A Comparative Study on PSⅡ Light Harvesting Chlorophyll a/b Protein Complexes Between Spinach and Cucumber

PS Ⅱ light harvesting chlorophyll a/b protein complexes (LHC Ⅱ ) were isolated from chloroplast of spinach (Spinacia oleracea Mill. ) and cucumber (Cucumis sativus L. ). Comparative studies were made on the polymerized forms. Chl a/b ratio, spectral characteristics and polypeptide components of these two kinds of LHC Ⅱ. Experimental results showed that the LHC Ⅱ from spinach had a Chl a/b ratio of 1.33 and the LHC Ⅱ from cucumber had a Chl a/b ratio of 1.77. The spectral characteristics of the LHC Ⅱ from cucumber also indicated the enrichment of Chl b in this LHC Ⅱ . There was also obvious differences in the polypeptide components between these two kinds of LHC Ⅱ, the LHC Ⅱ of spinach contained a 27 kD and a 25 kD polypeptides, while the LHC Ⅱ of cucumber contained only a 27 kD polypeptide. This showed that the 25 kD polypeptide contained less Chl b. The analysis of the chlorophyll protein complexes showed that the monomer, dimer and trimer of the LHC Ⅱ of spinach were composed of two polypeptides, while all the polymerized forms of cucumber’s LHC Ⅱ were composed of one polypeptide.     

铈对黄瓜叶绿体叶绿素蛋白质复合物形成的影响

黄瓜（Ｃｕｃｕｍ ｉｓｓａｔｉｖｕｓＬ．）叶片叶绿体中铈（Ｃｅ）含量随Ｈｏａｇｌａｎｄ 培养液中ＣｅＣｌ３ 浓度的增加而增加。Ｃｅ 对黄瓜叶片Ｃｈｌａ／ｂ 比值的影响与光强度有关,当植株生长在强光下,对照和处理叶片的Ｃｈｌａ／ｂ 比值均为３．０７;但在弱光下对照叶片的Ｃｈｌａ／ｂ 比值为２．７２,而处理叶片为２．８６。这说明只有在弱光下Ｃｅ才对叶片色素的组分有影响,Ｃｅ 使叶片中的Ｃｈｌｂ 略有下降。Ｃｅ能促进叶绿体光系统Ⅰ叶绿素蛋白质复合物及１１０ ｋＤ多肽的形成,并使捕光叶绿素ａ／ｂ 蛋白质复合物及其２７ ｋＤ多肽的含量减少。     

茄子（Solanum melongena L.）叶上表皮紫色花色素苷对光合机构的保护效应

以茄子同一叶片上紫斑区域和绿斑区域为材料,采用发光二极管LED发出的混合光谱(白光)和单色光谱(红、蓝、绿光)照射后,通过光合仪(CIRAS-2)和叶绿素荧光仪(PEA和Dual-PAM-100)测定了茄子叶上表皮紫色花色素苷(purple anthocyanin,PA)对光合机构的影响.结果表明,分布于茄子叶片上表皮的PA,主要截获约53.2%～73.6%的500～600 nm(黄绿光)可见光.紫斑区域的叶绿素a含量较低以及PA截获27%的400～480 nm(蓝光)和10%的630～700 nm(红光),可能是其最大净光合速率Pnmax降低的原因.随着白光照射强度(0～3000 μmol·m-2·s-1)的增大,茄子叶片的PSⅡ最大光化学效率Fv/Fm、单位面积的光合机构含有的反应中心数目RC/CS0和天线色素能量吸收驱动力DFABS下降,而初始荧光Fo、J相的相对可变荧光VJ和单位反应中心耗散的能量DI0/RC增加,但紫斑区域的上述参数变幅明显较小,表现为光抑制程度减轻.用2000 μmol·m-2·s-1的不同光质照射30 min后,茄子叶片Fv/Fm降低,但只有绿光和白光下紫斑区域的Fv/Fm显著的高于绿斑区域;同时白光照射后,茄子叶片的P700氧化还原动力学曲线降幅明显的大于PSⅡ动力学曲线,但紫斑区域的这两个光系统动力学曲线的下降幅度明显减小.这反映出茄子叶上表皮PA有效的保护了PSⅡ和PSⅠ反应中心,减轻了电子传递链的还原程度和热耗散机构的运转压力,较好地维持了PSⅡ与PSⅠ之间的功能协调性.这种通过PA截获500～600 nm(黄绿光)可见光对光合机构的保护效应属于生物物理水平的防御系统.     

