Response of in vitro pollen germination and pollen tube growth of almond (Prunus dulcis Mill.) to temperature,polyamines and polyamine synthesis inhibitor

Prunus dulcis L. ‘Mamaei’ is grown widely in souhtwest of Iran. It blooms in early spring when temperatures are still low. Based on our knowledge there are no reports in the literature regarding pollen behavior of this cultivar under specified condition. Thus, the possible factors for low germination percentage in this cultivar have not been reported. The effect of three different temperatures (10, 25, or 35 °C), polyamines (putrescine, spermidine, and spermine) and polyamine synthesis inhibitor, methylglyoxals-bis (guanyl-hydrazone) (MGBG) on in vitro pollen germination and pollen tube growth were investigated in P. dulcis L. ‘Mamaei’. All temperatures and chemicals significantly affected both pollen germination percentage and pollen tube growth. In general, different polyamines stimulated the pollen germination percentage compared to the control at all temperatures, but increasing the temperature, particularly to 35 °C, had demonstrated inhibitory effects on pollen germination. At a concentration of 0.05 mM putrescine and spermidine and 0.005 and 0.025 mM spermine revealed longer pollen tube growth than that of the control at 10 °C, while higher concentrations tended to inhibit pollen tube growth. At 25 °C, most of the treatments had an inhibitory effect on pollen tube growth except for 0.25 mM putrescine and 0.005 mM spermine, which slightly stimulated pollen tube growth. Pollen germination and pollen tube growth were inhibited by MGBG at all temperatures and in all concentrations.     

Do exogenous polyamines have an impact on the response of a salt-sensitive rice cultivar to NaCl?

In order to analyze the putative impact of polyamines (PAs) on the plant response to salt, seedlings from the salt-sensitive rice cultivar I Kong Pao (IKP) were exposed for 5, 12 and 19 days to 0, 50 or 100 mM NaCl in the absence, or in the presence of exogenous PAs (putrescine (Put), spermidine (Spd) or spermine (Spm) 1mM) or inhibitors of PA synthesis (methylglyoxalbis-guanyl hydrazone (MGBG) 1mM, cyclohexylammonium (CHA) 5mM and D-arginine (D-Arg) 5mM). The addition of PAs in nutritive solution reduced plant growth in the absence of NaCl and did not afford protection in the presence of salt. PA-treated plants exhibited a higher K+/Na+ ratio in the shoots, suggesting an improved discrimination among monovalent cations at the root level, especially at the sites of xylem loading. The diamine Put induced a decrease in the shoot water content in the presence of NaCl, while Spd and Spm had no effects on the plant water status. In contrast to Spd, Spm was efficiently translocated to the shoots. Both PAs (Spd and Spm) induced a decrease in cell membrane stability as suggested by a strong increase in malondialdehyde content of PA-treated plants exposed to NaCl. These results are discussed in relation to the putative functions of PAs in stressed plant metabolism.     

Inhibition of polyamine biosynthesis and growth in plant pathogenic fungi in vitro

Polyamine (PA) biosynthesis inhibitors, difluoromethylornithine (DFMO), difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and bis-(cyclohexylammonium) sulphate (BCHA) have been tested for their effects on colony diameters at different intervals after inoculation of four plant pathogenic fungi (Helminthosporium oryzae, Curvularia lunata, Pythium aphanidermatum and Colletotrichum capsici). All these inhibitors, except DFMA had strongly retarded the growth of four fungi in a dose- and species-dependent fashion, and H. oryzae and C. lunata were found to be most sensitive to the effects of PA inhibitors. P. aphanidermatum and C. capsici were relatively insensitive and required rather high concentrations of inhibitors to get greater inhibition of mycelial growth, except DFMA which had stimulatory effect on the growth of these two fungi. However DFMA had greatly suppressed the growth of H. oryzae and C. lunata. The effect was generally more pronounced with MGBG than with DFMO and BCHA, and 1 mM Put completely prevented the inhibitory effects of 1 and 5 mM DFMO. Analysis of free and conjugated PAs in two sensitive fungi (H. oryzae and C. lunata) revealed that Put was present in highest concentrations followed by Spd and Spm and their levels were greatly reduced by DFMO application, and such inhibitions were totally reversed by exogenously supplied Put; in fact, PA titers were considerably increased by 1 mM Put alone and in combination with 1 mM DFMO. These results suggest that PA inhibitors, particularly DFMO and MGBG may be useful as target-specific fungicides in plants.     

