How To Grow Tobacco At Home

Tobacco Growing Made Easy

Everything you need to know is explained in Tobacco Growing Made Easy. There is no time like the present to start your tobacco crop. You will however, need the information in this guide to get off to the best possible start. You could hunt the internet for months without even coming close to the amount of good information and tips in this guide. You will learn: Which seeds produce the best tobacco How to make a sand mixture to disperse tobacco seeds. How much light you should allow for optimum results. How to water your seedlings so they don't drown. The easiest way to germinate tobacco seeds Simple techniques for producing the largest tobacco plants Hands free maintenance allowing you to set it and forget it The very best time for harvesting Drying and curing for maximum flavour and quality The different types of tobacco available to you. How to choose the best seeds for the best plants. The truth about soil types and how they affect your plants. How to handle seedlings so that you do not damage them. How to avoid fungus and mould. Continue reading...

Tobacco Growing Made Easy Summary

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Distribution of Heavy Metals and Conjugating Ligands in Shoots

(e.g., Pb and As), Cd has a higher propensity to accumulate in shoots other than the roots. Still, there is normally more Cd in roots than in leaves, and even less in fruits and seeds (Wagner 1993). The tendency of tobacco plants to translocate Cd quite efficiently to the leaves contributes to the fact that tobacco smoke is an important Cd source for smokers (Lugon Moulin et al. 2004). But recently, some research showed that tobacco develops an original mechanism of metal detoxification by the exudation of metal Ca-containing particles through leaf trichomes (Choi et al. 2001 Choi and Harada 2005 Sarret et al. 2006).

Genetic manipulation for the improvement of NUE

Amenziane et al. (2000) introduced the gdhA gene from Escherichia coli encoding a NADPH-GDH into tobacco plants and expressed under the control of 35S promoter. The introduced NADPH-GDH was apparently able to assimilate some of the excess NH4+, resulting from the increase in free amino acids, carbohydrates, and biomass production in the transgenic plants grown under controlled conditions or in the field. Transgenic wheat plants expressing the a-isoform of the Chlorella NADPH-GDH gene (aminating GDH) showed an increase in grain yield (Schmidt R., personal communication). Since the gene product contains a chloroplast-targeting transit peptide, the translated a-isoform GDH is localised in chloroplasts in the transgenic wheat. It has been shown that NH3 emission from barley leaves (Mattsson and Schjoerring 1996) and oil seed rape (Mattsson et al. 1997) occurs in plants grown hydroponically with 2 mM NH+ in the light, but not with NO3-. An inhibitor of GS increased the emission, while an...

Modifications Induced by Stress Could be Inheritable

As discussed above, the rate of somatic recombination in plants increased in response to stress. Molinier et al. (2006) suggested that the transfer of epigenetic information between generations provided a memory of environmental stresses experienced in earlier generations. This memory of exposure to ultraviolet radiation and flagellin, a bacterially derived elicitor, resulted in increased somatic homologous recombination, which was inheritable in successive generations. Boyko et al. (2007) analysed the progeny of TMV-sensitive tobacco plants, the parental lines of which were infected with TMV. It was found that these plants had a higher frequency of rearrangements in the loci containing R-genes for pathogen recognition and alterations in global genome and R-gene loci-specific methylation. In those cases where ME change their position or are amplified, the variations promoted are inheritable, although the rate of reversion in some transposons can be high. Also the genomic changes which...

Small Molecules Involved In Signaling And Execution Of Hr Cell Death

In animal cells, nitric oxide (NO) is known to act as second messenger in concert with ROI in processes such as the innate immune response, inflammation, and PCD. Recently it was shown that NO might also play an important role in the regulation of defense responses in plants. Infection of resistant, but not susceptible, tobacco plants with tobacco mosaic virus (TMV) resulted in enhanced NO synthase activity (Durner et al., 1998). Furthermore, external application of NO induced salicylic acid accumulation and PR gene expression, and NO inhibitors blocked both effects. This suggests that several critical players of animal NO signaling, such as cyclic GMP or cyclic AMP-ribose, are also operative in plants (Durner et al., 1998 Klessig et al., 2000). In cultured soybean (Glycine max L.) cells, it was demonstrated that the efficient induction of HR cell death required a balance between ROI and NO production, whereas unregulated NO production was not sufficient to induce HR cell death...

Acclimation of Root Growth Towards Alterations of Light Intensity

Shown that a very pronounced and characteristic reaction of root growth occurs when light intensity is increased (Nagel et al. 2006) During the first three hours after increase of light intensity by a factor of five, a characteristic fluctuation of root tip growth velocity was observed that was connected to parallel decreases and increases of expansion within the meristematic zone and the zone of cell elongation. The fluctuations were caused by a superposition of a transient, hydraulic decrease of growth activity due to increased transpiration and an accelerating increase of growth activity induced by sucrose import. Experiments with tobacco plants that had a decreased activity of sucrose-phosphate phosphatase (Chen et al. 2005) showed far less pronounced growth reactions. As the total increase of root growth activity by a factor of four within four days exceeded the increase of shoot growth acceleration by far, a significant shift of root-shoot-ratio was detected as a consequence of...

Roprac Gtpase is a key regulator for innate immunity

In tobacco, expression of DN-OsRAC1 delays lesion formation in Ngene-mediated resistance to tobacco mosaic virus, which is correlated with reduced level of ROS production, altered PR gene expression, and reduction of salicylic acid accumulation (Moeder et al., 2005). In addition, DN-OsRAC1 also suppresses Pto gene-mediated hypersensitive responses and nonhost resistance to Pseudomonas syringae pv. maculicola (Moeder et al., 2005). However, systemic acquired resistance is not affected in the DN-OsRAC1 tobacco plants. Thus, the effects of ROP GTPase may change depending on kinds of defense responses and plant species. Transgenic tobacco plants carrying an antisense construct of a Medicago sativa RAC cDNA inhibit necrotic lesion that is induced by infiltration with a yeast elicitor (Schiene et al., 2000). Taken together, it is most likely that ROP genes have general roles in disease resistance in plants.

Temporal expression of the candidate pathways

The general phenylpropanoid pathway provides activated CoA esters of cinnamic acid derivatives for the biosynthesis of flavonoids, lignin and other phenolic compounds via specific branch pathways (Fig. 2). The first enzyme in the general pathway, phenylalanine ammonia-lyase (PAL), has been localised to the tapetum (Herdt, Stitfeld & Wiermann, 1978 Kehrel & Wiermann, 1985). The tapetum is also the site of flavonoid pigment synthesis (Herdt et al., 1978 Beerhues et al., 1989 van der Meer et al., 1992) indicating that the general pathway and the flavonoid branch are active in this tissue. This was also shown directly by the antisense RNA inhibition of chalcone synthase expression within the tapetum of petunia plants which prevented the synthesis of flavonoid pigments and caused male sterility due to an arrest in pollen development (van der Meer et al., 1992). Promoter-gus fusions of the Arabidopsis PALI gene (Ohl, 1990) and the parsley 4-coumarate coenzyme A ligase-1 gene (Hauffe...

The Role Of Cell Organelles In Hr Induction

Strengthened by the observation that the HR phenotype of several Arabidopsis lesion-mimic mutants, including lsdl and lsd3, is suppressed under short-day conditions (Dietrich et al., 1994). Likewise, catalase-deficient tobacco plants exposed to high light intensities developed necrotic lesions, which may be caused by excess ROI produced by chloroplast metabolism (Chamnongpol et al., 1996). In agreement with the assumption that perturbation of photosynthesis or other chloroplast functions may induce HR-like cell death is the recent characterization of the Arabidopsis ACD2 gene, which encodes a red chlorophyll catabolite reductase (Mach et al., 2001). It has been suggested that the cell death phenotype of acd2 mutant plants is caused by the accumulation of chlorophyll breakdown products, which might be specific triggers of cell death or might function by their ability to absorb light and generate free radicals (Mach et al., 2001). Recently, the plastid-localized protein DS9 was shown to...

General Anatomical Characteristics Of In Vitro Plants

The mesophyll layer of the leaf consists of parenchymatous tissues situated between the two epidermal layers of the leaf. It usually undergoes differentiation in order to form photosynthetic tissues and thus contains chloroplasts. Another important factor of the mesophyll layer is the presence of well-developed intercellular spaces which facilitates the exchange of gases. Thus for efficient photosynthesis not only the number of chloroplasts but the dimensions of intercellular spaces also play an important role. Zobayed et al. (2001b) showed that leaves of cauliflower and tobacco plants grown photomixotrophically in well sealed vessels exhibited a lack of well defined palisade and spongy mesophyll layers and the cells were more closely packed with smaller intercellular spaces compared to those grown in well aerated vessels (Figure 5). In contrast when grown photomixotrophically in aerated (diffusive and forced) vessels and in vivo both the species showed more structural integrity in...

Development Of Transgenic Plants Exploitation Of Hr For Disease Control

Although artificial generation of HR seems a promising approach to control biotrophic and hemibiotrophic pathogens, it may be counterproductive with respect to necrotrophic pathogens, which apparently depend on dead host cells for growth. Accordingly, suppression of HR cell death appears to be an appropriate measure to limit these types of pathogens. Experimental evidence for the validity of this concept has recently been obtained. Transgenic tobacco plants expressing negative regulators of apoptosis, such as the human Bcl-2, human Bcl-XL or nematode Ced-9 genes, exhibited resistance to several necro-trophic fungal pathogens, including Sclerotinia sclerotiorum, Botry-tis cinerea, and Cercospora nicotianae, as well as to tomato spotted wilt virus (Dickman et al., 2001). Plants harboring Bcl-XL with a loss-of-function point mutation did not protect against pathogen challenge, demonstrating that resistance was dependent on a functional transgene and was not due to unspecific stress...

Enzymatic Components of the ROSScavenging Pathways of Plants

Yoshimura et al. (2004) reported that in trans-genic tobacco plants expression of GPX protein in the chloroplast was more effective at providing stress tolerance than the expression of the same protein in the cytosol. Studies that aimed at developing transgenic plants expressing several chloroplast-targeted antioxidant enzymes simultaneously showed the effectiveness of manipulating more than one gene in improving plant resistance to stress. Two genes encoding MnSOD and GR were inserted into the plas-tome of tobacco plants. Transgenic plants over-expressing MnSOD showed an increased resistance to methyl viologen and UV-B stress, while GR over-expressing plants were more tolerant. The second gene chosen for this investigation was Escherichia coli gor encoding GR. From previous studies it seemed clear that GR of bacterial origin could function in plant chloroplasts to increase both the GSH GSSG ratio and the total glutathione pool (Noctor et al. 1998), and gluta-thione itself is an...

