high porous rate and pH sensitivity property, to co-load DHA and indocyanine green (ICG). Dynamic light scattering and transmission electron microscopy were used to characterize the prepared nanoparticles. The photothermal conversion and drug release performances of ICG&DHA@ZIF-8 were investigated. In vitro antitumor efficacy and cellular uptake were studied. The mechanism of the combination treatment was studied by reactive oxygen species level detection and western blot assays. In vivo antitumor assays were then studied with the guidance of ex vivo imaging. The results showed that the ICG&DHA@ZIF-8 based combination therapy could efficiently kill hepatic carcinoma cells and suppress tumor growth. This research provides a potential nanodrug for the treatment of hepatic carcinoma.

Selenium Deficiency Leads to Changes in Renal Fibrosis Marker Proteins and Wnt/ß-Catenin Signaling Pathway Components.

Renal fibrosis is the final result of the progression of chronic kidney disease (CKD) to end-stage renal disease (ESRD). Earlier studies confirmed that selenium (Se) displays a close association with kidney diseases. However, the correlation between Se and fibrosis has rarely been explored. Thus, this article mainly aimed to investigate the effect of Se deficiency on renal fibrosis and the Wnt/ß-catenin signaling pathway. Twenty BALB/c mice were fed a diet containing 0.02-mg/kg Se (Se-deficient diet) or 0.18-mg/kg Se (standard diet) for 20 weeks. A human glomerular mesangial cell (HMC) cell line was transfected with lentiviral TRNAU1AP-shRNA vector to establish a stable Se deficiency model in vitro. As indicated in this study, the glutathione (GSH) content in the Se-deficient group displayed an obvious decline compared with that in the control group, whereas the content of malondialdehyde (MDA) was obviously elevated. The results of Masson staining showed fibrosis around the renal tubules, and the results of immunohistochemistry showed that the area of positive fibronectin expression increased. In the Se-deficient group, the levels of collagen I, collagen III, matrix metalloproteinase 9 (MMP9), and other fibrosis-related proteins changed significantly in vivo and in vitro. Compared with the control group, the TRNAU1AP-shRNA group showed markedly reduced cell proliferation and migration abilities. Our data indicate that Se deficiency can cause kidney damage and renal fibrosis. Furthermore, the Wnt pathway is critical for the development of tissue and organ fibrosis. The data of this study demonstrated that the expression of Wnt5a, ß-catenin, and dishevelled 1 (Dvl-1) was significantly upregulated in the Se-deficient group. Therefore, the Wnt/ß-catenin pathway may play an important role in renal fibrosis caused by Se deficiency.

Selenium Deficiency-Induced Damage and Altered Expression of Mitochondrial Biogenesis Markers in the Kidneys of Mice.

Previous studies have raised concerns that kidney disease is often closely related to low serum Se levels in patients and that hyposelenemia may increase the vulnerability of patients to complications. However, few studies examining renal injury caused by Se deficiency have been conducted. To determine the effects of a selenium-deficient diet on renal function, a mouse model was fed a selenium-deficient diet (0.02 mg Se/kg) for 20 weeks. Meanwhile, mice in the control group (selenium-adequate) were fed a standard diet (0.18 mg Se/kg). The cellular models were established by lentiviral Trnau1ap-shRNA vectors transfected into mouse podocyte (MPC5) and mouse renal tubular epithelial (TCMK1) cell lines. Significant increases in serum creatinine levels and urinary protein/creatinine ratios were accompanied by increased MDA content in the Se-deficient group compared to the control group. The morphological observations of tissues showed widespread inflammation and ultrastructural changes in the Se-deficient group, such as swollen mitochondria and extensive podocyte fusion and renal tubular microvilli shedding. In addition, the expression of COXIV and cytochrome c was significantly downregulated in the Se-deficient group. Importantly, the mRNA levels of silent mating type information regulation 2 homolog 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and the protein levels of SIRT1 were increased in the Se-deficient group compared with the normal control group. Our data indicate that Se deficiency induces renal injury in mice. The elevated oxidative stress caused by Se deficiency may result in mitochondrial damage, which might affect renal function. Moreover, the SIRT1/PGC1α axis likely plays an important role in the compensatory mechanism of mitochondrial dysfunction.

