Spatial distribution of free and conjugated polyamines in leaves of Solanum melongena L. associated with differential morphogenetic capacity: efficient somatic embryogenesis with putrescine

In eggplant (Solanum melongena L., cv. Pusa Purple Long), explantsfrom different regions of the leaf showed significant differencesfor embryogenic potential. Discs from the apical region of leavesyielded more somatic embryos than those from the basal region.Apical discs showed consistently higher polya-mine titres thanthe basal discs. Putrescine treatment promoted somatic embryogenesisand at 0.5 mM it caused a remarkable increase (c. 6-fold) ina number of somatic embryos, accompanied by an increased putrescinetitre. On the other hand, spermidine and spermine had no stimulatoryeffect on embryogenesis; rather they were inhibitory at higherconcentrations. All tested inhibitors of polyamine biosynthesissuch as difluoromethylarginine, difluoromethylomithine, methylglyoxalbis (guanylhydrazone) and bis (cyclohex-ylammonium) sulphatesignificantly inhibited somatic embryogenesis. Difluoromethylarginineblocked somatic embryogenesis by lowering endogenous polyaminecontents (particularly putrescine) and such inhibitory effectswere totally restored by exogenous putrescine (0.5 mM), concomitantwith the revival of endogenous PA concentrations. These resultsdemonstrate (i) a positive correlation between the spatial distributionof free and conjugated polyamines and the embryogenic capacityof an explant and (ii) putrescine caused the promotion of somaticembryogenesis, suggesting the intricate regulatory role of polyamines,specifically putrescine, in somatic embryogenesis in eggplant. Key words: Solanum melongena, somatic embryogenesis, position effect, polyamines, putrescine, polyamine biosynthesis inhibitors, difluoromethylarginine     

Polyamines affect the cellular growth and structure of pro‐embryogenic masses in Araucaria angustifolia embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines

Polyamines (PAs) are abundant polycationic compounds involved in many physiological processes in plants, including somatic embryogenesis. This study investigates the role of PAs on cellular growth and structure of pro‐embryogenic masses (PEMs), endogenous PA and proton pump activities in embryogenic suspension cultures of Araucaria angustifolia. The embryogenic suspension cultures were incubated with putrescine (Put), spermidine (Spd), spermine (Spm) and the inhibitor methylglyoxal‐bis(guanylhydrazone) (MGBG), respectively (1 mM). After 24 h and 21 days, the cellular growth and structure of PEMs, endogenous PA contents and proton pump activities were analyzed. The addition of Spm reduced the cellular growth and promoted the development of PEMs in embryogenic cultures, which could be associated with a reduction in the activities of proton pumps, such as H⁺‐ATPase P‐ and V‐types and H⁺‐PPases, and alterations in the endogenous PA contents. Spm significantly affected the physiology of the A. angustifolia somatic embryogenesis suspension, as it potentially affects cellular growth and structure of PEMs through the modulation of proton pump activities. This work demonstrates the involvement of exogenous PAs in the modulation of cellular growth and structure of PEMs, endogenous PA levels and proton pump activities during somatic embryogenesis. To our knowledge, this study is the first to report a relationship between PAs and proton pump activities in these processes. The results obtained in this study offer new perspectives for studies addressing the role of PAs and proton pump on somatic embryogenesis in this species.     

Changes in endogenous polyamines during the formation of somatic embryos from isogenic lines of Dactylis glomerata L. with different regenerative capacities

The endogenous levels of polyamines (PAs) in leaf-base explants isolated from plants of two isogenic lines of Dactylis glomerata L., differing in their competence for somatic embryogenesis, were compared. Leaf-bases isolated from plants with a high level of competence for somatic embryogenesis (HEC) contained four times the level of polyamines compared to those isolated from plants with a low level of competence for somatic embryogenesis (LEC). When the levels of individual polyamines in the HEC and LEC lines were compared, leaf-bases from plants of the HEC line had much lower PUT/SPD ratios than those from the LEC line. When changes in the levels of PAs were monitored during the first 28 d of culture, on a medium which promotes initiation of somatic embryogenesis, leaf-base cultures from plants of the HEC line showed a 50% increase in the levels of PAs during the first 7 d of culture, after which time levels began to decline. By day 21, levels had dropped below those found in freshly isolated leaf bases. While PUT and SPM levels increased by about 30%, the greatest increase was shown by SPD, which increased by more than 100% during the first 7 d of culture, before declining. In contrast much smaller changes in PA levels were found when leaf-bases from plants of the LEC line were cultured.     

