Hybridization between Japanese and North American Chlamydomonas reinhardtii (Volvocales,Chlorophyceae)

The microalga Chlamydomonas reinhardtii is a model organism whose whole genome has been sequenced. Although considered a cosmopolitan species, only eastern North American isolates of C. reinhardtii were available before 2010, when new Japanese isolates were reported. In the study describing the new Japanese isolates, zygote formation between Japanese and North American strains was shown, but germination was not demonstrated. In this study, the germination of intercontinental hybrid zygotes was examined using wild‐type Japanese strains and mutant American strains that cannot utilize nitrate. Several clonal progeny strains were established, and the progeny strains were screened based on mating type and nitrate utilization to confirm their hybrid nature. The establishment of four intercontinental hybrid strains with different phenotypic combinations was confirmed by sequencing mating type‐specific and nitrate reductase‐related genes. The potential for hybrid formation between Japanese and North American strains suggests the existence of a worldwide mating population of C. reinhardtii.     

Effects of Calcium Ions and of Calcium Channel Blockers on Galvanotaxis of Chlamydomonas reinhardtii

The effects of calcium ions and of the calcium channel blockers verapamil, diltiazem and nifedipine on galvanotaxis in Chlamydomonas have been investigated using a fully automated and computerized population system. Galvanotaxis is a function of the voltage applied to the cell population. However, the galvanotactic orientation also depends on the external calcium concentration. In a calcium-deprived nutrient medium which still contains 6 × 10^?7M calcium, galvanotactic orientation is about 20% of orientation at optimal calcium concentration of 10^?4 M at 9 V. The higher the external calcium concentration is, the lower is the voltage necessary for optimal galvanotactic orientation. The calcium channel blockers diltiazem and nifedipine likewise inhibit galvanotaxis of Chlamydomonas very specifically without impairing motility. Verapamil is effective, but also inhibits motility by causing detachment or shortening of the flagella. Nevertheless, inhibition of galvanotaxis by verapamil is not the only result of decreased motility, because the galvanotactic orientation is impaired to a greater extent than motility. The effectiveness of the three blockers tested in inhibiting galvanotaxis depends on the concentration and on the voltage applied. At 10^?5 M, verapamil causes maximal inhibition of galvanotaxis at 9 V. At increasing concentrations up to 10^?4 M, diltiazem inhibits galvanotaxis more strongly than the other blockers. If the voltage is varied at a constant blocker concentration of 2 × 10^?5 M, nifedipine causes maximal inhibition at 3 V–6 V, diltiazem at 9 V and verapamil above 12 V.     

淀粉合成受限的莱茵衣藻的甘油酯酰基的变化

【目的】基于突变藻株本身属性和意义出发,考察在两种常用培养方式下莱茵衣藻淀粉突变株(CC-4326)与野生型藻株(CC-137)在甘油酯中酰基随生长的变化差异,为进一步认识莱茵衣藻突变株提供参考信息。【方法】分别在柱状鼓泡式反应器和摇瓶中培养CC-4326和CC-137,比较两株藻在正常培养和氮胁迫培养状态下甘油酯中酰基相对含量和其在甘油三酯(TAG)含量的差异。【结果】正常培养状态下,CC-4326和CC-137中多不饱和酰基C16:4和C18:3相对含量占总酰基45%左右,CC-4326在两种培养方式下这两个酰基含量及变化无差别,而CC-137在摇瓶中培养二者相对含量增加幅度和含量均高于反应器。缺氮条件下两种藻株积累TAG,但程度不同,CC-4326在反应器中培养TAG含量达到CC-137的1.5倍,在摇瓶中培养含量与CC-137无显著差异,两株藻的甘油酯和TAG中C18:1含量显著增加,CC-4326在反应器中培养C18:1增加幅度大于摇瓶,比摇瓶培养更快速积累TAG。而CC-137在摇瓶中培养TAG含量与反应器接近,单不饱和酰基增加幅度却高于反应器,表明CC-137在摇瓶中培养比反应器更利于积累TAG。最终,CC-4326在光生物反应器中缺氮培养实现了TAG 12倍的增加。【结论】通过对淀粉合成抑制,与CC-137相比,缺氮光生物反应器培养条件下,CC-4326能够实现TAG的高效积累。     

