【作者】 罗明华；

【导师】 林宏辉；

【作者基本信息】 四川大学 ， 植物学， 2007， 博士

【摘要】 水杨酸（salicylic acid，SA）是植物体内产生的一种简单的酚类物质。作为植物内源信号分子，在植物许多生理过程中的作用越来越受到重视。近些年来。陆续发现水杨酸能提高植物对非生物逆境的抗性，如提高植物的抗盐性、抗寒性、抗热性以及抗旱性等。我们这项研究的目的在于考察外源水杨酸预处理对干旱条件下大麦幼苗生理活动的影响，初步阐明水分胁迫下，光系统Ⅱ的主要功能蛋白D1，D2，LHCⅡ及其相应的基因表达与水杨酸，活性氧ROS之间的关系和相互作用机理。供试材料为大麦（Hordeum vulgate L）。直接用含PEG-6000（16％）的培养液胁迫的大麦幼苗为对照样（CK）；用0.25mmol／L的水杨酸浸泡大麦幼苗根部预处理1天再用含16％（w／v）PEG-6000的培养液进行水分胁迫（简称SA）；用0.25mmol／L的水杨酸+0.5mmol／L DMTU（二甲基硫脲，一种H₂O₂清除剂）预处理24小时后再用含16％（w／v）PEG-6000的培养液进行水分胁迫处理（简称SA+D）。以1d为间隔，三组处理时间为0h、24h、48、72h。用0～1.5mmol／L11种不同浓度的水杨酸进行预处理，水分胁迫2天后，测量电渗率和丙二醛含量后分析表明：0.25mmol／L的水杨酸预处理是最佳浓度，说明水杨酸提高植物的水分胁迫抗性与浓度密切相关。测定CK、SA、SA+D的RWC，电渗率，MDA的含量及叶总H₂O₂的含量表明，水杨酸预处理能使RWC的下降和电渗率，MDA的含量及叶总H₂O₂的含量的上升减缓。在SA+D中，清除了H₂O₂，则后来的胁迫中水杨酸的保护作用消失，说明水杨酸的保护作用与ROS有关。水杨酸+DMTU预处理后细胞总H₂O₂的变化与叶绿体H₂O₂的变化不同，叶绿体H₂O₂总是维持低水平。SDS-PAGE和Western杂交分析表明：CK的类囊体膜蛋白组分稳态水平随胁迫处理时间的延长而持续下降，尤其是光系统Ⅱ功能蛋白中的内周天线CP43，CP47，OEE1，CF1和光系统Ⅱ外周天线LHCⅡ（23-30kD范围多肽）下降较为明显。相比之下，SA和SA+D的类囊体膜蛋白组分稳态水平随胁迫处理时间的延长下降的水平轻微。Northern杂交分析表明：水杨酸同样可以在转录水平保护光系统，但核编码的cab和rbcS基因的转录水平与叶绿体编码的基因的转录水平是不一样的，在用水杨酸+DMTU处理的幼苗中，核编码的cab和rbcS基因随着水分胁迫的下降加剧，转录水平下降的最严重，甚至超过了CK。水杨酸预处理的下降程度明显减缓。核基因cab和rbcS转录水平的变化与细胞核，细胞浆内的ROS的变化一致。叶绿体编码的基因psbA，psbD在SA和SA+D中，在水分胁迫下仅表现出轻微的下降，这和叶绿体内ROS和蛋白的变化是一致的。比较Northern杂交和Western杂交的结果可以得知：SA+D处理中，cab转录的mRNA的下降和LHCⅡ蛋白保持稳定的情况表现的不一致，这是因为LHCⅡ维持含量不需要大量cab mRNA，而它的含量主要与叶绿体的ROS有关。经水杨酸预处理24h后，气孔导度降低，随之CO₂的同化速率降低，这是一种胁迫反应，在随后的水分胁迫中，气孔导度和CO₂的同化速率降低程度减缓，水杨酸提高了水分胁迫抗性。在SA+D中清除了H₂O₂，则水杨酸的保护作用消失，说明水杨酸提高水分胁迫抗性与ROS有关。用水杨酸或水杨酸+DMTU预处理大麦幼苗24小时，在随后的水分胁迫中可观察到Fv/Fm，Fv’/Fm’和Φ_PSⅡ的数值比CK更高。表明水杨酸对Fv/Fm，Fv’/Fm’和Φ_PSⅡ有保护作用。这种保护作用与水杨酸或水杨酸+DMTU预处理后叶绿体内低水平ROS相关。用水杨酸预处理大麦幼苗24小时，可观察到Fv’/Fm’的值比对照CK更高，即在有热耗散时，PSⅡ的最大光合效率增高；对应的NPQ的变化是：加水杨酸后，降低很明显，即：水杨酸能减少PSⅡ的热耗散。SA+D的变化介于CK和SA之间，说明水杨酸对PSⅡ热耗散的调节部分依赖于活性氧。水杨酸预处理提高大麦幼苗水分胁迫抗性与水杨酸预处理所用的浓度有密切的关系。水杨酸预处理提高大麦幼苗对水分胁迫的抗性与活性氧有关。光系统Ⅱ的主要功能蛋白D1，D2，LHCⅡ及其相应的基因转录在水分胁迫下会逐渐下降，而水杨酸预处理能缓解这种下降，水杨酸缓解渗透胁迫主要是通过ROS，而并非水杨酸本身作用。第二章中国象牙参属新分类群及花粉特征的研究本研究报道了姜科Zingiberaceae象牙参属Roscoea的一种新植物，以及在光学显微镜和扫描电镜下观察到的象牙参属Roscoea 11种3变种的花粉形态特征和研究结果。描述了云南姜科Zingiberaceae一新种，即苍山象牙参Roscoeacangshanensis M．H．Luo，X．F．Gao & H．H．Lin。该种与大理象牙参R．forrestiiCowley在体态上相近，但唇瓣深裂成2裂片，每个裂片再2裂，基部收缩成具白色条纹的柄，叶片较狭窄，（2-）7-24×1.5-2.5cm，叶片基部狭缩成叶柄状而不同。该新种叶片基部狭缩成叶柄状，唇瓣倒卵状楔形，长2.5-30.5cm，宽2.5-3.0cm，基部收缩成具白色条纹的柄，与长柄象牙参R．debilis Gagnep．相似，但苞片非管状，较短，长5-15mm，隐藏于叶鞘内，花冠管较长，10-12.5cm，唇瓣深裂成2裂片，每个裂片再2裂，可与后者明显区别。就目前来看，本研究发现了象牙参的一新种是中国特有种。花粉特征的研究支持新种苍山象牙参R．cangshanensis的建立。对本属花粉的研究表明：花粉类型属于无萌发孔型（Type Nonaperturate），具刺亚型（Subtype Spinate），长刺组（Group Long-spinate）。花粉为单粒。球形花粉粒大小变异幅度为47.2—88.5μm，属中小型花粉。花粉不具萌发孔。花粉壁厚为1.8—3.4μm。外壁薄，分层不明显，内壁厚。根据Hesse和Kubitzki的研究资料，将内壁分为外层及内层。外壁表面具刺状纹饰，刺长为2.0—4.6μm，刺间的外壁表面有皱褶，或比较平整而带有少数颗粒或有小突起。花粉特征的相似性表明本属是一个单起源的自然类群。根据花粉表面刺间是否皱褶的稳定特征分为外壁刺间皱褶亚组Subgroup crapy-surface和外壁刺间平整亚组Subgroup smooth-surface两个亚组。根据花粉特征和分亚组支持毛象牙参R．cautleoides Gagnepain var．pubescens Z．Y．Zhu独立成种，予以恢复。并可将微凹象牙参R．tibetica Batalin var．emarginata S．Q．Tong并入藏象牙参Roscoea tibetica Batalin。与邻近属花粉的相似点表明了它们相近的亲源关系。象牙参属Roscoea的花粉形态的研究为进一步研究该属乃至姜科植物系统演化提供了资料。更多还原

