【作者】 刘超；

【导师】 洪法水；

【作者基本信息】 苏州大学 ， 化学生物学， 2010， 博士

【摘要】 我国从上个世纪70年代就开始大规模推广稀土微肥农用,作用效果明显。相关实验室研究也证实适宜浓度的稀土元素处理能够刺激植物生长发育,增强光合能力,提高作物产量和改善品质。但是,目前有关稀土元素的植物生理效应研究还不够深入,特别是对稀土元素促进光合作用这一地球上最重要的化学反应的相应效应和机理缺乏系统研究。对稀土元素理化特性对光合作用所产生的化学生物效应也未得到阐明。鉴于此,我们选取镧（La）、铈（Ce）、钕（Nd）这三个稀土微肥中的主要组成元素,围绕光合作用能量传递转换过程,即光能依次向电能,活跃化学能,稳定化学能转换这一过程,系统研究了稀土元素提高菠菜（光合作用常用模式植物）光合作用效应的机制。并通过比较三种不同稀土元素的光合效应差异,旨在阐明稀土元素独特的4f层电子和变价特征以及与植物必需大量元素钙的相似性对光合作用的影响。本文不但为研究光合作用提供了新思路,且可为高效稀土农用寻求理论根据。实验发现:1)稀土元素可促进菠菜的光化学能力,促进菠菜光能的吸收、转运、分配、光电转换等各环节效率。La³⁺、Ce³⁺、Nd³⁺处理后,叶绿体膜特征吸收峰峰值增大,叶绿素a的Soret带蓝移,且Soret带与Q带峰强比增强,显示叶绿体内的色素捕光能力增加,特别是短波长的光更易吸收;荧光光谱也表明叶绿体捕光能力增加,且可促使叶绿素b和胡萝卜素吸收的光能迅速高效地传递到PSⅡ作用中心色素叶绿素a;使用双通道叶绿素荧光仪发现稀土元素同时促进两个光系统的光化学活性与电子传递效率,特别是光系统Ⅱ活性得到显著增加;同时还改善了光系统的光保护能力,增加其对光能的耐受能力;稀土元素还增加了叶绿体的全链电子传递活性、PSⅡ与PSⅠ的DCIP光化学还原活性和氧气释放速率,进一步说明稀土元素不但能增加光能吸收,且能将光能有效分配,促进激发能向PSⅡ大量分配。其中具有1个4f层电子和变价特征（+4）的Ce³⁺处理效果最为明显,其次为不具有变价特征,但有3个4f层电子的Nd³⁺,既不具有4f层电子结构又不具有变价特征的La³⁺处理增加幅度最小。提示稀土提高光合作用与其4f层电子结构和变价特征有密切关系。h2)稀土元素处理通过对LHCⅡ的调控显著增加PSⅡ的活力。适当浓度的La³⁺、Ce³⁺、Nd³⁺处理可使LHCⅡ去磷酸化,使光合机构向状态Ⅰ转换,从而相对增加PSⅡ的光吸收截面及光吸收,提高PSⅡ放氧活性。以拟南芥为实验材料,发现稀土元素处理对LHCⅡ的另一条调控机制:LhcⅡb得到大量表达,提示稀土处理可显著增加LHCⅡ在类囊体膜上的分布,促进了PSⅡ对光能的吸收,并促使LHCⅡ三聚体化,导致基粒堆积状况的调整,从而改变了PSⅠ和PSⅡ的电子流动次数,使得激发能由PSⅠ向PSⅡ分配。三种稀土元素处理效果仍为Ce³⁺>Nd³⁺>La³⁺。3)稀土元素处理增加光合碳同化能力。La³⁺、Ce³⁺、Nd³⁺处理后Rubisco羧化活力增强,干物质增加明显。这是因为稀土元素在体内诱导出Rubisco-Rubisco活化酶的超复合体:在稀土处理的菠菜体内纯化Rubisco时获得一大分子量蛋白,其光谱学性质,巯基含量及二级结构均与纯Rubisco不同,SDS-PAGE电泳分析发现除Rubisco的大小亚基（55kD,14.4kD）之外,还具有Rubisco活化酶的45和41 kD的两个亚基。非变性PAGE电泳分析证明除具有Rubisco的一条带（560kD）以外,还有一条分子量在1200 kD左右的蛋白带,即Rubisco全酶与16个Rubisco活化酶亚基组成的超复合体。使用Rubisco和Rubisco活化酶抗体经蛋白印迹实验证实了Rubisco-Rubisco活化酶超复合体的形成。Rubisco-Rubisco活化酶超复合体被认为是Rubisco在体内实行活性所必需的中间产物,但一直没得到证实。我们的发现首次为这一模型提供了实验证据。4)实验发现稀土元素处理诱导Rubisco-Rubisco活化酶超复合体产生的机制可能有两条。一是通过RT-PCR,Northern Blotting和Real-time PCR技术证明稀土元素能明显促进Rubisco大小亚基,特别是Rubisco活化酶亚基mRNA表达,使体内Rubisco活化酶蛋白的表达上调,提高Rubisco活化酶/Rubisco比例,从而增加了Rubisco-Rubisco活化酶超复合体的含量。Ce³⁺处理后这种增强作用比La³⁺和Nd³⁺处理更显著,显然这与Ce³⁺的4f电子层结构和变价特征有密切关系。二是稀土元素可能提供了Rubisco和Rubisco活化酶之间的“额外”连接,稳定Rubisco-Rubisco活化酶超复合体结构。Ce³⁺与Rubisco体外实验证明Ce³⁺与Rubisco直接结合:第一层与8个O原子配位,键长为2.55?;在第二配位层与6个O原子配位,键长为3.69?。这改善了蛋白的结构,最终提高了酶活性。5) Ce³⁺与大量元素Ca²⁺的化学性质极为接近,但Ce³⁺的电荷高于Ca²⁺,因而离子势大,对以离子键为主的化合物,则稀土离子的结合稳定性要高于钙离子,因而有人将稀土离子称为“超级钙”。它不但可以占据钙的位置,还将取代已结合的钙离子。实验证明,缺钙菠菜在施加适当浓度的CeCl₃后,缺钙症状得以缓解,干重和鲜重增加,叶绿素含量增加,光合效率和放氧活力增强,Rubisco羧化活力,氮代谢相关酶活性也得到一定恢复。Ce³⁺处理还能显著提高菠菜正常生长条件下以及缺钙条件下抗氧化系统活力,提高SOD、CAT、APX、GPX等抗氧化酶酶活性,降低超氧自由基、丙二醛含量,保持光合作用正常进行。体外实验发现Ce³⁺能与脱钙PSⅡ结合,与氨基酸的O原子配位,部分恢复脱钙PSⅡ的放氧活力。这再次证明Ce³⁺能够部分取代Ca²⁺的生理作用。更多还原

【Abstract】 Rare earth fertilizers have been widely used in China for nearly 40 years. It has been demonstrated in many experiments that rare earth elements （REEs） are able to promote the growth and development of plants, enhance photosynthesis and increase the yield of crops. However, the mechanisms underlying the biological effects of REEs on plants still remain elusive. Among all the questions, the effects of REEs on photosynthesis, is the most puzzling one. Specifically, very little is known about the relationships between the physiological effects and the unique characteristics of REEs. Thus, we selected three representative REEs, Lanthanum, Neodymium and Cerium (La³⁺, Ce³⁺ and Nd³⁺), to study the effects and mechanisms of REEs on photosynthesis. This study focuses on the energy transfer processes of photosynthesis——from light energy to electrical energy then active chemical energy, and stable chemical energy in the end. We also compare the different effects imposed by three REEs treatments to clarify the mechanism how unique characteristics of REEs, such as 4f electron characteristic, alternation valence and the similarity to the Ca²⁺, affect photosynthesis. This study will not only provide insights into photosynthesis mechanism study, but lay a solid ground for the efficient use of REEs in agriculture。