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Narrow-leafed lupin (Lupinus angustifolius L.) is a moderate-yielding legume crop known for its high grain protein content and contribution to soil improvement. It is cultivated under photoperiods ranging from 9 to 17 h, as a spring-sown (in colder locations) or as an autumn-sown crop (in warmer regions). Wild populations require a prolonged cold period, called vernalization, to induce flowering. The key achievement of L. angustifolius domestication was the discovery of two natural mutations (named Ku and Jul) conferring vernalization independence. These mutations are overlapping deletion variants in the promoter of LanFTc1, a homolog of the Arabidopsis thaliana FLOWERING LOCUS T (FT) gene. The third deletion, named here as Pal, was recently found in primitive germplasm. In this study, we genotyped L. angustifolius germplasm that differs in domestication status and geographical origin for LanFTc1 alleles, which we then phenotyped to establish flowering time and vernalization responsiveness. The Ku and Jul lines were vernalization-independent and early flowering, wild (ku) lines were vernalization-dependent and late flowering, whereas the Pal line conferred intermediate phenotype. Three lines representing ku, Pal, and Ku alleles were subjected to gene expression surveys under 8- and 16-h photoperiods. FT homologs (LanFTa1, LanFTa2, LanFTc1, and LanFTc2) and some genes selected by recent expression quantitative trait loci mapping were analyzed. Expression profiles of LanFTc1 and LanAGL8 (AGAMOUS-like 8) matched observed differences in flowering time between genotypes, highlighted by high induction after vernalization in the ku line. Moreover, these genes revealed altered circadian clock control in Pal line under short days. LanFD (FD) and LanCRLK1 (CALCIUM/CALMODULIN-REGULATED RECEPTOR-LIKE KINASE 1) were negatively responsive to vernalization in Ku and Pal lines but positively responsive or variable in ku, whereas LanUGT85A2 (UDP-GLUCOSYL TRANSFERASE 85A2) was significantly suppressed by vernalization in all lines. Such a pattern suggests the opposite regulation of these gene pairs in the vernalization pathway. LanCRLK1 and LanUGT85A2 are homologs of A. thaliana genes involved in the FLOWERING LOCUS C (FLC) vernalization pathway. Lupins, like many other legumes, do not have any FLC homologs. Therefore, candidate genes surveyed in this study, namely LanFTc1, LanAGL8, LanCRLK1, and LanUGT85A2, may constitute anchors for further elucidation of molecular components contributing to vernalization response in legumes.
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ORIGINAL RESEARCH
published: 26 October 2020
doi: 10.3389/fpls.2020.572135
Edited by:
Christian Jung,
University of Kiel, Germany
Reviewed by:
Dong-Hwan Kim,
Chung-Ang University, South Korea
Youbong Hyun,
Seoul National University,
South Korea
*Correspondence:
Michał Ksia¸ ˙
zkiewicz
mksi@igr.poznan.pl
Specialty section:
This article was submitted to
Plant Development and EvoDevo,
a section of the journal
Frontiers in Plant Science
Received: 12 June 2020
Accepted: 24 September 2020
Published: 26 October 2020
Citation:
Rychel-Bielska S, Plewi ´
nski P,
Kozak B, Galek R and Ksia¸ ˙
zkiewicz M
(2020) Photoperiod and Vernalization
Control of Flowering-Related Genes:
A Case Study of the Narrow-Leafed
Lupin (Lupinus angustifolius L.).
Front. Plant Sci. 11:572135.
doi: 10.3389/fpls.2020.572135
Photoperiod and Vernalization
Control of Flowering-Related Genes:
A Case Study of the Narrow-Leafed
Lupin (Lupinus angustifolius L.)
Sandra Rychel-Bielska1,2 , Piotr Plewi ´
nski2, Bartosz Kozak1, Renata Galek1and
Michał Ksia¸ ˙
zkiewicz2*
1Department of Genetics, Plant Breeding and Seed Production, Wrocław University of Environmental and Life Sciences,
Wrocław, Poland, 2Department of Genomics, Institute of Plant Genetics, Polish Academy of Sciences, Pozna´
n, Poland
Narrow-leafed lupin (Lupinus angustifolius L.) is a moderate-yielding legume crop
known for its high grain protein content and contribution to soil improvement. It is
cultivated under photoperiods ranging from 9 to 17 h, as a spring-sown (in colder
locations) or as an autumn-sown crop (in warmer regions). Wild populations require
a prolonged cold period, called vernalization, to induce flowering. The key achievement
of L. angustifolius domestication was the discovery of two natural mutations (named
Ku and Jul) conferring vernalization independence. These mutations are overlapping
deletion variants in the promoter of LanFTc1, a homolog of the Arabidopsis thaliana
FLOWERING LOCUS T (FT) gene. The third deletion, named here as Pal, was recently
found in primitive germplasm. In this study, we genotyped L. angustifolius germplasm
that differs in domestication status and geographical origin for LanFTc1 alleles, which
we then phenotyped to establish flowering time and vernalization responsiveness. The
Ku and Jul lines were vernalization-independent and early flowering, wild (ku) lines were
vernalization-dependent and late flowering, whereas the Pal line conferred intermediate
phenotype. Three lines representing Ku,Pal, and ku alleles were subjected to gene
expression surveys under 8- and 16-h photoperiods. FT homologs (LanFTa1,LanFTa2,
LanFTc1, and LanFTc2) and some genes selected by recent expression quantitative trait
loci mapping were analyzed. Expression profiles of LanFTc1 and LanAGL8 (AGAMOUS-
like 8) matched observed differences in flowering time between genotypes, highlighted
by high induction after vernalization in the ku line. Moreover, these genes revealed
altered circadian clock control in Pal line under short days. LanFD (FD) and LanCRLK1
(CALCIUM/CALMODULIN-REGULATED RECEPTOR-LIKE KINASE 1) were negatively
responsive to vernalization in Ku and Pal lines but positively responsive or variable in
ku, whereas LanUGT85A2 (UDP-GLUCOSYL TRANSFERASE 85A2) was significantly
suppressed by vernalization in all lines. Such a pattern suggests the opposite regulation
of these gene pairs in the vernalization pathway. LanCRLK1 and LanUGT85A2 are
homologs of A. thaliana genes involved in the FLOWERING LOCUS C (FLC) vernalization
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Rychel-Bielska et al. Photoperiod, Vernalization and Gene Expression
pathway. Lupins, like many other legumes, do not have any FLC homologs. Therefore,
candidate genes surveyed in this study, namely LanFTc1,LanAGL8, LanCRLK1, and
LanUGT85A2, may constitute anchors for further elucidation of molecular components
contributing to vernalization response in legumes.
Keywords: vernalization, photoperiod, flowering, expression, FLOWERING LOCUS T, duplication, deletion
INTRODUCTION
Narrow-leafed lupin (Lupinus angustifolius L.) is a legume plant
that is cultivated as “green” manure or as a grain crop for
animal feed or human consumption (Kurlovich, 2002). The
exploitation of L. angustifolius as a crop is advantageous in
many respects. First, L. angustifolius cultivation has a positive
influence on soil fertility due to the mobilization of soil-bound
phosphorus and diazotrophic nitrogen fixation (Evans et al.,
1987;Lambers et al., 2013). Moreover, L. angustifolius grains
are characterized by high protein content and the use of them
in livestock farming systems has many benefits in terms of the
economic and environmental impact (Abraham et al., 2019).
As seed alkaloid content was reduced by breeding below 0.01%
of seed dry weight, a bitter taste, a typical feature of lupins,
was fully eliminated from modern cultivars (Kamel et al., 2016).
Additionally, the postharvest stubble can be now safety grazed
by livestock, because the risk of lupinosis disease was diminished
by the introduction of Phomospis stem blight resistance genes
(Mulholland et al., 1976;Jago et al., 1982;Cowling et al., 1987;
Williamson et al., 1994;Shankar et al., 2002;Ksia¸˙
zkiewicz et al.,
2020). Furthermore, L. angustifolius is currently being promoted
in human food markets as a result of its nutritional, metabolomic,
and other health benefits (Foyer et al., 2016;Kouris-Blazos and
Belski, 2016).
Cultivated L. angustifolius germplasm primarily originated
from the western Mediterranean basin (Mousavi-Derazmahalleh
et al., 2018b). Natural adaptation of wild L. angustifolius
populations to the Mediterranean climate is the requirement of
the prolonged cold period (i.e., vernalization) during germination
or juvenile phase to induce flowering (Gladstones and Hill,
1969;Rahman and Gladstones, 1972;Landers, 1995;Adhikari
et al., 2012). The selection of early phenology was based on the
removal of the vernalization requirement, a major achievement
in the domestication of L. angustifolius, enabling temperature-
independent sowing (French and Buirchell, 2005). L. angustifolius
is grown in various environments ranging from the subtropics
(Australia) and Mediterranean regions (including Morocco,
Spain, southern France, and Italy), through temperate oceanic
climates (such as the United Kingdom, northern France, and
Benelux), humid continental climates (for example in Germany,
the Baltic countries, and Ukraine), though to the subarctic zone,
localized as far north as 60latitude (Russia). In warmer regions it
is usually autumn-sown, whereas in colder regions it is exclusively
spring-sown (Annicchiarico and Carroni, 2009).
Lupin crops are cultivated in various photoperiod conditions,
ranging from about 9 h (autumn sowing in Australia) to 16–
17 h (spring sowing in Baltic countries and Russia) (Ksia¸˙
zkiewicz
et al., 2017). The vernalization requirements of wild populations
are so demanding that they can only be completely fulfilled
by spring sowing in northern locations (Adhikari et al., 2012).
Worldwide, L. angustifolius cultivation is based on vernalization
independence selected from only two natural mutations (named
Ku and Jul) that were discovered in domesticated germplasm just
a little over half a century ago (Mikołajczyk, 1966;Gladstones
and Hill, 1969). The use of only two donors of early flowering
in L. angustifolius breeding, followed by a strong selection of key
agronomic traits, have resulted in a lack of phenological diversity
in domesticated germplasm (Stefanova and Buirchell, 2010;
Berger et al., 2012;Cowling, 2020). Therefore, further adaptation
of this crop to new agronomic conditions resulting from a rapidly
changing climate, may only be possible with the incorporation of
novel genetic resources of intermediate phenology.
