Effect of polyamines on in vitro anther culture of Citrus clementina Hort. ex Tan

The improvement of the induction rate in Citrus anther culture is important for taking practical advantage of the haploid potential in breeding. The influence of polyamines on anther culture of Citrus clementina, cv Nules, with particular attention to the free, soluble and insoluble-conjugated polyamine levels, has been investigated. Putrescine, spermidine and putrescine plus spermidine, were added to the standard induction medium. Before culture, spermidine was the most abundant among the free polyamines detected in anthers. The exogenous supply of either putrescine or spermidine, either independently or combined, effected greater uptake and accumulation of polyamines. The addition of 2 mM spermidine to the medium stimulated gametic embryogenesis in clementine Nules, whereas putrescine did not influence embryo production. Regenerants were mostly tri-haploids; a few doubled-haploids and no haploid plants were obtained.     

Polyamines reduce paraquat-induced soxS and its regulon expression in Escherichia coli

Polyamines, ubiquitous polycationic compounds, are involved in many cellular responses and relieve paraquat-induced cytotoxicity inEscherichia coli. We constructed a newE. coli mutant strain, JIL528, which is deficient in the biosynthesis of both putrescine and spermidine, to examine the physiological role of polyamines under oxidative stress caused by paraquat. Putrescine and spermidine downregulate the expression ofsoxS induced by paraquat in a concentration-dependent manner. The product of SoxS is a key regulator governing cellular responses against oxidative stress inE. coli. The downregulation ofsoxS expression by polyamines was not shown in thesoxR mutant background. Glucose-6-phosphate dehydrogenase (G6PDH; encoded byzwf) and manganese-containing superoxide dismutase (Mn-SOD; encoded bysodA) activities induced by paraquat were decreased by exogenous polyamines. The induction of thezwf expression by paraquat was also decreased by exogenous polyamines. The polyamine-deficient mutant strain JIL528 showed a highersoxS expression than its parent polyamine-proficient wild type BW1157, on exogenous supplementation of paraquat concentrations below 1 mol/L. While the growth rate of the mutant was decreased,soxS expression was increased in a concentration-dependent manner above 0.01 mol/L of paraquat. In contrast, growth inhibition of the mutant by paraquat was relieved, andsoxS was no longer induced by exogenous putrescine (1 mmol/L). In conclusion, polyamines protect against paraquat-induced toxicity but downregulatesoxS expression, suggesting that the protective role of polyamines against oxidative damage induced by paraquat results insoxS downregulation.     

生态因子对滇重楼花药开裂的影响

滇重楼为延龄草科重楼属植物,具有极高的药用价值,由于重楼传统药用部位生长缓慢、繁殖力低下,以及人们对野生重楼资源的过度采挖使其资源日趋枯竭。滇重楼的花药在整个花期中存在开裂－关闭的现象,花药的有效闭合应是保护花粉、延长花粉寿命、增强雄性适合度的一种适应机制。该研究以滇重楼为对象,通过设计正交实验和对比实验,观测其花药开裂过程中的光照强度、温度、湿度的变化,探究光照强度、温度、湿度等生态因子对滇重楼花药开裂的影响以及滇重楼花药开裂与生态因子变化的关系。结果表明：(1)在滇重楼花药开裂的过程中,光照强度增强、温度升高、相对湿度下降;(2)温度是影响滇重楼花药开裂时间的主导因子,升温促进花药开裂,降温促进花药关闭;(3)高湿度及黑暗推迟花药开裂,但并不能阻止花药开裂;(4)低温可使滇重楼花药持续关闭,而光照强度越高,花药持续关闭所需的温度越低。该研究有利于解释滇重楼花药白天开裂夜晚关闭的现象与环境因子的关系,对滇重楼的栽培育种提供理论指导。     

