Relationship Between Male Fertility and Endogenous Phytohormones in Photoperiod-Sensitive Genic Male-Sterile Rice

A spontaneous male sterile rice plant (Oryza sativa L. cv. Nongken 58S) "Photoperiod-sensitive Genic Male-sterile Rice has been found to be male sterile under long day cycles (LD) and fertile in short day cycles (SD). The period from secondary rachis-branch and spikelet primodia to pollen mother cell formation in the process of panical development was the photoperiod-sensitive stage for fertility alternation. The phenotype of this mutant was reported to be controlled by two pairs of recessive alleles. The research on relationship between the fertility alternation and phytohormone action in this mutant have been performed by Chinese scientists since 1985. In order to study the mechanism of fertility alternation in Nongken 58S, endogenous IAA, ABA, GA1 and GA4 in apical leaves and reproductive organs in different development stages under LD and SD conditions have been quantiatvely and qualitatively identified by GC-MS-SIM method. It was found that endogenous IAA in apical leaves at the stage of pistil and stamen primodia formation and in panicles at pollen mother cell stage of Nongken 58S with LD condition was deficient comparing with those in SD. Endogenous ABA level in panicles at pollen mother cell stage, in spikelets at uninucleate stage and in anthers at anthesis stage of Nongken 58S-LD were lower than those in SD. ABA levels in corresponding organs and developmental stages of wild type of rice, "Nongken 58" were always higher in LD treatment than those in SD. Endogenous IAA, GA1 and GA4 levels in anthers at anthesis stasge of "Nongken 58"-LD were increased obviously. Thus it appeared that "Nongken 58" possess stronger resistance to LD stress than Nongken 58S. It is concluded that IAA deficiency of reproductive organs at early developmental stage, ABA decrease implying poor resistance to LD stress and reduction of GAs in late developmental stages were the factors causing the anther sterility in Nongken 58S-LD.     

Freeze-fracture studies on barley plastid membranes. VI. Location of the P700-chlorophyll a-protein 1

The photosystem I mutant of barley, viridis-zb63, which totally lacks the P700-chlorophyll a-protein 1 was characterised by rotary shadowed, freeze-fracture electron microscopy. Objective measurements were made of particle density and size distribution for all four fracture faces, and compared with values for wild-type. A highly significant reduction in the size of the PFu particles was found, which could be attributed to the loss of a population of large particles from the mutant PFu face. A corresponding loss of EFu pits was also observed. It is concluded that the photosystem I reaction centre and two or three ancillary polypeptides are located in the large PFu particles which account for two-thirds of the total, and that these particles span the membrane. Since no differences were seen on the PFs and EFs faces, there was no evidence for the localisation of any photosystem I in appressed granal membranes, supporting the concept of extreme lateral heterogeneity. A model is presented of the localisation of different functional polypeptide units to different freeze-fracture particles within the membrane. A peculiar feature of viridis-zb63 thylakoids was the presence of EFs particle arrays in vivo.     

The Supramolecular Architecture in Chloroplast Thylakoid Membranes of Warigal Wheat

Thylakoid membranes of chloroplast from first leaf and flag leaf of wheat Warigal were examined by freeze-fracture and rotary shadowing etectron microscopy. The shape, size, density and size distribution of freeze-fracture partieles of their four faces were measured and plotted as three-dimensional histograms by a Hewlett-packard 9874 A digitizer with a HP 9845 B Computer and HP 9872 C plotter. When comparisons were made among different fracture faces and between the corresponding faces of the first leaf and the flag leaf, we found that the supramolecular architecture on the four fracture faces of the flag leaf differs from that on the corresponding faces of the chloroplast thytakoid membranes of the first leaf. The most significant difference was that the EFs particles contain the photosystem Ⅱ reaction centres associated with LHCP and the PFs particles were mostly light-harvesting complex. There was a 15% increase in EFs particle density, a 22% increase in PFs particle density and a 28% increase in EFu particle density. The large PFu particles contained the photosystem Ⅰ reaction centre and the flag lcaves contained 5% more than the first leaves. In addition, the stacking of thylakoid membranes in the flag leaf was 5% more than those in the first leaf.Thus, it provides theoretical basis for the fact that the flag leaf has higher photosynthetic rate.     