转基因衣藻对重金属的抗性及对镉离子的富集

目的：研究了转基因衣藻对不同重金属离子的抗性和对Cd^2＋的富集行为。方法：将MT-like基因在莱茵衣藻细胞中的表达后通过摇瓶实验测试其对重金属离子的抗性和富集能力。结果：转基因衣藻对Pb^2＋、Zn^2＋和Cd^2＋三种重金属离子的抗性得到明显增强,其中以对Zn^2＋的抗性增强最为显著。转基因藻对Cd^2＋的富集能力经MT-like蛋白表达后较野生藻细胞有较大增加,最大达到144．48μmol／g,为野生藻的8．3倍。结论：转基因衣藻对重金属离子的抗性和对Cd^2＋的富集能力较野生衣藻有较大提高。     

Effects of Chloroplast DNA Content on the Cell Proliferation and Aging in Chlamydomonas reinhardtii

Chlamydomonas is an unicellular green alga that contains one cup-shaped chloroplast with about 60 copies of cpDNA. Chloroplasts (cp) multiply in the cytoplasm of the plant cell by binary division, with multiple copies of cpDNA transmitted and maintained in successive generations. The effect of cpDNA copy number on cell proliferation and aging was investigated using a C. reinhardtii moc mutant, which has an undispersed cp-nucleoid and unequal segregation of cpDNA during cell division. When the mother cell divided into four daughters, one moc daughter cell chloroplast contained about 60 copies of cpDNA, and the chloroplasts in the three other daughter cells contained the 4–7 copies of cpDNA. In liquid medium, the number of moc cells at the period of stationary phase was about one-third that of the wild type. To observe the process of proliferation and aging in the mother cell, we used solid medium. Three out of four moc cell spores were preferentially degenerated 60 days after cell transfer. To confirm this, wild-type and moc mother cells containing four daughter cells were treated with novobiocin to inhibit cpDNA replication. Cell degeneration increased only in the moc strain following novobiocin introduction. In total, our results suggest that cells possessing smaller amounts of cpDNA degenerate and age more rapidly. Received 7 September 2000/ Accepted in revised form 14 February 2001     

Calmodulin Sensitivity of the Flagellar Membrane Adenylate Cyclase and Signaling of Motile Responses by cAMP in Gametes of Chlamydomonas reinhardtii

Cyclic AMP (cAMP) has been shown to be a primary signal of the agglutination-induced mating events of flagellar tip activation, cell wall loss, and mating structure activation in the unicellular alga Chlamydomonas reinhardtii (Pasquale and Goodenough, Cell Biol. 105 (1987), 2279–2293). The flagellar membrane adenylate cyclase of Chlamydomonas is here shown to be inhibited in vitro by EGTA, La³⁺, and trifluoperazine, and to be stimulated in the presence of calcium by incubation with exogenous calmodulin. Also, the motility of detergent-extracted models of Chlamydomonas is shown to be enhanced by cAMP. These observations suggest the hypothesis that the twitching motility characteristic of agglutinating Chlamydomonas gametes may be signaled by cAMP produced locally within the flagella by a calmodulin-sensitive adenylate cyclase.     

转衣藻染色质烟草细胞核的变化及衣藻核基因的表达

应用反复冻融法以衣藻原生质体为材料制备衣藻染色质,应用显微操作技术准确将衣藻染色质转移到烟草叶片外植体中,进行连续培养并镜检观察。结果表明,经染色质转移处理的烟草叶片外植体、衣藻细胞核与烟草细胞核均发生形态上的变化。同时烟草叶片外植体正常发芽并获得再生苗,经RT-PCR检测发现,有衣藻核基因(rbcs2)的表达。     

莱茵衣藻Nfr-4突变株中类胡萝卜素含量的变化及其对藻生长的影响

文章对相同条件下培养的莱茵衣藻野生型CC-137和八氢番茄红素脱氢酶(phytoene desaturase,PDS)基因突变株Nfr-4的生长进行分析;并用反相高效液相色谱分析总有色类胡萝卜素以及叶绿素含量变化,结果表明两者生长的差异明显;Nfr-4突变株的单细胞叶绿素和总有色类胡萝卜素含量高于野生型CC-137的。     

