Mario Rocha-Sosa

Construction of an Intron-Containing Marker Gene - Splicing of the Intron in Transgenic Plants and Its Use in Monitoring Early Events in Agrobacterium-Mediated Plant Transformation

SummaryAgrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to overcome this obstacle a modified prokaryotic indicator gene was constructed. The expression of this indicator gene and its use in analysing early events in Agrobacterium-mediated plant transformation are described. A portable intron, derived from a plant intron, was introduced into the β-glucuronidase (GUS) gene. In transgenic plants containing this chimaeric gene the intron is spliced efficiently, giving rise to GUS enzymatic activity. Mapping of the splice junction indicates the exact removal of the intron. No GUS activity is detected in agrobacteria containing this construct due to the lack of a eukaryotic splicing apparatus in prokaryotes. Early phases after transformation of Arabidopsis cotyledon explants were analysed using this GUS-intron chimaeric gene showing that as early as 36 h after Agrobacterium infection significant GUS activity is detected. In vivo GUS staining of transformed cells clearly shows that quickly proliferating calli expressing GUS activity are formed, mainly at the cut surface. Minor transformation events occur however throughout the whole cotyledon. These data indicate that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately. The intron-containing GUS gene can be used as an optimised marker gene in transient and stable transformation experiments.

Both developmental and metabolic signals activate the promoter of a class I patatin gene

Patatin is one of the major soluble proteins in potato tubers and is encoded by a multigene family. Based on structural considerations two classes of patatin genes are distinguished. The 5′‐upstream regulatory region of a class I gene contained within a 1.5 kb sequence is essential and sufficient to direct a high level of tuber‐specific gene activity which was on average 100‐ to 1000‐fold higher in tubers as compared to leaf, stem and roots in greenhouse grown transgenic potato plants when fused to the β‐glucuronidase reporter gene. Histochemical analysis revealed this activity to be present in parenchymatic tissue but not in the peripheral phellem cells of transgenic tubers. Furthermore the promoter fragment can be activated in leaves under conditions that simulate the need for the accumulation of starch in storage organs, i.e. high levels of sucrose. The expression is restricted to both mesophyll and epidermal cells in contrast to vascular tissue or hair cells.

Plant Lipoxygenases. Physiological and Molecular Features

Lipoxygenases (LOXs; EC[1.13.11.12][1]) are nonheme iron-containing dioxygenases widely distributed in plants and animals. LOX catalyzes the addition of molecular oxygen to polyunsaturated fatty acids containing a ( Z , Z )-1,4-pentadiene system to produce an unsaturated fatty acid hydroperoxide.

Systemic induction of proteinase-inhibitor-II gene expression in potato plants by wounding.

The systemic induction of expression of the gene for proteinase inhibitor II after wounding different parts of potato (Solanum tuberosum L.) plants was analysed at the RNA level. Wounding of either leaves or tubers led to an induction of expression of this gene in non-wounded upper and lower leaves as well as in the upper stem segment, whereas no expression was observed in nonwounded roots or in the lower stem segment. The signal mediating the systemic induction in nonwounded tissue must therefore be able to move both acropetally and basipetally. The systemic wound response is specific for the expression of the proteinase-inhibitor-II gene as no influence was observed for the expression of genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the tuber storage protein patatin which were examined in parallel with the proteinase-inhibitor-II gene.

Gene expression during tuber development in potato plants.

Potato tubers are modified stems that have differentiated into storage organs. Factors such as day‐length, nitrogen supply, and levels of the phytohormones cytokinin and gibberellic acid, are known to control tuberization. Morphological changes during tuber initiation are accompanied by the accumulation of a characteristic set of proteins, thought to be involved in N‐storage (i.e. patatin) or defense against microbial or insect attack (i.e. proteinase inhibitor II). Additionally, deposition of large amounts of starch occurs during tuber formation, which is paralleled by an increase in sucrose synthase and other enzymes involved in starch biosynthesis (i.e. ADP‐glucose pyrophosphorylase, starch synthases, and branching enzyme). Potential controlling mechanisms for genes expressed during tuberization are discussed.

