Rüdiger Hain

Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol

A gene from groundnut (Arachis hypogaea) coding for stilbene synthase was transferred together with a chimaeric kanamycin resistance gene. It was found to be rapidly expressed after induction with UV light and elicitor in tobacco cells (Nicotiana tabacum). Comparative studies of stilbene synthase mRNA synthesis in groudnut and transgenic tobacco suspension cultures revealed the same kinetics of gene expression. Stilbene synthase specific mRNA was detectable 30 minutes after elicitor induction and 10 minutes after UV irradiation. The maximum of mRNA accumulation was between 2 and 8 hours post induction. 24 hours after induction stilbene synthase mRNA accumulation ceased. Furthermore, in transgenic tobacco plants, the gene was found to be inducible in sterile roots, stems and leaves. Stilbene synthase was demonstrated in crude protein extracts from transgenic tobacco cell cultures using specific antibodies. Resveratrol, the product of stilbene synthase, was identified by HPLC and antisera raised against resveratrol.

Reduced virus infectivity inN. tabacum secreting a TMV-specific full-size antibody

We developed a new concept of controlling plant virus infection based on the expression and secretion of full-size antibodies in plants. The neotope-specific anti-Tobacco Mosaic Virus (anti-TMV) antibody mAb24 has a high affinity towards epitopes present only on the surface of intact tobacco mosaic virions. The infectivity of the virus is inhibited almost completely if TMV is adsorbedin vitro at ratios as low as 300 antibody molecules per virion prior to inoculation. Cloned full-size cDNAs of mAb24 heavy and light chains were integrated into the plant expression vector pSS in tandem array and used for transformation ofNicotiana tabacum. The resulting transgenic tobacco plants expressed heavy- and light-chains of mAb24 which were assembled into functional antibodies and exported to the intercellular space. TMV specificity and affinity of the plant-produced antibody (mAb24-P) was not altered when compared to the original murine mAb24. F1 progenies segregated 3:1 with respect to antibody secretion and showed up to two-fold higher expression levels compared to the F0 plants. Upon infection with TMV F1 plants producing mAb24-P showed a reduction of necrotic lesion numbers which is correlated with the amount of antibody produced in transgenic plants.

An ozone-responsive region of the grapevine resveratrol synthase promoter differs from the basal pathogen-responsive sequence.

Stilbene synthase (STS) is an enzyme involved in the biosynthesis of stilbenes, which are synthesized in various plants in response to pathogen attack, UV irradiation or exposure to ozone. We describe analysis of an ozone inducible STS transcript and its corresponding promoter (Vst1), combined with the β-glucuronidase (GUS) reporter gene. A single ozone pulse (0.1 µl/l, 10 h) resulted in 11-fold GUS expression. Histochemical localization of GUS activity revealed small spots distributed over the whole leaf. Cross-sections of leaf tissue showed that the Vst1 promoter was induced in palisade and spongy parenchyma cells and to a lesser extent in epidermal cells. Deletions at the 5′ end showed that a partial promoter sequence between position − 430 and− 280 constituted the ozone-responsive region, whereas for effective pathogen-inducibility sequences from− 280 to −140 have been shown to be necessary.

Functional Analysis of the Early Steps of Carotenoid Biosynthesis in Tobacco

Carotenoids contribute to energy transduction in the light harvesting complexes and serve in protection from excess light fluence. Because of the importance of carotenoids, the genes encoding enzymes of carotenoid biosynthesis in higher plants are potential targets for herbicides. To obtain further insight into tobacco carotenoid biosynthesis and to investigate and prioritize potential herbicide targets in the pathway, the effects of changed phytoene synthase (PSY) and phytoene desaturase (PDS) gene expression were studied in transgenic tobacco (Nicotiana tabacum Petit Havana SR1) plants. Genes for both enzymes were cloned from tobacco, and surprisingly two functional PSY genes were found. Transgenic tobacco plants constitutively expressing these genes in both sense and antisense orientations were examined regarding phenotype, carotenoid content and transcript levels of carotene biosynthesis genes. Overexpression of either psy gene resulted in severe phenotypic effects including dwarfism, altered leaf morphology, and pigmentation. A correlation among phenotype, transcript level, and metabolic profile was demonstrated by comparison of hemizygous and homozygous plants from the same transformation event. Antisense expression of PSY and PDS also caused lethal phenotypes. Transcript levels of other carotene biosynthesis genes remained unaltered in the transgenic mutant. Phytoene accumulated in plants expressing antisense RNA to pds. However, elevated levels of phytoene were detected suggesting an increase in metabolic flux into this pathway.

