Yeast 5‐aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco

Abstract

SummarySynthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA} in plants starts with glutamate and is a tRNAdependent 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.

Expression of yeast Hem1 gene controlled by Arabidopsis HemA1 promoter improves salt tolerance in Arabidopsis plants

“…, measured in the work was similar with the other reports (12,13). However, the ALA synthesizing capacity in Arabidopsis was found increased during salt stress (Fig.…”

“…In this work, we did not fuse a chloroplast-targeting peptide in the recombinant gene, and the expressed ALA-S protein was located in the mitochodria (Fig. 1e), where the enzyme could catalyze succinate and glycine to synthesize additional ALA, which is different from the previous reports (12,13). In the transgenic rice, the endogenous ALA content was 44-85% higher than that of the WT (13).…”

“…Until now, there were no papers which showed that the transgenic plants with ability to over-produce endogenous ALA can improve salt tolerance, although transgenic tobacco or rice with this ability was obtained several years ago (12)(13)(14)(15)(16)(17). In fact, the transgenic plants with a constitutive promoter could not grow under high light condition because the over-produced ALA under dark would be incorporated into protochlorophyllide, protoporphyrin IX, and Mg-protoporphyrin IX at the stage preceding chlorophyll biosynthesis, which are strong photosensitizers, and photooxidation would injury plants when they switched to light condition (14).…”

“…Therefore, the transgenic plants with yeast Hem1 controlled by a light-responsive promoter were guessed significant in agriculture. Furthermore, ALA biosynthesis in higher plants is located in chloroplasts (3, 4) and the exogenous ALA-S proteins in transgenic tobacco and rice were located in the stroma of chloroplasts (12,13). In this work, we did not fuse a chloroplast-targeting peptide in the recombinant gene, and the expressed ALA-S protein was located in the mitochodria (Fig.…”

“…GSA-AT, which codes the last enzyme in ALA biosynthesis in higher plants, was the first gene transformed into tobacco (11). Then, Zavgorodnyaya et al (12) transformed a gene coding aminolevulinate synthase (ALA-S) in yeast into tobacco. They found that the recombinant gene could be expressed in higher plants, and more endogenous ALA was biosynthesized because of the additional ALA-S activity.…”