光强调控三角褐指藻对海洋酸化的生理学响应

光和海洋酸化(CO₂浓度升高)分别对海洋硅藻的光合能力具有不同程度的影响, 但两者的耦合响应被较少关注。研究以三角褐指藻作为实验材料, 测定了不同光强下CO₂浓度升高对三角褐指藻的生长、净光合速率、生化组分、胞外碳酸酐酶(eCA)活性和核酮糖-1,5-二磷酸羧化/氧化酶(Rubisco)活性的影响。结果显示在低光下, CO₂浓度对三角褐指藻的生长和净光合速率(P_n)并没有显著影响, 而在高光下, 具有明显的影响。无论是在高光或是低光下, eCA活性、叶绿素和可溶性蛋白的含量都随着CO₂浓度的升高而降低。在低光下, 高浓度CO₂ (HC)培养下的Rubisco活性分别是低浓度CO₂ (LC)和中浓度CO₂ (MC)的2.42和1.39倍, 而在高光下, HC培养下的Rubisco活性分别是LC和MC的6.72和3.45倍。以上结果表明硅藻能够通过调节光合生理特征和CCM运行中能量的分配来适应环境中光强和CO₂浓度的变化。     

渗透胁迫对小麦幼苗光合作用中氧和二氧化碳交换的影响

小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．ｃｖ．１１６６）幼苗在不同渗透势的聚乙二醇溶液胁迫下,叶片的相对含水量和水势随渗透势下降递降,膜的相对透性增加,叶绿素和可溶性蛋白含量减少,核酮糖１,５－双磷酸浚化酶／加氧酶活性下降,而乙醇酸氧化酶活性则上升了。渗透胁迫对植物光合作用造成的不良影响,还表现为损害了光合放氧和二氧化碳的交换与代谢过程,其中二氧化碳受到的影响比氧敏感。     

杜氏盐藻rbcS启动子的克隆和功能分析

为提高转基因盐藻的表达效率,利用基因组步行方法和巢式PCR,从盐藻中克隆了1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）的小亚基基因rbcS 的5'上游调控序列,并对其进行序列分析和转基因功能分析。采用Dra I、EcoR V、Pvu II和Stu I四种平端限制内切酶分别酶切盐藻基因组DNA,并与接头连接,构建基因组步行文库GWL 1、GWL 2、GWL 3和GWL 4;设计特异引物从这四种文库中扩增rbcS基因的5'上游调控序列。在GWL 1、GWL 4中分别扩增出约1.2 kb的片段。对该序列的分析表明,它的3'端与已知盐藻rbcS cDNA 的5'端序列完全一致,说明是该基因的5'端上游区,并且包含多个与转录调控有关的保守序列（如TATA-box、CAAT-box）,富含GT的重复序列。此序列EcoR I下游的片段与除草剂抗性基因bar相融合,构建表达载体,电击法转化盐藻。通过对转化藻株的抗性筛选以及PCR和Southern blot检测,表明该区域能驱动外源基因bar在转基因盐藻中的表达,推断是盐藻rbcS基因的启动子调控区。     

外源海藻糖对PEG渗透胁迫下小麦Rubisco及其活化酶的影响

以抗旱品种‘晋麦47’和干旱敏感品种‘郑引1号’为材料,通过室内水培试验研究了外源海藻糖对PEG渗透胁迫下小麦叶片净光合速率、1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)和1,5-二磷酸核酮糖羧化酶/加氧酶活化酶(RCA)含量和相关基因表达特性的影响。结果表明:(1)外源海藻糖和渗透胁迫均能显著增加2个小麦品种叶片海藻糖含量。(2)渗透胁迫显著降低了2个品种小麦叶片的净光合速率,而外源海藻糖能显著缓解受胁迫小麦叶片净光合速率的降低幅度。(3)渗透胁迫仅使‘郑引1号’Rubisco大亚基基因(rbcL)相对表达量及相应蛋白含量显著降低;渗透胁迫显著降低了小麦RCAα和β亚基基因相对表达量,并显著降低RCA蛋白含量,而外源海藻糖不能缓解RCA蛋白含量的降低;渗透胁迫显著降低了Rubisco总活性、初始活性、活化状态及RCA活性,而外源海藻糖则能显著缓解上述酶活性的下降。(4)小麦叶片净光合速率与其rbcL、RCAα和β亚基基因相对表达量及Rubisco总活性、初始活性、活化状态及RCA活性均呈极显著正相关关系。研究发现,在渗透胁迫条件下,外源海藻糖主要从翻译后层面对小麦叶片Rubisco和RCA的活性发挥显著保护作用,从而缓解了小麦净光合速率的降低。     

