Microarray Identifies Transcription Factors Potentially Involved in Gravitropic Signal Transduction in Arabidopsis

On Earth, gravity is a constant stimulus governing a plant’s growth and orientation of its organs. A plant’s response to gravity can be separated into four phases: perception of the gravity stimulus, transduction of that perception event into a biochemical signal, transmission of that signal to the site of response, and the differential growth response of the plant organs (Morita and Tasaka, 2004). The gravity persistent signal (GPS) treatment involves a cold treatment to isolate the processes of signal transduction prior to auxin redistribution. When plants are reoriented with respect to gravity at 4°C, perception of the stimulus occurs, but auxin transport is virtually eliminated; however, when returned to vertical at room temperature (RT), auxin transport is restored and plants bend in response to the reorientation in the cold (Wyatt et al., 2002). The delayed curvature indicates that some component between perception and transmission of the signal is inhibited in the cold. Taking advantage of this GPS phenomenon, a gene expression microarray was conducted on plants subjected to the GPS treatment to identify additional molecular components or physiological processes involved in the gravitropic pathway. The goal of this ongoing research is to identify genes involved in the early events of gravitropic signal transduction. Here, we describe our initial work focused on transcription factors that are differentially expressed at early time points after reorientation of plants at 4°C.

A gene expression microarray analysis combined with the GPS treatment was used to isolate genes involved in the early events of gravity signal transduction. Samples were collected across a time course of the GPS response; RNA was isolated from inflorescence stem tissue at 2, 4, 10, and 30 min after plants were reoriented at 4°C. Transcription factors were selected for analysis because of their potential role in gene regulation of the gravitropic signaling pathway. Transcription factors can have large-scale downstream effects on gene regulation and are involved in the activation of regulatory cascades (Bolouri and Davidson, 2003). Of the 1,907 transcription factors identified in the microarray dataset, five (AtAIB, WRKY18, WRKY26, WRKY33, and BT2) were selected based on differential expression (LFC≤-1.0) at the 4 min time point (Figure 1). No transcription factors were found to be differentially expressed at 2 min. Although BT2 had a LFC<-1.0 at 4 min, its expression was not statistically significant (p-value<0.10); however, differential expression at 10 min was significant (p-value<0.10) (Table 1), so it was included for further analysis.

Figure 1.

Transcription factors in the WRKY family are expressed rapidly and transiently in response to many different environmental stresses (Rushton et al., 1996; Hara et al., 2000). In this study, WRKY26 and WRKY33 were significantly down-regulated only at 4 min, while WRKY18 was down-regulated at 4, 10, and 30 min (Table 1).

Although microarrays are capable of whole genome profiling, they can also generate numerous false positive signals (Wang et al., 2006). Thus, RT-qPCR was performed on RNA collected from wild type (WT) plants to independently assess the expression of AtAIB, WRKY18, WRKY26, WRKY33, and BT2. For all genes, RT-qPCR showed significant differential expression at 2, 4, and 10 min (Figure 2). RT-qPCR indicated that WKRY 26, WRKY 33, and AtAIB were all up-regulated at 2 min, which was not evident in the microarray analysis but may prove interesting.

Figure 2.

The GPS phenotype was assessed on five individual Arabidopsis lines, each containing a T-DNA insertion in one of the genes: AtAIB, WRKY18, WRKY26, WRKY33, or BT2. First, plants were bred for homozygosity of the T-DNA insertion in the target gene (Figure 3). Once homozygous lines were isolated, plants were subjected to the GPS treatment using the WT var. Columbia as the control. The inflorescence stems of bt2 curved less as compared to those of WT, and conversely inflorescence stems of ataib curved more than those of WT throughout the response phase (Figure 4). Interestingly, the three WKRY mutants responded differently. wrky18 showed no significant difference in bending of inflorescence stems compared to WT, curvature in the inflorescence stems of wrky26 only differed significantly from WT after 45 min, and inflorescence stems of wrky33 curved less when compared to those of WT (Figure 4).

Figure 3.

Figure 4.

