Effect of methylmercury chloride on the primary photosynthetic activity of higher plants

The effect of methylmercury chloride (MeHg) on the fluorescence characteristics of pea seedling leaves and thylakoids isolated from these leaves was studied by the pulse-amplitude-modulation (PAM) fluorometric method. In 3-4 days after the addition of MeHg (20 microM) to the nutritious solution, the maximal (Fv/Fm) and real (under steady state actinic light illumination) (deltaF/F'm) quantum photochemical yield of PS II decreased. The nonphotochemical fluorescence quenching coefficient in control (qN) decreased after its maximum value has been reached. In MeHg-treated samples, this decrease was not observed, possibly due to the disturbance of delta pH energy transducing processes in ATP synthase. This was confirmed by the results of experiments on isolated thylakoids. After MeHg (5 microM) treatment of thylakoids, the photophosphorylation rate and light-triggered Mg2+-dependent H+-ATPase activity were suppressed by 20-40%, depending on the duration of MeHg exposure. However, in experiments with isolated thylakoids, no decrease either in the electron transport rate or in the Fv/Fm ratio was observed. In total, the results obtained allow one to assume that MeHg at concentrations and time duration used directly damages the coupling complex. The PS II inactivation in leaves and algae cells may be a result of the oxidative stress processes.     

Pharmacological characterization of the effects of methylmercury and mercuric chloride on spontaneous noradrenaline release from rat hippocampal slices

The environmental contaminants methylmercury (MeHg) and mercuric chloride (HgCl2) stimulated the spontaneous release of [3H]noradrenaline ([3H]NA) from hippocampal slices in a time- and concentration-dependent manner. Both MeHg and HgCl2 were similarly potent, with an EC50 of 88.4 microM and 75.9 microM, respectively. The releasing effects of MeHg and HgCl2 increased in the presence of desipramine, showing that the mechanism does not involve reversal of the transmitter transporter, and were completely blocked by reserpine preincubation, indicating a vesicular origin of [3H]NA release. The voltage-gated Na+ channel blocker tetrodotoxin (TTX) did not affect the response to mercury compounds. [3H]NA release elicited by MeHg was partially dependent on extracellular Ca2+, since it decreased significantly in a Ca2+-free EGTA-containing medium whereas HgCl2 induced a release of [3H]NA independent of extracellular Ca2+. Neither Ca2+-channels blockers, cobalt chloride (CoCl2) and (omega-conotoxin-GVIA, nor the Na+/Ca2+-exchanger inhibitor benzamil reduced MeHg-evoked [3H]NA release. Moreover, thapsigargin or caffeine, endoplasmic reticulum Ca2+-depletors, did not modify metal-evoked [3H]NA release, whereas ruthenium red, which inhibits the mitochondrial Ca2+ transport, decreased the effect of both MeHg and HgCl2. All these data indicate that, in hippocampal slices, mercury compounds release [3H]NA from the vesicular pool by a mechanism involving Ca2+ mobilization from mitochondrial stores.     

Effect of mercuric chloride and methylmercury on photosynthetic activity of the diatom Thalassiosira weissflogii by a fluorescent method

It was shown by the pulse-amplitude modulation fluorescent method that, at a weak illumination (6 microE m-2.s-1), methylmercury at a concentration of 10(-6)-10(-7) M decreases the photochemical activity of the reaction centers of photosystem II in cells of microalgae Thalassiosira weissflogii after a prolonged lag phase. Cells resistant to methylmercury at these low concentrations were detected by the microfluorimetric method. Chloride mercury decreased the activity of photosystem II of the algae only when at higher concentrations. Both toxicants at a concentration of 10(-6) M decreased the rate of recovery of photoinduced damage of centers of photosystem II and led to an increase in the energization component of nonphotochemical fluorescence quenching. These results indicate that the complex of fluorescent methods can be used to monitor early changes in the photosynthetic apparatus of algae in response to the toxic action of heavy metals.     

