Identification and Characterization of a T-DNA Promoter Trap Line of Arabidopsis thaliana Uncovers an Embryo Sac-Specific Bi-directional Promoter

Abstract

The Arabidopsis thaliana promoter trap line showing uidA reporter gene expression specifically in mature embryo sacs was found to carry transfer DNA (T-DNA) insertion in the 5′ untranslated region (UTR) of At3g17150. At3g17140 and At3g17150 are a pair of head-tohead-oriented genes separated by a 668-bp intergenic region encoding for a putative pectin methyl esterase inhibitor protein. In the GUS-650 line, uidA reporter gene was inserted in head-to-head orientation to the At3g17140 gene. Presence of At3g17140 transcripts in the GUS-650 line indicated that T-DNA insertion did not affect this gene and the 668-bp intergenic region functions as a bi-directional promoter. The 672-bp fragment separating the uidA and At3g17140 genes was cloned and placed in forward or reverse orientation next to the uidA gene. Transgenic Arabidopsis plants carrying either of these constructs showed β-glucuronidase (GUS) activity in the ovules confirming that the 672-bp sequence is a bi-directional promoter specific to ovules. These results were also supported by the presence of cis-acting motifs such as TATA box, CAAT box, seed storage protein element (CAACAC), endosperm-specific elements (AACA), Skn-1 motif (GTCAT) and bZIP transcription factor binding element (ACGT) within the 672-bp sequence in both orientations. The significance of a promoter trap approach and the utility of such a promoter are discussed.

Gametophyte Development Needs Mitochondrial Coproporphyrinogen III Oxidase Function

“…The T-DNA insertion site was determined through the genome walking approach (Pratibha et al, 2013;Sharma et al, 2015). Briefly, genome walking libraries were constructed by digesting DNA with DraI, EcoRV, PvuII, or StuI restriction enzyme followed by adapter ligation.…”

Impaired embryo sac cellularization byPMEIgene mutation affects gamete specification and twin plants in Arabidopsis

“…An Arabidopsis thaliana mutant showing high seed sterility was identified in a T-DNA promoter trap mutant population. This line, designated as GUS-650, carries a promoter-less uidA reporter gene and showed GUS expression in the ovules (for details see Sharma et al 2015). Further, the T-DNA was found to be inserted in the 5′ UTR of At3g17150 gene coding for P ectin M ethyl E sterase Inhibitor ( PMEI ) and was specifically expressing in the mature embryo sacs.…”

“…In particular, the roles of embryo sac cellularization and cell wall composition in differentiation of gamete specification have not been investigated so far. Investigation of an Arabidopsis thaliana T-DNA promoter trap mutant showing GUS expression in the embryo sac (Sharma et al, 2015) revealed mis-specification of gamete cells leading to high seed sterility. Here we report that the loss-of-function mutation in the At3g17150 gene annotated to code for a “pectin methylesterase inhibitor” results in this shift in cell fate thereby revealing the role of cell-wall in determining cell fate of female gametophyte and subsequent embryo development.…”

The intergenic region of the maize defensin-like protein genesDef1 and Def2functions as an embryo-specific asymmetric bidirectional promoter

“…Most studies of natural bidirectional promoters have focused on the model plant Arabidopsis. Examples of bidirectional promoters identified in Arabidopsis include two gene pairs, cab1/cab2 and At5g06290/At5g06280 , involved in photosynthesis which have 2177- and 351-bp bidirectional promoters, respectively ( Bondino and Valle, 2009 ; Mitra et al , 2009 ); 668- and 461-bp intergenic regions in the At3g17140/At3g17150 and At4g10596/At4g110600 gene pairs identified by characterization of two T-DNA promoter trap lines ( Pratibha et al , 2013 ; Sharma et al , 2014 ); an asymmetric bidirectional promoter in the At1g71850 / At1g71860 gene pair ( Liu et al , 2015 ); and a heat-inducible bidirectional promoter in the AtClpB-C / AtCK2 gene pair ( Mishra and Grover, 2014 ).…”