有色膜遮光对烤烟生长和光合特性及其初烤品质的影响

以自然光为对照,采用红色、白色、蓝色、黄色4种有色薄膜于2010~2011年从团棵期开始对大田烤烟进行遮光处理,研究不同光质对烤烟生长、光合特性及初烤品质指标的影响。结果显示:(1)红膜处理最大叶长宽比最小、叶面积最大,黄膜处理则相反。(2)红、蓝膜处理烟叶净光合速率、气孔导度、蒸腾速率明显高于自然光处理,白、黄膜处理略高于对照或与对照持平,且遮膜处理前期红膜高于蓝膜处理,后期蓝膜高于红膜处理。(3)红、蓝膜处理有利于提高倒5叶SPAD值,黄膜处理则相反。(4)红膜处理显著降低了中部叶蛋白质、总氮含量和氮碱比,提高了施木克值,并显著提高了上部叶可溶性糖含量和氮碱比,降低了施木克值;蓝膜处理显著提高了中部叶烟碱和多酚含量,降低了可溶性糖含量、施木克值及氮碱比,并显著提高了上部叶蛋白质、总氮、烟碱和多酚含量,降低了施木克值,提高了氮碱比;黄膜处理显著降低了中上部叶蛋白质、总氮、烟碱和多酚含量,提高了上部叶施木克值、降低了氮碱比。研究表明,红、蓝膜处理更利于烟叶发育和光合特性的提高,初烤烟叶化学成分更协调,利于优质烟叶的形成。     

Expression of Mammalian Antiviral Enzymes from the 2–5A System in Transgenic Plants

The 2–5A system is an interferon-regulated antiviral RNA decay pathway present in cells of higher vertebrates. Two enzymes are essential, a 2–5A synthetase which produces 5′-phosphorylated, 2′,5′-linked oligoadenylates (2–5A) in response to doublestranded RNA, and the 2–5A-dependent RNase L. To determine if these human proteins would be functional in plants, we expressed the human cDNAs for a 2–5A synthetase and RNase L in separate tobacco plants. Both proteins were enzymatically active in extracts of transgenic plants while such activities were not detected in the control plants. Furthermore, activation by 2–5A of RNase L in the transgenic plant leaves was shown to cause degradation of ribosomal RNA. The requirement for both the synthetase and RNase L for antiviral activity was underscored by the observations that expression of human RNase L alone or 2–5A-synthetase alone was insufficient to protect plants against either tobacco etch virus or tobacco mosaic virus.     

Toxicity of Leaf and Stem Extracts to Tylenchorhynchus dubius

Plant extracts, made by grinding 2 g of fresh tissue in 5 ml of water, were toxic to Tylenchorhynchus dubius and Hoplolaimus spp. Such extracts from leaves and stems of bean (Phaseolus vulgaris L.) and leaves of tobacco (Nicotiana tabacum L.) were most toxic; those from leaves of corn (Zea mays L.), tomato (Lycopersicon esculentum Mill.) and rhododendron (Rhododendron catawbiense L.) were less toxic; and extracts of bean roots were nontoxic. Nematode movement slowed markedly within 1 hr in tobacco leaf extract, and within 4 hr in bean leaf extract; both extracts completely inactivated or killed 95% of the nematodes in 24 hr. Heating leaf extract 10 min at 80 C eliminated toxicity. Absorption of fusicoccin, a phytotoxin produced by Fusicoccum amygdali Del., increased the toxicity of tomato leaf extracts, whereas water extracts of acetone-extracted powder preparations of leaves were about 15-fold more toxic than water extracts of fresh tissue. Addition of homogenized leaves of bean, tobacco and tomato to soil significantly reduced nematode populations within 3 days.     

Three RNases in Senescent and Nonsenescent Wheat Leaves : Characterization by Activity Staining in Sodium Dodecyl Sulfate-Polyacrylamide Gels

We have described three RNases in wheat leaves (Triticum aestivum L. cv Chinese Spring) and developed assays for measuring each RNase individually in crude leaf extracts. We initially used activity staining in sodium dodecyl sulfate-polyacrylamide gels to characterize RNases in extracts of primary and flag leaves. We thus identified acid RNase (EC 3.1.27.1, here designated RNase WL_A), and two apparently novel enzymes, designated RNases WL_B and WL_C. RNase WL_B activity displays a distinctive isozyme pattern, a molecular mass of 26 kilodaltons (major species), a broad pH range with an optimum near neutrality, insensitivity to EDTA, and stimulation by moderate concentrations of KCl and by MgCl₂. RNase WL_C activity exhibits a molecular mass of 27 kilodaltons, a neutral pH optimum, insensitivity to EDTA, and inhibition by KCl, MgCl₂, and tri-(hydroxymethyl)aminomethane. Based on distinctive catalytic properties established in gels, we designed conventional solution assays for selective quantitation of each RNase activity. We used the assays to monitor the individual RNases after gel filtration chromatography and native gel electrophoresis of extracts. In accompanying work, we used the assays to monitor RNases WL_A, WL_B, and WL_C, which are present in senescent and nonsenescent leaves, during the course of leaf senescence.     