【Abstract】 Chapter 1 Physiological Effects of Salicylic Acid on Barley Photosystem II Under Osmotic StressSalicylic acid （SA） is a simple hydroxybenzene in plant. As an endogenesis signal molecule, the effects of SA on many physiological progresses in plant have been paid more and more attentions. In recent years, the ability of SA to improve the endurance of plants in abiotic stress, such as salt, cold, hot and drought has been found. We tried to study the effects of exogenous SA pretreatment on the physiological functions and photosynthesis system II of barley seedlings under osmotic stress, and primarily evaluated the effects of exogenous SA to PSII major proteins D1, D2 and LHC II and the transcripts of corresponding genes psbA, psbD and cab and the interaction of PSII, ROS and SA under osmotic stress.The material for study is barley （Hordeum vulgare L.）: the roots of control seedlings （CK） were submerged in half-strength Hoagland’s nutrient solution; the roots of SA pretreated seedlings （SA） were submerged in 0.25mmol/L SA+half-strength Hoagland’s nutrient solution; the roots of SA+D pretreated seedlings were submerged 0.25mmol/L SA+half-strength Hoagland’s nutrient solution+0.5mmol/L DMTU（dimethylthioutea）. Osmotic stress was initiated by submerging the roots of the seedlings into PEG solutions with an osmotic potential of - 0.6 MPa in beakers. All samples were treated for 0, 24, 48, and 72 h.Barley seedlings pretreated with different concentrations of SA pretreatment 24h were transferred into PEG solutions with an osmotic potential of - 0.6 MPa （16%） in beakers for 48 h. then the effects of different concentrations （0～1.5 mmol/L） of SA used in pretreatment were examined by the electrolyte leakage of membranes and Malonydialdehyde （MDA） Contents of leaves. It was shown that application of exogenous SA pretreatment at 0.25mmol/L most decreased the electrolyte leakage of membranes and MDA Contents of leaves, indicating that the effects of SA were related closely to concentration of SA used.The decrease in relative water content （RWC）, and increase in electrolyte leakage of plasma membranes and MDA in water-stressed leaves were alleviated by SA pretreatment. The protective effects of SA in SA+D barley seedlings disappeared because SA-induced H₂O₂ was eliminated by DMTU, indicating that protective effects of SA was associated with ROS. In SA+D leaves, leaf total H₂O₂ changed differently from plastid H₂O₂, which almost did not increase.SDS-PAGE and Western blotting analysis showed that decrease in the PSII major protein D1, D2, and LHCII were alleviated by SA and SA+D pretreatment under osmotic stress because of lower levels of ROS in plastids of both pretreatments.Northern blotting analysis indicated that the transcripts of nuclear-coded cab and rbcS were different from those of plastid-coded psbA and psbD. The decrease in transcripts of cab and rbcS in SA+D barley seedings was more serious than that in SA and CK, which was in accord with the changes of ROS in cytosol. Compared with CK, the transcripts of psbA and psbD in SA and SA+D decreased slightly, which were in accord with the changes of ROS in plastid. the decrease in transcripts of cab did not lead to decrease of level of LHCII, because sustainability of LHCII content did not need corresponding mRNA and was associated with inner ROS in plastid.After 24h of SA pretreatments, stomatal conductance and CO₂ assimilation rate decreased, which can be regarded as a stress response. As the water stress developed, a less decrease of stomatal conductance and CO₂ assimilation rate was observed in SA plants, indicating that SA pretreatment