Role of polyamines in regulating silymarin production in Silybum marianum (L.) Gaertn (Asteraceae) cell cultures under conditions of calcium deficiency

As part of our efforts to identify the possible role of polyamines (PAs) in silymarin (Sm) production, the effects of calcium deprivation on cell growth and on endogenous PAs levels and Sm production by milk thistle (Silybum marianum (L.) Gaertn) grown in cell cultures were examined. Young cultured cells of the H2 line of S. marianum were transferred to a medium without calcium and with ethylene glycol-bis-(β-aminoethyl) ether-N,N,N′,N′-tetraacetic acid present to chelate any free calcium in order to analyze the effects of this medium on the levels of PAs and Sm produced by the cells. During the 17 days of exposure to this calcium-free medium most of the cell populations were in the G0/G1 phase (from day 7 to day 14 of culture) while PA levels underwent a progressive decline up to day 17, after which they were no longer detectable. We observed that putrescine (Put) accumulation was always lower than that observed under normal conditions. The lack of calcium in the MS medium advances the onset of the stationary phase, whose beginning is marked by an increase in the Put/spermidine (Spd) index, raising the production of Sm; the suspensions were productive for a longer time and hence produced more of the substance. Our results indicate that under stress conditions the production of Sm in young-cell suspensions of S. marianum is not associated with high levels of PAs in the medium – contrary to what one would expect – allowing us to conclude that growth inhibition appears to be the factor responsible for the maximum Sm accumulation while PAs are not directly involved in the Sm synthesis pathway by milk thistle grown in culture.     

Drought-inhibited ribulose-1,5-bisphosphate carboxylase activity is mediated through increased release of ethylene and changes in the ratio of polyamines in pakchoi

To study the mechanisms of drought inhibiting photosynthesis and the role of PAs and ethylene, the photosynthetic rate (P_n), the maximal photochemical efficiency of PSII (F_v/F_m), the intercellular CO₂ concentration (C_i), photorespiratory rate (P_r), the amount of chlorophyll (chl), antioxidant enzyme activity, ethylene levels, RuBPC (ribulose-1,5-bisphosphate carboxylase) activity and endogenous polyamine levels of pakchoi were examined, and an inhibitor of S-adenosylmethionine decarboxylase (SAMDC) and an inhibitor of ethylene synthesis and spermidine (Spd) were used to induce the change of endogenous polyamine levels. The results show that drought induced a decrease in P_n and RuBPC activity, an increase in the intercellular CO₂ concentration (C_i), but no change in the actual photochemical efficiency of PSII (Φ_PSII), and chlorophyll content. In addition, drought caused an increase in the free putrescine (fPut), the ethylene levels, a decrease in the Spd and spermine (Spm) levels, and the PAs/fPut ratio in the leaves. The exogenous application of Spd and amino oxiacetic acid (AOAA, an inhibitor of ethylene synthesis) markedly reversed these drought-induced effects on polyamine, ethylene, P_n, the PAs/fPut ratio and RuBPCase activity in leaves. Methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of SAMDC resulting in the inability of activated cells to synthesize Spd and Spm, exacerbates the negative effects induced by drought. These results suggest that the decrease in P_n is at least partially attributed to the decrease of RuBPC activity under drought stress and that drought inhibits RuBPC activity by decreasing the ratio of PAs/fPut and increasing the release of ethylene.     