During recent years L. angustifolius was supplemented
with numerous molecular resources, including bacterial
artificial chromosome libraries carrying nuclear DNA inserts,
consecutively improved linkage maps with sequence-defined
markers, transcriptome assemblies, and a progressively updated
genome sequence of the reference cultivar Tanjil (Kasprzak
et al., 2006;Nelson et al., 2006;Gao et al., 2011;Yang et al.,
2013;Kamphuis et al., 2015;Wyrwa et al., 2016;Hane et al.,
2017;Zhou et al., 2018;Kozak et al., 2019). These resources
were harnessed to reveal the genetic identity of Ku, which was
found to be a homolog of a FLOWERING LOCUS T (FT) gene,
named LanFTc1 (Ksia¸˙
zkiewicz et al., 2016;Nelson et al., 2017).
FT gene is a well-recognized floral integrator gene, promoting
flowering in response to environmental conditions signaled
by photoperiod, vernalization, and circadian clock pathways
(Turck et al., 2008). The functional mutation underlying the
domesticated Ku allele in L. angustifolius was assigned to the
1423 bp deletion in the promoter region of LanFTc1, carrying
potential binding sites for several transcription factors acting
as FT gene repressors in Arabidopsis thaliana (Nelson et al.,
2017). Interestingly, the second L. angustifolius early phenology
mutation, Jul, was recently revealed to be the third LanFTc1
allele, in the form of a 5162 bp deletion in the promoter region,
fully encompassing that 1423 bp Ku deletion (Taylor et al.,
2019). Screening of germplasm resources with the LanFTc1
markers resulted in the identification of a fourth LanFTc1 allele
in wild population line originating from Palestine (a country
annotated as Israel in Australian collection or Egypt in Polish
gene bank), carrying 1208 bp deletion partially overlapping with
domesticated Ku deletion (Rychel, 2018;Taylor et al., 2019). This
Palestinian allele was named here as Pal.
Some genes involved in flowering time regulation are under
strict circadian clock control and their expression levels fluctuate
during a day (Shim et al., 2017). One such example in
Arabidopsis is the FT gene, which expression is correlated
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with the stability of CONSTANS (CO) protein and shows two
peaks, the first in the morning (lower) and the second in
the evening (higher) (Turck et al., 2008). This CO-dependent
regulation is facilitated by enhancer protein binding to specific
sites in the promoter sequence, located approximately 5 kbp
upstream of the first codon, which results in forming a DNA
loop bringing all the components together (Cao et al., 2014).
Interestingly, L. angustifolius allelic variants of the LanFTc1
promoter have the distal binding sequences (CCAAT-boxes)
preserved (Ksia¸˙
zkiewicz et al., 2016;Nelson et al., 2017;Taylor
et al., 2019). This study sought to explore whether the altered
length of the LanFTc1 promoter is associated with the altered
circadian clock control of LanFTc1 and other related genes.
Taking into consideration the (i) role of FT genes in
integrating key environmentally responsive pathways, (ii)
demonstrated allelic variability of LanFTc1 gene associated
with flowering time, and (iii) wide range of environmental
variables occurring at lupin cultivation sites, we decided to
explore the photoperiod and vernalization responsiveness in
L. angustifolius. Here, the L. angustifolius germplasm differing
in domestication status and geographical origin was genotyped
for LanFTc1 alleles and phenotyped for flowering time and
vernalization responsiveness. Then, the transcriptomic response
of candidate genes from the vernalization pathway in early
(Ku), intermediate (Pal), and late flowering (ku)L. angustifolius
germplasm was assayed under contrasting photoperiod and
vernalization conditions, accounting the influence of circadian
rhythm control. This study discusses the hypothetical function
of these genes in terms of flowering time regulation and response
to vernalization.
MATERIALS AND METHODS
Plant Material
Ninety-two L. angustifolius lines were subjected to genotyping
and phenotyping. Lines were derived from the European Lupin
Gene Resources Database maintained by the Pozna´
n Plant
Breeding Ltd. station located in Wiatrowo. The lines originated
from 13 countries, including Spain (32 lines), Poland (15),
Australia (11), Russia (11), Germany (7), Italy (4), Belarus (3),
Israel (2), Algeria, Morocco, Palestine, Portugal and Republic
of South Africa (1). Accession numbers and information on
the domestication status and country of origin are provided in
Supplementary Table 1.
Identification of LanFTc1 Alleles
Young leaf tissue from three biological replicates per plant was
sampled. Frozen (80C) plant tissue (50 mg) was homogenized
using TissueLyser II (Qiagen, Hilden, Germany) and two
stainless steel beads 5 mm) in 2 ml tubes (Eppendorf,
Hamburg, Germany). DNA was isolated using DNeasy Plant
Mini Kit (Qiagen). PCR was performed using GoTaq Long PCR
Master Mix (Promega, Mannheim, Germany) and published
LanFTc1_INDEL2 primers (Taylor et al., 2019), provided here
for reference in Supplementary Table 2. PCR conditions were
as follows: initial denaturation (94C for 2 min), then 35 cycles
composed of denaturation (94C for 30 s), annealing (62C
for 30 s), and elongation (72C for 5 min), followed by the
final extension (72C for 10 min). Products were resolved
by agarose gel electrophoresis and SYBR Safe DNA staining
(Invitrogen, Carlsbad, CA, United States) and visualized on UV-
transilluminator (Uvitec, Thermo Fisher Scientific, Waltham,
MA, United States). Wild P27255 allele (ku, without deletion) was
encoded as “A, Palestinian allele (Pal, 1208 bp deletion) as “B,
83A:476 allele (Ku, 1423 bp deletion) as “C, and Krasnolistny
allele (Jul, 5162 bp deletion) as “D.”
Evaluation of Vernalization
Responsiveness in Greenhouse
Vernalization was carried out by placing imbibed seeds for
21 days at 5C in darkness on moist filter paper in Petri dishes.
Non-vernalized control plants were sown five days before the end
of the vernalization procedure and kept at 21C to maintain a
similar thermal time (Huyghe, 1991). Plants were cultivated in
a greenhouse maintained by the Institute of Plant Genetics, the
Polish Academy of Sciences, Pozna´
n, Poland (52260N 16540E)
during growing seasons of 2014 (sowing of vernalized plants on
14.05) and 2015 (sowing of vernalized plants on 25.03) under
ambient long day photoperiod (12–17 h). The greenhouse was
equipped with automatic heating to keep the minimum air
temperature above 18C. Passive cooling was maintained by a
temperature-dependent ventilation system (activated at 22C).
Air temperature (daily mean and maximum) and daily sunshine
hours recorded by the nearby localized meteorological station
(Pozna´
n-Ławica, 5.1 km) as well as the theoretical photoperiod
hours calculated for this latitude (covering 120 days from
sowing date for both years) were provided for reference in
Supplementary Tables 3, 4. Flowering time was recorded as the
number of days from sowing date of vernalized plants until the
first fully colored petal was observed. The average number of
plants sampled in 2014 was 5.6 for the non-vernalized variant
(min. 3, max. 6) and 6.9 for the vernalized variant (min. 4, max.
7), whereas in 2015 it was 4.7 for the non-vernalized variant (min.
4, max. 5) and 5.0 for the vernalized variant.
Controlled Environment Experiment for
Gene Expression Profiling
Based on the results of LanFTc1 allele genotyping and
vernalization responsiveness phenotyping, three accessions were
selected for gene expression profiling: P27255 from Morocco
(96,234, carrying wild allele ku), 83A:476 from Australia (96,233,
carrying domesticated allele Ku), and Palestyna from Palestine
(95,799, carrying intermediate allele Pal). The vernalization of
lines was performed as described above. Non-vernalized control
plants were sown five days before the end of the vernalization
procedure. Plants from both variants were cultivated in climatic
chambers with controlled humidity (40–50% day, 70–80%
night) and temperature (22C day, 18C night). Two types of
photoperiods were studied, short day (SD, 8 h, from 8 AM to
4 PM) and long day (LD, 16 h, from 4 AM to 8 PM). Young
leaves from five biological replicates were sampled every week
at two times of day to follow a circadian rhythm, at 9 AM, and
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3 PM under SD or at 7 AM and 6 PM under LD. Plant material
was immediately frozen in liquid nitrogen and stored at 80C.
Three replicates with similar plant phenology (growth rate and
time to flowering) were subjected to gene expression profiling.
Taking into consideration observed flowering time, from 2 to 4
terms were selected for gene expression, representing the period
from about 2 weeks before flowering to the flowering date and/or
few days after flowering (Supplementary Table 5).
RNA Isolation and cDNA Synthesis
Frozen young leaf tissue (50 mg) was homogenized using
TissueLyser II (Qiagen) and two stainless steel beads 5 mm)
in 2 ml tubes (Eppendorf). RNA isolation was performed using
the SV Total RNA Isolation System (Promega) without any
alterations to the protocol. RNA concentration and purity were
measured using NanoDrop 2000 (Thermo Fisher Scientific) and
A260/A280 ratio. RNA integrity was visualized by 1% agarose
gel electrophoresis of denatured samples in 1 ×TAE buffer.
RNA concentration was equalized to 1000 ng/µl in nuclease-
free water. First-strand cDNA synthesis was performed using
GoScript (TM) Reverse Transcription System (Promega) and
5µg of total RNA per sample.