Somatic embryogenesis of holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.): ethylene production and polyamine content

The production of ethylene and the endogenous content of polyamines (PAs) have been recorded during the early development, maturation and germination of holm oak (Quercus ilex L.) somatic embryos. Ethylene production was high in embryogenic callus, immature somatic embryos and in explants showing secondary embryogenesis, while it was lower in mature and germinating somatic embryos. A higher ethylene production was also associated to the process of secondary embryogenesis. The exogenous application of 1-amino-1-cyclohexane carboxylic acid was not significantly effective on the production of ethylene by holm oak somatic embryos. Total PAs were more abundant in embryogenic callus and in both somatic and zygotic immature embryos, decreasing later on in the mature and germination phases. Immature somatic embryos of holm oak and immature zygotic embryos contain high levels of spermidine (Spd), which decreased during maturation and germination. Spermine (Spm) concentration was lower than that of Spd. Spm was more abundant in embryogenic callus and immature zygotic embryos than in mature embryos. Ethylene production did not seem to interfere with PA metabolism.     

Effect of polyamines on in vitro somatic embryogenesis in <Emphasis Type="Italic">Momordica</Emphasis><Emphasis Type="Italic">charantia</Emphasis> L.

The effects of exogenous polyamines (PAs) on enhancement of somatic embryogenic calli was investigated in Momordica charantia L. in vitro. Induction of somatic embryogenesis (SE) in leaf explants of M. charantia after 21 days of culture in Murashige and Skoog (MS) medium was determined using scanning electron microscopy. During induction of SE there were high titers of Putrescine (Put) as compared to Spermidine (Spd) and Spermine (Spm), a prerequisite for cell division. Addition of PAs to the embryogenic media resulted in an increase in fresh weights and number of somatic embryos of 21-day old embryogenic calli. Put at a concentration of 1 mM showed maximum increase in fresh weights of embryogenic calli (5 fold) and number of somatic embryos produced per 0.2 g of callus (2.5 fold). Moreover addition of PAs to the embryogenic media resulted in lowering of endogenous free PA level of 21-day old embryogenic calli. Thus, when the media was supplemented with exogenous PAs a positive correlation was found to exist between Somatic Embryogenesis enhancement and decrease in endogenous free PA levels.     

Polyamines as biomarkers for plant regeneration capacity: improvement of regeneration by modulation of polyamine metabolism in different genotypes of indica rice

The importance of cellular polyamine (PA) levels and the ratio of putrescine (Put) to spermidine (Spd) for plant regeneration ability via somatic embryogenesis in several commercially grown indica rice varieties is reported here. The genotypes namely NDR-624, IR-20, IR-36, BJ-1 (having Put:Spd ratio2.3) showed superior plant regeneration while KL, PB-1 and TN-1 (having Put:Spd ratio3.8) showed moderate plant regeneration ability. The genotypes namely HS, Bindli, DV-85, ACB-72, IR-64 and IR-72 (having Put:Spd ratio5.0) showed poor plant regeneration ability. In contrast KH-7 (Put:Spd ratio10.0) showed no response at all. Favorable modification of cellular PA titers and their Put:Spd ratio by the addition of exogenous PAs (Put, Spd) or their biosynthesis inhibitor, difluoromethylarginine (DFMA) led to the induction/promotion of plant regeneration in poorly responding genotypes. These results showed a close relationship between cellular PA levels and their Put:Spd ratio with in vitro morphogenetic capacity in indica rice and suggest that the cellular PAs and Put:Spd ratios are important determinants (biomarkers) of plant regeneration ability in indica rice, and the improvement/induction of plant regeneration in morphogenetically poor and recalcitrant species could be achieved by modulating PA metabolism.     

Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture

Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G₁ phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G₁ to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs.     

