Post on 16-May-2023
Potato CONSTANS is involved in photoperiodic tuberizationin a graft-transmissible manner
Nahuel D. Gonzalez-Schain, Mercedes Dıaz-Mendoza†, Marek _Zurczak‡ and Paula Suarez-Lopez*
Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, Campus UAB, Edifici CRAG, Bellaterra (Cerdanyola del
Valles), 08193 Barcelona, Spain
Received 10 June 2011; revised 10 January 2012; accepted 12 January 2012; published online 5 March 2012.*For correspondence (e-mail paula.suarez@cragenomica.es).
Sequence accession number for StCO cDNA: AM888389.†Present address: College of Natural and Agricultural Sciences, University of California, Riverside, CA, USA.‡Present address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
SUMMARY
CONSTANS (CO) is involved in the photoperiodic control of plant developmental processes, including
flowering in several species and seasonal growth cessation and bud set in trees. It has been proposed that CO
could also affect the day-length regulation of tuber induction in Solanum tuberosum (potato), a plant of great
agricultural relevance. To address this question, we examined the role of CO in potato. A potato CO-like gene,
StCO, was identified and found to be highly similar to a previously reported potato gene of unknown function.
Potato plants overexpressing StCO tuberized later than wild-type plants under a weakly inductive
photoperiod. StCO silencing promoted tuberization under both repressive and weakly inductive photoperiods,
but did not have any effect under strongly inductive short days, demonstrating that StCO represses
tuberization in a photoperiod-dependent manner. The effect of StCO on tuber induction was transmitted
through grafts. In addition, StCO affected the mRNA levels of StBEL5 – a tuberization promoter, the mRNA of
which moves long distances in potato plants – and StFT/StSP6A, a protein highly similar to FLOWERING
LOCUS T (FT), which is a key component of systemic flowering signals in other species. We also found that
StFT/StSP6A transcript levels correlate with the induction of tuber formation in wild-type plants. These results
show that StCO plays an important role in photoperiodic tuberization and, together with the recent
demonstration that StFT/StSP6A promotes tuberization, indicate that the CO/FT module participates in
controlling this process. Moreover, they support the notion that StCO is involved in the expression of long-
distance regulatory signals in potato, as CO does in other species.
Keywords: tuberization, potato, CONSTANS, photoperiod, long-distance signaling, flowering.
INTRODUCTION
The ability of organisms to perceive and respond to photo-
period is essential for adapting to seasonal environmental
changes. Among plant photoperiodic processes, flowering
has been extensively studied (Amasino, 2010). Flowering in
many species is promoted or inhibited under certain pho-
toperiods. Arabidopsis thaliana flowers earlier under long
days (LDs) than short days (SDs). By contrast, other species
promote flowering under SDs [e.g. Oryza sativa (rice) and
Pharbitis nil], and some are day-length neutral [e.g. Sola-
num lycopersicum (tomato) and some Nicotiana tabacum
(tobacco) varieties] and induce flowering in response to
other environmental cues, like temperature, or when a
particular size or developmental stage is reached (Bernier,
1988). Tuber formation also responds to day length. It is
induced by SDs in all Solanum tuberosum (potato) species
and varieties, but the behaviur under LDs is highly variable
(Rodrıguez-Falcon et al., 2006). Tuberization of S. tubero-
sum ssp. andigena is completely inhibited by LDs, whereas
other subspecies and cultivars tuberize under this photo-
period. Because SD responses are based on the perception
of night length, a short period of light during the night (a
night break, NB) can cause an LD-like response. Tuberization
of the andigena subspecies is partially repressed under
SD + NB conditions, such that it is delayed compared with
SDs (Gonzalez-Schain and Suarez-Lopez, 2008; Martin et al.,
2009).