Molecular biological studies of carotenoids

Developmentally regulated transcription was found to be the major mechanism that controls carotenogenesis in fruits and flowers. Merav et al. (2000) have cloned plant genes for carotenoid biosynthesis enzymes and analysed the regulation of their expression. Developmentally regulated transcription was found to be the major mechanism that controls carotenogenesis in fruits and flowers. To alter the accumulation of carotenoids they have genetically manipulated the pathway in tobacco and tomato. To this end they have over-expressed the following genes Ipi (isopentyl pyrophosphate isomerase), Psy (phytoene synthase), CrtB (phytoene synthase from cyanobacteria), CrtI (phytoene desaturase from Erivinia herbicola), Pds (phytoene desaturase from tomato) and CrtO ( 3-carotene ketolase from H. pluvialis). These genes were fused to various flower-specific promoters and a transit peptide from the tomato PDS was used in the case of the bacterial and algal genes. Petals of transgenic tobacco plants...

RNAinduced silencing complex RISC

A detailed analysis revealed that, under standard greenhouse growth conditions, spontaneous co-suppression was triggered at various frequencies (ranging from 5 to 42 ) between individual Class-II lines (Palauqui & Vaucheret, 1995). However, for all lines, triggering consistently occurred during a phenocritical period ranging from 15 days post-germination to flowering. In addition, the incidence of co-suppression between individuals from each line was increased if plants were grown in vitro prior to their transfer in greenhouse. Similar observations were made with co-suppressed lines of tobacco plants expressing a nitrite reductase (Nii) transgene (Palauqui & Vaucheret, 1995). These findings indicate that the physiological state of the plant (i.e. transition from vegetative to reproductive growth) and environmental factors exert a critical influence upon activation of spontaneous systemic silencing. In addition, triggering of Nia and Nii silencing was found to occur exclusively...

Exogenously induced systemic silencing

Upon bombardment, GFP silencing progressively radiated around those cells and formed macroscopically detectable red fluorescent foci resembling the chlorotic spots observed on mature leaves of the Class-II Nia co-suppressed plants (Voinnet et al., 1998). Silencing eventually reached a vein from which it was transmitted to remote parts of the plants that, eventually, became uniformly silenced for GFP. Biolistic delivery of Nia DNA constructs was also shown to trigger localized and systemic silencing in non-silenced Class-II tobacco plants (Palauqui & Balzergue,

Leaf Senescence and Proteomics

Studying leaf senescence with proteomics is just at the initial phase. Using TEM, Wilson et al. 26 observed that the most obvious ultrastructural changes during senescence occurred in chloroplasts of white clover leaves with progressive loss of thylakoid integrity and accumulation of osmiophilic globules in the stroma. The leaf proteins were separated by 2-DGE and verified by MALDI-TOF-MS. Out of 590 2D gel spots, 40 of them showed significant senescence-related changes in abundance. Moreover, approximately one-third of those senescence-responsive spots with decreased abundance appeared in the 2D gel profiles of the isolated chloroplasts. TMS analysis demonstrated those spots to be chloroplast proteins, including RuBisCO LSU and SSU, a RuBisCO activase, glutamine synthetase, and the 33-kDa protein of the PS II OEC. Since those proteins are key elements in photosynthesis, the decline in their expression levels emphasized the importance of proteolysis, chloroplast degradation, and...

Hydroxy Fatty Acids Which May Affect Fungal Growth

A number of long chain fatty acids were found in grass exudates (Dormarr et al. 2002) and Lupinus cultivar exudates (Lucas Garcia et al. 2000) and furthermore, a recent analysis has found decanoic acid, hexadecanoic acid, tetradecanoic acid, octade-cenoic acid some of their methyl esters in root extracts of transgenic tobacco plants (Mungur et al. 2005). Also found were some hydroperoxy derivatives and hydroxy stearic acid. A number of hydroxylated forms of these fatty acids were found in carrot root exudates and they were elevated in exudates of roots grown under Pi stress (Table 6). This is the first evidence that a variety of hydroxy fatty acids may be found in root exudates. Virtually nothing is known about hydroxy fatty acids in plant exudates but a hydroxylated form of palmitic acid, 2-hydroxyhexadecanoic acid (2-HHDA) is a functionally active component of sphingolipids which stimulate the production of fruiting bodies of the fungus Schizophyllum commune (Kawai et al. 1986).

Developmental regulation of late pollen gene promoters

The earliest examples of such analyses showed that the promoters of the tomato lat52 and lat59 genes were coordinately activated in developing pollen grains in close association with PMI (Twell, Yamaguchi & McCormick, 1990). Furthermore, the patterns of accumulation of GUS activity in developing pollen were very similar when identical promoter fusions were introduced into tomato and tobacco (Twell et al., 1990). Recently, more detailed histochemical analysis of transgenic tobacco plants showed that the lat52 and a 59 promoters were first activated soon after PMI prior to the migration of the generative cell into the interior of the vegetative cell (Fig. 3 Eady, Lindsey & Twell, 1994). Detailed developmental analysis of the promoter of the maize homologue of the lat52 gene, Zml3, in transgenic tobacco are in strict accord with these data (Guerrero et al., 1990), which suggests that regulatory mechanisms for late pollen promoter activation are conserved among species which shed...

Propagation of systemic RNA silencing 3341 Longdistance movement of RNA silencing

The speed of translocation of the silencing signal from leaf to vasculature was assessed in the GFP transgenic system by removal of the infiltrated leaf 1, 2, 3, 4 or 5 days after infiltration of the A. tumefaciens strain (Voinnet et al., 1998). There was systemic loss of GFP fluorescence in 10 of the plants if the infiltrated leaf was removed 2 days post-infiltration. A progressively higher proportion of plants exhibited systemic silencing when the infiltrated leaf was removed 3 days postinfiltration or later. Similar values were obtained with bombardment experiments carried out in the Class-II Nia tobacco plants (Palauqui & Balzergue, 1999). These observations indicate that 2-3 days are sufficient for the signal to accumulate and translocate into the phloem long-distance transport stream. Grafting experiments were also used to address the molecular requirements for the suppression effect of the phloem-transported signal in the Nia tobacco plants (Palauqui et al., 1997). It was...

Mitochondrial movement and cellular inheritance

These cell cycle-dependent changes in mitochondrial morphology are also evident in plants and algae. Using tobacco plants, Sheahan et al. (2004) showed that the size and morphology of mitochondria in cultured protoplasts varied with the cell cycle. Mitochondria are typically small and numerous when observed after protoplast isolation, but within 4 h of protoplast culture, the majority of mitochondria undergo MMF (see Section 1.4.2 Sheahan et al., 2005) to form a reticulum. While the volume of mitochondria (measured by GFP fluorescence) remained static, the number of individual organelles fell slightly for the first 24 h, indicating a net rise in fusion (Sheahan et al., 2004). From 48 h onwards, the reticulum began to fragment leading to a rise in the number of physically discrete mitochondria (Sheahan et al., 2004). This process continued so that by 72 h of culture (when protoplasts are ready to divide), there was a net doubling of the number of physically discrete mitochondria in the...

Identification of SAGs

SAG12 was first isolated by differential screen of an Arabidopsis leaf senescence cDNA library by Gan (1995). It encodes an apparent cysteine proteinase, and its expression is highly senescence-specific (Lohman et al. 1994 Gan 1995). IPT is an enzyme that catalyzes the first committed and rate-limiting step of cytokinin biosynthesis, condensation of dimethylallyl pyrophosphate (DMAPP) and 5'AMP to isopentenyladenosine (IPA) 5'-phosphate (Gan and Amasino 1996). The SAG12 promoter was fused to IPT to form an autoregulatory cytokinin production system (Gan and Amasino 1995). At the onset of leaf senescence, the senescence-specific promoter activates the expression of IPT, resulting in an increase in the cytokinin concentrations in turn, this prevents the leaf from senescing. The inhibition of leaf senescence will render the senescence-specific promoter inactive to prevent cyto-kinins from accumulating to very high levels overproduction of cytokinins may interfere with other aspects of...

Activities and mechanism of activase

The reactivation of Rubisco, which has been inhibited with CA1P or by an extended period of catalysis, has not been characterized as extensively. However, reactivation after CA1P inhibition was also confirmed in planta by analysis of the light-induced increase in Rubisco activity with transformed tobacco plants having reduced levels of activase (Hammond et al., 1998a,b). While more direct evidence of enhanced sugar phosphate release by the activase has not been obtained, there is no reason to suspect that the same fundamental mechanism does not occur. of carbamylation at suboptimal CO2 concentrations (Portis etal., 1995). Based on a characterization of transformed tobacco plants expressing reduced amounts of the activase, it has even been suggested that the activase may also increase the catalytic activity of fully activated Rubisco (He et al., 1997). However, obtaining further support for this idea with studies in vitro that can resolve an actual increase in the intrinsic maximal...

Responses of Pollinators

Sexual reproduction of many plants species depends on pollination by honey bees, bumble bees, solitary bees, syrphid flies or moths (Klein et al. 2006). Herbivory in early stages of plant growth reduces the photosynthetic area of the plant, and may result in smaller plants and a shorter flowering period. This is possibly due to allocation of resources to defenses, rather than growth and reproduction (Poveda et al. 2003). Herbivory may affect the production of pollen and nectar, the quality of nectar, morphology of flowers and may reduce seed production (Lehtila and Strauss 1997 Hamback 2001 Poveda et al. 2003). Both nectar quality and quantity are parameters that determine the number and type of pollinators that are attracted to the plants (Potts et al. 2003). While extrafloral nectar is known to increase after her-bivory, it remains unknown whether herbivory also affects floral nectar production (Adler et al. 2006). Just a handful of studies have addressed the effect of herbivory on...

Development Of Transgenic Plants That Display Broad Resistance Against Pathogens

Transgenic tobacco plants expressing the elicitor protein crypto-gein under the control of a pathogen-inducible promoter developed a HR in a normally compatible interaction with P. parasitica var. nicotiana (Keller et al., 1999). These transgenic plants also displayed enhanced resistance to fungal pathogens that were unrelated to Phy-tophthora species (Keller et al., 1999). Furthermore, the oomycete infl and fungal Avr9 genes confer avirulence to PVX on tobacco and Cf-9 tomato, respectively (Kamoun, Honee, et al., 1999). These re

Responses of Neighboring Plants

Plants may gain a fitness benefit from responding to herbivore-induced volatiles from neighboring plants that are being attacked. Such a signal is indicative of the imminent danger, and the receiving plant may profit from exploiting this information by readying its defense (Dicke and Bruin 2001). Herbivore-induced plant signals can be exploited by neighboring plants of the same and of different species alike (Engelberth et al. 2004 Baldwin et al. 2006). Strong signals may immediately activate plant defenses, while lower (more common in nature) concentrations of signaling molecules can prime the plant for attack (Turlings and Ton 2006). This was nicely shown in a field study by Kessler et al. (2006) reporting that plant volatiles from damaged sagebrush plants can prime the induction of proteinase inhibitors in nearby tobacco plants. The primed tobacco plants received less damage from subsequent herbivore attack as compared to non-exposed control plants. Not only volatile emission, but...

