Endogenous polyamine content during in vivo maturation and in vitro culture of maize pollen

Changes in polyamine content during in vivo maturation and in vitro culture of maize (Zea mays L.) pollen were studied. The endogenous content of free, conjugated and bound polyamines was analyzed during 30 days of pollen evolution, in both developmental pathways (microsporogenesis and androgenesis). The induction of androgenesis from cold-pretreated uninucleate pollen results, in most of cases, in a lower total polyamine content than that of the in vivo uninucleate pollen. These differences indicate that polyamine metabolism is altered during the induction of androgenesis, and this could be a consequence of increased polyamine assimilation. In general, pollen stages that involve cell division (tetrades, pre-anthesis pollen and four-day cultured pollen) are characterized by a predominance of free Spd. The increase of Spd and Spm in 15-day cultured pollen, when the first embryoids are formed, outline the possible implication of these polyamines in embryogenetic processes. Furthermore, these findings may contribute to the improvement of maize androgenesis yield, especially in recalcitrant genotypes, by the exogenous application of polyamines or polyamine-inhibitors to the culture medium.Abbreviations PAs polyamines - Put putrescine - Spd spermidine - Spm spermine - S free polyamine fraction - SH conjugated polyamine fraction - PH bound polyamine fraction     

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.Abbreviations IEC immature-embryo calluses - PAs polyamines - PEC pollen-embryo calluses - PH insoluble conjugated PA fraction - Put putrescine - S free PA fraction - SH soluble conjugated PA fraction - Spd spermidine - Spm spermine 2,4d-2,4 dichlorophenoxyacetic acid     

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.     

盐胁迫下大麦幼苗多胺的种类和状态与多胺氧化酶活性的关系

200 mmol/L的NaCl胁迫8 d大麦幼苗叶片和根系中的三种形态多胺都有不同程度地下降,其中游离态多胺含量的下降幅度最大;高氯酸不溶性结合态多胺含量变化较小.根系中PAO的活性先上升后下降,而叶片中PAO的活性先下降后上升.游离态多胺中,亚精胺和精胺(Spd Spm)的含量变化与相应部位PAO的活性变化趋势相反,表明PAO在盐胁迫下可能调节了游离态多胺的含量从而影响高氯酸可溶结合态与高氯酸不溶结合态多胺的含量.     

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.     

Factors affecting in vitro maturation of isolated maize microspores

Summary An in vitro method to simulate pollen development was developed in maize (Zea mays L.). Microspores at the late uninucleate to early binucleate stage were isolated and cultured under various conditions. Cell viability, starch content and the formation of the three nuclei as found in normal mature pollen were monitored during the course of the culture. Media composition was modified in order to promote starch accumulation and frequency of mitosis, while maintaining the viability of the microspores. Under the best conditions, up to 12% of the microspores matured in vitro into trinucleate, starch-filled viable pollen grains which were unable to germinate or produce seeds. At different stages during in vitro maturation, proteins patterns were analyzed and compared with their in vivo equivalent and the patterns were only partially similar.     

Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic Acid

Polyamine metabolism and its relation to the induction of α-amylase formation in the aleurone layers of barley seeds (Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA₃) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).

Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA₃ (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of α-amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA₃. Kinetic studies show that both applied [U-¹⁴C]ornithine and [U-¹⁴C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA₃. Additionally, added GA₃ does not affect the uptake and turnover of [1,4-¹⁴C]Put, nor does it affect the conversion of Put → Spd or Spd → Spm. Treatment of the aleurone layers with GA₃ for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase.

Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-¹⁴C] adenosine into ATP. The lower the level of Spd the less α-amylase formation is induced by added GA₃. The reduction of GA₃-induced α-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA₃, with respect to induction of α-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction.

     

Microsporogenesis and pollen formation in cassava

This article describes the complete microsporogenesis and pollen formation in cassava (Manihot esculenta Crantz) at the various developmental stages (pollen mother cell, meiosis, tetrads, early free spore, mid uninucleate, late uninucleate, binucleate and mature pollen grain). Light microscopy, transmission electron microscopy and confocal laser scanning microscopy were used for the study. Floral bud size and other inflorescence characteristics were correlated with specific stages of the microspore development. This association allows a rapid selection of floral buds with similar microspore developmental stages, useful when a large number of homogeneous cells are needed for analysis and for in vitro induction of androgenesis. This article also compares methods for digestion the exine wall in microspores.     