光呼吸代谢物乙醇酸、乙醛酸和草酸对烟草叶片硝酸还原的影响

乙醇酸、乙醛酸和草酸能明显促进烟草(Nicotiana rustica)叶片在黑暗中的硝酸还原,光呼吸抑制剂a-羟基吡啶甲烷磺酸能消除前二者的促进作用而不能完全消除草酸的作用。草酸+NAD~+能显著促进离体的硝酸还原。烟叶提取液加入草酸和NAD~+后生成NADH和CO_2认为活体内由乙醛酸氧化生成的草酸是经脱氢生成NADH供硝酸还原之用。未能证明在烟叶内存在乙醇酸脱氨酶,因此排除由乙醇酸直接脱氢以还原硝酸的可能。     

Virus-induced virus inhibitors from systemically virus-infected plants

After infection ofNicotiana tabacum cv. Samsun with tobacco mosaic virus (TMV) crude extracts from dark-green spots of upper leaves had a more strongly marked inhibitory effect upon TMV addedin vitro than crude extracts from the surrounding light-green tissue. Likewise, crude extracts from leaves ofNicotiana tabacum cv. Samsun showing recovery after infection with tobacco ringspot virus (TRV) were seen to have a marked inhibitory effect on TMV addedin vitro. The results obtained suggest that virus inhibitors are produced after virus infections not only in hypersensitive hosts but also in systemic hosts. Necrotizing processes are not an indispensable prerequisite of the production of virus-induced virus inhibitors.     

An N-acylamino acid acylase from Nicotiana tabacum leaves

An N-acylamino acid acylase was partially purified from tobacco (Nicotiana tabacum) leaves and some of its properties are described. It hydrolyses N-acetylarginine, N-acetylmethionine, N-acetylcysteine and to a lesser extent N-formylmethionine. It does not appreciably hydrolyse N-formyl peptides and is therefore unlikely to be involved in protein synthesis.     

Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.     

Fate of Tobacco Mosaic Virus after Entering the Host Cell

Tobacco leaves were inoculated with tobacco mosaic virus labeled with ³²P or ³⁵S. After various intervals, extracts of the leaves were prepared. In extracts from leaves infected for 5 to 360 min, about 40 to 60% of the virus retained on leaves was recovered in the pellet of the homogenate centrifuged at 12 000 × g. The virus associated with the 12 000 × g pellet was dissociable by treatment with pancreatic RNase, alkali or sodium dodecyl sulfate (SDS). The parental virus extracted by SDS from the pellet at 12 000 × g had a large amount of partially uncoated virus possessing naked RNA. Analysis by density gradient centrifugation suggested that, in addition to partially uncoated virus, some fragmented RNA was also associated with the 12 000 × g pellet. This fragmented RNA seemed to be derived from partially uncoated virus. Density gradient analysis of SDS extracts from the 12 000 × g pellet suggested that some of the virus underwent uncoating at the internal regions of the virus particle.     

Bioassay and attributes of a growth factor associated with crown gall tumors

An improved bioassay is described for a factor that promotes tumor growth which was first obtained from extracts of pinto bean leaves with crown gall tumors. Sixteen primary pinto bean leaves per sample are inoculated with sufficient Agrobacterium tumefaciens to initiate about 5 to 10 tumors per leaf and treated with tumor growth factor at day 3 after inoculation. The diameters of 30 to 48 round tumors (no more than 3 randomly selected per leaf) are measured per test sample at day 6. Mean tumor diameter increased linearly with the logarithm of the concentration of tumor growth factor applied. The tumor growth factor was separated by column chromatography from an ultraviolet light-absorbing compound previously reported to be associated with fractions having maximal tumor growth factor activity. Partly purified tumor growth factor showed no activity in a cytokinin bioassay or an auxin bioassay, and negligible activity in gibberellin bioassays. Representatives of these three classes of growth factors did not promote tumor growth. Extracts from crown gall tumors on primary pinto bean leaves, primary castor bean leaves, Bryophyllum leaves, carrot root slices, and tobacco stems showed tumor growth factor activity, whereas extracts from healthy control tissues did not. Extracts from actively growing parts of healthy pinto beans, Bryophyllum, and tobacco, however, showed tumor growth factor activity. Tumor growth factor is proposed to be a normal plant growth factor associated with rapidly growing tissues. Its synthesis may be activated in nongrowing tissues by infection with Agrobacterium sp.     