Role Of Hormones On Iron Deficiency Stress Responses By Strategy Ii Plants

There are, however, some experimental results suggesting that both Strategy I and Strategy II plants could share some common signals in the induction of their Fe deficiency stress responses. Higuchi et al. (2001) found that the barley HvNASl nicotianamine synthase involved in NA (nicotianamine) synthesis see Figure 12-2 promoter-gus fusion gene was induced in transgenic tobacco plants under Fe deficiency. Similarly, Yoshihara et al. (2003) showed that the Fe deficiency responsive gene, Ids2, of barley was induced under Fe deficiency in transgenic tobacco plants. These results clearly indicate that some signals involved in the induction of Fe deficiency stress responses by Strategy I plants could be recognized by the promoters of the genes involved in Fe deficiency stress responses by Strategy II plants. Curiously, the promoter region of some NAS genes of Arabidopsis thaliana has an ethylene-responsive element (Suzuki et al., 2001).

NR and photosynthesis

Negative feedback on NIA expression (Sivasankar and Oaks 1996, Coruzzi and Bush 2001). In leaf discs of wild type tobacco plants the NR mRNA level was low when discs were fed with glutamine, whereas feeding sucrose or a-ketoglutarate resulted in a high NR mRNA level. In transgenic plants expressing antisense GOGAT, and thereby possessing only 10 of wild type GOGAT activity, the NR mRNA level was high in spite of high in situ concentrations of glutamine (Ferrario-Mery et al. 2001). However, in these transgenic plants the concentration of a-ketoglutarate was also high. It appears that the expression of NR depends mostly on the ratio between glutamine and a-ketoglutarate, and the inhibiting effect of glutamine on transcription is overcome by high concentration of a-ketoglutarate. The experiments of Ferrario-Mery and co-workers are strongly supportive of PII (see above) being involved in regulation of nitrogen metabolism in plants, and PII may be a missing link between photosynthesis and...

MT Might Not Represent the Initial Target of MP

Interestingly, it was shown that transgenic tobacco plants overexpressing MP contain fibrous MP-associated material in the cavity of their PD (Ding et al. 1992a Lapidot et al. 1993 Moore et al. 1992), which may be consistent with an ability of MP to cause the formation of a cytoskeletal intercellular transport structure within PD. This hypothesis may be supported by the analysis of multicellular cyanobacteria overexpressing MP, where cytoskeletal, likely FtsZ-associated, filaments across the intercellular septa have been observed (Heinlein 2006 Heinlein et al. 1998b Boutant and Heinlein, unpublished results). Potentially related to a cytoskeleton-manipulating ability of MP may also be the observation that infected plant protoplasts, or protoplasts transiently expressing the MP, form plasma membrane protrusions into the medium (Heinlein 2002 Heinlein et al. 1998a), a behavior that may reflect a process by which MP causes the formation of a transport structure in the PD of walled cells....

Signals Originating from Photosynthetic Electron Transport

Accumulation which usually occurs after sugar depletion. This implies a connection between PET and sugar signalling in Arabidopsis. In winter rye Lhcb gene expression was found to be regulated by redox signals from PET which were induced by varying light and temperature regimes (Pursiheimo et al. 2001) . The authors concluded that the redox state of electron acceptors at the PSI (Photosystem I) acceptor site were a regulating parameter of nuclear gene expression under these conditions. In transgenic tobacco plants carrying a pea PetE gene construct it could be demonstrated that the PetE construct as well as endogenous Lhcb1 transcripts decreased upon DCMU treatment. In contrast, nuclear run-on transcription assays indicated up-regulation of the pea PetE construct expression suggesting multiple parallel influences at different levels of gene expression (Sullivan and Gray 2002). Redox regulation of post-transcriptional processes was also uncovered. In transgenic tobacco a pea...

Activator of NPK1 Mapkkk Nack1 a KLP

We postulated that overexpression of a truncated form of NACK1 (NACK ST) that contained the stalk (ST) region might have a phenotypic effect similar to overexpression of a kinase-defective NPK1 on cytokinesis if the direct binding of NACK1 is required for the localization and activation of NPK1. As expected, both BY-2 cells and tobacco plants overexpressing NACK ST have many multinucleated cells with incomplete cell plates (Fig. 4A Nishihama et al. 2002). In these BY-2 cells, time-lapse observations of cytokinesis reveals that expansion of cell plates is markedly suppressed (Nishihama et al. 2002). We therefore concluded that NACK1 regulates plant cytokinesis by activating NPK1 MAPKKK. Fig. 4 The involvement of the NACK-PQR pathway in plant cytokinesis. A Generation of bi- or multi-nucleate stomata and pavement cells upon expression of a motor domain-less NACK1 (+DEX) in tobacco plants (bottom panels). Bar, 10 im. Seeds of a plant transformed with the...

Identification of Other Components of the MAPK Cascade Activated by the Nacknpk1 Complex

The requirement of NQK1 for the expansion of the phragmoplast and for the formation of cell plates was demonstrated by overexpressing a kinase-defective mutant of NQK1 (NQK1 KW) in BY-2 cells and tobacco plants. The cells overexpressing NQK1 KW are multinucleated and form incomplete cell plates (Soyano et al. 2003). Also, the seedlings of the NQK1 KW transgenic plants are poorly developed, and its surfaces have bi- or un-nucleated guard and pavement cells, similar to those of plants overexpressing NACK ST. We observed consistent abnormalities in the cells in which the kinase-defective NQK1 was overexpressed therefore, NQK1 MAPKK seems to promote progression of cytokinesis downstream of NPK1 MAPKKK.

Mechanisms That Protect Pathogens From Pr Proteins

A gradual adaptation of the pathogen to PR proteins in the host is the other type of escape mechanism often suspected, especially when overexpression of the native PR-protein genes in the same host is attempted. It is a possible mechanism by which transgenic plants constitu-tively overexpressing a PR-protein gene may not exhibit adequate levels of resistance to infection and disease. Transgenic tobacco plants over-expressing a basic chitinase in large quantities did not show resistance to frog eye spot pathogen. It might be due to the adaptation of the fungal pathogen to the host defense mechanism (Neuhaus et al., 1991).

Involvement of Plant Antioxidant Systems in ROS Signaling

The activation of a MAPK (mitogen activated protein kinase)-like cascade by hydrogen peroxide is another characteristic that plants share with animals. The recognition of an extracellular signal might trigger GTP-binding proteins as effectors for the activation of a MAP kinase kinase kinase (MAPKKK) that phosphorylates a MAPKK that activated the phosphorylation of a MAPK. The phosphorylation of the MAPK induces its translocation to the nucleus, where it modulates gene expression through the phosphorylation and consequent activation of transcription factors (Hirt 1997). Among the genes induced by this pathway, those coding for glutathione transferase, glutathione peroxidase, ascorbate peroxidase as well as for heat shock proteins have been identified (Vranova et al. 2002, Noctor and Foyer 1998, Kovtun et al. 2000). The constituents of the MAPK cascade have been identified in tobacco plants (Wilson et al. 1998). Studies with tobacco mutants constitutively expressing MAPKK indicate that...

Regulation of Cytokinesis by a MAP Kinase Pathway

NPK1 cDNA has been cloned from tobacco cells and has been shown to encode a member of the MAPKKK family (Banno et al. 1993). The promoter of the gene for NPK1 is active mainly in shoot and root apices in the tobacco plant, suggesting a role in cell proliferation (Nakashima et al. 1998). The NPK1 protein is localized to the nucleus during interphase when the cells enter M-phase and the phragmoplast is formed from anaphase to cytokinesis, it is localized to the equatorial region of the phragmoplast (Nishi-hama et al. 2001 Ishikawa et al. 2002). The activity of the protein kinase is cell cycle-dependent and increases from anaphase to cytokinesis (Nishi-hama et al. 2001). The amount of protein and the activity of NPK1 decline markedly at the end of cytokinesis (Nishihama et al. 2001). Overexpression of the kinase-negative version of NPK1 in tobacco cells and plants results in multinucleate cells that contain stubs of cell walls but does not affect karyoki-nesis, suggesting that NPK1 plays...

Analysis of Glycopeptides

It is rather difficult to identify glycopeptides using MS in a complex protein digest. Glycopeptide signals are often suppressed in the presence of other peptides. For this reason, the peptide and glycopeptide mixture can be separated by RP chromatography. Fractions are collected and analyzed using MS. Samyn-Petit et al. 33 used this strategy for the comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants. The different HPLC fractions were collected and analyzed by MALDI-TOF. Glycopeptides were then identified, based on theoretical masses of peptides from the reduced and carboxamidomethylated lactoferrin with various additional N -glycans. After deglycosylation, the peptides were also analyzed by nESI-Q-TOF-MS MS to identify the glycosylation site. ESI-MS can also be used as an on-line HPLC detector for tryptic mapping of glycoproteins.

Evaluation Of The Biotechnological Use Of Plant Ferritin Overexpressors

Indeed, the influence of various soil conditions on the increase in leaf Fe content of various tobacco plant genotypes has been recently tested (Vansuyt et al., 2000). One control transgenic tobacco and two transgenic tobaccos overexpressing ferritin in the plastids or in the cytoplasm, respectively, were grown on five different soils, two of them being sewage sludge amended.

Airborne Induction ofResistance to Herbivores

Plant-plant communication was first reported from the Sitka willow Salix sitchensis (Rhoades 1983), poplar Populus x euroamericana, and sugar maple Acersaccharum (Baldwin and Schultz 1983). Since then, the phenomenon has been detected in the taxonomically unrelated species Arabidopsis thaliana, black alder (Alnus glutinosa), corn (Zea mays), lima bean (Phaseolus lunatus), sagebrush (Artemisia tridentata) and wild and cultivated tobacco (Nicotiana attenuata and N. tabacum) (Shulaev et al. 1997 Karban et al. 2000 Tscharntke et al. 2001 Engelberth et al. 2004 Choh et al. 2006 Karban et al. 2006 Kost and Heil 2006 Heil and Silva Bueno 2007b Ton et al. 2007 Godard et al. 2008). Most of these cases related to signaling among plants that belong to the same species, but plant-plant communication even occurs among different species for example, clipping sagebrush induced resistance in neighboring tobacco plants (Karban et al. 2000 Karban 2001 see Fig.1).

