ABSTRACT
MAIN CONCLUSION: Storage promotes carotenoid accumulation and converts amylochromoplasts into chromoplasts in winter squash. Such carotenoid enhancement is likely due to continuous biosynthesis along with reduced turnover and/or enhanced sequestration. Postharvest storage of fruits and vegetables is often required and frequently results in nutritional quality change. In this study, we investigated carotenoid storage plastids, carotenoid content, and its regulation during 3-month storage of winter squash butternut fruits. We showed that storage improved visual appearance of fruit flesh color from light to dark orange, and promoted continuous accumulation of carotenoids during the first 2-month storage. Such an increased carotenoid accumulation was found to be concomitant with starch breakdown, resulting in the conversion of amylochromoplasts into chromoplasts. The butternut fruits contained predominantly ß-carotene, lutein, and violaxanthin. Increased ratios of ß-carotene and violaxanthin to total carotenoids were noticed during the storage. Analysis of carotenoid metabolic gene expression and PSY protein level revealed a decreased expression of carotenogenic genes and PSY protein following the storage, indicating that the increased carotenoid level might not be due to increased biosynthesis. Instead, the increase likely resulted from a continuous biosynthesis with a possibly reduced turnover and/or enhanced sequestration, suggesting a complex regulation of carotenoid accumulation during fruit storage. This study provides important information to our understanding of carotenogenesis and its regulation during postharvest storage of fruits.
Subject(s)
Carotenoids/metabolism , Cucurbita/metabolism , Food Storage , Plastids/metabolism , Biosynthetic Pathways/genetics , Blotting, Western , Color , Cucurbita/genetics , Fruit/genetics , Fruit/metabolism , Gene Expression Regulation, Plant , Geranylgeranyl-Diphosphate Geranylgeranyltransferase/genetics , Geranylgeranyl-Diphosphate Geranylgeranyltransferase/metabolism , Hydrolysis , Plant Proteins/genetics , Plant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Starch/metabolism , Time Factors , Xanthophylls/metabolism , beta Carotene/metabolismABSTRACT
Radish floral bud abortion (FBA) is an adverse biological phenomenon that occurs during reproduction. Although FBA is a frequent occurrence, its molecular mechanism remains unknown. A transcript-derived fragment (TDF72), which was obtained by cDNA amplified fragment length polymorphism (cDNA-AFLP), was up-regulated in the aborted buds and exhibited 89% sequence homology with the AtγVPE gene. In this study, TDF72 was used to clarify the role of VPE in FBA by isolation of the VPE gene RsVPE1 from radish flower buds. The full-length genomic DNA was 2346 bp including nine exons and eight introns. The full-length cDNA was 1825 bp, containing a complete open reading frame (ORF) of 1470 bp, which encoded a predicted protein containing 489 amino acid residues, with a calculated molecular mass of 53.735 kDa. Expression analysis demonstrated that RsVPE1 was expressed in all tested organs of radish at different levels. Highest expression was detected in aborted flower buds, suggesting that RsVPE1 has a role in FBA. In order to analyze the role of RsVPE1 in FBA, RsVPE1 was overexpressed in transgenic Arabidopsis thaliana plants. Aborted flower buds appeared in transgenic plants subjected to heat stress. In addition, RsVPE1 expression in the transgenic plants reached a maximum when subjected to heat stress for 24 h and increased by 2.1-fold to 2.8-fold in three homozygous transgenic lines. These results indicated that RsVPE1 led to FBA when its expression levels exceeded a particular threshold, and provided evidence for the involvement of RsVPE1 in promoting FBA under heat stress.
