Biosynthesis of tobacco mosaic virus RNA in tobacco protoplasts

Changes in the number of protoplasts, viability, protein and chlorophyll contents and ribonucleases activity were studied in tobacco mesophyll protoplastsin vitro inoculated with tobacco mosaic virus (TMV). The number of protoplasts slowly increased during the cultivation period and the viability decreased from 95 to 67% in the control noninoculated protoplasts, and to 55% in the infected protoplasts. 30 h after inoculation the protein and chlorophyll contents strongly decreased to 25–30% and 17–19%, respectively, in comparison with contents 3 h after inoculation. The chlorophylla/b ratio decreased from 2.11 and 2.02 to 0.79 and 0.60 in healthy and infected protoplasts, respectively. The activities of ribonucleases in protoplasts quickly decreased during experiment but they were higher in infected than in noninfected protoplasts (between 20 to 30 h after inoculation they were 132 to 146% higher than that in healthy controls). These activities corresponded to the multiplication curve of TMV.     

Isolation,culture and regeneration of protoplasts from birdsfoot trefoil (Lotus corniculatus)

A method was developed for rapid plant regeneration from protoplasts of birdsfoot trefoil (Lotus corniculatus L. cv. Leo). Green cotyledons from in vitro grown seedlings were preplasmolyzed in CPW salts containing 13% mannitol (CPW 13 M) for 1 h prior to the enzyme treatment. The enzyme formula consisted of 2% (w/v) Onozuka Cellulase R-10, 1% (w/v) Macerase and 0.1% (w/v) Pectolyase Y-23 in CPW 13 M. This method produced high yields of viable protoplasts after purification. The procedure is reproducible and takes approximately 2.5 months from protoplast isolation to plantlet establishment in a greenhouse. More than 100 plantlets were grown in soil. Two somaclonal variants, a chimeric plant for chlorophyll production and an albino cell line, have been obtained by this procedure.     

Photosynthetic performance of regenerated protoplasts from disintegrated cells of <Emphasis Type="Italic">Bryopsis hypnoides</Emphasis> (Chlorophyta)

The photosynthetic performances of regenerated protoplasts of Bryopsis hypnoides, which were incubated in seawater for 1, 6, 12, and 24 h, were studied using chlorophyll (Chl) fluorescence and oxygen measurements. Results showed that for the regenerated protoplasts, the pigment content, the ratios of photosynthetic rate to respiration rate, the maximal photosystem II (PSII) quantum yield (F_v/F_m), and the effective PSII quantum yield (Φ_PSII) decreased gradually along with the regeneration progress, indicated that during 24 h of regeneration there was a remarkable reduction in PSII activity of those newly formed protoplasts. We assumed that during the cultivation progress the regenerated protoplasts had different photosynthetic vigor, with only some of them able to germinate and develop into mature thalli. The above results only reflected the photosynthetic features of the regenerated protoplasts at each time point as a whole, rather than the actual photosynthetic activity of individual aggregations. Further investigation suggested a relationship between the size of regenerated protoplasts and their viability. The results showed that the middle-sized group (diameter 20–60 μm) retained the largest number of protoplasts for 24 h of growth. The changes in F_v/F_m and Φ_PSII of the four groups of differently sized protoplasts (i.e. < 20, 20–60, 60–100, and > 100 μm) revealed that the protoplasts 20–60 μm in diameter had the highest potential activity of the photosynthetic light energy absorption and conversion for several hours.     

Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca²⁺, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C₃ leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C₃ protoplasts than in C₄ protoplasts or leaf tissue.Abbreviations CAM Crassulacean acid metabolism - Chl chlorophyll - CHM cycloheximide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - PEPCase PEP carboxylase     

The flexible interrelation between AOX respiratory pathway and photosynthesis in rice leaves.

