Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of L-phenylalanine ammonia-lyase

Tobacco plants over-expressing L-phenylalanine ammonia-lyase (PAL(+)) produce high levels of chlorogenic acid (CGA) and exhibit markedly reduced susceptibility to infection with the fungal pathogen Cercospora nicotianae, although their resistance to tobacco mosaic virus (TMV) is unchanged. Levels of the signal molecule salicylic acid (SA) were similar in uninfected PAL(+) and control plants and also following TMV infection. In crosses of PAL(+) tobacco with tobacco harboring the bacterial NahG salicylate hydroxylase gene, progeny harboring both transgenes lost resistance to TMV, indicating that SA is critical for resistance to TMV and that increased production of phenylpropanoid compounds such as CGA cannot substitute for the reduction in SA levels. In contrast, PAL(+)/NahG plants showed strongly reduced susceptibility to Cercospora nicotianae compared to the NahG parent line. These results are consistent with a recent report questioning the role of PAL in SA biosynthesis in Arabidopsis, and highlight the importance of phenylpropanoid compounds such as CGA in plant disease resistance.     

A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification

A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.     

Transgenic tobacco plants producing caffeine: a potential new strategy for insect pest control

Caffeine (1,3,7–trimethylxanthine) is one of the most widely used plant secondary metabolites, primarily as a stimulant and an ingredient in drugs. In nature, caffeine is believed to function in chemical defense, acting as an antiherbivory and allelopathic agent, and therefore it might be employed to protect agriculturally important crop plants. In coffee plants, caffeine is synthesized from the precursor xanthosine in four steps, three N-methylations and removal of ribose. We had previously isolated genes encoding three distinct N-methyltransferases, and we demonstrated production of recombinant enzymes that yielded caffeine in in vitro reconstitution experiments. When these caffeine biosynthetic pathway genes were simultaneously expressed in tobacco plants (Nicotiana tabacum), caffeine was successfully produced up to 5 μg/g fresh weight in leaves. The leaves were unpalatable to tobacco cutworms (Spodoptera litura). This repellent action appeared to be more widely␣applicable to lepidopteran caterpillars as observed with small white (Pieris rapae) fed on Chinese cabbages that had been top-treated with caffeine. Our recent results suggest a novel approach to strengthen anti-herbivore traits by producing caffeine in crop plants.     

Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.     

An extraction method for tobacco mosaic virus movement protein localizing in plasmodesmata

Summary. The intercellular communication by plasmodesmata (PD) is important for the growth and development of plants, and the transport of macromolecules through PD is likely to be regulated by developmental signals. While PD in the apical meristem transport macromolecules such as mRNAs, the branched PD in the mature leaf do not transport large macromolecules freely. The changes in PD during development might be important for sink-to-source changes in leaves, but the molecular mechanism is still unknown. Movement proteins (MPs) of the tobacco mosaic virus localize in the branched PD and increase the size exclusion limit, allowing transport of viral RNA. We developed a method for differential extraction of MP from isolated cell walls of transgenic tobacco leaves expressing MP or MP tagged with green-fluorescent protein. Lithium chloride at a concentration of 8 M removed filamentous structures in branched PD, the possible attachment site of MP. As some endogenous proteins were coeluted with MP by the treatment, this extraction method might be a powerful tool for investigating MP-interacting proteins in branched PD. Correspondence and reprints: Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.     

Phenolic compounds and the hypersensitivity reaction in nicotiana tabacum infected with tobacco mosaic virus

The hypersensitivity of Nicotiana tabacum cv. Xanthi to tobacco mosaic virus infection leads to the production and accumulation of a great number of phenolics (flavonol glycosides, caffeoylquinic, feruloylquinic and p-coumaroylquinic acids, glucose esters and glucosides of cinnamic and benzoic acids). An increase in temperature inhibits the hypersensitive reaction, resulting in the disappearance of these substances. The differences between the healthy and infected leaves become important when the synthesis of the virus is practically brought to completion and the hypersensitivity taken hold. The phenolic compounds do not appear to be responsible for the necrotic hypersensitivity and their production is one of the secondary effects of the virus infection.     

Insight into Types I and II nonhost resistance using expression patterns of defense-related genes in tobacco

Plants protect themselves against pathogens using a range of response mechanisms. There are two categories of nonhost resistance: Type I, which does not result in visible cell death; and Type II, which entails localized programmed cell death (or hypersensitive response) in response to nonhost pathogens. The genes responsible for these two systems have not yet been intensively investigated at the molecular level. Using tobacco plants (Nicotiana tabacum), we compared expression of 12 defense-related genes between a Type I (Xanthomonas axonopodis pv. glycines 8ra) nonhost interaction, and two Type II (Pseudomonas syringae pv. syringae 61 and P. syringae pv. phaseolicola NPS3121) nonhost interactions, as well as those expressed during R gene-mediated resistance to Tobacco mosaic virus. In general, expression of most defense-related genes during R gene-mediated resistance was activated 48 h after challenge by TMV; the same genes were upregulated as early as 9 h after infiltration by nonhost pathogens. Surprisingly, X. axonopodis pv. glycines (Type I) elicited the same set of defense-related genes as did two pathovars of P. syringae, despite the absence of visible cell death. In two examples of Type II nonhost interactions, P. syringae pv. phaseolicola NPS3121 produced an expression profile more closely resembling that of X. axonopodis pv. glycines 8ra, than that of P. syringae pv. syringae 61. These results suggest that Type I nonhost resistance may act as a mechanism providing a more specific and active defense response against a broad range of potential pathogens.     