Subject(s)
Cysteine Endopeptidases , Flowers , Heat-Shock Response/physiology , Plant Proteins , Raphanus , Arabidopsis/enzymology , Arabidopsis/genetics , Cysteine Endopeptidases/biosynthesis , Cysteine Endopeptidases/genetics , Flowers/enzymology , Flowers/genetics , Plant Proteins/biosynthesis , Plant Proteins/genetics , Plants, Genetically Modified/enzymology , Plants, Genetically Modified/genetics , Raphanus/enzymology , Raphanus/geneticsABSTRACT
BACKGROUND: Airway mucus hypersecretion is an important pathophysiological feature of chronic obstructive pulmonary disease, which is closely associated with cigarette smoking. However, the signal transduction pathway from the cell surface to the nucleus through which cigarette smoke causes upregulation of mucin gene expression is not well known. This study was designed to investigate the role of extracellular signal-regulated Kinase 1/2 (ERK 1/2) in airway mucus hypersecretion induced by cigarette smoke in rats. METHODS: A rat model of airway mucus hypersecretion was induced by exposure to cigarette smoke for 4 weeks.Rats exposed to inhalation of cigarette smoke or normal saline were given an intraperitoneal injection of U0126, a specific MEK1 kinase inhibitor, at doses of 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg for 14 days. Expression of MUC5AC mRNA and protein, ERK 1/2 and phosphorylated-ERK 1/2 (p-ERK 1/2) were detected by RT-PCR, immunohistochemistry and Western blotting. RESULTS: Cigarette smoke significantly increased airway goblet cells metaplasia, induced the overexpression of MUC5AC mRNA and protein in bronchial epithelia, and increased the ratio of p-ERK 1/2 and ERK 1/2. U0126 significantly attentuated the expression of MUC5AC mRNA and protein induced by cigarette smoke (P < 0.05). Moreover, there was a significant positive correlation between the ratio of p-ERK1/2 to ERK1/2 and the expression of MUC5AC mRNA and protein (P < 0.05). CONCLUSIONS: Inhibition of ERK 1/2 by U0126 decreased the ratio of p-ERK 1/2 to ERK 1/2 and expression of MUC5AC mRNA and protein. ERK 1/2 may play an essential role in cigarette smoke-induced mucus hypersecretion in vivo.
Subject(s)
Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Respiratory Mucosa/metabolism , Smoking/adverse effects , Animals , Blotting, Western , Bronchi/cytology , Bronchi/metabolism , Goblet Cells/drug effects , Goblet Cells/metabolism , Immunohistochemistry , Lung/drug effects , Lung/metabolism , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Mucin 5AC/genetics , Mucin 5AC/metabolism , Phosphorylation/drug effects , Rats , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
OBJECTIVE: To investigate the expression of interleukin (IL)-8 in lung tissues from patients with chronic obstructive pulmonary disease (COPD) and its association with stages of COPD. METHODS: The levels of mRNA and protein of IL-8 were measured with semi-quantitative polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in patients with mild COPD (n=21), patients with advanced COPD (n=15), and controls (n=15). The correlations between IL-8 levels and stages of COPD, lung function (FEV1/ FVC%, FEV1% pred) and cigarette smoking were analyzed with Pearson correlation analysis. RESULTS: The levels of IL-8 mRNA and protein in the lung tissues of COPD patients were significantly higher than those in the control group (P<0.05). The patients with advanced COPD had higher levels of IL-8 mRNA and protein than the patients with mild COPD (P<0.05). The COPD patients who smoked had higher levels of IL-8 mRNA and protein than those who did not smoke (P<0.05). But no significant differences in the levels of IL-8 mRNA and protein were found between smokers and and nonsmokers who did not have COPD (P>0.05). Increased expression of IL-8 in patients with COPD was positively correlated with stages of COPD (r=0.81, P<0.05); negatively correlated with lung function (FEV1/FVC%, FEV1% pred) (r=-0.62, -0.56, P<0.05), and positively correlated with volumes of cigarette smoking (r=0.53, P<0.05). CONCLUSION: IL-8 is associated with stages of COPD, which may serve as an indication for clinical progress. Cigarette smoking increases IL-8 expression in the lung tissues of COPD patients.
Subject(s)
Interleukin-8/metabolism , Lung/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Aged , Biomarkers , Female , Humans , Interleukin-8/genetics , Male , Middle Aged , Pulmonary Disease, Chronic Obstructive/classification , RNA, Messenger/genetics , RNA, Messenger/metabolism , Smoking/adverse effectsSubject(s)
Body Mass Index , Pulmonary Disease, Chronic Obstructive , Adult , Aged , Female , Humans , Male , Middle Aged , Respiratory Function TestsABSTRACT
In order to investigate the differential expression of the genes related to cytoplasmic male sterility in Chinese cabbage (Brassica campestris L. ssp. Penkinsis), a modified RNA fingerprinting technique was developed to compare the difference in the total RNA from flower bud of Chinese cabbage among cytoplasmic male sterility (CMS) lines, maintainer lines and F1 hybrids. Four stably differential fragments S47-412, S93-622, S176-343 and S199-904 were amplified, cloned and sequenced with primers selected from 186 random primers. Based on the nucleotide sequence of the four differential fragments, four pairs of specific primers were designed to validate the differential fragments. The validation showed S47-412 and S93-622 were false positives and S176-343 and S199-904 were confirmed by PCR with the specific primers. Sequence analysis revealed that both of two differential fragments had strong homology with the nucleotide sequence of orf224/atp6 site of Polima CMS and the nucleotide sequence of S176-343 and S199-904 had a superposed region. All these indicate that the two fragments probably have strong relationship with cytoplasmic male sterility in Chinese cabbage.