Alternative respiratory pathway was investigated in rice seedlings grown under total darkness, light/dark cycle, or continuous light. The capacity of the alternative pathway was relatively higher in leaves that had longer light exposure. An analysis of rice AOX1 multigene family revealed that AOX1c, but not AOX1a and AOX1b, had a light-independent expression. The alternative oxidase (AOX) inhibitor, salicylhydroxamic acid (SHAM, 1mM), inhibited nearly 68% of the capacity of the alternative pathway in leaves grown under different light conditions. The plants grown under different light periods were treated with SHAM and then were exposed to illumination for 4h. The transition from dark to 4h of light stimulated the capacity of alternative pathway in etiolated rice seedlings and in those grown under light/dark cycle, whereas the capacity of the alternative pathway was constant in seedlings grown under continuous light with additional 4h of illumination. Etiolated leaves did not show any CO(2) fixation after 4h of illumination, and the increase in chlorophyll content was delayed by the SHAM pretreatment. When seedlings grown under light/dark cycle were moved from dark and exposed to 4h of light, increases in chlorophyll content and CO(2) fixation rate were reduced by SHAM. Although these parameters were stable in plants grown under continuous light, SHAM decreased CO(2) fixation rate but not the chlorophyll content. These results indicate that the role and regulation of AOX in light are determined by the developmental stage of plant photosynthetic apparatus.     

Light- and sucrose-dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape (Brassica napus L.)

Light-dependent gene expression was analysed in photomixotrophic cell suspension cultures of rape (Brassica napus L.) growing in media containing either 2.0% or 0.6% sucrose. During growth in darkness phytochrome type I and NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) accumulated in both cell culture lines to a similar extent. Illumination with continuous white, blue or red light, but not with far-red light, resulted in disappearance of both chromoproteins within 24 h in both cell cultures. Further analysis showed that the phytochrome system of rape cell cultures reacts in a similar way to that of re-etiolated dicotyledonous plants, showing rapid P_fr destruction and rapid P_fr dark reversion. In contrast, the light-dependent expression of genes encoding the major chlorophyll a- and b-binding protein (CAB) and the re-accumulation of chlorophyll were found to be strongly dependent on sucrose concentration in culture media. Whereas cells grown in darkness in medium containing 2.0% sucrose showed, after exposure to continuous white light, a very weak re-induction of CAB mRNA, CAB protein and chlorophyll accumulation, the cells in medium containing 0.6% sucrose reacted very strongly. It was also possible to demonstrate that phytochrome (by high irradiance response, HIR, and by low fluence response, LF) and the blue/UV-A receptor are involved in the light-dependent gene expression of CAB. Similar to complete cells, protoplasts derived from the two different cell cultures showed an almost identical sucrose concentration-dependent and light-quality-dependent regulation of CAB mRNA accumulation. As the dark-grown photomixotrophic cells and protoplasts reflect some typical photoregulatory characteristics known from dark-grown plants it is supposed that this system will be an excellent tool for studying biochemical and molecular biological aspects of light-dependent signal transduction in cells of higher plants.     

Plant regeneration from protoplasts of peppermint (Mentha piperita L.)

Summary Enzymatically isolated leaf-derived protoplasts of peppermint (Mentha piperita L.) were cultured in modified B5 medium containing 1 mg/l NAA, 0.4 mg/l BA, 0.5% sucrose, 0.5 M mannitol and 0.1% Gelrite (first medium). After 30 d culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. Gelrite medium blocks were transferred into liquid medium to promote further growth. Colonies of 0.5 mm transferred to 0.2% Gelrite solidified medium (same components as first medium) formed green calli (1–2 mm) under incubation in the light. Green calli transferred to differentiation medium (B5, 0.1 mg/l NAA, 5 mg/l BA, 2% sucrose, 0.2 M mannitol, 0.2% Gelrite) developed shoot buds after 3–4 weeks. Whole plants were recovered following rooting of shoots in B5 medium without hormones.Abbreviations BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - KIN kinetin - ZEA zeatin - CPW cell and protoplast wash solution - B5 Gamborg et al. (1968) mineral elements - MS Murashige and Skoog (1962) mineral elements     

A co-culture system leads to the formation of microcalli derived from microspore protoplasts ofBrassica napus L. cv. Topas

