Effects of light intensity on leaf photosynthetic characteristics,chloroplast structure,and alkaloid content of <Emphasis Type="Italic">Mahonia bodinieri</Emphasis> (Gagnep.) Laferr.

The variation of light intensity has obvious effects on leaf external morphology, internal anatomy, and physiological characteristics; it even induces changes in secondary metabolite production. The effects of different irradiance levels on biomass, gas exchange parameters, and photosynthetic pigment contents in Mahonia bodinieri (Gagnep.) Laferr. were analyzed here. Combined analyses of physiology, cytology, and HPLC were used to study the differences in leaf morphology, structure, physiological characters, and alkaloid content in response to different irradiances. The results indicated that the highest foliar biomass was observed under I ₅₀ (50 % of full sunlight) followed by I ₃₀ (30 % of full sunlight), the highest net photosynthetic rate, stomatal conductance, transpiration rate values were observed under I ₃₀ followed by I ₅₀, and lower values occurred in I ₁₀ (10 % of full sunlight) and I ₁₀₀ (full sunlight). With increased light intensity, total leaf area and the contents of chlorophyll a (Chl a), chlorophyll b (Chl b), and chlorophyll (Chl a+b) per unit leaf area were clearly reduced, whereas leaf mass per area, carotenoid content, leaf thickness, thickness of palisade and spongy parenchyma, and stomatal density were all significantly increased. Electron microscopic observation revealed that the number of grana, stroma lamellae and the number of starch grains in chloroplasts were decreased, the number of plastoglobuli was increased when irradiance levels increased. The estimated total yield of alkaloids in a single plant was higher under I ₃₀ and I ₅₀ than under I ₁₀ or I ₁₀₀ as a result of the higher biomass of the plants. Therefore, I ₃₀ and I ₅₀ were not only beneficial to increase biomass, but also suitable for the synthesis and accumulation of the major secondary metabolites (alkaloids). Our findings provide valuable data for the determination and regulation of irradiance levels during artificial cultivation of M. bodinieri.     

Regulation of global protein translation and protein degradation in aerobic dormancy

We hypothesized that protein turnover would be substantially suppressed during estivation in the land snail, Otala lactea, as part of a wholesale move to conserve ATP in the hypometabolic state, and that decreased rates of protein synthesis and degradation would be mediated by altering the phosphorylation state of key proteins. Rates of protein translation, measured in vitro, decreased by ~80% in extracts of foot muscle and hepatopancreas after 2 days of estivation, and this reduction was associated with strong increases in the phosphorylation of ribosomal factors, eIF2α and eEF2, as well as decreased phosphorylation of 4E-BP1. Reductions in levels of markers of ribosomal biogenesis and a tissue-specific reduction in the phosphorylation state of eIF4E and eIF4GI were also evident after 14 days of estivation. Activity of the 20S proteasome decreased by 60–80% after 2 days of estivation and this decrease was mediated by protein kinase G in vitro, whereas protein phosphatase 2A activated the proteasome. Levels of protein carbonyls did not change in snail tissues during estivation whereas the expression heat shock proteins increased, suggesting that protein resistance to damage is enhanced in estivation. In conclusion, protein synthesis and degradation rates were coordinately suppressed during estivation in O. lactea and this is associated with the phosphorylation of ribosomal initiation and elongation factors and the 20S proteasome.     

Population genetic structure of <Emphasis Type="Italic">Helix pomatia</Emphasis> L. (Mollusca,Pulmonata) from the southeastern and eastern parts of the range

On the basis of the analysis of genetic variation detectable by ISSR-PCR, the state of the gene pools of 14 populations of Roman snail Helix pomatia L. in the conditions of urbanized landscapes of the southeastern and eastern parts of the current range was examined. According to the data obtained, the majority of the studied populations of this mollusk are in satisfactory condition. This is evidenced by the increased level of genetic diversity (H _e = 0.199 ± 0.025, I _sh = 0.306 ± 0.035) and the high values of effective population size, calculated, on the basis of the straight-line regression equation, between the pairwise genetic and geographic distances (Ne = 2.0–4.9) that are comparable with indigenous common species of terrestrial mollusks. Despite the high level of differentiation (G_st = 0.255, Φ_st = 0.233, N _m = 0.822), the population distribution was not random (R _m =–0.591, p = 0.0004) and corresponded to the model of isolation by distance. It is hypothesized that, in the adventitious colonies of this mollusk, effective formation of a balanced genetic structure takes place that, in the context of biological and ecological features, facilitates its adaptation to the conditions of an urban environment and the population of the new territories of Eastern Europe.     

