Development of chloroplast membranes during light induced greening ofChlorella fusca g-2

Summary The development of thylakoid protein complexes during light induced greening of a mutant ofChlorella fusca was studied. Separation of chlorophyll-protein complexes and thylakoid polypeptides by LDS-polyacrylamide gel electrophoresis show that cells grown in the dark contain proteins belonging to coupling factor and cytochrome f/b₆ complex. Parts of both reaction centers are present also. The antennae complexes are specifically lost in yellow cells. The changes in polypeptide pattern at different stages of development in the light are related to ultrastructural changes. The beginning of membrane appression can be correlated with the appearance of the light-harvesting complex II. While the average diameter of EF-particles increases throughout the greening process, their densitiesapart from the rearrangement due to membrane stacking-remain fairly constant. The kinetics of EF_u-particle enlargement are different from those of EF_s-particles.PF-faces in thylakoids grown in the dark contain particles of uniform diameter but some of them protrude more from the fracture plane than do their neighbors. During the first hours of greening, their density increases and two classes develop. From the beginning of membrane stacking, the composition of PF_u-faces remains constant and PF_s-particles increase in number for some time.Results are discussed on the basis of present knowledge of structurefunction relations in thylakoids.Abbreviations CF _o intrinsic membrane complex of the coupling factor - EF, EF _s ,EF _u exoplasmic fracture face, stacked and unstacked region, respectively - LDS lithium dodecyl sulfate - LHCII light-harvesting complex of Photosystem II - PF, PF _s ,PF _u protoplasmic fracture face, stacked and unstacked region, respectively - PS I andPS II Photosystem I and Photosystem II     

Heat shock response during anther culture of broccoli (Brassica oleracea var italica)

Embryo formation from microspores of Brassica oleracea var Italica (Broccoli) and other Brassica species is greatly enhanced by an initial incubation at elevated temperatures (eg 35°C) followed by continued incubation of 25°C. In the present study we observed that a three hour high temperature treatment induced the formation of heat shock proteins in cultured anthers. These were identified in two dimensional gels by silver staining, and labelled heat shock proteins were synthesised in vitro from isolated anther RNA. The appearance of heat shock proteins in anthers followed a similar pattern and displayed similar characteristics to that from leaves. Comparison of the heat shock proteins induced in isolated cultured anthers of known highly embryogenic and less embryogenic plans did not reveal obvious qualitative differences.     

Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress

The fast growing unicellular green microalgae Chlorella protothecoides has attracted interest as a promising organism for commercial production of a high-value carotenoid, lutein, by heterotrophic fermentation. Effects of two oxidant-forming reactive oxygen species (ROS) on the biomass concentration, and yield and content of lutein in batch culture of heterotrophic Chlorella protothecoides were investigated in this study. The addition of 0.1 mmol/L H₂O₂ and 0.01 mmol/L NaClO plus 0.5 mmol/L Fe²⁺ to the culture led to the generation of ^·OH and enhanced the lutein content from 1.75 to 1.90 and 1.95 mg/g, respectively. The lutein content further increased to 1.98 mg/g when 0.01 mmol/L H₂O₂ and 0.5 mmol/L NaClO were added to generate ¹O₂. The maximum yield of lutein (28.5, 29.8 and 31.4 mg/L) and a high biomass concentration (15.0, 15.3 and 15.9 g/L) were also achieved through the above treatments. The results indicated that 1O2 could promote lutein formation and enhance lutein production in heterotrophic Chlorella protothecoides. Moreover, ¹O₂ produced from the reaction of H₂O₂ and NaClO was more effective in enhancing lutein production and reducing biomass loss than ^·OH from the reaction of H₂O₂ or NaClO plus Fe²⁺. Supported by the National Key Project of Sci & Tech Supporting Programs Funded by Ministry of Science & Technology of China (Grant No. 2006BAD27B03), Sci & Tech Project of Guangzhou (Grant No. 2005Z3-E0331) and Sci & Tech Project of Guangdong (Grant No. 20052050166)     