A Circular Dichroism Spectroscopic Study Revealing the Cause of the Changes of Chlorophyll a Fluorescence Induction of Photosystem Ⅱ During Heat Treatment

Chlorophyll a fluorescence and circular dichroism (CD) spectra of photosystem Ⅱ (PSⅡ) membrane were measured after heat treatment. The chlorophyll fluorescence parameter Fo' remained stable after treatment at the temperatures from 30 ℃ to 40 ℃ and then reached a maximum after treatment at 55 ℃. In PSⅡ membranes and LHCⅡ (light-harvesting chlorophyll a/b binding complex)-enriched complexes, anomalous CD signals with extremely large amplitudes occurred during the heat treatment. The temperature corresponding to the maximum anomalous CD intensity peaking at 677 nm was 40 ℃. The results indicate that the aggregation state of the LHCⅡ in PSⅡ is related to the anomalous CD signal, and can be an important factor influencing Fo' in the heat treatment of PSⅡ membrane.     

Integrated regulation of the photosynthetic gene family from Arabidopsis thaliana in transformed tobacco cells

Summary Expression of the three chlorophyll a/b binding protein (cab) genes of Arabidopsis thaliana was studied in transformed tobacco tissues. For each cab gene, approximately 1000 bp of the promoter region plus a portion of the structural gene was inserted into a promoter-expression vector such that a translational fusion between the cab gene and the promoter-less chloramphenicol acetyltransferase (cat) gene was formed. The constructed molecules were introduced into either cultured tobacco cells or tobacco leaves and the promoter activity was monitored as chloramphenicol acetyltransferase activity. The light-grown tissues exhibited 1.5- to 60-fold greater promoter activity than did dark-grown tissues. Expression of the cab promoters was tissue specific: activities were much stronger in green leaves than other tissues. The cab promoters were almost equally active in transformed calli or shoots derived from leaves. However, in cultured tobacco cells, one promoter was two to three times stronger than the other two. The chimeric gene fusion, cab-cat, segregated in the F1 generation as a dominant Mendelian trait.     

Effects of Cadmium on Chlorophyll Content,Photochemical Efficiency,and Photosynthetic Intensity of Canna indica Linn.

The effects of cadmium (Cd²⁺) on growth status, chlorophyll (Chl) content, photochemical efficiency, and photosynthetic intensity were studied on Canna indica Linn. Plant specimens that were produced from a constructed wetland and precultivated hydroponically in 20 L of 1/10 Hoagland solution under greenhouse conditions for 1 week were exposed to cadmium in concentrations of 0, 0.4, 0.8, 1.6 and 3.2 mg L^―1 Cd²⁺, respectively. The results show that leaves were injured in the Cd²⁺ solution by the third day of exposure and the injury became more serious with an increase in the applied heavy metal. Under 3.2 mg L^―1 Cd²⁺ treatment, growth retardation, the decrease of chlorophyll content from 0.70 to 0.43 mg g^―1 FW, and a decrease in Chl a/b ratio from 2.0 to 1.2 were observed. Chl a was more sensitive than Chl b to Cd²⁺ stress. The decrease was the same with photochemical efficiency. Photosynthetic intensity decreased by 13.3% from 1.5×10⁴ μmol m^―2s^―1 CO₂ in control to 1.3×10⁴ μmol m^―2s^―1 CO₂ in the treatment of 3.2 mg L^―1. Because Canna species are used in heavy metal phytoremediation, these results show that C. indica can tolerate 0.4 to 0.8 mg L^―1 Cd²⁺. Therefore, it is a potential species for phytoremediation of cadmium with some limitations only at higher concentrations.     

