Scientia Horticulturae 286 (2021) 110223

Contents lists available at ScienceDirect

Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

The role of organ - and daylength - specific gene expression in bulb

development and resource management in onion (ALLIUM CEPA L.)
Wei Cheng a, c, Harun Ar Rashid b, Richard Stark a, Brian Thomas a, *
a
School of Life Sciences, University of Warwick, CV4 7AL, UK
b
Department of Horticulture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
c
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Saffron Walden CB10 1RQ, Hinxton, UK

A R T I C L E I N F O A B S T R A C T

Keywords: Onion development is a complex process with each developmental event depending on the adequate supply of
Onion resources. Also, these resource components themselves are crucial nutrients in onion when consumed as food. For
Bulbing higher yield and better quality in onion production, it is essential to not only have a high level of photosynthesis
Daylength
but also effective transportation of photosynthetic products. A comprehensive set of spatial and developmental
FT genes
Sugar sensing
time-course experiments detailed the gene expression in onion leaf and bulb tissues during the development of a
Resource allocation long-day onion cultivar under long-day and short-day conditions. Candidates included well characterised
functional clock genes in Arabidopsis flowering pathway, as well as some novel sequences differentially
expressed in bulb tissues. Both AcFT1 and AcFT4 showed significant increasing expression level during plant
development, with AcFT1 being expressed in long-days during bulb formation and AcFT4 in short-days where
bulbs are not formed. The data further support the role of AcFT1 as a promoter and AcFT4 as inhibitor in onion
bulb formation. A number of genes associated with onion carbohydrate metabolism, sulphur metabolism and
bulb development were identified as having different tissue-specific expression and provide targets for future
studies.

1. Introduction
The common onion varieties are now cultivated over a worldwide
geographic range from temperate to tropical regions. In terms of global
weight produced, onion presents as second in importance among vege­
tables, only exceeded by tomatoes (FAO statistics, 2018). Besides its
unique flavour, onion has a unique combination of diverse and highly
valuable families of phytochemicals: organosulfur, fructans and flavo­
noids. These families of compounds, working independently or in
combination, contribute to several salutary effects on human health as
well as, in some cases, helping to provide protection against pests and
disease.
Examples of organosulphur compounds include defensins (DFS),
which are small peptides that are rich in disulfide-linked cysteines and
are widely distributed throughout animals and plants . Plant defensins
were firstly isolated from wheat and barley by Mendez et al., 1990,
before being studied mainly with regard to their effects in enhancing the
ability of plants to counteract pathogens and insects. A second beneficial
organosulphur compound is lachrymatory factor (propanthial S-oxide).
The enzyme lachrymatory factor synthase (LFS) causes the characteristic
pungency of onion when chopped up, giving the savour and the ability
to defend against wild animals and microbes.
Plant defense compounds may also be beneficial to humans when
they include flavonoids, and health-related polyphenolic natural prod­
ucts (Koes et al., 1994; Gurjar et al., 2012). Onion is the richest source of
dietary flavonoids contributing to a large extent to the overall intake.
There are two types of flavonoid subgroups found in onion: the antho­
cyanins in red and purple varieties, and the flavonols such as quercetin
in yellow and brown varieties.
Fructans are long chain polymers of fructose that contribute to
human health through multiple mechanisms, despite their resistance to
hydrolysis by human digestive enzymes. They are the principal type of
carbohydrate storage in onion, making up 80% of the dry matter
(Brewster, 1994). In a study of 60 common vegetables, onions are re­
ported to have the highest quantity of fructans (Roberfroid, 2004).
Studies show fructans can help maintain gastrointestinal health by
sustaining beneficial bacteria (Franco-Robles and López, 2015). Estab­
lished beneficial properties of fructans also include antifungal,

* Corresponding author.
E-mail address: Brian.Thomas@warwick.ac.uk (B. Thomas).

