Detailed Description of EASE
As part of the efforts towards the identification of genes associated with apomixis, CAMBIA initiated a project to isolate regulatory elements and/or genes involved in the process of female gametophyte development by using particular enhancer detector lines with spatially restricted GUS expression patterns.
The egg apparatus-specific enhancer (EASE) was isolated and identified from an Arabidopsis enhancer detector line ET253, which shows specific GUS expression mainly in the egg apparatus and no GUS expression in anthers and any other tissues. There is only one Ds insertion in ET253 and its flanking sequences were analyzed by 5′ and 3′ deletion tests. A 77 bp sequence (ccacgatgca aatatatcga taacgttatt aaaaaaagta accgcatgat atattctctt tcgtatgata ttaaggc, GenBank accession no. AX100536) was identified as possessing the egg apparatus-specific enhancer activity in Arabidopsis and therefore named as EASE.
The sequence bearing the EASE is located on chromosome IV in Arabidopsis in an intergenic region and not associated with genes directly next to it. Without the use of enhancer detection, it might never have been uncovered. The EASE has the following features:
- There is only one copy in the Arabidopsis genome.
- The EASE sequence is conserved among different accessions of Arabidopsis.
- It acts in an orientation- and position-independent manner.
Although the endogenous function of the EASE in the Arabidopsis genome is still unknown, our experimental results show that its capability to control gene expression in the egg apparatus and early embryo is clear. Without the minimal promoter, the EASE itself is not functional. However, when it is fused to the CaMV 35S minimal promoter, the EASE can direct gusA or GFP gene expression in a very specific and highly efficient manner.
The EASE has also been tested in a genetic ablation system based on cell specific expression of the diphtheria toxin A-chain gene for embryo-specific ablation. Arabidopsis lines expressing the hybrid Gal4-VP16 transcription activator under the control of the EASE plus the minimal CaMV 35S promoter were generated. By crossing these lines with an Arabidopsis line harbouring the DTA gene controlled by an artificial promoter consisting of multiple copies of the upstream activating sequence (UAS) recognized by GAL4, linked to the minimal CaMV 35S promoter, embryo-specific ablation was achieved in the hybrid seeds. In these experiments, the silique development of the crossed flowers showed no difference from that of the selfed flowers. However, the ovules of the crossed flowers had the embryos arrest at early globular stage while the integument and endosperm development remained normal.
This demonstrated that the EASE could be used as a tool to study and manipulate gene expression in the female gametophyte and during early embryo development.
- Download vector maps of pWY093.1 (GFP), pWYO93.4 (GFP), pWYK105.1 (GUS) and pWY5D77 (GUS) constructs.
- Download Vector NTI viewer.
- Read the November 2005 publication: Yang W, Jefferson R A, Huttner E, Moore J M, Gagliano W B, Grossniklaus U (2005) An Egg Apparatus-Specific Enhancer of Arabidopsis, Identified by Enhancer Detection, Plant Physiology Download this paper