Photocontrol of somatic embryogenesis and polyamine content in Araujia sericifera petals

The effects of photoperiod and end-of-day phytochrome control on somatic embryogenesis and polyamine (PA) content in Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 16- and 8-h photoperiods. Far red (FR), red (R) and FR followed by R light treatments were applied at the end of the photoperiods for three weeks. The number of somatic embryos, callus weight and the levels of free and bound PAs in the cultured petal explants were determined 40 days after the beginning of light treatments. Long day (LD) promoted somatic embryogenesis but did not have any significant effect on PA content. Short day (SD) reduced somatic embryogenesis and enhanced total PAs, mainly in the form of bound spermidine. End-of-day FR treatment increased PA content and inhibited somatic embryogensis under LD but had no significant effect under SD. This effect of FR on PA levels was cancelled by R and was independent of the presence of silver thiosulphate in the medium. End-of-day R treatment reduced the total PA content under SD. However, end-of-day R increased or reduced somatic embryogenesis under SD depending on the presence or absence of silver in the medium. The results suggest a photoperiodic control of somatic embryogenesis and PA content in A. sericifera. The effects of end-of-day R and FR treatments depend on the length of the photoperiod. This finding and the FR/R photoreversibility of end-of-day treatments indicate that phytochrome may be involved in both somatic embryogenesis and accumulation of PA.     

Efficient plant regeneration from long-term callus cultures of rice by spermidine

A significant reduction in regeneration potential with increasing age (upto 12months) in rice (Oryza sativa L. cv.TN-1) embryogenic callus cultures was observed. Spermidine, while having an inhibitory effect on plant regeneration in fresh callus cultures, promoted morphogenesis in long-term callus cultures. A massive accumulation of polyamines, particularly putrescine (5-fold) was observed in 12 month old cultures resulting in a change of putrescine /spermidine ratio, which seems to be important for maintaining the morphogenetic response. Application of exogenous spermidine to 12 month old cultures showed increased levels of polyamines and restored the putrescine/spermidine ratio comparable to that found in freshly induced cultures, concomitantly, promoting the plant regeneration via somatic embryogenesis in long-term rice callus cultures.Abbreviations PA Polyamines - PCA Perchloric acid - PUT Putrescine - SPD Spermidine - SPM Spermine     

UV-B radiation induces changes in polyamine metabolism in the red seaweed <Emphasis Type="Italic">Porphyra cinnamomea</Emphasis>

Early investigations on the productivity of intertidal seaweeds found that, unlike some seaweeds, members of the genus Porphyra, a Rhodophyte, could tolerate physical stressors such as ultraviolet-B radiation (UV-B) both during immersion and when exposed to air. Increased stress tolerance was thought to be due to an unknown mechanism that operated at the thylakoid level. As recent research has shown that polyamines (PAs), bound to the thylakoid membranes of chloroplasts, play a critical role in protecting the photosynthetic apparatus from high-light and UV damage in both higher plants and in unicellular algae, we investigated PA metabolism in Porphyra cinnamomea exposed to UV-B. Our results show that PA biosynthesis was significantly upregulated in P. cinnamomea in response to UV-B, with the greatest proportional increases being in bound soluble putrescine (PUT), which increased by over 200%, in bound soluble spermidine (SPD) and spermine (SPM) which both increased by more than 150% and in bound insoluble SPM which increased by more than 120%. As PAs can be synthesised from ornithine via ornithine decarboxylase (ODC) or from arginine via arginine decarboxylase (ADC) we investigated the pathway via which polyamines were synthesised in P. cinnamomea. While exposure to UV-B caused increases in the activities of both ADC and ODC, the increase in ADC activity was 10 fold greater than that of ODC, suggesting that the ADC pathway was the principle route by which PA levels increased in response to UV-B. Mechanisms of PA mediated UV-B protection are discussed.     

