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In JoVE (1)
Other Publications (7)
Articles by Miao Wen in JoVE
JoVE General
Use of Arabidopsis eceriferum Mutants to Explore Plant Cuticle Biosynthesis
1Department of Botany, University of British Columbia - UBC, 2Department of Chemistry, University of British Columbia - UBC
The plant cuticle is a waxy outer covering on plants that has a primary role in water conservation but is also an important barrier against the entry of pathogenic microorganisms. In this video, we demonstrate the analysis of plant cuticle mutants identified by forward and reverse genetics approaches.
Other articles by Miao Wen on PubMed
Population Kinetics on K Alpha Lines of Partially Ionized Cl Atoms
A population kinetics code was developed to analyze K alpha emission from partially ionized chlorine atoms in hydrocarbon plasmas. Atomic processes are solved under collisional-radiative equilibrium for two-temperature plasmas. It is shown that the fast electrons dominantly contribute to ionize the K-shell bound electrons (i.e., inner-shell ionization) and the cold electrons to the outer-shell bound ones. Ratios of K alpha lines of partially ionized atoms are presented as a function of cold-electron temperature. The model was validated by observation of the K alpha lines from a chlorinated plastic target irradiated with 1 TW Ti:sapphire laser pulses at 1.5 x 10(17) W/cm(2), inferring a plasma temperature of about 100 eV on the target surface.
Nanotubules on Plant Surfaces: Chemical Composition of Epicuticular Wax Crystals on Needles of Taxus Baccata L
Needles of Taxus baccata L. were covered with tubular epicuticular wax crystals varying in diameters (100 and 250 nm) and lengths (300-500 and 500-1000 nm) on the abaxial and adaxial surfaces, respectively. Various sampling protocols were employed to study the chemical composition of the needle waxes on three different levels of spatial resolution. First, a dipping extraction of whole needles yielded the total cuticular wax mixture consisting of very long chain fatty acids (21%), alkanediols (19%), phenyl esters (15%), and secondary alcohols (9%) together with small amounts of aldehydes, primary alcohols, alkanes, alkyl esters, and tocopherols. Second, waxes from both sides of the needle were sampled separately by brushing with CHCl3-soaked fabric glass. Both sides showed very similar qualitative composition, but differed drastically in quantitative aspects, with nonacosan-10-ol (18%) and alkanediols (33%) dominating the abaxial and adaxial waxes, respectively. Third, the epi- and intracuticular wax layers were selectively sampled by a combination of mechanical wax removal and brushing extraction. This provided direct evidence that the tubular wax crystals contained high percentages of nonacosane-4,10-diol and nonacosane-5,10-diol on the abaxial surface, and nonacosan-10-ol on the adaxial surface of the needles. Together with these compounds, relatively large amounts of fatty acids and smaller percentages of aldehydes, primary alcohols, alkyl esters, and alkanes co-crystallized in the epicuticular layer. In comparison, the intracuticular wax consisted of higher portions of cyclic constituents and aliphatics with relatively high polarity. The formation of the tubular crystals is discussed as a spontaneous physico-chemical process, involving the establishment of gradients between the epi- and intracuticular wax layers and local phase separation.
Very-long-chain Secondary Alcohols and Alkanediols in Cuticular Waxes of Pisum Sativum Leaves
In cuticular waxes from leaves of Pisum sativum, 19 secondary alcohols, 10 primary/secondary alkanediols and three secondary/secondary alkanediols were identified by various chemical transformations with product assignment employing GC-MS. The homologous series of C29-C33 secondary alcohols (1.1 microg/cm2) was dominated by hentriacontanol isomers (94%). Only octacosanediols and trace amounts of hexacosanediols (< 1%) were detected in the primary/secondary alkanediol faction (0.7 microg/cm2). The secondary/secondary alkanediols (0.12 microg/cm2) contained a single homologue with chain length C31. All three compound classes showed characteristic isomer distributions with secondary functional groups predominantly located between C-14 and C-16. Based on the isomer compositions, the sequence of biosynthetic steps introducing the hydroxyl functions is discussed.
Very-long-chain Hydroxyaldehydes from the Cuticular Wax of Taxus Baccata Needles
In the cuticular wax of Taxus baccata needles, homologous series of very-long-chain 1,5-alkanediols and 5-hydroxyaldehydes were identified by various chemical transformations with product assignment using GC-MS. The 1,5-alkanediols had chain lengths ranging from C(28) to C(38), with strong predominance of even carbon numbers and a maximum at C(32) (29%). The series of 5-hydroxyaldehydes comprised chain lengths C(24) and C(26)-C(36), and showed a pronounced prevalence of even-numbered homologues. 5-Hydroxyoctacosanal was the most abundant compound of the series (42%). The 5-hydroxyaldehydes together amounted to 0.4 microg/cm(2), corresponding to 1.2% of total wax of the needles. A polyketide-like biosynthetic pathway is proposed based on the (similar) chain length distributions and functional group patterns for both compound classes.
