Identification of genes related to low-grade glioma progression and prognosis based on integrated transcriptome analysis.

Glioma is one of the most common types of human brain tumor, with high mortality in high-grade gliomas (HGG). Low-grade gliomas (LGG) can progress into HGG, leading to poor prognosis. However, it is unclear what factors affect the progression of LGG to HGG. This study aims to explore the function of the crosstalk genes on the progression and prognosis of LGG using bioinformatics analysis. Integrated transcriptome analysis was used to screen differentially expressed genes (DEGs). Then, gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to investigate the association between DEGs and gene functions and pathways by ClusterProfiler package and ClueGO plug-in. Protein-protein interaction (PPI) network analysis was applied to explore the connection between genes and biological processes. Subsequently, the gene clusters were analyzed using the Centiscape and molecular complex detection (MCODE) plug-in in Cytoscape software, where the crosstalk genes were identified for further study. Ultimately, the UALCAN website and Gene Expression Profiling Interactive Analysis (GEPIA) website were performed to visualize the expression levels and survival curves of genes, respectively. There were 74 DEGs identified in glioma, including 55 upregulated genes and 19 downregulated genes, which mainly were enriched in extracellular matrix (ECM)-receptor interaction, focal adhesion, PI3K-Akt signaling pathway, and so on. Then, six crosstalk genes were selected, including COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, and COL5A2 genes. Overall survival (OS) analysis of crosstalk genes was conducted on the GEPIA website. High expression levels of crosstalk genes were closely related to the low survival rate of patients with LGG. The overexpressed crosstalk genes, such as COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, and COL5A2 may participate in the progression and poor prognosis of LGG through the ECM-receptor interaction pathway.

Peptide targeting improves the delivery and therapeutic index of glucocorticoids to treat rheumatoid arthritis.

Rheumatoid arthritis (RA) is a prevalent autoimmune disease characterized by excessive inflammation in the joints. Glucocorticoid drugs are used clinically to manage RA symptoms, while their dosage and duration need to be tightly controlled due to severe adverse effects. Using dexamethasone (DEX) as a model drug, we explored here whether peptide-guided delivery could increase the safety and therapeutic index of glucocorticoids for RA treatment. Using multiple murine RA models such as collagen-induced arthritis (CIA), we found that CRV, a macrophage-targeting peptide, can selectively home to the inflammatory synovium of RA joints upon intravenous injection. The expression of the CRV receptor, retinoid X receptor beta (RXRB), was also elevated in the inflammatory synovium, likely being the basis of CRV targeting. CRV-conjugated DEX increased the accumulation of DEX in the inflamed synovium but not in healthy organs of CIA mice. Therefore, CRV-DEX demonstrated a stronger efficacy to suppress synovial inflammation and alleviate cartilage/bone destruction. Meanwhile, CRV conjugation reduced immune-related adverse effects of DEX even after a long-term use. Last, we found that RXRB expression was significantly elevated in human patient samples, demonstrating the potential of clinical translation. Taken together, we provide a novel, peptide-targeted strategy to improve the therapeutic efficacy and safety of glucocorticoids for RA treatment.

Free amino acids identification and process optimization in greengage wine fermentation and flavor formation.

Greengage wine with low alcohol content is increasing in popularity owing to its fruity taste and rich nutrition. The key to wine aroma and taste is flavor substances like free amino acids (FAAs), volatile fatty acids, higher alcohols, and esters. Amino acid (AA) metabolisms in yeast are an important source of these secondary compounds, which vary with the fermentation conditions. This study explored and optimized the impact of different parameters (carbon source, inoculum, pH, temperature) on FAA contents and dynamics in greengage wine. The results demonstrated that total and essential amino acid (EAA) content rose with a higher proportion of glucose, less yeast inoculation, higher temperature, and higher initial pH. With the results obtained it was concluded that the condition of 22.4°C, pH 4.5, and 3% inoculation was optimum for a 14.9-fold increase of EAAs in fermented greengage wine. In the long run, the research will aid in the development of the greengage brewing industry.

Ethanol promoting the upregulation of C-X-C Motif Chemokine Ligand 1（CXCL1） and C-X-C Motif Chemokine Ligand 6（CXCL6） in models of early alcoholic liver disease.