The biosynthesis of acetovanillone in tobacco cell-suspension cultures

A soluble enzyme, extracted from tobacco cell-suspension cultures 24 h after treatment with 100 μM methyl jasmonate, has been shown to synthesize acetovanillone (apocynin) from feruloyl-CoA in the presence of NAD. The enzyme displayed Michaelis-Menten kinetics with apparent K_m values of 5.6 μM for feruloyl-CoA and 260 μM for NAD and exhibited very high specificity for its substrates. The increase in acetovanillone synthase activity was followed by an increase in the concentration of both acetovanillone and acetosyringone in the culture medium. No intermediate could be detected when analysing the reaction medium by HPLC during the formation of acetovanillone in cell-free extracts. The apparent molecular mass estimated by gel permeation on an FPLC column was ca. 79 kDa. To our knowledge, this is the first report of an enzymic system catalysing the synthesis of an acetophenone. This work demonstrates that the biosynthesis of acetophenones in tobacco proceeds from hydroxycinnamic acids through a CoA-dependent β-oxidation pathway. Interestingly in methyl jasmonate-treated cells, which synthesize very large amounts of hydroxycinnamoylputrescines, inhibition of the synthesis of these conjugates increased the concentration of acetovanillone and acetosyringone in the culture medium, suggesting that the two metabolic pathways can compete for their common precursors, i.e. hydroxycinnamoyl-CoA thioesters.     

Molecular tagging of the tobacco chromosome carrying the TMV-resistance gene (N gene) by Agrobacterium-mediated transformation

Summary The hypersensitive response of tobacco to inoculation with tobacco mosaic virus (TMV) is controlled by a single dominant gene, the N gene. As a first step in localizing and transferring the N gene, we have prepared a line of tobacco plants in which the kanamycin-resistance (Km^r) gene is closely linked to the N gene. Nicotiana tabacum plants heterozygous for the N gene were transformed to Km^r by Agrobacterium carrying pMON200. Eighty-nine independent transformed clones were regenerated and were backcrossed with nontransformed, TMV-sensitive plants. Progeny from these crosses were screened first for Km^r; then the Km^r progeny were inoculated with TMV and scored for the hypersensitive response. Of the initial 89 clones, 68 appeared to have integrated a single functional Km^r gene. Initial tests for TMV resistance indicated possible linkage between Km^r and the N gene in 11 plants. With further testing, linkage has been established for two of these plant lines. In one of these lines, the two genes were 30–40 map units apart, and evidence of somatic instability in the linkage was obtained. However, in the second line, linkage between Km^r and the N gene was tight, and recombination between the genes in this case was only 5%. Southern hybridization revealed that this plant contained only a single copy of the Km^r gene. Linkage between Km^r and the N gene in this plant line has been verified in each of two additional backcross generations.Abbreviations nptII Neomycin phosphotransferase gene - Km^r kanamycin resistant - Km^s kanamycin sensitive - TMV tobacco mosaic virus - TMV-R TMV resistant - TMV-S TMV sensitive     

Interconversions of different forms of vitamin B6 in tobacco plants

There are six different vitamin B₆ (VB₆) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′-phosphate (PLP), pyridoxamine 5′-phosphate (PMP), and pyridoxine 5′-phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB₆ in the human diet, and VB₆ plays an important role in plants, the mechanisms underlying the interconversions of different VB₆ forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100 mg/L of PM, PL or PN and the abundance of the different B₆ vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB₆ was about 3.9 μg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB₆ by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B₆ vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of non-phosphorylated forms.     

H2O2参与AM真菌与烟草共生过程

以烟草((Nicotiana tabacum,品种CF90NF)为寄主,苗期接种丛枝菌根(AM)真菌摩西球囊霉(Glomus mosseae,G.m),测定G.m与烟草共生过程中烟草根部H2O2含量以及多胺氧化酶(PAO)和过氧化物酶(POD)活性;研究外源H2O2对G.m侵染烟草的影响以及H2O2清除剂和合成抑制剂对烟草侧根H2O2含量及烟草侧根和菌丝中H2O2荧光强度的影响,以探究H2O2在AM真菌侵染烟草过程中的作用。结果表明,接种G.m 20d后烟草侧根中出现H2O2含量的猝发,一定浓度的外源H2O2促进G.m对烟草的侵染,而H2O2清除剂抗坏血酸(AsA)显著削弱烟草侧根和菌丝中的H2O2荧光强度,降低G.m对烟草的侵染率,表明H2O2参与G.m与烟草共生过程;在G.m与烟草共生过程中,PAO和POD活性显著升高,PAO抑制剂二氨基十二烷(DADD)和POD抑制剂水杨羟肟酸(SHAM)显著降低烟草侧根中H2O2荧光强度,对菌丝中H2O2荧光强度无显著影响,表明烟草根部和G.m均可产生H2O2,PAO和POD参与烟草侧根中H2O2的合成,菌丝中可能存在其他来源的H2O2。     