https://doi.org/10.1016/j.scienta.2021.110223
Received 11 November 2020; Received in revised form 18 March 2021; Accepted 19 April 2021
Available online 13 May 2021
0304-4238/© 2021 Elsevier B.V. All rights reserved.
W. Cheng et al.
antibacterial, antitumor, anti-inflammatory, antithrombotic and, hypo­
cholesterolaemia (Lanzotti, 2006; Kumari and Augusti, 2007). They may
also play a part in treating diabetes as research shows that fructans in
onions can lower insulin levels and improve tolerance for glucose (Dey
et al., 2002; Liu, 2007).
The target of onion production is a high yield of high-quality prod­
uct, which is the result of many processes of plant growth and devel­
opment (Brewster, 2008). Each leaf is composed of a photosynthetic
blade and a non-photosynthetic storage leaf base or sheath. The edible
bulbs are formed from modified plant leaves (scale leaves) which do not
form blades but swell and develop into the storage organ. The change
from photosynthetic leaf production to scale and bulb formation is co­
ordinated with source-to-sink carbohydrate reallocation throughout the
plant. In onion, the source for bulb is the fructans produced from sucrose
and fructose in the leaf blade before being accumulated in the basal
scales (sink) during bulb formation (Pak et al., 1995).
Our aim in this work was to gain an improved understanding of how
the different pathways involved in bulb development and composition
were co-ordinated during the transition to bulbing by loking at expres­
sion across a range of genes with different functions. Twelve candidate
genes, including genes involved in leaf to bulb signalling, source and
sink activity and phytochemical content were selected and their
expression patterns were evaluated under inductive and non-inductive
daylengths. AcCOL2 (Rashid and Thomas, 2020) and AcFTs (Lee et al.,
2013) were selected for their proposed involvement in the photoperiodic
onion bulbing process, based on the well-established parallels with
Arabidopsis thaliana flowering pathway.
Two invertase genes (INV, or FFS) were selected, for their putative
function in catalysing sucrose hydrolysis to produce hexoses (fructose
and glucose) available for cellular activity (Benkeblia et al., 2004).
Additionally, three distinct sucrose 1-fructosyltransferase (SST1) genes,
coding for the key enzyme that catalyses fructan synthesis from sucrose
were selected (see Fig. S.1). For organosulfur metabolism, two genes,
Lachrymatory factor syhthase (LFS) and Defensin (DFS) were included.
By exploring the characteristics of these genes, the aim of this work was
to gain a better understanding of inter-organ co-ordination of develop­
ment and rsource management during the onion bulbing process.
2. Materials and methods
2.1. Plant material
Seeds of onion (Allium cepa L.) variety “Marco” F1 were obtained
from Marshall & Co Ltd, (Cambridgeshire, UK). Marco has a critical
daylength requirement of 15–16 h (information provided by seed
company) and is thus a LD variety. Marco plants were grown from seed
planted Dec 15th 2017 in School of Life Sciences Phytobiology Facility
(PBF) at the University of Warwick. At 37 days from sowing (DFS), they
were potted up into 7 cm pots and grown on in the PBF with a controlled
temperature of 22/18 ◦ C day/night and supplementary lighting to give a
16 h daylength. At 100 DFS, when basal swelling was first observed,
plants were separated into two groups and transferred to the different
daylength treatments. For one group, LD was provided in a compartment
with natural daylight (varied from 12 h 42 min to 15 h 24 min) plus 16 h
of supplementary light extension (400 W SONT lamps) from 03:00 to
19:00 GMT. For the other group, SD was provided in a compartment
fitted with photoperiod blackout blinds to give an 8 h daylength from
8:00 to 16:00 GMT, during which they received supplementary light
Table 1
Sampling timepoints and sample names. LB, LL, SB, SL represent long-day bulb, long-day leaf, short-day bulb and short-day leaf, respectively.
Days from sowing 102 109
Samples for bulbing ratio 1 2
Molecular samples LB1 LL1 LB2 LL2
SB1 SL1 SB2 SL2
Scientia Horticulturae 286 (2021) 110223
from 400 W SONT lamps. Plants in both groups were kept in these
condition for 42 days for sampling until the experiment finished.
Sampling was conducted two days after the transfer and at weekly
intervals seven times from 102 to 144 DFS in total (Table 1). For each
sampling time, five plants were taken from both LD and SD compart­
ments 10 h after the start of the light period (Zeitgeber (ZT)10). Plants
for harvesting were selected with a random number generator (Haahr,
2006). Measurements of bulb and neck diameter were conducted using
slide callipers and visible leaf number was counted. Bulb initiation was
quantified using ‘bulbing ratio’ (maximum bulb diameter/minimum
sheath diameter), which is an indication of onion bulbing when the
value greater than two (Clark and Heath, 1962).
2.2. Molecular analysis
At harvest, the middle 1 cm section of the second youngest leaf and a
cross section of basal tissue were excised using a scalpel and immedi­
ately frozen in liquid nitrogen. Total RNA was extracted from approxi­
mately 100 mg of frozen leaf and base tissue from specific daylengths
conditions using the Z6 buffer method, following the manufacturer’s
(Roche manufacturing Ltd., Republic of Ireland) guidelines. After the
isolation, samples were DNase treated using TURBO DNA-free™
(Ambion Inc, Cat. No. AM1907) to eliminate the genomic DNA
contamination. The quality and quantity of total RNA were measured
with the Thermo Scientific NanoDropTM 1000 Spectrophotometer
(NanoDrop Technologies, Inc., USA). First-strand cDNA was synthesised
using 2 μg total RNA with ThermoScriptTM Reverse transcription poly­
merase chain reaction System (Invitrogen by Life Technologies, Cat. No.
11,146–016) following the manufacturer’s guidelines.
2.3. RT-qPCR analysis
The expression of reference and candidate genes was carried out by
Real-time PCR quantification using the CFX384 TouchTM Real-time PCR
machine from BioRad (Bio-Rad Laboratories Ltd., UK) with the cDNAs
obtained. Each sample was run in triplicate and the average CT value
calculated. The optimisation and selection of dilution series for making
standard curves were conducted (acceptable PCR efficiency between 90
and 110%, R2 higher than 0.985 with a unique peak in melt curve). The
top three references genes (AcTUA, PP2A1 and UBL) out of seven tested
were chosen by Biogazelle qBase+ software based on the geNorm