Alcoholic liver disease (ALD) denotes a series of liver diseases caused by ethanol. Recently, immune-related genes (IRGs) play increasingly crucial role in diseases. However, it's unclear the role of IRGs in ALD. Bioinformatic analysis was used to discern the core immune-related differential genes (IRDGs) in the present study. Subsequently, Cell Counting Kit-8 say, oil red O staining, and triglyceride detection were employed to explore optimal experimental conditions of establishing hepatocellular models of early ALD. Ultimately, real-time reverse transcription-PCR and immunohistochemistry/immunocytochemistry methods were adopted to verify the expressions of mRNA and proteins of core IRDGs, respectively. C-X-C Motif Chemokine Ligand 1 (Cxcl1) and Cxcl6 were regarded as core IRDGs via integrated bioinformatics analysis. Besides, Lieber Decarli Ethanol feeding and 200 mM and 300 mM ethanol stimulating L02 cells for 36 h can both successfully hepatocellular model. In ethanol groups, the levels of CXCL1 and CXCL6 mRNA were significantly upregulated than pair-fed groups (P < 0.0001). Also, immunohistochemistry revealed that positive particles of CXCL1 and CXCL6 in mice model of early ALD were obviously more than control groups (P < 0.0001). Besides, in L02 hepatocytes stimulated by ethanol, CXCL1 and CXCL6 mRNA were over-expressed, compared with normal L02 cells (P < 0.0001). Meanwhile, immunocytochemistry indicated that CXCL1 and CXCL6 proteins in hepatocellular model of early ALD were higher than normal L02 hepatocytes stimulus (P < 0.0001). Ethanol promoted the upregulation of Cxcl1 and Cxcl6 mRNA and proteins in models of early ALD, denoting their potentiality of acting as biomarkers of ALD.

Integrated Analyses Identify Key Molecules and Reveal the Potential Mechanism of miR-182-5p/FOXO1 Axis in Alcoholic Liver Disease.

Background: Alcoholic liver disease (ALD) is one of the most common chronic liver diseases worldwide. However, the potential molecular mechanism in ALD development remains unclear. The objective of this work was to identify key molecules and demonstrate the underlying regulatory mechanisms. Methods: RNA-seq datasets were obtained from Gene Expression Omnibus (GEO), and key molecules in ALD development were identified with bioinformatics analysis. Alcoholic liver disease mouse and cell models were constructed using Lieber-DeCarli diets and alcohol medium, respectively. Quantitative real-time PCR and Western blotting were conducted to confirm the differential expression level. Dual-luciferase reporter assays were performed to explore the targeting regulatory relationship. Overexpression and knockdown experiments were applied to reveal the potential molecular mechanism in ALD development. Results: Between ALD patients and healthy controls, a total of 416 genes and 21 microRNAs (miRNAs) with significantly differential expression were screened. A comprehensive miRNA-mRNA network was established; within this network, the miR-182-5p/FOXO1 axis was considered a significant pathway in ALD lipid metabolism. Mouse and cell experiments validated that miR-182-5p was substantially higher in ALD than in normal livers, whereas the expression of FOXO1 was dramatically decreased by alcohol consumption (P < 0.05). Next, dual-luciferase reporter assays demonstrated that miR-182-5p directly targets the binding site of the FOXO1 3'UTR and inhibits its mRNA and protein expression. In addition, miR-182-5p was found to promote hepatic lipid accumulation via targeting the FOXO1 signaling pathway, and inhibition of the miR-182-5p/FOXO1 axis improved hepatic triglyceride (TG) deposition in ALD by regulating downstream genes involved in lipid metabolism. Conclusion: In summary, key molecules were identified in ALD development and a comprehensive miRNA-mRNA network was established. Meanwhile, our results suggested that miR-182-5p significantly increases lipid accumulation in ALD by targeting FOXO1, thereby providing novel scientific insights and potential therapeutic targets for ALD.

The value of microRNAs as the novel biomarkers for colorectal cancer diagnosis: A meta-analysis.

