Effects of polyamines on the early development of cystocarps,spore release,survival,and germination rate of Gracilariopsis lemaneiformis

Three polyamines(PAs)(spermidine(Spd),spermine(Spm),and putrescine(Put))were used as growth regulators to the marine macroalgae Gracilariopsis lemaneiformis and to female gametophyte and reproductive tissues(cystocarps were constructed),which led to early maturation of cystocarps and spore release.Laboratory cultivation revealed that exogenous PA application accelerated the development of cystocarps,and the mean sporeling growth rate was 20%–50%/d during the initial week of cultivation.The spore count(SC),survival rate(SR),and germination rate(GR)of G.lemaneiformis showed different responses to PA(10-6 mol/L and 10-3 mol/L)treatments under different temperatures(18,26,and 34℃),light intensities(30,60,and 90μmol photons/(m^(2)·s)),salinities(25,30,and 35),and nutrient levels(25꞉2.5,50꞉5.0,and 75꞉7.5μmol/L NH4Cl꞉K2HPO4),and the optimal conditions were 26℃,light intensity of 60μmol/(m^(2)·s),salinity of 35,and nutrient level of 50꞉5.0μmol/L NH4Cl꞉K2HPO4.Under the optimum conditions,the SC per cystocarp increased by 82.38%,and the spore SR and GR increased by 33.07%and 46.44%,respectively.Each experiment lasted for 7 days,with the highest SC on Day 4 and plateauing on Day 7.The results indicate that PAs could promote the early maturation,spore release,survival,and germination of G.lemaneiformis under laboratory conditions.

Transcriptome Analysis During Tetrasporogenesis of Gracilariopsis lemaneiformis and Preliminary Study of the Expressions of Its Meiotic Genes

The transcriptomes of three different parts of the fertile tetrasporophyte of Gracilariopsis lemaneiformis,including tip(T),middle(M),and subjacent(S)parts,with a gradual tetrasporangium maturity were analyzed and compared to identify the genes involved in the process of tetrasporogenesis.The number of differentially expressed genes(DEGs)for the Gple-S versus Gple-T comparison was 10296,and the numbers of DEGs for the Gple-S versus Gple-M and Gple-T versus Gple-M comparisons were 7435 and 1337,respectively.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed,and the results showed the enrichment of 132 KEGG pathways(corrected P<0.05).A total of 58 DEGs related to meiosis were screened and blasted against 18 meiosis-related genes(dmc1,mlh1,mnd1,msh4,msh2,msh6,mre11,pds5,pms1,rad21,rad50,rad51,smc1,smc2,smc4,smc5,smc6,and spo11),including four meiosis-specific genes.The transcriptome comparison indicated that in the T part,the meiosis,ribosome,and RNA transport-related genes were mostly up-regulated compared with those in the other two groups.In the M part,the genes related to ribosomes and the endoplasmic reticulum were also up-regulated compared with those in the lower part.Finally,in the S part,the genes associated with photosynthesis were mostly up-regulated,which might be helpful to the recovery from spore formation and release.

Transcriptome Analysis and Physiological Responses of Economic Macroalga Gracilariopsis lemaneiformis under Sulfate Deficiency

Sulfur is an essential macronutrient for the growth of all photosynthetic organisms and plays important roles in different metabolic pathways.However,sulfur metabolism and its related research on macroalgae with important ecological value is rather limited.In this study,marine ecological valued macroalga Gracilariopsis lemaneiformis was used to study the general physiological responses and transcriptome profiling under the sulfate deficiency.The relative growth rate of algae under sulfate deficiency was statistically significantly lower than that of control after 6 days.However,no significant differences were observed in the pigments content and Fv/Fm value,indicating that the photosynthesis was not obviously affected under the sulfate deficiency.Furthermore,the significantly increased soluble protein and carbohydrate contents,and indistinctively differentially expressed sulfate transporter/permease(ST)and ATP sulfurylase(ATPS)genes suggesting that G.lemaneiformis cells did not use sulfur from internal protein and carbohydrate pools but utilize stored sulfur from vacuole to satisfy their nutritional requirements during the sulfate deficiency.Transcriptome data showed that most annotated differentially expressed genes(DEGs)were down-regulated under the sulfate deficiency,including a large proportion of genes involved in the pathway of sulfur metabolism.Therefore,it was concluded that the pathway of sulfur metabolism was suppressed under the sulfate deficiency in G.lemaneiformis.The results and analysis in this study provide a comprehensive understanding for the physiological and molecular response of G.lemaneiformis to sulfate deficiency.

