Potential regulators of CpATFL1 initiate the floral transition in C. pallens
Global expression analysis of the leaves collected during the summer inductive conditions from tillers that subsequently flowered shows upregulation of crucial genes including CpFTIP1 and CpFDPthat are involved in the transport of florigen-like molecules from the leaves to the apex to subsequently activate the floral meristem gene(s) through formation of the florigen activation complex (FAC) (Kaneko-Suzuki et al., 2018). Calcium is also required to catalyse the formation of the florigen activation complex (Kawamoto, Sasabe, Endo, Machida, & Araki, 2015).The expression of CpCPK6 , a kinase required for calcium signalling, was also upregulated in the tillers that flowered in the next season. It is important to acknowledge that formation of FAC occurs at the shoot apical meristem. Even though the differential expression of these genes was observed in the leaves, it may suggest that CpATFL1 may form the FAC through the activity of CpFTIP1 and CpFDP, catalysed by CpCPK6 at the shoot apical meristem. Further investigation involving a yeast two hybrid assay or bimolecular fluorescence assay would reveal whether CpATFL1 and CpFTIP1 do indeed interact and could, therefore, transport ATFL1 to the apex.
Vernalisation responses to mediate flowering-time control in temperate monocots are controlled by the VERNALISATION (VRN) loci (Reams et al., 2014). The VRN2 locus encodes a CCT-domain protein that blocks the floral transition. Exposure to cold temperatures increases the expression of VRN1 , a repressor of VRN2 (Yan et al., 2004) and maintains the repressive state of VRN2even after vernalisation. Repression of VRN2 leads to activation of VRN3 , a homologue of FT and Hd3a, after plants are exposed to warm spring temperatures (Preston & Kellogg, 2008; Shimada et al., 2009; Woods, et al., 2016). Transcriptomic analysis showed an elevated expression of CpVRN1 in the tillers that flowered during the increase in summer temperature, which is also observed in other temperate cereals during the process of floral transition (Trevaskis, 2010). On the other hand, expression ofCpVRN2 , the repressor of FT -like genes (Alexandre & Hennig, 2007), was downregulated in the same samples. Greater expression of CpVRN1 during the spring season and which remained elevated through the summer may have blocked repressive signals fromCpVRN2 . This repression was potentially maintained until the next summer resulting in the activated transcription of CpATFL1 to induce flowering.
Transcriptomic analysis also revealed an increase in the expression of thermosensory genes, including CpPIF4 and CpPIF5 in the tillers associated with flowering in the next season. PIF genes are known to regulate responses to high temperatures (Choi & Oh, 2016) and are involved in the activation of the flowering process along with similar bHLH floral promoting proteins such as bHLH76 and bHLH80 (Ito et al., 2012). In addition to PIF -family genes, homologues ofbHLH76 and bHLH80 in C. pallens , were also upregulated in the above samples, which may interact with CpPIF4 to activate ATFL1 . Transient assays are further required to confirm this interaction. The summer temperature cue may also have blocked the expression of several floral repressors including homologues ofAP2-LIKE genes and SVP (Capovilla, Schmid, & Pose, 2015; Mateos et al., 2015).
Emphasis was also placed on the role of epigenetic modifiers known to regulate the reproductive transition in plants. Ambient temperatures have been shown to regulate the expression of floral repressors via epigenetic modification including either through deposition of repressive histone marks (to suppress gene expression) such as H3K27me3 or by removing acetyl groups from the histone tails at the gene loci (Bratzel & Turck, 2015; He, 2012). In contrast, high temperatures have been shown to lead to deposition of active histone marks (H3K4me3 or H3K36me3) at the loci of floral promoters (Avramova, 2015).CpREF6 and CpFLD , homologues of REF6 and FLDwhich are reported to promote flowering (He, 2012; Lu, Cui, Zhang, Jenuwein, & Cao, 2011), were upregulated in the leaves of plants that flowered in the next season. CpFLD may then interact with CpHDA6, a histone deacetylase complex, activating the floral promoting genes, a process well established in Arabidopsis (Yu, Chang, & Wu, 2016). Homologues of CpFLK , CpFVE , and CpFY , genes known to repress the expression of floral repressors epigenetically (Cho, Yoon, & An, 2017; He & Amasino, 2005), were upregulated in the tillers that subsequently flowered. Interestingly, homologues of epigenetic editors, including CpVEL1 and CpMSI1 , which are known to be involved in the activation of VRN1 and SOC1 , respectively through deposition of active histone marks (Higgins, Bailey, & Laurie, 2010; Oliver & Finnegan, 2011) were also upregulated in the tillers that flowered in the next season. This may suggest that an external signal such as summer temperatures may lead to certain epigenetic changes enabling the transcription of CpATFL1 to promote flowering.