Heat shock response in mulberry silkworm races with different thermotolerances

The thermal sensitivity and heat shock response of the different races of the mulberry silkwormBombyx mori have been analysed. The multivoltine race, strainsC. Nichi andPure Mysore showed better survival rates than the bivoltine race, strainNB4D2 exposed to 41°C and above. In general, the fifth instar larvae and the pupae exhibited maximum tolerance compared to the early larval instars, adult moths or the eggs. Exposure up to 39°C for 1 or 2 h was tolerated equally whereas temperatures above 43°C proved to be lethal for all. Treatment of larvae at 41°C for 1 h resulted in a variety of physiological alterations including increased heart beat rates, differential haemocyte counts, enlargement of granulocytes and the presence of additional protein species in the tissues and haemolymph. The appearance of a 93 kDa protein in the haemolymph, fat bodies and cuticle, following the heat shocking of larvaein vivo was a characteristic feature in all the three strains examined although the kinetics of their appearance itself was different. In haemolymph, the protein appeared immediately in response to heat shock inC. Nichi reaching the maximal levels in 2–4 h whereas its presence was noticeable only after 2–4 h recovery time inPure Mysore and bivoltine races. The fat body from bothC. Nichi andNB4D2 showed the presence of 93 kDa, 89 kDa and 70 kDa proteins on heat shock. The haemocytes, on the other hand, expressed only a 70 kDa protein consequent to heat shock. The 93 kDa protein in the haemolymph, therefore could have arisen from some other tissue, possibly the fat body. The 93 kDa protein was detected after heat shock in pupae and adult moths as well, although the presence of an additional (56 kDa) protein was also apparent in the adults. The presence of 46 kDa and 28 kDa bands in addition to the 93 kDa band in the cuticular proteins immediately following heat shock was clearly discernible. The 70 kDa band did not show much changes in the cuticular proteins on heat shock. In contrast to the changes in protein profiles seen in tissues and haemolymph following heat shockin vivo, the heat treatment of isolated fat body or haemolymphin vitro resulted in protein degradation.     

Characterization of the heat shock response inEnterococcus faecalis

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacteriumEnterococcus faecalis. The heat resistance (60°C or 62.5°C, 30 min) of log phase cells ofE. faecalis grown at 37°C was enhanced by exposing cells to a prior heat shock at 45°C or 50°C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72 kDa) and GroEL (63 kDa) heat shock proteins ofEscherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La)E. coli proteins and to theBacillus subtilis ⁴³ factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.     

Pyrolytic characteristics of heterotrophic Chlorella protothecoides for renewable bio-fuel production

Heterotrophic Chlorella protothecoides cells were pyrolyzed in athermogravimetric analyzer to investigate the pyrolytic characteristics anddetermine the kinetic parameters. Heating rates of 15, 40, 60 and 80 ^°C^-1 up to a final temperature of 800 ^°C wereused. The pyrolysis reactions mainly took place between 160–520^°C with a volatile yield of about 80%. The devolatilization stageconsisted of two main temperature zones (I and II) with a transition at300–320 ^°C. Crude lipid in cells decomposed at Zone II whileother main components at Zone I. The increase of heating rate caused alateral shift to higher temperatures in the thermograms, a decrease ofactivation energies for the devolatilization stage and an increase of both theinstantaneous maximum and average reaction rates. The difference ofactivation energies between two zones implied that more energy input forlipid pyrolysis seems needed in comparison with other main components.These data are useful for the design, operation, and modeling of thepyrolysis systems for microalgae.     

High yield of hydrocarbon gases resulting from pyrolysis of yellow heterotrophic and bacterially degradedChlorella protothecoides

Four kinds of cells ofChlorella protothecoides, green autotrophic cells, bacterially degraded green autotrophic cells, yellow heterotrophic cells and bacterially degraded yellow heterotrophic cells, were used to simulate thermal degradation and gas formation by heating without oxygen at 300°C for 100 h. The yield of pyrolysed hydrocarbon gases in yellow heterotrophic cells with bacterial degradation was 8.5 times higher than that of green autotrophic cells without bacterial degradation. The use of bacterially degraded yellow heterotrophic cells resulted in relatively more lipid and less protein. The results suggest that the hydrocarbon-producing potential of microplanktonic algae in nature may be greater than previously thought based on studies of green autotrophic cells.     