1) REEs are able to promote the light action of spinach, enhance the light absorption, transfer and distribution efficiency. Treated by La³⁺, Ce³⁺ and Nd³⁺, the characteristic absorbance peaks of the chloroplast are enhanced and blue shifted as well. The ratio of Soret band intensity and Q band intensity is also increased, indicating the ability to catch light of the chloroplast pigments is enhanced, especially in short wavelength. Fluorescence spectra further indicate that the light absorbed by chlorophyll b （chl b） and carotene could efficiently transfer to chlorophyll a （chl a） of photosystemⅡ（PSⅡ）. The results of Dual-PAM-100 showed that REEs treatments are able to promote both the photochemical activity and electron transfer ratio of two photosystems. However, PSⅡactivity is increased more efficiently. Based upon the chlorophyll fluorescence, the photo protective abilities of two photosystems are also increased, thus enhance the tolerance of light. The facts that the whole chain electron transport activity, PSⅡ, PSⅠDCPIP photo reduction and oxygen evolution of the chloroplast are all increased by treatments with REEs, further indicate that REEs are able to efficiently distribute the light energy and trigger the excitation energy mostly to PSⅡ. Among these three REEs, the effect of the Ce³⁺ treatment is the best, which is followed by the Nd³⁺, and the La³⁺ treatment is not as effective as other two elements. 4f electron characteristic and alternation valence of REEs might contribute to these observed differences, for Ce³⁺ has 1 4f electron and can change to +4, Nd³⁺ has 3 4f electrons but no alternation valence, whereas La has neither 4f electron nor alternation valence.2) REEs are able to promote the PSⅡactivity by regulating LHCⅡ（Light harvesting complexⅡ）. A suitable concentration of REEs treatment is able to change the redox state of PQ pool,whereby dephosphorylating LHCⅡand changing the photosystem to stateⅠ, and to certain degree enhancing the photoabsorption cross section and light absorption of PSⅡ. The real-time PCR experiments showed another mechanism of REEs to LHCⅡ. The results showed that REEs are able to enhance expression of LhcⅡb of arabidopsis and significantly increase LHCⅡcontent on the thylakoid membranes, thus inducing the LHCⅡto trimer formation and adjusting the grana distribution of the thylakoid membranes. This changes the electron transfer ratio of two photosystems and triggers the excitation energy mostly to PSⅡ. The effects of the three REEs are as follows : Ce³⁺>Nd³⁺>La³⁺>control.3) REEs are able to promote carbon assimilation. The La³⁺、Ce³⁺、Nd³⁺ treatments enhance the carboxylase activity of Rubisco （ribulose-1,5-bisphosphate carboxylase/oxygenase） and increase the dry matter content of spinach. It is resulted from the formation of the Rubisco- Rubisco activase super complex by REEs