Selection of Genes for Quantitative PCR
The set of genes selected for quantitative PCR included,
among others, four L. angustifolius representatives of FT clade,
namely LanFTa1 (Lup021189, XM_019571501.1), LanFTa2
(XM_019596455.1), LanFTc1 (Lup015264, XM_019601808.1),
and LanFTc2 (Lup005674, XM_019565316.1) (Ksia¸˙
zkiewicz
et al., 2016;Nelson et al., 2017). First names in parentheses
correspond to gene names provided in the L. angustifolius
pseudochromosome assembly paper (Hane et al., 2017), whereas
the second names address L. angustifolius NCBI Reference
Sequences LupAngTanjil_v1.0. Moreover, our assay endeavored
also some candidate genes revealed by recent L. angustifolius
(eQTL mapping) study (Plewi´
nski et al., 2019) to be associated
with the vernalization response, as follows: LanUGT85A2
(Lup002110, XM_019574900.1), LanCRLK1 (Lup011808,
XM_019603391.1), LanAGL8 (Lup018485, XM_019583439.1),
and LanFD (Lup018024, XM_019567853.1). Alignment of
coding sequences to the L. angustifolius genome assembly
provided evidence that LanAGL8 and LanFD are present
in single copies.
Moreover, some major A. thaliana components of the
flowering induction pathway were considered, as follows:
LEAFY (LFY, AT5G61850), APETALA1 (AP1, AT1G69120),
VERNALIZATION INSENSITIVE 3 (VIN3, AT5G57380),
and VERNALIZATION 5 (VRN5, AT3G24440). Multiple
sequence alignment revealed that all these genes have
putatively three copies in the L. angustifolius genome, namely
Lup006312 (XM_019602464.1), Lup012189 (XM_019558971.1)
and Lup027481 (XM_019607325.1) for LFY; Lup021855
(XM_019605469.1), Lup024348 (XM_019588203.1) and
Lup006876 (XM_019572960.1) for AP1; Lup009440
(XM_019586787.1), Lup013437 (XM_019598860.1) and
Lup026125 (XM_019608083.1) for VIN3; and Lup009144
(XM_019591910.1), Lup018692 (XM_019567058.1) and
Lup032778 (XM_019590742.1) for VRN5. Analysis of leaf
transcriptome data obtained for the L. angustifolius linkage
mapping population, covering developmental phases from
juvenile to generative growth after partial vernalization
(Plewi´
nski et al., 2019), revealed negligible expression of all
LFY and AP1 homologs (0.07 reads per kilobase million,
RPKM), very low expression of VRN5 (0.56 RPKM) and high
expression of VIN3 copies (25.07 RPKM) (Supplementary
Table 6). Therefore, VRN5 and VIN3 homologs were selected
for the expression assay, whereas LFY and AP1 homologs were
discarded. Reference genes validated in previous L. angustifolius
quantitative gene expression studies were selected for this
assay, namely LanDExH7 (Lup023733, XM_019579367.1),
and LanTUB6 (Lup032899, XM_019581544.1) (Taylor et al.,
2016, 2019;Nelson et al., 2017). Primers were designed in
Geneious Prime (Auckland, New Zealand) using Primer3
(Kearse et al., 2012;Untergasser et al., 2012). Due to the high
similarity between particular copies, all three VRN5 homologs
were analyzed together using one primer pair. The remaining
genes were profiled on one by one basis. Designed primers and
expected product sizes are provided in Supplementary Table 2.
Quantitative Gene Expression Analysis
In prior experiments, a CFX Connect Real-Time PCR Detection
System (Bio-Rad Polska, Warsaw, Poland) was calibrated
using Melt Calibration Kit (Bio-Rad Polska) according to the
manufacturer’s protocol. A standard curve was developed to
assess the performance of the quantitative PCR assay and
its dynamic range following recent recommendations (Svec
et al., 2015). Analyzed genes were amplified using GoTaq
G2 Flexi DNA Polymerase (Promega) and subjected to 1%
agarose gel electrophoresis. Amplicons were excised from a gel,
extracted with the aid of QIAquick Gel Extraction Kit (Qiagen),
quantified using NanoDrop 2000 (Thermo Fisher Scientific), and
outsourced (Genomed Ltd., Warsaw, Poland) for direct Sanger
sequencing on ABI PRISM 3130 Genetic Analyzer XL (Applied
Biosystems, Hitachi). A series of dilutions in concentrations
ranging from 1 to 1010 of the original templates were prepared
for every gene using an initial volume of 20 µl to reduce
the sampling error. 6 replicates per each concentration were
performed using the iTaq Universal SYBR Green Supermix (Bio-
Rad Polska). A two-step PCR protocol was exploited according
to the protocol. A calculation of R2and PCR efficiency values
was done in Bio-Rad CFX Manager 3.1. The values obtained are
provided in Supplementary Table 7.
The quantitative PCR analysis of gene expression was
performed using 96-well PCR plates and two reference
genes (LanDExH7 and LanTUB6). Inter-run calibration sample
(LanTUB6) and no template control were used on all plates. Three
biological replicates were analyzed for each time point, and all
samples were run in 3 technical repeats. High-resolution PCR
product melting in the range of temperature from 65 to 85C was
performed after PCR to control the specificity of amplification.
Melt profiles were inspected for the amplification of unspecific
products, highlighted by the presence of melting peaks at
different temperatures than those obtained during standard curve
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FIGURE 1 | Indel variation in the promoter sequence of LanFTc1 gene controlling flowering induction in L. angustifolius. Allele ku is typical for wild populations, alleles
Jul and Ku are present only in domesticated germplasm, whereas allele Pal was found only in wild germplasm from Palestine. The position is given in relation to the
first nucleotide of CCAAT-box (Ksia¸ ˙
zkiewicz et al., 2016;Nelson et al., 2017;Taylor et al., 2019).
preparation. Calculations of 11Cq were performed in Bio-
Rad CFX Manager 3.1 taking into consideration PCR efficiency
values and results obtained for both reference genes. The
final computations (mean value and standard deviation) and
visualization (graphs) were performed in Microsoft Excel 2010.
Statistical Analysis
Calculations were performed to check the influence of circadian
clock (expression in the evening divided by expression in the
morning), growth phase (maximum expression at analyzed date
divided by maximum expression at the first date), vernalization
(fold change of expression after vernalization), and genotype
(comparison of expression levels observed in studied lines,
including Ku/Pal,Ku/ku, and Pal/Ku, performed for all data
points). The values obtained are provided in Supplementary
Data Sheet 1. The statistical significance of these quotients was
tested using a t test for the mean ratio as proposed by Hauschke
et al. (1999),Tamhane and Logan (2004)). Calculations were
made in R (R Core Team, 2013) with custom script using
“t.test.ratio” function from the ratios package. In the first step, the
equal variance was tested. If this condition was satisficed classical
t-test formula was used, otherwise, Welch’s t-test formula was
used (Welch, 1947). P-values were rounded up to four decimal
places and are provided in the Supplementary Data Sheet 1.
RESULTS
European Lupin Gene Bank Preserves
the L. angustifolius Donors of Four
Alleles of LanFTc1 Promoter
Indel variation in the promoter region of the major flowering
time gene LanFTc1 (Figure 1) was recently revealed to be
associated with flowering time and vernalization responsiveness
in L. angustifolius (Nelson et al., 2017;Taylor et al., 2019).
To evaluate allelic composition in germplasm exploited by
European lupin breeders, ninety-two L. angustifolius accessions,
encompassing 43 primitive populations or landraces, 23 cross
derivatives or breeding lines, 25 cultivars, and one mutant,
derived from the European Lupin Gene Resources Database, were
screened with primers flanking polymorphic LanFTc1 promoter
region (Table 1). As expected, wild LanFTc1 allele (ku) was found
mainly in primitive accessions collected in the Mediterranean
Basin as well as in a few old domesticated materials originating
from Russia, Poland, Germany, and the Republic of South Africa.
One wild population line, Palestyna originating from Palestine,
was found to carry the shortest variant of deletion (1208 bp),
named here as Pal. Alleles Ku (1423 bp deletion) and Jul (5162 bp
deletion) were found only in domesticated germplasm the first
one was present mostly in released cultivars, whereas the second
one was typically in breeding materials at a different stage of
improvement. Accessions carrying Ku originated primarily from
Australia, Germany, and Poland, whereas those carrying Jul from
Poland and Russia. Results of marker screening are provided in
Supplementary Table 8.
Palestinian LanFTc1 Allele Confers
Intermediate Flowering Time Moderately
Responsive to Vernalization
Ninety-two L. angustifolius accessions with known LanFTc1
promoter allele composition were evaluated for phenotypic
response to vernalization in two consecutive years, 2014 and
TABLE 1 | Distribution of LanFTc1 alleles across wild and domesticated
L. angustifolius germplasm.
LanFTc1 allele Lines XC CV MU WP
ku, without deletion 48 3 3 42
Pal, 1208 bp deletion 1 1
Ku, 1423 bp deletion 24 7 17
Jul, 5162 bp deletion 19 13 5 1
XC, cross derivative or breeding line; CV, cultivar; MU, mutant; WP, wild population
or primitive landrace.
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TABLE 2 | Comparison of flowering time and vernalization responsiveness of
L. angustifolius germplasm carrying Jul,Ku,Pal, and ku LanFTc1 alleles, cultivated
in a greenhouse under natural long days (LD).
LanFTc1 allele Days to
flowering
‘2014
Vernalization
response
‘2014
Days to
flowering
‘2015
Vernalization
response
‘2015
ku, without deletion 88.8 ±15.6a41.1 ±13.8 88.0 ±5.9 21.8 ±6.0
Pal, 1208 bp deletion 53.5 ±1.9 14.5 ±2.7 68.2 ±5.6 14.8 ±3.4
Ku, 1423 bp deletion 40.5 ±2.6 3.3 ±2.6 56.7 ±2.1 3.0 ±2.5
Jul, 5162 bp deletion 38.8 ±1.1 0.2 ±2.2 55.6 ±1.9 2.2 ±2.2
aStandard deviation.
2015 (Table 2). Lines carrying wild ku allele revealed the longest
vegetative phase, ranging from 61.2 ±0.9 to 116.2 ±3.0 days
in ‘2014 and from 79.0 ±5.8 to 101.5 ±3.9 days in ‘2015.
