Role of Salts in Phase Transformation of Clathrate Hydrates under Brine Environments

Although ion exclusion is a naturally occurring and commonly observed phenomenon in clathrate hydrates, an understanding for the effect of salt ions on the stability of clathrate hydrates is still unclear. Here we report the first observation of phase transformation of structure I and structure II clathrate hydrates using solid-state ¹³C, ¹⁹F, and ²³Na magic-angle spinning nuclear magnetic resonance (NMR) spectroscopy, combined with X-ray diffraction and Raman spectroscopy. The phase transformation of clathrate hydrates in salt environments is found to be closely associated with the quadruple point of clathrate hydrate/hydrated salts and the eutectic point of ice/hydrated salts. The formation of the quasi-brine layer (QBL) is triggered at temperatures a little lower than the eutectic point, where an increasing salinity and QBL does not affect the stability of clathrate hydrates. However, at temperatures above the eutectic point, all hydrated salts and the QBL melt completely to form brine solutions, destabilizing the clathrate hydrate structures. Temperature-dependent in situ NMR spectroscopy under pressure also allows us to directly detect the quadruple point of clathrate hydrates in salt environments, which has been determined only by visual observations

Development of transgenic alfalfa expressing <i>IbOr</i> under the control of the <i>SWPA2</i> promoter (SOR plants).

<p>(A) Diagram of the oxidative stress-inducible <i>SWPA2</i> promoter: <i>IbOr</i> construct used for alfalfa transformation. Vertical bar shows the primer set (<i>A2pro</i>::<i>IbOr</i>) used for genomic PCR analysis. (B) Genomic DNA PCR analysis using the <i>A2pro</i>::<i>IbOr</i> primer set. Numbers (1–11) represent independent transgenic lines. M, size markers; NT, non-transgenic plant; PC, positive control. (C) RT-PCR analysis of 11 lines expressing stable <i>IbOr</i> gene integration in transgenic plants following 2 h of 5 μM MV treatment.</p

Quantitative HPLC analysis of total carotenoid contents and carotenoid compounds in NT and SOR plants.

<p>All levels are expressed as the mean (average content in grams dry weight) ± SD of two independent determinations with four biological repeats. Asterisks indicate a significant difference from that of NT at * <i>P < 0</i>.<i>05</i> or ** <i>P < 0</i>.<i>01</i> by <i>t</i>-test.</p

Enhanced tolerance to high salinity in SOR plants.

<p>(A) Plant growth under normal conditions (upper panel) and salt stress (250 mM NaCl) for 4 days (middle panel) and 7 days (lower panel). (B) Relative transcript levels of <i>IbOr</i> in leaves. (C) Relative chlorophyll contents of alfalfa plants after 3 days of salt treatment. (D) MDA contents in leaves after 3 days of salt treatment. (E) Proline contents of alfalfa plants after 3 days of salt treatment. (F) DAB staining for H₂O₂ accumulation in the third detached leaves after 3 days of 250 mM NaCl treatment. The values represent the mean ± SD of three independent replicates. Asterisks indicate a significant difference from that of NT at * <i>P < 0</i>.<i>05</i> or ** <i>P < 0</i>.<i>01</i> by <i>t</i>-test.</p

Gene-specific primers used for genomic and RT-PCR analysis.

<p>Gene-specific primers used for genomic and RT-PCR analysis.</p

Transcript analysis of carotenoid biosynthetic pathway genes in NT and SOR plants under drought stress.

<p>Leaves (from same position) of plants treated with drought stress for 2 days were utilized. The expression level of each gene was normalized to that of the <i>Actin</i> gene of alfalfa as the internal control. <i>MsPSY</i>, phytoene synthase; <i>MsCHY-β</i>, <i>β-</i>carotene hydroxylase; <i>MsLCY-β</i>, lycopene <i>β</i>-cyclase; <i>MsNCED</i>, 9-cis-epoxycarotenoid dioxygenase. The values represent the mean ± SD of three independent biological replicates. Asterisks indicate a significant difference from that of NT at * <i>P < 0</i>.<i>05</i> or ** <i>P < 0</i>.<i>01</i> by <i>t</i>-test.</p

Enhanced tolerance to drought stress in SOR plants.

<p>(A) Phenotypes of 1-month-old NT and SOR plants before treatment (upper panel), after withholding water for 4 days (middle panel), and recovered phenotype after re-watering for 7 days (lower panel). (B) Transcript levels of <i>IbOr</i> after withholding water for 2 days. (C) RWC (%) in leaves after 3 days of water withholding. (D) MDA contents in leaves after 3 days of drought treatment. (E) Proline contents in leaves after 3 days of drought treatment. (F) DAB staining for H₂O₂ accumulation in the third detached leaves of alfalfa plants after 3 days of drought treatment. Values represent the mean ± SD of three independent replicates. Asterisks indicate a significant difference from that of NT at * <i>P < 0</i>.<i>05</i> or ** <i>P < 0</i>.<i>01</i> by <i>t</i>-test.</p

Effects of MV-mediated oxidative stress on leaves of SOR and NT plants.

<p>(A) Visible damage in leaves after 5 μM MV treatment for 24 h. (B) Ion leakage was measured after 0, 12, 24, and 36 h of MV treatment. Percentage of relative membrane permeability was calculated using 100% to represent the values obtained after autoclaving. (C) <i>IbOr</i> transcript levels after 12 h of 5 μM MV treatment. The expression levels of <i>IbOr</i> were normalized to that of the alfalfa <i>Actin</i> gene as the internal control. Data are expressed as the mean ± SD of three independent biological replicates. <i>Bars</i> labeled with <i>asterisks</i> show significant differences from that of NT at * <i>P < 0</i>.<i>05</i> or ** <i>P < 0</i>.<i>01</i> by <i>t</i>-test.</p