Summary This paper describes a procedure in which protoplasts are obtained from microspores and pollen of rapeseed to induce callus formation aided by a feeder cell system with embryogenic microspores. Microspores at late unicellular stage and pollen at early bicellular stage were isolated and precultured for 24 h at 32 °C before enzymatic treatment. Eleven enzymes were tested in various combinations and concentrations. The optimal enzyme combination was 1.0% cellulase, 0.8% pectinase, 0.3% macerozyme, and 0.02% pectolyase, in which 26.3% of the microspores released protoplasts. A successful co-culture system was set up by employing embryogenic microspores as feeder cells. To this end, microspores were cultured in a medium with high osmotic pressure at 32 °C. Up to 37% of the microspores exhibited cell division and embryos developed to the heart-shape stage without changing medium. Microspore protoplasts were cultured in Millicells surrounded by the embryogenic microspores as feeder. In growth-regulator-free medium 14.5% of the protoplasts divided but only formed budding-like multicellular structures. Only after pretreatment with 4 mg of 2,4-dichlorophenoxyacetic acid and 1 mg of naphthaleneacetic acid per liter protoplasts divided and formed microcalli. Pollen tubes or tubelike structures were not observed. The experiments reveal that selection of the specific developmental stage of microspores, which is a prerequisite for microspore embryogenesis, is also important in microspore protoplast culture. Compared to other methods used before, microculture fed with embryogenic microspores has obvious superiority.Abbreviations CPW basic protoplast washing medium according to Power and Chapman - CPW972 CPW basic medium supplemented with 9% mannitol and 7.2% sorbitol - DAPI 4,6-diamidino-2-phenylindole - NLN nutrient medium according to Lichter modified by Pechan and Keller - NLN13 NLN medium supplemented with 13% sucrose - NLNP NLN13 supplemented with 7.2% sorbitol     

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L

A mass spectrometric method combining ¹⁶O/¹⁸O and ¹²C/¹³C isotopes was used to quantify the unidirectional fluxes of O₂ and CO₂ during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O₂ uptake and CO₂ evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O₂ (61 micromoles of O₂ per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O₂ per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO₂ in darkness at a rate of 27 micromoles of CO₂ per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O₂ evolution and CO₂ fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO₂ evolution by guard cell protoplasts was sharply decreased (37%), while O₂ uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO₂ assimilation and may be dissipated for other purposes such as ion uptake.     

Phosphorylation and Dephosphorylation of Guard-Cell Proteins from Vicia faba L. in Response to Light and Dark

Phosphorylation and dephosphorylation of proteins were investigated in guard-cell protoplasts from Vicia faba L. When guard-cell protoplasts were incubated with 32Pi in the dark for 80 min, several proteins, with molecular masses of 42, 40, 34, 32, 26, and 19 kD, were phosphorylated. Illumination of the dark-adapted protoplasts with red light caused dephosphorylation of the 26-kD protein, but there was no detectable change in levels of phosphorylation in other proteins. In the dephosphorylation of the 26-kD protein, far-red light of 730 nm was most effective, but when the light was turned off, the protein was phosphorylated to the original level within 10 min. Subcellular fractionation of guard-cell protoplasts indicated that the 26-kD protein was located in the chloroplast. The migration pattern of the 26-kD protein was exactly the same as the light-harvesting Chl a/b protein complex of photosystem II (LHCPII) from Vicia mesophyll cells on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dephosphorylated 26-kD protein was phosphorylated by adding sodium hydrosulfite, a strong reducing agent, under the far-red illumination of guard-cell protoplasts. The magnitude of dephosphorylation by red light (660 nm) was increased by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an electron transfer inhibitor of photosystem II (PSII). Light-induced dephosphorylation was inhibited by 1 nM okadaic acid, an inhibitor of serine/threonine protein phosphatase. From these results, it is concluded that the 26-kD protein is LHCPII and that LHCPII is present mostly in the phosphorylated form in the dark and is dephosphorylated by type 2A protein phosphatase under the light absorbed by photosystem I in Vicia guard-cell protoplasts.     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

利用荧光染色和流式细胞技术辅助卵孢小奥德蘑原生质体制备与再生研究

本研究以卵孢小奥德蘑液体培养菌丝作为实验材料,利用单因子变量法探索研究了菌丝培养时间、酶浓度、酶解时间、酶解温度、稳渗剂类型对卵孢小奥德蘑原生质体制备的影响,并对原生质体再生培养基进行选择和优化。通过荧光染色,利用激光共聚焦显微镜和流式细胞仪对原生质体的制备过程、得率和活力进行研究。结果表明,将卵孢小奥德蘑菌丝在液体培养基中培养5d收集菌丝体,以甘露醇作为渗透压稳定剂,在溶壁酶浓度2%、30℃条件下酶解5h,获得的原生质体得率最高,达2.0×10 ⁷个/mL;通过流式细胞仪分析,约57.69%的原生质体细胞为活细胞;在RM培养基中再生效果最好,再生率为(0.103±0.025)%。研究结果可以为卵孢小奥德蘑育种与食用菌原生质体制备再生提供研究基础。     