Transient breakdown in the selective permeability of the plasma membrane ofChlorella emersonii in response to hyperosmotic shock: Implications for cell water relations and osmotic adjustment

Summary In osmotic experiments involving cells of the euryhaline unicellular green algaChlorella emersonii exposed to hyperosmotic stress by immersion in a range of low molecular weight organic and inorganic solutes, a temporary breakdown in the selective permeability of the plasma membrane was observed during the initial phase of transfer to media of high osmotic strength (up to 2000 mosmol kg^–1). Thus, although the cells appeared to obey the Boyle-van't Hoff relationship in all cases, showing approximately linear changes in volume (at high salinity) as a function of the reciprocal of the external osmotic pressure, the extent of change was least for the triitols, propylene glycol and glycerol, intermediate for glucose, sorbitol, NaCl and KCl, with greatest changes in media containing the disaccharides sucrose and maltose. In NaCl-treated cells, uptake of external solute and loss of internal ions was observed in response to hyperosmotic treatment while sucrose-treated cells showed no significant uptake of external solute, although loss of intracellular K⁺ was observed. These observations suggest that the widely used technique of estimating cellular turgor, and osmotic/nonosmotic volume by means of the changes in volume that occur upon transfer to media containing increasing amounts of either a low molecular weight organic solute or an inorganic salt may be subject to error. The assumption that all algal cells behave as ideal osmometers, with outer membranes that are permeable to water but not to solutes, during the course of such experiments is therefore incorrect, and the data need to be adjusted to take account of hyperosmotically induced external solute penetration and/or loss of intracellular osmotica before meaningful estimates of cell turgor and osmotic volume can be obtained.     

Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes

Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM–N) in the dark rapidly degraded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and the synthesis of catabolites as did the addition to BM–N of cycloheximide or a chelator, 2,2-bipyridyl. In contrast, chloramphenicol or the protease inhibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation of cellular proteins such as light-harvesting complex II, cytochrome f and protochlorophyllide oxido-reductase. During growth in BM–N, protease activity increased and proteins immunologically detectable with an antibody against a senescence-enhanced cysteine protease accumulated. cDNAs from BM–N and BM+N cells were used for differential and subtractive screening to isolate cDNAs representing genes with degreening-enhanced expression (dee) in C. protothecoides. Several different dees were identified with different patterns of expression during Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4 was most abundant and its expression was exclusive in BM–N cultures. Analysis of the dee sequences showed that they encode different proteins including a novel amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a Ca²⁺-binding protein, MO25, ubiquinone-cytochrome c-reductase and several new proteins.     

Basic features of the Staphylococcal heat shock response

The major heat shock proteins of Staphylococcus aureus had apparent M_rs of 84,000, 76,000, and 60,000, and other prominent proteins of M_rs 66,000, 51,000, 43,000 and 24,000 were also induced. Staphylococcus epidermidis showed a similar response. These proteins were also induced by CdCl₂, ethanol and apparently osmotic stress (1.71 M NaCl or 2.25 M sucrose). Most of the proteins sedimented with the membrane fraction, but the M_r 60,000 protein remained in the cytoplasm.     

Effects of temperature and holding time on production of renewable fuels from pyrolysis of Chlorella protothecoides

To investigate the influence of temperature andholding time on the pyrolyzate yields of Chlorella protothecoides, the microalgal cells weresubjected to pyrolysis at 200, 300, 400, 500 and 600 ^°Cfor 5, 20, 60 and 120 min, separately. High oil yields above 40% dry weight cells wereobtained both at relatively low temperature (300 ^°C)with relatively long holding times (20–120min) and relatively high temperatures (400–500 ^°C)with relatively short holding times (5–20min). The maximum oil yield of 52.0% was achieved at500 ^°C for 5 min. The gas yield was generallyincreased with the increasing temperature and holdingtime. It could reach 63.3–76.0% at 600 ^°C.High pyrolytic rates of 72–87% were obtained at allexperiments except at 200 ^°C for 5–20 min or300 ^°C for 5 min. Thermogravimetric analysisindicated that the main thermal degradation of thismicroalga occurred at 200–520 ^°C. The resultsimply that C. protothecoides is a good candidatefor the production of renewable fuels by pyrolysis.     

