L. Willmitzer

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.

Expression of a yeast-derived invertase in the cell wall of tobacco and Arabidopsis plants leads to accumulation of carbohydrate and inhibition of photosynthesis and strongly influences growth and phenotype of transgenic tobacco plants.

Chimeric genes consisting of the coding sequence of the yeast invertase gene suc 2 and different N‐terminal portions of the potato‐derived vacuolar protein proteinase inhibitor II fused to the 35S CaMV promoter and the poly‐A site of the octopine synthase gene were transferred into tobacco and Arabidopsis thaliana plants using Agrobacterium based systems. Regenerated transgenic plants display a 50‐ to 500‐fold higher invertase activity compared to non‐transformed control plants. This invertase is N‐glycosylated and efficiently secreted from the plant cell leading to its apoplastic location. Whereas expression of the invertase does not lead to drastic changes in transgenic Arabidopsis thaliana plants, transgenic tobacco plants show dramatic changes with respect to development and phenotype. Expression of the invertase leads to stunted growth due to reduction of internodal distances, to development of bleached and/or necrotic regions in older leaves and to suppressed root formation. In mature leaves, high levels of soluble sugars and starch accumulate. These carbohydrates do not show a diurnal turnover. The accumulation of carbohydrate is accompanied by an inhibition of photosynthesis, and in tobacco, by an increase in the rate of respiration. Measurements in bleached versus green areas of the same leaf show that the bleached section contains high levels of carbohydrates and has lower photosynthesis and higher respiration than green sections. It is concluded that expression of invertase in the cell wall interrupts export and leads to an accumulation of carbohydrates and inhibition of photosynthesis.

Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.

Active loading of the phloem with sucrose in leaves is an essential part of the process of supplying non‐photosynthetic tissues with carbon and energy. The transport is protein mediated and coupled to proton‐symport, but so far no sucrose carrier gene has been identified. Using an engineered Saccharomyces cerevisiae strain, a cDNA from spinach encoding a sucrose carrier was identified by functional expression. Yeast strains that allow the phenotypic recognition of a sucrose carrier activity were constructed by expressing a cytoplasmic invertase from yeast, or the potato sucrose synthase gene, in a strain unable to transport or grow on sucrose due to a deletion in the SUC2 gene. A spinach cDNA expression library established from the poly(A)+ RNA from source leaves of spinach and cloned in a yeast expression vector yielded transformed yeast clones which were able to grow on media containing sucrose as the sole carbon source. This ability was strictly linked to the presence of the spinach cDNA clone pS21. Analysis of the sucrose uptake process in yeast strains transformed with this plasmid show a pH‐dependent uptake of sucrose with a Km of 1.5 mM, which can be inhibited by maltose, alpha‐phenylglucoside, carbonyl cyanide m‐chlorophenylhydrazone and p‐chloromercuribenzenesulfonic acid. These data are in accordance with measurements using both leaf discs and plasma membrane vesicles from leaves of higher plants. DNA sequence analysis of the pS21 clone reveals the presence of an open reading frame encoding a protein with a molecular mass of 55 kDa. The predicted protein contains several hydrophobic regions which could be assigned to 12 membrane‐spanning regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for an essential role of the sucrose transporter in phloem loading and assimilate partitioning.

Sucrose is the principal transport form of assimilates in most plants. In many species, translocation of assimilates from the mesophyll into the phloem for long distance transport is assumed to be carrier mediated. A putative sucrose proton cotransporter cDNA has been isolated from potato and shown to be expressed mainly in the phloem of mature exporting leaves. To study the in vivo role and function of the protein, potato plants were transformed with an antisense construct of the sucrose transporter cDNA under control of the CaMV 35S promoter. Upon maturation of the leaves, five transformants that expressed reduced levels of sucrose transporter mRNA developed local bleaching and curling of leaves. These leaves contained > 20‐fold higher concentrations of soluble carbohydrates and showed a 5‐fold increase in starch content as compared with wild type plants, as expected from a block in export. Transgenic plants with a reduced amount of sucrose carrier mRNA show a dramatic reduction in root development and tuber yield. Maximal photosynthetic activity was reduced at least in the strongly affected transformants. The effects observed in the antisense plants strongly support an apoplastic model for phloem loading, in which the sucrose transporter located at the phloem plasma membrane represents the primary route for sugar uptake into the long distance distribution network.

“Sink” regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin-cycle enzymes and an increase of glycolytic enzymes

Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the “sink” regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO2. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.