毛竹的花药发育研究

竹类植物因有着较长的开花周期,其生殖生物学研究的报道相对较少。该研究采用石蜡切片与野外观察的方法,对毛竹花药的发育以及花药发育与花序的关系进行了研究。结果表明:毛竹的花药壁结构包括4层细胞:表皮细胞、药室内壁细胞、中层细胞和绒毡层细胞。药室内壁和中层都只有一层细胞,而且细胞形状较扁,花药发育后期药室内壁会逐渐降解,而中层则会完全解体消失。花药壁的发育为单子叶型,绒毡层为腺质型,而且只有一层,细胞径向较长,最后也会消失。小孢子母细胞减数分裂时,胞质分裂方式为连续型。形成的小孢子经一次有丝分裂后逐渐形成成熟花粉粒,大多为二细胞型,很少产生三细胞型。此外,还发现毛竹花药的发育与花序形态变化存在着相对应的关系。野外连续观察和切片发现,随着花序形态的不断发育变化,首先花药开始形成并不断分化,药壁备层也逐渐形成;接着小孢子逐渐成熟,备层也慢慢随之解体、消失;最后花药逐渐开裂并开始散粉。该研究结果不仅丰富了毛竹和竹类生殖生物学的研究内容,而且对毛竹种质的研究也具有重要意义。     

Polyamines and protein kinase II. Effect of polyamines on cyclic AMP--dependent protein kinase from rat liver.

The effect of polyamines on the activity of cAMP-dependent protein kinase from rat liver was determined. It has been shown that polyamines inactivate the enzyme in the decreasing order of activity: spermine > spermidine > putrescine. This effect is due to inhibition of the catalytic subunit. On the other hand, binding of cAMP to the regulatory subunit of the enzyme is not affected by polyamines. It is suggested that the inhibition of protein kinase by polyamines is a general phenomenon.     

Comparative mapping of the barley genome with male and female recombination-derived, doubled haploid populations

Male (anther culture) and female (Hordeum bulbosum) derived, doubled haploid populations were used to map the barley genome and thus determine the different recombination rates occurring during meiosis in the F1 hybrid donor plants. The anther culture-derived (male recombination) population showed an 18% overall increase in recombination rate. This increased recombination rate was observed for every chromosome and most of the chromosome arms. Examination of linkage distances between individual markers revealed eight segments with significantly higher recombination in the anther culture-derived population, and one in the Hordeum bulbosum-derived population. Very strong distortions of single locus segregations were observed in the anther culture-derived population, but map distances were not affected significantly by these distortions. There were 1.047 and 0.912 recombinations per chromosome in the anther culture and Hordeum bulbosum-derived doubled haploid populations, respectively.     

Growth of anthers in Lilium longiflorum

The post-initiation growth of 64 anthers (1.1–17.4 mm long) in Lilium longiflorum Thumb. was examined by time-lapse marking experiments in combination with serial sections and the scanning electron microscope. Each anther was characterized by spatial and temporal variation in growth rate. Larger anthers had two, and occasionally three, series of peaks and troughs in local growth rate. Regions of negative growth rate were frequently encountered. When observed over several days, the growth maxima and minima were found to move basipetally as a waveform down the length of the anther. The wavelength was longer in taller anthers; amplitude and frequency were variable, and anthers of the same size were not always synchronous. Distribution patterns of cell division (and elongation, once division has ceased) recapitulate the growth data. Anther growth is a non-steady system, therefore, with growth centers constantly shifting. Implications for future studies in organ growth patterns are discussed.Abbreviation SEM scanning electron microscope     

Polyamine levels as related to growth,differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa

We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events.In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors -difluoromethyl-arginine (specific inhibitor of arginine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.     

