A Functional Genomics Approach to the Plant Soluble Pyrophosphatase Family

Although the activities of soluble pyrophosphatases were shown to be essential for a number of prokaryotes and eukaryotes, plants were assumed to lack cytoplasmic soluble pyrophosphatase activity and vacuolar membrane bound proton-pumping pyrophosphatase was accepted as the only enzyme responsible for the removal of pyrophosphate accumulated in the cytosol as a by-product of several biosynthetic pathways. On the contrary, Arabidopsis thaliana genome encodes six soluble pyrophosphatase isoforms (ASP1, ASP2A, ASP2B, ASP3, ASP4 and ASP5), which were shown to be highly conserved both in nucleic and in amino acid sequences. Microscopic analysis of leaves transiently transformed with C-terminal GFP tagged A. thaliana sPPase proteins revealed localization in the cytoplasm and/or nucleus for five isoforms (ASP1 to ASP4) and the in vivo plastidial localization of ASP5 could be shown. The analysis of stably transformed plants with promoter driven GUS expression revealed both tissue-specificity and developmental stage dependency of transcription for each A. thaliana soluble pyrophosphatase isoform. The promoter data were further confirmed by real time PCR analysis through which the active transcription of several A. thaliana soluble pyrophosphatase isoforms could be shown in all plant tissues, including later stages of development. The regulation of the expression of A. thaliana soluble pyrophosphatases by soluble sugars was analyzed using A. thaliana cell cultures and an in planta approach. A specific induction of the expression of ASP2B in response to sugar starvation was observed. Furthermore, the results indicate the possibility of sucrose regulation of the expression of ASP3, while the transcription of plastidial isoform (ASP5) was induced in response to 100 mM glucose in planta. The changes in the expression of A. thaliana soluble pyrophosphatases in response to ABA and different environmental stresses were analyzed using real time PCR. The data revealed an isoform- and/or tissue-specific and time dependent stress response suggesting specific roles for each isoform in vivo. Based on the data, the possible role of ASP3 in regulation of UGPase activity could be proposed. The wounding experiments using sugar beet (Beta vulgaris) plants overexpressing either Bsp1 (Beta vulgaris soluble pyrophosphatase isoform 1) or Bvp1 (Beta vulgaris vacuolar pyrophosphatase isoform 1) revealed the possibility of post-transcriptional regulation of mRNA levels of both soluble and vacuolar pyrophosphatases. Using recombinant Bsp1 protein expressed in Escherichia coli, the in vitro phosphorylation by protein kinase C could be shown and the possibility of post-translational regulation of plant soluble pyrophosphatase activity by phosphorylation was discussed. The overexpression of neither Bsp1 nor Bvp1 caused a significant effect on the sucrose accumulation of sugar beet taproots. On the other hand, the heterologous overexpression of not only Bvp1 but also Bsp1 revealed an impaired root growth and a possible contribution to the salt tolerance of A. thaliana. In summary, the results suggest that the plant soluble pyrophosphatases can perform multiple and vital functions during plant development and stress responses. In addition, the expression patterns of plant soluble pyrophosphatases indicate a strong link to the plant carbohydrate metabolism.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Heidelberg

Year

2006

Author dc:creator

• Ergen, Zahide Neslihan

Contributors dc:contributor

• Rausch, Thomas

Repository record source_url

http://www.ub.uni-heidelberg.de/archiv/6568

OAI identifier oai:identifier

oai:archiv.ub.uni-heidelberg.de:6568