These lines demonstrated also high vernalization responsiveness,
highlighted by the acceleration of flowering after vernalization
by 13.9 to 66.4 days in 2014, and by 11.6 to 35.6 days in 2015.
The line carrying Pal allele showed intermediate phenology and
flowered about 20-35 days quicker than the average ku genotype,
namely after 53 days from sowing in ‘2014 and 68 days
in ‘2015. The vernalization procedure advanced flowering
induction in this line by about two weeks. Accessions carrying
domesticated Ku allele were very early and started flowering after
37.2 ±0.4 to 46.2 ±8.9 days in 2014, and after 54.0 ±0.0
to 59.6 ±2.6 days in 2015. This set contained germplasm
low-responsive to vernalization, which accelerated flowering
time by up to 8.6 days, as well as some truly thermoneutral
accessions, which flowered at the same time regardless of the
vernalization procedure. Lines carrying domesticated European
Jul allele also revealed very early phenology, manifested by the
onset of flowering after 37.0 ±1.3 to 41.0 ±5.9 days in 2014, and
after 52.2 ±1.5 to 60.8 ±1.8 days in 2015. Most Jul lines were
fully thermoneutral. On average, Jul lines flowered earlier than Ku
lines in both years, however taking into consideration standard
errors resulting from variability between biological replicates,
differences between mean values were not statistically significant.
Results of time to flowering and vernalization responsiveness
in relation to LanFTc1_INDEL2 marker polymorphism are
provided in Supplementary Table 8 (2014) and Supplementary
Table 9 (2015).
Based on the results of LanFTc1_INDEL2 marker screening
and vernalization responsiveness, three lines were selected for
vernalization response phenotyping under 8-h (SD) and 16-
h (LD) photoperiods: 84A:476 carrying domesticated Ku allele
(early flowering, thermoneutral), Palestyna carrying wild Pal
allele (moderately flowering and responsive to vernalization),
and P27255 carrying wild ku allele (late flowering and highly
responsive to vernalization). 83A:476 was revealed to be the
earliest line in both photoperiods, followed by Palestyna
(Table 3). Under SD, 83A:476 accelerated transition between
phases in response to vernalization by about 5 days, whereas
under LD by about 3 days. These responses were higher
in Palestyna, amounting to about 12-19 days and 3-5 days,
respectively. P27255 did not flower during the experiment
(90 days) in all variants except vernalized plants under LD.
TABLE 3 | Number of days to first bud, flower, and pod in L. angustifolius
germplasm carrying Ku,Pal, and ku LanFTc1 alleles, cultivated under 8- and
16-h photoperiods.
LanFTc1 allele Vernalization
variant
Days to first
bud
Days to first
flower
Days to first
pod
8-h photoperiod
Ku - 51.3 ±1.6a57.2 ±4.3 63.3 ±9.8
+45.7 ±2.9 52.0 ±4.2 58.1 ±2.5
Pal - 65.3 ±3.7 71.2 ±3.8 83.5 ±2.7
+52.9 ±3.0 55.9 ±2.6 64.7 ±4.6
ku -
+–––
16-h photoperiod
Ku - 33.6 ±1.1 38.7 ±1.3 44.1 ±1.4
+29.8 ±2.7 35.3 ±1.0 41.2 ±1.0
Pal - 36.7 ±0.5 43.8 ±0.4 49.0 ±0.5
+40.0 ±0.0 48.4 ±1.4 52.0 ±0.8
ku -
+55.1 ±1.3 59.0 ±1.2 69.0 ±2.4
aStandard deviation.
LanFTc1 Expression Was High and
Thermoneutral in Ku, High and Partially
Vernalization-Independent in Pal,
Whereas Low and Positively Responsive
to Vernalization in ku
83A:476 (Ku), Palestyna (Pal), and P27255 (ku) grown under
SD and LD conditions were used for gene expression profiling
(Supplementary Data Sheet 1). The vernalization responsiveness
of analyzed genes was calculated as a mean fold change of
expression in vernalized plants compared to non-vernalized
control (averaged across all day terms and dates measured for
a particular line), as summarized in Table 4. The circadian
clock responsiveness were calculated as a mean fold change of
expression occurring between the morning and the evening terms
(averaged across all dates measured for a particular line) and are
provided in Table 5. The trend in expression level during plant
growth was calculated as a fold change of expression occurring
between the first and the last term (based on maximum daily
values) and is provided in Table 6.
First, we analyzed the expression of the LanFTc1 gene,
which is considered as the major controller of vernalization
responsiveness and early flowering in L. angustifolius (Nelson
et al., 2017;Taylor et al., 2019). The studied genotypes
showed different patterns of LanFTc1 expression in response to
photoperiod, vernalization, and circadian rhythm.
Under SD, LanFTc1 expression in non-vernalized plants was
the highest in 83A:476 and the lowest in P27255 (Figure 2A).
Indeed, LanFTc1 expression in 83A:476 was up to 2.4 times
higher than in Palestyna (P= 0.0016) and 460-2213 times
higher than in P27255 (P= 0.0000). After vernalization, LanFTc1
expression in 83A:476 was up to 4.5 times higher than in
Palestyna (P= 0.0064) and up to 686 times higher than in P27255
(P= 0.0029). The difference of LanFTc1 expression between
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83A:476 and P27255 increased during plant development in
both vernalization variants, whereas for the pair 83A:476 and
Palestyna it was decreasing. Under LD, LanFTc1 expression was
the highest in Palestyna and the lowest in P27255 (Figure 2B).
Namely, in non-vernalized plants, expression in Palestyna was
1.8-5.4 times higher than in 83A:476 (P= 0.0125) and 121-2642
times higher than in P27255 (P = 0.0085), whereas in vernalized
plants it was 1.5-13.8 times higher in Palestyna than in 83A:476
(not significant, NS) and 24-276 times higher than in P27255
(P= 0.0094). The difference to Palestyna was increasing during
plant growth for 83A:476 in both vernalization variants and
P27255 in the absence of vernalization.
Vernalization influence on LanFTc1 expression in P27255
was manifested by up to a 7.2-fold increase under SD (NS)
(Figure 2C) and up to 427-fold increase under LD (P= 0.0125)
(Figure 2D). The vernalization effect under SD was changing
in 83A:476 from a 0.5-fold decrease (P= 0.0115) to a 3.2-fold
increase (P= 0.0094) and similarly in Palestyna from a 0.5-
fold decrease (P= 0.0291) to 1.6-fold increase (NS). This effect
under LD was neutral in 83A:476 and positive in Palestyna, up to
5.1-fold increase (P= 0.0351).
The circadian clock regulation differed between genotypes and
partially between environments. In 83A:476, LanFTc1 expression
was generally higher in the evening than in the morning in all
combinations of the photoperiod and vernalization (up to 18.4-
fold increase, P= 0.0008). In Palestyna, LanFTc1 expression
under SD was usually higher in the morning (up to 2.9-fold
increase, P= 0.0172), whereas under LD this effect was variable.
In P27255, LanFTc1 expression was higher in the evening,
especially under LD after vernalization (P= 0.0046).
TABLE 4 | Vernalization responsiveness of analyzed genes in L. angustifolius germplasm carrying Ku,Pal, and ku alleles, cultivated under 8-h (SD) and 16-h
(LD) photoperiods.
Gene name Mean change of expression after vernalization under SD Mean change of expression after vernalization under LD
Ku Pal ku Ku Pal ku
LanAGL8 1.6a2.2 13.0 1.8 6.1 138.6
LanCRLK1 0.7 0.6 2.6 0.6 1.3 1.0
LanFD 0.5 0.5 3.6 0.3 0.7 1.0
LanFTa1 9.9 1.3 0.9 1.8 2.1 14.7
LanFTa2 1.1 0.3 13.3 3.2 1.1 1.2
LanFTc1 1.3 0.9 4.3 1.3 3.6 144.4
LanFTc2 0.7 0.7 3.5 1.1 3.2 1.4
LanUGT85A2 0.3 0.2 0.4 0.2 0.2 0.4
LanVIN3-1 0.9 0.9 1.9 0.8 1.0 1.1
LanVIN3-2 0.7 1.1 2.3 0.7 1.3 1.6
LanVIN3-3 0.9 1.7 2.3 0.6 1.3 1.0
LanVRN5 0.8 1.0 2.3 0.9 1.0 0.8
aFold change of expression occurring in response to vernalization, averaged across all data points (day terms and dates).
TABLE 5 | Circadian clock responsiveness of analyzed genes in L. angustifolius germplasm carrying Ku,Pal, and ku alleles, cultivated under 8-h (SD) and 16-h (LD)
photoperiods without vernalization and with vernalization.
Gene name Mean change of expression during light phase under SD Mean change of expression during light phase under LD
Ku Pal ku Ku Pal ku
LanAGL8 1.6 | 1.0a0.3 | 0.7 0.8 | 0.7 2.6 | 1.6 1.3 | 3.6 2.0 | 0.5
LanCRLK1 0.9 | 1.0 0.7 | 1.0 0.8 | 0.7 0.8 | 0.5 0.8 | 1.3 1.0 | 1.0
LanFD 0.3 | 0.8 1.7 | 3.1 1.2 | 1.6 0.3 | 0.4 1.6 | 2.4 1.9 | 1.3
LanFTa1 1.0 | 1.7 0.6 | 0.6 0.7 | 0.8 0.8 | 2.7 0.8 | 4.5 0.8 | 0.7
LanFTa2 0.6 | 0.9 0.9 | 0.4 0.4 | 0.4 0.4 | 2.2 0.7 | 0.4 0.6 | 0.6
LanFTc1 4.8 | 3.1 0.5 | 0.8 1.2 | 2.4 1.8 | 2.9 1.2 | 1.1 2.7 | 4.3
LanFTc2 2.0 | 4.3 2.0 | 11.1 5.6 | 24.3 2.7 | 1.7 2.0 | 0.5 3.4 | 4.2
LanUGT85A2 5.0 | 3.4 2.6 | 2.1 2.4 | 2.4 3.2 | 4.6 3.8 | 2.2 3.3 | 2.9
LanVIN3-1 1.2 | 1.5 0.7 | 1.1 1.4 | 0.9 1.4 | 1.4 1.9 | 2.7 1.5 | 1.5
LanVIN3-2 1.9 | 1.2 1.0 | 1.2 1.3 | 0.9 1.7 | 1.4 1.5 | 2.4 1.4 | 1.4
LanVIN3-3 1.4 | 1.3 0.8 | 1.0 1.2 | 0.9 2.1 | 1.5 1.5 | 2.0 1.4 | 1.7
LanVRN5 0.7 | 0.8 0.7 | 0.7 0.7 | 0.7 1.3 | 0.9 1.1 | 0.9 1.0 | 0.9
aFold change of expression occurring between the morning and the evening terms, averaged across all dates; the first number corresponds to the experiment without
vernalization, the second number to the experiment with vernalization.