Improved androgenesis of interspecific potato and efficiency of SSR markers to identify homozygous regenerants

Three interspecific diploid potato hybrids between selections of Solanum phureja Juz. & Buk. and S. chacoense Bitt. were used in anther culture experiments to construct a monoploid family. Different aspects of the anther culture process were affected by the treatments, such as: growing conditions of donor plants, ways of preparing the anther culture medium, and conditions of anthers in culture. Genotype and date of culture initiation were among the most significant sources of variation. Significant improvements in anther culture response were achieved by growing plants at 30°C and by a heat shock of 35°C for 12 h given to anthers in culture, which gave an increase of up to 40% in embryo yield. However, the heat shock reduced the plant regeneration rate. The majority of regenerated plants was diploid, suggesting that there were several recessive lethal alleles in heterozygous status in the anther-donor. Among the regenerants, the homozygotes could be successfully identified by simple sequence repeat analysis, using eight polymorphic primer pairs. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of polyamines in the cytokinin-dependent physiological processes. I. Effect of benzyladenine on polyamine levels during chloroplast differentiation in the tissue culture of Dianthus caryophyllus

Tissue culture of Dianthus caryophyllus L. (cv. William Sim.) obligatory requiring N⁶-benzyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry. Green callus growing on benzyladenine — containing medium showed decrease in the contents of free, conjugated and bound putrescine and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence of cytokinin. Spermidine was dominant polyamine bound to chromatin isolated from control callus. Chromatin isolated from green callus was characterized by a lower level of each polyamine in comparison to chlorophyll-less callus. Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine) in comparison to chlorophyll-less callus (− benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an inhibitor of S-adenosylmethionine decarboxylase depressed the greening process. Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic apparatus is discussed.     

Anther culture of kale (Brassica oleracea L. convar.acephala (DC.) Alef.)

The pollen development and androgenic ability of 18 kale (Brassica oleracea convar.acephala) genotypes was observed during an anther culture study. Anther culture was successful in 6 of the genotypes and the highest yield obtained was 17 embryos per 100 anthers plated. Two stages of anther development were identified as being responsive to anther culture. The first and most responsive was that corresponding to the late uninucleated stage and the second to the late binucleated stage. These stages correspond with the onset of mitotic events in the microspores. Pollen viability was studied and low viability was noted which declined to zero after 9 days of anther culture. The initial viability level however was not clearly related to androgenic ability. The significance of the production of haploid and dihaploid kale genotypes in the study and breeding of resistance to clubroot is discussed.     

Changes in endogenous polyamines during flower development in two diverse species of rose

Changes in the concentrations of endogenous free, conjugated and bound polyamine were determined in petals of two different species of rose, viz. Rosa damascena and Rosa bourboniana, from small bud (stage 1) till full bloom (stage 8). High free putrescine and spermidine concentrations were associated with early stages of flower development and then decreased in R. damascena. At full bloom, the concentration of free putrescine was higher than rest of the polyamines measured. A steady increase in conjugated putrescine, spermidine and spermine was observed during entire period of flower development with predominance of conjugated putrescine at full bloom. In R. damascena the bound spermine was higher than rest of the polyamines during full bloom. In R. bourboniana, during the early stages of flower development, similar situation was observed, however, at full bloom, free spermidine concentration was higher than rest of the polyamines. In this species, the concentration of conjugated and bound spermine was higher than rest of the polyamines during full bloom. Polyamine concentrations were generally lower in the petals of R. bourboniana than R. damascena which may be due to genotypic differences. The possible roles of the observed polyamines are discussed in relation to flower development.IHBT Communication no, 0345.     

Embryology of theaceae — Anther and ovule development ofAdinandra,Cleyera andEurya

Anther and ovule development of the theaceous Ternstroemioideae is reported for the first time on the basis of eight specles of three generaAdinandra, Cleyera andEurya. Anthers of these three genera are similar and can be characterized by the following traits: tapetum of glandular type, anther dehiscing latrorse-introrse, both connective and anther epidermis heavily tanniniferous, and connective and even anther wall layers having abundant druses. Their ovules are also very similar in being bitegmic and tenuinucellate, and in having a micropyle formed by the inner integument only, three cell-layered integuments, a raphe bundle terminating at chalaza, usually amphitropous or less often campylotropous ovule, embryo sac formation of Polygonum type, ephemeral antipodal cells, and tanniniferous ovule epidermis. Such ovules are readily distinguishable from those of Camellioideae and all other families. It is suggested that the three genera studied are closely related, and that the degree of embryological specialization is highest inAdinandra and lowest inEurya. On the basis of the significant embryological discrepacies, the Ternstroemioideae seem to have diverged rather distantly from the other core-subfamily Camellioideae of the Theaceae.     

Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines

Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.     

关苍术两性花与雌花花药的解剖学研究

苍术属(Atractylodes DC.)是菊科菜蓟族(Cynareae)刺苞亚族(Carlininae O. Hoffm.)的一个东亚特有属,世界上仅有7种,其中我国有5种。该研究以关苍术为材料,采用石蜡切片法比较研究了两性花和雌花的花药及雄配子体发育进程,并进一步探讨了其雌花产生花药退化的时期及原因。结果表明：(1)关苍术小孢子发育与花蕾长度间存在相关性,当花蕾长度在5 mm时进入花粉母细胞时期,花药壁已分化,在7~9 mm时处于四分体时期,大于11 mm时开始进入花粉粒时期。(2)关苍术花药5个,花粉囊4个,减数分裂属同时型,四分体以正四面体为主,属3-细胞型,萌发沟3个。(3)关苍术花粉囊壁发育属双子叶型,从外层的表皮、药室内壁,到内层的中层和绒毡层均由一层细胞构成,关苍术绒毡层为腺质绒毡层。(4)关苍术雌花花药退化发生在花药发育早期至四分体时期,表现为花药发育早期畸形、药壁分化异常、小孢子母细胞发育停滞在前期、绒毡层增生4个原因。该研究结果为苍术属植物的系统发育、物种形成和进化提供胚胎学依据。     

L-asparaginase of Tetrahymena pyriformis is associated with a kinase activity

Most of L-asparaginase activity of Tetrahymena pyriformis was found to be present in microsomal membranes from which it has been purified to homogeneity (Tsirka, S.A.E. and Kyriakidis, D.A. Mol. Cell. Biochem. 83: 147–155, 1988). The native enzyme has a relative molecular weight of approximately 200 kDa, while under denaturing conditions the enzyme exhibits. a subunit size of 39 kDa. Aminoacid analysis and an oligopeptide from N-terminal sequence have been determined. Dephosphorylation of L-asparaginase by alkaline phosphatase results in an activation of its catalytic activity. This enzyme also exhibits intrinsic phosphorylation activity with a Km value for ATP of 0.5 mM. Autophosphorylation with -^32P ATP of purified L-asparaginase results in the phosphorylation of tyrosine residues as well as in loss of its activity. Mg²⁺ and Ca²⁺ added together act synergistically to stimulate the kinase activity by more than 160%. The polyamines putrescine, spermidine and spermine activate the kinase approximately 100%, while neither cAMP or cGMP have any effect. These results indicate that this membrane protein with dual L-asparaginase/kinase activity must play an important role in regulating the intracellular levels of L-asparagine in Tetrahymena pyriformis.     

RNA,proteins and polyamines during gametophytic and androgenetic development of pollen in Nicotiana tabacum and Datura innoxia

Variations in polyamines, proteins and RNA during in vivo gametogenesis and in vitro androgenesis in Datura innoxia and in Nicotiana tabacum were studied. Spermidine was the major polyamine during gametogenesis in both species. Marked differences in proteins, RNA and polyamines were evident during meiosis and at the first haploid mitosis. In Nicotiana an unknown amine (X₆₀) appears at the beginning of the first haploid mitosis. At the same time a rapid increase in the concentrations of RNA and proteins is observed. In Datura, at the time of the first haploid mitosis there is large increase in amine and RNA levels followed by an arginine peak. During androgenesis, putrescine and spermidine were the major polyamines in both species. In Nicotiana during androgenesis an unknown amine (X₈₁) was observed together with putrescine and spermidine. This unknown compound peaks during the developmental stages of embryogenesis. In Datura androgenic induction was marked by an arginine peak followed by an increase in the putrescine and spermidine levels associated with maximum RNA. These biochemical events are tentatively correlated with structural changes during pollen development. The significance of these results is discussed in relation to the role of polyamines during gametogenesis and androgenesis.     

