Isolation of Chloroplasts from Marine Microalga Isochrysis galbana Parke for Their Lipid Composition Analysis

Marine microalga Isochrysis galbana is an important feed species with a high nutritional value.Different from other uni-cellular algae,its cell contains two chloroplasts which are the major sites for lipid synthesis.Here,we optimized a chloroplast isola-tion approach suitable for the isolation of I.galbana chloroplasts and determined the purity and integrity of the isolated chloroplasts through microscopic observations and enzyme activity assay.The chloroplast lipids were analyzed with a ultrahigh-performance li-quid chromatography-Q Exactive Orbitrap-mass spectrometry.This newly developed isolation approach is simple and reliable to isolate chloroplasts with high integrity and purity.The average yield of intact chloroplasts was 15.3%±0.1%.Glycolipids and acyl-glycerols were the main chloroplast lipids.Glycolipids accounted for 56.6%of chloroplast lipid.Digalactosyldiacylglycerol(DGDG),monogalactosyldiacylglycerol(MGDG)and sulfoquinovosyldiacylglycerol(SQDG)were the main glyceroglycolipids.The fatty acyl R1/R2 were mostly 18:4/16:1,18:3/16:1 and 18:4/18:5 in DGDGs,14:0/18:4,18:4/18:5,18:4/18:4 and 18:3/18:4 in MGDGs and 16:0/14:0,16:0/18:3,and 18:4/18:3 in SQDGs.In addition,diacylglycerol(DAG)was the most abundant acylglycerols;the content of 22:6/18:4-DAG was the highest.There was a little amount of glycosphingolipid(GSL)in chloroplast.Digalactosylmonoglyceride(DGMG),monogalactosylmonoglyceride(MGMG),sulfoquinovosylmonoacylglycerol(SQMG),monoglyceride(MAG),phospholi-pids(PLs),ceramide(Cer)and betaine lipids were nearly undetectable in chloroplast.The fatty acid proportions of DGDGs,MGDGs,SQDGs,DAGs,triglycerides(TAGs)and GSLs were either higher or lower than or similar to those of whole-cell.Collectively,our isolation approach is applicable to many aspects of chloroplast biology,and may offer a reference for the isolation of chloroplasts from other marine microalgae.     

Analysis of phospholipids in microalga Nitzschia closterium by UPLC-Q-TOF-MS

Precise structural identification of phospholipids in the microalga Nitzschia closterium has been established using ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) for direct analysis of total lipid extracts. Mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in negative mode. Phospholipid molecular species identification was based on the characteristic product ions and neutral loss yielded by different phospholipids under ESI-MS/MS mode. The molecular species were confirmed by the carboxylate anions produced by phospholipids in negative mode; the regiospecificity of the two acyl chains was determined from the ratio of sn-1 to sn-2 carboxylate anion abundances. As a result, 18 lipid molecular species were identified for the first time in this microalga, comprising seven phosphatidylcholines (PC), two phosphatidylethanolamines (PE), two phosphatidylinositols (PI), and seven phosphatidylglycerols (PG). Lipid standards of PC, PE, PI, and PG were added to the total lipids as internal standards for semiquantitative analysis, revealing concentrations of phospholipids in this species between 0.09 and 3.37 nmol/mg. This method can produce a full structural profile of intact phospholipid molecular species and can be used for study of the physiological and ecological functions of lipids by monitoring their individual changes over time.     

Effects of chilled storage and cryopreservation on sperm characteristics,antioxidant enzyme activities,and lipid peroxidation in Pacific cod <Emphasis Type="Italic">Gadus microcephalus</Emphasis>

The present study evaluated the effects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm motility and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (Gr), and lipid peroxidation (measured via malondialdehyde (MDA) content) were determined after the milt was stored at 4°C for 12 h, cryopreserved without cryoprotectant in 12% propylene glycol (PG), cryopreserved in 12% PG+0.1 mol/L trehalose, or cryopreserved in 12% PG spermatozoa but centrifuged to decant the supernatant prior to cryopreservation (only sperm cells were cryopreserved). After chilled storage or cryopreservation, the SOD, CAT and GPx activities were reduced in sperm cells and increased in seminal plasma in almost all treatments; sperm motility parameters were also decreased. However, the addition of trehalose into the cryoprotectant could significantly improve the postthaw sperm quality as revealed by the sperm average path velocity. This improvement might be attributed to the function of trehalose in scavenging reactive oxygen species. Chilled storage and cryopreservation had significant effects on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod.     