The Associated Regulatory Mechanisms of Zinc Lactate in Redox Balance and Mitochondrial Function of Intestinal Porcine Epithelial Cells.

Zinc lactate (ZnLA) is a new organic zinc salt which has antioxidant properties in mammals and can improve intestinal function. This study explored the effects of ZnLA and ZnSO4 on cell proliferation, Zn transport, antioxidant capacity, mitochondrial function, and their underlying molecular mechanisms in intestinal porcine epithelial cells (IPEC-J2). The results showed that addition of ZnLA promoted cell proliferation, inhibited cell apoptosis and IL-6 secretion, and upregulated the mRNA expression and concentration of MT-2B, ZNT-1, and CRIP, as well as affected the gene expression and activity of oxidation or antioxidant enzymes (e.g., CuZnSOD, CAT, and Gpx1, GSH-PX, LDH, and MDA), compared to ZnSO4 or control. Compared with the control, ZnLA treatment had no significant effect on mitochondrial membrane potential, whereas it markedly increased the mitochondrial basal OCR, nonmitochondrial respiratory capacity, and mitochondrial proton leakage and reduced spare respiratory capacity and mitochondrial reactive oxygen (ROS) production in IPEC-J2 cells. Furthermore, ZnLA treatment increased the protein expression of Nrf2 and phosphorylated AMPK, but reduced Keap1 and p62 protein expression and autophagy-related genes LC3B-1 and Beclin mRNA abundance. Under H2O2-induced oxidative stress conditions, ZnLA supplementation markedly reduced cell apoptosis and mitochondrial ROS levels in IPEC-J2 cells. Moreover, ZnLA administration increased the protein expression of Nrf2 and decreased the protein expression of caspase-3, Keap1, and p62 in H2O2-induced IPEC-J2 cells. In addition, when the activity of AMPK was inhibited by Compound C, ZnLA supplementation did not increase the protein expression of nuclear Nrf2, but when Compound C was removed, the activities of AMPK and Nfr2 were both increased by ZnLA treatment. Our results indicated that ZnLA could improve the antioxidant capacity and mitochondrial function in IPEC-J2 cells by activating the AMPK-Nrf2-p62 pathway under normal or oxidative stress conditions. Our novel finding also suggested that ZnLA, as a new feed additive for piglets, has the potential to be an alternative for ZnSO4.

Ulva prolifera Extract Alleviates Intestinal Oxidative Stress via Nrf2 Signaling in Weaned Piglets Challenged With Hydrogen Peroxide.

Background: Ulva prolifera extract contains a variety of functional active substances. Whether these substances had any beneficial effects on the small intestine of weaned piglets under oxidative stress remained unknown. Method: We explored the effects of U. prolifera extract on oxidative stress and related mechanisms in weaned piglets and intestinal porcine epithelial cells (IPEC-J2) challenged with hydrogen peroxide. Results: U. prolifera extract was found to mainly consist of polyphenols and unsaturated fatty acids. U. prolifera extract increased total antioxidant capacity and superoxide dismutase (SOD) activity, while it decreased malondialdehyde content, in the serum of weaned piglets challenged with hydrogen peroxide. Moreover, U. prolifera extract increased mRNA expression of SOD and catalase, as well as the intestinal expression of nuclear NF-E2-related factor 2 (Nrf2), both in vitro and in vivo. Furthermore, U. prolifera extract decreased reactive oxygen species and improved mitochondrial respiration in IPEC-J2 cells treated with hydrogen peroxide. However, AMPK inhibition did not affect nuclear Nrf2 expression and only partially affected the effects of U. prolifera extract on oxidative stress. Conclusion: We suggest that U. prolifera extract alleviates oxidative stress via Nrf2 signaling, but independent of AMPK pathway in weaned piglets challenged with hydrogen peroxide. These results shed new insight into the potential applications of U. prolifera extract as a therapeutic agent for the prevention and treatment of oxidative stress-induced intestinal diseases.

Comparative epigenomics reveals evolution of duplicated genes in potato and tomato.