生长光强对六个橡胶树品种幼苗光合特性的影响

研究了6个橡胶树品种幼苗(适应1年后)在不同生长光强(100%、50%、25%和5%自然光)下的叶片光合系统对光强和CO2浓度的响应特性。结果表明,6个橡胶树品种对不同的光环境均表现出较强的适应性。在不同生长光强下,橡胶树幼苗叶片的最大光合速率(Pmax)、光补偿点(LCP)、暗呼吸速率(Rd)、磷酸丙糖利用速率(TPU)、最大羧化速率(Vcmax)和最大电子传递速率(Jmax)以及叶绿素含量(Chl)均有显著差异(P<0.05),而光饱和点(LSP)和AQY(表观量子效率)则无显著差异。相同生长光强下,6个橡胶树品种间叶片的最大光合速率(Pmax)、暗呼吸速率(Rd)、磷酸丙糖利用速率(TPU)、最大电子传递速率(Jmax)和叶绿素含量(Chl)有显著差异(P<0.05),其光补偿点(LCP)、最大羧化速率(Vcmax)和表观量子效率(AQY)则无显著差异。综合比较各参数,RRIM600、云研77-4和PR107适宜于相对光强为100%~50%的植胶环境,而云研77-2、GT1和热研523适宜于相对光强为50%~25%的植胶环境。     

Mg2+对生长在不同光强下的小麦叶绿体光合功能的影响

研究结果表明,Mg~(2+)对生长在不同光强度下的小麦叶绿体光合功能有不同影响。与生长在低光强(2×10~8勒克斯)下的小麦叶绿体相比,Mg~(2+)更加明显地降低从生长在高光强(2×10~4勒克斯)下的小麦所分离的叶绿体的吸收光谱在红区和蓝区的吸收峰值;但它更大幅度地提高后者在低温(77K)下的PSⅡ相对荧光产量(F_(687))与PSⅠ相对荧光产量(F_(742))的比值,PSⅡ活性和PSⅡ原初光能转化效率。实验结果证明,更高的光强度可能有利于叶绿体形成更多可流动的LHC-Ⅱ和LHC-Ⅰ。     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.     

Kinetic Fluorescence Spectral Analysis of Core Antennas CP43 and CP47 of Photosystem Ⅱ with Ultrafast Time-resolved Technology

Ultrafast time-resolved fluorescence experiments have been performed with core antennas CP43 and CP47 of PS Ⅱ. Their dynamic fluorescence spectra were obtained with excitation wavelength 514.5 nm. For CP43, the emission spectrum was found to be from 640 to 780 nm with a peak at ～680 nm and the lifetime of fluorescence was 3.54 ns. For CP47, the emission spectrum was from 630 to 775 nm with a peak at ～691 nm and the fluorescence lifetime was 3.22 ns. The fluorescence emission efficiencies of Chl a in CP43 and CP47 were calculated to be approximately 38.3% and 40.6%, respectively. The energy transfer from β-Car to Chl a in CP43 and CP47 was discussed. The rates of energy transfer from β-Car to Chl a were measured to be about 9.6×1011 s-1 and 1.3×1012 s-1 and energy transfer efficiencies 47.5% and 66.5% respectively. The edge-edge distances between β-Car and Chl a in CP43 and CP47 were estimated to be ～0.110 nm and ～0.085nm respectively.     