不同叶位鸡蛋果叶片中核酮糖-1,5-二磷酸羧化酶/加氧酶的动力学特性

随着叶龄的增加,鸡蛋果Ru BP羧化酶/加氧酶的V_(max)(CO_2)值明显减小,K_m(O_2)值提高;K_m(CO_2)和V_(max)(O_2)值则保持相对的稳定。Ru BP羧化酶活性和Ru BP加氧酶活性均随叶龄增加而下降,但前者下降的速度高于后者,致使羧化/加氧此值也随叶龄增加而减小。     

短暂低温对佛手光合生理的影响

佛手(Citrus medica var. sarcodactylis Swingle)是一种对冷胁迫较为敏感的观果植物,在生产中普遍存在着冷害影响植物生长的现象.通过模拟浙中地区冬季设施种植中常见的短暂低温弱光条件,研究了佛手叶片的光合生理变化.研究表明,15℃低温即显著降低佛手光合速率、气孔导度,显著提高胞间CO2浓度;引起Fv/Fm显著性下降及初始荧光Fo显著上升的拐点温度为10℃,但延长处理时间至72h情况下,15℃亦显著降低Fv/Fm;低温处理还降低佛手光合羧化效率、最大光合速率,并导致光抑制现象发生时对应光强降低;低温条件下佛手叶片质膜透性及MDA含量高于对照,SOD、POD、CAT等抗氧化酶的活性则呈下降趋势;由此可见,短暂低温弱光胁迫首先是降低核酮糖1, 5-二磷酸羧化酶(Rubisco)等碳固定关键酶活性,引起氧自由基积聚,进而引发光抑制及光合速率的下降.     

各种因子对固定化烟草核酮糖1,5-二磷酸羧化酶/加氧酶解离作用的影响

pH,温度、离子强度及效应剂等对固定化烟草RuBP羧化酶在2.5mol/L尿素处理下的解离作用有各种不同的影响。在pH6.0时,仅小亚基从大亚基核(L_8)解离,当pH为中性偏碱时,大亚基核也解离。低温和低离子强度均促进酶的解离,而温度和离子强度对大亚基之间的解离的影响显著大于对大、小亚基之间的影响。这表明酶的亚基之间存在着不同的极性和疏水作用,而大亚基之间的疏水作用比大、小亚基之间的强。6-PG对大、小亚基之间解离的抑制作用表明大亚基上的催化位置与小亚基之间有一定的密切关系。     

两株绿藻响应CO2浓度变化的生长和生理特性的研究

以小球藻FACHB-1580和栅藻FACHB-1618为研究对象, 比较了两株绿藻在0.04% CO₂、5% CO₂和20% CO₂ (v/v)三种通气培养条件下的生长和生理特性的响应, 试图阐述与无机碳利用相关生理参数和微藻利用CO₂能力的关系。结果表明, 两株绿藻均能高效利用CO₂, 在5%(v/v)条件下均表现出最大生物量积累、最大比生长速率和最大二氧化碳固定速率。小球藻FACHB-1580和栅藻FACHB-1618最大生物量分别为3.5和5.4 g/L, 分别是0.04% CO₂ (v/v)条件的1.41和1.46倍。在高达20% CO₂ (v/v)条件下, 两株绿藻的生物量均显著高于空气组(P<0.05)。随着CO₂浓度的增加, 两株绿藻的无机碳亲和力、胞内和胞外CA活性、初始Rubisco活性, 及Rubisco活化度均有下降趋势, 总的Rubisco活性变化不明显。另外, 小球藻FACHB-1580存在较高的胞外和胞内CA活性; 而栅藻FACHB-1618胞外CA活性几乎为零, 胞内CA活性显著低于小球藻FACHB-1580。由此推测, 小球藻FACHB-1580能同时吸收介质中的${rm{HCO}}_3^ - $和CO₂, 其胞内CA催化胞内${rm{HCO}}_3^ - $快速转化为CO₂, 从而为Rubisco提供充足的CO₂来源; 而栅藻FACHB-1618主要吸收介质中的CO₂, 其胞内CA活性较低, 推测其通过提高胞内CA含量, 或增强Rubisco对CO₂的亲和力等促进光合固碳作用。     