Effects of doubled atmospheric CO2 concentration on the growth and photosynthesis of Chlamydomonas reinhardtii (Volvocales, Chlorophyceae)

The freshwater microalga, Chlamydomonas reinhardtii Dangeard, was cultured under 350 and 700 ppmv CO₂ to determine the impact of doubled atmospheric CO₂ concentration on its growth and photosynthesis. No significant difference was observed in the specific growth rate, photosynthetic efficiency, maximal net photo‐synthetic rate and light‐saturating point between the low and high CO₂ cultures. Both the low‐ and high‐CO₂‐grown cells showed reduced light‐dependent O₂ evolution rate and photochemical efficiency (F_v/F_m) owing to photoinhibition when exposed to high photon flux density. However, high‐CO₂‐grown cells were less photoinhibited, and showed better recovery in dim light or darkness during the initial period of the recovery process.     

7-Azido-Actinomycin D: A Photoaffinity Probe of the Sequence Specificity of DNA Binding by Actinomycin D

Abstract

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5′-dGpdC-3′ sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5′- flanking base was a pyrimidine and the 3′-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5′- TGGG-3′.     

Effect of culture conditions on the isocitrate dehydrogenase and isocitrate lyase activities in Chlamydomonas reinhardtii

In the green alga Chlamydomonas reinhardtii , nitrogen staravation induced a reversible increase (2-fold) in NAD-isocitrate dehydrogenase (NAD-IDH; EC 1.1.1.41) and NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO₂, the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD-IDH activity was detected, a 2-fold increase of NADP-IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD- and NADP-IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD-IDH increased in the last 4 h of the dark period, NADP-IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle.     

Effects of alginate oligomer on the expression of cell cycle- and stress-related genes in Chlamydomonas reinhardtii

Enzymatically prepared alginate oligomer (AO) promoted the growth of Chlamydomonas reinhardtii in a concentration-dependent manner. AO at 2.5 mg/mL induced increase in expression levels of cyclin A, cyclin B, and cyclin D in C. reinhardtii. CuSO₄ at 100 μM suppressed the growth of C. reinhardtiin, and AO at 2.5 mg/mL significantly alleviated the toxicity of CuSO₄. Increased intracellular reactive oxygen species level in C. reinhardtii induced by CuSO₄ was reduced by AO. After cultivation with CuSO₄ at 100 μM, expression levels of ascorbate peroxidase and superoxide dismutase in C. reinhardtii were increased, and AO reduced the increased levels of these enzymes. These results suggest that AO exhibits beneficial effects on C. reinhardtii through influencing the expression of various genes not only at normal growth condition but also under CuSO₄ stress.     

Effect of Immobilization on the Kinetic and Stability Properties of O-Acetyl-L-Serine Sulfhydrylase from: Chlamydomonas reinhardtii

The O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) from Chlamydomonas reinhardtii has been immobilized either by ionic binding to DEAE-cellulose or by covalent bonding to alkylamine silica and to vinyl acetate-divinylethylene urea copolymers. The immobilized enzyme had improved stability and showed sigmoidal kinetic behaviour with respect to O-acetyl-L-serine, but without major alterations in the corresponding apparent Km value or in the inhibitory effects of this substrate observed with the enzyme in solution. In addition, significant changes in optimum pH and reaction temperature for O-acetyl-L-serine sulfhydrylase activity were observed.     

Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii

Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation.     

小球藻和莱茵衣藻原生质体的电转化研究

以小球藻及莱茵衣藻原生质体为受体细胞,利用电击法将质粒p CAMBIA1301转入小球藻和莱茵衣藻,摸索电击转化条件并进行分子检测。结果表明:两类藻都对潮霉素敏感,小球藻及莱茵衣藻分别在含25 mg/L和100 mg/L潮霉素的固体培养基上的生长被完全抑制;小球藻和莱茵衣藻原生质体电击转化的最佳电击场强分别为0.8 k V/cm和0.6 k V/cm,最佳脉冲时间均为10 ms;制备原生质体和通过2-脱氧-D-葡萄糖处理可明显提高转化效率;分子检测说明GUS报告基因成功转入两种藻并可稳定遗传。     

Identification of a gene for UDP-sulfoquinovose synthase of a green alga, Chlamydomonas reinhardtii, and its phylogeny.

