A simple mechanism for the establishment of C₂‐specific gene expression in Brassicaceae

Abstract

SUMMARYThe transition of C 3 , via C 2 towards C 4 photosynthesis is an important example of stepwise evolution of a complex genetic trait. A common feature that was gradually emphasized during this trajectory is the evolution of a CO 2 concentration mechanism around Rubisco. In C 2 plants, this mechanism is based on tissue-specific accumulation of glycine decarboxylase (GDC) in bundle sheath (BS) cells, relative to global expression in the cells of C 3 leaves. This limits photorespiratory CO 2 release to BS cells. Because BS cells are surrounded by photosynthetically active mesophyll cells, this arrangement enhances the probability of re-fixation of CO 2 . The restriction of GDC to BS cells was mainly achieved by confinement of its P-subunit (GLDP). Here, we provide a mechanism for the establishment of C 2 -type gene expression by studying the upstream sequences of C 3 Gldp genes in Arabidopsis thaliana. Deletion of 59 bp in the upstream region of AtGldp1 restricted expression of a reporter gene to BS cells and the vasculature without affecting diurnal variation. This region was named the 'M box'. Similar results were obtained for the AtGldp2 gene. Fusion of the M box to endogenous or exogenous promoters supported mesophyll expression. Nucleosome densities at the M box were low, suggesting an open chromatin structure facilitating transcription factor binding. In silico analysis defined a possible consensus for the element that was conserved across the Brassicaceae, but not in Moricandia nitens, a C 2 plant. Collective results provide evidence that a simple mutation is sufficient for establishment of C 2 -specific gene expression in a C 3 plant.

The 5′UTR Intron of Arabidopsis GGT1 Aminotransferase Enhances Promoter Activity by Recruiting RNA Polymerase II

“…showed the reported expression pattern in the vascular system (Adwy et al, 2015), while CAT3 was expressed in the vascular system but also weakly in the entire leaf tissue as described by Zimmermann and colleagues (2006). As expected, the PEROXIDASE was proofed to be root-specific (Winter et al, 2007).…”

“…S8). As observed in GGT2 15I:: gusA lines, GGT1 5I activated leaf expression of GLDP1-P2, which is exclusively expressed in the vascular system (Adwy et al, 2015). However, GGT1 5I was not able to drive expression of the PEROXIDASE in leaves but enhanced its expression in roots.…”

“…The 5I substitution of the corresponding GGT isoform was performed by overlapping extension PCR (Supplemental Table S3). To test the effect of GGT1 5I on tissue specificity, primers were designed to amplify the 59 upstream sequences of CATALASE3 (At1g20620; TAIR Accession, Locus: 2034357), GLDP1-P2 (At4g33010; TAIR Accession, Locus: 2123777; Adwy et al, 2015), and a rootspecific peroxidase (At3g01190; TAIR Accession, Locus: 2102087). Furthermore, cloning GGT1 5I downstream of the promoter sequences necessitated the introduction of an MCS into the linker region between the promoter and the gusA gene.…”

“…To test whether GGT1 5I is able to drive leaf expression of other promoters than GGT2, the promoter and 59UTRs of three additional genes were cloned into the GUS expression vector. CATALASE3 (At1g20620, Zimmermann et al, 2006) and GLDP1-P2 (At4g33010, Adwy et al, 2015) are known to be mainly expressed in the vascular system, while the expression of a PEROXIDASE (At3g01190, Winter et al, 2007) is restricted to roots.…”

“…Intron-mediated enhancement (IME) of GGT1 5I was associated with higher abundance of RNA polymerase II on the gusA coding sequence. To investigate regulatory features of the GGT1 promoter, the GGT1 (At1g23310) 59 upstream sequence (1,454 bp) as well as the 59UTR (604 bp) was cloned upstream of the gusA gene into the pSAG vector (Adwy et al, 2015), resulting in GGT1::gusA. GusA, formally known as uidA, encodes for the bacterial reporter protein b-glucuronidase.…”