Coat proteins of two filamentous plant viruses display NTPase activity in vitro

Coat proteins (CPs) of plant viruses are involved in different stages of the viral life cycle such as virion assembly, replication, movement, vector transmission, and regulation of host defense responses. Here, we report that the CPs of two filamentous RNA viruses, potato virus X (PVX, Potexvirus) and potato virus A (PVA, Potyvirus) exhibit an enzyme activity. The CP isolated from PVX virions possesses ATP-binding and ATPase activities. Recombinant PVX and PVA CPs produced in Escherichia coli show Mg2+-dependent ATPase and UTPase activities inhibited by antibodies against virus particles. Deletion of the C-terminal regions of these proteins diminishes their ATPase activity.     

Enzyme systems of insecticide detoxication in the Colorado beetle

The activity of three enzymatic systems of xenobiotic metabolism (cytochrome P-450-dependent monooxygenases, non-specific esterases and glutathione S-transferases) was studied at different stages of development of the Colorado potato beetle (Leptinotarsa decemlineata). Significant sex and ontogenetic differences in the content of cytochrome P-450, position of maxima of CO-difference spectra of the cytochrome P-450 reduced form and substrate specificity of cytochrome P-450 were revealed. The activity of non-specific esterases was shown to increase depending on the age of larvae. The insecticide 1-naphtholenol methylcarbamate which is metabolized by the system of cytochrome P-450-dependent monooxygenases is more toxic in the larvae than at the imago stage, which is correlated with the activity of this system at different stages of ontogenesis. The microsomal monooxygenase inhibitor, piperonylbutoxide, increases the insecticide toxicity in the Colorado beetle imago more than 2-fold. The experimental results testify to different contribution of the detoxication systems to the sensitivity of the Colorado beetle to insecticides at various metamorphosis stages.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

Targeting of SPCSV-RNase3 via CRISPR-Cas13 confers resistance against sweet potato virus disease

Sweet potato (Ipomoea batatas) is one of the most important crops in the world, and its production rate is mainly decreased by the sweet potato virus disease (SPVD) caused by the co-infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus. However, methods for improving SPVD resistance have not been established. Thus, this study aimed to enhance SPVD resistance by targeting one of its important pathogenesis-related factors (i.e., SPCSV-RNase3) by using the CRISPR-Cas13 technique. First, the RNA targeting activity of four CRISPR-Cas13 variants were compared using a transient expression system in Nicotiana benthamiana. LwaCas13a and RfxCas13d had more efficient RNA and RNA virus targeting activity than PspCas13b and LshCas13a. Driven by the pCmYLCV promoter for the expression of gRNAs, RfxCas13d exhibited higher RNA targeting activity than that driven by the pAtU6 promoter. Furthermore, the targeting of SPCSV-RNase3 using the LwaCas13a system inhibited its RNA silencing suppressor activity and recovered the RNA silencing activity in N. benthamiana leaf cells. Compared with the wild type, transgenic N. benthamiana plants carrying an RNase3-targeted LwaCas13a system exhibited enhanced resistance against turnip mosaic virus TuMV-GFP and cucumber mosaic virus CMV-RNase3 co-infection. Moreover, transgenic sweet potato plants carrying an RNase3-targeted RfxCas13d system exhibited substantially improved SPVD resistance. This method may contribute to the development of SPVD immune germplasm and the enhancement of sweet potato production in SPVD-prevalent regions.     

Changes in the Activity of Polygalacturonase-Inhibiting Protein during Potato Development

The activity of a polygalacturonase-inhibiting protein was determined in growing potato plants and in stored potato tubers. The activity in leaves was higher than in stems, and it decreased by the end of the vegetative season. During the dormancy period, the inhibitory activity in tubers also changed. In the sprouting tubers, it was somewhat lower than in the nonsprouting ones, and, in sprouts, it was usually higher than in tubers. Both the plant polygalacturonase and the polygalacturonase secreted by phytopathogenic fungi after their penetration in plant tissues can serve as inhibitor's targets. Therefore, the inhibitor seems to control the resistance of plants to infection by particular pathogens, and this resistance is characteristic of definite developmental stages.     