Airborne Induction ofResistance to Pathogens

Pathogens, and its volatile derivative, methyl salicylate (MeSA), has been put forward as the most likely mobile signal (Park et al. 2007). In tobacco, MeSA is enzymatically converted back to SA by SA-binding protein 2 (SABP2), and SA then forms the active resistance-inducing compound (Kumar and Klessig 2003 Forouhar et al. 2005). In principle, this also opens up the possibility of airborne signaling in the context of pathogen resistance. Resistance expression has indeed been reported in tobacco plants that were exposed to the MeSA-rich air from infected plants (Shulaev et al. 1997) and in lima bean plants exposed to VOCs released from resistance-expressing conspecifics (Yi, Ryu, Heil, unpublished data).

Plants and Atmospheric Pollution

Injury to vegetation by photochemical smog in the Los Angeles basin has been a long standing problem, and the most important phytotoxic agent in the smog has been found to be ozone. Extensive ozone damage has been observed in many parts of the USA (Heggestad and Middleton, 1959 Bormann, 1982). It is now accepted that ozone is the most damaging of the air pollutants to vegetation (Ashmore et al., 1987). In recent years it has become apparent that damage affecting forests in Germany has not been caused solely by acid depositions, as was once believed. Ozone concentrations have been shown to reach values that cause damage to trees in the USA, and it has been proposed that ozone and frost damage lead to a predisposition to attack by acid aerosols (Smith, 1986). Ozone toxicity appears as a brown or white flecking on the leaves. There is a wide variation in the sensitivity of species to ozone toxicity and in the sensitivities of cultivars of the same species. The high sensitivity of one...

Regulation of the Translation of Isoforms The Catalase

In many cases, there is a lack of information not only about the effect of oxidation on protein activity or function but also in the form that plant metabolism copes with protein oxidative modification. In this regard, an interesting mechanism consisting of increasing the synthesis of protein subunits less sensible to oxidation has been described in plants for the enzyme catalase. Catalase (CAT EC 1.11.1.6) is one of the main antioxidant enzymes that catalyzes the conversion of hydrogen peroxide to O2 and H2O, which in plants is localized inside peroxisomes. In plants, catalase activity shows a great degree of susceptibility to metal stress (Gallego et al. 1996, 2002 Balestrasse et al. 2001 Pandey and Sharma 2002 Singh et al. 2006). Moreover, catalase has been involved in Cd tolerance enhancement in plants, as was demonstrated in transgenic tobacco over-expressing a Cd-induced catalase cDNA from the hyperaccumulator species Brassica juncea (BjCAT3) (Guan et al. 2009). In contrast,...

Salicylic acid and jasmonic acid are signal molecules in pathogen defense

The biosynthesis of salicylic acid (SA) is described in section 18.2 and that of jasmonic acid (JA) in section 15.7. Jasmonic acid and salicylic acid are both involved in signal cascades induced during pathogen attack. SA plays a crucial role in defense responses against biotrophic pathogens (which keep the cell alive), and hemi-biotrophic pathogens (which initially keep the cell alive but kill them at a later stage). Mutants of transgenic tobacco plants,

Function of NDH in Photoprotection

Development of successful chloroplast transformation made it possible to analyze the function of NDH using gene disruption in higher plants. Four groups independently succeeded in the production of NDH-deficient transformants of tobacco (Burrows et al., 1998 Kofer et al., 1998 Joet et al., 1998 Shikanai et al., 1998). Unlike the Synechocystis mutants, the NDH-deficient tobacco plants can grow normally, at least under growth chamber conditions below 100 mol photons m-2 s-1. The common phenotype of all of these transformants is that they lack a transient increase in chlorophyll fluorescence in the dark after actinic illumination, which represents the transient reduction of the plastoquinone pool by reducing equivalents accumulated in the stroma during illumination (Asada et al., 1993 Mano et al., 1995). Thus, the lack of a post-illumination increase in chlorophyll fluorescence indicates that the NDH-deficient mutant does not have a pathway of electron transfer from the stromal reducing...

Initiation and progression of mitosis by CDKs

Overexpression of a dominant-negative CDKA of Arabidopsis in tobacco plants produced smaller plants, which is a result of reduced rate of cell division (Hemerly et al., 1995). Recently, it has been reported that the mutant of CDKA in Arabidopsis exhibited a defect in pollen mitosis during male game-togenesis (Iwakawa et al., 2006 Nowack et al., 2006). These results show that plant CDKA is required for entry into mitosis. Overexpression of a dominantnegative of Arabidopsis CDKB1 1 caused delayed G2 M transition in tobacco cells, suggesting that CDKB1 is also involved in mitotic entry (Porceddu et al, 2001).

Polyamines as stress protectants

Symptoms of extreme salinisation (Flores 1991). Similar results have been obtained in transgenic tobacco plants overexpressing oat ADC, under the control of a tetracycline-inducible promoter (Masgrau et al. 1997). Diverse mechanisms have been suggested to explain the protective effect of Spm and Spd. Plants sensitive to drought (Wang et al. 1990) or salinity (Morgan and Drew 1997) increase ethylene biosynthesis when exposed to stress. The fact that Spd and Spm reduce ethylene biosynthesis by inhibiting ACC synthase (Davis et al. 1991) could explain their protective effect. On the other hand, both Spd and Spm can interact with membranes inhibiting the movement of phospholipids through the lipidic bilayer (Bratton 1994), stabilising molecular complexes at the thylakoid membranes (Besford et al. 1993) and or inhibiting lipid peroxidation in osmotically-stressed oat leaves (Borrell et al. 1997).

Physiological Role of the Water Water Cycle

When activation of the Calvin cycle and steady state CO2-fixation have been attained, the flux through the water-water cycle is decreased to 10-30 (Asada, 2000), or below 10 of the total electron flux from PS II (Foyer and Noctor, 2000 Badger et al., 2000), although the level varies depending on the plant species and environmental conditions. Whether ATP production and PS II control by the water-water cycle is physiologically significant has been under debate (Osmond and Grace, 1995 Asada, 1999, 2000 Heber, 2002). Based on the generally low electron flux rates observed under steady state photosynthesis, Heber (2002) has proposed that the water-water cycle does not contribute much to controlling of PS II through the formation of a proton gradient across the thylakoid membranes. Even so, the water-water cycle is indispensable for the rapid scavenging of superoxide and hydrogen peroxide to protect plants from photoinhibition due to ROS. Tobacco plants in which expression of the key...

Primary metabolism and GSH

Malate is involved in a wide variety of primary physiological processes. Malate oxidation is accomplished through various enzymes including the NADP-dependent malic enzyme (E.C. 1.1.1.40.), which catalyses the oxidative decarboxylation of malate to pyruvate, producing C02 and NADPH. A promoter fragment of the gene encoding the malic enzyme was prepared from bean and fused to a gene encoding p-glucuronidase (GUS reporter gene). Tobacco plants were transformed by this chimeric gene construct and the regulatory properties of the promoter sequence were studied by detecting the histochemical staining of GUS activity. GSH (10 mM) strongly induced the GUS activity, i.e. the transcription from the promoter of malic enzyme, but GSSG, ascorbic acid, and a fungal elicitor were also effective promoter-stimulating agents (Schaaf et al. 1995). By the activation of malic enzyme the production of reducing equivalents (NADPH) can be increased. The rapid access to NADPH is of crucial...

Novel Strategies For Controlling Plantparasitic Nematodes

There has been considerable interest in using the strategy of silencing nematode genes by expression in planta of dsRNA corresponding to nematode genes in plants (Gheysen and Vanholme, 2007 Lilley et al., 2007). This approach was stimulated by evidence from several independent groups that genes can be silenced in both RKNs and cyst nematodes by soaking infective J2 in solutions of dsRNA corresponding to that gene (Chen et al., 2005 Rosso et al, 2005 Urwin et al., 2002). Because PPNs normally feed only on the cytoplasm of their feeding cells, a chemical stimulus was often used to facilitate the uptake of the dsRNA from the solution. These experiments stimulated efforts to test whether resistance to nematodes could be produced by in planta production of dsRNA. Yadav et al. (2006) found that expression of dsRNA corresponding to specific RKN genes could confer resistance in tobacco plants. Similarly, reduced reproduction of root-knot species was found in Arabidopsis and of H. glycines on...

Genes involved in the transport of iron chelators

The necessity for NA in the transport of Fe, including phloem loading unloading and the distribution of Fe from cells adjacent to the veins to the leaf lamina, has also been demonstrated in non-graminaceous plants through the observation of the tomato mutant chloronerva, which lacks NA as a result of a point mutation in the NAS gene (Becker et al., 1995 Higuchi et al., 1996 Ling et al., 1999). Moreover, transgenic tobacco plants overexpressing the HvNAAT gene exhibited phenotypes similar to that of the chloronerva mutant, including severe interveinal chlorosis in young leaves, flower deformity, and sterility, owing to the exhaustion of NA (Takahashi et al., 2003). The characterization of the transformants suggested the essential function of NA in intercellular and intracellular metal transfer in both vegetative and reproductive organs.

GST in bacterial infections

The role of GST was investigated also in pear and tobacco plants infected with Erwinia amylovora, the causal agent of fire blight disease. The bacterium caused a sustained oxidative stress and GST induction in both pear and tobacco leaves (in compatible and incompatible interactions, respectively). The unexpected ability of E. amylovora to generate oxidative stress even in a compatible situation was linked to its functional hrp gene cluster. It was suggested that E. amylovora utilizes the production of ROS as a tool to provoke host cell death to invade plant tissues (Venisse et al. 2001).

Chlorophyll Biosynthesis

Porphyrin pathway leading to heme and chlorophylls (Hu et al. 1998) (Fig. 1). In a similar vein, the down-regulation of tobacco, through an antisense technology led to the formation of necrotic lesions that were correlated with the accumulation of uroporphyrinogen due to the formation of reactive oxygen species under light conditions (Mock and Grimm 1997) . A very similar phenomenon was observed in transgenic tobacco plants, when the conversion of coproporphyrinogen III to pro-toporphyrinogen IX was inhibited through the antisense expression of copropor-phyrinogen oxidase III gene (CPO) (Mock et al. 1999) (Fig. 1). A characteristic feature of tobacco expressing antisense UROD and CPO is the accumulation of the fluorescent coumarin phytoalexin scopolin and the pathogenesis-related protein PR-1, which normally participate in defense responses (Mock et al. 1999) . This trend was reinforced by the fact that the level of salicylic acid was concomitantly increased. The same trend was...