Nucleotide sequence of proteinase inhibitor II encoding cDNA of potato (Solanum tuberosum) and its mode of expression

SummaryTwo cDNA clones containing the complete coding region of a developmentally controlled (tuber-specific) as well as environmentally inducible (wound-inducible) gene from potato (Solanum tuberosum) have been sequenced. The open reading frame codes for 154 amino acids. Its sequence is highly homologous to the proteinase inhibitor II from tomato, indicating that the cDNAs encode the corresponding proteinase inhibitor II of potato. In addition the putative potato proteinase inhibitor II contains a sequence which is completely homologous with that of another small peptide proteinase inhibitor from potato, called PCI-I. Evidence is presented that this small peptide is probably derived from the proteinase inhibitor II by posttranslational processing.Northern type experiments using RNA from wounded and nonwounded leaves demonstrate that RNA homologous to the putative proteinase inhibitor II cDNAs accumulates in leaves as a consequence of wounding, whereas normally the expression of this gene is under strict developmental control, since it is detected only in tubers of potato (Rosahl et al. 1986). In addition the induction of this gene in leaves can also be achieved by the addition of different polysaccharides such as poly galacturonic acid or chitosan. In contrast to the induction of its expression by wounding in leaves, wounding of tubers results in a disappearance of the proteinase II inhibitor m-RNA from these organs.

Effect of antisense repression of the chloroplast triose-phosphate translocator on photosynthetic metabolism in transgenic potato plants

The introduction of an antisense DNA into transgenic potato (Solanum tuberosum L.) plants decreased the expression of the chloroplast triose-phosphate translocator and lowered its activity by 20–30%. With plants propagated from tubers, the effect of the transformation on photosynthetic metabolism was analysed by measuring photosynthesis, the formation of leaf starch, and the total and subcellular metabolite contents in leaves. Although the transformants, in contrast to those propagated from cell cultures, did not differ from the wild-type plants in respect to rates of photosynthesis, plant appearance, growth and tuber production, their photosynthetic metabolism was found to be severely affected. The results show that the decrease in activity of the triose-phosphate translocator in the transformants caused a fourfold increase in the level of 3-phosphoglycerate and a corresponding decrease in inorganic phosphate in the stromal compartment, resulting in a large increase in the synthesis of starch. Whereas during a 12-h day period wild-type plants deposited 43% of their CO2 assimilate into starch, this value rose to 61–89% in the transformants. In contrast to the wild-type plants, where the rate of assimilate export from the leaves during the night period was about 75% of that during the day, the export rate from leaves of transformants appeared to be much higher during the night than during the day. As the mobilisation of starch occurs in part hydrolytically, resulting in the formation of glucose, the triose-phosphate translocator loses its exclusive function in the export of carbohydrates from the chloroplasts when the photoassimilates are temporarily deposited as starch. It appears that by directing the CO2 assimilates mainly into starch, the transformants compensate for the deficiency in triose-phosphate translocator activity in such a way that the productivity of the plants is not affected by the transformation.

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.

Heterosis manifestation during early Arabidopsis seedling development is characterized by intermediate gene expression and enhanced metabolic activity in the hybrids

Heterosis-associated cellular and molecular processes were analyzed in seeds and seedlings of Arabidopsis thaliana accessions Col-0 and C24 and their heterotic hybrids. Microscopic examination revealed no advantages in terms of hybrid mature embryo organ sizes or cell numbers. Increased cotyledon sizes were detectable 4 days after sowing. Growth heterosis results from elevated cell sizes and numbers, and is well established at 10 days after sowing. The relative growth rates of hybrid seedlings were most enhanced between 3 and 4 days after sowing. Global metabolite profiling and targeted fatty acid analysis revealed maternal inheritance patterns for a large proportion of metabolites in the very early stages. During developmental progression, the distribution shifts to dominant, intermediate and heterotic patterns, with most changes occurring between 4 and 6 days after sowing. The highest incidence of heterotic patterns coincides with establishment of size differences at 4 days after sowing. In contrast, overall transcript patterns at 4, 6 and 10 days after sowing are characterized by intermediate to dominant patterns, with parental transcript levels showing the largest differences. Overall, the results suggest that, during early developmental stages, intermediate gene expression and higher metabolic activity in the hybrids compared to the parents lead to better resource efficiency, and therefore enhanced performance in the hybrids.

Expression of the triose phosphate translocator gene from potato is light dependent and restricted to green tissues

The export of primary photosynthesis products from chloroplasts into the cytoplasm is mediated by the triose phosphate translocator. The transporter is an integral membrane protein localized at the inner envelope of chloroplasts. In order to study the expression of the major chloroplast envelope protein gene E29, which is assumed to function as the translocator, we have isolated corresponding cDNA clones from potato. A full-length clone was sequenced and shown to be highly homologous to the E29 gene from spinach. Expression on the RNA level is restricted to green tissues, is light dependent and cannot be induced by sucrose in darkness. The presence of a single-copy gene argues for the existence of different translocator systems responsible for import and export of carbohydrates in chloroplasts and amyloplasts.