(Vandesompele et al., 2002) and qBase technology (Hellemans et al.,
2007). Normalisation was achieved by dividing the expression of the
gene of interest against the expression of the mean of three reference
genes at that same sampling-point.
2.4. Gene sequences and primers
The gene sequences were obtained in one of two ways. The genes
from Arabidopsis photoperiodic flowering homologs including COL2 and
FTs were taken from published papers (Rashid and Thomas, 2019).
Others were selected from the onion transcriptome RNA-seq database
(Rashid and Thomas, 2019) and putative functions confirmed by
BLASTing gainst the NCBI database. The sequences and q-qRT-PCR
primers used are included in the supplementary material. Candidates
included invertases (beta-fructofuranosidase, FFS) FFS-1 and FFS-2; su­
crose 1-fructosyltransferase (SST1) SST1-B1 (putative bulb specific
sequence), SST1-B2 and SST1-L (putative leaf specific sequence);
116 123 130 137 144
3 4 5 6 7
LB3 LL3 LB4 LL4 LB5 LL5 LB6 LL6
SB3 SL3 SB4 SL4 SB5 SL5 SB6 SL6
2
W. Cheng et al.
defensins (DFS); and lachrymatory factor synthase (LFS).
2.5. Bioinformatic analysis of the relation between tissue specificity and
daylength sensitivity
To interpret biological roles of genes changing expression in
response to day length, transcripts were first annotated using TRAPID
against a reference of Lilopsidia genomes. The resulting GOterm asso­
ciations were then tested for enrichment in day-length differential
expression groups of genes using hypergeometric test in the goseq
package of the R Bioconductor library. BLAST searching of the tran­
scripts against the NCBI non-redundant database of proteins was also
done to find potential functions in more distant homologs.
2.6. Statistical analyses
Means, standard deviations and standard errors were calculated in
Microsoft Excel and the significance of the differences was assessed by
using factorial analysis of variance (ANOVA) with repeated measures.
ANOVA was carried out using statistical software package SPSS.
3. Results
3.1. Bulbing ratio and leaf number
Measurements of bulbing ratio and leaf number were made as the
differences in bulbing responses. Fig. 1 shows the bulbing ratio and
visible leaf number during plant development in Marco grown in SD and
LD. There were slight differences at the starting point for both figures
due to the randomization when separating the plants. Plants in which
bulbing was just beginning to take place, as judged visually, were
preferentially placed in SD.
Along with no obvious change in bulbing ratio, an increase in visible
leaf number can be seen after 4 harvests in SD plants (Fig. 2), indicating
an interrupted bulbing process followed by a restart of leaf production.
In LD plants, a clear increase in the bulbing ratio can be seen, coupled
with the cessation of leaf production. The apparent reduction in leaf
number is because the oldest leaves had senesced and died back. The
bulbing ratio for LD plants was less than that of SD plants at the
beginning of the sampling timepoints, but progressively increased after
around 127 days from sowing. The reverse was true for leaf number and
crossover of the values for visible leaf number between SD and LD
occurring at approximately the same time as the bulbing ratios.
Fig. 1. Bulbing ratio and visible leaf number after plants transfer. The red arrow indicates the transferring point (2 days before the first sampling date). A indicates
SD plants’ bulbing ratio and leaf number; B indicates LD plants’ bulbing ratio and leaf number. Sampling was then conducted at weekly intervals. Error bars represent
the SEM (n = 5).
3
Scientia Horticulturae 286 (2021) 110223
3.2. Relative expression of photoperiod genes
Marco plants were grown in LD or SD as described in Table 2 and the
expression of target genes was measured by RT-qPCR. AcCOL2 expres­
sion was highly leaf specific and expressed in both daylengths, although
at a higher level in the SD treatment (Table S.1). No obvious trend was
observed during onion leaf development in either daylength (Fig. 2).
AcFT1 was expressed only in LD leaf with increasing transcripts during
plant development (Fig. 2). The expression of the gene is highly selective
between tissues and very sensitive to daylength (Table S.2). The gene
showed almost no transcripts in bulb material nor under SD, although
foliage leaves continued to be produced. In addition, if the SD expression
data is plotted on an expanded scale (Fig. S.1), some expression of AcFT1
can be observed in the newly transferred material, that was just begin­
ning to bulb, but this was sharply reduced after the plants were trans­
ferred from LD to SD. AcFT4 expression was also significantly selective
in plant sites and daylengths (Table S.3). It was only expressed in the SD
leaf in contrast with AcFT1, though also with a rising trend in transcript
levels during with Marco development (Fig. 2). There was no expression
of AcFT4 detected in the basal tissue (leaf bases and scale leaves), and
very limited expression in the leaf grown in LD.
AcFT5 mRNA was highly expressed in LD leaf material but very low
expression in SD leaf (Fig. 3). There was barely any detectable expres­
sion in bulb tissues. ANOVA further confirmed AcFT5 expression was
significantly affected by daylengths (Table S.4). AcFT6, by contrast to
AcFT5, was expressed mostly in the SD leaf material with an increasing
pattern during the onion development (Fig. 3). Low level of transcripts
was presented in basal tissue for both daylengths. The expression of
AcFT6 was also significantly affected by daylength and tissue - specific
(Table S.5). A reducing amount of mRNA in SD bulb material was
observed after the plants being transferred from LD.
3.3. Relative expression of invertases (beta-fructofuranosidase, FFS)
genes
The expression of FFS-1 was mostly detected in leaves, but only in the
first two harvests (Fig. 4) although overall, the difference between leaf
and bulb was not statistically significant (Table S.6). FFS-2 expression
was detected throughout the plant under both daylengths throughout
plant development (Fig. 4), with significantly more transcripts in bulb
tissue (Table S.7). The gene is not sensitive to daylength, and no obvious
trend can be seen over time. As FFS-2 was predicted to code for an
insoluble invertase, its higher expression in leaf base may be related to
thickening of cell walls in sink tissue during onion bulb formation.
W. Cheng et al. Scientia Horticulturae 286 (2021) 110223