Overexpression of <Emphasis Type="Italic">IrlVHA</Emphasis>-<Emphasis Type="Italic">c</Emphasis>, A Vacuolar-Type H<Superscript>+</Superscript>-ATPase c Subunit Gene from <Emphasis Type="Italic">Iris lactea</Emphasis>, Enhances Salt Tolerance in Tobacco

Vacuolar-type H⁺-ATPase (V-ATPase), a multi-subunit endomembrane proton pump, plays an important role in plant growth and response to environmental stresses. In the present study, transgenic tobacco that overexpressed the V-ATPase c subunit gene from Iris lactea (IrlVHA-c) was used to determine the function of IrlVHA-c. Quantitative PCR analysis showed that IrlVHA-c expression was induced by salt stress in I. lactea roots and leaves. Subcellular localization of green fluorescent protein (GFP) as marker combined with FM4-64 staining showed that the IrlVHA-c-GFP was localized to the endosomal compartment in tobacco cells. Compared with the wild-type, the IrlVHA-c transgenic tobacco plants exhibited greater seed germination rates, root length, fresh weight, and higher relative water content (RWC) of leaves under salt stress. Furthermore, the IrlVHA-c transgenic tobacco leaves have lower stomatal densities and larger stomatal apertures than wild-type. Under salt stress, superoxide dismutase (SOD) activity in the transgenic tobacco was significantly enhanced. Moreover, the level of malondialdehyde (MDA) in the transgenic tobacco was significantly lower than that in wild-type plants under salt stress. Taken together, these results suggested that the IrlVHA-c plays an important role in salt tolerance in transgenic tobacco by influencing stomatal movement and physiological changes.     

Performance of multi-trait genomic selection for <Emphasis Type="Italic">Eucalyptus robusta</Emphasis> breeding program

In forest tree genetic improvement, multi-trait genomic selection (GS) may have advantages in improving the accuracy of the genotype estimation and shortening selection cycles. For the breeding of Eucalyptus robusta, one of the most exotic planted species in Madagascar, volume at 49 months (V49), total lignin (TL), and holo-cellulose (Holo) were considered. For GS, 2919 single nucleotide polymorphisms (SNP) were used with the genomic best linear unbiased predictor (GBLUP) method, which was as efficient as the reproducing kernel Hilbert space (RKHS) and elastic net methods (EN), but more adapted to multi-trait modeling. The efficiency of individual I model, including the genomic data, was much higher than the provenance effect P model. For example, with V49, mean goodness-of-fit was: r_{I_Full} =?0.79, r_{P_Full} =?0.37 for I and P, respectively. The prediction accuracies using the cross-validation procedure were lower for V49: r_I =?0.29 r_P =?0.28. The genetic gains resulting from the indexes associating (V49, TL) and (V49, Holo) were higher using I than for the P model; for V49, the relative genetic gain was 37 and 20%, respectively, with 5% of selection intensity. The single-trait approach was as efficient as the multi-trait approach given the weak correlations between V49 and TL or Holo. The I model also brings greater diversity: for V49 the number of provenances represented in a selected population was two and three with the P model, and 6 and 16 with the I model.     

The regulation of thapsigargin-sensitive sarcoendoplasmic reticulum Ca<Superscript>2+</Superscript>-ATPase activity in estivation

Estivation (aerobic dormancy) is characterized by sustained metabolic rate depression, which is crucial to survival in the face of unfavorable environmental conditions and enables the preservation of endogenous fuel reserves. Ion pumping is one of the most energetically taxing physiological processes in cells, and ion motive ATPases are likely loci to be differentially regulated in models of metabolic arrest. We proposed that the sarcoendoplasmic reticulum (SER) calcium-ATPase (SERCA) would be deactivated in the estivating desert snail Otala lactea, potentially contributing to the overall suppression of metabolism. SERCA kinetic parameters [decreased maximal velocities, increased substrate K _m values, increased Arrhenius activation energy (E _a)] were indicative of a less active enzyme in the estivated state. Interestingly, the less active SERCA population in dormant snails featured greater kinetic (K _m Mg.ATP versus temperature) and conformational (resistance to urea denaturation) stability than that in active snails. Western blotting confirmed that SERCA protein content did not change during estivation. In light of this observation, we proposed that estivation-dependent changes in SERCA activity was due to changes in SERCA phosphorylation state. In vitro studies promoting specific kinase or phosphatase action indicated that decreased SERCA activity in estivation was linked with endogenous kinase activity whereas reactivation of SERCA was facilitated by endogenous protein phosphatases (PP).     

Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail <Emphasis Type="Italic">Theba pisana</Emphasis> in relation to genetic structure

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.     

Species divergence in <Emphasis Type="Italic">Iris</Emphasis> series <Emphasis Type="Italic">Lacteae</Emphasis> (Iridaceae) in Russia and adjacent countries based on chloroplast DNA sequence data

In Russia, the species of the genus Iris L., series Lacteae Doronkin, the taxonomic structure of which still remains controversial, are found in the south of Siberia and the Russian Far East, as well as in other regions of Asia. Sequence analysis of three chloroplast DNA regions (rps4, trnL–trnF, and trnS–trnG) shows that, in Russia and adjacent countries, there are two genetically and geographically isolated Lacteae species. I. oxypetala Bunge grows in the south of the Russian Far East, and I. lactea Pall. grows in Siberia, Mongolia, and Kazakhstan. Genetic differentiation between the populations of I. lactea is extremely low and statistically insignificant (the fixation index Φ_ST = 0.057, P > 0.05), pointing to the unity of the gene pool and the absence of other Lacteae species in this area.     

Molecular and Functional Characterization of <Emphasis Type="Italic">ZmNF-YC14</Emphasis> in Transgenic Arabidopsis

The optimum transition stage from vegetative to reproductive development is important for flowering plants to obtain the desired plant architecture to maximize yield. In this study, we overexpressed the maize NUCLEAR FACTOR Y, SUBUNIT C 14 (ZmNF-YC14) gene in Arabidopsis to investigate its potential functions in the regulation of plant transition. Overexpression of ZmNF-YC14 in Arabidopsis inhibited plant flowering and retarded the duration of the branch-producing inflorescence phase 1 (I₁). These plants exhibited increased length of I₁ and higher ratio of inflorescence phase 1 to inflorescence phase (I) (I₁:I) under long-day conditions. As a consequence, these transgenic plants exhibited dramatic changes in their overall inflorescence morphology. In addition, the phenotypes of inflorescence morphology caused by ZmNF-YC14 overexpression were enhanced by exogenous gibberellin (GA) treatment, which obtained a significant increase in I₁:I, inflorescence phase 1 to inflorescence phase 2 (I₂) (I₁:I₂), and remarkable decrease in I₂ and I₂:I in transgenic plants compared to those under normal conditions. Taken together, the results of this study suggest that ZmNFYC14 negatively regulates flowering and controls flower formation under long-day conditions in Arabidopsis and may be involved in a GA regulation pathway.     

Effect of low temperature on globin expression,respiratory metabolic enzyme activities,and gill structure of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

Low temperature frequently influences growth, development, and even survival of aquatic animals. In the present study, physiological and molecular responses to low temperature in Litopenaeus vannamei were investigated. The cDNA sequences of two oxygen-carrying proteins, cytoglobin (Cygb) and neuroglobin (Ngb), were isolated. Protein structure analysis revealed that both proteins share a globin superfamily domain. Real-time PCR analysis indicated that Cygb and Ngb mRNA levels gradually increased during decrease in temperatures from 25 to 15°C and then decreased at 10°C in muscle, brain, stomach, and heart, except for a continuing increase in gills, whereas they showed a different expression trend in the hepatopancreas. Hemocyanin concentration gradually reduced as the temperature decreased. Moreover, the activities of respiratory metabolic enzymes including lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) were measured, and it was found that LDH activity gradually increased while SDH activity decreased after low-temperature treatment. Finally, damage to gill structure at low temperature was also observed, and this intensified with further decrease in temperature. Taken together, these results show that low temperature has an adverse influence in L. vannamei, which contributes to systematic understanding of the adaptation mechanisms of shrimp at low temperature.     

