Molecular Biology Reports (2020) 47:2171–2179

https://doi.org/10.1007/s11033-020-05316-7

ORIGINAL ARTICLE

A flowering inhibitor of the temperature‑dependent pathway

in Crocus sativus L.
Roya Haghighi1 · Badraldin Ebrahim Sayed Tabatabaei2 · Sayed Ali Mohammad Mirmohammadi Maibody1 ·
Majid Talebi2 · R. V. Molina3 · Sergio G. Nebauer3 · Begoña Renau‑Morata3

Received: 15 October 2019 / Accepted: 7 February 2020 / Published online: 17 February 2020
© Springer Nature B.V. 2020

Abstract
Saffron is the world highest-priced spice because its production requires intensive hand labour. Reduce saffron production
costs require containerised plant production under controlled conditions and expand the flowering period. Controlling the
flowering process and identify the factors involved in saffron flowering is crucial to introduce technical improvements. The
research carried out so far in saffron has allowed an extensive knowledge of the influence of temperature on the flower induc-
tion, but the molecular mechanisms controlling flowering induction processes are largely unknown. The present study is the
first conducted to isolate and characterize a regulator gene of saffron floral induction the Short Vegetative Phase (SVP) gene,
which represses the floral initiation genes in the temperature response pathway, which involved in saffron flower induction.
The results obtained from both phylogenetic analysis and T-coffee alignment confirms that the isolated sequence belongs
to the SVP gene clades of MADS-box gene family. Gene expression analysis in different developmental stages revealed the
highest expression of SVP transcript (CsSVP) during the dormancy and the vegetative stages, but decrease when flower
development initiated and it was the least in late September when flower primordia are developed. Furthermore, its expression
increased in the apical bud when corms are storage at 9–10 ºC, thus inhibiting flower induction. Additionally, comparison
of the CsSVP transcript in apical buds from big and small corms, differing in their flowering capacity, indicates that the
CsSVP transcript is present only in vegetative buds. Taken together, these results suggested inhibitory role of the SVP gene.

Keywords Crocus sativus · Short vegetative phase gene · Flowering inhibitor · Gene isolation · Gene expression

Introduction [3, 4]. Saffron is universally known for the delicate aroma
and attractive colour of its stigmas that have found wide-
Saffron (Crocus sativus L.) is a perennial monocot triploid spread applications as a food additive, as a colorant in textile
plant belonging to the Iridaceae family [1]. The origin of dyes and as a medicinal component for their anti-depression,
saffron is not exactly clear, but evidences indicate two possi- anti-carcinogenic and anti-tumour properties [5, 6].
ble sites: One in Greece in the Mediterranean area, the other The high price of this valuable plant may be explained
at East in Turkey-Iran-India [2]. Saffron has been cultivated by the fact that technology of saffron production has not
for 3500 years for its stigmas. Nowadays, about ninety per- changed from the ancient times. The picking of C. sativus
cent of the total saffron used in the world is produced in Iran flowers and the separation of the stigmata require an inten-
sive hand labour. A technological breakthrough to increase
the profitability of saffron cultivation would be the cultiva-
* Badraldin Ebrahim Sayed Tabatabaei
[email protected] tion under greenhouse conditions. It would be much easier
to mechanise blossom collection in containerised plants
1
Department of Agronomy and Plant Breeding, College than in those grown in the soil. The development of saffron
of Agriculture, Isfahan University of Technology, industrial production under greenhouse requires an accurate
Isfahan 8415683111, Iran
control of the flowering time for this species and the flow-
2
Department of Biotechnology, College of Agriculture, ering period should be extended [7]. Saffron only blooms
Isfahan University of Technology, Isfahan 8415683111, Iran
once a year over a short period of 15 to 30 days in autumn
3
Departamento de Producciόn Vegetal, Universitat Politècnica (the length of this period depends on weather conditions in
de València, Camino de vera s.n, 46022 Valencia, Spain