水稻细胞质雄性不育系幼穗发育过程中多胺与乙烯的关系初探(英文 )

利用ＨＰＬＣ和ＧＣ分别测定了水稻细胞质雄性不育系及其保持系幼穗多胺（ 腐胺,亚精胺和精胺） 含量和乙烯释放速率,并研究了外施多胺合成抑制剂ＭＧＢＧ 和乙烯前体ＡＣＣ生成抑制剂ＡＶＧ 对两系幼穗多胺含量和乙烯释放速率以及花粉育性的影响。结果表明, 不育系幼穗乙烯释放速率显著高于其保持系幼穗, 外施ＡＶＧ 引起两系幼穗乙烯释放速率下降,并使不育系花粉育性得以部分恢复; 不育系幼穗多胺含量显著低于保持系幼穗, 外施ＭＧＢＧ 使两系幼穗Ｓｐｄ 和Ｓｐｍ 含量下降, 并使保持系花粉育性降低。外施ＡＶＧ 抑制乙烯释放,促进多胺合成;而外施ＭＧＢＧ 抑制Ｓｐｄ和Ｓｐｍ 合成, 却促进乙烯的释放; 而且,乙烯释放速率与多胺（精胺和亚精胺） 含量呈显著负相关。提示在水稻ＣＭＳ 系及其保持系幼穗发育过程中乙烯与多胺（ 精胺和亚精胺） 的生物合成竞争ＳＡＭ。     

Enhanced susceptibility of photosynthesis to low-temperature photoinhibition due to interruption of chill-induced increase of S-adenosylmethionine decarboxylase activity in leaves of spinach (Spinacia oleracea L.)

The possible involvement of polyamines in the chilling tolerance of spinach (Spinacia oleracea L.) was investigated focusing on photosynthesis. During chilling at 8/5C (day/night) for 6 d, S-adenosylmethionine decarboxylase (SAMDC) activity increased significantly in leaves in parallel with the increase in putrescine and spermidine (Spd) content in leaves and chloroplasts. Treatment of leaves with methylglyoxal-bis(guanylhydrazone) (MGBG), an SAMDC inhibitor, resulted in the deterioration of plant growth and photosynthesis under chilling conditions, which was reversed by the concomitant treatment with Spd through the roots. Plants treated with MGBG showed lower photochemical efficiency of PSII than either the control or plants treated with MGBG plus Spd during chilling and even after transfer to warm conditions, suggesting an increase of photoinhibition due to low Spd in chloroplasts. Indeed, MGBG-treated plants had much lower activities of thylakoid electron transport and enzymes in carbon metabolism as well as higher degrees of lipid peroxidation of thylakoid membranes compared to the control. These results indicate that the enhanced activity of SAMDC with a consequential rise of Spd in chloroplasts is crucial for the cold acclimation of the photosynthetic apparatus in spinach leaves.     