龙须菜(Gracilariopsis)/江蓠(Gracilaria)的生长和种质特性分析

龙须菜(Gracilariopsislemaneiformis)是具有重要经济价值和生态效益的大型红藻,主要用作琼胶提取原料和鲍的饵料。本研究利用生理生化、液相色谱-质谱联用和氨基酸分析等方法,比较了龙须菜(Gp.lemaneiformis)(wt、981、Gl-1、Gl-s、Gl-g)、异枝龙须菜(Gp.heterocla,Gh)和细基江蓠繁枝变种(Gracilaria tenuistipitata var.liu,Gt)的生长及藻胆蛋白、琼胶、红藻糖苷和氨基酸等的差异,以期为龙须菜/江蓠栽培中的种质区分及选择提供参考。结果表明Gt在23℃和30℃条件下生长均最快,其相对生长速率分别为野生型龙须菜(wt)的2.19倍和2.49倍。龙须菜Gl-s中藻胆蛋白浓度最高,为wt的1.91倍。除了Gt之外,其余6种藻中琼胶产率较高(16.22%~18.91%)。Gt中红藻糖苷和海藻糖积累最多,分别为wt的3.50倍和1.81倍。Gl-g、Gl-s、Gt和Gh多糖丰富,在36.89%~40.23%;龙须菜981、Gl-1、wt和Gl-s总氨基酸浓度较高,在152.35~161.32mg·g^(-1)干质量之间,并且981、Gl-1、Gl-s氨基酸评分较优。综合以上结果,龙须菜981、Gl-1和Gl-s的藻胆蛋白、琼胶和氨基酸等均显著优于其他藻,并且生长较快,可用于琼胶、藻胆蛋白及多糖的提取或鲍的饵料;而细基江蓠繁枝变种生长快、红藻糖苷和海藻糖丰富,可大规模栽培用作鲍的饵料。该研究为丰富及开发利用中国大型海藻种质资源提供了重要的资料。

A Comparison of Different Gracilariopsis lemaneiformis(Rhodophyta) Parts in Biochemical Characteristics, Protoplast Formation and Regeneration

Gracilariopsis lemaneiformis is a commercially exploited alga. Its filaceous thallus can be divided into three parts, holdfast, middle segment and tip. The growth and branch forming trend and agar content of these three parts were analyzed, respectively, in this study. The results showed that the tip had the highest growth rate and branched most, although it was the last part with branch forming ability. The holdfast formed branches earliest but slowly. Holdfast had the highest agar content. We also assessed the difference in protoplast formation and regeneration among three parts. The middle segment displayed the shortest enzymolysis time and the highest protoplast yield; whereas the tip had the strongest vitality of protoplasts formation. Juvenile plants were only obtained from the protoplasts generated from the tip. These results suggested that the differentiation and function of G. lemaneiformis was different.