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different regions). Within this context, identifying the fac- genes FLOWERING LOCUS T (FT), TWIN SISTER OF
tors involved in flower induction and development acquires FT (TSF), and SUPPRESSOR OF OVEREXPRESSION OF
a great interest. CONSTANS1 (SOC1), but also interacts consistently with
Studies of model plants as arabidopsis and rice have the FLOWERING LOCUS C (FLC) to repress flower induc-
shown that genes controlling flowering time occur in a net- tion. During the floral transition, SVP and FLC are down
work of six major pathways. The photoperiod and vernali- regulated so that the plant is induced to change from the
zation pathways control flowering in response to seasonal vegetative to reproductive phase [25, 26].
changes in day length and temperature. The ambient tem- In Arabidopsis thaliana svp mutant plants were observed
perature pathway responds to daily growth temperatures. to exhibit early flowering, while SVP overexpression ones
The age, autonomous and gibberellin pathways act more were found to have late flowering, indicating that SVP is an
independently of environmental stimuli [8]. These six path- inhibitor of flowering [23]. In winter cereals such as barley,
ways converge to regulate a small number of “floral integra- three SVP-like genes include BM1 and BM10 are induce
tor genes” encoded by different classes of proteins, which by cold and expressed during vegetative growth [27]. Other
govern flowering time by merging signals from multiple SVP-like gene, BcSVP (homologous to the AtSVP in arabi-
pathways. These integrator genes include FLOWERING dopsis) was isolated from Chinese cabbage. This gene is
LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION responsible for flowering time control and preventing early
OF CONSTANS 1 (SOC1), which both rapidly promote floral bolting. Similar to BM1 and BM10 in barley, BcSVP in Chi-
development. nese cabbage is a flowering repressor, but it is not affected
Most of the studies with the aim to identify the network of by cold [28]. SVP in Arabidopsis is not only involved in the
major pathways controlling flowering have been performed temperature pathway, but together with AP1 and AGL24,
on plant species from cold regions, where dormancy is regu- it also controls floral meristem identity [29]. On the other
lated by short photoperiod and low temperature. The ques- hand, a possible involvement of SVP in the hormonal control
tion of internal and environmental regulation of flowering of flowering has been suggested, taking into account that
in bulbous plants with summer dormancy at the biochemical the GA20ox2 gene is up-regulated in svp mutant plants to
and molecular levels has been far less studied. However, promote early flowering [30].
the homologues of FT and LFY, the key conserved genes Taken together, the studies cited above suggest that SVP-
in floral initiation have been found in a few geophytes and like genes could be key genes in a temperature-dependent
their spatio-temporal expression has been studied in Nar- pathway of flowering in saffron. However, no study is
cissus and Allium sativus [9–13]. Very few work about the reported in the literature about it. In the present work, we
molecular biology of flowering in saffron has been pub- have isolated and characterized the SVP gene in C. sativus.
lished. Although previous research about the MADS BOX In addition, we have studied its expression in different tis-
gene family controlling the organ identity genes has been sues, developmental stages and in corms differing in their
carried out [14–16], the molecular mechanism controlling flowering capacity in order to unveil its involvement in the
flower induction in this species remains largely unknow. flowering pathway of saffron.
Temperature is the most potent factor affecting floro-
genesis in many flower bulbs [17, 18] and also in saffron.
According to Molina et al. [7], the best temperature range Materials and methods
for flower initiation in C. sativus is from 23 to 27 °C, taking
place during summer after the senescence and death of the Plant materials
above ground organs. Similar to Tulipa [19], Freesia [20],
Hyacinthus, Iris, Muscari [21], Narcissus [22] and like most Saffron corms (C. sativus L.) obtained from a local farmer in
geophytes, saffron is thermo-periodic. After flower initiation Albacete, Spain (> 25 g in weight), were used in this study.
in summer, a drop in temperatures (until reaching values Plants were cultured in the experimental fields of an agricul-
between 10 and 17 °C) is required to promote flower bud tural cooperative in eastern Spain (ANECOOP, Valencia).
development and escape elongation. The vegetative growth Corms were planted in furrows, at a depth of 15 cm, with
takes place in autumn and winter, followed by dormancy 15 cm between plants and 50 cm between furrows. The cul-
beginning in the middle of the spring. tivation practices used were those commonly used for this
One of the most important transcription factor family crop, and an organic fertiliser (mature manure) was applied.
involved in controlling the flowering process is the MADS- Reined conditions met the water requirements at the start of
box gene family. SVP is one of the MADS-box genes that growth (October to November), and plants were drip irri-
responds to ambient temperature and plays a key role as a gated from December to April.
repressor in this pathway [23, 24]. At the vegetative phase, In order to measure differences in expression among
SVP not only controls negatively the three floral integrator organs, samples of buds, leaves, flowers, and corms were