降水对新疆马铃薯甲虫分布的影响

为明确降水在中国新疆地区对马铃薯甲虫分布的影响,揭示制约马铃薯甲虫分布扩散的关键环境因子,为马铃薯甲虫的持续防控和综合治理提供理论依据。该研究结合新疆历史降水数据,对马铃薯甲虫现有分布区内的降水时空格局展开分析,比较了马铃薯甲虫危害程度与降水时空格局的关系。结果表明:马铃薯甲虫现主要分布于新疆年降水量在150 mm以上地区,早期定殖的地区降水量大于后期定殖区,其扩散方向为自西向东,同时年降水量也逐渐减少。马铃薯甲虫危害程度也随着经度增加而递减,早期发现马铃薯甲虫的地区受危害程度较重。降水量减少导致的水分缺乏对马铃薯甲虫的分布扩散具有一定的制约作用。     

Impact of invertase overexpression on cell size, starch granule formation and cell wall properties during tuber development in potatoes with modified carbon allocation patterns

Transgenic potato tubers that overexpressed either a cytosolic or an apoplastic invertase in the wild type or AGPase antisense background were used to analyse the effect of invertase activity on cell expansion, starch granule formation and turgor pressure during tuber development. Although the transgenic plants did not develop a visible phenotype in aerial regions the size and number of tubers were significantly modified in the various lines. Transmission electron and light microscopy were performed to monitor starch grain size and number, cell size and cell wall thickness. Water potential, osmotic pressure, and indirectly, turgor pressure were determined during the final stages of tuber development. Glucose levels were high in transgenic tubers that overexpressed a yeast-derived invertase. The number of starch grains per cell was almost identical in all transgenic lines. However, the amount of starch was modified in the transgenics as compared to the wild type. As expected, the size of starch grains was reduced in all lines that expressed an AGPase antisense mRNA. These results indicate that invertase activity and glucose levels do not affect initiation of starch grain formation during the early stages of tuber development, but growth of starch corns in the later stages of tuber maturation.     

A bioluminescence method of determining the activity of NAD-dependent hydrogenase]

An analytical multienzyme system composed of NAD-dependent hydrogenase of Alcaligenes eutrophus, and reductase and luciferase from luminous bacteria was studied. The rate of luminescence increase of this system was found to be proportional to hydrogenase activity. The apparent Michaelis constants for NAD and hydrogen were determined (5 and 40 microM, respectively). The pH optimum is 7.5-9.0. Over the NAD concentration range from 20 to 100 microM, the rate of luminescence increase changed by less than 10%. At higher concentrations of NAD a monotonous decreasing of the rate of luminescence increase was observed. The proposed multienzyme system can be used for measuring the hydrogenase activity and hydrogen concentration. The high sensitivity to hydrogen (0.1 nmol in sample) and to hydrogenase (0.5 mU) and specificity of the system enable its application in the development of a biosensor for rapid detection of hydrogen in a medium.     

Dehydrogenase activity in skeletal muscles of rats after long-term exposure to weightlessness

Malate- and isocytratedehydrogenase activity in mitochondrial and cytoplasmic fractions and lactate dehydrogenase activity in hindlimb muscles have been studied at different stages after 18.5-day flight on a biosatellite "Cosmos-1129" and after 20-day hypokinesia. A decrease in dehydrogenase activity has been found on the first postflight day. The enzyme activities returned to the control values in mitochondria, and in the cytoplasm they were greater by day 6 postflight. It was concluded that hypokinesia did not reveal all the effects of microgravity on the whole system but some enzyme alterations in the muscle resembled those observed during the flight. The effects may be caused by the inhibition of both aerobic and anaerobic metabolic pathways under the effect of microgravity.     

Influence of temperature on the life-cycle of Globodera spp

The potato cyst nematodes (PCN) G. rostochiensis (Woll.) and G. pallida (Stone) are the most economically important pests of potato (Solanum tuberosum L.) in the UK and are widespread in ware potato growing regions in Europe. The new EU directive 2007/33/EC which came into effect July 1, 2010 aims to control their spread and limit further increases in populations. We are investigating the role of temperature in the life cycle of PCN to assess how this effects population multiplication in relation to managing PCN. Hatching and nematode development are stages in the life cycle that are affected by temperature and thus are important life stages that can be examined to determine the impact of temperature on the length of time required for one generation to be completed and the potential for final populations to increase on different potato genetic backgrounds. In some conditions a partial or complete second generation has also been observed within the growing season. Females have been observed on the surface of tubers and "pecking" skin damage can occur which may be a result of a second generation. We are investigating the influence of temperature on the potential for a second generation or the induction of diapause.     