BACKGROUND: Numerous studies have reported that microRNAs (miRNAs) hold great potential as the biomarkers for colorectal cancer (CRC). However, inconsistent results have made it challenging to evaluate their diagnostic performance. Thus, the aim of this meta-analysis was to systematically assess the pooled efficacy of miRNAs for CRC diagnosis. METHODS: A search for eligible studies up to October 30, 2019 was conducted using PubMed, Web of Science and EMBASE databases. A random-effects model was used to evaluate the pooled sensitivity and specificity. The summary receiver operator characteristic (SROC) curve and area under the curve (AUC) were calculated to assess the overall diagnostic efficacy. RESULTS: A total of 3258 CRC patients and 2683 healthy controls were identified in 35 included studies. The overall diagnostic accuracy was as follows: sensitivity, 0.80 [95 % confidence interval (CI) 0.75-0.83]; specificity, 0.80 (95 % CI, 0.75-0.84); positive likelihood ratio (PLR), 4.0 (95 % CI, 3.2-5.0); negative likelihood ratio (NLR), 0.26 (95 % CI, 0.21-0.31); diagnostic odds ratio (DOR), 16 (95 % CI, 11-23); and AUC, 0.87 (95 % CI, 0.83-0.89). CONCLUSION: The results indicated that miRNAs, particularly serum-derived miRNAs, can serve as the powerful and promising biomarkers for early CRC screening.

A Nomogram for the Prediction of Progression and Overall Survival in Childhood Acute Lymphoblastic Leukemia.

Background: Advances in treatment and supportive care have significantly improved the overall survival (OS) of pediatric patients with acute lymphoblastic leukemia (ALL). However, there is a large number of these patients who continue to relapse after receiving standard treatment. Accurate identification of patients at high risk of relapse and targeted therapy may significantly improve their prognosis. Therefore, the aim of this study was to identify significant prognostic factors for pediatric ALL and establish a novel nomogram for the prediction of survival. Methods: The ALL clinical data of Phases I and II of the Therapeutic Applicable Research to Generate Effective Treatments (TARGET) project were merged and randomly divided into training and validation groups. The LASSO regression model was used to select the specific factors related to the OS of the training group and generate prognostic nomograms according to the selected characteristics. The predictive accuracy of the nomogram for OS was verified using the concordance index of the training and validation groups, the area under the receiver operating characteristic curve for prognostic diagnosis, and the calibration curve. Results: A total of 1,000 children with ALL were included in the TARGET project. Of those, 489 patients had complete follow-up data for further analysis. The data were randomly divided into the training group (n = 345) and the validation group (n = 144). Seven clinical characteristics, namely age at diagnosis, peripheral white blood cells, bone marrow and CNS site of relapse, ETV6/RUNX1 fusion, TCF3/PBX1, and BCR/ABL1 status, were selected to construct the nomogram. The concordance indices of the training and validation groups were 0.809 (95% confidence interval: 0.766-0.852) and 0.826 (95% confidence interval: 0.767-0.885), respectively. The areas under the receiver operating characteristic curve of the 3-year, 5-year, and 10-year OS in the training group were 0.804, 0.848, and 0.885, respectively, while that of the validation group were 0.777, 0.825, and 0.863, respectively. Moreover, the calibration curves demonstrated a favorable consistency between the predicted and actual survival probabilities. Conclusions: Independent predictors of OS in children with ALL included age at diagnosis, white blood cells, bone marrow site of relapse, CNS site of relapse, ETV6/RUNX1 fusion, TCF3/PBX1, and BCR/ABL1 status. The nomograms developed using these high-risk factors can more simply, accurately, and quantitatively predict the survival of children, and improve treatment and prognosis.

Identification of Circulating MicroRNAs as a Promising Diagnostic Biomarker for Cervical Intraepithelial Neoplasia and Early Cancer: A Meta-Analysis.