The evolution of duplicated genes after polyploidization has been the subject of many evolutionary biology studies. Potato (Solanum tuberosum) and tomato (Solanum lycopersicum) are the first two sequenced genomes of asterids, and share a common polyploidization event. However, the epigenetic role of DNA methylation on the evolution of duplicated genes derived from polyploidization is not fully understood. Here, we explore the role of the DNA methylation in the evolution of duplicated genes in potato and tomato. The overall levels of DNA methylation are different, although patterns of DNA methylation are similar in potato and tomato. Different types of duplicated genes can display different methylation patterns in potato and tomato. In addition, we found that differences in the methylation levels between duplicated genes were associated with gene expression divergence. In particular, for the majority of duplicated gene pairs, one copy is always hyper- or hypo-methylated compared with the other copy across different tomato fruit ripening stages, and these genes are enriched for specific function related to transcription factor (TF) activity. Furthermore, transcription of hundreds of duplicated TFs was shown to be regulated by DNA methylation during fruit ripening stages in tomato, some of which are well-known fruit ripening TFs. Taken together, our results support the notion that DNA methylation may facilitate divergent evolution of duplicated genes and play roles in important biological processes such as tomato fruit ripening.

MicroRNA-328 is involved in the effect of selenium on hydrogen peroxide-induced injury in H9c2 cells.

Oxidative stress induces apoptosis in cardiac cells, and antioxidants attenuate the injury. MicroRNAs (miRNAs) are also involved in cell death; therefore, this study aimed to investigate the role of miRNAs in the effect of selenium on oxidative stress-induced apoptosis. The effects of sodium selenite were analyzed via cell viability, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration. Flow cytometry was used to evaluate cell apoptosis. Fura-2AM was used to calculate intracellular Ca2+ concentration. Sodium selenite could ameliorate hydrogen peroxide (H2 O2 )-induced cell apoptosis and improve expression levels of glutathione peroxidase and thioredoxin reductase. Pretreatment with sodium selenite improved SOD activity and reduced MDA concentration. Treatments with H2 O2 or sodium selenite decreased miR-328 levels. MiR-328 overexpression enhanced cell apoptosis, reduced ATP2A2 levels, and increased intracellular Ca2+ concentration, while inhibition produced opposite effects. MiR-328 might be involved in the effect of sodium selenite on H2 O2 -induced cell death in H9c2 cells.

Application of alkyl polyglycoside surfactant in ultrasonic-assisted extraction followed by macroporous resin enrichment for the separation of vitexin-2″-O-rhamnoside and vitexin from Crataegus pinnatifida leaves.

An alkyl polyglycoside (APG) surfactant was used in ultrasonic-assisted extraction to effectively extract vitexin-2″-O-rhamnoside (VOR) and vitexin (VIT) from Crataegus pinnatifida leaves. APG0810 was selected as the surfactant. The extraction process was optimized for ultrasonic power, the APG concentration, ultrasonic time, soaking time, and liquid-solid ratio. The proposed approach showed good recovery (99.80-102.50% for VOR and 98.83-103.19% for VIT) and reproducibility (relative standard deviation, n=5; 3.7% for VOR and 4.2% for VIT) for both components. The proposed sample preparation method is both simple and effective. The use of APG for extraction of key herbal ingredients shows great potential. Ten widely used commercial macroporous resins were evaluated in a screening study to identify a suitable resin for the separation and purification of VOR and VIT. After comparing static and dynamic adsorption and desorption processes, HPD100B was selected as the most suitable resin. After column adsorption and desorption on this resin, the target compounds VOR and VIT can be effectively separated from the APG0810 extraction solution. Recoveries of VOR and VIT were 89.27%±0.42% and 85.29%±0.36%, respectively. The purity of VOR increased from 35.0% to 58.3% and the purity of VIT increased from 12.5% to 19.9%.

Meta-analysis: low-dose intake of vitamin E combined with other vitamins or minerals may decrease all-cause mortality.