Differences in the ribosome state of nerve cells detectable by acridine orange fluorochrome staining

Acridine orange (AO) binding with ribosomal RNA in the cytoplasm of nerve cells depends on the animal functional state to differ in active and hibernating ground squirrels. These differences are expressed as changes in the ratio of red and green AO luminescence intensities. The transition of squirrels from the activity to hibernation provokes a five-fold fall in the red luminescence, while the green one shifts no more than by 10%, thus indicating the change in AO interaction with one-helical regions of rRNA only. The red luminescence decreases simultaneously with the rise of monoribosomes portion and dissociation of polysomes, but does not depend on RNA concentration in the cytoplasm. The dependence of AO binding on protein-RNA interaction in the one-helical regions and its relation to the capacity of ribosomes of protein synthesis are discussed.     

Comparative study of metabolism of the purple photosynthetic bacteria grown in the light and in the dark under anaerobic and aerobic conditions

For three species of anoxygenic phototrophic alphaproteobacteria differing in their reaction to oxygen and light, physiological characteristics (capacity for acetate assimilation, activity of the tricarboxylic acid (TCA) cycle enzymes, respiration, and the properties of the oxidase systems) were studied. Nonsulfur purple bacteria Rhodobacter sphaeroides, Rhodobaca bogoriensis, and aerobic anoxygenic phototrophic bacteria Roseinatronobacter thiooxidans were the subjects of investigation. All of these organisms were able to grow under aerobic conditions in the dark using the respiratory system with cytochrome aa ₃ as the terminal oxidase. They differed, however, in their capacity for growth in the light, bacteriochlorophyll synthesis, and regulation of activity of the TCA cycle enzymes. Oxygen suppressed bacteriochlorophyll synthesis by Rha. sphaeroides and Rbc. bogoriensis both in the dark and in the light. Bacteriochlorophyll synthesis in Rna. thiooxidans occurred only in the dark and was suppressed by light. The results on acetate assimilation by the studied strains reflected the degree of their adaptation to aerobic growth in the dark. Acetate assimilation by light-grown Rha. sphaeroides was significantly higher than by the dark-grown ones. Unlike Rha. sphaeroides, acetate assimilation by Rbc. bogoriensis in the light under anaerobic and aerobic conditions was much less dependent on the growth conditions. Aerobic acetate assimilation by all studied bacteria was promoted by light. In Rha. sphaeroides, activity of the TCA cycle enzymes increased significantly in the cells grown aerobically in the dark. In Rbc. bogoriensis, activity of most of the TCA cycle enzymes under aerobic conditions either decreased or remained unchanged. Our results confirm the origin of modern chemoorganotrophs from anoxygenic phototrophic bacteria. The evolution from anoxygenic photoorganotrophs to aerobic chemoorganotrophs included several stages: nonsulfur purple bacteria → nonsulfur purple bacteria similar to Rbc. bogoriensis → aerobic anoxygenic phototrophs → chemoorganotrophs.     

Cloning and characterization of a cathepsin D inhibitor gene from Solanum tuberosum L.

A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.     

致病疫霉拮抗菌株YR-7 的分离鉴定及其活性物质

【目的】从黄河边的农田土壤中分离筛选拮抗致病疫霉的粘细菌,鉴定目标菌株,分析其发酵上清液的稳定性及对马铃薯晚疫病菌的抑制效果,为活性物质分离鉴定及抗马铃薯晚疫病菌生物农药的研发奠定基础。【方法】采用兔粪诱导法分离菌株,通过平板对峙法筛选对马铃薯晚疫病菌有拮抗作用的粘细菌,通过形态特征、生理生化特征以及16S r RNA基因序列分析对菌株进行鉴定。采用称重法测定菌株生长曲线,通过平皿法测定菌株不同生长时期发酵上清液对致病疫霉的菌丝生长抑制率和浓缩发酵上清液的稳定性。通过马铃薯离体叶片涂布浓缩发酵上清液和接种病原菌孢子悬浮液法,测定该菌株对马铃薯晚疫病的防病作用。【结果】从土壤样品中共分离获得7株粘细菌,其中4株拮抗致病疫霉,拮抗效果最强的为YR-7菌株,菌丝的生长抑制率为96%,该菌株被鉴定为Myxococcus xanthus。培养7 d后,菌株发酵上清液对致病疫霉的抑制活性趋于稳定。浓缩发酵上清液经30-50°C处理后,对致病疫霉菌丝的生长抑制率可达50.90%,高于50°C时抑菌活性逐渐下降,90°C处理后菌丝的生长抑制率仍可达25.45%。浓缩发酵上清液在p H 4.0-9.0条件下比较稳定,保持40.21%以上菌丝的生长抑制率,当p H4.0或p H9.0时,抗菌活性显著降低。活性物质不能被蛋白酶降解,其抗菌活性不受紫外线、自然光照射的影响。对马铃薯离体叶片的生防效果检测表明,YR-7的浓缩发酵上清液处理组叶片相对病斑面积仅为0.35%,对照组的相对病斑面积高达68.19%。【结论】粘细菌菌株YR-7可以产生抗马铃薯晚疫病菌的次生代谢产物,抗菌活性物质具有较好的稳定性,可以有效抑制致病疫霉侵染马铃薯叶片,具有开发成抗马铃薯晚疫病生物农药的潜在价值。     