Protein kinase involvement in land snail aestivation and anoxia: Protein kinase A kinetic properties and changes in second messenger compounds during depressed metabolism

In response to environmental stress (low water, low oxygen) snails sharply suppress their metabolic rate, a process that is coordinated at the molecular level by reversible protein phosphorylation of key enzymes and functional proteins. Factors affecting protein kinase activity are, therefore, critical to metabolic suppression. Changes in the concentration of protein kinase second messenger compounds were followed over the first 24 h of aestivation and anoxia exposure in the terrestrial snail Otala lactea (Muller) (Pulmonata, Helicidae). The results showed declining concentrations of cyclic AMP over the first 24 h of anoxia exposure and aestivation in foot. Cyclic AMP concentrations in hepatopancreas transiently decreased with the lowest concentration observed at 4 h in both anoxic and aestivating animals. A transient increase in foot muscle cyclic GMP concentrations was apparent 4 h after the start of aestivation whereas a slow, steady increase was seen in anoxic foot muscle. Foot muscle 1,4,5-inositol triphosphate (IP3) concentrations decreased transiently during anoxia exposure and aestivation. Hepatopancreas IP₃ concentrations were significantly lower in 24 h anoxic snails and foot IP3 concentrations were significantly lower in 24 h aestivating snails. Kinetic characterization of purified PKA catalytic subunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg²⁺ ion but was inhibited at Mg²⁺ concentrations above 0.5 mM. Increasing concentrations of neutral salts and phosphate also inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (I₅₀ = 39 mM) was much lower than that of the neutral salts (I₅₀ 240 mM). The enzyme exhibited a broad pH optimum between pH 6.5–8.5. Arrhenius plots gave an activation energy of 13.3 kcal/mol corresponding to a Q₁₀ value of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed.Abbreviations CAMP adenosine 3:5-cyclic monophosphate - cGMP-guanosine 3:5-cyclic monophosphate - H-89N [2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCI - IP₃ d-myo-inositol 1,4,5-triphosphate - I₅₀ the concentration of inhibitor required to reduce the velocity to one half its original value - PKA cAMP dependent protein kinase - PKAc PKA catalytic subunit - PKA-I PKA inhibitor protein - PKC calcium and phospholipid-dependent protein kinase - PKC-I PKC inhibitor protein - PKG cGMP dependent protein kinase - mU nmol of phosphate transferred per minute     

Enhanced pyruvate production in <Emphasis Type="Italic">Candida glabrata</Emphasis> by overexpressing the <Emphasis Type="Italic">CgAMD1</Emphasis> gene to improve acid tolerance

Objectives

To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism.

Results

The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 _(H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l.

Conclusion

CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

     

Dark adaptation and conformations of carotenoids in the cells of <Emphasis Type="Italic">Cladophora aegagropila</Emphasis> (L). Rabenh.

Intact cells of freshwater algae Cladophora aegagropila (L). Rabenh. (synonymous to Aegagropila linnaei Kutz.) were investigated by resonance Raman spectroscopy. It was found that incubation in the dark (up to 24 h) leads to changes in the Raman spectroscopy spectrum of this species, namely to changes in the ratio of amplitudes of the I₁₅₂₃/I₁₁₅₅ and I₉₆₀/I₁₀₀₄ bands and in the half width of band in the region of 1523 cm–1. We suggested that the adaptation of algae to the dark alters the conformation of the molecule of the carotenoid by delocalization of π-electrons in the polyene chain of the molecule and changes the orientation of the ring. Moreover, the composition of carotenoids, as well as their location in the cell and microenvironment in the pigment–protein complexes can change: in the absence of illumination, the distribution of carotenoids in algal cells is more uniform. These changes are probably caused either by changes in the location of cell organelles or by carotenoid redistribution between photosynthetic membranes, plastoglobules, and lipophilic formations in the cytoplasm.     

M3 cholinoreceptors alter electrical activity of rat left atrium via suppression of L-type Ca<Superscript>2+</Superscript> current without affecting K<Superscript>+</Superscript> conductance

Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10^?5 M) in the presence of selective M2 antagonist methoctramine (10^?7 M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10^?8 M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K⁺ currents I _to, I _Kur, and I _Kir. In the absence of M2 blocker methoctramine, pilocarpine (10^?5 M) produced stronger attenuation of I _CaL and induced an increase in I _Kir. This additive inward rectifier current could be abolished by highly selective blocker of K_ir3.1/3.4 channels tertiapin-Q (10^?6 M) and therefore was identified as I _KACh. Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of I _CaL, but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of I _KACh.     