The Cytochrome P450 Enzyme CYP96A15 is the Midchain Alkane Hydroxylase Responsible for Formation of Secondary Alcohols and Ketones in Stem Cuticular Wax of Arabidopsis
Most aerial surfaces of plants are covered by cuticular wax that is synthesized in epidermal cells. The wax mixture on the inflorescence stems of Arabidopsis (Arabidopsis thaliana) is dominated by alkanes, secondary alcohols, and ketones, all thought to be formed sequentially in the decarbonylation pathway of wax biosynthesis. Here, we used a reverse-genetic approach to identify a cytochrome P450 enzyme (CYP96A15) involved in wax biosynthesis and characterized it as a midchain alkane hydroxylase (MAH1). Stem wax of T-DNA insertional mutant alleles was found to be devoid of secondary alcohols and ketones (mah1-1) or to contain much lower levels of these components (mah1-2 and mah1-3) than wild type. All mutant lines also had increased alkane amounts, partially or fully compensating for the loss of other compound classes. In spite of the chemical variation between mutant and wild-type waxes, there were no discernible differences in the epicuticular wax crystals on the stem surfaces. Mutant stem wax phenotypes could be partially rescued by expression of wild-type MAH1 under the control of the native promoter as well as the cauliflower mosaic virus 35S promoter. Cauliflower mosaic virus 35S-driven overexpression of MAH1 led to ectopic accumulation of secondary alcohols and ketones in Arabidopsis leaf wax, where only traces of these compounds are found in the wild type. The newly formed leaf alcohols and ketones had midchain functional groups on or next to the central carbon, thus matching those compounds in wild-type stem wax. Taken together, mutant analyses and ectopic expression of MAH1 in leaves suggest that this enzyme can catalyze the hydroxylation reaction leading from alkanes to secondary alcohols and possibly also a second hydroxylation leading to the corresponding ketones. MAH1 expression was largely restricted to the expanding regions of the inflorescence stems, specifically to the epidermal pavement cells, but not in trichomes and guard cells. MAH1-green fluorescent protein fusion proteins localized to the endoplasmic reticulum, providing evidence that both intermediate and final products of the decarbonylation pathway are generated in this subcellular compartment and must subsequently be delivered to the plasma membrane for export toward the cuticle.
Composition of Secondary Alcohols, Ketones, Alkanediols, and Ketols in Arabidopsis Thaliana Cuticular Waxes
Arabidopsis wax components containing secondary functional groups were examined (i) to test the biosynthetic relationship between secondary alcohols and ketols and (ii) to determine the regiospecificity and substrate preference of the enzyme involved in ketol biosynthesis. The stem wax of Arabidopsis wild type contained homologous series of C(27) to C(31) secondary alcohols (2.4 microg cm(-2)) and C(28) to C(30) ketones (6.0 microg cm(-2)) dominated by C(29) homologues. In addition, compound classes containing two secondary functional groups were identified as C(29) diols (approximately 0.05 microg cm(-2)) and ketols (approximately 0.16 microg cm(-2)). All four compound classes showed characteristic isomer distributions, with functional groups located between C-14 and C-16. In the mah1 mutant stem wax, diols and ketols could not be detected, while the amounts of secondary alcohols and ketones were drastically reduced. In two MAH1-overexpressing lines, equal amounts of C(29) and C(31) secondary alcohols were detected. Based on the comparison of homologue and isomer compositions between the different genotypes, it can be concluded that biosynthetic pathways lead from alkanes to secondary alcohols, and via ketones or diols to ketols. It seems plausible that MAH1 is the hydroxylase enzyme involved in all these conversions in Arabidopsis thaliana.
High Mobility Group Box Protein-1 Correlates with Microinflammatory State and Nutritional Status in Continuous Ambulatory Peritoneal Dialysis Patients
High mobility group box protein-1 (HMGB-1) was recently identified as a new type of inflammatory cytokine. Inflammation can lead to malnutrition to some extent. Our study was aimed to clarify the relationship between serum HMGB-1 level with microinflammatory state and nutritional status in continuous ambulatory peritoneal dialysis (CAPD) patients. Patients in the treatment of maintenance of peritoneal dialysis for >6 months were included. HMGB-1, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). High-sensitivity C-reactive-protein (hs-CRP), prealbumin (PA), serum albumin (S-Alb), hemoglobin (Hb), subjective global nutritional assessment (SGA), and CAPD presents' urea clearance rate (Kt/V), creatinine clearance (CrCl), residual glomerular filtration rate (rGFR), and dialysate-to-plasma ratio of creatinine after 4 h (D/P(4Cr)) were analyzed. The Independent-samples t test and Pearson's rank correlation test were used. Serum HMGB-1, IL-6, and TNF-α of CAPD patients were significantly higher than in the control group (P < 0.05); Serum HMGB-1 levels had positive relationships with TNF-α (r = 0.730, P < 0.01), hs-CRP (r = 0.361, P < 0.01), and IL-6 (r = 0.865, P < 0.01), and had negative relationships with Hb (r = -0.59, P < 0.01), Alb (r = -0.34, P < 0.05), and PA (r = -0.44, P < 0.01); no significant relationships were found between serum HMGB-1 with SGA, peritoneal dialysis age, Kt/V, CrCl, rGFR, and D/P(4Cr). Our study revealed that HMGB-1 was elevated significantly in CAPD patients and correlated with indicators of inflammation and malnutrition. Serum HMGB-1 could be used as a marker for evaluating inflammation and malnutrition in CAPD patients.