BACKGROUND: Cervical cancer (CC) is one of the most common female malignant tumors. And cervical intraepithelial neoplasia (CIN) is the precancerous lesion of CC, which can progress to invasive CC. MicroRNAs (miRNAs) have been found to be potential diagnostic biomarkers for CIN or CC. However, recently, the lack of sufficient studies about the diagnostic value of miRNAs for CIN made it challenging to separately investigate the diagnostic efficacy of miRNAs for CIN. Likewise, the conclusions among those studies were discordant. Therefore, we conducted this meta-analysis, aimed at evaluating the diagnostic efficacy of miRNAs for CIN and CC patients. METHODS: Literature search was performed in PubMed, Embase, and Web of Science databases. Pooled sensitivity, specificity, and other diagnostic parameters were calculated through Stata 14.0 software. Furthermore, subgroup analyses and metaregression analysis were conducted to explore the main sources of heterogeneity. RESULTS: Ten articles covering 50 studies were eligible, which included 5,908 patients and 4,819 healthy individuals. The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under the curve (AUC) were 0.81 (95% CI, 0.77-0.85), 0.86 (95% CI, 0.83-0.89), 5.9 (95% CI, 4.5-7.7), 0.22 (95% CI, 0.17-0.28), 27 (95% CI, 17-44), and 0.91 (95% CI, 0.88-0.93), respectively. Additionally, the ethnicity and internal reference were the main sources of heterogeneity. CONCLUSIONS: Circulating miRNAs can be a promising noninvasive diagnostic biomarker for CIN and early CC, especially miR-9 and miR-205, which need to be verified by large-scale studies.

The Diagnostic Value of MicroRNAs as a Biomarker for Hepatocellular Carcinoma: A Meta-Analysis.

BACKGROUND: Recently, the role of microRNAs (miRNAs) in diagnosing cancer has been attracted increasing attention. However, few miRNAs have been applied in clinical practice. The purpose of this study was to evaluate the diagnostic efficacy of miRNAs for hepatocellular carcinoma (HCC) at early stages clinically. METHODS: A literature search was carried out using PubMed, Web of Science, and EMBASE databases. We explored the diagnostic value of miRNAs in distinguishing HCC from healthy individuals. The quality assessment was performed in Review Manager 5.3 software. The overall sensitivity and specificity and 95% confidence intervals (CIs) were obtained with random-effects models through Stata 14.0 software. And heterogeneity was assessed using Q test and I 2 statistics. Meta-regression and subgroup analyses were conducted based on the sample, nation, quality of studies, and miRNA profiling. The publication bias was evaluated through Deeks' funnel plot. RESULTS: A total of 34 studies, involving in 2747 HCC patients and 2053 healthy individuals, met the inclusion criteria in the 33 included literature studies. In the summary receiver operating characteristic (sROC) curve, AUC was 0.92 (95% CI, 0.90-0.94), with 0.84 (95% CI, 0.79-0.88) sensitivity and 0.87 (95% CI, 0.83-0.90) specificity. There was no publication bias (P=0.48). CONCLUSION: miRNAs in vivo can be acted as a potential diagnostic biomarker for HCC, which can facilitate the early diagnosis of HCC in clinical practice.

Measuring specific interaction of transcription factor ZmDREB1A with its DNA responsive element at the molecular level.

Specific interactions between transcription factors and DNA responsive elements are of fundamental importance in understanding how genetic regulatory proteins control gene transcription. Here we have developed a new method of using atomic force microscopy (AFM) to quantitatively study the single molecular specific interaction between ZmDREB1A, a transcription factor from maize, and its DNA responsive element, dehydration-responsive element (DRE) with core sequence A/GCCGAC. It was found that ZmDREB1A bound to both DRE ACCGAC and GCCGAC efficiently. The single molecular interaction forces of ZmDREB1A with DRE A/GCCGAC were determined to be 101 +/- 5 and 108 +/- 3 pN, respectively. The point mutation of ZmDREB1A in its DNA-binding domain or single base substitution of the DRE core sequence greatly reduced the binding affinity, demonstrating the high sensitivity of the AFM measurements. AFM is expected to be a simple, quick, sensitive and reliable method that offers valuable information for the characterization of transcription factors and the identification of their potential DNA responsive elements in functional genomics research.

Expression of p21(WAF1) and p53 and polymorphism of p21(WAF1) gene in gastric carcinoma.

