Submitted to: Journal of Experimental Botany
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/18/2010
Publication Date: 2/18/2010
Citation: Hung, C., Sun, Y., Chen, J., Darlington, D., Williams, A., Burkey, K.O., Xie, J. 2010. Identification of a Mg-protoporphyrin IX monomethyl ester cyclase homologue, EaZIP, involved in leaf-variegation of Epipremnum aureum 'Golden Pothos' is achieved through a unique method of comparative study using tissue cultu. Journal of Experimental Botany. 61:1483-1493. Interpretive Summary: Photosynthesis in higher plants occurs in special organelles called chloroplasts present in cells of leaves and other green tissues. Formation of chloroplasts requires the expression of genes from both the cell nucleus and the organelle itself. The complexity of this process is illustrated in naturally variegated plants that develop leaves containing both green tissue with normal chloroplasts and white or yellow tissue where chloroplast development is stopped at an early stage of organelle formation. In this study, gene expression was compared in green and yellow plants derived from tissue culture of either green or yellow leaf sectors, respectively, from the variegated house plant Epipremnum aureum ‘Golden Pothos’. The expression of a gene encoding an enzyme required for chlorophyll biosynthesis was severely inhibited in both the yellow plants and yellow sectors of variegated leaves from the parent plant. The results demonstrated that pigment synthesis serves a regulatory function in chloroplast formation. The green and yellow plants will serve as a model system for future research on the interactions of nuclear and chloroplast genes for the purpose of manipulating chloroplasts to facilitate the manufacture of biopharmaceuticals.
Technical Abstract: Variegated-leaf plants provide a great tool for studying chloroplast biogenesis by allowing comparative studies of chloroplast development in green and white/yellow sectors on the same leaves. Comparative studies of different color sectors typically employ dissecting techniques that are subject to problems of cross-contamination. An alternative approach is to regenerate plantlets from these sectors using tissue culture techniques. We explored this method and successfully regenerated stable green and pale yellow plants from a natural variegated Epipremnum aureum ‘Golden Pothos’. Comparing the gene expression abundance between green and pale yellow plants by suppression subtractive hybridization, a total of nine cDNA clones were found to be down-regulated with most of them encoded by the nucleus; but no nuclear-encoded cDNA clones were found to be up-regulated in pale yellow plants. The EaZIP that was reduced more than 4,000-fold in qRT-PCR analysis is a homologue of tobacco NTZIP and Arabidopsis CHL27, which encodes for Mg-protoporphyrin IX monomethyl ester (MPE) cyclase, an enzyme involved in chlorophyll synthesis. This was supported by the alignment of deduced full length amino acid sequence, cross-reactivity with antibodies to a MPE cyclase homologue from Chlamydomonas reinhardtii, and 5-aminolevulinic acid feeding experiments. Examining the expression levels of EaZIP in variegated ‘Golden Pothos’ leaves confirmed EaZIP plays a major role in the loss of chlorophyll. EaZIP, together with the other differentially expressed nuclear- and plastid-encoded genes, may directly or indirectly orchestrate the formation of leaf-variegation.