A comparative study of the toxicity of mercury dichloride and methylmercury, assayed by the Frog Embryo Teratogenesis Assay--Xenopus (FETAX)

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a powerful and flexible bioassay that makes use of the embryos of the anuran amphibian Xenopus laevis. The FETAX can detect xenobiotics that affect embryonic development, when mortality, teratogenicity and growth inhibition are used as endpoints. The FETAX was used to compare the embryotoxic and teratogenic potentials of two chemical species of mercury, inorganic mercury(II) chloride (HgCl2) and organic methylmercury chloride (MeHgCl). A higher toxicity of MeHgCl (the estimated median lethal concentration [LC50] and median teratogenic concentration [TC50] were 0.313microM and 0.236microM, respectively) over HgCl2, with estimated LC50 and TC50 values of 0.601microM and 0.513microM, respectively). On the basis of these results, HgCl2 and MeHgCl can be classified as "slightly teratogenic compounds", as the ratio of LC50/TC50 is less than 1.5. There was a significant deviation from the commonly described monotonic behaviour of the concentration-response curves, suggesting a hormetic effect of both species of mercury. Uptake experiments, followed by neutron activation analysis, showed a higher incorporation of mercury in embryos exposed to MeHgCl compared with those exposed to HgCl2. Interestingly, Hg- exposed embryos showed a higher content of selenium and zinc than did control embryos.     

不同品种美国山核桃叶绿素荧光参数日变化的研究

以湖南省永州市冷水滩采穗圃中的美国山核桃为试材,研究了叶绿素荧光参数的日变化规律。结果表明:初始荧光(Fo)、最大荧光(Fm)、PSII原初光能转化效率(Fv/Fm)、光合量子产额(Yield)、光化学猝灭系数(qP)、非光化学猝灭系数(qN)和表观电子传递速率(ETR)均存在着明显的日变化。其中Fv/Fm、Fm、Yield、qP均呈先下降后上升的趋势,在中午强光下降低到最低值;qN则呈先上升后下降的趋势,在中午时分达到峰值;Fo呈下降趋势,部分品种傍晚稍有回升,但仍比早晨低;ETR日变化呈双峰曲线。不同品种间Fv/Fm、Yield、ETR、qP、qN对光强和温度的响应也存在着明显差异,可作为鉴定品种耐光抑制能力大小的指标。     

低温下壳聚糖处理对黄瓜幼苗生理生化特性的影响

50 mg/L的壳聚糖处理减轻了低温对黄瓜幼苗膜的伤害,低温(6℃)处理4 d后,幼苗膜透性和MDA含量明显低于对照,并且显著减缓了低温导致的光合速率、实际光化学效率(ФPS Ⅱ)、光化学猝灭系数(qP)和1光系数(F’v /Em’)的下降;同时,处理使抗氧化酶SOD、POD、CAT和APX活性提高,可溶性蛋白和脯胺酸含量增加.因此,壳聚糖处理增强了黄瓜幼苗的抗冷性,保护了膜系统,提高了活性氧清除能力,减轻了低温对光合机构的破坏.     

倪氏拟多甲藻叶绿素荧光活性对环境因子的响应

采用叶绿素荧光分析技术探讨了温度、pH、光强对水华优势种倪氏拟多甲藻光合活性的影响。结果表明, 倪氏拟多甲藻光系统Ⅱ的量子产量Y(Ⅱ)和最大光化学效率(Fv/Fm)随温度(7.5-20.0℃)升高而显著增加(P0.05), 在低温下电子传递速率未受阻, 细胞在7.5-20.0℃内均有高光合活性; 10.0 ℃下的光合活性随pH增大先升高后降低, 峰值出现在pH 7.3时, 光合活性顺序为: 弱碱性 中性 酸性; 快速叶绿素荧光动力学曲线分析显示pH 7.3下的光合活性为典型OJIP曲线, 其他pH下PSⅡ反应中心、电子受体库受损, 显示该藻适应较窄的pH范围, pH7.0-8.0内是其适宜的条件; 快速光响应曲线显示其半饱和光强Ek为385.52 mol photons/(m2s), 表明其具有高光饱和点, 耐受高光强。研究表明藻细胞光合活性对温度和光强变化有较强适应性, 对pH的变化敏感, 弱碱性条件是其光合作用的适宜条件; 低温时细胞通过环式电子链提高光化学效率, 降低高光强可能带来的光损伤; 弱酸性(pH 5.0)会同时损伤其光系统Ⅰ和光系统Ⅱ, 造成其光化学效率的显著下降;倪氏拟多甲藻在低温和高光强下的独特光合特性使其在春季淡水水体中占据竞争优势, 是其形成水华的内在原因。     