The GPS treatment involves a cold treatment to isolate the processes of signal transduction prior to auxin redistribution and may help to shed light on early events which link the perception of the biophysical stimulus and the biochemical response. Because transcription factors are essential elements of gene regulation and can have large-scale downstream effects on the expression of other genes, here, we focused on analysis of transcription factors from a microarray gene expression study across the GPS treatment. Early time points after reorientation of plants was of particular interest and since the presentation time of Arabidopsis has been estimated to be between 30 sec and 5 min (Sack et al., 1985; Kiss et al., 1989), the 2 and 4 min time points were chosen to survey the earliest transcription factors induced by gravity stimulus. Of the 1,907 transcription factors identified across the microarray data, and as annotated in the Database of Arabidopsis Transcription Factors, none were differentially expressed at 2 min. Five genes were selected for analysis based on differential expression at the 4 min time point: AtAIB, WRKY18, WRKY26, WRKY33, and BT2 (Figure 1). All transcription factors selected were down-regulated at the 4 min time point (Figure 1, Figure 2) but showed more variable expression at other time points (Figure 2).

Initial analysis of the GPS phenotype of mutants defective in each of the transcription factors showed a potentially complex story. Curvature of inflorescence stems of ataib increased after GPS treatment as compared to WT. AtAIB is an abscisic acid (ABA)-induced basic helix-loop-helix-type (bHLH) protein which has been shown to positively regulate ABA response (Li et al., 2007). In the roots, curvature in response to gravity is known to be influenced by ABA (Pilet and Barlow, 1987), as well as other hormonal regulators. Application of exogenous ABA to maize roots also promotes differential growth in response to gravity stimulus in darkness (Wilkins and Wain, 1975). Our results may indicate a role of ABA in curvature of stems in response to gravity.

Conversely, inflorescence stems of bt2 bent less after the GPS treatment as compared to WT. BT2 has been found to be involved in telomerase regulation (Ren et al., 2007) and in multiple plant responses to exogenous stimulations and stresses, including ABA (Mandadi et al., 2009). BT2 is believed to be a key player in multiple signal transduction pathways, and now we provide evidence to suggest a role in gravitropic signal transduction.

In our study, three WRKY transcription factors (WRKY18, 26, and 33) were all down-regulated at 4 min after reorientation in the cold. However, mutants defective in these genes do not share a phenotypic response; curvature of inflorescence stems of wrky18 was not significantly different from WT; whereas those of wrky26 over responded after 45 min, and inflorescence stems of wrky33 bend less than those of WT. The WRKY superfamily is one of the largest groups of transcriptional regulators in plants. These genes share the highly conserved WRKYGQK amino acid sequence at the N-terminus and function in various signaling pathways in plants. Potential targets of downstream regulation by WRKYs contain the W-box promoter element. WRKY18 was previously found to be pathogen-induced, forming protein heterocomplexes with WRKY40 and WRKY60 that function as bacterial pathogen defenses (Xu et al., 2006). These paralogous WRKYs have also been implicated in the regulation of ABA-induced genes (Shang et al., 2010; Chen et al., 2010; Liu et al., 2012). WRKY33 is also known to function in response to pathogen defense (Zheng et al., 2006; Birkenbihl et al., 2012) and in abiotic stress (Jiyang and Deyholos, 2009). WRKY33 and WRKY26 also appear to be involved in coordinating induction of plant thermo tolerance (Li et al., 2011). In space-flown experiments, WRKY18 was upregulated over 2-fold in the hypocotyls of 12-day-old Arabidopsis (Paul et al., 2013) and several WRKY genes (including homologs of WRKY 18 and 33) have been identified in Brassica rapa flown for 27 days aboard the International Space Station (ISS) (Sugimoto et al., 2014). Although only three WRKY genes were differentially expressed in our dataset, these and others have been implicated in other gravity-related studies, including microgravity, hypergravity, and parabolic flight (Martzivanou et al., 2006; Fengler et al., 2015; Soh et al., 2015).

The role of transcription factors is complex. In these preliminary studies, we focused on expression at early time points after reorientation hoping to identify transcriptional regulation of early events in gravitropic signaling. However, transcription factors are necessary for long-term responses to alterations in the gravity field or the spaceflight environment, and this adds to the complexity of the system. Further analysis of gravitropic phenotypes in the mutants will provide a better indication if these transcription factors are involved in early signaling events throughout the plant. Additional biochemical, physiological, and protein-protein interaction studies will be necessary to sort out the role these, and other, transcription factors play in gravitropic signaling and response.