水稻叶片在自然衰老过程中1，5-二磷酸核酮糖羧化酶/加氧酶的降解(英文)

１,５二磷酸核酮糖羧化酶／加氧酶（Ｒｕｂｉｓｃｏ）是光合碳同化的关键酶,研究其降解机理对合理调控水稻生长后期光合衰退具有重要意义。前人用人为诱导植物衰老的方法,研究了Ｒｕｂｉｓｃｏ的降解机理,认为该酶降解之前,必需发生亚基间的交联聚合和向类囊体膜转移,这样在结构和空间上有利于水解酶的作用。我们用自然衰老叶片进行研究的结果表明：Ｒｕｂｉｓｃｏ在降解过程中其比活基本保持恒定,意味着未发生酶的失活,也就是说酶结构未发生根本性改变,由此也可初步判断酶未发生亚基间的交联聚合（已证明亚基交联可导致酶失活）。接着用ＳＤＳＰＡＧＥ和蛋白印迹技术证实了上述观点：Ｒｕｂｉｓｃｏ降解之前只有极少量的大亚基聚合体,随后同未聚合大亚基一起很快降解。此外,研究结果进一步表明酶分子在降解之前有少量与叶绿体膜结合,但降解过程中并未见膜结合蛋白增加。根据上述结果我们认为,亚基间交联聚合和向膜转移并非水稻叶片自然衰老时Ｒｕｂｉｓｃｏ降解的必要条件。     

不同初始氮浓度下尖状栅藻同化硝态氮和CO2的研究

尖状栅藻（Scenedesmus acuminatus（Lagerheim） Chodat）是一种高产油淡水单细胞绿藻,该藻在较低氮素浓度下能显著提高产油效率,是生产生物柴油的理想藻株。本研究以尖状栅藻为实验材料,通过测定藻细胞硝酸还原酶和Rubisco活性、碳氮元素含量和培养液硝酸根离子浓度,分析18.0、9.0、6.0、3.6 mmol·L^-1初始氮浓度下尖状栅藻的碳氮同化特征和时相变化规律。结果显示,18.0 mmol·L^-1组尖状栅藻细胞密度最高,为7.9×10⁷ cells·mL^-1,硝酸还原酶和Rubisco的活性高,且持续时间长。培养液中产生的NO₂^-随初始氮浓度的升高而增多,18.0 mmol·L^-1组至培养期结束仍保持相对较高的NO₂^-水平。4个实验组（初始氮从高到低）培养10 d藻细胞的氮含量依次为7.2%、4.1%、2.8%和1.9% DW,碳含量为46%、52%、54.5%和57.4% DW,细胞的C/N摩尔比值为：8.0、16.7、24.3和35.2。研究结果表明初始氮浓度影响尖状栅藻的生长繁殖,藻细胞的碳氮同化相互影响,适宜的低氮浓度可促进藻细胞碳固定。     

Seasonal and tidal variability of environmental carbon related physico-chemical variables and inorganic C acquisition in Gracilariopsis longissima and Enteromorpha intestinalis from Los Toruños salt marsh (Cádiz Bay, Spain)