高温对仁用杏光合特性及PSⅡ光化学活性的影响

为探讨高温胁迫下仁用杏叶片的光合适应机制,以科尔沁沙地生长的4年生'超仁'仁用杏为试材,设置环境温度为25℃、30℃、40℃和50℃处理,利用气体交换技术和快速叶绿素荧光诱导动力学曲线分析技术(JIP-test),研究了仁用杏叶片光合特性和PSⅡ光化学活性.结果表明:在一定温度范围内,随着温度升高,仁用杏通过提高光合色素含量和比例来维持光能的吸收、传递和转换能力,从而保证光合机构正常运转;当高温超过叶片自身生理调节限度后,叶绿素发生分解、净光合速率(Pn)明显下降、胞间CO2浓度(Ci)上升,说明光合作用的下降是由叶肉因素造成的.温度40℃导致单位面积有活性反应中心数量(RC/CSo)显著下降;而50℃高温下荧光诱导曲线中K点(Wk)和J点(Vj)明显增加,高温对仁用杏叶片放氧复合体(OEC)、受体侧和PsⅡ反应中心造成了伤害.此外,50℃高温还导致初始荧光(Fo)显著升高,为对照的2.26倍,PSⅡ最大光化学效率(Fv/Fm)和光化学性能指数(PI/ABS)分别下降为对照的37.9%和10.3%.高温损害了PSⅡ供体侧和受体侧的功能,造成光合效率下降,这是高温胁迫对仁用杏叶片光合机构伤害的主要机制之一.     

铀对两种小球藻生长和光合作用的影响

为了探究铀对藻类生长及光合作用的影响,筛选新的基于光合作用的水体铀污染生态风险评价指标,本试验采用不同浓度铀（0、0.5、1、5、10、20mg U·L-1）分别处理普通小球藻(Cholorella vulgaris)和黄龙普通小球藻两种来自不同生境的微藻,在处理后的第3、5、7、10、14d进行相对生长速率、光合放氧速率、叶绿素含量和叶绿素荧光动力学参数等指标的测定。结果表明：（1）0.5mg·L-1低浓度铀处理显著促进两种小球藻的生长和光合作用效率,表现为两种微藻的相对生长速率、光合放氧速率、光系统II最大光化学量子产量Fv/Fm、实际光化学量子产量Y（Ⅱ）、相对电子传递速率rETR等叶绿素荧光参数等指标均显著高于对照, 而5-20 mg·L-1高浓度铀处理则显著抑制两种小球藻的生长和光合作用;（2）黄龙普通小球藻比普通小球藻对铀处理更敏感,在1mg·L-1处理浓度下生长与光合作用就受到显著抑制,可以用来作为水体铀污染生物监测的指示生物;（3）回归分析表明,不同浓度铀处理下,叶绿素荧光参数Y(II)和rETR的响应速度快于相对生长速率、光合放氧速率、叶绿素含量和Fv/Fm等指标的变化,可以作为水体铀污染生态风险评价的敏感指标。     

Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components – calculation of qP and Fv-/Fm-; without measuring Fo-;

Imaging of chlorophyll a fluorescence from leaves has enabled the spatial resolution of the fluorescence parameter, F/Fm-;. Although this parameter provides a reliable estimate of photosynthetic efficiency under most conditions, the extent to which this efficiency is defined by (i) competition with other energy-dissipating processes operating at photosystem II and (ii) by processes on the reducing side of photosystem II, such as carbon assimilation, requires the use of additional parameters. Of particular value are qP, which quantifies the photochemical capacity of photosystem II, and Fv-;/Fm-;, which quantifies the extent to which photochemistry at photosystem II is limited by competition with thermal decay processes. Imaging of both qP and Fv-;/Fm-; requires measurement of Fo-; (the minimum fluorescence yield in the light-adapted state), which cannot be imaged with existing systems. In this paper, a method is described which estimates Fo-; through a simple equation involving the minimum fluorescence yield in the dark-adapted state (Fo), the maximum fluorescence yield in the dark-adapted state (Fm), and the maximum fluorescence yield in the light-adapted state (Fm-;). This method is tested here, through comparison of measured and calculated values of Fo-;. An example of the application of this method to analysis of photosynthetic performance in leaves, from images of chlorophyll a fluorescence, is also presented.     