The Response of Bio-Component Production of Nannochloris oculata to the Combinations of Monochromatic Light

Illuminating conditions are crucial factors affecting the microalgal growth and biosynthesis.The effects of illuminating spectral quality on the growth and bio-component production of Nannochloris oculata were investigated.The results indicated that a high proportion of Red-light enhanced the pigments and carbohydrate production but reduced those of the biomass and lipid.Mono-chromatic Blue-light has advantageous effects on lipid production compared with Red-and White-light.The optimal light spectrum for the protein production was the combination of Red-and Blue-light at a ratio of 4:3 or 5:2.Among the seven fatty acids identified from N.oculata,the contents of C16:0,C18:0,and C18:3(n-3)in the lipid were inhibited by the increased proportion of Red-light while those of C18:2(n-9),C16:2(n-6),and C20:0 were inhibited by Blue-light.Monochromatic Red-and Blue-light and their com-binations were proposed as a promising illuminating strategy for the large-scale cultivation aiming for various bio-components.     

Isolation and characterization of an enoyl-acyl carrier protein reductase gene from microalga Isochrysis galbana

In most bacteria,plants and algae,fatty acid biosynthesis is catalyzed by a group of freely dissociable proteins known as the type II fatty acid synthase(FAS II) system.In the FAS II system,enoylacyl carrier protein reductase(ENR) acts as a determinant for completing the cycles of fatty acid elongation.In this study,the cDNA sequence of ENR,designated as IgENR,was isolated from the microalga Isochrysis galbana CCMM5001.RACE(rapid amplification of cDNA ends) was used to isolate the full-length cDNA of IgENR(1 503 bp),which contains an open reading frame(ORF) of 1 044 bp and encodes a protein of 347 amino acids.The genomic DNA sequence of IgENR is interrupted by four introns.The putative amino acid sequence is homologous to the ENRs of seed plants and algae,and they contain common coenzymebinding sites and active site motifs.Under different stress conditions,real-time quantitative polymerase chain reaction(RT-qPCR) showed the expression of IgENR was upregulated by high temperature(35℃),and downregulated by depleted nitrogen(0 mol/L).To clarify the mechanism of lipids accumulating lipids,other genes involved in lipids accumulation should be studied.     

Effects of Dietary Carbohydrates Sources on Lipids Compositions in Abalone, Haliotis discus hannai Ino

A study was conducted to evaluate the effects of dietary carbohydrates on triglyceride, cholesterol and fatty acid concentrations in abalone, Haliotis discus hannai Ino. Six semi-purified diets with different carbohydrates (dextrin, heat-treated wheat starch, wheat starch, corn starch, tapioca starch and potato starch, respectively), all containing a carbohydrate level of 33.5%, were fed to abalone (initial shell length: 29.98 mm±0.09 mm; initial weight. 3.42 g±0.02 g) for 24 weeks in a recirculation system. The results indicate that serum triglyceride concentrations were significantly (P<0.05) higher in the abalone fed with dextrin, heat-treated wheat starch and wheat starch than those fed with corn starch, and serum cholesterol concentrations were significantly (P<0.05)higher in the abalone fed with dextrin, heat-treated wheat starch than those fed with corn starch. Fatty acid C20:4n-6 in the foot muscles were significantly (P<0.05) lower in the abalone fed with dextrin than those fed with wheat starch, corn starch, tapioca starch and potato starch. Fatty acid C20:4n-6 in hepatopancreas was significantly (P<0.05) lower in abalone fed with heat-treated wheat starch than those fed with corn starch, tapioca starch and potato starch. Fatty acid C22:6n-3 in the foot muscles were significantly (P< 0.05) lower in the abalone fed with dextrin and heat-treated wheat starch than those fed with wheat starch and potato starch.     