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TABLE 6 | Change of expression level of analyzed genes in L. angustifolius germplasm carrying Ku,Pal, and ku alleles, during plant growth under 8-h (SD) and 16-h
(LD) photoperiods.
Gene name Change of expression during experiment under SD Change of expression during experiment under LD
Ku Pal ku Ku Pal ku
LanAGL8 1.5 | 2.3a3.5 | 6.3 0.4 | 6.4 1.6 | 1.0 16.6 | 6.8 4.0 | 805.9
LanCRLK1 0.5 | 1.2 0.9 | 1.2 0.6 | 0.5 0.8 | 0.8 0.6 | 2.7 0.6 | 1.2
LanFD 0.2 | 0.6 0.7 | 1.2 0.4 | 1.9 0.4 | 0.2 0.3 | 0.4 1.7 | 4.7
LanFTa1 0.7 | 1.3 1.7 | 5.6 0.5 | 1.4 23.6 | 7.3 404.5 | 340.2 2.2 | 765.0
LanFTa2 0.2 | 0.4 7.6 | 1.6 0.09 | 9.2 0.05 | 0.5 0.4 | 1.9 0.2 | 2.6
LanFTc1 0.4 | 1.2 0.9 | 3.0 0.08 | 0.3 1.5 | 2.2 15.7 | 10.2 0.7 | 118.8
LanFTc2 0.4 | 1.5 1.2 | 2.5 0.5 | - 1.1 | 0.9 3.8 | 5.9 0.7 | 1.1
LanUGT85A2 0.1 | 0.1 0.3 | 0.3 0.4 | 1.2 0.3 | 0.1 0.02 | 0.02 0.5 | 0.08
LanVIN3-1 0.3 | 3.0 1.0 | 1.2 0.8 | 1.4 2.3 | 1.2 1.0 | 1.1 1.0 | 1.0
LanVIN3-2 0.4 | 2.6 1.3 | 1.3 0.9 | 1.9 3.3 | 1.1 1.0 | 1.8 0.9 | 1.9
LanVIN3-3 0.2 | 3.5 1.2 | 3.2 0.4 | 1.3 2.5 | 0.9 1.2 | 1.8 0.9 | 0.9
LanVRN5 0.3 | 1.5 1.4 | 1.1 0.3 | 0.9 3.7 | 2.2 1.1 | 1.0 1.0 | 0.8
aFold change in expression occurring between the first and the last term, based on the maximum daily values; the first number corresponds to the experiment without
vernalization, the second number to the experiment with vernalization.
LanFTa1 Expression Was Low and
Uniform in All Lines Under SD, However,
It Was Highly Induced Just Before
Flowering, Especially in Pal and ku
Genotypes Under LD
Besides LanFTc1, three other FT genes, namely LanFTa1,
LanFTa2, and LanFTc2, were analyzed to complement our
perspective on FT clade transcriptional activity in L. angustifolius
response to major environmental cues. LanFTa1 gene expression
under SD did not reveal any significant trend during plant
growth and differences between genotypes were also usually
not significant (Figure 3A). The relative level of expression
was rather low (mean 0.38). A different pattern was observed
under LD, when the expression was highly induced before
flowering compared to the first term, namely 24-fold (P= 0.045)
in non-vernalized 83A:476 (7-fold in vernalized, NS), 405-fold
(P= 0.0051) in non-vernalized Palestyna (340-fold in vernalized,
P= 0.0015), and 765-fold in vernalized P27255 (Figure 3B). In
non-vernalized P27255 LanFTa1 expression remained at a low
level but this genotype did not flower in such conditions.
The vernalization effect on LanFTa1 expression under LD in
83A:476 and Palestyna was slightly positive, up to 2.5-fold (NS)
and 2.8-fold (P= 0.0011) increase, respectively (Figures 3C,D).
In P27255, the vernalization influence was initially moderately
negative (0.14-fold and 0.35-fold decrease, P= 0.0002) but
became highly positive just before flowering (43.6-fold increase).
The circadian regulation was not stable across terms and
genotypes, however, when LanFTa1 was induced before flowering
in Palestyna under LD, its expression was significantly higher
in the evening than in the morning (up to 25.1-fold increase,
P= 0.0004).
LanFTa2 and LanFTc2 Expression Was
Low in Both Photoperiods in All Lines
LanFTa2 gene expression was very low in both photoperiods,
amounting to mean values of 0.095 under SD and 0.004 under LD
(Supplementary Figures 1A,B). There was remarkable induction
of expression in P27255 after vernalization under SD (up to
38-fold, P= 0.0002), however, the relative expression values
achieved were much lower than those observed for LanFTc1 and
LanFTa1 (Supplementary Figures 1C,D). This pattern was not
recreated under LD.
LanFTc2 expression was on a low level in all lines under
both photoperiods (Supplementary Figures 2A,B). A moderate
induction by vernalization in P27255 under SD (up to 7.3-fold
increase, P= 0.0187) and in Palestyna under LD (up to 4.7-
fold increase, P= 0.0023) was observed but obtained levels were
less than half of the mean expression obtained for control genes
(Supplementary Figures 2C,D).
LanAGL8 Expression Profile Reflected
Observed Differences in Plant Phenology
and Was Similar to LanFTc1
Besides homologs constituting the L. angustifolius FT clade
(Ksia¸˙
zkiewicz et al., 2016;Nelson et al., 2017), four novel
candidate genes (LanAGL8, LanFD, LanUGT85A2, and
LanCRLK1), recently considered to be putatively involved in
Ku-based response (Plewi´
nski et al., 2019), were profiled in
this study. LanAGL8 is an L. angustifolius homolog of the
A. thaliana FRUITFULL gene participating in flowering time
control, meristem identity, and fruit development (Mandel
and Yanofsky, 1995;Gu et al., 1998). LanAGL8 expression was
consecutively increasing during plant growth for all studied
combinations of lines, photoperiod, and vernalization variants
except non-vernalized P27255 under SD. However, there were
considerable differences in the observed expression levels
between genotypes. Under SD without vernalization, expression
of the LanAGL8 gene in 83A:476 (Ku) was approximately 7-17
times higher (P= 0.0028) than in Palestyna (Pal) and 1420-6090
times higher (P= 0.0035) than in P27255 (ku) (Figure 4A). The
influence of vernalization resulted in a reduction of differences
in LanAGL8 expression between 83A:476 and Palestyna by
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FIGURE 2 | Gene expression profile of the LanFTc1 gene in response to photoperiod and vernalization in three lines (83A:476, Palestyna, and P27255) carrying
different LanFTc1 alleles (Ku,Pal, and ku). (A) expression under an 8-h photoperiod, (B) expression under 16-h photoperiod, (C) vernalization response under 8-h
photoperiod, (D) vernalization response under 16-h photoperiod. T1-T4 stands for sampling terms (Supplementary Table 5), V for vernalized plants, and N for
non-vernalized plants. Timespan of photoperiods: 8-h from 4 AM to 8 PM, 16-h from 4 AM to 8 PM. Two references were used for normalization (LanDExH7 and
LanTUB6) and one sample (LanTUB6) for inter-run calibration. Error bars indicate a standard deviation of 3 biological replicates, each representing a mean of 3
technical replicates. A logarithmic scale was used to accommodate observed large differences in gene expression values.
11–23%, and between 83A:476 and P27255 by 30–92%. Under
LD without vernalization, LanAGL8 expression in 83A:476
was higher up to 12-fold (P= 0.0086) than in Palestyna and
up to 1805-fold (P= 0.0001) than in P27255 (Figure 4B).
The application of vernalization reduced these differences
considerably. To summarize, Palestyna revealed an intermediate
LanAGL8 expression profile, however, it was much more like
83A:476 than P27255.
The LanAGL8 gene revealed high positive responsiveness
to vernalization in P27255 in both photoperiods, and this
effect was consecutively increasing during plant growth until
flowering. Namely, the change of LanAGL8 expression in
P27255 after vernalization was raised from 2.1-fold (NS)
to 29.8-fold (P= 0.0175) under SD (Figure 4C), and
from 4.3-fold (P= 0.0121) to 398.2-fold (P= 0.0004)
under LD (Figure 4D). Vernalization was also inductive in
Palestyna, providing an up to 2.6-fold increase (P= 0.0329) of
LanAGL8 expression under SD (effect stable across sampling
terms) and up to 11.8-fold increase (P= 0.0297) under
LD (effect consecutively decreasing). In the 83A:476 line,
the influence of vernalization was the lowest, yielding up
to a 2.9-fold increase of LanAGL8 expression under SD
(P= 0.0066) and up to a 2.0-fold increase under LD
(P= 0.0315).
The LanAGL8 gene revealed diversified circadian clock
regulation between genotypes and environments. Under SD, its
expression was higher in the morning than in the evening in
Palestyna and P27255, whereas in 83A:476 this relation was
the opposite. Moreover, vernalization partially diminished these
differences. Under LD, LanAGL8 expression was usually higher
in the evening terms for all lines, however, during the growth of
vernalized P27255 this trend reversed.