Relation to other micronutrients

We observed an interesting 'mitigation effect' of Fe-deficiency symptoms in tobacco plants grown under a combined deficiency of Fe and other micronutrients (Kobayashi et al., 2003b). Tobacco plants grown under these conditions showed better growth and higher chlorophyll concentrations, as well as reduced levels of Fe-deficiency-induced expression of the HvIDS2 promoter, than did plants grown in media deficient only in Fe, even though the measured Fe concentration in plant tissues was essentially the same. This phenomenon is likely to be the opposite to the induced deficiency described above. Through physiological dissection of these plants, we deduced that this effect could be explained in part by the reduction of chelate competition, suggesting the importance of chelating molecules inside the plant body in metal homeostasis (Kobayashi et al., 2003b).

Regulation of Aquaporin Activity

Different results were obtained via reverse genetic strategies. When the specific hydraulic conductivity of tobacco plants with reduced expression of NtAQP1 and respective control plants was analyzed, a strong correlation between cellular water permeability and specific root hydraulic conductivity was observed (Siefritz et al. 2002). This indicates that the transcellular pathway is very important for vascular and long distance water transport and that strictly apoplastic transport through cell walls is of minor impact, at least in roots.

Reversible Male Sterility

Metabolic engineering of primary metabolites has targeted glutamine, carbohydrates, and pyruvate. The amino acid glutamine is essential for pollen development (Benito Moreno et al. 1988). Transferring into tobacco plants a gene driven by the tapetum-specific promoter TA29 and encoding a dominant-negative version of glutamine synthetase (dnGS) resulted in male sterility (Ribarits et al. 2007). Spraying plants with glutamine restored male fertility. Anthers of the male-sterile lines contained viable microspores, and restoration was also achieved by isolating the microspores, maturing these in vitro in the presence of glutamine, and using the in vitro matured pollen for pollination of emasculated flowers in situ (Touraev and Heberle-Bors 1999). A similar strategy has been tried in tobacco plants. Here, knocking down an extracellular invertase gene by an anti-sense construct resulted in carbohydrate deficiency of pollen (Goetz et al. 2001), but no seeds were obtained after pollination...

Induced Male Sterility

An alternative to reversible male sterility is induced male sterility. The strategy described by Kriete et al. (1996) involved transgenic tobacco plants expressing the bacterial argE gene specifically in the tapetum. When these plants were treated with the non-toxic compound N-acetyl-phosphinothricin, it was converted into the herbicide phosphinotricin, thereby destroying the tapetal cells. F1-hybrid breeding based on induced male sterility employs two male-fertile inbred lines, one of which is sprayed with N-acetyl-phosphinothricin for F1-hybrid seed production.

Plant Movements and Rhythms

The resistance of biological membranes to gas transport was hardly recognized as a factor limiting metabolic function. However, and in contrast to the mammalian aquaporins (Cooper et al. 2002), a clear role of CO2 conducting aquaporins in plant physiology could be shown. Tobacco plants with modified aquaporin expression were changed in water transport characteristics (Siefritz et al. 2002), as well as in CO2 dependent processes like rate of photosynthetic CO2 fixation and stomatal movement (Uehlein et al. 2003). Also Aharon et al. (2003) reported a beneficial effect of aquaporin expression on stomata and photosynthesis. Under favorable growth conditions, tobacco plants overexpressing the aquaporin PIP1 2 from Arabidopsis showed a significant increase in transpiration rate and photo-synthetic efficiency, as well as a higher stomatal density (Aharon et al. 2003). However, the authors had not analyzed the membrane transport of CO2 and therefore, related the effects to an improved water...

Regulation of intracellular Ca2 1 stores

Recent studies have suggested that at least two inositol phosphates can act as second messengers releasing Ca2+ in the ABA modulation of stomatal closure (Hunt et al, 2003 Lemtiri-Chlieh et al, 2000,2003). InsP3 is generated from phosphatidylinositol-4,5-bisphosphate PtdIns(4,5)P2 by the action of phos-pholipase C (PLC) (see Chapter 8). Immunolocalization suggests the presence of a Ca2+-activated PLC in tobacco guard cells (Hunt et al., 2003). In transgenic tobacco plants with reduced levels of PLC (due to cosupression induced by the full-length NrPLC2 cDNA expressed under the control of a guard cell-specific promoter), the guard cells appeared partially defective in ABA induction of aperture closing. These results imply the involvement of PLC in the amplification of the Ca2+ signal responsible for reduction of stomatal aperture in response to ABA (Hunt et al., 2003). Other inositol phosphates may also act as second messengers in ABA signal transduction pathways. The myo-inositol...

Reverse Genetics Analysis Using Gene Down Regulation

Muschietti et al. (1994) were the first to design antisense experiments with the pollen-specific tomato gene LAT52. Plants with reduced amounts of LAT52 mRNAs and protein showed smaller pollen grains segregating 1 1. In vivo pollination experiments suggested pollen tube growth arrest in the style, for a proportion of the transformants pollen. In order to elucidate the function of another pollen-specific gene from tobacco, NTP303 (Weterings et al. 1992), de Groot et al. (2004) have transformed tobacco plants with NTP303 co-suppression and anti-sense gene constructs and shown that the

Requisite Role of Plastid During Senescence and Signaling for Reproduction

During plant senescence degenerative processes as well as nutrient remobilization take place in an orderly fashion. Several regulatory factors associated to plastids play a decisive role. For instance, it has been observed that angiosperms displaying developmental leaf senescence possess plastid genes that code for a NADH-specific dehydrogenase complex (ndh). The ndh genes are apparently absent from several evergreen gymnosperms (Sabater et al. 2002). This observation is reinforced by the fact that transgenic tobacco plants in which the plastid ndhF has been knocked-down exhibit delayed senescence (Zapata et al. 2005).

Plastid transformation to generate transgenic plants is advantageous for the environment

Certain cases this can also be achieved by the transformation of the nuclear genome. Another advantage of the plastid transformation is that proteins with a disulfide bridge can be formed in the plastids, which is not possible in the cytosol. As described earlier, chloroplast enzymes are regulated by the oxidation of adjacent -SH groups (Fig. 6.25). Because of this ability the plastid compartment is well suited to produce, after genetic transformation, animal proteins, such as antibodies or oral vaccines, where disulfide bridges are responsible for the correct folding. The expression of genes originating from bacteria, e.g., Bt toxin (section 22.6), is made easy since bacteria and chloroplasts employ the same triplet codons. Transplastome tobacco plants have been generated. The plastid transformation of other crop plants is still a big challenge, as it would allow generating transformants which harmonize with the environment, as they cannot spread their foreign genes to other plants...

Plants are protected against some insects by the BT protein

Of the inhibitors is not restricted to the wound site, but often occurs in large parts of the plant and thereby protects them from further attacks. The introduction of suitable foreign genes in transgenic potato, lucerne (alfalfa), and tobacco plants enabled a high expression of proteinase inhibitors in these plants, protecting them efficiently from being eaten by insects. This strategy has the advantage that the proteinase inhibitors are not specific to certain insect groups. These proteinase inhibitors are contained naturally in many of our foods, sometimes in relatively high concentrations, but they are destroyed by cooking.

Reengineering The Carbonnitrogen Interface

Nevertheless, in some contrasting cases, alterations in the expression of GS and of glutamate dehydrogenase (GDH) (chapter 2) have resulted in substantial growth changes in some plant species (Table 1). In a particularly dramatic instance, a 42 increase in the cytosolic isoform of GS (GS1) in poplar trees resulted in an increase in height of 76 over controls after two months of growth, although this increase declined to 21 after six months (Gallardo et al. 1999). The potential for increasing GS activity to significantly increase yield in rice is suggested by the observation that GS1 overexpression experiments with another cereal grain, wheat, resulted in about 20 higher grain yield on a mass basis (Habash et al. 2001). GS1 overexpression also resulted in biomass increases in the legume Lotus japonicus (Hirel et al. 1997) and in tobacco (Fuentes et al. 2001) and, similarly, overexpression of GS2, the plastidic isoform, was shown to have positive effects on seedling biomass in tobacco...

CisMotif Context Modified the Centre for Synthetic Promoter Engineering

Modification of cis-regulatory context can be performed by two basic 'cut-and-paste' strategies firstly, by rearrangement (also known as 'swapping' or 'shuffling') of defined modules, each containing a number of cis-motifs within native context, and secondly, by construction of novel synthetic modules by cis-motif insertion (of plant or non-plant origin). Both strategies can be used to develop synthetic promoters with additional features and or enhanced transcriptional activity (or at least functionally equivalent) when compared to wild-type counterparts, but with minimum sequence homology in an attempt to reduce HDGS, as discussed above in Section 20.2.4. Bhullar et al. (2003) evaluated both strategies (domain swapping versus novel cis-regulatory context) using domain A (core promoter plus subdomain A1) of the CaMV 35S promoter as model (see Sect. 20.2.3) compared to expression levels of the wild-type 35S promoter in transient and stably transformed tobacco plants. Results from this...

Plants can be protected against viruses by gene technology

It has long been known that after infection with a weak pathogenic strain of a certain virus, a plant may be protected against infection by a more aggressive strain. This phenomenon has been applied successfully in the biological plant protection of squash plants. It was presumed that a single molecular constituent of the viruses caused this protective function, and this has been verified by molecular biology the introduction of the coat protein gene of the tobacco mosaic virus into the genome of tobacco plants makes them resistant to this virus. This has been confirmed for many other viruses if a gene for a coat protein of a particular plant virus is expressed sufficiently in a plant, the plant usually becomes resistant to infection by this pathogen. This principle has already been used several times with success to generate virus-resistant plants by genetic engineering. In the United States, a virus-resistant squash variety generated in this way has been licensed for cultivation. In...

Activation Of Plant Defense Responses

Ing the hypersensitive response, and PR-protein and phytoalexin production (Heath, 2000 McDowell and Dangl, 2000 Shirasu and Schulze-Lefert, 2000). A gene encoding the elicitor cryptogein (a small basic protein, 98 amino acids in length) from the pathogen Phytophthora cryptogea was cloned and expressed in transgenic tobacco under control of a pathogen-inducible promoter (Keller et al., 1999). Challenge inoculation with a range of fungi induced the HR as well as several defense genes, and growth of the pathogens was concomitantly restricted (Table 7.1). Resistance to the pathogens was not complete, possibly because of the time needed for production of the transgenic elicitor following initial infection (Keller et al., 1999). Another elicitor, INF1, was shown to act as an avirulence factor in the to-bacco-Phytophthora infestans interaction and triggered the onset of the HR (Kamoun et al., 1998). Expression of the gene encoding the AVR9 peptide elicitor from Cladosporium fulvum in...