Fine structure of the chloroplast membranes ofEuglena gracilis as revealed by freeze-cleaving and deep-etching techniques

Summary The chloroplasts ofEuglena gracilis have been examined by freeze-cleaving and deep-etching techniques.The two chloroplast envelope membranes exhibit distinct fracture faces which do not resemble any of the thylakoid fracture faces.Freeze-cleaved thylakoid membranes reveal four split inner faces. Two of these faces correspond to stacked membrane regions, and two to unstacked regions. Analysis of particle sizes on the exposed faces has revealed certain differences from other chloroplast systems, which are discussed. Thylakoid membranes inEuglena are shown to reveal a constant number of particles per unit area (based on the total particle number for both complementary faces) whether they are stacked or unstacked.Deep-etchedEuglena thylakoid membranes show two additional faces, which correspond to true inner and outer thylakoid surfaces. Both of these surfaces carry very uniform populations of particles. Those on the external surface (the A surface) are round and possess a diameter of approximately 9.5 nm. Those on the inner surface (the D surface) appear rectangular (as paired subunits) and measure approximately 10 nm in width and 18 nm in length. Distribution counts of particles show that the number of particles per unit area revealed by freeze-cleaving within the thylakoid membrane approximates closely the number of particles exposed on the external thylakoid surface (the A surface) by deep-etching. The possible significance of this correlation is discussed. The distribution of rectangular particles on the inner surface of the thylakoid sac (D surface) seems to be the same in both stacked and unstacked membrane regions. We have found no correlation between the D surface particles and any clearly defined population of particles on internal, freeze-cleaved membrane faces. These and other observations suggest that stacked and unstacked membranes are similar, if not identical in internal structure.     

A chloroplast membrane lacking Photosystem I. Changes in unstacked membrane regions

The structure and polypeptide composition of the photosynthetic membrane of a mutant of maize has been investigated. The thylakoid membranes of the mutant plants are deficient in Photosystem I activity, although Photosystem II is at near normal levels. SDS polyacrylamide gel electrophoresis of thylakoid membranes from the mutant shows them to be deficient in two polypeptide bands which have been associated with Photosystem I. Freeze-fracture studies of the membrane show that the absence of these polypeptides is associated with a measurable reduction in particle diameter on the unstacked protoplasmic fracture face. This fracture face is derived from the splitting of membranes in unstacked regions of the thylakoid membrane system. It is suggested that in membranes stacked by salts in vitro, Photosystem I activity may be confined to this region.     

Structural analysis and identification of cis-elements of rice osRACD gene

Sexual reproduction in many plants is sensitive to thechange of day-long and photoperiod. It was discoveredby Shi et al. [1] in 1973 that the natural mutant Nongken58S is infertile under long day while being fertile undershort day. In recent two decades, much work has beendone to probe into its molecular mechanism from variousperspectives, such as physiology, genetics as well asmolecular biology [2,3]. The previous study showed thatphysiological changes of 58S rice could be dependent oncombi…     

Changes of Freeze-Fracture Ultrastructure and Light Harvesting Chlorophyll Protein Complex in Thylakoid Membranes During Ontogeny of Maize

