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,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（phyA）的含量。从农垦58S的二次技梗原基分化期开始,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）沉淀一,上清液供ELISA分析。以燕麦phyA的多克隆隆抗体、单克隆抗体分别作ELISA的一抗和检测抗体。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含量有较大的影响。要相同的光周期下,农垦58S的phyA在较长时间暗期中的合成速度明显比农垦58快,这证实了前人的推测,即农垦58S甲基化程度较低的phyA基因的表达可能比农垦59更活跃。许多研究指出,pohyA含量的高低必然影响有关的生理过程。两种基因型相同的大麦品种对光周期、远红光（FR）敏感性的不同反应正是phyA含量差异所致。因此,SD下叶片积累较多的phA可能是农垦58S花粉育性恢复所必需的。另外,在农垦58S育性转变敏感期内每天的光周期结束时（EOD）进行短暂（15分钟）的FR照射实验,以推测光敏色素(PhyB)在育性转变中的作用,因为EODFR反应由phyB介导。10次EODFR处理后,农垦58S的抽穗、开花期均比SD对照相应地推迟2天。这与穗、开花（LD效应）,其花粉败育、种了结实率却没有变化（图1）,说明开花与花粉育性的调控机制有所不同。从上述结果我们认为,可能是phyA,而不是pohyB参与光周期诱导的农垦58s花粉育性转变过程,并且phyA可能通过其含量的变化起调节作用;phyB与水稻的抽穗、开花等现象有关。农垦58S可能通过其含量的变化起调节作用;phyB与水稻的抽穗、开花等现象有关。农垦58s控制花粉正常发育的phyA信号传导链可能存在某些缺陷,从而导致对光周期的敏感性增加,SD下积累较多的phyA则可以弥补这些缺陷。     

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。     

ADH在光敏感核不育水稻中反应特征的研究

乙醇脱氢酶(ADH)活性在湖北光敏感核不育水稻(HPGMR)幼穗发育的育性诱导阶段对光周期反应非常敏感。此时,在短日照或远红光处理的条件下,乙醇脱氢酶同工酶AdhII活性高,湖北光敏感核不育水稻原始不育株农垦58雄性可育;在长日照或红光间断长暗期的条件下,AdhII活性陡降,表现雄性不育。因此认为AdhII与湖北光敏感核不育水稻育性转换有关,可能是它参予了育性基因表达的调控作用。     

光周期敏感核不育水稻叶绿体的特异性蛋白质

用双向聚丙烯酰胺凝胶电泳技术,将光周期敏感核不育水稻“农垦58S”和其对照品种“农垦58”苗期及育性转换光周期敏感期的叶绿体蛋白质分离为大约90个蛋白质点。“农垦58S”的叶绿体内有一个45kD(pI_(6.7))和一个61kD(pI_(6.0))的特异性蛋白质点,而“农垦58”没有。“农垦58S”的另一个61kD(pI_(6.2))蛋白质点的含量明显高于“农垦58”。不同光周期(长日照和短日照)处理不影响光敏感期的这种叶绿体蛋白质的差异。     

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。     

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。     

光敏核不育水稻的育性与^45Ca的关系（简报）

光敏核不育水稻农垦58s育性转换始期,以涂叶法和灌根法对长日、短日处理植株饲喂的45Ca可自叶片或根部转入功能器官（叶、幼穗）,转入量随标记强度的增加而增加;植株吸收的45Ca可明显提高长日照下稻株的花粉可育率和自交结实率,而短日照下稻株的效应则相反。     

光温条件对短光低温不育水稻育性转换的影响

短光低温不育水稻是一类与长光高温不育水稻育性转换特性相反的新种质,通过对分期播种材料自然条件下的育性及人工短光处理后的育性比较分析表明,宜DIS的光敏期为第一苞分化期至第二次枝梗原基和小穗原基分化期（幼穗分化I－III期）,在常温下光敏期的短日处理可使育性趋于不育,但在高温下光敏期短日不育效应被削弱,宜DIS的花粉育性在高,低温作用下育性的均出现下降,根据高,低温影响育性的资料分析得到的敏感期基本一致,即花粉母细胞形成期至花粉内容物充实期（幼穗分化V－Ⅶ期）。     

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     

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.     

Effect of End-of-day far-red light exposures on fertility alteration and flowering in photoperiod-sensitive genic male-sterile rice

The rice photoperiod-sensitive genic male-sterile mutant (PGMR) is sterile under long days, but fertile in short days. Phytochrome is involved in the photoperiod-induced male-sterile process. To investigate the mechanisms, of phytochrome action in PGMR, end-of-day (EOD) experiments were carried out. Flowering in PGMR was delayed considerably by EOD far-red light exposures following a short day of 10 hr, whereas its fertility decreased to the same extent as the original line. This result suggests that photoperiod response mediating fertility alteration in PGMR somewhat differed from that in flowering,i.e., fertility alteration and flowering might be under the separate phytochrome signaling control.     

Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh.

The long-day plant Arabidopsis thaliana (L.) Heynh. flowers early in response to brief end-of-day (EOD) exposures to far-red light (FR) following a fluorescent short day of 8 h. FR promotion of flowering was nullified by subsequent brief red light (R) EOD exposure, indicating phytochrome involvement. The EOD response to R or FR is a robust measure of phytochrome action. Along with their wild-type (WT) parents, mutants deficient in either phytochrome A or B responded similarly to the EOD treatments. Thus, neither phytochrome A nor B exclusively regulated flowering, although phytochrome B controlled hypocotyl elongation. Perhaps a third phytochrome species is important for the EOD responses of the mutants and/or their flowering is regulated by the amount of the FR-absorbing form of phytochrome, irrespective of the phytochrome species. Overexpression of phytochrome A or phytochrome B resulted in differing photoperiod and EOD responses among the genotypes. The day-neutral overexpressor of phytochrome A had an EOD response similar to all of the mutants and WTs, whereas R EOD exposure promoted flowering in the overexpressor of phytochrome B and FR EOD exposure inhibited this promotion. The comparisons between relative flowering times and leaf numbers at flowering of the over-expressors and their WTs were not consistent across photoperiods and light treatments, although both phytochromes A and B contributed to regulating flowering of the transgenic plants.     

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。     

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.     

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.     

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.     

The rosette habit of Arabidopsis thaliana is dependent upon phytochrome action: novel phytochromes control internode elongation and flowering time

A major function of phytochromes in light-grown plants involves the perception of changes in the relative amounts of red and far-red light (R:FR ratio) and the initiation of the shade-avoidance response. In Arabidopsis thaliana, this response is typified by increased elongation growth of petioles and accelerated flowering and can be fully induced by end-of-day far-red light (EOD FR) treatments. Phytochrome B-deficient (phyB) mutants, which have a constitutive elongated-petiole and early-flowering phenotype, do not display a petiole elongation growth response to EOD FR, but they do respond to EOD FR by earlier flowering. Seedlings deficient in both phytochrome A and phytochrome B (phyA phyB), have a greatly reduced stature compared with wild-type or either monogenic mutant. The phyA phyB double null mutants also respond to EOD FR treatments by flowering early, suggesting the operation of novel phytochromes. Contrary to the behaviour of wild-type or monogenic phyA or phyB seedlings, petiole elongation in phyA phyB seedlings is reduced in response to EOD FR treatments. This reduction in petiole elongation is accompanied by the appearance of elongated internodes such that under these conditions the plants no longer display a rosette habit.