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FIGURE 3 | Gene expression profile of the LanFTa1 gene in response to photoperiod and vernalization in three lines (83A:476, Palestyna, and P27255) carrying
different LanFTc1 alleles (Ku,Pal, and ku). (A) expression under 8-h photoperiod, (B) expression under 16-h photoperiod, (C) vernalization response under 8-h
photoperiod, (D) vernalization response under 16-h photoperiod. T1-T4 stands for sampling terms (Supplementary Table 5), V for vernalized plants, and N for
non-vernalized plants. Timespan of photoperiods: 8-h from 4 AM to 8 PM, 16-h from 4 AM to 8 PM. Two references were used for normalization (LanDExH7 and
LanTUB6) and one sample (LanTUB6) for inter-run calibration. Error bars indicate a standard deviation of 3 biological replicates, each representing a mean of 3
technical replicates.
LanCRLK1 Expression Was Higher Under
SD in All Lines and Negatively
Responsive to Vernalization in
Domesticated Ku Line
LanCRLK1 is an L. angustifolius homolog of the A. thaliana
CRLK1 gene which participates in response to low temperature
in a calcium-dependent manner (Yang et al., 2010). Changes
in LanCRLK1 expression level were usually not related to
the progress of plant growth, except P27255 cultivated under
SD, which revealed a stable decrease of LanCRLK1 both
in non-vernalized and vernalized variants. The expression
of LanCRLK1 was higher under SD than under LD.
Moreover, under SD without vernalization, LanCRLK1
expression was the highest in Palestyna (up to 2.7-fold
increase compared to 83A:476, P= 0.0002) and the lowest
in P27255 (about 0.4-fold decrease, P= 0.0099). However,
after vernalization, expression was highest in P27255 (up to a
3.5-fold increase compared to 83A:476, P= 0.0005) and lowest
in 83A:476 (Figure 5A). The differences in the LanCRLK1
expression between genotypes under LD were generally not
significant (Figure 5B).
The effect of vernalization on LanCRLK1 expression under
SD was negative (from 0.4-fold to 0.7-fold decrease, P= 0.0009)
or neutral in 83A:476, negative in Palestyna (from 0.6-fold
to 0.8-fold decrease, P= 0.0000), and positive in P27255 (up
to 3.0 fold increase, P= 0.0000) (Figure 5C). Under LD,
the vernalization effect was also negative in 83A:476 (0.6-fold
decrease, NS), however, in the remaining two lines, the effect
changed during plant growth from negative to positive: from a
0.7-fold decrease to 1.9-fold increase in Palestyna (all NS) and
from 0.6-fold decrease (P= 0.0001) to 1.4-fold increase in P27255
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FIGURE 4 | Gene expression profile of the LanAGL8 gene in response to photoperiod and vernalization in three lines (83A:476, Palestyna, and P27255) carrying
different LanFTc1 alleles (Ku,Pal, and ku). (A) expression under 8-h photoperiod, (B) expression under 16-h photoperiod, (C) vernalization response under 8-h
photoperiod, (D) vernalization response under 16-h photoperiod. T1-T4 stands for sampling terms (Supplementary Table 5), V for vernalized plants, and N for
non-vernalized plants. Timespan of photoperiods: 8-h from 4 AM to 8 PM, 16-h from 4 AM to 8 PM. Two references were used for normalization (LanDExH7 and
LanTUB6) and one sample (LanTUB6) for inter-run calibration. Error bars indicate a standard deviation of 3 biological replicates, each representing a mean of 3
technical replicates. A logarithmic scale was used to accommodate observed large differences in gene expression values.
(P= 0.0107) (Figure 5D). The influence of the circadian clock
on LanCRLK1 expression was variable in both photoperiods and
usually not significant.
LanFD Expression Was Negatively
Responsive to Vernalization in Ku and
Pal Lines but Positively Responsive or
Variable in ku
LanFD is an L. angustifolius homolog of the A. thaliana
FD gene which triggers flowering based on FT-mediated
signaling (Abe et al., 2005). LanFD expression in non-vernalized
plants was usually the highest at the first sampling date and
decreased during plant growth in both photoperiods, except
in P27255 cultivated under LD. However, in vernalized plants,
this decreasing trend was diminished or even reversed, as
observed for P27255. Under SD, LanFD expression was the
highest in Palestyna (1.1–5.6 times higher than in 83A:476,
P= 0.0024) and the lowest in P27255 (0.3–0.9 times lower
than in 83A:476, P= 0.0123) (Figure 6A). Interestingly,
these relations were changed after vernalization as follows:
LanFD expression in P27255 compared to 83A:476 was 3.2–
12.2 times higher (P= 0.0011), whereas in Palestyna 2.1–
7.4 times higher than in 83A:476 (P= 0.0001). Under LD
without vernalization, differences in LanFD expression between
genotypes were rather low, accounting for up to a 2.3-fold
increase in Palestyna (P= 0.0205) and up to a 5.0-fold
increase in P27255 (P= 0.0000) compared to 83A:476. However,
vernalization exaggerated these contrasts and observed LanFD
expression in P27255 was up to 25.7 times higher than in 83A:476
(P= 0.0078) and up to 5.5 times higher than in Palestyna
(P= 0.0032) (Figure 6B).
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FIGURE 5 | Gene expression profile of the LanCRLK1 gene in response to photoperiod and vernalization in three lines (83A:476, Palestyna, and P27255) carrying
different LanFTc1 alleles (Ku,Pal, and ku). (A) expression under 8-h photoperiod, (B) expression under 16-h photoperiod, (C) vernalization response under 8-h
photoperiod, (D) vernalization response under 16-h photoperiod. T1-T4 stands for sampling terms (Supplementary Table 5), V for vernalized plants, and N for
non-vernalized plants. Timespan of photoperiods: 8-h from 4 AM to 8 PM, 16-h from 4 AM to 8 PM. Two references were used for normalization (LanDExH7 and
LanTUB6) and one sample (LanTUB6) for inter-run calibration. Error bars indicate a standard deviation of 3 biological replicates, each representing a mean of 3
technical replicates.
The effect of vernalization on LanFD expression was negative
in 83A:476 and Palestyna under both photoperiods (0.17-fold
to 0.85-fold decrease), positive in P27255 under SD (up to 6.2-
fold increase), and unstable in P27255 under LD (from 0.5-fold
decrease to 1.4-fold increase) (Figures 6C,D). Genotypes differed
in the circadian clock control of LanFD expression. Generally, in
83A:476 the levels were higher in the morning (up to 4.4 fold-
increase), whereas in Palestine and P27255 in the evening (up to
4.5-fold and 3.1-fold increase, respectively).
LanUGT85A2 Expression Revealed a
Growth-Dependent Decreasing Trend,
Strong Circadian Clock Control, and
Negative Response to Vernalization in All
Lines Under Both Photoperiods
LanUGT85A2 is considered as an L. angustifolius homolog of the
A. thaliana UDP-glycosyltransferase 85A2 gene. The expression of
this gene was the highest at the first sampling date in all lines
and consecutively decreased during the experiment. Palestyna
was revealed to have the biggest decrease of LanUGT85A2 during
the transition from juvenile to generative phase, whereas P27255
had the lowest. Indeed, this decrease of LanUGT85A2 expression
during plant growth under SD reached approximately 0.1-fold for
83A:476 (P= 0.0114), 0.3-fold for Palestyna (P= 0.0033) and 0.4-
fold for P27255 (P= 0.0003), whereas under LD about 0.3-fold
for 83A:476 (P= 0.0501), 0.02-fold for Palestyna (P= 0.0182) and
0.5-fold for P27255 (P= 0.0196).
Under SD, the highest first-term LanUGT85A2 expression
was observed in 83A:476, about 1.7-fold higher than in
Palestyna (P= 0.0448) and P27255 (NS) (Figure 7A). These
differences were doubled by vernalization. Contrary, under
LD, the highest first-term expression was in P27255, 1.1-fold
higher than in Palestyna (NS) and 3.7-fold higher than in
83A:476 (P= 0.0006), and these differences were tripled by
vernalization (Figure 7B).
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FIGURE 6 | Gene expression profile of the LanFD gene in response to photoperiod and vernalization in three lines (83A:476, Palestyna, and P27255) carrying
different LanFTc1 alleles (Ku,Pal, and ku). (A) expression under 8-h photoperiod, (B) expression under 16-h photoperiod, (C) vernalization response under 8-h
photoperiod, (D) vernalization response under 16-h photoperiod. T1-T4 stands for sampling terms (Supplementary Table 5), V for vernalized plants, and N for
non-vernalized plants. Timespan of photoperiods: 8-h from 4 AM to 8 PM, 16-h from 4 AM to 8 PM. Two references were used for normalization (LanDExH7 and
LanTUB6) and one sample (LanTUB6) for inter-run calibration. Error bars indicate a standard deviation of 3 biological replicates, each representing a mean of 3
technical replicates. A logarithmic scale was used to accommodate observed large differences in gene expression values.
Vernalization was observed to have a stable negative effect on
LanUGT85A2 gene expression, highlighted by an average 0.29-
fold decrease under SD (Figure 7C) and a 0.28-fold decrease
under LD (Figure 7D). Response to vernalization was the weakest
in P27255 (0.38-fold) and the strongest in Palestyna (0.21-
fold). Such a disproportion in vernalization response, combined
with the observed difference in decreasing trend during plant
growth, resulted in the highest LanUGT85A2 expression in
the P27255 line at the end of the experiment under both
photoperiods (up to 31-fold increase compared to 83A:476 under
LD, P= 0.0233).
The direction of circadian clock control was coherent across
all genotypes and environments. LanUGT85A2 expression was
higher in the evening than in the morning for all terms, both
with and without vernalization. Under SD, it increased during
the day up to 13.9-fold in 83A:476 (P= 0.0165), 8.3-fold in
Palestyna (P= 0.0075) and 28.2-fold in P27255 (P= 0.0000).