Effect of end-of-day irradiations on polyamine accumulation in petal cultures of Araujia sericifera

We have studied photoperiodic control and the effect of phytochrome photoconversion at the end-of-day (EOD) on polyamine (PA) accumulation in petal explants of Araujia sericifera . Petals from immature flowers were cultured under long (LD) and short (SD) days. Light was provided by Gro-lux fluorescent lamps (90–100 µmol m⁻² s⁻¹). Red (R), far red (FR), red followed by far-red (R-FR) and far-red followed by red (FR-R) light treatments were applied daily at the end of the photoperiod. The free and bound putrescine (Put), spermidine (Spd) and spermine (Spm) fractions in petal explants were determined 40 days after the beginning of the culture. We also aimed to clarify the involvement of PA changes by using two inhibitors of PA biosynthesis: D- l -α-difluoromethylarginine (DFMA) and methylglyoxal bis (guanylhydrazone) (MGBG). We found PA accumulation to be under photoperiodic control, and the inhibitory effect of DFMA on this accumulation suggests that arginine decarboxylase (ADC) is the major pathway for Put biosynthesis. Polyamine levels were higher under LD, mainly as a result of the accumulation of free and bound Put. FR-EOD treatment, which dramatically reduced the R : FR ratio after LD, increased the accumulation of PA, mainly as free Put and free and bound Spd. Sequential R-FR and FR-R-EOD treatments strongly increased bound Spd. The concentration of MGBG used increased total PA accumulation, mainly as Put. However, all EOD light treatments dramatically reduced Put accumulation in the presence of MGBG. This may be due to a dual role of FR light in PA accumulation: (1) FR per se stimulates PA production, probably via ADC, and (2) in the presence of MGBG, FR inhibits Put accumulation, probably via ethylene production.     

Reactive oxygen species are involved in pollen tube initiation in kiwifruit

The role of reactive oxygen species (ROS) during pollen tube growth has been well established, but its involvement in the early germination stage is poorly understood. ROS production has been reported in germinating tobacco pollen, but evidence for a clear correlation between ROS and germination success remains elusive. Here, we show that ROS are involved in germination and pollen tube formation in kiwifruit. Using labelling with dihydrofluorescein diacetate (H(2) FDA) and nitroblue tetrazolium (NBT), endogenous ROS were detected immediately following pollen rehydration and during the lag phase preceding pollen tube emergence. Furthermore, extracellular H(2) O(2) was found to accumulate, beginning a few minutes after pollen suspension in liquid medium. ROS production was essential for kiwifruit pollen performance, since in the presence of compounds acting as superoxide dismutase/catalase mimic (Mn-5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H,23H-porphin, Mn-TMPP) or as NADPH oxidase inhibitor (diphenyleneiodonium chloride, DPI), ROS levels were reduced and pollen tube emergence was severely or completely inhibited. Moreover, ROS production was substantially decreased in the absence of calcium, and by chromium and bisphenol A, which inhibit germination in kiwifruit. Peroxidase activity was cytochemically revealed after rehydration and during germination. In parallel, superoxide dismutase enzymes, particularly the Cu/Zn-dependent subtype - which function as superoxide radical scavengers - were detected by immunoblotting and by an in-gel activity assay in kiwifruit pollen, suggesting that ROS levels may be tightly regulated. Timing of ROS appearance, early localisation at the germination aperture and strict requirement for germination clearly suggest an important role for ROS in pollen grain activation and pollen tube initiation.     

Effects of spermidine and spermine levels on salt tolerance associated with tonoplast H+-ATPase and H+-PPase activities in barley roots

The effects of polyamines (Putrescine— Put; Spermidine—Spd; and Spermine—Spm) on␣salt tolerance of seedlings of two barley (Hordeum vulgare L.) cultivars (J4, salt-tolerant; KP7, salt-sensitive) were investigated. The results showed that, the salt-tolerant cultivar J4 seedlings accumulated much higher levels of Spd and Spm and lower Put than the salt-sensitive cultivar KP7␣under salt stress. At the same time, the dry weight of KP7 decreased significantly than that of␣J4. After methylglyoxal bis(guanylhydrazone) [MGBG, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC)] treatment, Spd and Spm levels together with the dry weight of both cultivars were reduced, but the salt-caused dry weight reduction in two cultivars could be reversed by the concomitant treatment with Spd. MGBG decreased the activities of tonoplast H⁺-ATPase and H⁺-PPase too, but the experiments in vitro indicated that MGBG was not able to affect the above two enzyme activities. However, the polyamines, especially Spd, promoted their activities obviously. These results suggested that the conversion of Put to Spd and Spm and maintenance of higher levels of Spd and Spm were necessary for plant salt tolerance.     