雄性不育无籽刺梨花药发育过程中生理特性的初步研究

为探究无籽刺梨(Rosasterilis)雄性不育原因,采用1%I2-KI染色法观察花粉活性,并对无籽刺梨和正常可育刺梨(R.roburghii)花药不同发育时期的生理生化指标进行了研究。结果表明,无籽刺梨的败育花粉占95.5%,刺梨的正常花粉占99%。刺梨花药的可溶性蛋白质、可溶性糖和脯氨酸含量在各时期的总体变化趋势相似,可溶性淀粉含量呈上升趋势,而无籽刺梨花药的可溶性蛋白质、可溶性糖、淀粉和脯氨酸含量在各时期的变化无规律可循,且花粉成熟期这4种物质的含量均明显低于刺梨,即花粉成熟期缺少各营养物质的积累。在花药发育过程中,无籽刺梨的SOD活性均低于刺梨;MDA含量呈上升趋势,且上升幅度比刺梨大;MDA含量和POD活性均高于刺梨。因此,营养物质的匮乏和酶系统的紊乱可能是导致无籽刺梨雄性不育的原因。     

长花柱型滇丁香小孢子发生及雄配子体发育

利用常规石蜡切片法和细胞学压片法,对异型花柱植物滇丁香的长花柱型植株的小孢子发生、雄配子体发育及花粉萌发进行观察。结果表明:(1)长花柱型滇丁香具5枚花药,花药4室。(2)花药壁由1层表皮、1层花药内壁、2层中层和1~3层绒毡层组成;花药壁发育方式为基本型,绒毡层类型为腺质绒毡层。(3)小孢子母细胞减数分裂的胞质分裂为同时型,四分体排列方式为四面体型,偶有左右对称型;不同药室间小孢子母细胞减数分裂不同步。(4)成熟花粉粒为二细胞型。(5)小孢子发生和雄配子体发育过程正常,表明长花柱型滇丁香属于发育正常的两性花。(6)授粉4h后,长花柱型滇丁香的花粉在长、短两种花柱柱头上的萌发率分别达(91.8±1.6)%和(93.2±1.1)%,且两者间无显著性差异(t=1.585,df=8,p=0.152),表明长花柱型滇丁香的成熟花粉粒在长、短两种花柱柱头上的萌发均正常。     

中国特有种苦绳(广义夹竹桃科)的大小孢子发生和雌雄配子体发育及其分类学意义

近年来的分子系统学把狭义萝藦科和狭义夹竹桃科合并为广义夹竹桃科,包括5个亚科和25个族,但亚科和族间的亲缘关系较为复杂,亟待多学科证据澄清。本文利用常规石蜡切片技术观察了马利筋亚科南山藤属中的中国特有植物苦绳(Dregea sinensis var. sinensis)的孢子发生和配子体发育,结合已有资料比较了5个亚科的胚胎学特征。结果表明:(1)苦绳的花药由一对侧生并列药室组成,各有一个花粉团。(2)花药壁有6层,由外至内分别为表皮、2层药室内壁、中层和2层绒毡层,花药壁发育模式属于多层型。(3)绒毡层细胞单核,排成2列,为腺质型; 在小孢子四分体形成时期,药室内壁发生明显纤维状加厚; 花药成熟时,位于药室远轴最外侧处的花药壁发生断裂,准备散粉。(4)小孢子母细胞减数分裂中,胞质分裂方式为连续型,小孢子四分体排列方式为左右对称; 成熟花粉粒为3-细胞型,排列紧密,形成花粉团。(5)雌蕊含有两枚离生心皮,具边缘胎座,胚珠倒生,单珠被,薄珠心,蓼型胚囊。本文观察到的这些胚胎学特征为牛奶菜族提供了新资料。同时,胚胎学特征在5个亚科间的区别和联系,支持广义夹竹桃科的成立。