GS and photorespiration

(Oliveira et al. 2002), GS2 in tobacco (Kozaki and Takeba 1996), and GS2 in rice (Hoshida et al. 2000) has suggested that GS is a limiting factor in this pathway. In all of these studies, there was a positive correlation between GS activity and the amount of photorespiration (chapter 6). In conjunction with the increased growth rates reported in the tobacco plants, these findings are striking in that they appear to contradict the idea that photorespiration is a wasteful process, in which both fixed C and N can be lost in substantial quantities from the plant (Ehleringer and Monson 1993, Mattsson et al. 1997). Such waste, however, might be minimised if GS overexpressors are able to refix the N released in the glycine decarboxylation step of photorespiration, and if C released in the same step can also be effectively refixed, possibly by phosphoenolpyruvate carboxylase (PEPcase, see below and chapter 6), which is up-regulated by glutamine levels these in turn might be increased by...

QQ of Pectobacterium pathogenicity

Plants were genetically modified to gain the capacity to produce or inactivate N-AHSL signals. A first series of these transgenic plants were developed to activate QS functions of pathogens at an inappropriate time a second type of plants was designed to block the initiation of the QS regulatory cascade. Transgenic tobacco plants, into which the yenl gene of Yersinia enterolitica encoding N-AHSL synthase was introduced, were able to produce C6-HSL and 30,C6-HSL (Fray et al. 1999). The N-AHSL-pro-ducing plants was able to complement the virulence of an N-AHSL-defective mutant of P. carotovorum, as well as the biocontrol activity of an N-AHSL-defective mutant of Pseudomonas aureofaciens. However, while a decrease in the virulence of a wild-type P. carotovorum strain on the non-host tobacco plant expressing expl was reported (Mae et al. 2001), an increase in the virulence was observed when wild-type P. carotovorum was inoculated on the host potato plant expressing the yenl gene (Toth et...

Down Regulation of the Antioxidant Network Evidence from Lesion Mimic Mutants

Similar conclusions can be drawn from the study of mutants and transgenic plants that are suppressed in the expression of components of antioxidant defenses. The best studied example is the suppression of CAT1 expression in transgenic tobacco plants. These plants develop light intensity-dependent spreading chlorosis on their leaves (Changmonnpol et al., 1996 Willekens et al., 1997 Mittler et al., 1999 Dat et al., 2003). This phenotype is caused by failure of such plants to scavenge photorespiratory cycle-produced H2O2, which then triggers the expression of a wide range of defense genes associated with pathogen infection.

Regulation of Transcription by PP2C

Tobacco DNA-binding protein phosphatase (DBP1) was isolated by interaction with the promoter region of tomato citrus exocortis viroid (CEVI1) gene, which is induced during the course of compatible plant-virus interactions. The C-terminal part of DBP1 shows homology to the PP2C catalytic domain, whereas the N-terminal region contains DNA binding sequences (Carrasco et al. 2003). Correspondingly, DBP1 shows Mg2+-dependent phosphatase activity and contains a functional nuclear localization signal. DBP1 negatively controls transcription of CEVI1 as demonstrated by constitutive up-regulation of the CEVI1 gene in DBP1-antisense transgenic tobacco plants. DBP1 and its distantly related Arabidopsis AtDBP1 possess in vitro DNA binding activity, mediated by the N-terminal region via the conserved DNC (DBP N-terminal core) motif (Carrasco et al. 2005). Interestingly, the 14-3-3 isoform G from tobacco and the 14-3-3 X GRF6 from Arabidopsis were identified by screenings in yeast using DBP1 and...

Compatible Solutes And Drought Stress

Leaves close their stomata to avoid evaporation of water during drought, and consequently, the in-flow of CO2 into the leaves stops. As a result, the sun's energy cannot be used for CO2 fixation and instead is used for formation of active oxygen molecules in the chloroplasts. Superoxide and hydrogen peroxide are decomposed by enzymes specific to these active oxygen species. However, no enzyme has been shown to decompose hydroxyl radicals, the most dangerous of all active oxygen species. Some compatible solutes function as scavengers of hydroxyl radicals (Akashi et al., 2001 Shen et al., 1997). For example, it has been reported that levels of free radicals are decreased in tobacco plants transformed to accumulate more proline (Hong et al., 2000). The reactivity of citrulline and mannitol to hydroxyl radicals is much higher that that of proline citrulline can promptly decompose all hydroxyl radical molecules at the formation site (Table 2) (Akashi et al., 2001).

Transgenic Plants With Combinations Of Pr Proteins

Transgenic tobacco plants expressing a barley ribosome inactivating protein cDNA, under control of a wound-inducible promoter, were significantly protected against the soil-borne pathogen R. solani when compared to nontransgenic controls (Logemann et al., 1991). Higher levels of resistance to R. solani infection were observed in tobacco plants expressing three barley proteins, namely a class II chitinase, a 3-1,3-glucanase, and a type I RIP. This observation supports the synergistic interaction of the PR proteins with other anti-fungal proteins, leading to enhanced levels of plant protection (Jach et al., 1995)

Conclusions and Future Perspectives in Phytoremediation

Hyperaccumulators or high biomass green plants. Symbiosis between plants and bacteria or fungi together with genetically modified plants are of the greatest chance for phytoremediation development. The major concern of the process efficiency is plants' resistance to environmental stress factors represented by the pollutant its self (heavy metal, xenobiotic), as well as by accompanying stressors (air pollution at contaminated site, fungal infections, etc.). Thus, the understanding of oxidative stress and defence mechanisms of plants used in phytoremediation is of a great importance. In our studies, tropospheric ozone induced the biosynthesis of salicylic acid in ozone-sensitive tobacco plants which was strongly correlated with the level of injuries observed on leaves after exposure in ambient air conditions. We could assume, that ozone has a strong negative impact on plants causing the ozone-induced oxidative burst.

Plant Growth During Moderate Water Stress Episodes

Affect cell wall proteins such as expansins, XET (xyloglucan endotransgly-cosylase) and cell wall polysaccharide linkages, thereby effecting wall loosening and increased rates of cell expansion (Cosgrove, 2005 Fry, 1986 Hager et al, 1971 Rayle and Cleland, 1970, 1992). Zorb et al. (2005) and Pitann et al. (2009) provided further evidence for the involvement of proton-pumping ATPases in leaf growth inhibition by the osmotic component of salinity stress. Finally, Gevaudant et al. (2007) genetically engineered increased expression in tobacco plants of either a wild-type H+ ATPase or of a modified H+ ATPase in which the autoinhibitory domain was removed in order to facilitate constitutive expression. The latter transformant showed aberrant growth but also revealed increases in resistance to growth inhibition by salt stress.

The Application Of Induced Resistance

Ever since White (1979) found that treatment of tobacco plants with salicylic acid or aspirin would induce disease resistance, it was clear that the external application of a chemical could be used to protect plants by enhancing disease resistance. Based on the principles of induced disease resistance and what properties a resistance activator should have, the first synthetic resistance inducers were developed (Tally et al., 1999). The first of these, 2,6-dichloroisonicotinic acid (Figure 6.5), was capable of inducing resistance in a number of crops against a fairly wide range of pathogens and had characteristics that suggested it functioned as an analog of salicylic acid (Kessmann et al., 1994). Another functional analog of salicylic acid, ASM (Figure 6.5), has been developed into a commercial product (Actigard or Boost) that is registered on several crops (Tally et al., 1999). ASM has been shown to be effective as a foliar spray (Oostendorp et al., 2001 Tally et al., 1999), as well...

Compartmentation of peptidases

With Arabidopsis, these proteases are encoded by gene families, making their functional analysis quite challenging. It is known that Clp protease(s) are essential for chloroplast function, as genetic interference with some Clp proteins is detrimental to plant viability (Adam and Clarke, 2002). However, no evidence for a major role of Clp proteins in N remobilization during leaf senescence has been found to date. Similarly, while some specific functions start to be associated with some of the other mentioned plastidial proteases, they have so far not evolved as good candidates for the degradation of photosynthetic proteins at the onset of the senescence process. On the other hand, Kato et al. (2004) have recently reported that tobacco plants with lower levels of the DNA-binding protease CND41 had delayed leaf senescence and Rubisco degradation, giving, to the knowledge of the author of this chapter for the first time, direct evidence for the functional involvement of a defined protease...

Sucrose Biosynthesis in Source Leaves

There is growing evidence that higher plants contain more than one gene encoding SPS. An inspection of the Arabidopsis genome revealed the presence of four genes putatively encoding SPS enzymes, all of which appear to be both transcribed (Lunn and MacRae, 2003) and enzymatically active (F. Bornke, unpublished data). Phylogenetically, the four SPS sequences from Arabidopsis and all those known from other dicot species fall into three families A, B and C (Lunn and MacRae, 2003). Recently, it was shown that monocot species contain an additional D family that probably arose after monocots and dicots diverged (Castleden et al., 2004). The number of SPS genes in plants raises the question about functional specialisation of particular isoforms. A-family members have been the subject of most expression studies and most of the expressed sequence tags (ESTs) examined belong to the A-family, implying that A-family genes are more abundantly expressed than those belonging to other families....

For Engineering Tolerance

As examples, transgenic rice plants developed with choline oxidase (codA), d-pyrroline-5-cor-boxylate synthase (P5CS), LEA protein group 3 (HVA1), alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) genes exhibited drought tolerance (Datta 2002 Soren et al. 2010). Potato and rice (Yeo et al. 2000 and Garg et al. 2002, respectively) transformed with trehalose synthesis genes displayed tolerance to drought (in case of potato), and salt, drought, and low-temperature stress (in case of rice). Tobacco plants transformed with ectoine biosynthesis genes from the halo-philic bacterium Halomonas elongate showed enhanced salt tolerance. Also transformation with genes for sorbitol (Sheveleva et al. 1997) or man-nitol (Shen et al. 1997) resulted in an increased accumulation of these osmolytes and tolerance to high salinity (Table 1.1). Overexpression of genes encoding the enzymes pyrroline-5-carboxylate As previously stated, abiotic stress generates an increase in reactive oxygen species...