Fig. 2. Relative expression of AcCOL2, ACFT1 and ACFT4 in Marco leaf and bulb tissue under LD and SD. Error bars represent the SEM. Reference genes were
AcTUA, PP2A1 and UBL.

3.4. Relative expression of sucrose 1-fructosyltransferase (SST1) genes
SST1-B1 was expressed throughout the developmental stages in all
tissues under both daylengths (Fig. 5). Statistics suggested the gene is
not selective between tissues and not sensitive to daylengths treatments
(Table S.8). SST1-B2 showed greater abundance in onion bulb materials
under both daylengths (Fig. 5), ANOVA confirmed the gene is signifi­
cantly bulb specific (Table S.9). There was no obvious differential
expression in response to daylengths. In addition, the gene seemed to
have an increasing number of transcripts in all tissues over time, indi­
cating it may have a role in fructan accumulation in both source and sink
materials. SST1-L presented a significant leaf-specific pattern with very
limited mRNAs detected in the basal tissue (Fig. 5). Statistics confirmed
the gene is not only tissue selective, but also daylength sensitive with
higher expression under SD (Table A.10).

4
W. Cheng et al. Scientia Horticulturae 286 (2021) 110223

Table 2 protein existing in this part of the plant. The expression was also
Comparison of numbers of the genes with tissue specificity or daylength sensi­ detected in leaves but much lower than that in bulb. ANOVA showed LFS
tivity as found by RNA-seq (Rashid and Thomas, 2019). Tissue selective genes is tissue specific but not sensitive to daylength (Table S.12).
are those which show different expression betweel leaf and bulb. Daylength
responsive genes are those that show differential expression in LD and SD.
3.6. Analysis of the relation between tissue specificity and daylength
Tissue selective genes sensitivity
Leaf: LD- Leaf: SD- Leaf daylength-
enhanced enhanced insensitive
Bulb LD-enhanced 8 3 315 It is noteworthy that, when comparing tissue specificity and day­
Bulb SD-enhanced 3 6 334 length sensitivity of twelve candidate genes, all daylength sensitive
Bulb-daylength 181 148 22,282 genes showed significant tissue selection, including the sugar meta­
insensitive
bolism gene SST1-L; whereas the ones with tissue specificity did not
Daylength-responsive genes necessarily respond to daylength e.g. AcCOL2. We further tested this
SD-Bulb SD-Leaf SD-tissue selective with the RNA-seq experiment resported in (Thomas and Rashid 2019).
LD-Bulb 2375 17 1666 From the total 23,280 sequences, only 4.29% of the tissue specific genes
LD-Leaf 29 5002 2136 were expressed differentially in LD and SD, whilst 68.34% of the day­
LD- tissue selective 2422 2262 7371
Total: 23,280
length sensitive genes were tissue-selective (Table 2). The evidence of
organ specific responses to daylength consistent with interorgan sig­
nalling between the site of perception in the leaf and response in the
3.5. Relative expression of onion organosulphur metabolism genes bulbing tissues.

DFS presented a clear bulb-specific pattern visually (Fig. 6) and this
was confirmed statistically (Table S.11). The transcripts were also
detected in leaf material but much lower than in bulb. DFS had no
daylength sensitivity nor an accumulation in transcription level
(Table S.11). LFS mRNAs were evident throughout basal tissues for both
SD and LD presenting a clear increasing trend in both bulb materials
over time (Fig. 6). It is an indication of the accumulation of its functional
4. Discussion
4.1. Daylength sensitivity of photoperiod genes and their possible roles in
bulb formation
For the genes also found in the Arabidopsis photoperiodic flowering
pathway, all the targets other than AcCOL2, showed significant

Fig. 3. Relative expression of ACFT5 and AcFT6 in Marco leaf and bulb tissue under LD and SD. Error bars represent the SEM. Reference genes were AcTUA, PP2A1
and UBL.