Reproductive cycle and seasonal variations in lipid content and fatty acid composition in gonad of the cockle Fulvia mutica in relation to temperature and food

From March 2004 to February 2005,seasonal variations in lipid content and fatty acid composition of gonad of the cockle Fulvia mutica(Reeve) were studied on the eastern coast of China in relation to the reproductive cycle and environment conditions(e.g.,temperature and food availability).Histological analysis as well as lipid and fatty acid analyses were performed on neutral and polar lipids of the gonad.Results showed that gametogenesis occurred in winter and spring at the expense of lipids previously accumulated in summer and autumn,whereas spawning occurred in summer(20.4-24.6℃).The seasonal variation in lipid content was similar to that of the mean oocyte diameter.In both neutral and polar lipids,the 20:5n-3 and 22:6n-3 levels were relatively higher than saturated fatty acids,and polyunsaturated fatty acids were abundant,with series n-3 as the predominant component.Seasonal variations in the 20:5n-3 and 22:6n-3 levels and the principal n-3 fatty acids were clearly related to the reproductive cycle.The ∑(n-3) and ∑(n-6) values were relatively high during January-May,and the associated unsaturation index was significantly higher than that in other months.The results suggest that fatty acids play an important role in the gametogenesis of F.mutica.     

Effects of dietary carbohydrates sources on lipids compositions in abalone, Haliotis discus hannai Ino

A study was conducted to evaluate the effects of dietary carbohydrates on triglyceride, cholesterol and fatty acid concentrations in abalone, Haliotis discus hannai Ino. Six semi-purified diets with different carbohydrates (dextrin, heat-treated wheat starch, wheat starch, corn starch, tapioca starch and potato starch, respectively), all containing a carbohydrate level of 33.5%, were fed to abalone (initial shell length: 29.98 mm ± 0.09 mm; initial weight: 3.42 g ± 0.02 g) for 24 weeks in a recirculation system. The results indicate that serum triglyceride concentrations were significantly (P < 0.05) higher in the abalone fed with dextrin, heat-treated wheat starch and wheat starch than those fed with corn starch, and serum cholesterol concentrations were significantly (P < 0.05) higher in the abalone fed with dextrin, heat-treated wheat starch than those fed with corn starch. Fatty acid C20:4n-6 in the foot muscles were significantly (P < 0.05) lower in the abalone fed with dextrin than those fed with wheat starch, corn starch, tapioca starch and potato starch. Fatty acid C20:4n-6 in hepatopancreas was significantly (P < 0.05) lower in abalone fed with heat-treated wheat starch than those fed with corn starch, tapioca starch and potato starch. Fatty acid C22:6n-3 in the foot muscles were significantly (P < 0.05) lower in the abalone fed with dextrin and heat-treated wheat starch than those fed with wheat starch and potato starch.     

Comparison of lipids in organs of the starfish Asterias amurensis associated with different treatments

Lipids were extracted from organs of the starfish Asterias amurensis associated with different treatments(raw-control,boiling and heating),and then analyzed for lipid content,lipid oxidation index,lipid classes and fatty acid composition.Results showed that boiling softened the hard starfish shells,thus facilitating the collection of starfish organs.As compared with raw organs,the boiled organs had lower water content and higher lipid content,possibly due to the loss of water-holding capacity caused by protein denaturation.Both boiling and heating increased the peroxide value(PV),thiobarbituric acid(TBA) value and carbon value(CV) of lipids.Despite slight increases in the content of complex lipids,associated lipid composition had no substantial variations upon boiling and heating.For simple lipids,the content of 1,2-diglyceride decreased in boiled and heated organs,with free fatty acids observed on thin layer chromatography(TLC).However,neither boiling nor heating significantly changed the fatty acid compositions of simple or complex lipids in starfish organs,suggesting that these two treatments had no significant effects on complex lipids in starfish organs.Together,our results indicated that boiling of starfish soon after capture facilitated the handling and extraction of useful complex lipids consisting of abundant glucosylceramide and eicosapentaenoic acid(EPA)-bounded phospholipids.     