原始小球藻AP2转录因子的生物信息学分析

为了揭示原始小球藻AP2基因家族编码蛋白的理化特性、分子功能和遗传进化特征,该文对原始小球藻AP2基因家族的蛋白成员进行了详细的生物信息学预测和分析,并利用PrtoParam、Pfam 3.20、Protscale等在线工具分析了原始小球藻AP2家族所包含的8个蛋白成员。结果表明:该家族成员含有的氨基酸数为247(XP_011395646.1)到715(XP_011401904.1),理论等电点最大为9.39(XP_011396011.1)、最小为5.81(XP_011398158.1); 家族中各个成员所含有的保守结构域的位置和数量都各不相同; 所有蛋白成员均无信号肽,均不包含跨膜螺旋,不具有跨膜区域; 蛋白成员中最大亲水性值为-3.222(XP_011398158.1),最大疏水性值为2.333(XP_011401904.1),且所有成员的平均亲疏水性值均小于0; 成员中有多个磷酸化位点值远超标准值0.5,最大磷酸化位点值为0.998; 该基因家族的蛋白成员二级结构的组分含量从大到小排序均为无规则卷曲>α-螺旋>延伸链>β-转角,推测α-螺旋和无规则卷曲是其二级结构的主要方式,延伸链和β-转角则分散在所有蛋白成员的氨基酸链中; 所有成员的三级结构分析中均能观察到α-螺旋、β-折叠、β-转角以及N端和C端; 系统进化分析结果表明,在其余15个物种中,与原始小球藻亲缘关系最远的是金牛介球菌(Ostreococcus tauri),亲缘关系最接近的是小球藻(Chlorella variabilis NC64A)和螺旋孢子虫(Helicosporidium),较接近的是苦参3号(Picochlorum sp. SENEW3)。该研究较系统地分析了原始小球藻AP2蛋白家族的理化特性、保守基序及系统进化关系,为进一步研究原始小球藻AP2转录因子功能和互作关系提供一定参考依据。     

Developmental study of thermotolerance and the heat shock response inLucilia cuprina (Weidemann)

The ability to withstand thermal stress in a laboratory population of the blowflyLucilia cuprina (measured as per cent adult survival following varying periods of exposure to elevated temperature up to a maximum of 48°C) was in the order pupa > larva > adult. Pre-exposure to a mild heat shock (37°C) induced tolerance to temperatures which were otherwise lethal. An analysis of heat shock-induced protein synthesis during development at similar elevated temperatures presented patterns corresponding to the above observations on thermotolerance. The induced level of synthesis of major heat shock proteins (viz., 79, 69, 28, 20 and 19 kDa) were greater in larval tissues than in most of the adult tissues except gonads. The response varied between young (2 days) and old (30 days) adults in a tissue-specific manner. In general, heat shock protein 69 kDa was most abundant in all the tissues studied. Control as well as heat shocked Malpighian tubules of adults uniquely showed two major [³⁵S]methionine labelled bands corresponding to approximately 62 and 64 kDa. Immunoblots showed the 62 kDa protein to cross react with an antibody againstHelioihis HSP60. Although the synthesis of the 62 kDa polypeptide was prominent only in Malpighian tubules of adult blowflies, nearly equal levels of this HSP60 family polypeptide were present in all tissues (control as well heat shocked) except the larval salivary glands.     

