A 100 bp GAGA motif-containing sequence in AGAMOUS second intron is able to suppress the activity of CaMV35S enhancer in vegetative tissues

Abstract

Flower-specific promoters enable genetic manipulation of floral organs to improve crop yield and quality without affecting vegetative growth. However, the identification of strong tissuespecific promoters is a challenge. In addition, information on cis elements that is able to repress gene expression in vegetative tissues remains limited. Here, we report that fusing a 35S enhancer to the stamen-and carpel-specific NtAGIP1 promoter derived from the tobacco AGAMOUS second intron (AGI) can significantly increase the promoter activity. Interestingly, although the activity of the new promoter extends to sepals and pedicles, it does not cross the boundary of the reproductive organs. Serial deletion of the AGI and chromatin immunoprecipitation (ChIP) assay reveal a 100-bp fragment that contains a conserved GAGA factor binding motif contributes to the flower specificity by mediating histone H3 lysine 27 trimethylation (H3K27me3) modification of the promoter. Furthermore, this fragment shows significant suppressive effect on the activity of the 35S enhancer in vegetative tissues, consequently, resulting in a significant increase of the activity of 35S enhancer: AGI chimeric promoter without sacrifice of its specificity in inflorescence. A 100 bp GAGA motif-containing sequence in AGAMOUS second intron is able to suppress the activity of CaMV35S enhancer in vegetative tissues. PLoS ONE 15(3): e0230203. https://doi.org/ 10.The plasmid pBI121 [29] that contain CaMV35S::GUS was used as backbone to construct vectors used for plant transformation in this study. The second intron of NtAG-1 gene was fused with a 45 bp minimal 35S promoter to create a functional NtAGIP1 promoter [28]. NtAGIP1:: PLOS ONEA GAGA-containing sequence in AGAMOUS intron suppress the activity of CaMV35S enhancer in vegetative tissues PLOS ONE | https://doi.org/10.A GAGA-containing sequence in AGAMOUS intron suppress the activity of CaMV35S enhancer in vegetative tissues PLOS ONE | https://doi.org/10. Formal analysis: Ruochen Liu.Funding acquisition: Yan Pei.Investigation: Ruochen Liu, Xiuping Zou, You Wang, Qin Long. Fig 8. ChIP assay for H3K27me3 levels in NtAGI-1 intron and chimeric promoters. (A) ChIP assay for H3K27me3 levels at the NtAGI-1 intron in leaves of wild-type tobacco. (B) ChIP assay for H3K27me3 levels in leaves of the -2835 and -2735 lines. The genomic fragment from immunoprecipitation for qRT-PCR detection is depicted as black horizontal line. Enrichment was represented as percentage of Input (% Input). Error bars represent the standard deviation of three biological replicates.A GAGA-containing sequence in AGAMOUS intron suppress the activity of CaMV35S enhancer in vegetative tissues PLOS ONE | https://doi.org/10.

Cis-regulatory sequences in plants: Their importance, discovery, and future challenges

“…They are crucial for establishing precise, tissue-specific expression patterns by blocking expression in cell types and tissues where the gene should be silenced; they prevent ectopic gene expression ( Ogbourne and Antalis, 1998 ). For example, a 100-bp fragment in the second intron of the tobacco AG gene represses AG expression in nonfloral tissues, promoting flower-specific AG expression ( Liu et al, 2020 ). Similarly, TACPyAT repeats in the Petunia hybrida chalcone synthase A gene promoter hamper ectopic chalcone synthase A expression ( van der Meer et al, 1992 ).…”

“…Larger intron size might result from selection favoring insertions over deletions as a way to preserve functional CREs. CRMs are, for example, found in large introns in FLOWERING LOCUS C and AGAMOUS in Arabidopsis and knotted1 in maize ( Zea mays ; Greene et al, 1994 ; Sieburth and Meyerowitz, 1997 ; Busch et al, 1999 ; Hong et al, 2003 ; Qüesta et al, 2016 ; Yuan et al, 2016 ; Liu et al, 2020 ).…”

Characterization of Transcription Regulatory Domains of OsMADS29: Identification of Proximal Auxin-Responsive Domains and a Strong Distal Negative Element

“…These experiments helped in narrowing down the most effective negative regulatory region to a 186 bp region between –1,290 and –1,104 bases upstream of the M29 TSS. Similar approaches have been used in the past to ascertain the specific response of promoter regions and cis -elements appended to either a minimal or a complete 35S CaMV promoter with either luciferase or GUS reporter genes ( Liu et al, 1990 , 2020 ; Quayle et al, 1991 ; Treuter et al, 1993 ; Morita et al, 1998 ; Ishige et al, 1999 ).…”