Light quality influences the polyamine content of lettuce (Lactuca sativa L.) cotyledon explants during shoot production <Emphasis Type="Italic">in vitro</Emphasis>

Light quality has previously been shown to influence morphogenesis in lettuce cotyledon explants, with white or red light promoting adventitious shoot production, and blue light inhibiting it. Endogenous polyamine (PA) concentrations were compared between explants cultured under different light qualities. Explants cultured under white or red light accumulated PAs during shoot primordia production, with a 5.6-fold increase compared to initial concentrations under white light, and 6.7-fold increase under red light. These results suggest polyamines are involved in the formation of shoot primordia. After 18 days in culture PA concentrations decreased under white light, and to a lesser extent under red light, signaling a shift in polyamine metabolism that correlates with shoot expansion, which occurs more readily under white light. Explants cultured under blue light accumulated polyamines for the first 7 days, to a level 1.3 times greater than initial values, followed by a gradual decline during the remainder of the culture period. Explants cultured under blue light also contained a greater proportion of PCA-insoluble conjugated PAs, compared to explants under white or red light, which contained greater proportions of free or PCA-soluble conjugated polyamines. The ratio of putrescine to spermidine was also different with a lower Put:Spd ratio being associated with shoot production under white or red light, and higher Put:Spd ratio being associated with culture under blue light.     

外源亚精胺对盐胁迫下黄瓜幼苗游离态多胺含量和多胺合成酶基因表达的影响

以黄瓜品种‘津春2号’（Cucumis sativusL.cv.Jinchun No.2）为材料,采用营养液栽培,研究了外源亚精胺（Spd）对NaCl胁迫下黄瓜幼苗叶片游离态多胺含量和多胺合成酶基因表达的影响。结果表明,75 mmol/LNaCl胁迫下,幼苗株高、茎粗和干鲜重显著降低,外源喷施1 mmol/L Spd处理可明显缓解盐胁迫对幼苗生长的抑制。盐胁迫下叶片游离态多胺含量显著增加,外源Spd进一步促进了游离态Spd和精胺（Spm）的积累,降低了游离态腐胺（Put）的积累。多胺合成酶基因表达分析表明,盐胁迫上调了adc、odc、samdc和spds基因的表达,施用外源Spd后进一步上调了samdc基因,下调了adc、odc、spds基因的表达。表明外源Spd参与了黄瓜幼苗体内多胺代谢的调节,通过下调盐胁迫下adc、odc基因的表达,抑制游离态Put的积累,上调samdc基因的表达促进游离态Spd和Spm的积累,进而缓解盐胁迫对植物生长的抑制。     

Cadaverine: a common polyamine in zygotic embryos and somatic embryos of the species Capsicum chinense Jacq.

The behavior of endogenous polyamines was studied in somatic embryos and zygotic embryos of Habanero pepper (Capsicum chinense). In the first part of the work, the polyamine content was evaluated in both types of embryos (somatic and zygotic). As a result, in addition to the common polyamines (putrescine, spermidine and spermine), it was also possible to detect cadaverine, a polyamine rarely found in plants. In general, all the polyamines were found to be more abundant in somatic embryos than in zygotic embryos, with significantly higher contents of putrescine and cadaverine. Subsequently, the content of putrescine, spermidine, spermine and cadaverine, in their different forms (free, bound and conjugated) was determined in somatic embryos which were cultured in non-ventilated and ventilated containers. Detection of polyamines was carried out at 28 and 42 days of culture by the HPLC method. The ethylene content was monitored during the process in both culture conditions (ventilated and non-ventilated). As a result of the analysis, cadaverine was always found present, indicating that it is a common polyamine in the species. Ethylene was detected in containers without ventilation throughout the culture, except during replenishment of the culture medium (R1, R2 and R3). The behavior pattern of each polyamine, analyzed under different culture conditions (ventilated and non-ventilated) and at different moments of culture (28 and 42 days of culture), show that the polyamines are not only involved in morphogenic processes in plants; polyamines are also significantly affected by the surrounding environment. However, the most novel result, presented for the first time in this paper, is that cadaverine is found to be a common polyamine in C. chinense since it is present in both zygotic embryos and somatic embryos.

     

NaCl胁迫对菜用大豆种子多胺代谢的影响

采用蛭石栽培,在100 mmol·L-1NaCl胁迫下,对耐盐性不同的两个品种菜用大豆种子的丙二醛(MDA)含量和多胺(PAs)代谢进行了研究.结果表明:NaCl胁迫显著增加了菜用大豆种子的MDA含量,但耐盐品种‘绿领特早’(LL)的增幅低于盐敏感品种‘理想高产95-1’(LX).与LX相比,LL种子在整个NaCl胁迫期间均维持了相对较高的游离态精胺(Spm)、结合态Spm、结合态亚精胺(Spd)、束缚态Spd和束缚态腐胺(Put)含量,较高的(Spd +Spm )/Put 和(cPAs+bPAs)/fPAs值及较低的Put/PAs值,在胁迫中、后期(9～15 d)维持了相对较高的游离态Spd含量;胁迫期间,LL的精胺酸脱羧酶(ADC)长时期(6～15 d)保持相对较高的活性,而多胺氧化酶(PAO)则长时期(6～15 d)维持相对较低的活性.综上,LL具有较强的多胺合成能力及较强的Put向Spd和Spm以及游离态多胺向结合态和束缚态多胺转化的能力,进而有效抑制了细胞的膜脂过氧化,这可能是其耐盐性较强的重要原因之一.     

