Transplastomic expression of bacterial l-aspartate-α-decarboxylase enhances photosynthesis and biomass production in response to high temperature stress

Metabolic engineering for beta-alanine over-production in plants is expected to enhance environmental stress tolerance. The Escherichia coli l-aspartate-alpha-decarboxylase (AspDC) encoded by the panD gene, catalyzes the decarboxylation of l-aspartate to generate beta-alanine and carbon dioxide. The constitutive E. coli panD expression cassette was co-introduced with the constitutive, selectable aadA expression cassette into the chloroplast genome of tobacco via biolistic gene transfer and homologous recombination. Site specific integration of the E. coli panD expression cassette into the chloroplast genome and generation of homotransplastomic plants were confirmed by PCR and Southern blot analysis, respectively, following plant regeneration and germination of seedlings on selective media. PanD expression was verified by assays based on transcript detection and in vitro enzyme activity. The AspDC activities in transplastomic plants expressing panD were drastically increased by high-temperature stress. beta-Alanine accumulated in transplastomic plants at levels four times higher than in wildtype plants. Analysis of chlorophyll fluorescence on plants subjected to severe heat stress at 45A degrees C under light verified that photosystem II (PSII) in transgenic plants had higher thermotolerance than in wildtype plants. The CO2 assimilation of transplastomic plants expressing panD was more tolerant to high temperature stress than that of wildtype plants, resulting in the production of 30-40% more above ground biomass than wildtype control. The results presented indicate that chloroplast engineering of the beta-alanine pathway by over-expression of the E. coli panD enhances thermotolerance of photosynthesis and biomass production following high temperature stress.