Antisense-overexpression of the MsCOMT gene induces changes in lignin and total phenol contents in transgenic tobacco plants

Initially, we isolated the caffeic acid O-methyltransferase (COMT) gene from Miscanthus sinensis (accession number HM062766.1). Next, we produced transgenic tobacco plants with down-regulated COMT gene expression to study its control of total phenol and lignin content and to perform morphological analysis. These transgenic plants were found to have reduced PAL and ascorbate peroxidases expression, which are related to the phenylpropanoid pathway and antioxidant activity. The MsCOMT-down-regulated plants had decreased total lignin in the leaves and stem compared with control plants. Reduced flavonol concentrations were confirmed in MsCOMT-down-regulated transgenic plants. We also observed a morphological difference, with reduced plant cell number in transgenic plants harboring antisense MsCOMT. The transgenic tobacco plants with down-regulated COMT gene expression demonstrate that COMT plays a crucial role related to controlling lignin and phenol content in plants. Also, COMT activity may be related to flavonoid production in the plant lignin pathway.     

Changes in the chlorophylls and carotenoids of leaves of Nicotiana tabacum during senescence

In addition to chlorophylls a and b, β-carotene, lutein, violaxanthin and neoxanthin, leaves of tobacco (Nicotiana tabacum L. cv. Virginia Gold) contain antheraxanthin in some harvests. In lower leaves, chlorophylls decreased more rapidly than carotenoids during senescence, but both types of pigment decreased at equal rates in upper leaves. The chlorophyll a:b ratio decreased only in post-mature leaves. Total carotenoid decreased with age, with the relative proportion of β-carotene increasing in lower leaves. Seasonal influences rather than age of leaf determines whether antheraxanthin is present. No esterified xanthophylls were found in senescent leaves.     

Targeting of castor bean glyoxysomal isocitrate lyase to tobacco leaf peroxisomes

The cDNA encoding castor bean endosperm isocitrate lyase (ICL) was expressed under the control of the promoter of the small subunit of pea ribulose bisphosphate carboxylase in transformed tobacco. ICL protein was detected using anti-ICL antibodies on immunoblots of total leaf protein extracts. Nycodenz density gradient separation of the extracts from the transgenic tobacco leaves showed ICL co-fractionated with hydroxypyruvate reductase, a peroxisomal matrix marker protein, and away from lactate dehydrogenase, a cytosolic marker protein. Immunoelectron microscopy of ultrathin leaf sections demonstrated the location of ICL within the matrix of the leaf peroxisomes of the transgenic plants. In vitro transcribed and translated ICL was also imported into leaf peroxisomes isolated from germinating sunflower seeds. The in vivo and in vitro import of this protein into leaf peroxisomes provides strong support for the notion that the import machinery of glyoxysomes and peroxisomes is very similar.     

Properties of some defective strains of tobacco mosaic virus and their behaviour as affected by inhibitors during storage in sap

From the type strain of tobacco mosaic virus, defective strains were isolated that produced chlorotic or ringspot type symptoms in tobacco and were difficult to transmit without carborundum in the inoculum. Their concentration was less than 0–1 μg/ml of sap instead of the usual 2 mg/ml with the type strain. Phenol extracts of infected leaves were a little more infective than extracts in buffer, whereas phenol extracts of leaves infected with type strain were very much less infective than extracts in buffer. Electron microscopy of infective sap rarely showed any virus particles, but preparations concentrated by ultracentrifugation contained virus particles, many of which were broken or seemed inadequately assembled. Changing the ambient temperature at which infected plants were kept from 20 to 35°C did not increase the amount or improve the appearance of the virus. Some of the strains were inactivated during heating for 10 min between 70 and 80 °C. Undiluted sap lost its infectivity in 3 days at 20 °C, as did the type strain when diluted to 0–1 μg/ml in sap from healthy leaves. This is because substances that inhibit infection were produced by microbes in the sap. The ability of sap from healthy leaves to inhibit infection increased by more than twenty-five times when left 3 days at 20 °C. Infectivity of appropriate mixtures of type strain and aged sap was restored by diluting them in buffer. Sodium azide at 0·02% in sap prevented formation of the inhibitor. The infectivity of the defective strains increased when inoculated together with the type strain.     

