Transcriptomic analysis identifies potential regulators ofCpATFL1
To study the transcriptional changes associated with the induction of
flowering in more depth, differential expression (DE) profiling, using
RNA-seq, was performed. Leaf samples (January 2017) from 17Hot
transplants located at UC which flowered heavily the next season were
compared to the control plants (17Control; January 2017) that remained
vegetative in the next season which was a non-masting year.
High-throughput 150 bp paired-end sequencing yielded 32 GB of raw data
with 120 million average read counts for each replicate. Reads were
assembled into a reference transcriptomic assembly using the Trinity
pipeline. The generated de novo assembly yielded 140,826
transcripts, comprised of 383,092 contigs with an average length of
1428.92 bp and an N50 length of 2414 bp (Table S3). A total of 29,566
contigs (adj P < 0.01) were significantly differentially
expressed (DE) with 14,514 and 15,052 transcripts significantly up and
downregulated, respectively (Fig. 3).
Gene Ontology analysis: To gain insights into the function of
the genes that were DE, contigs were functionally categorised on the
basis of putative biological processes, molecular function, and cellular
localisation. Out of 29,566 DE contigs, 15,974 (54.09 %) were annotated
against the B. distachyon protein database with an E-value of
10-5. The DE genes were further categorised based on
gene ontology using a hypergeometric test with a significance threshold
of 0.05 to identify key correlations between the internal cellular
activity and the phenotypic differences.
Upregulated genes in the leaf samples associated with tillers that
flowered in the next season were significantly enriched in the cellular
components belonging to the cytoplasm, organelle, and intracellular
organelle as the top three categories. Proteins encoded by the
upregulated genes were further clustered into separate functional
categories belonging to protein binding, transferase activity and anion
binding. These genes were found to be involved in biological processes
enriched in response to stimuli, oxidation-reduction processes and
cellular response to stimulus (Fig. 3d). Similarly, gene ontology
analysis was also carried out for the proteins encoded by the
downregulated transcripts. The downregulated transcripts were
significantly enriched in the cellular components assigned to the
cytoplasm, cell periphery and plasma membrane. These transcripts were
then further clustered into distinct molecular functions with most of
them belonging to transferase activity, anion binding, and small
molecule binding. Finally, these proteins were assigned to the category
of biological processes involved in organo-nitrogen compound metabolic
process, protein metabolic process, and biological regulation as the top
three classes (Fig. 3d).
2,786 downregulated transcripts when mapped to the KEGG database were
enriched in biosynthesis of secondary metabolites, metabolic pathways
and circadian rhythms with a false discovery rate of less than 0.05
(Fig. S5). About 3,788 (12.9%) transcripts of the upregulated genes
were found to be associated with the KEGG pathways. The genes were
significantly enriched in metabolic pathways (44.1%), biosynthesis of
secondary metabolites (26.1%) and protein processing in endoplasmic
reticulum (4.75%) as the top three categories.
Differentially expressed orthologues of floral genes in C.
pallens: Out of the 29,567 DE contigs, 200 homologous floral protein
sequences (from A. thaliana and B. distachyon ) were
significantly differentially expressed in the leaves of the tillers that
flowered in the next season compared with tillers that remained
vegetative (Table S4). Floral integrator genes, including CpMADS1and CpATFL1 , were highly expressed in the tillers that flowered
in the next season which also aligns with the quantitative PCR analysis
performed earlier (Fig. 3c; Appendix S2). FRIGIDA (CpFRI ), a
known floral repressor in A. thaliana (Choi et al., 2011) andB. distachyon and other CpFRI -interacting genes were
downregulated in the tillers that flowered in the next season (Table
S4).
Leaf samples from 17Hot transplants that flowered in the next season
also showed an increase in the expression of thermosensory genes
including CpPIF4 , CpPIF5 and CpbHLH80 relative to
plants at the control site. These genes also act as floral promoters in
response to high temperatures (Kumar et al., 2012). CpSPL15 a
known floral promoter in perennial plants (Hyun et al., 2019), was also
upregulated in the 17Hot transplants compared to the control plants.CpVRN2 , another temperature regulated floral repressor (Yan et
al., 2004) was also downregulated in the tillers during the inductive
summer period that subsequently flowered (Fig. 3c).
Two gibberellin catabolism gene family members (CpGA2ox1 andCpGA2ox8 ) were also downregulated, while genes involved in
gibberellin synthesis, including CpKS (kaurene synthase) andCpGA20ox2 were upregulated in the tillers that subsequently
flowered. Gibberellins have been shown to promote flowering in plants
either by the activation of FT through SPL-family proteins or by
activation of SOC1 , independent of FT (Yu et al., 2012).
Several epigenetic editor genes, known to deposit active methylation
marks to activate the expression of flowering promoting genes, includingCpREF6 , CpMSI1 , CpFLD , and CpEBS (He, 2012),
were upregulated in the tillers that flowered in the next season
compared to the tillers from the plants at the control site that had
remained vegetative (Table S4). Additionally, genes involved in the
epigenetic repression of floral repressors such as CpFLK ,CpFY , CpFPA and CpVEL1 (Qüesta, Song, Geraldo, An,
& Dean, 2016) were also upregulated in the tillers that subsequently
flowered (Table S4).