Potential regulators of CpATFL1 initiate the floral
transition in C. pallens
RNA-seq analysis of the leaves collected during the summer inductive
conditions from tillers that subsequently flowered shows upregulation of
crucial genes including CpFTIP1 and CpFDP that 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 the formation of the
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 (Ream 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 repressed 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
(RNA-seq) showed an elevated expression of CpVRN1 in the tillers
that flowered during the increase in summer temperature, which is also
observed in B. distachyon and temperate cereals during the
process of floral transition (Ream et al., 2014; Trevaskis, 2010). On
the other hand, expression of CpVRN2 , the repressor ofFT -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 from CpVRN2 . This repression was potentially
maintained until the next summer resulting in the activated
transcription of CpATFL1 to induce flowering.
Global transcriptomic analysis also revealed an increase in the
expression of thermosensory genes, including CpPIF4 andCpPIF5 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 toPIF -family genes, homologues of bHLH76 and bHLH80in C. pallens , were also upregulated in the above samples, which
may interact with CpPIF4 to activate ATFL1 . However, transient
assays are required to confirm this interaction. The summer temperature
cue may also have blocked the expression of several floral repressors,
including homologues of AP2-LIKE genes and SVP (Capovilla,
Schmid, & Pose, 2015; Mateos et al., 2015).
As warmer summer temperatures were shown to promote flowering, emphasis
was also placed on the role of epigenetic modifiers known to regulate
the reproductive transition in plants in response to temperature change.
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 the deposition of active histone
marks (H3K4me3 or H3K36me3) at the loci of floral promoters (Avramova,
2015). CpREF6 and CpFLD , homologues of REF6 andFLD, which 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 FLK , FVE , and FY , 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 VIL3 and MSI1 , which are known to be involved in
the activation of VRN1 and SOC1 , respectively, through
deposition of active histone marks (Higgins et al., 2010; Oliver &
Finnegan, 2011; An et al., 2015) 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.