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collected. Leaves and corms were collected in March and Three nested-PCRs were performed to amplify 5′ end
different parts of the flowers were collected in October. of the CsSVP cDNA. For this purpose, a specific forward
With the purpose of analysing differences in gene expres- primer (SPF2: 5′-AGA​TGG​CGA​GGG​AGA​AGA​TAC​AAA​
sion during vegetative stage and the diverse developmental TAA-3′) was designed based on the SVP cDNA in plant
stages of flower development, apical buds were sampled in species that exhibited similarities to the sequencing result
January, May, June, July, and September. To measure gene alignment of CsSVP 3′-end. Three reverse primers were
expression in buds coming from corms stored both at tem- also designed based on 3′-end sequencing result (SPR1:5′-
perature allowing flowering and at temperature inhibiting GTC​ACA​AGT​TTT​TCT​CAC​CCA​ACT​-3′), (SPR2:5′-AAT​
floral induction. One hundred and twenty big corms (> 25 g) AAG​T TC​TAG​T CC​ATC​AGC​ACA​G -3′), and (SPR3:5′-
were harvested in June. One 60 group of corms were stored GCA​T TA​GTG​ACT​GAT​T CA​GAG​GAT​T-3′). The first
at 9–10 °C and the other group were stored at 25 °C. After round amplification reaction was conducted using the
50 days, bud tissues were collected in July. In addition, with SPF2 and SPR1 primers and the diluted (1/50) PCR prod-
the purpose of studying differential expression in buds com- uct was used as a template for the second round PCR with
ing from small (< 8 g) and big corms (> 25 g) differing in SPF2 and SPR2 primers. The PCR product thus obtained
their flowering ability, buds from both corm sizes were col- was diluted (1/10) to be used as a template for the third
lected in mid July. All the samples were frozen in liquid round PCR with SPF2 and SPR3 as primers. The 600 bp
nitrogen and stored at ‒ 80 °C until analysing gene expres- fragment obtained was cloned into the pJET1.2/blunt
sion by means of qPCR analysis. Cloning Vector and sequenced.