Under LD, it increased up to 7.4-fold in 83A:476 (P= 0.0326),
12.8-fold in Palestyna (P= 0.0005) and 4.4-fold in P27255
(P= 0.002).
LanVIN3-1,LanVIN3-2, and LanVIN3-3
Genes Revealed High Expression in Ku
Without Vernalization
The L. angustifolius genome contains three homologs (named
here as LanVIN3-1,LanVIN3-2, and LanVIN3-3) a VIN3 gene
that is involved in the vernalization response in A. thaliana (Sung
and Amasino, 2004). Therefore, the transcriptional activity of
these genes was also profiled. In general, LanVIN3-1 revealed
the highest expression, whereas LanVIN3-3 the lowest (see
Supplementary Figures 3–5).
All three copies revealed differences in expression levels
between genotypes, usually in descending order, 83A:476
Palestyna P27255. Maximum differences between 83A:476
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FIGURE 7 | Gene expression profile of the LanUGT85A2 gene in response to photoperiod and vernalization in three lines (83A:476, Palestyna, and P27255) carrying
different LanFTc1 alleles (Ku,Pal, and ku). (A) expression under 8-hphotoperiod, (B) expression under 16-h photoperiod, (C) vernalization response under 8-h
photoperiod, (D) vernalization response under 16-h photoperiod. T1-T4 stands for sampling terms (Supplementary Table 5), V for vernalized plants, and N for
non-vernalized plants. Timespan of photoperiods: 8-h from 4 AM to 8 PM, 16-h from 4 AM to 8 PM. Two references were used for normalization (LanDExH7 and
LanTUB6) and one sample (LanTUB6) for inter-run calibration. Error bars indicate a standard deviation of 3 biological replicates, each representing a mean of 3
technical replicates.
and P27255 expression under LD reached 5.9-fold (LanVIN3-
1,P= 0.0016), 25.6-fold (LanVIN3-2,P= 0.0061) and 8.4-fold
(LanVIN3-3,P= 0.0059), whereas under SD these values were as
follows: 4.8-fold (LanVIN3-1,P= 0.0135), 10.8-fold (LanVIN3-2,
P= 0.0036) and 5.5-fold (LanVIN3-3,P= 0.0072). The expression
levels of all three genes under LD were usually significantly
higher in the evening than in the morning in all lines, whereas
under SD, diurnal variations were frequently not significant or
variable. Photoperiod conditions had a significant influence on
expression changes during plant growth, especially for 83A:476.
The expression levels of these genes in non-vernalized 83A:476
under LD increased by 2.3-fold (LanVIN3-1,P= 0.0008), 3.3-fold
(LanVIN3-2,P= 0.0048) and 2.5-fold (LanVIN3-3,P= 0.0036)
whereas under SD they decreased by up to 0.32-fold (LanVIN3-
1,P= 0.0000), 0.37-fold (LanVIN3-2,P= 0.0110), and 0.24-
fold (LanVIN3-3,P= 0.0009). The vernalization effect differed
between genotypes and photoperiods. Under LD it was significant
for 83A:476 (repression of all three genes, up to 0.43-fold) and
Palestyna (induction of LanVIN3-2 and LanVIN3-3, up to 2.0-
fold), whereas under SD it was significant for 83A:476 (changing
from repression, up to 0.18-fold, to induction, up to 2.3-fold)
and P27255 (induction, up to 3.6-fold). To summarize, all
LanVIN3 homologs revealed different transcriptomic responses
to vernalization than the LanFTc1 and LanAGL8 genes.
Besides VIN3, VRN5 protein is also considered to participate
in the vernalization-induced epigenetic silencing in A. thaliana
(Greb et al., 2007). Therefore, the expression of the latter gene was
also profiled to supplement the analysis. All three L. angustifolius
copies, LanVRN5-1,LanVRN5-2, and LanVRN5-3, were analyzed
using one universal primer pair (Supplementary Figure 6).
LanVRN5 clade revealed an expression profile relatively similar
to LanVIN3, especially to LanVIN3-2. Like LanVIN3 genes,
LanVRN5 in 83A:476 revealed an increase of expression during
plant growth under LD and decrease under SD. All lines showed
a significant decrease of expression during the light phase under
SD and usually not a significant diurnal trend under LD. In
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both photoperiods without pre-sowing vernalization, 83A:476
revealed a significantly higher expression of LanVRN5 than
Palestyna and P27255. However, the vernalization responsiveness
of LanVRN5 considerably differed between photoperiods and
genotypes. Under LD, only P27255 was responsive (showing
repression), whereas under SD all lines responded but in
different ways and with changing response during plant growth:
namely from repression to induction in 83A:476, from induction
to repression in Palestyna, and by continuously increasing
induction in P27255. In vernalized plants under SD, LanVRN5
expression was the highest in P27255.
DISCUSSION
INDEL Polymorphism in the LanFTc1
Promoter as a Legume Model for
Functional Studies Aiming Vernalization
Responsiveness
FT is a major flowering time integrator gene, gathering signals
from several pathways that detect environmental conditions
(Turck et al., 2008). First studies on the conservation of
A. thaliana genes in legumes have revealed the presence of
the well-preserved homologs of many flowering time regulatory
genes, including some representatives of FT clade found in
Pisum sativum L., Medicago truncatula L., and Glicyne max
(L.), Merrill (Hecht et al., 2005). When the first gene-based
linkage map of L. angustifolius was published, it highlighted
a conserved collinear block shared between the fragment of
M. truncatula chromosome 7 and the so-called linkage group
LG01 (currently NLL-10), carrying major early flowering locus
Ku (Nelson et al., 2006). It was later revealed that the
syntenic region in M. truncatula contained several FT homologs
(Young et al., 2011). The development of a bacterial artificial
chromosome library for the L. angustifolius nuclear genome
opened a possibility of gene cloning by DNA hybridization
and the sequencing of selected clones (Kasprzak et al., 2006).
Such an approach, combined with novel high-throughput
sequencing techniques and gene expression profiling, resulted in
the identification of a candidate gene for Ku and enabled the
formulation of the hypothesis that a 1423 bp deletion in the
promoter region of this gene is a causal mutation conferring
early flowering phenotype (Nelson et al., 2017). Then, two
other overlapping deletion variants, covering 1208 bp (Pal)
and 5162 bp (Jul) were identified (Taylor et al., 2019). In
the A. thaliana, a variation in promoter length was found to
modulate the photoperiodic response of FT, and some important
regulatory blocks contributing to this response were identified
(Adrian et al., 2010;Liu et al., 2014). However, comparative
mapping revealed a low sequence similarity between LanFTc1
and A. thaliana FT promoters, except RE-alpha and CCAAT
boxes which were found at expected positions and indicated that
FT promoter length in L. angustifolius may be at least as big as in
A. thaliana (up to 7 kb) (Ksia¸˙
zkiewicz et al., 2016). All recognized
LanFTc1 deletions conferring Ku,Pal, and Jul alleles are located
downstream of the pair of CCAAT boxes marking a putative
beginning of the functional promoter (Ksia¸˙
zkiewicz et al., 2016;
Nelson et al., 2017;Taylor et al., 2019). Thus, L. angustifolius
Ku,Pal, and Jul alleles have both distal and proximal regions
preserved (Taylor et al., 2019). However, deletion sequences
encompassed candidate binding sites for many transcription
factors, including those already evidenced to be involved in
FT regulation in model plants (Nelson et al., 2017;Taylor
et al., 2019). In A. thaliana, similarly to L. angustifolius, the
functional FT promoter indels also retained distal and proximal
regions (Adrian et al., 2010;Liu et al., 2014). On the contrary,
the study involving the FT promoter from cotton provided
evidence that the proximal region might play an important role
in this species (Sang et al., 2019). To our knowledge, a model
revealed for L. angustifolius is the only known legume example
of FT promoter indel variation. However, in many legumes,
FT genes were found to be associated with flowering traits.
In the economically most important legume crop worldwide, a
soybean, Glycine max, mutations in FT genes are responsible
for at least three loci conferring early/late flowering, namely E9
(GmFT2a), E10 (GmFT4), and qDTF-J1 (GmFT5a) (Takeshima
et al., 2016;Zhao et al., 2016;Samanfar et al., 2017). Moreover,
natural variations of the GmFT2b sequence are associated with
soybean adaption to highlatitude regions (Chen et al., 2020). In
M. truncatula, vernalization responsiveness and early flowering
are conferred by the FTa1 gene, whereas photoperiod response
by the FTb gene (Laurie et al., 2011;Putterill et al., 2013). In
pea (Pisum sativum), the FTa1 gene corresponds to the pea
GIGAS locus, which is essential for flowering under LD and
promoting flowering under SD (Hecht et al., 2011). In chickpea, a
major quantitative trait locus (QTL) for the flowering time under
SD conditions was mapped in the region containing a cluster
of three FT genes (FTa1-FTa2-FTc), which collectively showed
upregulated expression in domesticated germplasm (Ortega et al.,
2019). In the sister lupin crop species, white lupin (L. albus
L.), one of the four major QTLs conferring early flowering and
partial vernalization independence was found associated with
the FTa1 gene (Rychel et al., 2019). L. angustifolius genome
contains two FTa and two FTc genes, which putatively arose
from single copies by lineage-specific duplication, whereas the
whole FTb subclade is absent (Ksia¸˙
zkiewicz et al., 2016). Indeed,
L. angustifolius was recently used as a reference species in
several phylogenetic studies addressing the influence of whole-
genome and local duplications on the evolutionary fate of selected
legume-specific and plant-wide gene clades (Przysiecka et al.,
2015;Naro˙
zna et al., 2017;Szczepaniak et al., 2018;Czy˙
z et al.,
2020). The differences in the expression profiles for FTa and
FTc genes, as established in the present study, provided novel
evidence supporting the hypothesis on a functional divergence of
particular duplicates.