Ethylene and Growth under Limiting Environmental Conditions

When growing at high densities with competition for light, plants show several shade-avoidance responses, including enhanced elongation, elevated leaf angles, and early flowering (Smith 2000). Interestingly, it was found that ethylene controls the timing of these shade-avoidance responses (Pierik et al. 2003). Moreover, recent evidence shows that ethyl-ene-sensitive tobacco plants exhibit shade-avoidance responses when blue light is omitted from the light spectrum, whereas the ethylene-insensitive plants are unresponsive to the absence of blue light (Pierik et al. 2004). These results suggest that the ethylene-transduction pathway interacts with light perception and that ethylene is required to maintain growth during competition for light. 1994). Loss of chlorophyll under salt stress has been reported due to ethylene-induced reduction in intermediates of chlorophyll biosynthesis (Khan 2003). Salt-tolerant rice species synthesize more ethylene compared to less tolerant...

In Plant Reaction to Stress

Electromagnetic Spectrum Black And White

Tobacco plants (Nicotiana tabacum L.) of both cultivars were exposed to ambient air of the city of Poznan (a city located in west-central Poland) and surrounding rural areas according to the standard VDI methodology (2000) applied in the biomonitoring of air contamination with ozone in European countries in years 1999-2002 (Klumpp et al. 2006) . The exposure of the Bel-W3 plants to ozone caused a significant increase, on average fourfold, in the content of free salicylic acid (SA) and a nearly 20-fold increase in the total salicylic acid (TSA - free and in the form of glucoside) in leaves showing ozone-caused injuries (compared with control plants). The exposure of the ozone-tolerant Bel-B cultivar resulted in a slight, insignificant increase in the salicylic acid content only. SA observed for the tobacco leaves of the Bel-W3 cultivar exhibiting almost 100 injuries was at the concentration level close to nearly 20 mg g-1 FW. This was several times lower than the salicylic acid content...

Two Component Transactivated Gene Switches Promising Systems for Flexible Transgene Expression

In the last few years, numerous studies (see references in Sect. 20.3.2.2) have reported on the design and use of chimeric two-component systems integrating combinations of core promoter and multimeric repeats or specific combinations of upstream heterologous c s-motifs of prokaryotic, insect and or mammalian origin. One of the most widely used examples is the pOp LhG4 transactivation system (Moore et al. 1998, 2006). This system comprises a synthetic pOp-promoter (consisting of two regulatory E.col lac operator elements upstream of a CaMV 35S core promoter) known as the 'reporter' unit, and a transcription 'activator' unit constitu-tively or spatially expressing a chimeric LhG4 TF, which is a fusion between a mutant lac-repressor and the transcription-activation domain II from the Saccharo-myces cerev s ae Gal4 protein (Moore et al. 1998). The pOp-promoter is transcrip-tionally inactive when introduced alone in reporter plant lines devoid of the chimeric LhG4 TF. However, transgene...

GR As a Detoxifying Gene in Transgenic Plants

A critical component of the ROI-scavenging system is the maintenance of ascorbate and glutathi-one pools in a reduced state. Of all the enzymes that are involved in this process, GR has been most extensively studied (Pooja et al. 2008). Initial attempts to enhance the GR expression in transgenic tobacco plants using a GR gene from E. coli resulted in a 3.5-fold increase in extract-able GR activity and leaves of these plants were found to have reduced visible damage after MV exposure but no increase in ozone tolerance (Aono et al. 1991). Further analyses of transgenic plants that express the E. coli GR gene showed that they contained more reduced ascorbate after MV exposure than control plants (Foyer et al. 1991). The introduction of the E. coli GR to chloroplasts in were noticed in the leaves of tobacco plants that expressed a chimeric gene derived from a pea GR cDNA (Creissen et al. 1995). Interestingly, some transgenic plant lines that expressed pea GR without the transit peptide or...

Chieen Ka Kaac Ka

Addy HD, Schaffer GF, Miller MH, Peterson RL (1994) Survival of the extraradical mycelium of a VAM fungus in frozen soil over winter. Mycorrhiza 5 1-5 Almeida AM, Silva AB, Araujo SS, Cardoso LA, Santos DM (2007) Responses to water withdrawal of tobacco plants genetically engineered with the AtTPS1 gene a plants. Plant Physiol 144 3-5 Han SE, Park SR, Kwon HB, Yi BY, Lee GB, Byun MO (2005) Genetic engineering of drought-resistant tobacco plants by introducing the trehalose phospho-rylase (TP) gene from Pleurotus sajor-caju. Plant Cell Tissue Organ Cult 82 151-158 Higashiyama T (2002) Novel functions and applications Phillips DV, Wilson DO, Dougherty DE (1984) Soluble carbohydrates in legumes and nodulated nonlegumes. J Agric Food Chem 32 1289-1291 Pilon-Smits EAH, Terry N, Seors T, Kim H, Zayed A, Seongbin H, Van Dun K, Voogd E, Verwoerd TC, Krutwagen RW, Goddijn OJM (1998) Trehalose-producing transgenic tobacco plants show improved growth and performance under drought stress. J Plant...

The Tobacco Thin CellLayer System TCL

Explants consisting of a few of the outermost cell layers of the stem have been used by several groups to study the action of various substances on development. When taken from stems of flowering tobacco plants, these TCLs produced roots, shoots or flowers with no apparent intermediate callus (Tranh Than Van et al., 1974). Four types of organ-inducing media (distinguished by auxinrcytokinin concentrations) have been defined, namely root, vegetative shoot, and flower media and a transition medium on which few organs develop (Mohnen et al., 1990). Apart from variations in the IBA and kinetin concentrations in the medium, de novo flower formation without callus has been shown to be influenced by pectic cell-wall fragments (Eberhard et al., 1989) and by polyamines (Galston and Kaur-Sawhney, 1990). The flowering response is also strongly dependent on the developmental state of the source tissue (Rajeevan and Lang, 1993). flowering when applied to TCLs derived from vegetative tobacco...

Genes And Metabolism

Xanthosine into caffeine (Uefuji et al., 2003). Silencing and overexpression approaches led to an overview of metabolic engineering of the caffeine bio-synthetic pathway (Ogita et al., 2004). Tobacco plants (caffeine free in natural conditions) that simultaneously expressed the three methylation genes also produced caffeine (Uefuji et al., 2005).

Plant Microtubules as Mechanosensors

It has long been speculated that calcium fluxes are involved in the signal transduction that culminates in thigmomorphogenesis. By generating transgenic tobacco plants that expressed the luminescent calcium reporter ae-quorin (Knight et al. 1991), it became possible to observe these fluxes directly and to demonstrate that the signature triggered by a touch stimulus was specifically different from those induced by other stimulation qualities such as cold. Since these changes of intracellular calcium levels occur rapidly after stimulation (Legue et al. 1997), mechanosensitive calcium channels have been postulated as the primary element of mechanosignalling.

Zinctransporting Genes In Plants

The expression of IRT1 is strongly induced in plants under conditions of iron deficiency and is repressed in iron-replete plants. This correlates well with the finding that cadmium uptake is enhanced in iron-deficient pea seedlings. However, these transporters are tightly regulated at the transcriptional and post-transcriptional levels, and, to date, no reports on plants engineered to overexpress transporters of the ZIP family have been issued. Tobacco plants engineered to contain increased amounts of NtCBP4 protein (TC 1.A.1.5.1), a putative cyclic-nucleotide and calmodulin-regulated cation channel in the plasma membrane, displayed an increased sensitivity to lead, a 1.5-to 2.0-fold shoot accumulation of lead, and an increased nickel tolerance. Yeast cells expressing the wheat LCT1 cDNA (TC 9.A.20.1.1), encoding a low-affinity cation transporter, were hypersensitive to Cd2+ and accumulated increased amounts of cadmium 84 . Plants overexpressing AtNramp3 (TC 2.A.55), a member of the...

Sources of Singlet Oxygen

The effect of dark and light on tobacco leaf ascorbate and dehydroascorbate contents. The leaf ascorbate and dehydroascorbate contents of 6-week-old tobacco plants was measured before and after a dark period of 16h and then during a period of 24 h continuous light (350 xmol m-2 s-1). Photosynthetic CO2 assimilation rates in the light are also shown. For further information see Pignocchi (2000). Fig. 3. The effect of dark and light on tobacco leaf ascorbate and dehydroascorbate contents. The leaf ascorbate and dehydroascorbate contents of 6-week-old tobacco plants was measured before and after a dark period of 16h and then during a period of 24 h continuous light (350 xmol m-2 s-1). Photosynthetic CO2 assimilation rates in the light are also shown. For further information see Pignocchi (2000).

Celltocell movement of RNA silencing

Maintenance of silencing has also been observed with the GFP transgenic N. ben-thamiana,as tissues taken from silenced scions were able to sustain signal production when used as rootstocks (Voinnet et al., 1998). Analysis of silenced and non-silenced tissues in those plants indicated that entry of the systemic signal into cells was correlated with de novo methylation of the GFP transgene coding region (Jones et al., 1999). Analysis carried out in systemically silenced GUS tobacco plants provided a similar conclusion (Mallory et al., 2001). Work in Arabidopsis has also linked methylation of transgene coding region to PTGS (Elmayan et al., 1998 Mourrain et al., 2000). Moreover, a recent study of plants carrying a mutation in the gene for the major DNA methyltransferase MET1 revealed that this protein was necessary for maintenance rather than initiation of PTGS, as the new growth of met1 plants exhibiting PTGS of a GUS transgene progressively lost the silencing phenotype (Morel et al.,...

Molecular Regulation Of Ion Homeostasis And Involvement Of Intermediary Signalling Components

ABA-induced H2O2 activates Ca2+ channels in guard cells and mediates stomatal closure (Pei et al., 2000). In Arabidopsis, it was shown that H2O2 activates the MAP kinase ANP1 and is involved in the regulation of gene expression in plants (GST6, HSP18 2 and GH3). Interestingly, tobacco plants over-expressing the tobacco ANP1 orthologue NPK1, exhibit an increased tolerance to heat shock, freezing and salt stress. However, the expression of the stress-responsive gene RD29A is not affected by either H2O2 or the activated MAPK cascade (Kovtun et al., 2000), suggesting that the pathways for the regulation of DRE CRT genes (Shinozaki and Yamaguchi-Shinozaki, 2000) are activated through different mechanisms (Xiong and Zhu, 2002c). Similarly, the osmotic stress activated MAPK pathways react to other stresses such as UV and g irradiation, cytokines, certain mitogens and oxidative stress. These pathways play a major role in apoptosis, cytokine production, transcriptional regulation and...