RNA isolation and cDNA synthesis

Expression analysis
Total RNA was extracted from leaves, buds, petals, stigmas,
Total RNA was extracted from the leaf, corm, root, petal,
stamens, roots, and corms using the RNeasy plant mini kit
stigma, and stamen according to the Landolino’s protocol
(Qiagen). Following the manufacturer’s protocol, the con-
[31]. RNA was quantified on a UV/VIS spectrophotometer.
taminating genomic DNA was eliminated in a DNase treat-
First-strand cDNA was synthesized using 1 µg of total
ment step using Turbo DNase (Ambion). First-strand cDNA
RNA employing the First Strand cDNA Synthesis Kit
was synthesized using the SuperScript™ cDNA Synthesis
SuperScript II one-step RT-PCR (Invitrogen) for real-time
Kit according to the manufacturer’s instructions.
PCR (RT-PCR) following the manufacturer’s protocol. The
specific primers RT-F (5′-ATC​C CT​CAA​GTT​AGG​T CT​
Gene isolation TCCAT-3′) and RT-R (5′-GTC​ACA​AGT​T TT​T CT​CAC​
CCA​ACT​-3′) were designed for real-time PCR by primer3
One specific forward primer (SPF1: 5′-GAT​GAG​GGG​AGA​ plus (https​://prime​r3plu​s.com/) and Integrate DNA Tech-
AGA​CCT​TCA​AGG​-3′) was designed based on the align- nology, oligo analyzer tools (https​://eu.idtdn​a.com/calc/
ment of the nucleotide sequences of SVP cDNA sequence analy​zer) database. The C. sativus tubulin primers (Tub-
in other plant species available at the NCBI database (www. F: 5′-TGA​TTT​CCA​ACT​CGA​CCA​GTGTC-3′) and (Tub-
ncbi.nlm.nih.gov). The 3′-end of CsSVP cDNA was ampli- R:5′-ATA​CTC​ATC​ACC​CTC​GTC​ACC​ATC​-3′) were used
fied using the 3′ RACE procedure. Briefly, ten percent of the as the reference genes [32–34].
synthesized cDNA was used as a PCR template with 0.8 μM Diluted cDNA was used as the template in a reaction
of SPF1 and oligo (dT)-anchor primers, 0.2 mM dNTPs, and volume of 20 μl containing 0.3 µM of each forward and
1U Dream Taq DNA Polymerase (Thermo Scientific) in a reverse primers and 10 µl of SYBR Green PCR master
reaction volume of 20 μl. Reactions were performed under mixture (Power SYBR®Green PCR Master Mix; Applied
the following conditions: 95 °C for 3 min and 30 cycles of Biosystems). Expression analysis of the CsSVP gene was
94 °C for 1 min, 65 °C for 1 min and 72 °C for 2 min, and a performed with real-time PCR using a CFX Connect (Bio-
final extension step of 7 min at 72 °C. The first round PCR Rad).The cycling parameters were incubated at 50 °C for
products were diluted (1/50) and used as a template for the 2 min and 95 °C for 10 min followed by 40 amplification
second round PCR using with 0.4 μM of both SPF1 and cycles (95 °C for 15 s plus 60 °C for 1 min). Three inde-
anchor (5′-GACC ​ ACG​ CGT ​ ATC
​ GAT ​ GTC
​ GAC-3′) primers. pendent biological and technical replicates of each sample
Amplification conditions were same as for the first round. were used for RT-PCR analysis. Relative expression lev-
The 500 bp fragment obtained was cloned into the pJET1.2/ els of the target genes were calculated using the 2­ −ΔΔCT
blunt Cloning Vector (Fermentas). Several individual clones method [35]. Statistical analyses were performed using the
were screened by colony PCR and sequenced by the DNA Statgraphics software.
sequencing unit at the IBMCP-UPV (Valencia, Spain).

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Bioinformatics analysis Expression analysis

