The WRKY transcription factor family and senescence in switchgrass

Rinerson et al. BMC Genomics (2015) 16:912 DOI 10.1186/s12864-015-2057-4 RESEARCH ARTICLE Open Access The WRKY transcription factor family and senescence in switchgrass Charles I. Rinerson1†, Erin D. Scully2†, Nathan A. Palmer2†, Teresa Donze-Reiner3, Roel C. Rabara1, Prateek Tripathi4, Qingxi J Shen5, Scott E. Sattler2, Jai S. Rohila6, Gautam Sarath2* and Paul J. Rushton1,7* Abstract Background: Early aerial senescence in switchgrass (Panicum virgatum) can significantly limit biomass yields. WRKY transcription factors that can regulate senescence could be used to reprogram senescence and enhance biomass yields. Methods: All potential WRKY genes present in the version 1.0 of the switchgrass genome were identified and curated using manual and bioinformatic methods. Expression profiles of WRKY genes in switchgrass flag leaf RNASeq datasets were analyzed using clustering and network analyses tools to identify both WRKY and WRKYassociated gene co-expression networks during leaf development and senescence onset. Results: We identified 240 switchgrass WRKY genes including members of the RW5 and RW6 families of resistance proteins. Weighted gene co-expression network analysis of the flag leaf transcriptomes across development readily separated clusters of co-expressed genes into thirteen modules. A visualization highlighted separation of modules associated with the early and senescence-onset phases of flag leaf growth. The senescence-associated module contained 3000 genes including 23 WRKYs. Putative promoter regions of senescence-associated WRKY genes contained several cis-element-like sequences suggestive of responsiveness to both senescence and stress signaling pathways. A phylogenetic comparison of senescence-associated WRKY genes from switchgrass flag leaf with senescence-associated WRKY genes from other plants revealed notable hotspots in Group I, IIb, and IIe of the phylogenetic tree. Conclusions: We have identified and named 240 WRKY genes in the switchgrass genome. Twenty three of these genes show elevated mRNA levels during the onset of flag leaf senescence. Eleven of the WRKY genes were found in hotspots of related senescence-associated genes from multiple species and thus represent promising targets for future switchgrass genetic improvement. Overall, individual WRKY gene expression profiles could be readily linked to developmental stages of flag leaves. Background Switchgrass (Panicum virgatum) is a temperate, warmseason perennial that is being developed as a cellulosic biofuel crop [1, 2]. Tetraploid switchgrass populations and cultivars have higher yields as compared to octaploid populations [3]. Thus, most current breeding efforts are focused on improving biomass yields and * Correspondence: ; † Equal contributors 2 Grain, Forage and Bioenergy Research Unit USDA-ARS UNL, Lincoln, NE 68583-0937, USA 1 Texas A&M AgriLife Research and Extension Center, Dallas, TX 75252, USA Full list of author information is available at the end of the article quality in tetraploid lines [4, 5]. Tetraploid populations can occur as upland and lowland ecotypes, with the lowland plants significantly out-yielding the upland lines across several locations [5]. However, the latitudinal adaptation of these different ecotypes presents challenges, since most of southerly-adapted, high-yielding, lowland germplasm suffers from significant winter-kill at more northern sites of the USA [6]. Some crosses of upland x lowland plants show heterosis for yields [7], and this approach appears to hold promise in the continuing development of switchgrass as a biomass crop [8]. Nevertheless, extending the period of carbon assimilation by delaying aerial senescence could be a means to © 2015 Rinerson et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rinerson et al. BMC Genomics (2015) 16:912 significantly improve yields, as long as other plant attributes, such as dormancy onset and nutrient remobilization are not impaired [6]. Senescence is a genetically programmed trait that can potentially be reprogrammed by several molecular breeding strategies such as marker-assisted selection. To develop switchgrass cultivars with delayed senescence, it is critical to determine the key molecular events that occur during senescence to identify the regulators that trigger this process. Senescence is the final stage of plant development and is tightly controlled to increase the fitness of the whole plant [9]. Transcriptome analysis of Arabidopsis thaliana (A. thaliana; At) leaf senescence suggests that several families of transcription factors play major roles in the cellular reprogramming associated with senescence. The major transcription factor families associated with A. thaliana leaf senescence are NACs, WRKYs, C2H2 zinc finger proteins, AP2/ERFs, MYBs, homeobox proteins, bZIPs, bHLHs, and C3H zinc finger proteins. WRKY TFs were the second largest TF family to be induced during senescence in this study [10]. WRKY transcription factors are key regulators of many plant processes, including responses to biotic and abiotic stresses, wounding, senescence, seed dormancy, and seed germination [11]. They are components of intracellualar signaling webs, for example many are phosphorylated by MAP kinase cascades [12]. The defining feature of WRKY transcription factors is their DNA binding domain referred to as the WRKY domain, which is named after the almost invariant WRKY amino acid sequence within the N-terminal region [13]. The WRKY domain is about 60 residues in length and also possesses a Cx4– 5Cx22–23HxH or Cx7Cx23HxC zinc-finger structure at the C-terminus [11]. Structural determination of the WRKY domain bound to its W box cis-acting element revealed that part of a four-stranded β-sheet enters the major groove of DNA almost perpendicular to the DNA helical axis in a β-wedge. Amino acids in the conserved WRKYGQK signature motif contact the W box DNA bases [14]. Functional genomic studies of individual WRKY transcription factors have provided clear evidence that specific WRKY proteins are regulators of senescence, although some of these transcription factors play multiple roles in planta [15–17]. The first evidence supporting a role of WRKY transcription factors in the senescence process came from studies of A.thaliana AtWRKY6 [18, 19]. One target gene for AtWRKY6 is FLG22-induced receptor-like kinase 1 (FRK1 previously called SIRK) whose expression is strongly induced du (...truncated)