5
W. Cheng et al.
Fig. 4. Relative expression of FFS-1 and FFS-2 in Marco leaf and bulb tissue under LD and SD. Error bars represent the SEM. Reference genes were AcTUA, PP2A1
and UBL.
daylength sensitivities and leaf-specific pattern, indicating their role in
light perception in sugar source organs. AcCOL2 forms part of the day­
length perception mechanism in Arabidopsis and, is also expressed in
leaves in both LD and SD (Reference here). AcFT1 was expressed only in
the LD leaf material, with a notable increasing trend of transcripts
during plant development under inductive daylength conditions. In the
expanded scale of expression, a quickly down-regulated AcFT1 mRNA
level was observed when plant transferred to non-inductive daylength.
By contrast, AcFT4 was expressed only in the SD leaf material, also with
an increasing mRNA level during plant growth. The gene was clearly
upregulated after the plants were transferred from LD to SD. All these
traits of AcFT1 and AcFT4 expression in this time-course experiment can
further support their functional role as a promoter and inhibitor in onion
bulbing process (Lee et al., 2013).
Additionally, the reverse pattern of AcFT1 and AcFT4 may suggest
there is a negative correlation between the two. Besides being a circa­
dian regulator, the FT family has been reported to play extended roles in
modifying source-sink interactions. A recent study in potato tuberization
suggested that the switch to tuber development may be mediated by the
interaction between the tuberization specific FT homolog StSP6A and
the sucrose efflux transporter StSWEET11 (Abelenda et al., 2019). The
FT homolog is therefore likely to promote symplastic sucrose transport
in potato. The link between the sugar and photoperiodic pathways
during tuberization extends the role of proteins of FT family from mobile
signals to mediators of source-sink partitioning, which could also be the
case in onion, although that would need to be tested.
Additionally, an opposite expression pattern between AcFT5 and
6
Scientia Horticulturae 286 (2021) 110223
AcFT6 was also observed in this experiment. AcFT5 was found mostly in
LD leaf while AcFT6 was only expressed in SD leaf, both showed an
accumulation of transcripts during maturity at the required daylength.
The result indicates they are also daylength regulated in Marco and play
a role in leaf tissues under inductive or non-inductive daylength
although the functions remain unknown.
4.2. Diversity of invertase-related genes and their specific role in
respective organs
In higher plants, the catabolism of sucrose is to allow the subsequent
utilization of the component hexoses. This catabolism is mediated by
two enzyme activities, invertase and sucrose synthase (Ap Rees, 1988).
We chose two invertase related sequences the RNA-seq database. FFS-1
is predicted to be a soluble acid invertase, which means the enzyme
functions in the vacuole. The experiment showed the gene was highly
expressed in leaf during earlier stage and the mRNA reduced with plant
maturity. The reason for its leaf-specific pattern may be that there is a
requirement for sucrose catabolism to support heterotrophic tissues (e.g.
epidermal cells) or to provide respiratory substrates at night in leaves. In
addition, a possible sensing role of invertase in leaves has also been
suggested (Kingston-Smith et al., 1999). The mRNA abundance of FFS-1
is sensed and transduced into altered patterns of gene expression, and
further alter patterns of the genes modulating sucrose catabolism in sink
tissues. FFS-2 was predicted to translate an insoluble isoenzyme,
meaning it is one type of cell wall-bound invertase. The experiment
suggested it is a bulb-specific invertase gene, indicating its role in
W. Cheng et al.
Fig. 5. Relative expression of SST1-B1, SST1-B2 and SST1-L1 in Marco leaf and bulb tissue under LD and SD. Error bars represent the SEM. Reference genes were
AcTUA, PP2A1 and UBL.
carbohydrate accumulation at basal tissue during onion bulb formation
and growth. It has been suggested in various studies that invertase is
closely correlated with growth (Arai et al., 1991) and expansion in many
sink tissues (Schaffer et al., 1986). No daylength sensitivities were seen
in either FFS.
The organ-specific expression pattern of invertase seen in this
experiment has been found in many other plant species. In poplar, there
are six cell wall invertase genes (PtCWINVs) differentially expressed in
leaf, stem and root tissue (Chen et al., 2015). In carrots and tomatoes,
7
Scientia Horticulturae 286 (2021) 110223
some cell wall or vacuolar invertase genes showed markedly different
organ specific expression patterns with plant development (Sturm et al.,
1995; Godt and Roitsch, 1997). This experiment also showed tissue