Polyamine changes during the development of zygotic embryos in Prunus avium

A study of the polyamine profile was carried out during zygotic embryo development in Prunus avium. Zygotic embryos were collected from 2 donor trees and sorted into 3 size classes: C1 [2.5 to 3.5 mm]; C2 [3.6 to 4.5 mm] and C3 [5.5 to 7 mm]. Evolution of the various polyamines was similar for the two donor trees. Changes in the relative amount of the various free polyamines were observed during zygotic embryo development. Agmatine and spermine levels increased from C1 to C3. Spermidine, the predominant polyamine, showed a two-fold decrease in C3 compared with C1 and C2; the evolution of putrescine was opposed, showing an increase in the last developmental stage. The putrescine/spermidine ratio could be a marker for these 3 developmental stages with a higher ratio in C3 compared with C1 and C2. Polyamine changes in cotyledons from class C1 were investigated during in vitro culture. A 10-day induction on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin caused a strong decline in free spermidine levels and a dramatic increase in free putrescine. The formation of conjugated putrescine occurred simultaneously, and twenty days after removal of growth regulators, the various polyamine contents were still at the same level.Abbreviations Agm agmatine - Dap diaminopropane - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine - Spm spermine     

Antisense inhibition of a spermidine synthase gene highlights the role of polyamines for stress alleviation in pear shoots subjected to salinity and cadmium

Three transgenic European pear (Pyrus communis L.) lines with reduced spermidine synthase (SPDS) expression and spermidine (Spd) titers were developed using a construct containing an apple SPDS gene (MdSPDS1) in antisense orientation. After exposure to either salt or cadmium stress, growth inhibition was more severe in the antisense lines than in the wild-type (WT). The antioxidant system, as shown by glutathione (GSH) content, activity of glutathione reductase (GR) and superoxide dismutase (SOD), and proline accumulation, was not effectively induced under stress in the antisense lines as compared with the WT. The reduction in antioxidant system function in the antisense lines was accompanied by a greater accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation. Growth inhibition, Spd level, and parameters indicative of the antioxidant system were significantly ameliorated by exogenous Spd application. Under either salt or cadmium stress, GSH content, GR and SOD activity, and proline accumulation were positively correlated with Spd, putrescine (Put), and total polyamine titers. Conversely, MDA level showed a significantly negative correlation with these polyamines under both stress conditions. Thus, the responses to stress treatments were first identified in the SPDS antisense European pears, and the results provide further evidence for the important role of polyamines in both salt and cadmium stress tolerance, in which the polyamines act, at least in part, by influencing the antioxidant system.     

Observation of Androgenesis in Cultured Wheat Anthers at Meiosis,Tetrad, Early Uninucleate and Trinucleate Stage

The obtaining of calluses and plantlets from cultured wheat anthersat the stages from pollen mother cell to trinucleate microspore has been reported previously. Haploids as well as diploids existed among the regenerated plantlets derivedfrom anthers at these stages. Present paper reports the study on androgenesis patter-ns of cultured anthers at meiosis, tetrad, early mid- and late uninucleate and trinucleate stage. Cytological evidence of pollen-origin of calluses produced by anthers atthese stages was given. Observation showed that meiosis of wheat anthers was able tocomplete under culture conditions, resulting in releasing microspores, from which multinucleate and multicellular pollen grains formed. In meiosis anthers, abnormal cells,including syncytium and two kinds of binueleate calls were sometimes observed. Theymight be products of abnormal meiosis and abnormal development of tapetum cells. Itwas noted that failure and/or uncomplction of forming callus wall and/or pollen wallin in vitro anthers at meiosis, tetrad and early uninucleate stage occured often. Itmight lead to the low frequency of callus induction. Mature wheat anthers (trinucleate stage) contained both normal and abnormal pollen grains (pollen dimorphism); onlythe abnormal pollen grains developed into embryoids while all the normai trinucleatepollen grains degenerated rapidly. However, the date of the frequency of equal divisionof microspores suggested that abnormal pollen (N pollen, small pollen) could not be theonly source of androgenic pollens in cultured anthers at late uninucleate and other earlier stages.     

Polyamines control the growth of the fish-killing dinoflagellate Karenia mikimotoi in culture

Effects of intracellular polyamines on the growth of the harmful dinoflagellate Karenia mikimotoi were investigated in culture experiments using an axenic culture. Polyamines were analyzed with HPLC. Free norspermine was the most abundant polyamine during growth of K. mikimotoi. Cellular norspermidine contents increased significantly during the exponential growth phase with increasing growth rate. The maximum growth yield of K. mikimotoi was reduced by the polyamine biosynthetic inhibitor, d,l-alpha-difluoromethylornithine (DFMO) which inhibits ornithine decarboxylase. These results suggest that polyamines, especially free norspermine, play significant roles in the growth of K. mikimotoi.