Prevention of thermal aggregation of an allosteric protein by small molecules: some mechanistic insights

Detailed knowledge of conformation and dynamics of native, intermediate and unfolded states of a protein is essential in searching for effective small molecules to prevent its aggregation. In a recent study we have demonstrated how allosteric effectors may influence protein-protein interactions at high temperatures using glutamate dehydrogenase (GDH) as a model allosteric protein. In the present study, thermal aggregation of this well-characterized enzyme was investigated in the presence of a number of amino acids (including Gly, Glu, Trp, Pro, Lys, Arg), polyamines (putrescine and spermidine) and chaperone-like molecules (cyclodextrins and caseins) as non-specific effectors. It was shown that some amino acids and polyamines may suppress aggregation via interaction with native species and may preserve the activity of the enzyme while cyclodextrins and caseins may exert their anti-aggregation potential via binding to aggregation-prone intermediates, without having any capacity to protect its native structure from unfolding. Observations describing the similarities and differences between the specific ligands and non-specific small molecules related to their interaction with native and aggregation-prone states of GDH are presented and discussed. It is argued that the type of studies described in the present communication is useful for the development of effective strategies for prevention of aggregation by small molecules.     

Polyamine catabolism is involved in response to salt stress in soybean hypocotyls

The possible relationship between polyamine catabolism mediated by copper-containing amine oxidase and the elongation of soybean hypocotyls from plants exposed to NaCl has been studied. Salt treatment reduced values of all hypocotyl growth parameters. In vitro, copper-containing amine oxidase activity was up to 77-fold higher than that of polyamine oxidase. This enzyme preferred cadaverine over putrescine and it was active even under the saline condition. On the other hand, saline stress increased spermine and cadaverine levels, and the in vivo copper-containing amine oxidase activity in the elongation zone of hypocotyls. The last effect was negatively modulated by the addition of the copper-containing amine oxidase inhibitor N,N'-diaminoguanidine. In turn, plants treated with the inhibitor showed a significant reduction of reactive oxygen species in the elongation zone, even in the saline situation. In addition, plants grown in cadaverine-amended culture medium showed increased hypocotyl length either in saline or control conditions and this effect was also abolished by N,N'-diaminoguanidine. Taken together, our results suggest that the activity of the copper-containing amine oxidase may be partially contributing to hypocotyl growth under saline stress, through the production of hydrogen peroxide by polyamine catabolism and reinforce the importance of polyamine catabolism and hydrogen peroxide production in the induction of salt tolerance in plants.     

Salt stress impact on the molecular structure and function of the photosynthetic apparatus—The protective role of polyamines

In the present study the green alga Scenedesmus obliquus was used to assess the effects of high salinity (high NaCl-concentration) on the structure and function of the photosynthetic apparatus and the possibility for alleviation by exogenous putrescine (Put). Chlorophyll fluorescence data revealed the range of the changes induced in the photosynthetic apparatus by different NaCl concentrations, which altogether pointed towards an increased excitation pressure. At the same time, changes in the levels of endogenous polyamine concentrations, both in cell and in isolated thylakoid preparations were also evidenced. Certain polyamine changes (Put reduction) were correlated with changes in the structure and function of the photosynthetic apparatus, such as the increase in the functional size of the antenna and the reduction in the density of active photosystem II reaction centers. Thus, exogenously added Put was used to compensate for this stress condition and to adjust the above mentioned changes, so that to confer some kind of tolerance to the photosynthetic apparatus against enhanced NaCl-salinity and permit cell growth even in NaCl concentrations that under natural conditions would be toxic.     

Enhancement of spermidine content and antioxidant capacity in transgenic pear shoots overexpressing apple spermidine synthase in response to salinity and hyperosmosis

In our previous work, an apple spermidine synthase (SPDS)-overexpressing transgenic European pear (Pyrus communis L. 'Ballad'), line no. 32 (#32), demonstrated attenuated susceptibility to stress treatment. In the current paper, changes in enzymatic and non-enzymatic antioxidant capacity of the transgenic pear (line #32) were investigated in response to NaCl or mannitol stress. Under non-stressed conditions (before stress treatment), spermidine (Spd) contents and SPDS activity of line #32 were higher than those of the non-transformant (wild type). However, no significant differences were detected between line #32 and the wild type as regards contents of malondialdehyde (MDA) and H2O2, and activities of antioxidant enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). When exposed to NaCl or mannitol stress, both the wild type and line #32 exhibited accumulation of Spd with the latter accumulating more. The transgenic line contained higher antioxidant enzyme activities, less MDA and H2O2 than the wild, implying it suffered from less injury. These results suggested that increase of Spd content in the transgenic line could, at least in part, lead to enhancing enzymatic and non-enzymatic antioxidant capacity.     

Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development

Arginine decarboxylase (ADC) is a rate-limiting enzyme that catalyzes the first step of polyamine (PA) biosynthesis in Arabidopsis thaliana. We generated a double mutant deficient in Arabidopsis two ADC genes (ADC1-/- ADC2-/-) and examined their roles in seed development. None of the F2 seedlings from crosses of adc1-1 and adc2-2 had the ADC1-/- ADC2-/- genotype. In addition, some abnormal seeds were observed among the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- siliques. Viable offspring with the ADC1-/- ADC2-/- genotype could not be obtained from the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- plants. These results indicate that AtADC genes are required for production of polyamines that are essential for normal seed development in Arabidopsis.     

Spermidine alleviates the growth of saline-stressed ginseng seedlings through antioxidative defense system

Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150 mM NaCl) for 7 d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1 mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1 mM Spd concentrations, but a lower concentration (0.01 mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress.     

Remodeling of tobacco thylakoids by over-expression of maize plastidial transglutaminase

Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing ?-(γ-glutamyl) links and covalent conjugation of polyamines. In chloroplast it is well established that TGases specifically polyaminylate the light-harvesting antenna of Photosystem (PS) II (LHCII, CP29, CP26, CP24) and therefore a role in photosynthesis has been hypothesised (Della Mea et al. [23] and refs therein). However, the role of TGases in chloroplast is not yet fully understood. Here we report the effect of the over-expression of maize (Zea mays) chloroplast TGase in tobacco (Nicotiana tabacum var. Petit Havana) chloroplasts. The transglutaminase activity in over-expressers was increased 4 times in comparison to the wild-type tobacco plants, which in turn increased the thylakoid associated polyamines about 90%. Functional comparison between Wt tobacco and tgz over-expressers is shown in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both in vivo probing and electron microscopy studies verified thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIα) at the expense of centers located mainly in stroma thylakoids (PSIIβ). A major increase in the granum size (i.e. increase of the number of stacked layers) with a concomitant decrease of stroma thylakoids is reported for the TGase over-expressers.     

盐适应过程中香根草内源游离态、结合态、束缚态多胺含量的变化

研究了不同浓度NaCl胁迫下,香根草（Vetiteria zizanioides）根、叶中的游离态、结合态、束缚态多胺（PAs）[包括腐胺（Put）,尸胺（Cad）,亚精胺（Sod）和精胺（Spm）]含量的变化。在中度盐胁迫（100,200mmol L^-1NaCl）9天时,香根草基本能够正常生长,但在重度盐胁迫（300mmol L^-1NaCl）下,其生长受到严重抑制。在上述3个不同浓度的NaCl胁迫下,香根草根、叶中游离态Put,Cad,spd,Stma和总的游离态PAs含量明显下降,在高盐浓度下下降幅更大;结合态Put,Cad,Sod,Spm和总的结合态PAs含量显著上升,但在重度盐胁迫下升幅较小或与对照相当;束缚态Put,Cad和总的束缚态PAs含量均减少,而束缚态Spd和Spm含量在叶中是下降的,在根中则增加,且在中度盐胁迫下更明显。对根和叶片而言,除游离态（Spd＋Spm）,Put比值在重度盐胁迫下较对照显著下降外,其它游离态、结合态、束缚态和总的（Spd＋Spm）／Put比值在不同盐胁迫下均上升,在中度盐胁迫下更明显。这表明,维持多胺总量的稳态和较高的（Spd＋Spm）／Put比值是香根草适应中度盐胁迫的一个重要机制。