Lipid and Fatty Acid Compositions of Cod(Gadus morhua),Haddock(Melanogrammus aeglefinus)and Halibut(Hippogiossus hippoglossus)

This study was conducted to compare lipid and fatty acid composition of cod,haddock and halibut.Three groups of cod(276 g±61 g),haddock(538 g±83 g)and halibut(3704 g±221 g)were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling.The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters(FAME).Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak(R).The phospholipid fraction was further separated by high-performance thin-layer chromatography(HPTLC)and the FAME profile was obtained.Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%,respectively,with phospholipid constituting 83.6% and 87.5% of the total muscle lipid,respectively.Halibut was a medium-fat fish and its muscle lipid content was 8%,with 84% of the total muscle lipid being neutral lipid.Total liver lipid contents of cod,haddock and halibut were 36.9%,67.2% and 30.7%,respectively,of which the neutral lipids accounted for the major fraction(88.1%-97.1%).Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid.Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid.Fatty acid compositions of phospholipid were relatively constant.In summary,the liver of cod and haddock as lean fish was the main lipid reserve organ,and structural phospholipid is the major lipid form in flesh.However,as a medium-fat fish,halibut stored lipid in both their liver and muscle.     

Lipid and fatty acid compositions of cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>), haddock (<Emphasis Type="Italic">Melanogrammus aeglefinus</Emphasis>) and halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis>)

This study was conducted to compare lipid and fatty acid composition of cod, haddock and halibut. Three groups of cod (276 g ± 61 g), haddock (538 g ± 83 g) and halibut (3704 g ± 221 g) were maintained with commercial feeds mainly based on fish meal and marine fish oil for 12 weeks prior to sampling. The fatty acid compositions of muscle and liver were determined by GC/FID after derivatization of extracted lipids into fatty acid methyl esters (FAME). Lipids were also fractionated into neutral and polar lipids using Waters silica Sep-Pak?. The phospholipid fraction was further separated by high-performance thin-layer chromatography (HPTLC) and the FAME profile was obtained. Results of the present study showed that cod and haddock were lean fish and their total muscle lipid contents were 0.8% and 0.7%, respectively, with phospholipid constituting 83.6% and 87.5% of the total muscle lipid, respectively. Halibut was a medium-fat fish and its muscle lipid content was 8%, with 84% of the total muscle lipid being neutral lipid. Total liver lipid contents of cod, haddock and halibut were 36.9%, 67.2% and 30.7%, respectively, of which the neutral lipids accounted for the major fraction (88.1%–97.1%). Polyunsaturated fatty acids were the most abundant in cod and haddock muscle neutral lipid. Monounsaturated fatty acid level was the highest in halibut muscle neutral lipid. Fatty acid compositions of phospholipid were relatively constant. In summary, the liver of cod and haddock as lean fish was the main lipid reserve organ, and structural phospholipid is the major lipid form in flesh. However, as a medium-fat fish, halibut stored lipid in both their liver and muscle.     

Lipids and fatty acids in Calanus sinicus during over-summering in the southern Yellow Sea

Over-summering is a crucial period for Calanus sinicus in the southern Yellow Sea,where it is a key member of the zooplankton community.Lipids play an important role in copepod diapause,which is part of their over-summering strategy.We investigated how different fatty acids and lipid classes,including wax esters,changed during over-summering of C.sinicus during three cruises in June and August 2011 and November 2010,corresponding to the pre-,during and post-diapause periods,respectively.Large amounts of lipids were accumulated,mainly wax esters as previously found in C.finmarchicus during its diapause,and most of the storage lipids were used during over-summering.Wax ester polyunsaturated fatty acids(PUFAs) showed the most variation of the fatty acids(FAs),while the percentage composition of FAs in polar lipids was relatively stable.Selective use of wax ester PUFAs has already been shown to play important roles in the winter diapause of Calanus species in other regions,and our FA results show that this is the case for the Yellow Sea Cold Bottom Water(YSCBW) population that diapauses in summer.     

