Jerry S. Marshall

Two Genes Encode Highly Similar Chloroplastic NADP-Malic Enzymes in Flaveria (Implications for the Evolution of C4 Photosynthesis)

To gain an understanding of the molecular events underlying the evolution of C4 photosynthesis, we have undertaken a detailed study of the NADP-malic enzyme gene family in C4 and C3 species of Flaveria. Three genomic clones from the C4 species Flaveria bidentis were characterized and found to encode two highly similar chloroplastic forms of NADP-malic enzyme, termed ME1 and ME2. Genomic Southern blotting with gene-specific probes showed that both Me1 and Me2 are found in Flaveria trinervia (C4) and Flaveria pringlei (C3) as well as in F. bidentis. Northern blots demonstrated that Me1 expression in leaves parallels the degree of C4 photosynthesis in seven Flaveria species. Furthermore, whereas Me2 was expressed at a low level in both roots and leaves of F. bidentis, Me1 expression was seen only in leaves and was light-regulated. We discuss these results in the context of the evolution of C4 photosynthesis in Flaveria.

Expression of the C4 Me1 Gene from Flaveria bidentis Requires an Interaction between 5[prime] and 3[prime] Sequences.

The efficient functioning of C4 photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle sheath cells. To determine the mechanism controlling bundle sheath cell-specific expression of the NADP-malic enzyme, we made a set of chimeric constructs using the 5[prime] and 3[prime] regions of the Flaveria bidentis Me1 gene fused to the [beta]-glucuronidase gusA reporter gene. The pattern of GUS activity in stably transformed F. bidentis plants was analyzed by histochemical and cell separation techniques. We conclude that the 5[prime] region of Me1 determines bundle sheath specificity, whereas the 3[prime] region contains an apparent enhancer-like element that confers high-level expression in leaves. The interaction of 5[prime] and 3[prime] sequences was dependent on factors that are present in the C4 plant but not found in tobacco.

Expression of the C4 Me1 Gene from Flaveria bidentis Requires an Interaction between 5 and 3 Sequences

The efficient functioning of C4 photosynthesis requires the strict compartmentation of a suite of enzymes in either mesophyll or bundle sheath cells. To determine the mechanism controlling bundle sheath cell-specific expression of the NADP-malic enzyme, we made a set of chimeric constructs using the 5’ and 3’ regions of the F/ayeria bidentis Mel gene fused to the P-glucuronidase gusA reporter gene. The pattern of GUS activity in stably transformed F. bidentis plants was analyzed by histochemical and cell separation techniques. We conclude that the 5‘ region of Mel determines bundle sheath specificity, whereas the 3’ region contains an apparent enhancer-like element that confers highleve1 expression in leaves. The interaction of 5‘ and 3’ sequences was dependent on factors that are present in the C4 plant but not found in tobacco.