Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco.
Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria. A gene encoding a fusion protein of yeast ALA-S with an aminoterminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA. Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis. ALA-S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT. Steady-state RNA and protein levels and consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S. In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression. The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.
Zavgorodnyaya
A
A
Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany.
Papenbrock
J
J
Grimm
B
B
D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002734:Chlorophyll / Q000096:biosynthesis* / Q000172:deficiency; D017931:DNA Primers; D008928:Mitochondria / Q000201:enzymology; D010641:Phenotype; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D015398:Signal Transduction; D014026:Nicotiana / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D002734:Chlorophyll; D000624:5-Aminolevulinate Synthetase; D012273:Ribulose-Bisphosphate Carboxylase