AIM: To investigate the relationship between expression of p21(WAF1) and p53 gene, and to evaluate the deletion and polymorphism of p21(WAF1) gene in gastric carcinoma (GC). METHODS: Expression of p21 and p53 proteins, and deletion and polymorphism of p21 gene in GC were examined by streptavidin-peroxidase conjugated method (SP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) respectively. RESULTS: The expression of p21 and p53 was found in 100% (20/20) and 0% (0/20) of normal gastric mucosae(NGM), 92.5% (37/40) and 15.0% (6/40) of dysplasia (DP) and 39.8% (43/108) and 56.5% (61/108) of GC, respectively. The positive rate of p21 in GC was lower than that in NGM and DP (P<0.05), while the positive rate of p53 in GC was higher than that in NGM and DP (P<0.05). p21 and p53 were significantly expressed in 63.3% (19/30) and 36.7% (11/30), 35.0% (14/40) and 77.5% (31/40), 26.7% (4/15) and 80.0% (12/15), 30.8% (4/13) and 30.8% (4/13), and 20.0% (2/10) and 30.0% (3/10) of well-differentiated, poorly-differentiated, undifferentiated carcinomas, mucoid carcinomas and signet ring cell carcinomas. The expression of p21 in well-differentiated carcinomas was significantly higher than that in poorly-differentiated, un-differentiated, mucoid carcinomas and signet ring cell carcinomas (P<0.05). Contrarily, The expression of p53 was increased from well-differentiated to poorly-differentiated and un-differentiated carcinomas (P<0.05). The expression of p21 and p53 in paired primary and metastatic GC (35.3% and 70.6%) was different from non-metastatic GC (62.5% and 42.5%) markedly (P<0.05). The expression of p21 in invasive superficial muscle (60.0%) was higher than that in invasive deep muscle or total layer (35.2%) (P<0.05) and was higher in TNM stages I (60.0%) and II (56.2%) than in stages III (27.9%) and IV (22.2%) (P<0.05), whereas the expression of p53 did not correlate to invasion depth or TNM staging (P>0.05). The exoression patterns of p53+/p21-, and of p53-/p21+ were found in 5.0% and 82.5% of DP. There was a significant correlation between expression of p21 and p53 (P<0.05). But there was no significant correlation between expression of both in GC (P>0.05). There was no deletion in exon 2 of p21 gene in 30 cases of GC and 45 cases of non-GC, but polymorphism of p21 gene at exon 2 was found in 26.7% (8/30) of GC and 8.9% (4/45) of non-GC, a significant difference was found between GC and non-GC (P<0.05). There was no significant relation between p21 expression of polymorphism (37.5%, 3/8) and non-polymorphism (45.5%, 10/22) in GC (P>0.05). CONCLUSION: The loss of p21 protein and abnormal expression of p53 are related to carcinogenesis, differentiation and metastasis of GC. The expression of p21 is related to invasion and clinical staging in GC intimately. The expression of p21 protein depends on p53 protein largely in NGM and DP, but not in GC. No deletion of p21 gene in exon 2 can be found in GC. The polymorphism of p21 gene might be involved in gastric carcinogenesis.There is no significant association between polymorphism of p21 gene and expression of p21 protein.

Boron influences pollen germination and pollen tube growth in Picea meyeri.

To study the role of boron in pollen germination and pollen tube growth of Picea meyeri Rehd. et Wils., pollen grains were cultured in standard medium or boron-deficient medium. Effects of boron on the localization of pectins and callose in the walls of pollen tubes were observed by laser scanning confocal microscopy after staining with aniline blue or immunolabeling with antibodies JIM5 and JIM7. Changes in the structures of pectins and phenolics were investigated by fourier transform infrared (FTIR) microspectroscopy. Pollen germination in boron-deficient medium ranged from 18 to 24%, whereas pollen germination in standard medium reached 61%. Callose accumulated in the tip-regions of pollen tubes cultured in boron-deficient medium, but not in standard medium. Immunolabeling with antibody JIM5 revealed that acidic pectin preferentially accumulated in the tip regions of pollen tubes cultured in boron-deficient medium, whereas acidic pectin was weakly distributed along the entire lengths of pollen tubes cultured in standard medium. Esterified pectin, detected by immunolabeling with antibody JIM7, showed a similar distribution pattern in pollen tubes in both the boron-deficient and standard treatments. The FTIR spectra indicated slight increases in contents of phenolics and carboxylic acids and a substantial decrease in the content of saturated esters in boron-deficient pollen tubes compared with normal pollen tubes. The FTIR spectra confirmed that boron deficiency enhanced acidic pectin accumulation in pollen tubes, which may be associated with the increased content of carboxylic acid. We conclude that boron has a regulatory role in pollen germination and pollen tube growth.

