Impaired photoassimilate partitioning caused by phloem-specific removal of pyrophosphate can be complemented by a phloem-specific cytosolic yeast-derived invertase in transgenic plants.

Constitutive expression of the Escherichia coli ppa gene encoding inorganic pyrophosphatase resulted in sugar accumulation in source leaves and stunted growth of transgenic tobacco plants. The reason for this phenotype was hypothesized to be reduced sucrose utilization and loading into the phloem. To study the role of PPi in phloem cells, a chimeric gene was constructed using the phloem-specific rolC promoter of Agrobacterium rhizogenes to drive the expression of the ppa gene. Removal of cytosolic PPi in those cells resulted in photoassimilate accumulation in source leaves, chlorophyll loss, and reduced plant growth. From these data, it was postulated that sucrose hydrolysis via sucrose synthase is essential for assimilate partitioning. To bypass the PPi-dependent sucrose synthase step, transgenic plants were produced that express various levels of the yeast suc2 gene, which encodes cytosolic invertase, in their phloem cells. To combine the phloem-specific expression of the ppa gene and the suc2 gene, crosses between invertase- and pyrophosphatase-containing transgenic plants were performed. Analysis of their offspring revealed that invertase can complement the phenotypic effects caused by the removal of PPi in phloem cells.

[1]  M. Bevan,et al.  Binary Agrobacterium vectors for plant transformation. , 1984, Nucleic acids research.

[2]  T. Schmülling,et al.  Promoters of the rolA, B, and C genes of Agrobacterium rhizogenesare differentially regulated in transgenic plants. , 1989, The Plant cell.

[3]  R. Taussig,et al.  Nucleotide sequence of the yeast SUC2 gene for invertase. , 1983, Nucleic acids research.

[4]  R. Lahti,et al.  Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12 , 1988, Journal of bacteriology.

[5]  M. Stitt,et al.  Metabolite levels in specific cells and subcellular compartments of plant leaves , 1989 .

[6]  P. Turner,et al.  A comparison of eukaryotic viral 5'-leader sequences as enhancers of mRNA expression in vivo. , 1987, Nucleic acids research.

[7]  M. Stitt,et al.  Phloem‐specific expression of pyrophosphatase inhibits long distance transport of carbohydrates and amino acids in tobacco plants , 1996 .

[8]  L. Jouanin,et al.  Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading frames. , 1986, The Journal of biological chemistry.

[9]  Marc Van Montagu,et al.  Efficient octopine Ti plasmid-derived vectors for Agrobacterium- mediated gene transfer to plants , 1985, Nucleic Acids Res..

[10]  M. Stitt,et al.  Regulation of fructose 2,6-bisphosphate concentration in spinach leaves. , 1984, European journal of biochemistry.

[11]  R. Giaquinta.  Phloem Loading of Sucrose , 1983 .

[12]  W. Frommer,et al.  Evidence for an essential role of the sucrose transporter in phloem loading and assimilate partitioning. , 1994, The EMBO journal.

[13]  M. Stitt,et al.  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. , 1990, The EMBO journal.

[14]  L. Willmitzer,et al.  Expression of a tuber‐specific storage protein in transgenic tobacco plants: demonstration of an esterase activity , 1987, The EMBO journal.

[15]  M. Van Montagu,et al.  Characterization of different plaque-forming and defective temperate phages in Agrobacterium. , 1975, The Journal of general virology.

[16]  N. Yang,et al.  Maize sucrose synthase-1 promoter directs phloem cell-specific expression of Gus gene in transgenic tobacco plants. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[17]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[18]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[19]  W. Frommer,et al.  Potato sucrose transporter expression in minor veins indicates a role in phloem loading. , 1993, The Plant cell.

[20]  W. Frommer,et al.  Expression of an Arabidopsis sucrose synthase gene indicates a role in metabolization of sucrose both during phloem loading and in sink organs. , 1993, The Plant journal : for cell and molecular biology.

[21]  F. Nagy,et al.  Multiple cis regulatory elements for maximal expression of the cauliflower mosaic virus 35S promoter in transgenic plants. , 1989, The Plant cell.

[22]  L. Willmitzer,et al.  Improved method for the isolation of RNA from plant tissues. , 1987, Analytical biochemistry.

[23]  U. Sonnewald,et al.  Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning. , 1992, The Plant journal : for cell and molecular biology.

[24]  W. Frommer,et al.  Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast. , 1992, The EMBO journal.

[25]  J. Bonnemain,et al.  Immunolocalization of the Plasma Membrane H+ -ATPase in Minor Veins of Vicia faba in Relation to Phloem Loading , 1994, Plant physiology.

[26]  M. Stitt,et al.  On a possible role of fructose 2,6‐bisphosphate in regulating photosynthetic metabolism in leaves , 1982 .

[27]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[28]  W. Bullock.  XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. , 1987 .

[29]  S. Delrot,et al.  Involvement of Protons as a Substrate for the Sucrose Carrier during Phloem Loading in Vicia faba Leaves. , 1981, Plant physiology.

[30]  L. Hirth,et al.  Nucleotide sequence of cauliflower mosaic virus DNA , 1980, Cell.

[31]  M. Van Montagu,et al.  The complete nucleotide sequence of the TL‐DNA of the Agrobacterium tumefaciens plasmid pTiAch5. , 1984, The EMBO journal.

[32]  Robert Turgeon,et al.  The Sink-Source Transition in Leaves , 1989 .

[33]  M. Stitt.  Rising Co2 Levels and Their Potential Significance for Carbon Flow in Photosynthetic Cells , 1991 .

[34]  R. Amasino.  Acceleration of nucleic acid hybridization rate by polyethylene glycol. , 1986, Analytical biochemistry.

[35]  W. Eschrich.  Sealing Systems in Phloem , 1975 .

[36]  B. Loveys,et al.  Comparative investigations on the distribution of sucrose synthase activity and invertase activity within growing, mature and old leaves of some C3 and C4 plant species , 1985 .