Co-Production of Lipids,Eicosapentaenoic Acid,Fucoxanthin, and Chrysolaminarin by Phaeodactylum tricornutum Cultured in a Flat-Plate Photobioreactor Under Varying Nitrogen Conditions

The marine diatom Phaeodactylum tricornutum is a polymorphological, ecologically significant, and well-studied model of unicellular microalga. This diatom can accumulate diverse important metabolites. Herein, we cultured P. tricornutum in an internally installed tie-piece flat-plate photobioreactor under 14.5 m mol L~(-1)(high nitrogen, HN) and 2.9 m mol L~(-1)(low nitrogen, LN) of KNO_3 and assessed its time-resolved changes in biochemical compositions. The results showed that HN was inductive to accumulate high biomass(4.1 g L~(-1)). However, the LN condition could accelerate lipid accumulation in P. tricornutum. The maximum total lipid(TL) content under LN was up to 42.5% of biomass on day 12. Finally, neutral lipids(NLs) were 63.8% and 75.7% of TLs under HN and LN, respectively. The content of EPA ranged from 2.3% to 1.5% of dry weight during the growth period under the two culture conditions. Peak volumetric lipid productivity of 128.4 mg L~(-1)d~(-1) was achieved in the HN group(on day 9). The highest volumetric productivity values of EPA, chrysolaminarin, and fucoxanthin were obtained in the exponential phase(on day 6) under HN, which were 9.6, 93.6, and 4.7 mg L~(-1)d~(-1), respectively. In conclusion, extractable amounts of lipids, EPA, fucoxanthin, and chrysolaminarin could be obtained from P. tricornutum by regulating the culture conditions.     

Preservation of Lipid Molecular Fossils in the Sediments from an Oil-Gas Releasing Zone in the South China Sea

In order to explore the standing stock and preservation of organic carbon, different forms of compound specific lipids were analyzed, based on a 1.8 m sediment core collected in the northern continental shelf of South China Sea in May, 2016. In the core sediments, 21 species of fatty acids, 6 species of alcohols, 8 species of sterols and phytol were detected. Fatty acids were dominated components with the percent of 67%–91% in the total lipids, in which saturated fatty acid C16:0 accounted for about 50%. The contents of lipid species generally decreased from the sediments at surface to those deep, except alcohols waving in a certain range at the same time. Fatty acids primarily existed in free form, followed by base hydrolytic form, and then acid hydrolytic form. Free alcohols were obviously dominated, while base and acid hydrolytic forms contributed small comparably. Phytol gave a significant priority to base hydrolytic form, while acid hydrolytic form could be neglected. Similar to phytol, sterols mostly presented in base hydrolytic form, and then in free form. The compositions of lipids and their forms suggested that organic carbon basically originated from primary production in upper waters in studying area, and were well preserved in the sediments.     

Strain and culture medium optimization for production enhancement of prodiginines from marine-derived Streptomyces sp. GQQ-10

A mutant (GQQ-M6) of a Sponge-Derived streptomyces sp. GQQ-10 obtained by UV-induced mutation was used for producing prodiginines (PGs). Single factor experiments and orthogonal array design (OAD) methods were employed for medium optimization. In the single factor method, the effects of soluble starch, glucose, soybean flour, yeast extract and sodium acetate on PGs production were investigated individually. In the subsequent OAD experiments, the concentrations of these 5 key nutritional components combined with salinity were further adjusted. The mutant strain GQQ-M6 gave a 2.2-fold higher PGs production than that of the parent strain; OAD experiments offered a PGs yield of 61mg L-1, which was 10 times higher than that of the initial GQQ-10 strain under the original cultivation mode.     