TINY, a dehydration-responsive element (DRE)-binding protein-like transcription factor connecting the DRE- and ethylene-responsive element-mediated signaling pathways in Arabidopsis.

Dehydration-responsive element-binding proteins (DREBs) and ethylene-responsive element (ERE) binding factors are two major subfamilies of the AP2/ethylene-responsive element-binding protein family and play crucial roles in the regulation of abiotic- and biotic-stress responses, respectively. In the present work, we have reported a previously identified DREB-like factor, TINY, that was involved in both abiotic- and biotic-stress signaling pathways. TINY was capable of binding to both DRE and ERE with similar affinity and could activate the expression of reporter genes driven by either of these two elements in tobacco cells. The 15th amino acid in the APETALA2 (AP2)/ethylene-responsive element-binding factor domain was demonstrated to be essential for its specific binding to ERE, whereas the 14th and 19th amino acids were responsible for the binding to DRE. The expression of TINY was greatly activated by drought, cold, ethylene, and slightly by methyl jasmonate. Additionally, overexpression of TINY in Arabidopsis resulted in elevated expressions of both the DRE- and the ERE-containing genes. Moreover, the expression of DRE-regulated genes, such as COR6.6 and ERD10, was up-regulated upon ethylene treatment, and the expression of ERE-regulated genes, such as HLS1, was also increased by cold stress, when the expression of TINY was being induced. These results strongly suggested that TINY might play a role in the cross-talk between abiotic- and biotic-stress-responsive gene expressions by connecting the DRE- and ERE-mediated signaling pathways. The results herein might promote the understanding of the mechanisms of specific DNA recognition and gene expression regulation by DREBs.

An ankyrin repeat-containing protein, characterized as a ubiquitin ligase, is closely associated with membrane-enclosed organelles and required for pollen germination and pollen tube growth in lily.

Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth.

Inhibition of RNA and protein synthesis in pollen tube development of Pinus bungeana by actinomycin D and cycloheximide.

* The effects of actinomycin D and cycloheximide on RNA and protein synthesis were investigated during pollen tube development of Pinus bungeana. * RNA and protein contents, protein expression patterns, cell wall components and ultrastructural changes of pollen tubes were studied using spectrophotometry, SDS-PAGE electrophoresis, Fourier transformed infrared (FTIR) microspectroscopy and transmission electron microscopy (TEM). * Pollen grains germinated in the presence of actinomycin D, but tube elongation and RNA synthesis were inhibited. By contrast, cycloheximide inhibited pollen germination and protein synthesis, induced abnormal tube morphology, and retarded the tube growth rate. SDS-PAGE analysis showed that protein expression patterns changed distinctly, with some proteins being specific for each phase. FTIR microspectroscopy established significant changes in the chemical composition of pollen tube walls. TEM analysis revealed the inhibitors caused disintegration of organelles involved in the secretory system. * These results suggested RNA necessary for pollen germination and early tube growth were present already in the pollen grains before germination, while the initiation of germination and the maintenance of pollen tube elongation depended on continuous protein synthesis.

Molecular cloning of two exo-beta-glucanases and their in vivo substrates in the cell walls of lily pollen tubes.

Full-length cDNA sequences of two exo-beta-glucanases, LP-ExoI and LP-ExoII, secreted into cell walls of lily (Lilium longiflorum) pollen tube, were determined by RT-PCR. LP-ExoI exhibited over 80% similarity to LP-ExoII at both DNA and amino acid levels. RT-PCR showed that LP-ExoI transcripts were abundant in pollen grains and tubes, but could not be detected in leaf, stem, stigma, style, ovary, petal, filament, young root, young bud, and scale leaf of bulb. However, LP-ExoII transcripts ubiquitously existed in all the tissues tested. To determine the potential substrates of exo-beta-glucanases, cell wall components of lily tissues were analyzed. Linkage analysis revealed that pollen tubes contained high levels of 3-Glc in hemicellulose (44.3%), while pollen grains had no detectable 3-Glc. The hemicellulose fraction of pollen tubes was treated with lichenase and the product was analyzed by HPLC-PAD to determine the origin of 3-Glc. Specific tetra-saccharide was liberated from hemicellulose of pollen tubes, suggesting the presence of 1,3 : 1,4-beta-glucan in lily pollen tube hemicellulose. The structure of this 1,3 : 1,4-beta-glucan may be different from cereal plant 1,3 : 1,4-beta-glucan, since tri-saccharide was not detected in hemicellulose fraction after lichenase treatment. LP-ExoI and LP-ExoII, expressed in pollen grains and tubes, may be involved in the regulation of pollen tube elongation by hydrolyzing callose and 1,3 : 1,4-beta-glucan within pollen tube walls.