specific invertase activity of FFSs, revealing that these genes play an
important role in their respective organs in terms of providing carbo­
hydrates for growth and development, and provides a basis for under­
standing the function of invertases in onion.
W. Cheng et al.
Fig. 6. Relative expression of DFS and LFS in Marco leaf and bulb tissue under LD and SD. Error bars represent the SEM. Reference genes were AcTUA, PP2A1
and UBL.
4.3. Fructans are synthesised and stored in different specialized organs
Fructosyltransferases transfer fructose from sucrose to the growing
fructan chain. According to the classical model of Edelman et al. (1968),
two enzymes (SST1 and FFT1) are involved in the synthesis of the
simplest form of fructan, and this experiment only focused on SST1s
(Fig. 5). The three SST1s had different tissue-specific sites of expression.
SST1-B1 was expressed in all tissues throughout development. SST1-B2
was preferentially expressed in bulb material while SST1-L expression
was limited to photosynthetic leaves. The source for onion bulb is the
fructans produced from sucrose and fructose then accumulated in sinks,
namely basal scales, during bulbing (Pak et al., 1995). There are also
fructans in leaf tissues, thus fructans may be translocated to the sink
tissues directly through the phloem, or be hydrolysed first, then trans­
ported and resynthesized (Sharma et al., 2015). Vijn et al. (1997) cloned
SST1 from onion, and found if placing onion leaves under continuous
illumination, the excessive sucrose content would induce fructan syn­
thesis and SST1 mRNA accumulation. This experiment provided a sup­
portive result showing a statistically significant daylength sensitivity of
SST1-L.
Fructans are often stored in different specialized organs, for example
in the taproot of chicory (Cichorium intybus), the tubers of dahlia (Dahlia
variabilis), and the bulbs of tulip (Tulipa gesneriana) (Ritsema and
Smeekens, 2003). In this experiment, both SST1-B1 and SST1-B2 also
were abundant in bulb materials as seen in tulip. Van den Ende et al.
(2000) isolated the cDNA for SST1 from Taraxacum officinale, and the
data showed the mRNA concentrations of SST1 were very low in leaves
8
Scientia Horticulturae 286 (2021) 110223
but abundant in young roots. Fructans can also play a role in the
expansion of petals. Studies showed petals of daylily (Hemerocallis) and
Campanula rapunculoides rapidly degraded fructan during flower open­
ing stage (Bieleski, 1993; Vergauwen et al., 2000). In cereals, fructans
temporarily accumulate in stems and early in seed development (Van
den Ende et al., 2011; Joudi et al., 2012). Fructans therefore have a
range of functions in addition to their role as the major storage carbo­
hydrates in leaves and bulbs in onions.
We also looked at the expression of LFS and DFS, genes involved in
organosulfur metabolism. These originally showed up as bulb-specific
genes in RNA-seq analysis (Rashid PhD thesis). This was confirmed by
qRT-PCR, where both were more highly expressed in the leaf bases or
scales and were not daylength-sensitive. As bulbs are a major source of
stored nutrients, they are vulnerable to predation by a range of pests and
diseases. LFS catalyses the generation of lachyratomory factor during
tissue damage (Silvaroli et al., 2017), which would deter predators. DFS
genes code for cys-rich peptides that show potent activity against a
range of microbial pathogens, including fungi and bacteria (Sathoff
et al., 2019) and are proposed to be involved in the defence against pests
and diseases (Garcia-Mina, 2012). DFS is commonly expressed in a tissue
specific manner, including the peripheral cells of potato tubers and
radishes, where they are thought to constiture the first line of defence
against pathogens (Broekaert et al., 1995). The tissue specificity of these
genes suggests that their role is to help protect the basal storage tissues
in onion, which might be particularly vulnerable to soil-borne disease.
W. Cheng et al.
5. Conclusion
The data in this paper help us to understand how the transition to
bulbing involves the coordinated tissue and daylength specific expres­
sion of a genes with a range of functions. The daylength-specific
expression of AcFT1 and AcFT4 genes in Marco confirms results found
in othe LD onions. In addition, AcFT5 and AcFT6 were found to have
daylength-specific expression unlike in previous studies with Renate
(Rashid and Thomas, 2019). This may indicate that there are varietal
differences in thedaylength responses in LD onions. The results also
indicate that genes that are daylength-sensitive tend to be tissue specific.
This is not surprising as differential development of leaf and basal tissues
is the primary response to daylenth. Further studies of these
