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.     

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。     

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.     

光周期调节光敏感核不育水稻叶片抗环血酸过氧化物酶的活性

从第二次枝梗原基分化期开始用长日照(LD)或短日照(SD)处理光敏感核不育水稻农垦58S 和常规水稻农垦58。与 SD 处理比较,LD 处理明显抑制农垦58S 和农垦58的抗坏血酸过氧化物酶(AsAPOD)的活性,对农垦58S 的 AsA POD 活性的抑制效应较之农垦58的大。随着 AsAPOD 活性下降,抗坏血酸(AsA)和丙二醛(MDA)的含量逐渐增加,AsA POD 活性与 AsA 和MDA 含量之间呈负相关。LD 抑制 ASA POD 活性和抑制幼穗发育的时间有一定的一致性。推测在 LD 处理下 AsA POD 活性下降与幼穗发育受阻有某些内在的联系。     

光周期调节光敏感不育水稻叶片抗坏血酸过氧化物酶的活性

从第二次枝梗原基分化期开始用长日照(LD)或短日照(SD)处理光敏感核不育水稻农垦58S和常规水稻农垦58。与SD处理比较,LD处理明显抑制农垦58S和农垦58的抗坏血酸过氧化物酶(AsAPOD)的活性,对农垦58S的AsA POD活性的抑制效应较之农垦58的大。随着AsA POD活性下降,抗坏血酸(AsA)和丙二醛(MDA)的含量逐渐增加,AsA POD活性与AsA和MDA含量之间呈负相关。LD抑制ASA POD活性和抑制幼穗发育的时间有一定的一致性。推测在LD处理下AsA POD活性下降与幼穗发育受阻有某些内在的联系。     

免疫测定光敏核不育水稻中光敏色素Ⅰ含量

光敏核不育水稻（农垦５８Ｓ）是中国水稻研究中的一个重要发现,在水稻杂交育种上有巨大潜力。研究表明：农垦５８Ｓ的雄性不育性受光周期调控,光敏色素是育性转变过程中光周期反应的光受体,然而,鉴于高等植物中存在至少两种不同的光敏色素分子,并且它们同时存在于水稻中,人们对调节农垦５８Ｓ雄性不育过程的光敏色素分子种类及作用方式仍然不清楚。本文采用酶联免疫吸附测定法（ＥＬＩＳＡ）,比较了不同光周期下农垦５８Ｓ和对照品种农垦５８叶片（光周期感受器官）中光敏色素Ｉ（ｐｈｙＡ）的含量。从农垦５８Ｓ的二次枝梗原基分化期开始,进行１０天的光周期处理。一组为短日照（ＳＤ）,另一组为长日照（ＬＤ）。在第１０个暗期结束前,于暗绿光下收获每株水稻的最上部两片新展叶,立即保存在液氮中。样品在研钵中磨成液氮粉,然后加入提取液（含５０ｍｍｏｌ／ＬＴｒｉｓ－ＨＣＩ和０．２ｍｏｌ／Ｌ巯基乙醇,ｐＨ８．５）匀浆。粗提液经０．５％聚乙烯亚胺（ＰＥＩ）沉淀后,上清液供ＥＬＩＳＡ分析。以燕麦ｐｈｙＡ的多克隆抗体、单克隆抗体分别作ＥＬＩＳＡ的一抗和检测抗体。采用ＥＬＩＳＡ可以专一性地检测水稻ｐｈｙＡ（Ｔａｂｌｅ１）。几次独立的实验说明,光周期处理对ｐｈｙＡ含     

P61 Protein from a Male Sterile Mutant of Rice is an Isoform of the Chloroplast ATPase β Subunit

P61 was a protein identified from chloroplasts of Nongken 58S, a male sterile mutant of rice (Oryza sativa L. ssp. japonica). Microsequence analysis has revealed that its N-terminal sequence was identical to N-termini of ATPase β subunits of chloroplasts from rice and barley. The antiserum produced using ATPase β subunit from maize specifically recognized P61. P61 had the same molecular weight as the chloroplast ATPase β subunit of wild-type rice “Nongken 58”, but had different isoelectric point (pI) from this β　subunit. P61 was more basic than this β subunit. Thus, P61 would be identified as an isoform of the chloroplast ATPase β subunit of rice, named β1. Genetic analysis with a F2 population of Nongken 58SדNongken 58” showed that a single recessive genic gene regulated the formation of β1.     