Involvement of polyamines in the post-anthesis development of inferior and superior spikelets in rice

Early-flowered superior spikelets usually exhibit a faster grain filling rate and heavier grain weight than late-flowered inferior spikelets in rice (Oryza sativa L.). But the intrinsic factors responsible for the variations between the two types of spikelets are unclear. This study investigated whether and how polyamines (PAs) are involved in regulating post-anthesis development of rice spikelets. Six rice genotypes differing in grain filling rate were field grown, and PA levels and activities of the enzymes involved in PA biosynthesis were measured in both superior and inferior spikelets. The results showed that superior spikelets exhibited higher levels of free spermidine (Spd) and free spermine (Spm) and higher activities of arginine decarboxylase (ADC, EC 4.1.1.19), S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and Spd synthase (EC 2.5.1.16) than inferior spikelets at the early endosperm cell division and grain filling stage. The maximum concentrations of free Spd and free Spm and the maximum activities of ADC, SAMDC and Spd synthase were significantly correlated with the maximum cell division and grain filling rates, maximum cell number and grain weight. Application of Spd and Spm to panicles resulted in significantly higher rates of endosperm cell division and grain filling in inferior spikelets along with the activities of sucrose synthase (EC 2.4.1.13), ADP glucose pyrophosphorylase (EC 2.7.7.27) and soluble starch synthase (EC 2.4.1.21), suggesting that these PAs are involved in the sucrose-starch metabolic pathway. The results indicate that the poor development of inferior spikelets is attributed, at least partly, to the low PA level and its low biosynthetic activity.     

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity of Allium cepa root tips

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.     

水稻细胞质雄性不育系及其保持系幼穗发育过程中的多胺代谢

在幼穗发育过程中,不育系和保持系幼穗多胺含量先剧降后稳定或略回升,精氨酸脱羧酶活性快速下降,而二胶和多胺氧化酶活性缓慢下降。从雌雄蕊形成期到花粉母细胞形成期,不育系的多胺含量和精氨酸脱羧酶活性明显低于保持系;不过,两系二胺氧化酶和多胺氧化酶活性却差别不大。外施D-Arg抑制两系Put合成,也抑制以Put为前体的Spd的合成;外施MGBG抑制Spd和Spm的合成;同时,D-Arg或MGBG对不育系花粉育性影响不大,但明显降低保持系花粉育性,D-Arg＋MGBG对花粉育性的降低效应更强;Put和pd＋Spm可抵消（或部分抵消）D-Arg和MGBG的降低效应。且Put＋Spd＋Spm能使不育系花粉的育性得以轻度恢复。     

Inhibition of Uredospore Germination and Germ Tube Growth by Inhibitors of Polyamine Metabolism in Uromyces phaseoli L

We have studied the effects of two polyamine biosynthetic inhibitors,-difluoromethylor-nithine (DFMO) and -difluoromethylarginine(DFMA), and of polyamines (PAs), alone and in combination onuredospore germination and germ tube growth in Uromyces phaseoliL, race O. Both the inhibitors at concentrations 0.01, 0.1 and1.0 mM produce successively inhibition of uredospore germinationin vitro. The inhibitors also delay the timing of spore germinationfor 15–30 min and restrict germ tube elongation. Stimulationof spore germination and germ tube growth was noticed in culturescontaining PAs (putrescine or spermidine) alone, while culturesfortified with inhibitor plus PA resulted in a partial reversionof the inhibitory effect, suggesting that PAs may be requiredfor normal germination and outgrowth of fungal spores. Sporegermination was completely inhibited on the surface of unifoliolatebean leaves treated with 0.5 mM or higher DFMO 1 d before inoculationwith pathogen, while DFMO treated 1 d after inoculation showedgreater damage of uredosporelings. In contrast, DFMA confersno effect even at 5 mM. Spores collected from bean plants givena pre- and post-inoculatory treatments with DFMO and DFMA showno significant differences in germination and pathogenicity,however, the higher doses caused significant decrease. (Received April 25, 1988; Accepted October 20, 1988)     

