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Title: ANALYSIS OF THE ARABIDOPSIS RCA MUTANT COMPLEMENTED WITH ENGINEERED RUBISCOACTIVASE AT A NUCLEOTIDE BINDING SITE

Author
item KALLIS, RUSS - LIFE SCIENCES UNIV OF ILL
item PORTIS JR, ARCHIE

Submitted to: Arabidopsis Research International Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 7/28/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: In site directed mutagenesis experiments we have altered Arabidopsis cDNA coding for the 42kD form of Arabidopsis rubisco activase in a nucleotide binding region at amino acid position #111. Recombinant enzymes with amino acid changes Q111D and Q111E had a greater rate and extend of rubisco activation, while recombinant enzyme Q111S showed a diminished rate and extent of rubisco activation when compared to the 42kD recombinant wild type. Rubisco activation by recombinant enzymes was also measured in response to various ADP/ATP ratios. The recombinant enzymes Q111D and Q111E demonstrated rubisco activation at ADP/ATP ratios that inhibited activation by the 42kD recombinant wild type enzyme to the level of spontaneous activation. Agrobacterium tumefaciens EHA101 was transformed with binary vector pCGN1547 carrying altered Arabidopsis rubisco activase cDNA and the NPTII gene coding for kanamycin resistance, and introduced into Arabidopsis rca by vacuum infiltration. (Bent et al. Science 265:1856-1860, 1994). Seeds from infiltrated plants were plated on selection media containing 50mg/L kanamycin and germinated at 100-150 uE m-2 s-1 light and 1 percent CO2. Rubisco activase polypetide was detected in soluble leaf extracts from putative transformants by immunoblotting using spinach rubisco activase polyclonal antibodies. Arabidopsis rca complemented with Q111E have recovered a photosynthesis rate comparable to the wild type plant. The possibility that small differences in photosynthesis rate effect plant growth will be analyzed in homozygous plants. The impact of variations in rubisco activation state and RuBP levels upon regulations of photosynthesis and rubisco activity is under current study.