It has been suggested that vitamin E alone or combined with other vitamins or minerals can prevent oxidative stress and slow oxidative injury-related diseases, such as cardiovascular disease and cancer. A comprehensive search of PubMed/MEDLINE, EMBASE and the Cochrane Library was performed. Relative risk was used as an effect measure to compare the intervention and control groups. A total of 33 trials were included in the meta-analysis. Neither vitamin E intake alone (RR=1.01; 95% CI, 0.97 to 1.04; p=0.77) nor vitamin E intake combined with other agents (RR=0.97; 95% CI, 0.89 to 1.06; p=0.55) was correlated with all-cause mortality. Subgroup analyses revealed that low-dose vitamin E supplementation combined with other agents is associated with a statistically significant reduction in all-cause mortality (RR=0.92; 95% CI, 0.86 to 0.98; p=0.01), and vitamin E intake combined with other agents is associated with a statistically significant reduction in mortality rates among individuals without probable or confirmed diseases (RR=0.92; 95% CI, 0.86 to 0.99; p=0.02). Neither vitamin E intake alone nor combined with other agents is associated with a reduction in all-cause mortality. But a low dose (<400 IU/d) of vitamin E combined with other agents is correlated with a reduction in all-cause mortality, and vitamin E intake combined with other agents is correlated with a reduction in the mortality rate among individuals without probable or confirmed diseases.

The cysteine protease CEP1, a key executor involved in tapetal programmed cell death, regulates pollen development in Arabidopsis.

Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation.

Dishevelled-1 (Dvl-1) protein: a potential participant of oxidative stress induced by selenium deficiency.

Oxidative stress induced by selenium deficiency has been shown to be associated with cardiovascular diseases. Nevertheless, the mechanism associated with oxidative stress induced by selenium deficiency is poorly understood. In the present study, 36 weaning C57BL/6 mice were randomly divided into 4 groups as follows: control (n =9), 4-week selenium deficiency (n =9), 8-week selenium deficiency (n = 9), and 12-week selenium deficiency (n =9). The levels of myocardial glutathione peroxidase (GPx), superoxide dismutase (SOD), and malondialdehyde (MDA) were determined by Western blotting or commercial kits. Real-time PCR was performed to detect the mRNA expression of dishevelled-1 (Dvl-1) protein. Western blotting was conducted to evaluate the protein expression levels of Dvl-1 and ß-catenin. Our results demonstrated that the levels of GPx and SOD were significantly reduced, along with an increase in MDA in selenium-deficient mice. Importantly, Dvl-1 and ß-catenin were clearly upregulated under oxidative stress. Collectively, our findings indicate that Dvl-1 may be an underlying participant of oxidative stress induced by selenium deficiency.

Two novel diacylglycerol acyltransferase genes from Xanthoceras sorbifolia are responsible for its seed oil content.

Xanthoceras sorbifolia is an excellent model system for studying triacylglycerol (TAG) biosynthesis in woody oilseed plants due to the high amount of seed oil, which is important for food and industrial uses. TAG is the major form of stored lipids in seeds and diacylglycerol acyltransferase (DGAT; EC 2. 3. 1. 20) catalyzes the final and critical step of TAG synthesis. Here, two novel DGAT genes, designated XsDGAT1 and XsDGAT2, were cloned from developing X. sorbifolia embryos. Sequence analysis showed that XsDGAT1 had little sequence homology to XsDGAT2. Heterologous expression of XsDGAT1 and XsDGAT2 in TAG-deficient yeast mutants restored TAG synthesis, confirming their biological activity. Expression of the two genes in wild-type Arabidopsis led to TAG synthesis and an increase in total seed oil in transgenic plants, with XsDGAT1 appearing to contribute to TAG synthesis at a greater level. Comparison of the expression patterns revealed that both XsDGAT1 and XsDGAT2 were expressed in the examined tissues and had similar spatiotemporal expression patterns with higher expression in embryos than in leaves and petals. The expression patterns of both XsDGAT1 and XsDGAT2 correlated with oil accumulation in developing X. sorbifolia embryos. These data suggest that XsDGAT1 and XsDGAT2 are both responsible for TAG synthesis in X. sorbifolia seeds.

Exon skipping of AGAMOUS homolog PrseAG in developing double flowers of Prunus lannesiana (Rosaceae).