Sulfoquinovosyl diacylglycerol is responsible for the structural and functional integrity of the photosystem II complex of a green alga, Chlamydomonas reinhardtii. We cloned a cDNA of C. reinhardtii containing an open reading frame for a protein 36-64% identical in the primary structure to known UDP-sulfoquinovose synthases, which are required for SQDG synthesis, in other organisms. Through the introduction of the cDNA, a cyanobacterial disruptant as to the UDP-sulfoquinovose synthase gene recovered the ability to synthesize sulfoquinovosyl diacylglycerol, thus confirming that the cDNA encodes the UDP-sulfoquinovose synthase. On the genome, the cDNA was divided into 14 exons, and the gene designated as SQD1 was present as one copy. The molecular phylogenetic tree for the UDP-sulfoquinovose synthase showed grouping of C. reinhardtii together with species that require sulfoquinovosyl diacylglycerol for the functioning of the PSII complex, but not with those that do not utilize the lipid for photosynthesis. The role of sulfoquinovosyl diacylglycerol in the functioning of the photosynthetic membranes might evolve in harmony with the system of the membrane lipid synthesis such as UDP-sulfoquinovose synthase gene.     

基于液质联用的莱茵衣藻极性甘油酯组定性定量分析

以模式藻株莱茵衣藻(Chlamydomonas reinhardtii)为材料, 基于液质联用技术对其极性甘油酯组进行定性定量分析。通过综合利用UPLC-ESI-Q-Trap/MS的一级质谱扫描(中性丢失或母离子扫描)及UPLC-ESI-Orbitrap/MS²的二级碎片信息扫描, 共鉴定出109种极性甘油酯分子; 再通过外标法利用UPLC-ESI-Q-Trap/MS在多级反应监测模式下对各分子进行靶向定量分析。结果表明, 莱茵衣藻的极性脂以糖脂MGDG、DGDG及甜菜碱脂DGTS为主, 所有极性脂的分子组成表明, DGDG、SQDG、DGTS及PI是C18脂肪酸的去饱和载体。该研究利用液质联用技术建立了莱茵衣藻极性甘油酯组的结构图谱及定量分析技术平台, 为微藻极性脂生物学功能及脂质代谢研究奠定了基础。     

The dynamic behaviour of mitochondria in living zygotes during maturation and meiosis in Chlamydomonas reinhardtii

To understand fully the function of mitochondria during the development of cells and organs, it is important to elucidate the dynamics of their morphology. However, the detailed morphology of mitochondria during meiosis has not yet been studied in algae. We examined the mitochondrial morphology of Chlamydomonas reinhardtii and classified zygotes into seven types by mitochondrial morphology in order to analyse the morphological change in mature and meiotic zygotes. We also investigated the oxygen consumption of living zygotes and the effects of tubulin and actin polymerization inhibitors on mitochondria, using fluorescence microscopy and oxygen electrodes. During zygote maturation, mitochondria fragmented into small particles, with a large decrease in oxygen consumption. When mature zygotes were exposed to light, mitochondria became tubular and formed a network, and oxygen consumption gradually recovered. At the same time, particle-like mitochondrial nucleoids became stringy and produced new nucleoid particles. Tubular mitochondria accumulated around the cell nucleus and then spread throughout the cell. Cell division followed (first and second rounds), and the resultant daughter cells had tubular mitochondria in a mesh-like arrangement. An inhibitor of tubulin polymerization, demecolcine, inhibited the assembly of mitochondria around the cell nucleus, whereas an inhibitor of actin polymerization, latrunculin B, inhibited the formation of tubular mitochondria. These results suggest that microtubules are probably involved in mitochondrial accumulation around the cell nucleus, whereas microfilaments may maintain the tubular network of mitochondria.