基于降水利用比较分析的四川省种植制度优化

比较分析四川8个农业生态区典型站点及其主要种植模式的降水盈亏产量降低率、产量降低率风险指数、降水利用效率和降水经济效率。结果表明:(1)四川省不同区域、不同种植模式、不同作物及其不同生育阶段基于降水盈亏的产量降低率多年均值差异较大。区域分布上,雅安最低,仅23％,攀西最高,达50％以上,其余地区30％-40％;种植制度上,麦-玉-苕等旱三熟低于麦-稻等水旱轮作两熟制;作物种类上,冬小麦、冬油菜、秋播马铃薯等作物普遍高于水稻、玉米、棉花、红薯和大豆作物;生育阶段上,冬小麦、冬油菜、秋播马铃薯作物开花前后普遍较高,各种作物生育末期较低。(2)基于自然降水,攀西地区遭遇旱灾的风险极大,麦-玉-苕等旱三熟的产量降低率风险指数相对较小;雅安等盆地内部多数区域由于阶段性降水过多引起湿害偏重,导致麦-稻等水旱轮作两熟制略优于旱三熟。基于降水利用效率和降水经济效率,各地比较一致,较优的种植制度首先是麦(油、薯)-稻两熟制,其次才是麦(油)-玉-苕(豆)旱三熟。(3)综合旱涝灾害风险、降水利用效率和降水经济效率,以及复杂地形等因素,有较好灌溉条件的农田应以麦(油、薯)-稻水旱轮作两熟制为主,而无水源保障的旱地则以麦(油)-玉-苕(豆)旱三熟为主。     

Sensibilized dimer luminescence of singlet molecular oxygen in solutions

With the use of mechanical phosphoroscope the "universal" delayed emission has been found in aerobic solutions of different sensitizers in CCl4. The spectrum of this emission has the main maximum at 703 nm. The luminescence intensity is proportional to the square of the intensity of the exciting light. Removal of oxygen or addition of 10% of acetone led to disappearance of the luminescence. At equal intensities of singlet oxygen generation relative intensities of the 1272 and 703 nm bands differed by several orders of magnitude in solutions of different sensitizers. The energy migration from the molecules responsible for the luminescence to bacteriopheophytin and phtalocyanine has been observed. The luminescence is interpreted as dimol emission of solvated singlet molecular oxygen activated by sensitizer molecules.     

Cysteine proteinase forms in sprouting potato tuber

Transformation of plants with exogenous proteinase inhibitor genes represents an attractive strategy for the biological control of insect pests. However, such a strategy necessitates a thorough characterization of endogenous proteinases. which represent potential target enzymes for the exogenous inhibitors produced. In the present study. changes in general endoproteolytic activity were monitored during sprouting of potato ( Solanum tuberosum L. cv. Kennebec) tuber. Quantitative data obtained using standard procedures showed that an increase in cysteine proteinase (EC 3.4.22) activity occurs during sprouting. This increased activity results from the gradual appearance of new cysteine proteinase forms, as demonstrated by the use of class-specific proteinase activity gels. While only one cysteine proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, oryzacystatins I and II. two cysteine proleinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase delected. Similar results were obtained with leaf, stem and stolon proteinases. This apparent absence of direct interference supports the potential of oryzacystatin genes for production of insect-tolerant transgenie potato plants.     

Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.)

Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber-specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full-length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.