treatment. A higher molecular weight protein is found during the purifying procedure of Rubisco in REEs-reated spinach. Its spectral characteristic, sulfhydryl groups and secondary structure are quite different from the pure Rubisco. SDS-PAGE analysis showed that this higher molecular weight protein contains not only 52 kD and 14 kD subunits of Rubisco, but 2 extra isoforms at 41 and 45 kD as well, which was supposed to be the isoforms of Rubisco activase. The native-PAGE result suggested the higher molecular weight protein is the Rubisco- Rubisco activase super complex and the molecular weight is about 1200 kD （a 560 kD Rubisco holoenzyme plus 16 isoforms of Rubisco activase）. It was examined with the antibodies of both Rubisco and Rubisco activase in Western-blotting. This is the first evidence to confirm the formation of the Rubisco- Rubisco activase super complex, which is the hypothesis for activating the carboxylase activity of Rubisco in vivo.4) The mechanism for REEs to induce the formation of the Rubisco- Rubisco activase super complex may be complicated. RT-PCR, Northern blotting and real-time PCR strongly indicate that the mRNA levels of the Rubisco and Rubisco activase were enhanced by the REEs treatment. However, rbca is highly expressed which may increase both the protein content of Rubisco activase and the ratio of the Rubisco activase to Rubisco. It is still the Ce that has the strongest influence on the gene expression, then follwed by Nd, and La is the least. These results suggested that 4f electron characteristics and alternation valence of REEs have a close relationship with carbon assimilation. The second mechanism to form the super complex is found by EXAFS. Nd is coordinates with Rubisco by 4 Nd-N（O）bond（2.46 （A|°））and 2 Nd-S bond（3.47 （A|°）） in vivo, which suggests REEs ion may provide the extra linkage between Rubisco and Rubisco activase, which stabilize the super complex structure. The in vitro study also showed that that Ce³⁺ could directly bind Rubisco, coordinates with 8 oxygen atoms in first shells and 6 oxygen atoms in second shells, change the spectra characteristic of Rubisco, which enhances the enzyme activity.5) Ce³⁺ has similar chemical property to Ca²⁺, but the charge and potential energy in Ce³⁺ are higher than Ca²⁺. So Ce³⁺ could not only occupy a Ca²⁺ position, but also substitute for bound Ca²⁺. Thus Ce ³⁺ are known a“supercalcium”. It was showed that Ce ³⁺ could relieve calcium-deficiency symptoms in spinach. Ce treatment could raise the dry weight and fresh weight of calcium deficient spinach, increase the chlorophyll content and photosynthetic rate, and the oxygen release rate and enhance the activities of Rubisco and the nitrogen metabolism related enzymes system. Ce treatment could also enhance the activities of the antioxidation system both in the normal and calcium-deficiency condition. Specifically, the activities of superoxide dismutase （SOD）, catalase（CAT） and peroxidase（POD） are enhanced and the membrane permeability, while reactive oxygen species （ROS） and malond ialdehyde（MDA） are reduced, which would maintain photosynthesis. The in vitro study showed that Ce could bind the Ca-depleted PSⅡparticle, coordinate with oxygen atoms and partially recover the activity of Ca-depleted PSⅡ. It also suggests that Ce³⁺ could replace Ca²⁺.更多还原