Sequencing results were blasted by NCBI database (https​ The qRT-PCR was performed to derive the expression pat-
://www.ncbi.nlm.nih.gov/) to evaluate the accuracy of terns of the SVP gene in the different organs of C. sativus
the sequences obtained. Prediction of the amino acid including the leaf, corm, root, vegetative bud, petal, stigma,
sequence was carried out through the translate tool of the and stamen. As shown in Fig. 2a, the CsSVP transcript was
ExPASy database (https​://web.expas​y.org/trans​late/). To expressed in all vegetative organs, but the expression level
compare the amino acid alignment thus deduced, T-Coffe in floral organs including the petals, stamens, and stigmas,
multiple alignment computer program Version_11.00 [36] was very low or absent. The highest CsSVP gene expression
was used and visualized with JalView version 2 [37]. Phy- was observed in leaves.
logenetic relationships were performed using the Neigh- In order to determine whether the CsSVP gene is related
bour-Joining Method with the p-distance correction and to flowering inhibition also in saffron, its expression was
the phylogenetic tree was constructed using the MEGA analysed by qPCR in apical buds throughout the different
7 software [38]. developmental stages of the plant (Fig. 3x). Significant dif-
ferences were observed between the vegetative and repro-
ductive stages (Fig. 2b). The higher CsSVP expression was
found during vegetative development, but also when the
Results corm entered into dormancy after leaf senescence. However,
that expression declined during flower initiation and devel-
CsSVP isolation from C. sativus opment, showing the lowest expression during September,
when flowering took place (Fig. 2b).
Prior to cloning, the SVP homologue in C. sativus (CsSVP) To unveil if CsSVP is also related with the competence of
was amplified from the cDNA collected from the bud tis- the bud to flower, the expression of the gene was analysed
sue using a general primer designed based on the con- by qPCR in buds of big corms able to flower, and in buds
served domain of the SVP genes in monocot flowering of small corms, lacking flower induction capacity (Fig. 3y).
plants. Sequencing result blasts revealed a 52% similarity Our results pointed out that CsSVP could have an inhibitor
with the SVP nucleotide sequence in Dendrobium cat- role in the flower induction process, showing a high level
enatum. Thus, nested PCR was employed to isolate the of expression in small corms, meanwhile it is lower in big
5′-end of the CsSVP gene using a Dendrobium specific corms having a size enough to flower (Fig. 2c).
primer. C. sativus cDNA has a length of 850 bp and an Differences in corm storage temperature affecting floral
open reading frame of 675 bp (Fig. 1a) that encodes a induction (25 ºC vs 9 ºC) also affect CsSVP expression. In
225 amino acid predicted polypeptide (Gen Bank Acc. Fig. 3z differences in bud development can be observed.
No. MN015609). Blasting with the NCBI database of the Saffron corms that were exposed to 25 °C for 50 days and
nucleotide sequence thus obtained, indicated that the iso- showing flower development had lower expression in CsSVP
lated gene encoded a SVP-like protein. gene, than corms that were exposed to 9 °C (Fig. 2d). There-
Multiple alignment (Fig. 1b) revealed great similarity fore, it can be concluded that the expression of SVP gene in
in the MADS box and K box motifs between the CsSVP saffron plant is directly affected by temperature and this gene
deduced amino acid and the SVP amino acid sequence could be involved in the flowering control through tempera-
in other plant species, regardless of whether the plants ture pathway.
were monocots or dicots. The CsSVP deduced amino acid
exhibited 72%, 61%, and 59% homologies with Narcis-
sus tazetta, Hordeum vulgare and Arabidopsis thaliana,
respectively. Discussion
The phylogenetic tree derived by the Neighbor-Joining
method to gain knowledge of the SVP relationships between The vegetative-to-reproductive phase change in the apical
C. sativus and the plant species present in Fig. 1b. It revealed meristem of saffron corm is promoted by warm temperatures
a distinctly dichotomous branching of the SVP gene whereby during summer. To shed light on the regulation of this pro-
the SVP gene from monocots clustered together, as expected cess, the isolation and characterization of the Short Vegeta-
(Fig. 1c). The monocot clade is subdivided into two further tive Phase (SVP) gene, which represses the floral initiation
clades. CsSVP was placed in a group with species belonging genes in the temperature response pathway, has been carried
to the Asparagales order containing many geophytes. The out. As far as we know, this is the first work showing the
other group contains species belonging to the Poales order involvement of a saffron transcription factor in the floral
including grasses (Poaceae) and bromeliads (Bromeliaceae). induction process.

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Fig. 1  CsSVP gene sequence, multiple alignment, and phylogenetic ticosa), AST22412.1 (Lansium domesticum), AHC92621.1 NSVP1
tree in C. sativus. a CsSVP cDNA complete sequence (the CDs (Narcissus tazetta), AHC92622.1 NSVP2, (Narcissus tazetta),
highlighted in sequence). b Multiple alignments of SVP protein AHC92623.1 NSVP3, (Narcissus tazetta), XP_020692709.1 SVP-
sequence in MN015609 (Crocus sativus), ALD15665.1 (Erythranthe like isoform X1 (Dendrobium catenatum), XP_020692711.1 SVP-
guttata), AHY82603.1 (Brassica juncea), ABG24233.1 (Brassica like isoform X2 (Dendrobium catenatum), ABM21529.1 (Hordeum
rapa), AUN50241.1 (Triticum aestivum), AAX47170.1 (Pisum sati- vulgare), ABU95408.1 (Arabidopsis thaliana) on the T-COFFEE
vum), AKN56865.1 (Betula platyphylla), AIY25021.1 (Dimocarpus Multiple Sequence Alignment Server. The highlighted sequence rep-
longan),BAN89455.1 (Shorea beccariana), AFG73587.1 (Brassica resents MADS-box (2–73) and K-box (93–168) conserved domain. c
napus), AOW72912.1 (Epimedium sagittatum), ABY78023.1 (Gly- Phylogenetic tree of SVP-like and MADS-like gene sequences in the
cine max), NP_001275284.1 (Solanum tuberosum), AHM25245.1 plant species present in Fig. 1b. (Color figure online)
SVP-2 (Paeonia suffruticosa), AHM25244.1 SVP-1 (Paeonia suffru-