daylength-sensitive genes will help to understand the molecular
mechisms underlyin bulb formation.
CRediT authorship contribution statement
Wei Cheng: Investigation, Writing – original draft, Visualization.
Harun Ar Rashid: Investigation, Resources, Writing – review & editing.
Richard Stark: Formal analysis, Writing – review & editing. Brian
Thomas: Conceptualization, Supervision, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors are pleased to thank the Chancellors’ International
Scholarship/Midland Integrative Biosciences Training Partnership
(MIBTP) award at the University of Warwick, Coventry, CV4 7AL, UK for
funding H.R. in this research.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.scienta.2021.110223.
References
Ap Rees, T., 1988. Hexose phosphate metabolism by nonphotosynthetic tissues of higher
plants. Biochem. Plants 14, 1–33.
Abelenda, J.A., Bergonzi, S., Oortwijn, M., Sonnewald, S., Du, M., Visser, R.G.,
Sonnewald, U., Bachem, C.W., 2019. Source-sink regulation is mediated by
interaction of an FT homolog with a sweet protein in potato. Curr. Biol. 29 (7),
1178–1186. https://doi.org/10.1016/j.cub.2019.02.018.
Arai, M., Mori, H., Imaseki, H., 1991. Roles of sucrose-metabolizing enzymes in growth
of seedlings. Purification of acid invertase from growing hypocotyls of mung bean
seedlings. Plant Cell Physiol. 32 (8), 1291–1298. https://doi.org/10.1093/
oxfordjournals.pcp.a078208.
Benkeblia, N., Onodera, S., Yoshihira, T., Kosaka, S., Shiomi, N., 2004. Effect of
temperature on soluble invertase activity, and glucose, fructose and sucrose status of
onion bulbs (Allium cepa) in store. Int. J. Food Sci. Nutr. 55 (4), 325–331. https://
doi.org/10.1080/09637480412331290512.
Bieleski, R.L., 1993. Fructan hydrolysis drives petal expansion in the ephemeral daylily
flower. Plant Physiol. 103 (1), 213–219. https://doi.org/10.1104/pp.103.1.213.
Brewster, J.L., 1994. Environmental physiology of the onion: towards quantitative
models for the effects of photoperiod, temperature and irradiance on bulbing,
flowering and growth. In: Proceedings of the I International Symposium on Edible
Alliaceae, 433, pp. 347–374. https://doi.org/10.17660/ActaHortic.1997.433.37.
Broekaert, W.F., Terras’ F, R.G., Cammue, B.P.A., Osborn, R.W., 1995. Plant Defensins:
novel antimicrobial peptides as components of the host defense system. Plant
Physiol. 108, 1353–1358. https://doi.org/10.1104/pp. 108.4.1353.
Brewster, J.L., 2008. Onions and Other Vegetable Alliums (Vol. 15). CABI.
Chen, Z., Gao, K., Su, X., Rao, P., An, X., 2015. Genome-wide identification of the
invertase gene family in Populus. PLoS One 10 (9). https://doi.org/10.1371/journal.
pone.0138540.
Clark, J.E., Heath, O.V.S., 1962. Studies in the physiology of the onion plant: V. An
investigation into the growth substance content of bulbing onions. J. Exp. Bot. 13
(2), 227–249. https://doi.org/10.1093/jxb/13.2.227.
9
Scientia Horticulturae 286 (2021) 110223
Dey, L., Attele, A.S., Yuan, C.S., 2002. Alternative therapies for type 2 diabetes. Altern.
Med. Rev. 7 (1), 45–58. PMID: 11896745.
Edelman, J., Jefford, T.G., Singh, S.P., 1968. Studies on the biochemical basis of
physiological processes in the potato tuber. Planta 84 (1), 48–56. https://doi.org/
10.1007/BF00384821.
Haahr, M., 2006. RANDOM. ORG: True Random Number Service. https://www.random.
org.
FAOSTAT, 2018. Food and Agriculture Organisation of the United Nations-Agricultural
Data. http://faostat.fao.org/site/291/default.aspx (Last Accessed: 30th May
2019)</Dataset>.
Franco-Robles, E., López, M.G., 2015. Implication of fructans in health:
immunomodulatory and antioxidant mechanisms. Sci.c World J. 2015 https://doi.
org/10.1155/2015/289267.
Garcia-Mina, J.M., 2012. Plant nutrition and defense mechanism: frontier knowledge.
Advances in Citrus Nutrition. Springer, pp. 1–12.
Godt, D.E., Roitsch, T., 1997. Regulation and tissue-specific distribution of mRNAs for
three extracellular invertase isoenzymes of tomato suggests an important function in
establishing and maintaining sink metabolism. Plant Physiol. 115 (1), 273–282.
https://doi.org/10.1104/pp.115.1.273.
Gurjar, M.S., Ali, S., Akhtar, M., Singh, K.S., 2012. Efficacy of Plant Extracts in Plant
Disease Management. https://doi.org/10.4236/as.2012.33050.
Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., Vandesompele, J., 2007. qBase
relative quantification framework and software for management and automated
analysis of real-time quantitative PCR data. Genome Biol. 8 (2) https://doi.org/
10.1186/gb-2007-8-2-r19 p.R19.
Joudi, M., Ahmadi, A., Mohamadi, V., Abbasi, A., Vergauwen, R., Mohammadi, H., Van
den Ende, W., 2012. Comparison of fructan dynamics in two wheat cultivars with
different capacities of accumulation and remobilization under drought stress.
