Promoter analysis of the WRKY transcription factors CaWRKY1a and CaWRKY1b homoeologous genes in coffee (Coffea arabica)

Abstract

The regulation of the CaWRKY1 homoeologous genes were analyzed through the characterization of their promoters. The pW1a promoter is proposed as a new tool for coffee plant biotechnologies. WRKY transcription factors are important elements of the plant immune response. The CaWRKY1 gene from Coffea arabica is induced by several biotic and abiotic stresses, including challenge by the rust fungus Hemileia vastatrix. Two homoeologous CaWRKY1 genes, named CaWRKY1a and CaWRKY1b, were previously identified in the C. arabica allotetraploid genome. To gain insight into the transcriptional regulation of these genes, their promoter sequences, named pW1a and pW1b, respectively, were cloned and characterized in this study. In silico analysis revealed some important defense-associated regulatory elements, including W-boxes and as-1 elements. Promoter activities were analyzed in transient assays conducted by agroinfiltration of tobacco leaves. Exogenous salicylic acid (SA) treatments increased promoter activities corroborating the presence of as-1 regulatory elements. Transactivation assays with the CaWRKY1 protein showed the reduction of both pW1a and pW1b promoter activities, indicating that the CaWRKY1 protein may negatively regulate its own promoters. Stable transgenic C. arabica lines expressing a pW1a::GUS construct were obtained by Agrobacterium-mediated transformation and high GUS activity was observed in leaves subjected to mechanical wounding. Hence, the ability of pW1a to drive transgene expression in coffee plants as well as to enhance expression in response to stresses opens possibilities for using this promoter as a new tool for biotechnological approaches in coffee plants.

bZIPs and WRKYs: two large transcription factor families executing two different functional strategies

“…In agreement with the W-box enrichment in their own promoters, it has been demonstrated that the WRKY proteins act on the promoters of their own genes and on other WRKY genes in cotransfection assays resulting in activation or repression of a reporter gene (Robatzek and Somssich, 2002; Petitot et al, 2013; Potschin et al, 2013). In addition, a pull-down analysis of WRKY53 with genomic DNA resulted in a list of putative target genes of WRKY53 including eight different WRKY genes (Miao et al, 2004).…”

“…For example, two W-boxes were found in the promoter of AtWRKY6 , four and five W-boxes in the promoters of the two homologous genes of Coffea arabica , five W-boxes in the promoter of AtWRKY18 , and three perfect W-boxes plus an additional TGAC cluster in the promoter of AtWRKY53 (Robatzek and Somssich, 2001; Petitot et al, 2013; Potschin et al, 2013). Some WRKYs even carry 11 or 12 ( AtWRKY66 , AtWRKY17 ) TTGAC core elements in an analyzed 1.5 kb promoter fragment (Dong et al, 2003).…”

Somatic Embryogenesis in Coffea spp.

“…The cloning of genes involved in the SE process, with the goal of increasing the efficiency of the process (Arroyo-Herrera et al 2008), and the production of plants with resistance to different kind of stresses (Leroy et al 1999(Leroy et al , 2000Perthuis et al 2005). During the last years our knowledge on coffee pests and disease resistance genes (Lashermes et al 2010;Noir et al 2003;Ribas et al 2011b), drought resistance genes (Marraccini et al 2011), and specific promoters (Marraccini et al 1999;Petitot et al 2013) has made possible to target specific genes to improve Coffea spp.…”

A systems biology perspective on the role of WRKY transcription factors in drought responses in plants

“…In many cases, comparative genomics leads to new findings in agriculturally and economically important crops (Marchive et al 2007;Mare et al 2004;Petitot et al 2013;Shekhawat et al 2011). Recently, Luo et al (2013) have described that in wild soybean, Glycine soja, GswRKY20 promotes the expression of negative regulators of ABA signaling while repressing positive regulators.…”