Freeze-fracture electron microscopy enables us to observe and count the freeze-fracture particles which correspond to the different functional components of thylakoid membranes. The present paper reports the observation on freeze-fracture ultrastructure of thylakoid membranes and the analysis of proteins by the SDS-polyacrylamide gel electrophoresis within the membranes from differenly located leaves of maize. In the past, we found that the leaies subtending the ear of maize had a much higher chlorophyll content, a lower chlorophyll a/b ratio and more staking thylakoid membranes and provided the photosynthetic energy used to fill the maize seeds more than that of other leaves. Recently, we have further found that the particle densities of all four faces of thylakoid membranes from the ear leaf were the highest, than those, successively, from the terminal leaf, and the fifth leaf (from the base of the plant). The particle densities on all four fracture faces of thylakoid membranes isolated from the ear leaves of maize were significantly higher than those from the terminal leaves with the increases of 19% in EFs, 28% in PFs and 20% in PFu. Increases in particle densities on the PFs, EFs and PFu faces result in increased densities of LHCP II, PSⅡ and PSI reactions centres, respectively. It is significant that this supramolecular architecture of the ear leaves is consistent with our analytical results of the SDS-polyacrylamide gel electrophoresis within the membranes (a detailed report in another paper). The contents of major polypeptides of 21 kD (LHCP Ⅰ) and 25 kD (LHCP Ⅱ) in thylakoid membranes from the ear leaves were more than those from the terminal leaves. The characteristics of both supramolecular architecture and polypeptide components are in favour of absorbing, transferring, distributing and conversing light energy in the course of photosynthesis of the ear leaves in maize.     

光周期对光敏核不育水稻光敏色素A含量及其mRNA丰度的影响

光敏核不育水稻(农垦58S)是我国特有的水稻(Oryza sativa L.)种质材料,光敏色素是光周期诱导其育性转变的受体。报道了育性转换敏感期间的光周期处理对农垦58S及对照“农垦58”叶片中光敏色素A(Phy A)含量及其mRNA丰度的影响。在10个光周期处理的最后一个暗期结束前,收获每株水稻的上部两片叶,用酶联免疫吸附测定法测定Phy A。和长日照(LD)相比,短日照(SD)处理导致农垦58SPhy A相对含量增加38.5％;而“农垦58”只增加18.5％。显然,在较长的暗期中,农垦58S中Phy A的积累比对照快。在水稻幼苗中也得出相似的结果。以光敏色素A基因(phy A)的特异性片段RPA3作探针,用RNA斑点杂交的方法对叶片中Phy A mRNA丰度进行分析的结果表明,光周期处理5d和10d时,两品种水稻的Phy A mRNA丰度都是SD处理的比LD的高,而且SD下农垦58S Phy A mRNA的丰度均比“农垦58”的高。这些结果表明,甲基化水平较低的农垦58S phy A可能比“农垦58”的phy A更活跃地表达。另外,在育性转换敏感期每日主光期结束时(EOD)进行10次短暂的远红光(FR)照射。结果表明,农垦58S植株抽穗和开花期比SD处理推迟2d,而花粉败育率、种子结实率却无变化。暗示农垦58S开花和育性转变过程的光周期反应可能不同。     

核酸代谢抑制剂对水稻光敏核不育性的影响

在稻穗分化的二次枝梗期后,观察比较了叶片喷施核酸代谢抑制剂5-FU对水稻光敏核不育性及幼穗发育的影响,结果表明：长日照下,5-FU在使农垦58S穗长缩短、颖花数减少的同时,却使其抽穗提前,可育性得到一定程度的恢复;5-FU处理还使农垦58的抽穗被完全阻止。在短日照下,5-FU处理则使农垦58S与农垦58在穗长与颖花数减少的同时,其抽穗反被延迟,可育性受到抑制而结实率显著降低,上述效应还可被随后施用的恢复剂乳清酸所弱化。因此可以认为光敏核不育现象与稻穗发育中核酸代谢的改变有关。     

Effect of Photoperiod Treatments on Phytochrome a Levels and Its mRNA Abundance in Photoperiod-Sensitive Genic Male-Sterile Rice Mutant and the Wild Type