Pal Allele Carrying Intermediate
Phenology and Light Vernalization
Responsiveness Provides Worldwide
Opportunities for L. angustifolius
Breeding
In the present study, based on experiments performed in
a greenhouse under natural LD (12–17 h photoperiod), Ku
and Jul alleles were found to be associated with early
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Rychel-Bielska et al. Photoperiod, Vernalization and Gene Expression
flowering and vernalization independence, Pal with slightly
delayed flowering and partial vernalization responsiveness, and
ku with late flowering and high vernalization dependence.
The same phenomenon was reported for the recent study
performed in phytotron under natural 10–12 h photoperiod
(intermediate between SD and LD) in Australia (Taylor
et al., 2019). However, we observed relatively high differences
in flowering time for early lines (Ku,Pal, and Jul) in
a greenhouse between 2014 and 2015 repeats, accounting
for about two weeks on average (earlier in 2014). These
differences can be attributed to the variation in temperature
which occurred during these experiments. During the first
70 days of the 2014 experiment, we observed 31 days
with maximum temperature 25C and 9 days with 30C,
whereas in ‘2015 these numbers were much lower, 3 and
0 days, respectively. The higher temperature could advance
flowering because it is shown to strongly accelerate flowering
in model plants as well as in many other plant species
(Parmesan and Yohe, 2003;Thines et al., 2014). Moreover,
the average photoperiod in 2014 was about 2 h longer than
in 2015 due to differences in sowing terms. Indeed, our
subsequent controlled-environment study revealed that even
early L. angustifolius germplasm is responsive to LD conditions
and accelerated transition between particular developmental
phases by about 18–25 days compared to SD. The observed
phenology of the Pal allele can be very beneficial for
L. angustifolius cultivation in the era of changing climate,
especially in Europe and Australia where the majority of
worldwide lupin production occurs. Thus, the European land
climate experienced rapid warming in recent decades, resulting
in the mean year temperature surge to approximately 2C
above the 1910–1960 average (NOAA, 2020). Climate warming
raised multiple challenging issues for grain legume breeders,
including higher water deficits and severe drought periods,
propagation of pests and diseases as well as de-regulation
of temperature-based control of growth and development
processes (Vadez et al., 2012;Scheelbeek et al., 2018;Lippmann
et al., 2019). Affected regulatory pathways include, among
others, the flowering time control (Nelson et al., 2010).
The rapid flowering of domesticated germplasm may favor
drought escape and adaptation for spring sowing in higher
latitudes (Annicchiarico et al., 2010, 2018;Berger et al., 2017).
However, the observed extension of the vegetation period
has raised the demand for germplasm with intermediate
phenology and cross-environment adaptation. Such research
was recently initiated in L. albus in three European locations
contrasting sowing time (autumn or spring) and climate
type (Annicchiarico et al., 2019). Climatic variables were
also addressed in an L. angustifolius genome-wide association
study, providing some candidate polymorphisms that await
further exploitation (Mousavi-Derazmahalleh et al., 2018a).
L. angustifolius Pal allele confers flowering time and vernalization
responsiveness phenotype intermediating between domesticated
and wild lines. As this phenotype is consistent within the
large range of photoperiod conditions (8, 10–12, and 16–17 h),
it may be found applicable for all regions where lupins are
currently cultivated.
LanFTc1,LanAGL8,LanCRLK1, and
LanUGT85A2 Are Candidate Genes
Involved in the Vernalization
Responsiveness of L. angustifolius
Previous studies have highlighted the negative association of
LanFTc1 and LanAGL8 gene expression with the number
of days to flowering in L. angustifolius, mapping population
and the positive direction of such association for LanFD,
LanCRLK1, and LanUGT85A2 genes (Nelson et al., 2017;
Plewi´
nski et al., 2019). The present study revealed that these
genes differ in vernalization responsiveness between genotypes
and photoperiods. LanFTc1 and LanAGL8 genes were found to
be highly induced by vernalization in wild germplasm, whereas
LanUGT85A2 was found to be significantly suppressed (Table 4).
LanAGL8 protein sequence revealed the highest similarity to
A. thaliana FRUITFULL (FUL,AGAMOUS-LIKE 8, AT5G60910)
and APETALA1 (AP1,AGAMOUS-LIKE 7, AT1G69120) genes.
Both AP1 and FUL play a role in floral meristem identity
but have different functions. AP1 controls the formation of
sepals and petals whereas FUL is involved in cauline leaf
and fruit development (Irish and Sussex, 1990;Gu et al.,
1998). These genes revealed tissue-specific expression during
generative organ development (Irish and Sussex, 1990;Mandel
and Yanofsky, 1995;Klepikova et al., 2016). In the present
study, high levels of LanAGL8 expression were revealed in
leaf tissue. Moreover, the expression profiles and vernalization
responsiveness of LanAGL8 and LanFTc1 were very similar.
Both genes revealed comparable circadian clock control, i.e.,
morning induction in Pal line under short days. Confronting
these observations with the information on a 100% association
between the LanFTc1 genotype and flowering time phenotype in
a large germplasm collection (Nelson et al., 2017;Taylor et al.,
2019), the conclusion can be raised that LanAGL8 acts putatively
downstream of LanFTc1 in L. angustifolius, in which LanAGL8
may perform a similar function, like its homolog in cereals,
an AP1-like gene called VRN1, which regulates the transition
from vegetative to generative phase in response to vernalization
and is expressed in many organs, including leaves (Trevaskis
et al., 2003;Yan et al., 2003). Indeed, the wheat homolog of FT,
(VRN3), activates expression of VRN1 in leaves and shoot apical
meristem, promoting flowering under inductive long days (Li
et al., 2015). As a MADS box transcription factor, VRN1 binds
to many targets in the genome and may regulate many genes,
linking vernalization and photoperiod pathways (Deng et al.,
2015). Moreover, the allelic diversity of VRN1 copies provides
wide plasticity of temperature-based responses in winter wheat
(Dixon et al., 2019).
In this study, we also revealed differences in the vernalization
responsiveness of a LanFD gene between early and late flowering
germplasm. In wheat, FDlike, VRN3, and 1433 proteins
form together a florigen activation complex which can bind the
VRN1 promoter, therefore a variation in FD expression may
modulate the effect of mobile florigen signal (Li et al., 2015).
Results obtained in this study, are supported by a significant
correlation between the LanFD gene expression profile and
vernalization responsiveness in the L. angustifolius mapping
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population (Plewi´
nski et al., 2019), which indicates that LanFD
may contribute to FT regulatory function, especially in the wild,
vernalization-responsive germplasm.
Our recent expression quantitative trait loci (eQTL) mapping
study provided transcriptomic evidence for the contribution
of several genes acting in C-repeat binding factor (CBF) cold
responsiveness (LanCRLK1), and in UDP-glycosyltransferases
(LanUGT85A2) pathways in the vernalization response via
LanFTc1 in L. angustifolius (Plewi´
nski et al., 2019). LanCRLK1
is a homolog of A. thaliana CALCIUM/CALMODULIN-
REGULATED RECEPTOR-LIKE KINASE 1, which is the first
component in the cold responsiveness pathway (Yang et al.,
2010). Downstream genes in this pathway, the C-repeat binding
factors (CBF) and INDUCER OF CBF EXPRESSION 1 (ICE1),
provide regulatory links to FLOWERING LOCUS C (FLC)
(Kim et al., 2004;Lee et al., 2015). The present study has
highlighted the positive vernalization responsiveness of the
LanCRLK1 gene but only in wild germplasm under SD. In
other genotype x environment combinations, the response
was quasi thermoneutral. This finding is coherent with the
general observation that CBF cold responsiveness pathway is
downregulated and less effective under LD conditions than
under SD (Lee and Thomashow, 2012). The expression profile
of LanCRLK1 did not provide convincing evidence on the
contribution of this gene in the vernalization responsiveness
of L. angustifolius. Nevertheless, the reduction of LanCRLK1
expression in the evening, combined with the decreasing
trend during development that was revealed in this study for
vernalized ku line under LD, may explain the direction of the
association between the LanCRLK1 expression pattern and the
vernalization responsiveness observed in the L. angustifolius
mapping population e-QTL study, which was also performed
under LD with partial vernalization (Plewi´
nski et al., 2019). The
question arises as to whether these differences in the LanCRLK1
gene expression profile between the Ku/Pal and the ku lines,
may have consequences in terms of cold acclimation and freezing
tolerance of early flowering lines. A negative correlation between
early phenology and cold acclimation could be a very undesirable
trait hampering the autumn sowing of L. angustifolius in many
regions of Southern Europe.
This study evidenced the negative response of LanUGT85A2
to vernalization in all genotypes under both photoperiods. The
genotypes explored in this study revealed different responses
to photoperiod, and under SD, the LanUGT85A2 expression
was highest in the early flowering line, whereas under LD,
it was in late flowering. Indeed, during the L. angustifolius
mapping population e-QTL assay, which was performed under
LD with mild vernalization, LanUGT85A2 expression revealed a
significant positive correlation with the late-flowering phenotype
(Plewi´
nski et al., 2019). LanUGT85A2 is a representative of
the UDP-glycosyltransferases protein family. In A. thaliana,
a relatively close homolog of this gene, UGT87A2, promotes
flowering in the vernalization and gibberellin pathways by
repression of FLC (Wang B. et al., 2012). Similarly, ectopic
over-expression in tobacco of a putative glycosyltransferase
gene 1, PtGT1, derived from poplar (Populus tomentosa Carr.),
resulted in an early flowering phenotype (Wang Y.-W. et al.,
2012). Contrary, another A. thaliana homolog, UGT84A2, delays
flowering by activation of the indole-3-butyric acid (IBA)
pathway, leading to down-regulation of AUXIN RESPONSE
FACTOR 6 (ARF6) and ARF8 genes, and, consequently FT
(Zhang et al., 2017). Taking into consideration the direction of
the association between LanUGT85A2 expression and time to
flowering, the latter mechanism seems to be more probable in
L. angustifolius than those of UGT87A2 and PtGT1.
This research highlighted the hypothetical involvement of
FLC-related genes in L. angustifolius vernalization-dependent
flowering time regulation. However, legume genomes, except for
soybean, generally do not h