Ferritin Overexpression In Transgenic Plants And Its Consequences

Iron and oxygen metabolisms can interact to promote oxidative stress. Therefore, Fe sequestration in ferritin of transformed plants could have a beneficial effect against Fe-mediated oxidative stress. Methylviologen, the active molecule of the herbicide paraquat, acts by promoting an oxidative stress in the chloroplast, leading to proteolysis, lipid peroxidation and ultimately to cell death (Dodge, 1994). The toxic effect of methyl viologen requires free Fe to take place, and can be antagonized by Fe chelators such as desferrioxamine (Korbaschi et al., 1986 Zer et al., 1994). Indeed plants overexpressing ferritin are more resistant to methylviologen toxicity, confirming that the transgenic ferritins were functional in vivo - i.e. able to sequester Fe atoms (Deak et al., 1999 Van Wuytswinkel et al., 1999). However, it has been documented in animal cells, that ferritin can act either as anti- or pro-oxidant (Cairo et al., 1995). Therefore, the increased resistance to paraquat treatment...

Natural And Synthetic Elicitors Of Prprotein Genes

Salicylic acid is an endogenous signal molecule that very often can induce PR proteins in plants. SA is postulated to bind to a receptor which, in turn, may trigger the signal transduction cascade leading to the production of transcription factors regulating PR-protein expression or other defense proteins (Metraux, 2001). In tobacco plants, SA induces the expression of at least nine different PR-protein genes, and interestingly, all of them were also induced by TMV infection. The endogenous levels of SA increased 50-fold in TMV-inoculated leaves followed by accumulation of PR proteins in resistant plants. Neither response was detected in susceptible plants. However, SA treatment of susceptible plants did lead to the triggering of PR-protein gene expression, suggesting that TMV infection could not enhance SA levels in susceptible plants and, therefore, failed to trigger PR-gene expression (Malamy et al., 1990). Tobacco plants expressing the NahG gene (salicylate hydroxylase gene from...

Occurrence in the Solanaceae Tables 31 and

As already mentioned, most of the commercial tobaccos produced in the world belong to Nicotiana tabacum L., which is assumed to be an allotetraploid, natural hybrid of two wild species, N. sylvestris (maternal genome) and N. tomentosiformis (paternal genome), also supported by phylogenetic molecular analysis based on ITS regions of nuclear ribosomal DNA (Chase et al. 2003). Alternatively, though less likely the latter is assumed to be N. otophora. However, a molecular analysis based on five genes encoding putrescine N-methyltransferase supported the hypothesis of Kenton et al. (1993) that the progenitors are N. sylvestris and an introgressed hybrid between N. tomentosiformis and N. otophora (Riechers and Timko 1999 and references therein). There are innumerable cultivars, e.g., over 1500 entries in a United States Department of Agriculture inventory. The only other species used on a limited scale is N. rustica (Tso 1999), which is assumed to be a hybrid of N. undulata and N....

Physiological and Molecular Aspects for Improving NUE

Nitrate ion and not its downstream metabolite came from NR-deficient mutants of Arabidopsis and N. plumbaginifolia (Krapp et al. 1998 Lejay et al. 1999). Studies in the last decade have shown that enhancing the uptake of N by overexpressing transporters may not necessarily improve NUE. For example, transgenic overexpression of a CHL1 cDNA (representing the constitutive HATS) driven by the cauliflower mosaic virus 35S promoter in a chl1 mutant, recovered the phenotype for the constitutive phase but not for the induced phase (Liu et al. 2003) . Similarly, the NO3- contents in transgenic tobacco plants over-expressing the NpNRT2.1 gene (encoding HATS), were remarkably similar to their wildtype levels, despite an increase in the NO3- influx. These findings indicate that genetic manipulation of nitrate uptake may not necessarily lead to associated improvement in nitrate retention, utilization, or NUE, though it remains to be seen whether different plants respond differently to the...

Role of GST activity in infected plants

Tmv Xanthi Tobacco

Summarizing the above reports on GST, it seems that the role of GSTs in infected plants is the suppression of necrotic disease symptoms by the detoxification of toxic lipid hydroperoxides that derive from peroxidation of cell membranes. In the future, this supposition may be confirmed by the study of transgenic plants overexpressing GST isoenzymes and of transformants expressing GSTs in antisense direction. Tobacco plants have been transformed with genes encoding GST (Roxas et al. 1997, Thompson et al. 1998). These transgenic tobacco plants overexpressing GST were more resistant towards abiotic stress effects than wild type plants. The effects of microbial infections on these transgenic plants have not been reported yet.

Cell Axis and Plant Development

The susceptibility of the crops to lodging and windbreak (Luib and Schott 1990). Most crop plants are typical sun plants, i.e. they exhibit a pronounced shade-avoidance response when grown in dense canopies (Smith 1981). They are able to sense their neighbours through subtle changes in the ratio between red and far-red light utilizing the photoreversible plant photoreceptor phy-tochrome. They respond to this change in red far-red ratio by enhanced stem and petiole elongation. The shade-avoidance response is supposed to protect these plants against overgrowth by neighbouring plants. Indeed, this has been confirmed in field trials, where photoreceptor mutants of Arabidopsis thaliana that were not able to trigger shade avoidance were monitored under field conditions and found to be less competitive as compared to the respective wild type (Ballare and Scopel 1997). As useful as this response may be for the survival of a weed like thale cress in a canopy, it is undesired for a crop plant....

The transActing siRNA Pathway

The discovery of RNA silencing also serving as a natural antiviral defence mechanism in plants has provided the plant scientific community with a new avenue for the development of antiviral strategies. The expression of transgenes encoding hpRNA has been shown to form a powerful tool for engineering virus resistance in plants. Wang et al. (2000) have been able to induce complete resistance to Barley yellow dwarf virus (BYDV) in barley (Hordeum vulgare) plants transformed with such a hpRNA transgene. Similar results were achieved in tobacco plants expressing a hpRNA construct against PVY (Smith et al. 2000). More recently, an alternate approach has been employed to control virus infection in plants. Artificial miRNA (amiRNA) vectors consisting of a sequence of the Arabidopsis miR159a pre-miRNA transcript in which the endogenous miRNA sequence had been replaced with an amiRNA specific to the viral suppressor genes p69 and HC-Pro were shown to provide resistance against Turnip yellow...

NO emission from nitrate reductase

Typical pattern of NO-emission from detached tobacco leaves. The upper curve shows data from nitrate-fed plants. NO emission is low in the dark (grey or black bars on top of the figure), up to 10-fold higher in the light and increases transiently after light off. This is due to a transient overshoot of nitrate reductase leading to some nitrite accumulation, which stops after NR has been down regulated in the dark, requiring 5 -15 min. In contrast to NR, nitrite reduction stops immediately after light off. NO emission in the dark is drastically stimulated under anoxia, (black bar) because NR is activated, NiR does not work, leading to a strong accumulation of nitrite. Probably, the absence of reactive oxygen species under anoxia also contributes to the high NO-emission under dark-anoxic conditions (compare to Figure 5). Also shown (lower curve with open symbols) is the complete absence of NO-emission from leaves of ammonium-grown hydroponic tobacco plants, which contain no...

Wu Jiang Xielong Flax Mill Mail

New Phytol 167 171-180 Cheong YH, Chang H-S, Gupta R, Wang X, Zhu T, Luan S (2002) Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol 129 661-677 Choi C-S, Sano H (2007) Abiotic-stress induces demethylation and transcriptional activation of a gene encoding a glycerolphosphodiesterase-like protein in tobacco plants. Mol Genet Geno-mics 277 589-600

Seed Maturation

The biosynthesis of seed storage proteins is subject to tissue-specific and developmental regulation. Significant amounts accumulate only in the storage parenchyma of embryos or in the cereal endosperm during mid- and late seed maturation. The process is integrated into the seed maturation program. Therefore, a profound understanding of storage protein formation requires knowledge of the regulatory network of seed maturation, which has been studied in some detail in Arabidopsis (reviewed in Finkelstein et al. 2002 Gutierrez et al. 2007), legumes (Wobus and Weber 1999 Weber et al. 1997a, 1998a, 2005) and, based on transcriptional profiling, in the barley endosperm (Sreenivasulu et al. 2004, 2006). Seed maturation is controlled by a complex transcriptional network. Its spatial and temporal regulation requires the concerted action of several signalling pathways integrating information from genetic programs, from hormonal signals with greatest importance of abscisic acid (ABA), as well as...

Genetic Control

Particularly for quarantine pathogens. Increasingly, modern diagnostic tools are being based on the DNA characteristics of the pathogen as they present adequate diversity to distinguish species, strains, substrains, isolates, and even individuals and offer convenience of detection using modern bio-techniques such as polymerase chain reaction (PCR) or Reverse Transcription (RT)-PCR 122 . Using RT-PCR technique detection of Cherry green ring mottle virus and Cherry necrotic rusty mottle virus in Prunus spp. has been done 123 . Further advancement in detection methods has been done by developing micro-array technology which provides the next generation of DNA diagnostics to measure different pathogens in a massively parallel manner on a single chip and avoid laborious confirmation procedures. A porous capillary solid phase micro-array system is shown for plant-pathogenic Phytophthora spp. multiplex detection 124 . Besides diagnostic methodology transformation techniques have been used to...

Biotic Stresses

The transcription of a DGK gene of rice, OsBIDK1, was shown to be induced by treatment with benzothiadiazole, a structural analog of salicylic acid (SA), which is involved in the induced resistance response during infection by virulent pathogens, such as the blast fungus Magnaporthe grisea (Zhang et al. 2008). Transgenic tobacco plants, overexpressing OsBIDK1 exhibited enhanced resistance against tobacco mosaic virus and Phytophtora parasitica. Also, the AtDGK5 gene of Arabidopsis is suggested to be involved in the response to virulent Pseudomonas bacteria, since a Atdgk5 knock-out mutant failed to induce PR1 gene expression during either pathogen infection or treatment with SA (S. Van Wees, B. Van Schooten, T. Munnik, pers. com.). Moreover, the basal resistance against the bacteria was decreased. Both OsBIDK1 and AtDGK5 are members of cluster III. As a GFP-fusion of the latter localizes to the plasma membrane, it will be interesting to learn whether this is due to the CBD domain...

Hcr94D

Necrosis-inducing activity (NIA) of AVR4 and AVR4E elicitor proteins. Agrobacterium cultures carrying Avr4 or Avr4E were coinfiltrated into leaves of six-week-old tobacco plants in a 1 1 ratio with Agrobacterium cultures carrying the resistance genes Hcr9-4D or Hcr9-4E. NIA was scored three days postinfiltration (dpi) and photographs were taken at 7 dpi. Note that AVR4 and AVR4E induce a necrotic response only in the presence of the matching resistance proteins, Hcr9-4D and Hcr9-4E, respectively.

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