Characterization of target site of aluminum phytotoxicity in photosynthetic electron transport by fluorescence techniques in tobacco leaves

Aluminum (Al) toxicity limits crop yield in acidic soil through affecting diverse metabolic processes, especially photosynthesis. The aim of this work was to examine the effect of Al on photosynthetic electron transport in vivo as determined by chlorophyll fluorescence and delayed fluorescence of tobacco leaves. Results showed that Al treatment inhibited the photosynthetic rate and electron transfer, and decreased photosystem (PS) II photochemical activity in a time- and concentration-dependent manner, which could not be obviously alleviated by the addition of the reactive oxygen species (ROS) scavenger ascorbic acid (AsA). These results suggested that photosynthetic electron transfer chain components, especially PSII, might be directly damaged by Al instead of in an ROS-dependent manner. Furthermore, the fluorescence imaging and biochemical analysis exhibited that Al, after entering the cells, could accumulate in the chloroplasts, which paralleled the decreased content of Fe in the chloroplast. The changes in the chlorophyll fluorescence decay curve, the delayed fluorescence decay curve and the chlorophyll fluorescence parameters indicated that Al, through interacting with or replacing the non-heme iron between Q(A) and Q(B), caused the inhibition of electron transfer between Q(A) and Q(B), resulting in PSII photochemical damage and inhibition of the photosynthetic rate. In summary, our results characterized the target site of Al phytotoxicity in photosynthetic electron transport, providing new insight into the mechanism of Al phytotoxicity-induced chloroplast dysfunction and photosynthetic damage.     

Nondestructive evaluation of the photosynthetic properties of micropropagated plantlets by imaging photochemical reflectance index under low light intensity

The photochemical reflectance index (PRI) of micropropagated potato leaves was estimated nondestructively from outside the culture vessel using a PRI imaging system developed by the present group. The PRI was determined under low light intensity conditions after dark treatment and compared with the chlorophyll fluorescence parameter Fv/Fm, which denotes photosystem II maximum quantum yield. Short-term high-light treatment decreased Fv/Fm of the plantlets. Culture conditions such as temperature and sucrose concentration also affected Fv/Fm. A linear relationship between the PRI and Fv/Fm was observed in both cases of high-light treatment and different culture conditions, suggesting the potential of the PRI to be used as a substitute for Fv/Fm. PRI estimated from reflection images under low light intensity conditions may be used for rapid and noninvasive evaluation of photosynthetic properties of micropropagated plantlets in a similar manner to Fv/Fm.     

Photosynthesis of Prochlorothrix hollandica under Sulfide-Rich Anoxic Conditions

The photosynthetic activity and photosystem II fluorescence of Prochlorothrix hollandica were studied under anoxic, sulfide-rich conditions. Oxygenic photosynthetic activity with water as the electron donor was highly resistant to inhibition by sulfide. Cells still retained 50% of their oxygenic photosynthetic activity at >1 mM sulfide. In the presence of DCMU [N-(3,4-dichlorophenyl)-N(prm1)-dimethylurea], an inhibitor of photosystem II activity, P. hollandica cells exhibited a low but significant anoxygenic photosynthetic activity when sulfide was present. This activity increased with higher sulfide concentrations and reached maximal rates at concentrations exceeding 1 mM sulfide. The effects of hydroxylamine on both oxygen evolution and fluorescence induction kinetics were similar to those observed for sulfide. It was concluded that the oxidizing site of photosystem II was the site of sulfide action leading to reduced or even fully inhibited electron donation to photosystem II. These observations bear similarity to the situation in some cyanobacteria in which both hydroxylamine and sulfide inhibit electron donation from H(inf2)O to P(inf680). The high resistance of photosystem II to sulfide is related to the hydrophobic nature of the manganese-stabilizing protein in P. hollandica (T. S. Mor, A. F. Post, and I. Ohad, Biochim. Biophys. Acta 1141:206-212, 1993). The observed sulfide tolerance of P. hollandica may confer a competitive advantage in its natural environment, where it forms a dominant fraction of phytoplankton in waters in which sulfide presence is a recurring phenomenon.     