Changes in the Photosynthetic Pigment Contents and Transcription Levels of Phycoerythrin-Related Genes in Three Gracilariopsis lemaneiformis Strains Under Different Light Intensities

Three Gracilariopsis lemaneiformis strains,including wild type and high-temperature-resistant cultivars 981 and 2007,were studied with the changes in their photosynthetic pigment contents and related gene transcription levels under different light intensities(10,60,100,and 200μmolm^(−2)s^(−1)).The three G.lemaneiformis strains had the following photosynthetic pigments with high-to-low contents:phycoerythrin(PE),phycocyanin(PC),allophycocyanin(APC),and chlorophyll a(Chl a).Among the three strains,cultivar 981 had the highest PE content,followed by cultivar 2007.The PC and APC contents were similar among the three strains,but they were higher in cultivars 981 and 2007 than in the wild type.The Chl a contents in the three G.lemaneiformis strains were equal.A low light intensity(10μmolm^(−2)s^(−1))promoted photosynthetic pigment accumulation in G.lemaneiformis and improved the relative PE gene transcription(peA and peB)in a short period(≤6 d).A high light intensity decreased the PE content.PebA and PebB,which catalyzed phycoerythrobilin synthesis,showed no compensatory upregulation at a low light intensity among the strains except for the wild type.At a high light intensity,transcription levels of pebA and pebB in the three strains were upregulated.This study provided an experimental basis for elucidating the photosynthesis of G.lemaneiformis.As key genes of algal growth,photo-synthesis-related genes served as useful gene markers for screening elite varieties with good traits in breeding.Cultivar 2007 was superior to cultivar 981 in terms of maintaining high pigment levels in a wide range of light intensities,which is the most suitable for aquaculture.

Eff ects of temperature on photosynthetic performance and nitrate reductase activity in vivo assay in Gracilariopsis lemaneiformis (Rhodophyta)

Gracilariopsis lemaneiformis is an economically-valued species and widely cultured in China at present.After being acclimated to diff erent growth temperatures(15,20,25,and 30°C)for 7 days,the relative growth rate(RGR),nitrate reductase activity,soluble protein content and chlorophyll a fl uorescence of G.lemaneiformis were examined.Results show that RGR was markedly aff ected by temperature especially at 20°C at which G.lemaneiformis exhibited the highest eff ective quantum yield of PSII[Y(II)]and lightsaturated electron transport rate(ETR max),but the lowest non-photochemical quenching.Irrespective of growth temperature,the nitrate reductase activity increased with the incubation temperature from 15 to 30°C.In addition,the greatest nitrate reductase activity was found in the thalli grown at 20°C.The value of temperature coeffi cient Q10 of alga cultured in 15°C was the greatest among those of other temperatures tested.Results indicate that the optimum temperature for nitrate reductase synthesis was relatively lower than that for nitrate reductase activity,and the relationship among growth,photosynthesis,and nitrate reductase activity showed that the optimum temperature for activity of nitrate reductase in vivo assay should be the same to the optimal growth temperature.

Biomass and carbon storage of Gracilariopsis lemaneiformis(Rhodophyta) in Zhanshan Bay, Qingdao, China

Marine macroalgae can absorb carbon and play an important role in carbon sequestration. As an important economic macroalga, Gracilariopsis lemaneiformis has the potential to significantly affect carbon absorption and storage in wave-sheltered intertidal reef systems. However, detailed knowledge on seasonal biomass changes and carbon storage of G. lemaneiformis is lacking, especially in many small and scattered ecosystems. Considering the influence of human activities on wild distribution of G. lemaneiformis, the understanding of seasonal dynamics of an economically important species in nature is necessary. In this study, we first investigated seasonal variations in biomass, coverage area, and carbon storage during low tide from August 2011 to July 2012 in Zhanshan Bay, Qingdao, China. Furthermore, we estimated the carbon storage potential of wild G. lemaneiformis using light use efficiency （LUE）. The results show that the standing biomass and coverage area changed significantly with season. However, seasonal variations in carbon content and water content were not obvious, with an average content of 35.1% and 83.64%, respectively. Moreover, carbon storage in individual months varied between 0.67 and 47.03 g C/mE, and the value of carbon storage was the highest in August and June and the lowest in February. In Zhanshan Bay, LUE of G. lemaneiformis was only 0.23%. If it is increased to the theoretical maximum （5%-6%）, the carbon storage will have an increase of at least 21 times compared with the current, which suggested that carbon storage of wild G. lemaneiformis had a high enhancement potential. The study will help to assess a potential role of G. lemaneiformis in reducing atmospheric CO2