Somatic embryogenesis in saffron (<Emphasis Type="Italic">Crocus sativus</Emphasis> L.). Histological differentiation and implication of some components of the antioxidant enzymatic system

The ontogenetic developmental stages of saffron somatic embryogenesis have been studied and characterized using light microscopy and the biochemical determination of the antioxidant enzymatic system. The embryogenic callus underwent internal segmented divisions with the formation of globular embryos that were attached to the callus surface by a broad multicellular structure. Further development of the embryoids was characterized by the emergence of a shoot apical meristem and cotyledon (monopolar stage) with the subsequent differentiation of a minicorm at the basal part of the somatic embryo (dipolar stage). During the morphological differentiation of the somatic embryos changes in the antioxidant enzymatic system with increased superoxide dismutase (SOD) and catalase (CAT) activities were detected at the initial stages of somatic embryogenesis. The isoforms of SOD, including two Mn-SODs and four Cu, Zn-SODs, were also detected. Although all the isoforms were expressed during the successive stages of somatic embryogenesis, an increase in Mn-SODs and a decrease in Cu, Zn-SODs during the last two stages was observed. Significant changes were also detected in the antioxidant activities ascorbate peroxidase, dehydroascorbic acid reductase and glutathione reductase.     

Early Cellular Events During Direct Somatic Embryogenesis in Cotyledon Explants of Solanum aviculare Forst.

A detailed light and electron microscope study of early cellularevents at the onset of somatic embryogenesis in cotyledon explantsof Solanum aviculare Forst., cultured on MS medium supplementedwith 1 mg l^–1 2,4-dichloro-phenoxyacetic acid (2,4-D)for periods of 0–12 d in darkness, is described. Examinationsof longitudinal sections in a plane offset from the centralveins indicated that the earliest embryogenic events in explantstook place within the first 3–4 d of culture in the parenchymacells associated with the vascular traces closest to the cutbasal ends of cotyledons. Thereafter, parenchyma associatedwith more distal vascular traces became active in an apparentlysequential manner such that, by the second week of culture,progressive stages of embryogenesis could be observed alongthe lengths of cotyledon sections. Despite the fact that epidermalcells and palisade tissues were exposed directly to the 2,4-Dmedium, initiation of embryogenic development was never observedin cells other than those directly associated with vasculartraces. None of the embryogenic events characterized at theultrastructural level were observed in cotyledons cultured onMS medium in the absence of 2,4-D with the exception that starchaccumulated in decreasing amounts from the wounded basal endto the distal end of each cotyledon. This system provides a valuable model with which to study earlybiochemical and molecular events occurring in explants duringthe onset of somatic embryogenesis because they occur in a predictablefashion at sequentially situated sites along the explant tissues. Somatic embryogenesis, Solanum aviculare, cotyledon explants, cellular changes     

Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture

Polyamine levels and the activities of two polyamine biosynthetic enzymes, arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50), were determined during somatic embryogenesis of carrot (Daucus carota L.) cell cultures. Embryogenic cultures showed severalfold increases in polyamine levels over nondifferentiating controls. A mutant cell line that failed to form embryos but grew at the same rate as the wild-type line also failed to show increases in polyamine levels, thus providing evidence that this increased polyamine content was in fact associated with the development of embryos. Furthermore, inhibition of these increases in polyamines caused by drugs inhibited embryogenesis and the effect was reversible with spermidine. The activities of arginine decarboxylase and Sadenosylmethionine decarboxylase were found to be suppressed by auxin; however, the specific effects differed between exogenous 2,4-dichlorophenoxyacetic acid and endogenous indole-3-acetic acid. The results indicate that increased polyamine levels are required for cellular differentiation and development occurring during somatic embryogenesis in carrot cell cultures.Abbreviations ADC arginine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DCHAS dicyclohexylammonium sulfate - SAMDC S-adenosylmethionine decarboxylase     