Ozone-induced gene expression occurs via ethylene-dependent and -independent signalling

Recent studies suggest that ethylene is involved in signalling ozone-induced gene expression. We show here that application of ozone increased glucuronidase (GUS) expression of chimeric reporter genes regulated by the promoters of the tobacco class I β-1,3-glucanases (GLB and Gln2) and the grapevine resveratrol synthase (Vst1) genes in transgenic tobacco leaves. 5′-deletion analysis of the class I β-1,3-glucanase promoter revealed that ozone-induced gene regulation is mainly mediated by the distal enhancer region containing the positively acting ethylene-responsive element (ERE). In addition, application of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, blocked ozone-induced class I β-1,3-glucanase promoter activity. Enhancer activity and ethylene-responsiveness depended on the integrity of the GCC boxes, cis-acting elements present in the ERE of the class I β-1,3-glucanase and the basic-type pathogenesis-related PR-1 protein (PRB-1b) gene promoters. The minimal PRB-1b promoter containing only the ERE with intact GCC boxes, was sufficient to confer 10-fold ozone inducibility to a GUS-reporter gene, while a substitution mutation in the GCC box abolished ozone responsiveness. The ERE region of the class I β-1,3-glucanase promoter containing two intact GCC boxes confered strong ozone inducibility to a minimal cauliflower mosaic virus (CaMV) 35S RNA promoter, whereas two single-base substitution in the GCC boxes resulted in a complete loss of ozone inducibility. Taken together, these data strongly suggest that ethylene is signalling ozone-induced expression of class I β-1,3-glucanase and PRB-1b genes. Promoter analysis of the stilbene synthase Vst1 gene unravelled different regions for ozone and ethylene-responsiveness. Application of 1-MCP blocked ethylene-induced Vst1 induction, but ozone induction was not affected. This shows that ozone-induced gene expression occurs via at least two different signalling mechanisms and suggests an additional ethylene independent signalling pathway for ozone-induced expression of genes involved in phytoalexin biosynthesis.

Recombinant proteins from transgenic plants

Transgenic plants can express a wide variety of foreign genes and offer the opportunity of large-scale protein production in agricultural systems. The recombinant protein can serve both ex situ and in situ purposes. Due to significant progress in plant molecular biology, many different plant species can now be transformed and are even capable of producing very complex proteins such as antibodies or vaccines. Furthermore, recombinant proteins can mediate resistance against microbial pathogens, such as fungi or viruses, or protect transgenic plants from insect pests.

Ozone- and ethylene-induced regulation of a grapevine resveratrol synthase promoter in transgenic tobacco

Stilbene synthases (STSs) are enzymes that play a critical role in the biosynthesis of stilbene, phytoalexins in a small number of unrelated plant species, and are induced by various biotic and abiotic stressors like pathogen attack, UV-irradiation or ozone exposure. To investigate the molecular basis for ozone-induced plant stress responses, we have examined the promoter of the grapevine resveratrol synthase (Vst1). In this report we summarize the influence of ozone on gene regulation. In transgenic tobacco a chimeric gene construct, containing the Vst1 promoter combined with the β-glucuronidase (GUS) reporter gene, is rapidly induced by ozone (0.1 µl·l−1, 12 h). The same construct is also strongly induced by ethylene (20 µl·l−1, 12 h). Promoter deletion analysis of the 5′ flanking sequence identified a positive regulatory element between −430 bp and −280 bp. This region contains ethylene-responsive enhancer elements, as well as an elicitor-responsive sequence in inverse orientation.

Overexpression of the potential herbicide target sedoheptulose-1,7-bisphosphatase from Spinacia oleracea in transgenic tobacco

Several proteins are recalcitrant to expression in Escherichiacoli. To explore transgenic plants as an alternative expressionsystem, the gene encoding the potential herbicide target sedoheptulose-1,7-bisphosphatase (SBPase, EC 3.1.3.37) was expressed in transgenic tobacco(Nicotiana tabaccum) under the control of a duplicatedCaMV 35S RNA promoter. The active protein, a key enzyme in the Calvin cycle,accumulated to approximately 1.2% of total soluble protein. In order to purifyrecombinant SBPase, a sequence encoding six histidine residues was insertedC-terminally which allows a one step purification via Ni2+-NTAaffinity chromatography. N-terminal amino acid sequence analysis of the purifiedprotein confirmed processing of the transit peptide and revealed the previouslyunknown cleavage site. The transit peptide consists of 67 amino acids followedby the mature SBPase subunit of 342 amino acids including the C-terminalfusion. Purified SBPase was found to be enzymatically active after reduction with DTTand showed many biochemical properties of the native enzyme such as thedependence on Mg2+ and a pH optimum of 8.3. Subsequently, SBPaseproduced in transgenic tobacco was used in large-scale screening for thediscovery of novel herbicides.

Chapter 29 Tobacco Protoplast Transformation and Use for Functional Analysis of Newly Isolated Genes and Gene Constructs

Publisher Summary This chapter discusses the tobacco protoplast transformation and use for the functional analysis of newly isolated genes and gene constructs. It has been known for about two decades that, under certain conditions, plant protoplasts can take up viruses, microorganisms, and nucleic acids. After the development of chimeric genes, direct gene transfer (DGT) and the generation of transgenic plants were reported. Since then, numerous reports of DGT into plant protoplasts have been published. DGT is not restricted to certain plant species, but the breadth of its application depends on the ability of protoplasts to divide subsequently and regenerate. DNA uptake methods are a valuable tool for the rapid analysis of promoter/reporter gene constructs and can be used for studying promoter or other regulatory elements that influence transcription. If protoplast regeneration is possible, the expression of the transferred DNA can also be analyzed at the level of the regenerated callus or plant.