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 the 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 floral protein sequences
homologous to Arabidopsis 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 CpMADS1 andCpATFL1 , were highly expressed in the tillers that flowered in
the next season, which also aligns with the RT-qPCR analysis described
earlier (Fig. 3c; Appendix S2). FRIGIDA (CpFRI ), a known floral
repressor in Arabidopsis (Choi et al., 2011) and B. distachyon(Higgins, Bailey, & Laurie, 2010) and other CpFRI -interacting
genes were downregulated in the tillers that flowered in the next season
(Table S4).
The data derived from the RNA-seq suggests that 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 ,
a temperature regulated floral repressor (Yan et al., 2004), was
downregulated during the inductive summer period in the tillers that
subsequently flowered (Fig. 3c).
The RNA-seq data also revealed that two gibberellin catabolism gene
family members (CpGA2ox1 and CpGA2ox8 ) were downregulated,
while genes involved in gibberellin synthesis, including CpKS(kaurene synthase) and CpGA20ox2, 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
(Tilmes et al., 2019; 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 CpVIL3 (An, Guo, Liu, & An, 2015),
were also upregulated in the tillers that subsequently flowered (Table
S4).