Polyamines and cytokinins in celery embryogenic cell cultures

The effect of inhibitors of polyamine biosynthesis on the development of embryogenic cell cultures of celery (Apium graveolus L.) was studied. Several developmental stages of somatic embryos were compared for differences in the content and biosynthesis of free polyamines and for cytokinin content. Cyclohexylamine and particularly methylglyoxal bis(guanylhydrazone), inhibited both cell division and the organization of polar embryos from globular embryos. Difluoromethylornithine slightly promoted embryo development, especially cell division.The free putrescine content of globular embryos was 6-fold that of fully differentiated plantlets, and that of spermidine 2-fold. Only a slight increase in the spermine content was found with embryo development. These differences were confirmed by data from polyamine biosynthesis. Incorporation of ¹⁴C-arginine into polyamines was slightly higher than that of ¹⁴C-ornithine. Over 96% of this incorporation was detected in the putrescine fraction. Incorporation of ¹⁴C into putrescine in globular embryos was 3 to 4-fold that in fully-differentiated plantlets. Incorporation into spermidine and spermine was, however, higher in plantlets than in globular embryos.Cytokinin analysis revealed considerable differences in the biological activity between the developmental stages of embryogenesis. This could be due to endogenous cytokinins and/or BA taken up from the maintenance medium. Cytokinin levels decreased with increased embryo development. Most of the detected cytokinin-like activity co-chromatographed with BA and its metabolites. Some as yet unidentified peaks of activity were recorded in the globular embryos.The results are considered with respect to the possible participation of polyamines and cytokinins in the development of embryogenic cell cultures of celery. It is suggested that the onset of embryogenesis is characterized by a high content of putrescine and cytokinins, while a decrease in putrescine synthesis and cytokinin content, and an increase in spermidine and spermine content, accompany further embryo development and plantlet formation.Abbreviation ADC arginine decarboxylase - ODC ornithine decarboxylase - 2,4-D dichlorophenoxyacetic acid - DFMA difluoromethylarginine - DFMO difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone) - CHA cyclohexylamine - BA benzyladenine - BAR benzyladenine riboside     

Regulation of polyamine metabolism in Pyropia cinnamomea (W.A. Nelson), an important mechanism for reducing UV‐B‐induced oxidative damage

It is generally accepted that ultraviolet (UV) radiation can have adverse affects on phototrophic organisms, independent of ozone depletion. The red intertidal seaweed Pyropia cinnamomea W.A. Nelson (previously Porphyra cinnamomea Sutherland et al. 2011), similar to many other intertidal macrophytes, is exposed to high levels of UV radiation on a daily basis due to emersion in the upper littoral zone. It has been shown that seaweeds, like higher plants, respond to an increased activity of antioxidative enzymes when exposed to stress. However, earlier investigations have shown that P. cinnamomea also compensates for stress due to UV radiation by increasing polyamine (PA) levels, especially bound‐soluble and bound‐insoluble PAs. The PA precursor putrescine (PUT) can be synthesized via two enzymatic pathways: arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). Both of these enzymes showed increased activity in P. cinnamomea under UV stress. In higher plants, ADC is the enzyme responsible for increased PA levels during stress exposure, while ODC is correlated with cell division and reproduction. However, there are contrary findings in the literature. Using two irreversible inhibitors, we identified the enzyme most likely responsible for increased PUT synthesis and therefore increased stress tolerance in P. cinnamomea. Our results show that changes in the PA synthesis pathway in P. cinnamomea under UV stress are based on an increased activity of ADC. When either inhibitor was added, lipid hydroperoxide levels increased even under photosynthetically active radiation, suggesting that PAs are involved in protection mechanisms under normal light conditions as well. We also show that under optimum or low‐stress conditions, ODC activity is correlated with PUT synthesis.     

Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana

In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.     

ABA对枸杞体细胞胚发生的调节作用

Using Enzyme Linked Immunosorbent Assay (ELISA) method, we determined the ABA contents of different stages in somatic embryogenesis. The results showed that endogenous ABA contents increased to maximum value twice during somatic embryogenesis. After first maximum value of ABA contents embryogenic cells were observed in callus, and simultaneously, there was a specific protein of somatic embryogenesis investigated by SDS-PAGE. This protein accumulates preferentially in embryogenic callus but not in transferred callus. So it is suggested that ABA could promote the expression of specific genes and the synthesis of embryogenic protein during somatic embryogenesis in Lycium barbarum L. and ABA play an important role in globular stage as well. In addition, treatment of non-embryogenic activity callus with 4 mumol/L exogenous ABA could stimulate somatic embryogenesis. And the ABA function mechanism in relation to somatic embryogenesis was discussed.