The Mechanism of Lipids Extraction from Wet Microalgae Scenedesmus sp. by Ionic Liquid Assisted Subcritical Water

In this paper, the total sugar concentration, protein concentration, lipid yield and morphology characteristics of the algae residue were determined to explain the mechanism of lipids extraction from wet microalgae Scenedesmus sp. by ionic liquid assisted subcritical water. The results showed similar variation for the sugar, protein and lipid. However, the total sugar was more similar to lipids yield, so the results showed that the reaction between ionic liquid and cellulose and hemicellulose in cell wall was the most important step which determined the lipids extration directly. And the total sugar variation may be representing the lipids yield. For later lipids extraction, we can determine the total sugar concentration to predict the extraction end product.     

Changes of Protein and Lipid Contents,Amino Acid and Fatty Acid Compositions in Eggs and Yolk-Sac Larvae of American Shad(Alosa sapidissima)

To investigate the changes of the biochemical composition of American shad(Alosa sapidissima) eggs and larvae at embryonic and early larval stages, samples were collected at different development stages from artificial fertilization to the end of yolk absorption including 2 h, 12 h and 30 h after fertilization and newly hatched larvae including 1 and 3 days after hatching. The composition of lipid, fatty acids, protein and amino acids were analyzed. The content of total protein exhibited a decreasing trend during embryogenesis and larval development, and a significant reduction was detected after hatching(P 0.05). The total lipid content remained relative stable. A significant reduction was detected in almost all amino acids after hatching except for glycine(P 0.05), while a significant decrease was found in the content of cysteine, proline, tyrosine, valine, isoleucine, leucine and phenylalanine during the yolk-sac phase(P 0.05). On the other hand, all the groups of fatty acids remained stable during the period of embryogenesis. But after hatching, a significant decrease was found in the content of C18:2n-6, C18:3n-6, SFA and ratio of EPA/ARA(P 0.05), while a significant increase was found in the content of C18:3n-3, C20:4n-6, C22:6n-3 and ratio of n-3/n-6(P 0.05). In conclusion, the combined data suggested that American shad utilizes the protein content as preferential energy substrates during embryonic and early larval developments with some specificity in the consumption of different amino acids.     

Amylase production by the marine yeast Aureobasidium pullulans N13d

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pacific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identification methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 °C, aeration rate 6 Lmin⁻¹ and agitation speed 250 rmin⁻¹. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.     

Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensiss,cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exous and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca2+ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.     

Effect of soil salinity on cold tolerance of mangrove Kandelia candel

Analysis of cold tolerance on mangroveKandelia candel leaf growing in different soil salinity along Jiulong River Estuary in South China showed that the cold tolerance decreased as the increase of soil salinity. The lethal temperatures ofK. candel leaf were −10.4, −9.9 and −8.6 °C in Liaodong, Baijiao and Aotou, respectively. Under 1–2°C cold stress treatment on detached leaves ofK. candel, their caloric value gradually decreased, while electrolyte leakage gradually increased. The leaf's caloric value and electrolyte leakage in Aotou with higher soil salinity varied more largely than those in Liaodong with lower soil salinity. InK. candel leaf, total water content lowered a little, bound water content rose significantly and free water content dropped significantly with duration of cold stress. At the same time, reduction sugar, soluble sugar and starch content gradually decreased and sucrose content gradually increased. Bound water, free water and sucrose content inK. candel leaf from Aotou with higher soil salinity changed more slowly than those from Liaodong with lower soil salinity, but reduction sugar, soluble sugar and starch content inK. candel leaf from Aotou had faster variations than those from Liaodong. These data indicated that soil salinity can reduce cold tolerance ofK. candel leaf by increasing negative effect of salt ions in cell membrane, inhibiting variations of water content, and aggravating consumption of material and energy. Supported by the Found for Outstanding Young Teacher, the Ministry of Education, China.