The 850 bp isolated SVP-like gene in C. sativus was SVP maintains plants in the vegetative stage by repressing
classified as one neighbouring the two clades of Poaceae flower-promoting genes.
and geophytes in the monocot group. The SVP serves The study of the SVP expressions in the different organs
varied functions in different plants in the Poaceae family of diverse species like Arabidopsis [23], Brassica rapa [28],
for example; TaVRT2 in wheat is inhibited by vernaliza- Hordeum vulgare [27], Oryza sativa [41], or Narcissus
tion [39, 40]. BM1, BM10, and HvVRT2 are induced by tazetta [22] revealed the presence of the transcripts during
cold in barley (Hordium vulgar) [27], and the SVP-like the vegetative and dormancy stages. Similarly, in C. sati-
(OsMADS47) gene in rice is a negative regulator of brassi- vus CsSVP gene expression was only detected in vegetative
nosteroids (BR) [41]. However, they all share the common organs, with most of the expression observed in leaves. A
role of floral inhibition. Also in monocot geophytes like great decrease of CsSVP expression takes place in stamen,
Narcissus tazzeta, SVP play a role in flower development stigma, and petals.
[22]. Unusual flower structures have been reported in The CsSVP expression in apical buds throughout the
NSVP1 overexpressing plants of Arabidopsis. It seems that different developmental stages reported in this study is in

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Fig. 2  SVP expression in C. sativus by qRT-PCR. a CsSVP gene coming from small (no competent to flower) corms and corms big
expression in the apical bud at the different developmental stages; b enough to flower (d) CsSVP expression in buds of corms stored at
CsSVP expression in different organs (c) CsSVP expression in buds different temperature (25 °C and 9–10 °C)

agreement with what it has been observed in other species. between CsSVP expression and the competence of the bud
The highest CsSVP expression was found during vegeta- to flower due to the corm size has been unveiled.
tive development, but also when the corm entered into The most important environmental factor related to flower
dormancy after leaf senescence. However, that expression induction in saffron is temperature. Flower differentiation
declined during flower initiation and development. In Nar- requires high temperatures (23–30 °C) to take place (Molina
cissus tazetta, one of the species closest to saffron from et al. 2005) and temperatures around 9–10 °C inhibit flower
those that has been studied, NSVP1 has a high expression development. Our results point out that the suppression of
in the shoot meristem during its vegetative and dormancy flower development due to low temperatures could be also
stages, with minimum expressions reported at the begin- mediated by an increased in CsSVP expression. These find-
ning of flowering differentiation and release dormancy ings provide support for the idea that SVP could act as a
stages [22]. Also in other monocots like barley, the BM1, floral inhibitor in saffron.
BM10, and HvRT2 genes are inhibited during spike devel-
opment [27].
Taking together all these results about the differences on
Conclusion
the expression of CsSVP, we could suggest that this gene
like its orthologue genes, acts as a flowering inhibitor in
In conclusion, we have identified a possible flowering inhibi-
saffron, and keeps the plant in the vegetative stage. Confirm-
tor gene in saffron (CsSVP), which could play an important
ing this hypothesis, we observed higher expression of the
role in temperature pathway. This gene could be exploited
gene in small corms, lacking flower induction capacity, when
as a marker in saffron to accurately examine the effects of
comparing to big corms able to flower. Thus, a correlation
environmental factors on flowering. Moreover, it could be
beneficially exploited to control flowering time in saffron.

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Fig. 3  A scheme of saffron bud in different development of stage. x corms (a) or big corms (b). z Differences in bud development from
Vegetative stage and the diverse flower development stages studied corms that have been stored at 9 °C (a) and 25 °C (b)
in this work. y Differences in bud development coming from small

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