<i>In Vitro</i> Propagation,Isolation and Expression Studies of <i>Suaeda edulis</i> Genes Involved in the Osmoprotectants Biosynthesis

Halophytes are an excellent choice for the study of genes conferring salt tolerance to salt-sensitive plants and, they are suitable for reclamation and remediation of saline soil. We develop an in vitro plant propagation protocol and studies of genes involved with GB and Pro biosynthesis in Suaeda edulis. Axillary buds were used as explants and cultured in different treatments on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. The highest number of multiple shoots was on MS medium containing 1 mg/L Benzyladenine (BA) and / or 2 g/L activated carbon with 5.5 ± 06 shoots per explant. The identification and expression analysis of genes involved in glycine betaine (GB) biosynthesis were S-adenosylmethionine synthetase (SAMS), choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), and for proline (Pro) was pyrroline 5-carboxylate synthetase (P5CS). These sequences shared 90–95% of identity with others plant homologous in public databases. The amino acids sequence analysis showed that all these peptides contain some of the conserved motifs of those kinds of enzymes. The qRT-PCR analysis revealed a higher expression of SeBADH, SeCMO, and, SeP5CS genes in the roots and leaves from plants collected in the field in contrast with from in vitro plants. However, the expression level of SeSAMS was higher only in the leaves of plants collected in the field when compared to those cultivated in vitro.     

三疣梭子蟹肌肉组织中甜菜碱、糖原及无机盐变化研究

为了探讨甜菜碱、糖原及无机盐含量对三疣梭子蟹肌肉口感品质的影响,采用可见分光光度法、蒽酮比色法和原子分光光度法,分别测定了3个发育阶段（8月份幼蟹阶段、10月份交配前和12月份交配后）的野生三疣梭子蟹肌肉组织中甜菜碱、糖原及无机盐含量。结果表明：1）肌肉中甜菜碱含量在第一阶段（8月幼蟹）甜菜碱含量最低,为172 mg/100 g,10月交配前雌蟹肌肉甜菜碱含量最高,达473 mg/100 g,但12月份交配后含量减半;雄蟹肌肉则表现为从第1到第3阶段逐渐升高趋势。2）幼蟹的糖原含量极低,仅为0.8μg/mg,交配前雌雄蟹仍保持较低水平,交配后糖原含量急剧升高,雌雄蟹分别上升至11.26μg/mg和9.61μg/mg,各是交配前的6.5倍和10倍。3）肌肉无机盐中K、Na是宏量元素,含量最高,K含量336~457 mg/100 g,Na含量131~185 mg/100 g,Fe为微量元素,含量最低,仅有1.12~1.26 mg/100 g。三疣梭子蟹肌肉中的甜菜碱含量远高于一般水产品,故蟹肉入口爽滑带有甜味,且随着肌肉中糖原含量不断增加,其风味也更佳。     

Influence of Triadimefon on the Metabolism of NaCl Stressed Radish

The effect of triadimefon (TDM) on various biochemical parameters was studied in NaCl stressed radish (Raphanus sativus L.). Stress imposed by 80 mM NaCl decreased the protein content and proline oxidase activity, and increased the proline and glycine betaine contents, and protease, -glutamyl kinase and ATPase activities. The TDM treatment alleviated the stress by increasing protein, and glycine betaine contents, and by decreasing proline accumulation, and proline oxidase and ATPase activities.     

Osmotic adjustment in cyanobacteria from hypersaline environments

The intracellular concentrations of the monovalent inorganic cations K⁺ and Na⁺, low molecular weight carbohydrates and quaternary ammonium compounds have been determined for 4 strains of cyanobacteria (Aphanothece halophytica, Coccochloris elabens, Dactylococcopsis salina and Synechocystis DUN52) originally isolated from hypersaline habitats (i.e. habitats with a salinity greater than that of seawater) over a range of external salt concentration (from 50% to 400% seawater). Intracellular cation levels (Na⁺ and K⁺) were determined to be within the range 80–320 mmol · dm^-3 (cell volume), showing only minor changes in response to salinity. Intracellular carbohydrates were found to comprise a negligible component of the intracellular osmotic potential [at 2–19 mmol · dm^-3 (cell volume)], throughout the salinity range. Quaternary ammonium compounds, however, were recorded in osmotically significant quantities [up to 1,640 mmol · dm^-3 (cell volume)] in these strains, showing major variation in response to salinity. Thus Synechocystis DUN 52 showed an increase in quaternary ammonium compounds in the oder of 1,200 mmol · dm^-3 between 50% and 400% seawater medium, accounting for a significant proportion of the change in external osmotic potential.Examination of intact cells and cell extracts using ¹³C and ¹H nuclear magnetic resonance (NMR) spectroscopy confirmed the presence of the quaternary ammonium compound glycine betaine as the major osmoticum in the 4 strains; no other compounds were detected during NMR assays. These results suggest a common mechanism of osmotic adjustment, involving quaternary ammonium compounds, in cyanobacteria from hypersaline environments.     

Responses of sugar beet (Beta vulgaris L.) to drought and nutrient deficiency stress

The responses of two sugar beet genotypes, 24367 (putative droughttolerant) and N6 (putative drought intolerant), to drought and nutrientdeficiency stress were investigated in an attempt to identify reliable andsensitive indicators of stress tolerance. In glasshouse-grown plants of bothgenotypes, relative water content (RWC) of the leaves decreased and leaftemperature increased in response to drought stress. Genotype differences inresponse to drought included leaf RWC, glycine betaine accumulation, alterationof shoot/root ratio and production of fibrous roots. Thus, in comparison to N6,genotype 24367 lost less water from leaves, produced more fibrous roots,produced more glycine betaine in shoots and tap roots and had a much reducedshoot/root ratio in response to withholding water for up to 215 h.The hydraulic conductance and sap flow of sugar beet seedlings grown innutrientculture decreased when subjected to nitrogen deficiency stress. Under nitrogensufficient conditions sap flow was greater in 24367 than in N6. The resultsindicate that genotype 24367 is more tolerant to stresses induced by water andnitrogen deficiency and that increased fibrous root development may be a majorfactor in increasing sap flow via a concomitant enhancement of aquaporinactivity.     