Photoinduced reactivation of photosystem II in Chlorella after prolong incubation without light

The light-dependent reactivation of photosystem II in Chlorella pyrenoidosa Chick, CALU-175 cells, inactivated with supraoptimal temperatures (40-43 degrees C) in the dark or during heterotrophic growth was studied. It was shown that the inactivation of photosystem II after incubation in the dark at 41-42 degrees C, which showed up in the suppression of relative yield of variable chlorophyll fluorescence Fv due to an increase in yield F0 could be completely reversed by light. The inactivation of photosystem II at 43 degrees C in the dark could not be reversed by subsequent irradiation. In this case, the suppression of Fv/Fm was related not only to the growth of F0 but also with the decrease in Fm. The light dependences of the rate and extent of reactivation of yield Fv after heterotrophic growth or incubation of chlorella at 41 degrees C in the dark completely coincided. The full light-induced reactivation of photosystem II took place as the rate of photoinduced electron transport reached the rate of nonphotochemical reduction of plastoquinone in the dark. These results suggest that the light-reversed inactivation of photosystem II after heterotrophic growth or incubation at 41 degrees C in the dark is due to the redox-interaction of the primary quinone acceptor with plastoquinone reduced by the electron flux from the substrates of chlororespiration.     

Inhibition of the human thioredoxin system. A molecular mechanism of mercury toxicity

Mercury toxicity mediated by different forms of mercury is a major health problem; however, the molecular mechanisms underlying toxicity remain elusive. We analyzed the effects of mercuric chloride (HgCl(2)) and monomethylmercury (MeHg) on the proteins of the mammalian thioredoxin system, thioredoxin reductase (TrxR) and thioredoxin (Trx), and of the glutaredoxin system, glutathione reductase (GR) and glutaredoxin (Grx). HgCl(2) and MeHg inhibited recombinant rat TrxR with IC(50) values of 7.2 and 19.7 nm, respectively. Fully reduced human Trx1 bound mercury and lost all five free thiols and activity after incubation with HgCl(2) or MeHg, but only HgCl(2) generated dimers. Mass spectra analysis demonstrated binding of 2.5 mol of Hg(2+) and 5 mol of MeHg(+)/mol of Trx1 with the very strong Hg(2+) complexes involving active site and structural disulfides. Inhibition of both TrxR and Trx activity was observed in HeLa and HEK 293 cells treated with HgCl(2) or MeHg. GR was inhibited by HgCl(2) and MeHg in vitro, but no decrease in GR activity was detected in cell extracts treated with mercurials. Human Grx1 showed similar reactivity as Trx1 with both mercurial compounds, with the loss of all free thiols and Grx dimerization in the presence of HgCl(2), but no inhibition of Grx activity was observed in lysates of HeLa cells exposed to mercury. Overall, mercury inhibition was selective toward the thioredoxin system. In particular, the remarkable potency of the mercury compounds to bind to the selenol-thiol in the active site of TrxR should be a major molecular mechanism of mercury toxicity.     

施氮和大气CO2浓度升高对小麦旗叶光合电子传递和分配的影响

采用开顶式气室盆栽培养小麦,设计2个大气CO2浓度(正常:400 μmol.mol-1;高:760 μmol·mol-1)、2个氮素水平(0和200 mg·kg-1土)的组合处理,通过测定小麦抽穗期旗叶氮素和叶绿素浓度、光合速率(Pn)-胞间CO2浓度(C1)响应曲线及荧光动力学参数,来测算小麦叶片光合电子传递速率等,研究了高大气CO2浓度下施氮对小麦旗叶光合能量分配的影响.结果表明:与正常大气CO2浓度相比,高大气CO2浓度下小麦叶片氮浓度和叶绿素浓度降低,高氮处理的小麦叶片叶绿素a/b升高.施氮后小麦叶片PSⅡ最大光化学效率(Fv/Fm)、PSⅡ反应中心最大量子产额(Fv'/Fm')、PSⅡ反应中心的开放比例(qr)和PSⅡ反应中心实际光化学效率(φPSⅡ)在大气CO2浓度升高后无明显变化,虽然叶片非光化学猝灭系数(NPQ)显著降低,但PSⅡ总电子传递速率(JF)无明显增加;不施氮处理的Fv'/Fm'、φPSⅡ和NPQ在高大气CO2浓度下显著降低,尽管Fv/Fm和qp无明显变化,JF仍显著下降.施氮后小麦叶片JF增加,参与光化学反应的非环式电子流传递速率(Jc)明显升高.大气CO2浓度升高使参与光呼吸的非环式电子流传递速率(J0)、Rubisco氧化速率(V0)、光合电子的光呼吸/光化学传递速率比(J0/Jc)和Rubisco氧化/羧化比(V0/Vc)降低,但使Jc和Rubisco羧化速率(Vc)增加.因此,高大气CO2浓度下小麦叶片氮浓度和叶绿素浓度降低,而增施氮素使通过PSⅡ反应中心的电子流速率显著增加,促进了光合电子流向光化学方向的传递,使更多的电子进入Rubisco羧化过程,Pn显著升高.     