毛竹PSⅡ大量捕光天线蛋白基因克隆及其表达分析

采用RT-PCR和RACE技术,从一年生毛竹(Phyllostachys edulis)新鲜叶片中分离到3个捕光色素结合蛋白基因cab-PhE2、cab-PhE3和cab-PhE6,GenBank登记号分别为EF207230、EF405877和EF628209.序列分析表明,3个基因分别与其它植物如水稻、玉米、大麦等PSⅡ的Ihcb2、Ihcb1、Ihcb3基因有着较高的一致性,其编码的蛋白属于大量捕光天线.蛋白结构预测表明,3个基因编码的蛋白均由导肽和成熟蛋白组成,其中成熟蛋白的二级结构均包含4个α螺旋结构、3个类胡萝卜素结合位点,以及相应的叶绿素a、b结合位点.组织特异性表达检测表明,3个基因在叶片、叶鞘和幼茎中均有表达,且在叶片中最高,而在根中未检测到表达.在强光照射( 1500μmol·m-2·s-1)条件下,3个基因的表达量均呈下调趋势,不同基因间存在着一定的差异,其中cab-PhE3和cab-PhE2的表达丰度在光照4h内下降较快,至6 h cab-PhE2接近于零,而cab-PhE6经光照4h表达丰度仍为对照的70％以上,但之后2h后很快降至对照的5％以下.     

Isolation of an oxygen-evolving Photosystem II preparation containing only one tightly bound calcium atom from a chlorophyll b-deficient mutant of rice

Oxygen-evolving Photosystem II (PS II) particles were prepared from the thylakoid membranes of a chlorophyll b-less rice mutant, which totally lacks light-harvesting chlorophyll a/b proteins, after solubilization with β-octylglucoside. The preparation was essentially free of Photosystem I as judged from its low-temperature fluorescence spectrum and polypeptide composition. The PS II particles contained all the major subunit polypeptides of the PS II reaction center core complexes and the three extrinsic proteins related to oxygen evolution. The relative abundances of the 33, 21 and 15 kDa proteins were 100, 64 and 20%, respectively, of the corresponding proteins in the mutant thylakoids. The chlorophyll-to-Q_A ratio was 53 and there was only one bound Ca²⁺ per Q_A. Thus, one of the two bound Ca²⁺ present in the oxygen-evolving PS II membrane preparations from wild-type rice (Shen J.-R., Satoh, K. and Katoh, S. (1988) Biochim. Biophys. Acta 933, 358–364) is missing. The mutant PS II particles were highly active in oxygen evolution in the absence of exogenously added Ca²⁺, although addition of 5 mM Ca²⁺ enhanced the activity by 30%. When the 21 and 15 kDa proteins were supplemented to the particles, the Ca²⁺-effect disappeared and the rate of oxygen evolution increased to a level exceeding 1000 μmol O₂ per mg chlorophyll per h. The results indicate that the number of Ca²⁺ needed to promote a high rate of oxygen evolution is one per PS II in higher plants.     

Study on the Binding State of Calcium in Photosystem ⅡOxygen-Evolution Complex

Washing spinach PSII oxygen-evolution complex (OEC) with 2 mmol/L EGTA or extraction medium caused a 28.4% and 25.0% loss of oxygen evolution activities respectively, but the loss of polypeptide components of OEC did not take place, whereas washing with 1 mol/L NaCI caused both a 90.0% loss of oxygen evolution activity and loss of 17, 23kD polypeptides. Adding 5–10 mmol/L CaC12 could restore oxygen evolution activities of OEC by various washing to a great extent, but had no effect on control OEC, whereas adding 5–10 mmol/L EGTA had no effect on the OEC by various' washing, but caused the loss of oxygen evolution mixtures, which could induce the release of of 17, 23kD polypeptides from OEC, caused 54.3% loss of oxygen evolution activity, under this circumstance, adding 2 mmol/L of EGTA could only maintain a weak oxygen evolution activity of OEC, but adding 10 mmol/L of CaCl2 could restore oxygen evolution activity of OEC to the control level. These findings' suggest a two way loose binding of Ga2+ to PSⅡ OEC in one way Ca2+ is loose bound to the surface of PSⅡOEC and in other, the Ca2+-binding site is wrapped by 17, 23kD polypeptides. Both of them have effect on oxygen evolution activity of PSⅡ OEC. By way, Mn2+ can antagonize the restoration of oxygen evolution activity by Ca2+ to the NaCl-washing PSⅡ OEC.