The effect of photoperiod treatments on phytochrome A (Phy A) level and its mRNA abundance in the leaves of a photoperiod-sensitive genic male-sterile mutant (Nongken 58S) and its wild type ("Nongken 58') of Oryza sativa L. was investigated. The top two leaves of each rice shoot were harvested at the end of the last dark phase of 10 cycles during photoperiod-sensitive stage for fertility alteration of the mutant. Phy A was measured by a sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal and mouse monoclonal antibodies. Compared with longday (LD) treatment,short day (SD) resulted in 38.5% increase of relative Phy A content in the mutant, only 18.5% increase in the wild type. In an extended darkness (25 h), the accumulation of Phy A also appeared to be more rapid in the mutant seedlings than in its wild type. RNA dot blot analyses with RPA3 (a cDNA clone of rice Phy A) as a probe showed:the abundances of Phy A mRNA in top leaves of Nongken 58S and "Nongken 58" were obviously higher in SD than those in ID at the end of dark phase of 5 d and 10 d photoperiod treatments. Moreover, under SD Phy A mRNA contents in Nongken 58S were more than those in "Nongken 58" during the whole photoperi- od-sensitive stage for fertility alteration. In addition, after 10 cycles of end-of-day far-red irradiations (EOD FR), the heading and flowering date of the mutant was delayed for 2 d. However, EOD FR had little or no effect on male fertility of the mutant.     

Pms1 is not the Locus Relevant to Fertility Difference Between the Photoperiod-Sensitive Male Sterile Rice Nongken 58S and Normal Rice

The photoperiod-sensitive male sterile rice, Nongken 58S, was obtained as a spontaneous mutant of the Oryza sativa L. ssp. japonica cultivar "Nongken 58". To determine the chromosomal location of the locus related to the fertility difference between Nongken 58S and its wild-type ancestor, the authors assayed the DNA polymorphisms between these two varieties using a total of over 300 RFLP probes covering the entire molecular marker linkage map. Seven probes detected polymor- phisms between "Nongken 58" and Nongken 58S. Two probes, RG30 and RZ626, both from chromosome 7, happened to be located in the genomic region of pmsl, a locus for photoperiod-sensitive male sterility identified in the authors' previous study. These two probes were used to assay a random sample of 140 individuals from a F2 population of a cross between Nongken 58S and "Nongken 58", in which the fertility segregated in a typical 3: 1 ratio. An analysis of variance of the fertility using the RFLP genotypes as the groups clearly evidenced that these two marker loci are not linked to the locus associated fertility segregation in this population. It is concluded that the locus relevant to fertility difference between Nongken 58S and "Nongken 58" is not in the vicinity of the pmsl region.     

光敏核不育水稻农垦58S与正常品种“农垦58”在pmsl区段无育性基因分离

光敏核不育水稻农垦58S系由正常品种“农垦58”(Oryza sativa L.ssp.japonica)自然突变产生。为弄清该突变基因在染色体上的位置,曾用覆盖整个水稻基因组的300余个RFLP探针对农垦58S和“农垦58”进行了对比分析,得到了7个具多态性的探针,其中2个探针RG30和RZ626正好落在第7染色体上以前定位的光敏核不育基因pmsl所在的区段。以这两个标记对农垦58S/“农垦58”组合F_2随机群体140单株进行了RFLP分析,按RFLP基因型分组对育性作方差分析,结果表明,这2个标记位点与此群体中引起育性分离的位点无连锁关系。说明由正常“农垦58”变为光敏核不育农垦58S的突变基因不在pmsl区段。     

氮素形态对烤烟光合特性影响的研究

1996～1997年连续二年进行了固定氮素用量,改变铵态、硝态氮肥的施用比例试验,对烤烟的一些光合生理特性进行了研究。试验结果表明,在烤烟生长前、中期,增施铵态氮施用比例,烤烟的叶绿素含量增加,但达100％铵态氮时,叶绿素含量反而下降,低于施用50％和75％铵态氮的处理;在烤烟生长后期．功能叶片叶绿素含量基本上与施用铵态氮比例高低—致,以100％铵态氮处理的叶绿素含量最高。另外增加铵态氮施用比例,烤烟功能叶片的希尔反应活性及光合磷酸化活性有所提高,但P/O比值基本不变。从叶绿素诱导荧光动力学特征参数及比值情况来看：增加铵态氮施用比例,Fm和Fv值升高;几种荧光参数的比值如Fd/Fs、Fv/Fo、Fv/Fm比值有所上升,但均以25％硝态氮+75％铵态氮的处理最高;并且其烤后烟叶的产量最高、品质最好。     