农垦58s育性转换与吲哚乙酸氧化酶和过氧化物酶的关系

本文报道了农垦58s和农垦58幼穗发育期长短日下吲哚乙酸氧化酶和过氧化物酶的活性变化。结果表明:58s LD叶片中吲哚乙酸氧化酶活性从花粉母细胞形成期至花粉内容物充实期显著低于58s SD,与游离IAA的积累密切相关,可能影响花粉育性;但过氧化物酶的活性在上述时期显著高于58s SD,与游离IAA的积累无关,其作用尚不清楚。58s LD幼穗及花药中两酶活性在减数分裂期和花粉内容物充实期均较58s SD高,与同期幼穗及花药中游离IAA的亏缺有一定联系,从而可能在一定程度上影响花粉育性。对照农垦58叶片和幼穗及花药中两酶活性变化都不呈现上述现象。     

Effects of Photoperiod on Supramolecular Architecture of Thylakoid Membranes from a Rice Mutant (Oryza sativa Nongken 58S)

Plants of a rice mutant (Hubei photoperiod-sensitive genic male-sterile rice, Oryza sativa L. Nongken 58S) and its wild type cv. Nongken 58 were cultured in natural summer conditions in Beijing. After induction of proper photoperiods small panicle at the stem tip emerged and developed to the stage of secondary rachis-branch and spikelet primordium formation. Subsequently, part of the rice plants received long day (LD), i.e. 10 h of day-light treatment followed by 5 h of white fluorescent illumination with 1～2 Wm-2) . The others were exposed to daylight for 10 h alternating with a 14 h of dark period as short day (SD) treatment. After 10 days of the photoperiodic treatments, the chloroplast ultrastructure of the first leave below the flag leaf was examined by freeze-fracture rotary and unidirectionally shadowed electron microscopy. At anthesis stage, Nongken 58S plants with LD treatment showed complete pollen sterility, while the same plants with SD treatment exhibited normal fertility. And fertility of Nongken 58 was not affected by photoperiod treatments. The results from electron microscopic observation showed no significant effects of either SD or LD treatment on the freeze-fractured uhrastructure of thylakoid membranes in Nongken 58. No significant difference in particle density and size distribution was found on stacked and unstacked thylakoid membrane regions of the Nongken 58S-SD and those of Nongken 58 rice. However, the particle density of the endoplasmic fracture face in the staked region (EFs) and protoplasmic fracture face in the staked region (PFs) faces detected from the leaf thylakoid membranes of Nongken 58S-SD rice was significantly higher than that of the corresponding faces from Nongken 58S-LD. In some cases much more particles on EFs faces of thylakoid membranes isolated from Nongken 58S-SD rice appeared as paracrystalline particle array, indicating increases in the number of PS Ⅱ reaction centres, LHC I and Cyt b6/f per unit area of thylakoid membrane. The particle density of the endoplasmic fracture face in the unstaked region (EFu) and protoplasmic fracture face in the unstaked region (PFu) faces from unstacked thylakoid membranes of Nongken 58S-LD was less than that of the corresponding faces from Nongken 58S-SD. And the particle density of PFu faces from margin and end of the membranes of the grana thylakoids of LD-treated Nongken 58S leaves was also less than that of unstacked thylakoid membranes from SDtreated rice. In severe cases, most of the particles on endoplasmic fracture face in the unstaked region (EFu) and protoplasmic fracture face in the unstaked region (PFu) faces were even missing, indicating a decrease in the numbers of photosystem Ⅰ , LHCⅠ , Cyt b6/f and ATPase per unit area of' thylakoid membrane. The above results could further provide an augmentation for explaning the photoperiod-sensitive genic male-sterility.     