Analysis of DNA Methylation of Gracilariopsis lemaneiformis Under Temperature Stress Using the Methylation Sensitive Amplification Polymorphism(MSAP) Technique

Gracilariopsis lemaneiformis is an economically important agarophyte, which contains high quality gel and shows a high growth rate. Wild population of G. lemaneiformis displayed resident divergence, though with a low genetic diversity as was revealed by amplified fragment length polymorphism(AFLP) and simple sequence repeat(SSR) analyses. In addition, different strains of G. lemaneiformis are diverse in morphology. The highly inconsistence between genetic background and physiological characteristics recommends strongly to the regulation at epigenetic level. In this study, the DNA methylation change in G. lemaneiformis among different generation branches and under different temperature stresses was assessed using methylation sensitive amplified polymorphism(MSAP) technique. It was shown that DNA methylation level among different generation branches was diverse. The full and total methylated DNA level was the lowest in the second generation branch and the highest in the third generation. The total methylation level was 61.11%, 60.88% and 64.12% at 15℃, 22℃ and 26℃, respectively. Compared with the control group(22℃), the fully methylated and totally methylated ratios were increased in both experiment groups(15℃ and 26℃). All of the cytosine methylation/demethylation transform(CMDT) was further analyzed. High temperature treatment could induce more CMDT than low temperature treatment did.

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.

Isolation and callus formation of Gracilariopsis bailiniae(Gracilariales, Rhodophyta) protoplasts

This paper reports the first successful isolation of protoplasts from G racilariopsis bailiniae and their callus formation. The base solution type, concentration of isolating enzymes, concentration of sorbitol, incubation time, temperature and pH of the enzyme solution were tested to optimize the protoplast yield. The optimized isolation conditions were: 40% base solution 3(deionized water containing 25 mmol/L MESTris and 25 mmol/L CaCl 2 ·2 H 2 O) and 60% crude Marinomonas sp. YS-70 agarase solution, containing 2% w/v cellulase, 1% w/v macerozyme R-10 and 0.4 mol/L sorbitol, with incubation for 4 h at 28°C and pH 6.5. The highest yield of viable protoplasts, which was obtained in these conditions, was(1.75±0.25)×10 6 cells/g fresh weight. Cell wall regeneration of most protoplasts from G. bailiniae was complete within 60 h and the first division of cells happened after ≥3 days. Two division types were observed in the first division of protoplasts from G. bailiniae— asymmetric division and symmetric division. After the first division, the cells underwent a series of divisions to form callus cell masses.

Structure of reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis (Gracilariaceae,Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial devel-opment revealed the similar cytodynamic pattern followed by all the Gracilariaceae members devel-oped from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released.