光强对烟草幼苗形态和生理指标的影响

通过白纱布遮荫模拟不同光生境条件(透光率分别为100%、68.2%、35.4%和16.7%),研究了光强因子对烟草幼苗形态和生理指标的影响.结果表明:随相对光强的减弱,幼苗高度增加,茎粗、干鲜比、叶片厚度和单位叶面积质量均呈降低趋势,幼苗干物质积累减少,但其对叶数的影响不大.弱光条件下,叶片自由水、叶绿素、总氮和蛋白质含量增加,束缚水含量降低,叶绿素a/b值减小,转化酶活性降低;烟草幼苗根系相对不发达,根冠比和根生物量减小,根系活力降低.表明弱光条件不利于培育烟草壮苗,生产中应尽可能改善苗床的光照条件.     

Evaluation of UV damage at DNA level in <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> protoplasts using single cell gel electrophoresis

The aim of the present study was to observe the induction and repair of single strand breaks (Ssbs) and double strand breaks (Dsbs) in mesophyll protoplasts of Nicotiana plumbaginifolia, irradiated with UV-C and cultured under light or dark conditions. DNA damage and repair was determined by the neutral and alkaline comet assay to reveal Dsbs and Ssbs respectively. Subculturing protoplasts for 4 h at low temperature was essential to reduce the amount of Dsbs to the detection limit of the assay procedure. Light-cultured protoplasts showed a significant increase of Ssbs and Dsbs compared to dark cultured protoplasts, in which the number of Ssbs and Dsbs remained very constant throughout the experiments. UV treatment significantly enhanced the levels of Ssbs and Dsbs in light and dark cultured protoplasts. On average, equal levels of DNA damage were observed under light or dark conditions. Formulations introduced to evaluate the contribution of UV-C or light treatment in repair kinetics of DNA damage, showed that the number of Ssbs, but not of Dsbs, evolved differently for light and dark cultured protoplasts. DNA repair was more rapidly observed under light conditions and occurred in different repair phases. Observations are discussed in relation to the involvement of chromatin remodelling, photosynthetic active radiation and DNA repair mechanisms.     

三倍体‘银中杨’叶肉原生质体制备的优化

以三倍体杨树品种‘银中杨’（Populus alba×P.berolinensis Yinzhong）无菌苗叶片为材料,对其原生质体分离及纯化条件进行研究,为进一步通过细胞融合、基因工程等进行品种改良探索新的途径。结果表明：酶的种类及浓度、渗透压、酶解时间对‘银中杨’叶肉原生质体分离效果有显著影响,适宜的分离条件为CPW+3% Cellulase RS+0.5% Macerozyme R-10+0.3% Pectinse Y-23+0.6 mol/L甘露醇+0.6 g/L MES+1 g/L BAS,酶解时间为8 h,原生质体产量和活力分别为2.13×10⁷个/g和80.18%;‘银中杨’叶肉原生质体纯化最佳方法为上浮法蔗糖等密度离心,且蔗糖浓度为40%时原生质体产量最高（1.06×10⁷个/g）,可满足进一步的原生质体培养等技术的要求。     

Isolation and culture of protoplasts from embryogenic suspension cultures of red fescue (Festuca rubva L.)

Protoplasts were isolated from fast-growing embryogenic suspension cultures of red fescue cv. Dawson (Festuca rubra L.) without agitation. The enzyme isolation solution was highly efficient at releasing protoplasts of greater than 95% viability (5×10⁶–10⁷ protoplasts per ml of packed cell volume). A three step procedure was followed for washing and transferring protoplasts from a solution high in inorganic salts to a medium containing glucose and sucrose. The addition of 30 mM sodium thiosulfate to the wash and culture media was found to be helpful in reducing the number of lysed protoplasts. Isolated protoplasts began to divide within 48–72 h when protoplasts were plated in agarose squares and surrounded by nurse cells (mixed nurse plating technique). Maximum colony formation (plating efficiency) was approximately 1%. Many of the colonies continued to grow and produced embryos when transferred to a medium consisting of half-strength MS salts, 4 mg/l 2,4-D, 3 g/l casein hydrolysate and 30 g/l sucrose. Upon transfer to hormone-free medium and exposure to light 16 h/day, many of the embryos germinated to produce green leaves and roots.Abbreviations BA Benzylaminopurine - 2,4-D 2,4-dicholorophenoxyacetic acid - DMSO dimethyl sulfoxide - MES 2-(N-morpholino)-ethanesulfonicn acid - MS Murashige and Skoog medium (1962) - UGC Ultraclone Growth Chamber - KM Kao and Michayluk medium (1975) - NAA Naphthalene acetic acid     