Examination of the structures of several glycerolipids from marine macroalgae by NMR and GC‐MS

Several classes of glycerolipids were isolated from the total lipids of the algae Saccharina cichorioides, Eualaria fistulosa, Fucus evanescens, Sargassum pallidum, Silvetia babingtonii (Ochrophyta, Phaeophyceae), Tichocarpus crinitus, and Neorhodomela larix (Rhodophyta, Florideophyceae). The structures of these lipids were examined by nuclear magnetic resonance (NMR) spectroscopy, including 1D (¹H and ¹³C) and 2D (COSY, HSQC and HMBC) experiments. All of the investigated algae included common galactolipids and sulfonoglycolipids as the major glycolipids. Minor glycolipids isolated from S. cichorioides, T. crinitus, and N. laris were identified as lyso‐galactolipids with a polar group consisted of the galactose. Comparison of the ¹H NMR data of minor nonpolar lipids isolated from the extracts of the brown algae S. pallidum and F. evanescens with the ¹H NMR data of other lipids allowed them to be identified as diacylglycerols. The structures of betaine lipids isolated from brown algae were confirmed by NMR for the first time. The fatty acid compositions of the isolated lipids were determined by gas chromatography‐mass spectrometry.     

Molecular cloning and characterization of betaine aldehyde dehydrogenase gene from Suaeda liaotungensis and its use in improved tolerance to salinity in transgenic tobacco

cDNA encoding betaine aldehyde dehydrogenase (BADH) from the halophyte Suaeda liaotungensis has been cloned, sequenced and expressed in tobacco (Nictiana tabacum 89). The full-length cDNA is 1506 base pairs (bp) long and encodes a 502 amino-acid polypeptide. The cDNA fragment coding for the mature enzyme was cloned into vector pCAMBIA-1301 for expression in tobacco. Southern blotting analysis showed that that the Badh gene was integrated into the genome of tobacco. Tobacco expressing BADH survived on MS medium containing 200 mM NaCl, whereas the untransformed plants turned yellow after about 20 d and died.     

Variation in Osmoregulation in Differentially Drought-Sensitive Wheat Genotypes Involves Calcium

Two wheat (Triticum aestivum L.) genotypes differing in their sensitivity to water deficit (stress tolerant - C306 and stress susceptible - HD2329) were subjected to osmotic stress for 7 d using polyethylene glycol (PEG-6000; osmotic potential –1.0 MPa), at initial vegetative growth. The plants were either supplemented with 1 mM CaCl₂ (Ca²⁺) alone or along with verapamil (VP; calcium channel blocker) to investigate the involvement of calcium in governing osmoregulation. Relative elongation rate (RER), dry matter (DM) production, water potential (_w), electrolyte leakage (EL), contents of proline (Pro) and glycine betaine (GB) and activities of -glutamyl kinase (GK) and proline oxidase (PO) in shoots and roots were examined during stress period. C306 showed relatively higher accumulation of Pro while HD2329 accumulated more GB under stress. RER, DM and _w were relatively higher in C306 than HD2329. Roots compared to shoots showed lower content of osmolytes but had faster rate of their accumulation. Presence of Ca²⁺ in the medium increased the activity of GK and decreased that of PO while in the presence of its inhibitor, decrease in activity of both the enzymes was observed. Ca²⁺ appeared to reduce the damaging effect of stress by elevating the content of Pro and GB, improving the water status and growth of seedlings and minimizing the injury to membranes. The protective effect of Ca²⁺ was observed to be more in HD2329 than C306.     

Biosynthesis and function of trehalose inEctothiorhodospira halochloris

Trehalose-6-phosphate synthase, catalyzing the reaction between UDP-glucose and glucose 6-phosphate and forming trehalose 6-phosphate, was isolated and partially purified (30-fold) from the phototrophic, haloalkaliphilic bacteriumEctothiorhodospira halochloris. The activity is stabilized by 20mM MgCl₂, 50mM NaCe and 2M glycine betaine. The molecular weight was 63000.The enriched enzyme had a MgCl₂ optimum at 3–6mM, a pH optimum at 7.5 (in Tris-HCl buffer) and a temperature optimum at 50°C. The K_m-values were 1.5×10^–3M for UDP-glucose and 2×10^–3M for glucose 6-phosphate. The enzyme showed a salinity dependence with optimal concentrations between 100 and 300mM salt. Higher concentrations of salt resulted in a decrease in activity. In the presence of inhibitory salt concentrations the compatible solute glycine betaine had a protective effect with a maximum between 0.5 and 2.0M.     

Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy

Abstract Natural abundance ¹³C NMR spectroscopy has identified sucrose and trehalose as the principle compatible solutes accumulated by non-halophilic purple and green sulphur bacteria respectively, in response to osmotic stress. Synthesis of glycine betaine as a compatible solute was rare in non-halophilic phototrophic sulphur bacteria and appears to be limited almost exclusively to halotolerant isolates, although all isolates tested were able to accumulate exogenous glycine betaine from the growth medium in response to osmotic stress. These data support the hypothesis that the degree of halotolerance of a microorganism may be due, at least in part, to the metabolic effects of the compatible solute(s) accumulated.