Ultrastructural studies on the membrane systems and cell inclusions of the filamentous prochlorophyte Prochlorothrix hollandica

The outer membrane of Prochlorothrix hollandica is covered with a network of fine fibrils on its surface and separated from the cytoplasmic membrane by an electrondense peptidoglycan layer (8 to 20 nm thick). The thylakoid membranes are arranged in stacked and unstacked regions which present four characteristic fracture faces with different numbers and sizes of intramembrane particles. Cell inclusions such as polyhedral bodies (carboxysomes), ribosomes, and polyphosphate granules were found in Prochlorothrix hollandica. Another type of cell inclusions was identified by its characteristic shape (a cylindre with conical caps) and a regular striation as gas vesicles. It is concluded that the organism is in its morphological structure similar to the cyanobacteria.Abbreviations C carboxysome - CM cytoplasmic membrane - EF_s, EF_u exoplasmic fracture face of stacked and unstacked membrane area, respectively - ES exoplasmic surface - PF_s, PF_u plasmic fracture face of stacked and unstacked membrane area, respectively - PG peptidoglycan layer - TM thylakoid membrane Dedicated to Prof. Dr. D. Peters, Hamburg, on the occasion of his 75th birthday     

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

A chloroplast membrane lacking photosystem II. Thylakoid stacking in the absence of the photosystem II particle.

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

FREEZE FRACTURE STUDY OF THYLAKOIDS OF FUCUS SERRATUS1

Freeze-fractured thylakoids of Fucus serratus L. exhibit three types of faces with a particle density analogous to that of EFs, EFu and PF faces of green plants. However the particle size distribution is unimodal in the three types with a mean of about 8 nm. No obvious distinction between PFs and PFu faces could be detected. The absence on EFs faces of the distinct class of large particles (>13 nm) existing in green plant thylakoids implies a unique organization of pigment proteins, especially of the light-harvesting complexes.     

湖北光敏核不育水稻农垦58S叶片60 kD蛋白特异性及其功能的讨论

湖北光敏核不育水稻(Hubei photoperiod-sensitive genic male-sterile rice, HPGMR)是在我国发现并开始应用研究的。人们在应用研究中发现的许多问题,如光敏不育特性转育到籼稻上多变成温敏不育等,很难达成共识。加强机理研究已成为光敏水稻研究深入发展的当务之急。除了对不同时期“农垦58”与58S的代谢做各种分析外,寻找特异蛋白与特异基因是光敏水稻机理研究中两个重要方面。在蛋白研究方面,目前已报道发现35kD、45kD、60kD、61kD等与光敏不育材料有关的特异蛋白。这些结果表明,在“农垦58”与58S之间是可以找到特异蛋白的,但还不能确定这些蛋白与光敏不育性状的关系。为了解决已发现的特异蛋白与光敏不育性状的相关性问题,我们采用多对照的方法,对已知在农垦58S中稳定表达而在“农垦58”中不出现的60kD蛋白做了进一步的研究。     

Freeze-fracture studies on barley plastid membranes. VII. Structural changes associated with phosphorylation of the light-harvesting complex

Chloroplast thylakoid membranes were isolated from barley at room temperature under redox conditions which ensured that the light-harvesting complex was either non-phosphorylated or phosphorylated. The ultrastructural appearance of these membranes was characterised by rotary shadowed, freeze-fracture electron microscopy. Upon phosphorylation, there was a slight (5%) decrease in the extent of thylakoid stacking, as evidenced by an increase in EFu face particle density. It was concluded from detailed measurements of particle density and size distribution that phosphorylation of the light-harvesting complex results in the movement of some of the Photosystem II EFs particles and some of the PFs particles containing the light-harvesting complex from grana to stroma membranes. There was also a slight increase in PFs particle size and the appearance of a population of large particles on this face, which may be due to conformational changes in the light-harvesting complex or to the movement of some Photosystem I particles from stroma to grana membranes.