Inhibitory effect of fucoidan from brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus in tobacco leaves of two cultivars

The effect of fucoidan from the brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus (TMV) was investigated in the leaves of tobacco (Nicotiana tabacum L.) of two cultivars (Ksanti-nk and Samsun). In the leaves of cv. Ksanti-nk inoculated with a mixture of TMV preparation (2 μg/ml) and fucoidan (1 mg/ml), the number of local necrotic lesions induced by the virus decreased by more than 90% as compared with the leaves inoculated with the virus alone. In tobacco leaves of cv. Samsun, virulence and the concentration of the virus 3 days after inoculation with the same mixture of TMV and fucoidan were by 62 and 66%, respectively, lower than in the leaves inoculated with TMV alone. As the infection spread, the inhibitory effect of fucoidan decreased. When the leaves were treated with fucoidan before and after the inoculation with TMV, its antiviral activity was less pronounced than when a mixture of the virus and the polysaccharide was used as inoculum. Electron microscopic investigation of TMV mixed with fucoidan often showed agglutinated virions. The highest virulence of the mixture (TMV preparation, 12 μg/ml, plus fucoidan, 1 mg/ml) was observed upon its twofold dilution, and after that it decreased. It was concluded that, when the leaves were inoculated with the mixture of TMV and fucoidan, the latter affected not only the plant but the virus as well. Treatment of tobacco leaves, cv. Ksanti-nk, with actinomycin D (10 μg/ml) 24 h before the inoculation with TMV almost completely suppressed the effect of fucoidan, indicating that fucoidan acted at a gene level.     

RNA from callus cultures and leaves of Nicotiana tabacum

MAK column chromatography has been used to analyse RNA from normal and crown gall callus cultures and leaves of Nicotiana tabacum. To determine the elution behaviour of well-defined DNA-like RNAs with different GC content, complementary RNAs (c-RNA) synthesized on Agrobacterium tumefaciens DNA and crown gall DNA were used. The elution profile of the RNA from all three tissues followed a similar pattern. By salt gradient elution the RNA in the tRNA region showed a remarkably high CMP content which was significantly higher for the normal tissues than for crown gall tissue. RNA from the callus cultures contained more DNA-like RNA (D-RNA) with a higher turnover rate than RNA from leaves. Because of its relatively low poly A content, measured as RNase A + T₁ resistance, as well as its high turnover rate, the salt-eluted D-RNA is thought to be heterogeneous nuclear RNA (Hn-RNA) and not mRNA. RNA molecules that might represent the mRNA population, having intramolecular poly A tracts, were subsequently eluted by a salt gradient, a low salt buffer and with the chaotropic agent guanidine thiocyanate, which removed tenaciously bound (TB-RNA) in two fractions, α and β. Crown gall RNA showed both a different labelling behaviour and a higher poly A content in the α and β fractions compared to the normal tissues. c-RNAs may be eluted at different salt concentrations because of their different GC content. They give rise to a considerable fraction of TB-RNA which in the presence of tobacco leaf RNA was split into fractions similar to α and β. No fraction was found amongst these RNAs which did have intramolecular poly A tracts.     

Establishing a Corky Ringspot Disease Plot for Research Purposes

A method to establish two experimental corky ringspot disease (CRS) plots that had no prior CRS history is described. CRS is a serious disease of potato in the Pacific Northwest caused by tobacco rattle virus (TRV) and transmitted primarily by Paratrichodorus allius. ‘Samsun NN’ tobacco seedlings were inoculated with viruliferous P. allius in the greenhouse before they were transplanted into the field soil at the rate of 3,000 plus seedlings/ha. Care was taken to keep soil around plants in the greenhouse and transplants in the field moist to avoid vector mortality. The vector population in the soil of one of the fields was monitored by extraction, examination under microscope and bioassay on tobacco seedlings to ascertain that they were virus carriers. Presence of virus in tobacco bioassay plants was determined by visual symptoms on tobacco leaves and by testing leaves and roots using ELISA. Although TRV transmission was rapid, there was loss of infectivity in the first winter which necessitated a re-inoculation. After two years of planting infected tobacco seedlings, 100% of soil samples collected from this field contained viruliferous P. allius. In the second field, all five commercial potato cultivars, known to be susceptible, expressed symptoms of CRS disease indicating that the procedure was successful.