Heat shock response in Actinobacillus actinomycetemcomitans

Abstract The heat shock response in Actinobacillus actinomycetemcomitans , a capnophilic Gram-negative bacterial species that is implicated in the development of certain forms of periodontitis, was characterized. Different strains of A. actinomycetemcomitans were grown at 37, 42 and 48°C in the presence of ³⁵S-methionine. The bacterial cells were lysed, run on SDS-PAGE and subsequently blotted on nitrocellulose paper. After autoradiography of the blots, several protein bands from the cultures at 42°C showed an increased intensity; major bands were observed at 90, 70, and 60 kDa, but increased protein synthesis was also detected at 54, 28 and 17 kDa. Nitrocellulose blots were also incubated with a panel of monoclonal and polyclonal antibodies directed to epitopes on different heat shock proteins. Strong reactivity was found with several antibodies at the position corresponding to a molecular mass of 60 kDa. The protein is probably the GroEL homologue in A. actinomycetemcomitans , a member of the ‘common bacterial antigen’ family.     

Cold shock response inLactococcus lactis ssp.diacetylactis

The acquired freeze-thaw tolerance was investigated forLactococcus lactis ssp.diacetylactis. Pretreatment of microorganisms at less severe temperatures to initiate cold tolerance gaveL. lactis ssp.diacetylactis improved cell viability after successive freezings and thawings. The ability of cells to survive freeze-thaw was dependent on factors experienced prior to freezing. Factors affecting lactic acid bacteria survival during freeze-thaw cycles were found to be different diluents, growth phase, and different cold temperatures. Viability experiments showed that this strain displaying cold shock cryotolerance had an improved survival capacity in stationary phase. The plasmid contents of lactic acid bacteria isolated from different types, DRC-2 and DRC-2C, were examined and compared with the plasmid contents of culture collection strains both before and after cold shock treatment. Using agarose gel electrophoresis, no obvious correlation between the cold shock response and the number of plasmids in the cell could be observed.     

Heat shock proteins induce pores in membranes

Human heat shock protein (hsp) 70 and bacterial protein groEL promote leakage of calcein from liposomes induced by human serum albumin signal peptide, byS. aureus toxin or by diphtheria toxin. Hsp 70 and groEL, as well as two mycobacterial homologues hsp 71 and hsp 65, induce ion conducting pores across planar lipid bilayers at low or neutral pH. It is concluded that hsp induce pores in membranes and that this may contribute to their action within cells.     

Preliminary study of heat shock proteins in nemerteans

Nemerteans experience varying environmental temperatures during low tide exposures. Inducible heat shock proteins (hsps) have been reported for most organisms following both artificial heat stress and natural environmental temperature variations. This preliminary study reports the presence of hsps in the phylum Nemertea. A lethal temperature of 36 °C was determined for Paranemertes peregrina Coe, 1901. The nemerteans were exposed to a temperature of 34 °C for 2 h. After a 2 h recovery time, the worms were then analyzed for hsps by SDS–PAGE and western immunoblot protocols. Control worms were allowed to acclimate to ambient temperatures (13–15 °C) before hsp analysis. Hsp70 and hsp90 were detected in both the control and heat-shocked worms in highly variable concentrations, and the latter group had significantly elevated hsp70 levels. In addition, the analysis detected different isoforms of hsp70. The detection of hsps indicates a possible role in nemertean physiology during response to thermal stress, and potentially to other environmental challenges.     

Heat shock proteins and effects of heat shock in plants

Soybean seedlings when exposed to a heat shock respond in a manner very similar to that exhibited by cultured cells, and reported earlier [2]. Maximum synthesis of heat shock proteins (HSPs) occurs at 40C. The heat shock response is maintained for a relatively short time under continuous high temperature. After 2.5 hr at 40 C the synthesis of HSPs decreases reaching a very low level by 6 hr. The HSPs synthesized by cultured cells and seedlings are identical and there is a large degree of similarity in HSPs synthesized between the taxonomically widely separated species, soybean and corn. Storage protein synthesis in the developing soybean embryo is not inhibited but is actually stimulated during a heat shock, unlike most other non-HSPs, whose synthesis is greatly reduced. Seedlings respond differently to a gradual increase in temperature than they do a sudden heat shock. There is an upward shift of several degrees in the temperature at which maximum protein synthesis occurs and before it begins to be inhibited. In addition, there appears to be a protection of normal protein synthesis from heat shock inhibition when the temperature increase is gradual. An additional function of the heat shock phenomenon might be the protection of seedlings from death caused by extreme heat stress. The heat shock response appears to have relevance to plants in the field.