温度和氮营养盐对龙须菜生长与光合生理的影响

以栽培龙须菜(Gracilariopsis lemaneiformis)为试验材料,设置2个温度水平(LT:20℃;HT:24℃)和3个氮营养盐浓度(LN:3.1μmol/L;MN:30.1μmol/L;HN:103.1μmol/L),研究2个环境因子变化对龙须菜生长和光合生理的影响。结果表明,温度、氮营养盐及二者交互作用对龙须菜的相对生长速率(RGR)均具有显著影响(P<0.05),RGR随着温度的升高、氮营养盐浓度的增加而升高。叶绿素荧光参数分析显示,在同一氮营养盐浓度下,HT处理组的有效光合量子产量[Y(Ⅱ)]在0.18~0.24内波动,均高于LT处理组,在同一温度培养条件下,HN处理组的Y(Ⅱ)普遍高于LN处理组;温度对龙须菜的最大相对电子传递速率(rETRmax)存在显著影响(P<0.05);氮营养盐对不同处理下龙须菜的rETRmax、光能利用效率(α)、饱和光强(Ik)均存在显著影响(P<0.05),二者交互作用对α存在显著影响(P<0.05)。此外,温度、氮营养盐及二者交互作用对龙须菜的光合色素含量均具有显著影响(P<0.05);氮营养盐及二者交互作用对其可溶性蛋白质(SP)含量存在显著影响(P<0.05);在LT或HT条件下,氮营养盐浓度的增加显著促进叶绿素a(Chl a)、类胡萝卜素(Car)和SP的合成(P<0.05)。HN和HT促进了龙须菜的生长,结果为解析龙须菜应对核电站温排水区的环境变化提供了理论数据。

热锻炼对龙须菜高温胁迫响应的影响

夏季高温是限制龙须菜(Gracilariopsis lemaneiformis)生长和发育的一个主要问题。本研究以野生龙须菜作为材料,经过28℃的热锻炼预处理72 h,再分别置于32、36、40℃高温胁迫24 h,并测定了高温胁迫条件下龙须菜的丙二醛(MDA)、过氧化氢(H2O2)和可溶性蛋白质的含量,以及超氧化物歧化酶(SOD)活性和抗坏血酸(AsA)水平等生理指标,研究了龙须菜对高温胁迫的响应。结果表明:热锻炼能维持藻体较低的MDA含量,降低H2O2的产生速率,缓解藻体的膜脂过氧化伤害;同时热锻炼缓解了可溶性蛋白质的降解,增加了SOD酶的比活力,缓解了AsA的下降速率,说明热锻炼能缓解高温胁迫对藻体的伤害,有效地提高了龙须菜对高温胁迫响应的能力。

Comparative analysis of four essential Gracilariaceae species in China based on whole transcriptomic sequencing

Three Gracilaria species, G. chouae, G. blodgettii, G. vermiculophylla and a close relative species, Gracilari-opsis lemaneiformis which is now nominated as Gracilaria lemaneiformis, are the typically indigenous spe-cies which are important resources for the production of special proteins, phycobilisomes, special carbo-hydrates, and agar in China. In this study, de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time. Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G. lema-neiformis and other three Gracilaria species. Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species. The genes encoding allophycocyanin, phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G. lemaneiformis had close relationship to genus Gracilaria, as well as that within genus Gracilaria, G. chouae had closer relationship to G. vermiculophylla rather than to G. blodgettii. The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.

Study of Morphology and Agar Contents in Some Important <i>Gracilaria</i>Species of Indian Coasts

This study reports the morphological, anatomical and agar content difference among various species of Gracilaria. The cortex was found 1-2 layered in G. edulis and G. eucheumatoides whereas 5-6 layered in G. foliifera and G. crassa. The medulla was 6-8 layered in G. edulis, 10-11 layered in G. foliifera, 14-15 layered in G. eucheumatoides and 8-10 layered in G. crassa. Similarly, distinct variations were observed in the structure of cystocarp of these taxa. The outer pericarp was 6-8 layered in G. verrucosa (attached type), 8-9 in Graciriopsis megaspora, 12-13 layered cells in G. edulis, 9-11 layered in G. foliifera, 12-15 layered in G. eucheumatoides and 11-14 layered in G. crassa. The tubular nutritive filaments were radiating type in G. edulis whereas these were penetrating and radiating type in G. foliifera. In G. crassa, the same was produced from a small swollen short base and in G. verrucosa (attached type) it was long. Further, in G. eucheumatoides and Gracilariopsis megaspore, nutritive filaments were not observed. Among various species of Gracilariales studied in this work, agar from Gracilaria verrucosa (floating type or sterile type) showed highest yield with gel strength 260 gm/cm2 and viscosity 10 milipass. Based on the agar quality,