improved water stress tolerance. and the effects of stomatal closure induced by SA pretreatment disappeared in SA+D pretreated plants because H₂O₂ was eliminated, showing that water stress tolerance promoted by SA pretreatment was relative to ROS. After 1 day pretreatment with SA or with SA+D, higher values of Fv/Fm, Fv’/Fm’ andΦ_PSII were observed during subsequent osmotic stress, showing that SA had a protective role of the efficiency of Fv/Fm, Fv’/Fm’ andΦ_PSII. These effects were associated with the low level of ROS in plastid.After 24h pretreatment with SA and before osmotic stress, a higher value of Fv’/Fm’ revealed the efficiency of excitaion energy capture by open reaction centres （Fv’/Fm’） increased in the case of heat dissipation, meanwhile, corresponding value of NPQ was remarkbly lower than that in CK. In other words, pretreatment with SA could reduce the heat dissipation of PS II. After 1 day pretreatment with SA+D and before osmatic stress, the values of Fv’/Fm’ and NPQ distributed between that of SA and CK, indicating that the modulating effect of SA for heat dissipation of PSII partly depended on ROS.As a whole, SA pretreatment enhanced water stress tolerance of barley seedlings, and the effects of SA were related closely to concentration of SA pretreatment. Osmotic stress tolerance enhanced by SA pretreatment was associated with ROS. The decrease in PSII major proteins D1, D2 and LHC II and the transcripts of corresponding genes psbA, psbD and cab were alleviated by SA pretreatment under water stress. We found that enhanced osmotic stress tolerance in barley seedings by SA pretreatment was mainly mediated with ROS, rather than the SA itself.Chapter 2 Studies on Pollen Morphology and A New Species of Roscoea （Zingiberaceae） in ChinaPollen morphology of 11 species and 3 varieties of Roscoea （Zingiberaceae） in China was studied under both light microscope and scanning electron microscope, and a new species of Roscoea was also reported in this paper.Roscoea cangshanensis M. H. Luo, X. F. Gao & H. H. Lin, a new species of the Zingiberaceae from Yunnan, China, is described and illustrated. The new species is related to R. forrestii Cowley in habit, but differs in having 2-lobed labellum, each lobe 2-lobulate, base narrowed to a stalk with white lines, and narrower leaf blade, （2-）7-24×1.5-2.5 cm, with base narrowed to petiolelike. The new species is also similar to R. debilis Gagnep. in having leaf base narrowed to petiolike, labellum obovate-cuneate, 2.5-3.5×2.5-3.0 cm, with white lines at throat, but differs in having bracts non-tubular, shorter, 5-15 mm long, concealed in leaf sheaths, corolla tubes longer, 10-12.5 cm long, labellum 2-lobed with each lobe further 2-lobulate.Pollen grains of Roscoea are spherical, subspherical, 47.2—88.5μm in size, nonaperturate. The wall is composed of a very thin exine and a thick intine. The exine is spinate, crapy or smooth between spines, and spinas are about 1.8—3.4μm.According to pollen morphology, the pollen of Roscoea belonged to Group Long-spinate , Subtype Spinate, Type Nonaperturate, and could be considered as a natural group, and was divided into two subgroups: Subgroup crapy-surface and Subgroup smooth-surface. The taxonomic significance of the pollen types in Roscoea was also discussed.更多还原