Detection and localization of pectin methylesterase isoforms in pollen tubes of Nicotiana tabacum L.

Pectin methylesterases (PMEs) were detected in tobacco ( Nicotiana tabacum) pollen tubes grown in vitro. Seven PME isoforms exhibiting a wide isoelectric-point (pI) range (5.3-9.1) were found in crude extracts of pollen tubes. These isoforms were mainly retrieved in supernatants after low- and high-speed separation of the crude extract. Two isoforms, with pIs 5.5 and 7.3 and molecular weight about 158 kDa, were detected by immunoblotting with anti-flax PME antiserum. Localization of pectins and PME isoforms in pollen tubes was investigated by immunogold labelling with JIM5 monoclonal antibodies and anti-flax PME antiserum, respectively. In germinated pollen grains, two PME isoforms were mainly detected in the exine, Golgi apparatus and secretory vesicles. In pollen tubes the same two PME isoforms were distributed along the outer face of the plasma membrane in the vicinity of the inner layer of the cell wall, in the Golgi and around secretory vesicles. In pollen grains, PME isoforms were, in some cases, mixed with acidic pectins in proximity to the outer surface of the plasma membrane. In pollen tubes the presence of PMEs inside secretory vesicles carrying esterified pectins supports the hypothesis that, during pollen tube growth, PMEs could be transferred by secretory vesicles in a precursor form and be activated at the tip where exocytosis takes place.

Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L.

The transcription factors DREB1s/CBFs specifically interact with the DRE/CRT cis-acting element (core motif: G/ACCGAC) and control the expression of many stress-inducible genes in Arabidopsis. We isolated a cDNA for a DREB1/CBF homolog, ZmDREB1A in maize using a yeast one-hybrid system. The ZmDREB1A proteins specifically bound to DRE and the highly conserved valine at the 14th residue in the ERF/AP2 DNA binding domain was a key to determining the specific interaction between this protein and the DRE sequence. Expression of ZmDREB1A was induced by cold stress and slightly increased by high-salinity stress. This gene was also transiently expressed by mechanical attack. ZmDREB1A activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts. Overexpression of ZmDREB1A in transgenic Arabidopsis induced overexpression of target stress-inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought and freezing stresses. This indicated that ZmDREB1A has functional similarity to DREB1s/CBFs in Arabidopsis. The structure of the ERF/AP2 domain of ZmDREB1A in maize is closely related to DREB1-type ERF/AP2 domains in the monocots as compared with that in the dicots. ZmDREB1A is suggested to be potentially useful for producing transgenic plants that is tolerant to drought, high-salinity and/or cold stresses.

Nectar production and transportation in the nectaries of the female Cucumis sativus L. flower during anthesis.

In an effort to gain a greater understanding of nectar production, we studied the dynamic mechanisms of starch accumulation and transformation and nectar transportation in the Cucumis sativus L. female flower. Starch, which is the main precursor of nectar, accumulates in the epidermis and underlying parenchyma, with the most active accumulation occurring in the parenchyma cells within 3 days prior to anthesis. Thereafter, the starch was successively hydrolyzed and the hydrolyte was transported from the amyloplasts to vacuoles, suggesting that amyloplasts and vacuoles are the centers of nectar production. In addition, we observed few plasmodesmata and the presence of invaginated plasmalemma and electron-dense material in the intercellular spaces, suggesting that the apoplast system is involved in nectar transportation in an ATPase-dependent fashion.