A class II patatin promoter is under developmental control in both transgenic potato and tobacco plants

SummaryA new member of the patatin gene family belonging to the class II subfamily was isolated and characterized by DNA sequencing. In order to study the expression profile of this gene, the promoter was fused to the β-glucuronidase gene and transferred to potato and tobacco. Histochemical analysis revealed high expression in a few defined cells in potato tubers and in a specific layer of both potato and tobacco root tips. In contrast to the developmentally and metabolically regulated class I patatin gene B33 this gene was not inducible by elevated levels of sucrose. Expression of this chimaeric gene was also found in callus and suspension cultures of potato.

Immunocytochemical localization of patatin, the major glycoprotein in potato (Solanum tuberosum L.) tubers

Patatin is a family of glycoproteins with an apparent molecular weight of 40 kDa. The protein is synthesized as a pre-protein with a hydrophobic signal sequence of 23 amino acids. Using different immunocytochemical methods we determined the tissue-specific as well as subcellular localization of the patatin protein. Since antibodies raised against patatin showed crossreactivity with glycans of other glycoproteins, antibodies specific for the protein portion of the glycoprotein were purified. Using these antibodies for electron-microscopical immunocytochemistry, the protein was found to be localized mainly in the vacuoles of both tubers and leaves of potatoes (Solanum tuberosum L.) induced for patatin expression. Neither cell walls nor the intercellular space contained detectable levels of patatin protein. Concerning the tissue specificity, patatin was mainly found in parenchyma cells of potato tubers. The same distribution was observed for the esterase activity in potato tubers.

A detailed study of the regulation and evolution of the two classes of patatin genes in Solanum tuberosum L.

The class-specific expression of patatin genes was investigated by analysing four new patatin genes. A class I patatin gene from cv. Berolina as well as a class I and two class II patatin genes from the monohaploid cultivar AM 80/5793 were isolated and partially sequenced. Sequence comparison indicates rearrangements as the major source for the generation of diversity between the different members of the classes. The expression of single genes was studied in potato plants transformed with chimaeric genes where the putative patatin promoters were fused to the GUS reporter gene. A detailed histochemical analysis reveals that both class I genes are expressed as the previously described class I patatin gene B33 from cv. Berolina [1], i.e. in the starch-containing cells of potato tubers and in sucrose-induced leaves. The class II gene pgT12 shows the same pattern as the previously described class II gene pgT2 [2], i.e. expression in root tips and in the vascular tissue of tubers, whereas no activity was detectable for pgT4. Thus the expression pattern of both classes of genes seems to be stable at least within or even between different cultivars.

Presence of a Transposon-Like Element in the Promoter Region of an Inactive Patatin Gene in Solanum-Tuberosum-L

The promoter of the PGT3 patatin gene belonging to the class II subfamily is highly homologous to other class II patatin genes except for a 736 bp insertion in front of the putative transcription start site. The insertion is characterized by structural features resembling a transposable element such as an 11 bp inverted repeat at the termini and an 8 bp duplication flanking the insertion site. Despite the high homology to active patatin genes, fusion of its promoter to the β-glucuronidase reporter gene does not lead to detectable β-glucuronidase (GUS) activity in transgenic potato or tobacco plants, suggesting that the inactivation of this gene might be caused by the insertion of the transposon like element.

Characterization of novel F-box proteins in plants induced by biotic and abiotic stress

Plants protect against pathogen infections by a combination of constitutive and induced strategies. The induction of plant defense involves the recognition of compounds derived from the pathogen or the plant itself, called elicitors. Looking for new genes involved in plant defense responses, we isolated a cDNA clone corresponding to an elicitor-induced mRNA from Phaseolus vulgaris cell suspension cultures. This clone, PvFBS1, encodes a protein with an F-box, therefore a putative component of an SCF ubiquitin ligase complex. PvFBS1 mRNA accumulates in leaves of whole plants in response to wounding or osmotic stress, as well as, following the application of methyl jasmonate (MeJA). salicylic acid (SA) or abscisic acid (ABA). Several sequences related to PvFBS1 were found in the GenBank. In Arabidopsis thaliana there are 4 genomic sequences coding for proteins with similarity to PvFBS1. One of them, AtFBS1, displays a pattern of induction analogous to the one observed for PvFBS1. A yeast two-hybrid assay proved that AtFBS1 was able to interact with ASK1, the component of the SCF complex that binds the F-box. A deletion of the F-box in AtFBS1 abolishes the ability of this protein to interact with ASK1. This demonstrates the functionality of the F-box contained in AtFBS1. Gene fusions to the GUS reporter gene revealed a complex regulation for AtFBS1 expression.