The seasonal and tidal variability of inorganic C acquisition mechanisms, photosynthesis, internal composition and growth were studied in two co-occurring macroalgae in Los Toruños salt marsh (Cádiz Bay), Gracilariopsis longissima and Enteromorpha intestinalis. This variability was monitored together with physico-chemical variables affecting carbon availability, photosynthesis, and growth. The environmental variables, such as light, temperature, pH, salinity, oxygen, alkalinity, dissolved inorganic carbon (DIC) and CO₂, displayed not only an expected seasonal cycle but also a daily (tidal) variability, with abrupt and rapid changes influenced by biological activities, physical variables, tidal state and tidal timing. In contrast to environmental variables, photosynthesis, pigments and C:N composition were affected by seasonal changes but not by tidal regimes, as organisms integrated these short-term fluctuations in physico-chemical variables. Photosynthesis, pigments and internal N composition were maximal in autumn and minimal in summer for both species. Growth showed a seasonal trend, displaying a summer drop with negative values. This response can be the result of extreme values of environmental variables (temperature, light, pH, nutrients, and the shortage of DIC) in summer, in comparison with higher growth rates in September onwards. The use of inhibitors of carbon acquisition in situ at normal DIC concentrations (2.2. mM) revealed species-dependent differences. While the external carbonic anhydrase (CA) activity showed a constitutive character in G. longissima, it showed little effect in E. intestinalis, which relies on internal CA activity. The 4, 4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS)-sensitive bicarbonate transport in G. longissima was effective in winter. In contrast, DIDS stimulated photosynthesis in summer, and relieved AZ inhibition. This response could suggest a stimulation of a H⁺ extrusion mediated-CO₂ transport in periods of low CO₂ availability.     

氮磷营养限制影响三角褐指藻光合无机碳利用和碳酸酐酶活性

以海洋硅藻三角褐指藻为实验材料, 研究了不同氮磷比培养对其光合无机碳利用和碳酸酐酶活性的影响, 结果显示三角褐指藻生长速率在N:P=16:1时最大, 高于或低于16:1时明显下降, 表明其最适生长受到氮磷的限制。氮限制(N:P=4:1或1:1)导致叶绿素a含量分别下降30.1% 和47.6%, 磷限制(N:P=64:1或256:1)下降39.1%和52.4%, 但氮或磷限制对叶绿素c含量并没有明显影响。不同营养水平培养对光饱和光合速率具有明显的影响, 与营养充足培养相比, 在严重氮磷限制(N:P=1:1或256:1)培养下光饱和光合速率分别下降39.7%和48.0%, 光合效率与暗呼吸速率也明显下降。在氮磷限制培养下藻细胞pH补偿点明显下降; K0.5CO2值在磷限制下降低30%, 表明磷限制有助于提高细胞对CO2的亲和力, 但氮限制并没有明显影响。在氮磷限制培养的细胞反应液中Fe (CN)63-浓度下降速率较慢, 表明在氮磷限制环境中生长的细胞质膜氧化还原能力明显低于营养充足条件下生长的细胞。氮磷限制也导致胞内、外碳酸酐酶活性明显下降, 其中在氮限制下胞外碳酸酐酶活性分别下降50%和37.5%, 在磷限制下下降22.3%和42.1%。严重的氮(N:P=1:1)或磷(N:P=256:1)限制导致胞内碳酸酐酶活性下降36.5%和42.9%。研究结果表明, 三角褐指藻细胞在氮磷营养限制的环境中, 可以通过调节叶绿素含量、无机碳的利用方式和碳酸酐酶的活性以维持适度的生长。     

Plastidic phosphoglycerate kinase from Phaeodactylum tricornutum: on the critical role of cysteine residues for the enzyme function

Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.     

Structural characterization of beta-D-(1-->3)-glucans from different growth phases of the marine diatoms Chaetoceros mülleri and Thalassiosira weissflogii

We have investigated the content and structure of the chrysolaminarans isolated from the two marine diatoms Chaetoceros mülleri and Thalassiosira weissflogii. Samples were taken from different phases of growth, and the structure of the chrysolaminaran was seen in relation to the specific growth rate of the diatoms. The structure determined for the glucan from C. mülleri was found not to vary with different specific growth rates. T. weissflogii showed some variance in the structure, both throughout the different stages of growth and between samples taken from the stationary phase. C. mülleri was found to have a chrysolaminaran with a degree of polymerization (DP) of 22-24 and a degree of beta-(1-->6) branching of 0.006-0.009. These results corresponded well with previous results obtained in our laboratories. The chrysolaminaran isolated from T. weissflogii was found to have a DP of 5-13 and no beta-(1-->6) branching. This is to our knowledge the first characterization of the chrysolaminaran from T. weissflogii.     

