Expression of ATFL1 correlates with the induction of
flowering in C. pallens
Leaf samples from the control plants at 1070 m and transplants that
flowered in the next season collected during and after the
transplantation period were analysed for the expression of selected
flowering-time genes using RT-qPCR (Table 1).
Leaf samples collected from both the 17Hot transplants that flowered in
2018 and control plants (17control) that had remained vegetative showed
a seasonal expression pattern (P < 0.05; Table S5; Fig. S2).
The seasonal expression patterns for CpFT3, CpFT4, CpFT5, CpATFL1,
CpVRN1, CpMADS50, CpMADS1 and CpHd1 (Fig. S2) were similar
between the control plants at 1070 m, all of which had remained
vegetative, and the transplants that flowered in the next season (Table
S5). Greater expression of FT-like genes was observed in the
spring season (October samples), aligning with studies in model plant
species (Nagano et al., 2019). Expression of FT-like genes was
similar during the spring season in both leaf sample sets, whether from
tillers that flowered in the next season or remained vegetative.
However, CpFT1 transcript was not detected in any of the samples
due to its expression either being below the limit of detection or
because it was not expressed in the leaves of the plants during the time
when leaf samples were collected. An increased expression ofCpVRN1 (Fig. S2), another floral promoter, was observed during
and after the winter season similar to its known seasonal expression
pattern in B. distachyon and temperate cereal species (Distelfeld
et al., 2009; Woods, Ream, & Amasino, 2014).
When the expression pattern is compared between the two sets of plants
during the inductive summer conditions (January 2017), CpATFL1had a significantly greater expression in the leaf samples of the 17Hot
transplants that flowered in the next season compared to the control
plants at 1070 m that remained vegetative. Along with CpATFL1,other floral promoters including CpTPS1, CpMADS1 ,CpMADS50, and CpVRN1 were also highly expressed in the
leaf samples associated with flowering tillers (Fig. 2a, Fig S3).
Similarly using RT-qPCR, the expression of flowering-time genes in the
leaf samples from the transplanted plants that had flowered in 2017
(16Hot transplants and 16Cool transplants) was also compared to the
control plants at 1070 m (16Control), none of which flowered. All the
gene(s) showed a seasonally variable expression pattern (P <
0.001; Table S5) (Fig. S4). The expression pattern of CpFT2 ,CpFT3 , CpFT4 , CpATFL1 , CpMADS1 ,CpEhd3 and CpTPS1 in the leaves of tillers that flowered
in the next season at both the sites, UC and 1520 m (16Hot transplants
and 16Cool transplants, respectively) showed a similar expression
pattern to that observed in the leaf samples associated with flowering
in 17Hot transplants (comparing Fig. S3 and Fig. S4). Even thoughCpFT5 had a seasonal expression pattern, plants at UC showed
greater expression during autumn. All the genes showed a significant
differential expression between tillers which subsequently flowered vs
tillers that remained vegetative during the inductive summer period (P
< 0.001), except for CpFT4 in the 16Cool transplants
(Table S5).
Similar to the 17Hot transplants, CpATFL1 had a significantly
greater expression in the tillers that flowered at both the sites (UC
and 1520 m; 16Hot transplants and 16Cool transplants, respectively)
during the inductive summer period (January-2016) (Fig. 2a). Expression
analysis of CpVRN1 also correlated with its known seasonal
expression pattern with a higher peak in expression post winter (Shimada
et al., 2009). The expression of CpVRN1 was greater in the
tillers at both the sites (UC and 1520 m) that flowered in the next
season compared to the plants at the control site that had remained
vegetative (Fig. S3). Targeted expression analysis of CpHd1 ,CpGI , CpEhd3 , CpMADS50 and CpMADS1 showed
expression was greater for each of these genes in the leaf samples of
the transplanted plants which subsequently flowered compared to the
control site plants that remained vegetative (Fig. S3).
Leaf samples associated with tillers that remained vegetative in both
the transplants, (16Hot transplant and 16Cool transplants) were found to
have significantly lower expression of CpATFL1 during the
inductive summer temperature (January 2016) compared to the leaf samples
from the same groups of transplants that flowered in the next season
(Fig. 2b). In addition to the transplants, leaf samples collected from
the control plants during January 2018 (18 Control) that flowered in the
masting year 2019 also showed a significantly greater expression ofCpATFL1 compared to the leaf samples from plants that remained
vegetative in the year 2019 (Fig. 2b). In summary, expression studies
suggest that an elevated expression of CpATFL1 during the
inductive summer temperature period is associated with the induction of
flowering in C. pallens .