A system in which anthocyanin synthesis is induced in regenerated torenia shoots

A system in which anthocyanin synthesis can be induced under defined conditions was established in regenerated torenia shoots. Leaf discs prepared from torenia plantlets grown under sterile conditions were placed on solidified half-strength MS medium containing 3% sucrose and 4.4×10^–6 M benzyladenine (BA) and cultured under 16 h light/8 h dark (standard light) conditions for 10 days, then in the dark for a further 10 days. The discs were transferred to medium containing 7% sucrose without BA and cultured under standard light conditions. Six days after transfer, anthocyanin synthesis started in the regenerated shoots, and thereafter, anthocyanin accumulation increased while chlorophyll content decreased. Experiments in which either the timing of illumination was altered or shoots were retransferred to medium containing 1.5% sucrose or other sugars as well as sucrose indicated that both osmotic stress and light are required to induce anthocyanin synthesis. Once anthocyanin synthesis was induced in the torenia shoots 6 days after transfer, the shoots were fated to the synthesis of anthocyanins and the degradation of chlorophylls, and could not revert to the developmental pathway of shoot regeneration. This system may provide a good model for the investigation of the mechanisms underlying the induction of anthocyanin synthesis.     

沙冬青子叶原生质体瞬时表达体系的建立及其AmDREB1蛋白的亚细胞定位

以沙冬青(Ammopiptanthus mongolicus(Maxim.ex Kom.)Cheng f.)幼苗的子叶为材料,对其原生质体的分离、纯化和瞬时表达体系进行了研究。结果表明,子叶原生质体分离的最佳酶解液组成为CPW溶液+3.0%纤维素酶R-10+0.5%离析酶R-10+0.3%半纤维素酶+9.0%甘露醇(p H5.8);最佳酶解条件为室温、避光、40 r/min轻摇14 h。采用W5溶液作为漂洗液将酶解物稀释后进行过滤,将过滤液在4℃、700 r/min条件下离心5 min,所得纯化原生质体的产量约为2.50×106cells/g,活力达到90%;以纯化的原生质体作为受体,利用聚乙二醇(PEG)介导法成功将植物瞬时表达载体p BI-GFP导入其中,转化效率达到50.8%。利用本研究建立的原生质体瞬时表达体系,检测到沙冬青脱水应答转录因子Am DREB1定位于细胞核内。     

ACCLIMATION OF PROROCENTRUM MINIMUM (DINOPHYCEAE) TO PROLONGED DARKNESS BY USE OF AN ALTERNATIVE CARBON SOURCE FROM TRIACYLGLYCERIDES AND GALACTOLIPIDS

The dinoflagellate Prorocentrum minimum (Pavillard) Schiller is known to be a major bloom-causing microalga in the southern ocean of the Korean peninsula. The acclimation of this alga to darkness for 10 days was investigated by analyzing the content of various lipids, such as phospholipid (PL), galactolipid (GL), and triacylglyceride (TAG). Actively growing cultures of the alga under normal growth conditions (14:10 h LD [light:dark] cycle) were transferred to a growth chamber under conditions of no light and no carbon sources in the medium, and the culture was continued for another 10 days. The results showed that the content of TAG and GL decreased gradually during dark incubation, whereas the total PL content changed little; PC, PE, and PG decreased; and PS, PA, and PI increased. An increase in the activity of β-oxidation and isocitrate lyase (ICL, a glyoxylate cycle enzyme) paralleled the decrease of TAG and GL. These observations strongly suggested that TAG and GL were utilized as alternative carbon sources by the cells under the prolonged dark cultivation. Light treatment of the cells cultivated in the dark for 10 days allowed them to attain the lipid composition that was observed in cells grown in light. These results strongly suggested that the cells maintained their metabolic integrity without unrecoverable cellular damages or cell death during 10 days of dark cultivation.