A multi-generation analysis of the stability of transgenic virus resistance in doubled-haploid tobacco lines

Plants can be genetically engineered for virus resistance by transformation with a viral gene. We transformed tobacco with the tomato spotted wilt virus (TSWV) nucleocapsid gene from the Hawaiian L isolate in order to obtain TSWV resistant breeding lines. Doubled-haploid lines were produced from primary transgenic plants that were selected for resistance to the virus. Several of these lines showed very high levels of resistance and were symptomless after inoculation with the Hawaiian L isolate of TSWV. The accumulation of only low levels of full-length transgene RNA and protein observed in these lines is consistent with an RNA-mediated mechanism of resistance. The lines that were highly resistant to the Hawaiian L isolate of TSWV were also found to be highly resistant to several other isolates of TSWV, while lines that were only moderately resistant to the Hawaiian L isolate were often susceptible to the other isolates. The highly resistant lines were advanced over several generations by self-pollination. Although these lines were fully homozygous, several lines lost resistance in later generations, indicating that the resistance was unstable. Selection for resistance in these unstable lines did not prevent the occurrence of susceptible progeny in subsequent generations. Therefore, testing over several generations is required to determine the stability of resistance when breeding crops with transgenic virus resistance.     

Characterisation and immunolocation of an 87 kDa polypeptide associated with UDP-glucuronic acid decarboxylase activity from differentiating tobacco cells (Nicotiana tabacum L.)

UDP-glucuronic acid decarboxylase catalyses the reaction responsible for the formation of UDP-xylose and commits assimilate for the biosynthesis of cell wall polysaccharides and glycosylation of proteins. Xylose-rich polymers such as xylans are a feature of dicot secondary walls. Thus a cell culture system of tobacco transformed with the ipt gene from Agrobacterium tumefaciens for cytokinin production and which when manipulated with auxin and sucrose leads to induction of xylogenesis, has been used as a source for purification of the enzyme. UDP-glucuronic acid decarboxylase was purified by ion-exchange, gel filtration and affinity chromatography on Reactive Brown-Agarose. The native enzyme had an apparent M(r) of 220,000 which yielded a single subunit of 87,000 when analysed on SDS-PAGE using silver staining. This appears to be a novel form of the enzyme since a gene family encoding polypeptides around M(r) 40,000 with homology to the fungal enzyme also exists in plants. Using an antibody raised to the native 87 kDa form of the enzyme, this decarboxylase was localised mainly to to cambium and differentiating vascular tissue in tobacco stem, consistent with a role in the provision of UDP-xylose for the synthesis of secondary wall xylan. Further analysis using immunogold electron microscopy localised the 87 kDa UDP-glucuronic acid decarboxylase to the cytosol of developing vascular tissue.     

Expression of tobacco mosaic virus RNA in transgenic plants

Summary Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in planta.     

Purple acid phosphatase in the walls of tobacco cells

Purple acid phosphatase isolated from the walls of tobacco cells appears to be a 220 kDa homotetramer composed of 60 kDa subunits, which is purple in color and which contains iron as its only metal ion. Although the phosphatase did not require dithiothreitol for activity and was not inhibited by phenylarsine oxide, the enzyme showed a higher catalytic efficiency (k_cat/K_m) for phosphotyrosine-containing peptides than for other substrates including p-nitrophenyl-phosphate and ATP. The phosphatase formed as a 120 kDa dimer in the cytoplasm and as a 220 kDa tetramer in the walls, where Brefeldin A blocked its secretion during wall regeneration. According to our double-immunofluorescence labeling results, the enzyme might be translocated through the Golgi apparatus to the walls at the interphase and to the cell plate during cytokinesis.     

Antibody-mediated improved resistance to ClYVV and PVY infections in transgenic tobacco plants expressing a single-chain variable region antibody

Transgenic Nicotiana tabacum plants expressing a single-chain variable region antibody fragment derived from a broad-spectrum monoclonal antibody 3-17 showed suppression of virus infection following challenge by two distinct potyviruses: potato virus Y strain D, and clover yellow vein virus strain 300. Monoclonal antibody 3-17, which was raised against the potyvirus Johnsongrass mosaic virus, was shown to react strongly with 14 potyvirus species. Two different single-chain antibody constructs were used to produce chimeric genes encoding recombinant proteins designed to be targeted either to the apoplasm or to the cytoplasm. Transgenic plant lines showed reduced numbers of local lesions and systemic symptoms when challenged with potato virus Y, strain D and reduced local lesions following challenge with clover yellow vein virus, strain 300. The level of suppression conferred by the transgene when plants were challenged under laboratory conditions with high concentrations of virus, together with the ability of the transgene to partially protect plants against distinct viruses suggest that one single-chain gene construct might be used to protect plants from distinct potyviruses.