The environmental endocrine disruptor, bisphenol A, affects germination, elicits stress response and alters steroid hormone production in kiwifruit pollen

In vitro toxicity of the endocrine disruptor bisphenol A (BPA) to pollen, the male haploid generation of higher plants, was studied. BPA caused significant inhibition of both tube emergence and elongation of kiwifruit pollen in a dose-dependent manner, beginning at 10 mg · l(-1); morphological changes to tubes were also detected. Despite strong inhibition of pollen tube production and growth, a large percentage of treated cells remained viable. Immunoblotting experiments indicated that levels of BiP and 14-3-3, which are proteins involved in stress response, substantially increased in BPA-treated pollen compared to controls. The increases were dose-dependent in the range 10-50 mg · l(-1) BPA, i.e. even when germination ability was completely blocked. Steroid hormones (17 β-estradiol, progesterone and testosterone) were detected in kiwifruit pollen, and their levels increased during germination in basal medium. In a BPA treatment of 30 mg · l(-1), larger increases in both estrogen and testosterone concentrations were detected, in particular, a six-fold increase of 17 β-estradiol over control concentration (30 min). The increased hormone levels were maintained for at least the 90 min incubation. Increasing concentrations of exogenous testosterone and 17 β-estradiol increasingly inhibited pollen tube emergence and elongation. Current data for BPA-exposed kiwifruit pollen suggest a toxicity mechanism that is at least in part based on a dramatic imbalance of steroid hormone production during tube organisation, emergence and elongation. It may be concluded that BPA, a widespread environmental contaminant, can cause serious adverse effects to essential pollen functions. On a broader scale, this chemical poses a potential risk to the reproductive success of higher plants.     

Role of polyamines in adventitious shoot morphogenesis from cotyledons of cucumber <Emphasis Type="Italic">in vitro</Emphasis>

The relationship between polyamines (PAs) metabolism and adventitious shoot morphogenesis from cotyledons of cucumber was investigated in vitro. The endogenous levels of free putrescine (Put) and spermidine (Spd) in the explants decreased sharply, whereas endogenous spermine (Spm) increased during adventitious shoot morphogenesis. The presence of 1–15 mM Put, 1–2 mM Spd, 0.05–1 mM Spm, 5–10 M aminoethoxyvinylglycine (AVG) or 5 M AVG together with 50 M 1-aminocyclopropane-1-carboxylic acid (ACC) in the regeneration medium could promote adventitious shoot formation. Conversely, 1–5 mM D-arginine (D-Arg) or 0.01–0.1 mM methylglyoxal bis-guganylhydrazone (MGBG) inhibited regeneration; and 0.005–0.05 mM ACC displayed little or no evident effects. The explants growing on medium containing 5 M AVG produced higher levels of free Put and Spm, and on medium containing 5 mM Put the explants responded similarly to the AVG-treated explants. However, the exogenous use of 1 mM D-Arg reduced the levels of Put, Spd and Spm, and 0.1 mM MGBG reduced the levels of free Spd and Spm. Moreover, although the explants cultured on medium containing Put and MGBG enhanced ethylene production, AVG and D-Arg inhibited ethylene biosynthesis. This study shows the PAs requirement for the formation of adventitious shoot from cotyledons of cucumber in vitro and the enhanced adventitious shoot morphogenesis may be associated with the elevated level of endogenous free Spm, albeit the promotive effect of PAs on adventitious shoot morphogenesis may not be related to ethylene metabolism.     