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。     

光周期诱导光敏感核不育水稻花药蛋白变化的研究

采用双向电脉技术对不同光周期条件下,光敏感核不育水稻（农垦５８ｓ）的可育与不育花药蛋白的变化进行分析,发现花粉发育的不同阶段中,不育花药具有四个特异蛋白ｐＩ６．２／ｂＭＷ７０ＫＤ,ｐＩ６．２／ＭＷ６８ＫＤ,ｐＩ６．２／ＭＷ３８ＫＤ和ｐＩ７．４／ＭＷ３７ＫＤ．对游离组蛋白的分析表明．长日照诱导的不育花药中游离组蛋白的相对百分率均明显低于短日照下的可育花药．据此推测长日照诱导不百花药蛋白质组成和代谢变化．不育花药中游离组蛋白含量低,可能受ＤＮＡ合成数量少的影响．     

光敏核不育水稻幼穗的乙烯生成与育性转换

研究了在不同光温组合下光敏核不育水稻（ＰＧＭＲ）农垦５８５（ＮＫ５８Ｓ）及普通水稻品种农垦５８（ＮＫ５８Ｂ）的幼穗乙烯生物合成变化及其与育性表达的关系。结果表明ＮＫ５８Ｓ幼穗乙烯生成受光周期和温度的共同调节,而光周期对ＮＫ５８Ｂ幼穗乙烯生成无明显影响。ＮＫ５８Ｓ幼穗乙烯生成的变化与育性转换光温作用模式完全吻合。其幼穗乙烯释放速率（ＥＲＲ）与花粉可育度（ｌｎｙ）呈极显著负相关。用ＡＶＧ抑制乙烯生成可使ＮＫ５８Ｓ（ＬＤ）不育性明显逆转,而用ＡＣＣ促进乙烯生成又可急剧降低ＮＫ５８Ｓ（ＳＤ）的可育水平。表明幼穗乙烯生成与ＰＧＭＲ育性表达密切相关,乙烯参与育性转换的调控并可能起着重要作用。     

乌龙岭’龙眼胚胎发育时期特异性蛋白质的变化

应用IEF-SDS-PAGE技术分析龙眼胚胎分化发育过程中蛋白质组分的变化。结果表明,在各发育阶段大多数蛋白质组分的电泳图谱基本一致,但也有变化。其中花后38d存在TE1(27．1kD、p,7．3),TE2(17．5kD、pI8．2)2个特异蛋白,45d存在TE3(11．4kD、pI7．6),TE4(13．2kD、pI9．9)2个特异蛋白,52d存在TE5(22．6kD、pI7．2),TE6(18．6kD、pI8．3),TE,(23．5kD、pI3．6)3个特异蛋白。31d胚胎电泳图谱中的蛋白质点数相对较多,表明此时蛋白质旺盛合成与积累,这与蛋白含量的变化基本一致。龙眼胚胎发育过程中特异蛋白的出现或消失．对胚胎的分化发育具有重要作用。     

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。     

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。     

光敏核不育水稻花药IAA的免疫组织化学分析

首次应用免疫组织化学分析方法对长日和短日处理后的农垦５８Ｓ和对照农垦５８（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．ｓｕｂｓｐ．ｊａｐｏｎｉｃａ）花药中的ＩＡＡ进行了定位研究和相对水平的比较。结果表明,此方法可反映游离态ＩＡＡ在花药中的分布及其相对水平的变化。从雌雄蕊原基形成期至单核晚期的５个时期中,经长日照处理的农垦５８Ｓ花药中的ＩＡＡ水平都低于短日照处理的农垦５８Ｓ及在不同光周期处理下的农垦５８花药。对花     

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。     

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。