Physiol. Plant. 144 (1), 1–12. https://doi.org/10.1111/j.1399-3054.2011.01517.x.
Kingston-Smith, A.H., Walker, R.P., Pollock, C.J., 1999. Invertase in leaves: conundrum
or control point? J. Exp. Bot. 50 (335), 735–743. https://doi.org/10.1093/jxb/
50.335.735.
Koes, R.E., Quattrocchio, F., Mol, J.N., 1994. The flavonoid biosynthetic pathway in
plants: function and evolution. Bioessays 16 (2), 123–132. https://doi.org/10.1002/
bies.950160209.
Kumari, K., Augusti, K., 2007. Lipid lowering effect of S-methyl cysteine sulfoxide from
Allium cepa Linn in high cholesterol diet fed rats. J. Ethnopharmacol. 109 (3),
367–371. https://doi.org/10.1016/j.jep.2006.07.045.
Lanzotti, V., 2006. The analysis of onion and garlic. J. Chromatogr. A 1112 (1–2), 3–22.
https://doi.org/10.1016/j.chroma.2005.12.016.
Lee, R., Baldwin, S., Kenel, F., McCallum, J., Macknight, R., 2013. FLOWERING LOCUS T
genes control onion bulb formation and flowering. Nat. Commun. 4 (1), 1–9. https://
doi.org/10.1038/ncomms3884.
Liu, R.H., 2007. Whole grain phytochemicals and health. J. Cereal Sci. 46 (3), 207–219.
https://doi.org/10.1016/j.jcs.2007.06.010.
Mendez, E., MORENO, A., COLILLA, F., PELAEZ, F., LIMAS, G.G., MENDEZ, R., ..., de
HARO, C., 1990. Primary structure and inhibition of protein synthesis in eukaryotic
cell-free system of a novel thionin, γ-hordothionin, from barley endosperm. Eur. J.
Biochem. 194 (2), 533–539. https://doi.org/10.1111/j.1432-1033.1990.tb15649.x.
Pak, C., van der Plas, L.H., De Boer, A.D., 1995. Importance of dormancy and sink
strength in sprouting of onions (Allium cepa) during storage. Physiol. Plant. 94 (2),
277–283. https://doi.org/10.1111/j.1399-3054.1995.tb05312.x.
Rashid, M.H.A., Thomas, B., 2019. Diurnal expression of Arabidopsis gene homologs
during daylength-regulated bulb formation in onion (Allium cepa L.). Sci. Hortic.
261, 108946 https://doi.org/10.1007/BF00195721.
Ritsema, T., Smeekens, S., 2003. Fructans: beneficial for plants and humans. Curr. Opin.
Plant Biol. 6 (3), 223–230.
Roberfroid, M., 2004. Inulin: A Fructan, Inulin-Type Fructans; Functional Food
Ingredients. CRC Press. https://doi.org/10.1093/jn/137.11.2493S.
Schaffer, A.A., Liu, K.C., Goldschmidt, E.E., Boyer, C.D., Goren, R., 1986. Citrus leaf
chlorosis induced by sink removal: starch, nitrogen, and chloroplast ultrastructure.
J. Plant Physiol. 124 (1–2), 111–121. https://doi.org/10.1016/S0176-1617(86)
80183-3.
Sharma, K., Assefa, A.D., Ko, E.Y., Lee, E.T., Park, S.W., 2015. Quantitative analysis of
flavonoids, sugars, phenylalanine and tryptophan in onion scales during storage
under ambient conditions. J. Food Sci. Technol. 52 (4), 2157–2165. https://doi.org/
10.1007/s13197-013-1225-2.
Silvaroli, J.A., Pleshinger, M.J., Banerjee, S., Kiser, P.D., Golczak, M., 2017. Enzyme that
makes you cry–crystal structure of lachrymatory factor synthase from Allium cepa.
ACS Chem. Biol. 12 (9), 2296–2304.
Sturm, A., Šebková, V., Lorenz, K., Hardegger, M., Lienhard, S., Unger, C., 1995.
Development-and organ-specific expression of the genes for sucrose synthase and
three isoenzymes of acid β-fructofuranosidase in carrot. Planta 195 (4), 601–610.
https://doi.org/10.1007/BF00195721.
Sathoff, A.E., Velivelli, S., Shah, D.M., Samac, D.A., 2019. Plant defensin peptides have
antifungal and antibacterial activity against human and plant pathogens.
Phytopathology 109, 402–408. https://doi.org/10.1094/phyto-09-18-0331-r.
Van den Ende, W., Michiels, A., Van Wonterghem, D., Vergauwen, R., Van Laere, A.,
2000. Cloning, developmental, and tissue-specific expression of sucrose: sucrose 1-
fructosyl transferase from Taraxacum officinale. Fructan localization in roots. Plant
Physiol. 123 (1), 71–80. https://doi.org/10.1104/pp.123.1.71.
Van den Ende, W., Peshev, D., De Gara, L., 2011. Disease prevention by natural
antioxidants and prebiotics acting as ROS scavengers in the gastrointestinal tract.
Trends Food Sci. Technol. 22 (12), 689–697. https://doi.org/10.1016/j.
tifs.2011.07.005.
W. Cheng et al.
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A.,
Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data
by geometric averaging of multiple internal control genes. Genome Biol. 3 (7)
https://doi.org/10.1186/gb-2002-3-7-research0034 research0034-1.
Vergauwen, R., Van den Ende, W., Van Laere, A., 2000. The role of fructan in flowering
of Campanula rapunculoides. J. Exp. Bot. 51 (348), 1261–1266. https://doi.org/
10.1093/jexbot/51.348.1261.
10
Scientia Horticulturae 286 (2021) 110223
Vijn, I., Van Dijken, A., Sprenger, N., Van Dun, K., Weisbeek, P., Wiemken, A.,
Smeekens, S., 1997. Fructan of the inulin neoseries is synthesized in transgenic
chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:
fructan 6G-fructosyltransferase. Plant J. 11 (3), 387–398. https://doi.org/10.1046/
j.1365-313X.1997.11030387.