Backbone and side-chain resonance assignments of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> SUMO

One of the most debilitating diseases Malaria, in its different forms, is caused by protozoan of Plasmodium species. Deadliest among these forms is the “cerebral malaria” which is afflicted upon by Plasmodium falciparum. Plasmodium adopts numerous strategies including various post-translational modifications (PTMs) to infect and survive in the human host. These PTMs have proven their critical requirement in the Plasmodium biology. Recently, sumoylation has been characterized as one of the important PTMs and many of its putative substrates have been identified in Plasmodium. Sumoylation is the covalent attachment of SUMO protein to the substrate protein, which is mediated by an enzyme cascade involving activating (E1), conjugating (E2), and ligating enzymes (E3). Here, we report resonance assignment for ¹H, ¹³C and ¹⁵N of Plasmodium falciparum SUMO (Pf-SUMO) protein determined by various 2D and 3D heteronuclear NMR experiments along with predicted secondary structures.     

Life history traits determine the differential vulnerability of native and invasive apple snails (<Emphasis Type="Italic">Pomacea</Emphasis> spp.) to a shared juvenile-stage predator

The vulnerability of gastropods to their predators varies with life history traits such as morphology, body size, behavior, and growth rates as well as predator size. A recent study suggested that the invasive apple snail, Pomacea maculata, was considerably more vulnerable to crayfish predators than the native Florida apple snail, P. paludosa. The difference was hypothesized to be caused by the relatively small hatchling size of P. maculata. To test this hypothesis, we conducted a series of feeding assays designed to quantify maximum feeding rates and selective foraging of crayfish on apple snails. The rate at which crayfish killed individual P. maculata (i.e., kill rates) decreased with snail size, and kill rates on both species increased with crayfish size. Kill rates on juvenile P. maculata were higher than kill rates on size-matched hatchling P. paludosa, and crayfish fed selectively on P. maculata when offered mixed groups of size-matched snails. Further analyses revealed that hatchling P. paludosa possess shells 1.8× heavier than size-matched P. maculata suggesting differences in vulnerability to crayfish were consistent with interspecific differences in shell defenses. Differences in hatchling size and defensive traits in combination make crayfish kill rates on hatchling P. maculata approximately 15.4× faster than on hatchling P. paludosa, but the relative contribution of hatchling size to differences in apple snail vulnerability was >3× greater than the contribution of defensive traits.     

Adenosine monophosphate-activated protein kinase from the mud crab, <Emphasis Type="Italic">Scylla paramamosain</Emphasis>: cDNA cloning and profiles under cold stress

Adenosine monophosphate-activated protein kinase (AMPK), an important energy sensor, is crucial for organism survival under adverse conditions. In this study, the roles of this gene under cold stress in a warm-water mud crab, Scylla paramamosain was investigated. The full-length cDNA (SpAMPK) was 1884 bp and its open reading frame of 1566 bp was isolated and characterized. The expressions of SpAMPK detected by quantitative real-time PCR (qRT-PCR) in various tissues revealed that the highest expression was in the hepatopancreas. The profiles of SpAMPK gene in the hepatopancreas, chela muscle and gill were detected when the subadult crabs were exposed to the four temperature conditions of 10, 15, 20 and 25^°C. The results showed that the expression patterns of SpAMPK mRNA in the three tissues were significantly higher when crabs were exposed to 15^°C than the other three temperature treatments, while at 10^°C treatment, the SpAMPK mRNA was lowest among the four temperature treatments. These findings suggested that the high expression of SpAMPK mRNA might initiate ATP-producing pathways to generate energy to cope with cold stress at 15^°C treatment, which was slightly below the range of optimum temperatures; while treatment at 10^°C, far lower than optima, the low expression of SpAMPK mRNA could reduce the energy expenditure and thus induce the crabs into cold anesthesia. The results of SpAMPK in this study might contribute to the understanding of the molecular mechanism of acclimation to cold hardiness in S. paramamosain.