Induction of intracellular carbonic anhydrases during the adaptation to low inorganic carbon concentrations in wild-type and ca-1 mutant cells of Chlamydomonas reinhardtii

Mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang, wild type and the ca-1 mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO₂ (high inorganic carbon, C_i) to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-C_i grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO₂ supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-C_igrown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-C_i cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70–80% of total chloroplastic CA activity was found to be in the insoluble fraction while 17–20% remained in the soluble fraction. Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-C_i algae. After that time their activities were similar to the corresponding CA values in low-C_i-grown cells. In contrast, plastids from high-C_i cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-C_i and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-C_i air-adapted and low-C_i cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.This work is dedicated to Professor A. Wild on the occasion of his 65th birthday     

Purification and properties of the carbonic anhydrase of Rhodospirillum rubrum

The carbonic anhydrase (EC 4.2.1.1) of Rhodospirillum rubrum has been purified to apparent homogeneity and some of its properties have been determined. The enzyme was cytoplasmic and was found only in photosynthetically grown cells. It had a molecular weight of about 28,000, and was apparently composed of two equal subunits. The amino acid composition was similar to that of other reported carbonic anhydrases except that the R. rubrum enzyme contained no arginine. The isoelectric point of the enzyme was 6.2 and the pH optimum was 7.5. It required Zn(II) for stability and enzymatic activity. The K _m(CO₂) was 80 mM. Typical carbonic anhydrase inhibition patterns were found with the R. rubrum enzyme. Strong acetazolamide and sulfanilamide inhibition confirmed the importance of Zn(II) for enzymatic activity as did the anionic inhibitors iodide, and azide. Other inhibitors indicated that histidine, sulfhydryl, lysine and serine residues were important for enzymatic activity.Abbreviation CA carbonic anhydrase In memory of R. Y. Stanier     

The first activation study of a δ-carbonic anhydrase: TweCAδ from the diatom Thalassiosira weissflogii is effectively activated by amines and amino acids

The activation of the δ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii (TweCAδ) was investigated using a panel of natural and non-natural amino acids and amines. The most effective activator of TweCAδ was d-Tyr (K_A of 51?nM), whereas several other amino acids and amines, such as L-His, L-Trp, d-Trp, dopamine and serotonin were submicromolar activators (K_As from 0.51 to 0.93?µM). The most ineffective activator of TweCAδ was 4-amino-l-Phe (18.9?µM), whereas d-His, l-/d-Phe, l-/d-DOPA, l-Tyr, histamine, some pyridyl-alkylamines, l-adrenaline and aminoethyl-piperazine/morpholine were moderately potent activators (K_As from 1.34 to 8.16?µM). For any δ-CA, there are no data on the crystal structure, homology modelling and the amino acid residues that are responsible for proton transfer to the active site are currently unknown making it challenging to provide a detailed rational for these findings. However, these data provide further evidence that this class of underexplored CA deserves more attention.     

Biosynthesis of Phytochelatins and Arsenic Accumulation in the Marine Microalga <Emphasis Type="BoldItalic">Phaeodactylum tricornutum</Emphasis> in Response to Arsenate Exposure

The arsenate-induced synthesis of phytochelatins (PC), intracellular cysteine-rich metal-binding peptides, and its relationship with toxicity and with As accumulation in the cell have been studied in laboratory cultures of the marine microalga Phaeodactylum tricornutum. The time course of cellular PC and As in short-term exposures showed that the involvement of PC in the As detoxification as well as the pathway of cellular As depend on the extent of As accumulation and on the rate of PC synthesis. At arsenate concentrations causing As accumulation at a rate exceeding that of PC synthesis, cells seem to activate a mechanism of release of As mainly in a chemical form not complexed with PC. At arsenate concentrations at which the synthesis of PC occurs at a rate sufficient to allow a significant portion of As accumulated in the cell to be bound, the fate of cellular As seems to be mainly controlled by PC. The occurrence of these different pathways of As detoxification was discussed to explain the pattern of cellular As and PC in cells grown for three days at growth-inhibitory and at no growth-inhibitory concentration of arsenate.