The cell wall of kiwifruit pollen tubes is a target for chromium toxicity: alterations to morphology, callose pattern and arabinogalactan protein distribution

Trivalent chromium has previously been found to effectively inhibit kiwifruit pollen tube emergence and elongation in vitro . In the present study, a photometric measure of increases in tube wall production during germination showed that 25 and 50 μ m CrCl₃ treatment induced a substantial reduction in levels of polysaccharides in walls over those in controls. Moreover, chromium-treated kiwifruit pollen tubes had irregular and indented cell walls. Callose, the major tube wall polysaccharide, was deposited in an anomalous punctuate pattern. Arabinogalactan proteins (AGPs), which are integral in maintaining correct tube growth and shape in kiwifruit pollen, were found to be strongly altered in their distribution after CrCl₃ treatment compared to control tube walls. Transmission electron microscopy–immunogold analysis using four monoclonal antibodies (JIM8, JIM13, JIM14 and MAC207) revealed discontinuous AGP distribution within the treated tube walls. Such clearly discernable alterations in the molecular and morphological architecture of pollen tube walls may be detrimental in vivo for the male gametophyte to accomplish its vital role in the fertilisation process.     

Spermidine and methylglyoxal bis(guanylhydrazone) affect maturation and endogenous polyamine content of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and methylglyoxal bis(guanylhydrazone) (MGBG), a putative inhibitor of Spd synthesis, improved somatic embryo formation of Scots pine (Pinus sylvestris L.). The induced maturation due to MGBG and Spd was accompanied by significantly retarded proliferation growth and by reduction in the concentration of free polyamines compared to the control cultures. The action of MGBG revealed that it has a non-specific effect on the whole polyamine metabolism of Scots pine. Furthermore, at certain concentrations it may induce plant differentiation as well.     

Polyamines and ethylene in the removal of embryonal dormancy in apple seeds

Putrescine (Put), spermidine (Spd) and spermine (Spm) were found in seeds of apple ( Malus domestica Borkh. cv. Antonovka), in amounts that increased in the order given. The levels slowly decreased during 30 days of stratification. Exogenous polyamines (PAs) affected germination of isolated embryos in a way dependent on the type of polyamine, its concentration, and the state of the embryo dormancy. The effect of Put and Spd on germination was stimulatory, while that of Spm was inhibitory. Stimulation of germination was also observed when embryos were treated with arginine, ornithine and methionine. Canavanine inhibited germination, and this effect was reversed by arginine or Put. Ethephon, aminooxyacetic acid (AOA) and aminoethoxyvinylglycine (AVG) present during seed stratification had no effect on the levels of endogenous PAs. Put and Spd did not change ethylene production, neither during seed stratification nor during embryo germination, whereas Spm reduced ethylene evolution. The data suggest that Spm plays a role in the maintenance of dormancy by preventing ethylene production, while Put and Spd participate in dormancy removal, independently of ethylene.     

Modulation of SAMDC expression in Arabidopsis thaliana alters in vitro shoot organogenesis

S -Adenosylmethionine decarboxylase (SAMDC, EC 4.1.4.50) is a rate-limiting enzyme in the biosynthesis of polyamines (PAs) from putrescine. To gain more insight into the role of PAs in shoot organogenesis, a reverse genetic approach has been used to study in vitro shoot organogenesis by manipulating SAMDC expression in Arabidopsis . Up- and downregulation of SAMDC expression was achieved by transferring sense, antisense and double-stranded Arabidopsis SAMDC complementary DNA constructs back into Arabidopsis via Agrobacterium tumefaciens . Results show that the biosynthesis of PAs and ethylene is mutually antagonistic by manipulation of SAMDC expression. Further results demonstrate that increased shoot organogenesis seems to be directly related to PA accumulation. This effect of PA may be further enhanced with reduced ethylene. It also suggests that spermidine is involved in the process of acquiring organogenesis competence through downregulation of ethylene production and shoot organogenesis, which might result from the concerted action of PAs and plant hormones such as auxin, cytokinins and ethylene.