KEY MESSAGE : Two transcript isoforms of AGAMOUS homologs, from single and double flower Prunus lannesiana, respectively, showed different functions. The Arabidopsis floral homeotic C function gene AGAMOUS (AG) confers stamen and carpel identity. Loss of AG function results in homeotic conversions of stamens into petals and formation of double flowers. In order to present a molecular dissection of a double-flower cultivar in Prunus lannesiana (Rosaceae), we isolated and identified a single-copy gene, AG homolog from two genetically cognate P. lannesiana bearing single and double flowers, respectively. Sequence analysis revealed that the AG homolog, prseag-1, from double flowers showed a 170-bp exon skipping as compared to PrseAG (Prunus serrulata AGAMOUS) from the single flowers. Genomic DNA sequence revealed that abnormal splicing resulted in mutant prseag-1 protein with the C-terminal AG motifs I and II deletions. In addition, protein sequence alignment and phylogenetic analyses revealed that the PrseAG was grouped into the euAG lineage. A semi-quantitative PCR analysis showed that the expression of PrseAG was restricted to reproductive organs of stamens and carpels in single flowers of P. lannesiana 'speciosa', while the prseag-1 mRNA was highly transcribed throughout the petals, stamens, and carpels in double flowers from 'Albo-rosea'. The transgenic Arabidopsis containing 35S::PrseAG displayed extremely early flowering, bigger stamens and carpels and homeotic conversion of petals into staminoid organs, but ectopic expression of prseag-1 could not mimic the phenotypic ectopic expression of PrseAG in Arabidopsis. In general, this study provides evidences to show that double flower 'Albo-rosea' is a putative C functional ag mutant in P. lannesiana.

In vitro adventitious shoot regeneration via indirect organogenesis from inflorescence explants and peroxidase involvement in morphogenesis of Populus euphratica Olivier.

The inflorescences as explants for rapid propagation in vitro remained unknown in Populus euphratica Olivier. Here, we reported that multiple shoots were initiation from calli of both male and female inflorescences. The optimum medium for shoot induction from male inflorescences was lactose sulfite medium containing 1.0 mg L(-1) 6-benzylaminopurine (BA) and 0.5 mg L(-1) α-naphthalene acetic acid (NAA) or Murashige and Skoog (MS) medium containing 0.5 mg L(-1) BA and 0.2 mg L(-1) NAA. The optimum medium of shoot induction from female inflorescence calli was the MS medium containing 0.5 mg L(-1) BA and 0.2 mg L(-1) NAA. Rooting of regenerated shoots was obtained on 1/2 MS medium supplemented with 0.5∼1.0 mg L(-1) indole-3-butyric acid (IBA) and the highest frequency rooting was on medium containing 0.5 mg L(-1) IBA. No shoots were obtained on medium without BA and NAA. Peroxidase (POD) activity was measured by polyacrylamide gel electrophoresis during shoot induction and differentiation stages. The results showed that two bands of POD (2a and 2b) activity appeared lowest during the early 8 days at the dedifferentiation phase of leaves inducing calli, whereas POD 2a, 2b activity appeared to be increasing at the homeochronous dedifferentiation phase of inflorescence. Five most intensive bands, POD 1a, 1b, 1c, 2a, and ab, appeared in 8th and 28th days at the redifferentiation phase during shoot morphogenesis. These results demonstrated that the POD was involved in shoot morphogenesis from both leaf and inflorescence explants of Populus euphratica.

NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development.

Proteinases play a critical role in developmental homeostasis and in response to environ-mental stimuli. Our present research reports that a new cysteine protease, NtCP56, is involved in the development of pollen grains in Nicotiana tabacum L. The NtCP56 gene, which encodes a protein of 361 amino acid residues with a calculated molecular mass of 40 kDa, is strongly expressed in anthers. The recombinant NtCP56 showed a high activity towards casein. Kinetic analysis revealed a K(m) of 2.20 mg ml(-1) and V(max) of 11.07 microg ml(-1) min(-1). The recombinant NtCP56 retained more than 50% of its maximum enzymatic activity from 20 degrees C to 60 degrees C with an optimum Tm range of 30-50 degrees C. The enzyme had a maximum activity at approximately pH 6.5. Suppression of the NtCP56 gene in anti-sense transgenic tobaccos resulted in the sterility of pollen grains. Our data indicated that, as a cysteine protease, NtCP56 might play an important role in pollen development.