温州蜜柑叶片光合作用光抑制的保护机理

晴天条件下,使用便携式调制荧光仪和分光光度计观察了温州蜜柑叶片光合作用光抑制发生过程中几个主要荧光参数（初始荧光F0、最大荧光Fm、PSⅡ的光化学效率Fv/Fm、非光化学猝灭qN及其快相qNf和慢相qNs）、电子传递速率（ETR）和玉米黄素相对含量的日变化,结果表明,随着光强的增强,ETR、qN及其qNr与qNs以及玉米黄素相对含量升高,Fv/Fm、Fm和F0下降。用DTT处理后,qNs较对照明显下降,F0较对照明显上升,可以认为,柑橘在光合作用日变化中存在依赖于叶黄素循环和类囊体膜质子梯度两种非辐射能量耗散方式,而且它们在防御光破坏方面起着重要作用。     

Selective inhibition of photosystem II in spinach by tobacco mosaic virus: an effect of the viral coat protein

Leaves of Spinacia oleracea inoculated with tobacco mosaic virus (TMV) strain PV230 develop mild chlorotic and mosaic symptoms of infection. Thylakoid membranes isolated from these infected leaves showed a reduced Fv/Fm ratio for chlorophyll fluorescence kinetics, at 25 degrees C. The photosystem II (PS II)-mediated electron-transport rate was inhibited 50%, whereas PS I activity was unaffected by virus infection. Protein analysis indicated that TMV coat protein was associated with thylakoids, in particular with the PS II fraction. The results demonstrate that TMV-infected S. oleracea shows inhibition of photosynthetic electron transport through PS II. We propose that the inhibition of photosynthetic activity results from the association of viral coat protein with the PS II complex.     

花后不同时期弱光和高温胁迫对小麦旗叶荧光特性及籽粒灌浆进程的影响

在田间试验条件下研究了花后不同时期弱光和高温胁迫对小麦旗叶荧光特性及籽粒灌浆进程的影响.结果表明,弱光处理3 d后,旗叶PSⅡ最大光化学效率（Fv/Fm）和光合速率（Pn）显著降低,但PSⅡ实际光化学效率（ΦPSⅡ）、荧光光化学猝灭系数（qP）和非光化学猝灭（NPQ）与对照相比差异较小;高温处理3 d后,Fv/Fm、Pn、ΦPSⅡ和qP均极显著降低, NPQ升高幅度较小.胁迫解除后,灌浆前期（花后8～10 d）弱光和高温处理后的旗叶荧光参数和光合速率略有恢复,但灌浆中期（花后15～17 d）处理后的各参数始终呈下降趋势, 说明前期处理效应是可逆的,而中期处理加速其衰老进程.用Logistic方程对各处理的籽粒灌浆过程模拟明,弱光和高温处理后籽粒粒重的降低主要是平均灌浆速率、最大灌浆速率和渐增期灌浆速率显著降低所致.灌浆持续期、最大灌浆速率出现时间、缓增持续期和缓增期灌浆速率受弱光和高温影响较小.     

Inhibition of Photosystem II in Isolated Chloroplasts by Lead

Inhibition of photosynthetic electron transport in isolated chloroplasts by lead salts has been demonstrated. Photosystem I activity, as measured by electron transfer from dichlorophenol indophenol to methylviologen, was not reduced by such treatment. However, photosystem II was inhibited by lead salts when electron flow was measured from water to methylviologen and Hill reaction or by chlorophyll fluorescence. Fluorescence induction curves indicated the primary site of inhibition was on the oxidizing side of photosystem II. That this site was between the primary electron donor of photosystem II and the site of water oxidation could be demonstrated by hydroxylamine restoration of normal fluorescence following lead inhibition.