678 ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd
The Plant Journal (2012) 70, 678–690 doi: 10.1111/j.1365-313X.2012.04909.x
The CONTANS/FLOWERING LOCUS T (CO/FT) module is
central for the photoperiodic regulation of plant develop-
mental processes (Turck et al., 2008). CO, first identified in
Arabidopsis, is the founding member of a protein family
comprising transcriptional regulators that contain two
conserved sequence regions: a B-box domain at their N
terminus and a CONSTANS, CONSTANS-like, TOC 1 (CCT)
region at their C terminus (Putterill et al., 1995; Robson
et al., 2001; Griffiths et al., 2003). In Arabidopsis, CO
promotes flowering under inductive photoperiods (Putterill
et al., 1995). Within the CO family, the group containing CO
also includes Heading date 1 (Hd1), which accelerates
flowering under SDs and delays it under LDs in rice, and
PtCO2, involved in the photoperiodic regulation of seasonal
growth cessation and bud set in aspen (Yano et al., 2000;
Griffiths et al., 2003; Hayama et al., 2003; Bohlenius et al.,
2006). Therefore, several genes belonging to the same CO
family group play an important role in photoperiodic
responses. Although three CO-like genes from this group
have been isolated from day length-neutral tomato,
whether they play a role in flowering-time control in this
species remains uncertain (Ben-Naim et al., 2006). A CO
family member also affects photoperiod-regulated pro-
cesses in the unicellular green alga Chlamydomonas rein-
hardtii (Serrano et al., 2009), and another member has been
proposed to be involved in the control of reproduction by
day length in the moss Physcomitrella patens (Shimizu
et al., 2004), indicating an evolutionary conservation of CO
function.
The photoperiodic regulation of flowering by CO relies
on the daily oscillation pattern of its mRNA and the control
of CO protein levels by light (Suarez-Lopez et al., 2001;
Roden et al., 2002; Yanovsky and Kay, 2002; Valverde et al.,
2004). When the CO protein coincides with light, it is
stabilized, and it activates the transcription of FT, which
promotes flowering (Valverde et al., 2004; Turck et al.,
2008). This occurs only under inductive LDs, when CO
mRNA levels peak at the end of the light period and during
the night, whereas under SDs the CO mRNA only accumu-
lates during the dark period (Suarez-Lopez et al., 2001). FT
plays a key role in flowering-time control by integrating
signals from different pathways regulating flowering in
several species. Once FT is transcribed in the leaves in
response to CO activation, the FT protein is transported
through the phloem to the shoot apical meristem, where it
interacts with other proteins to induce the transition to
flowering (Turck et al., 2008).
Recent results suggest that a potato FT-like protein might
act as a systemic tuberization signal (Navarro et al., 2011). In
addition, two RNA molecules are linked to long-distance
signaling for tuber induction. StBEL5 is a homeodomain
transcription factor that promotes potato tuberization when
it is overexpressed (Chen et al., 2003; Banerjee et al., 2006a).
StBEL5 mRNA has been shown to be graft transmissible,
and its movement to stolons correlates with tuber induction
(Banerjee et al., 2006a). In addition, the effect of the
microRNA miR172, which also acts as a tuberization
inducer, is transmitted through graft junctions. This miRNA,
present in the vasculature of potato plants, has been
proposed as a possible mobile tuberization signal (Martin
et al., 2009).
Overexpression of Arabidopsis CO in potato delays tub-
erization under SD and SD + NB conditions (Martınez-Garcıa
et al., 2002; Gonzalez-Schain and Suarez-Lopez, 2008), sug-
gesting the potential involvement of a potato CO gene in the
control of tuber induction. Here we show that a potato CO
family member, StCO, is involved in the photoperiodic
regulation of tuberization. Grafting experiments suggest
that StCO acts upstream of long-distance signaling mole-
cules. This is supported by the fact that StCO influences the
level of StBEL5 mRNA, a mobile transcript. Finally, StCO
affects the expression of a potato FT homolog recently
shown to promote tuberization (Navarro et al., 2011), indi-
cating that the CO/FT module has been recruited for the
photoperiodic control of tuberization in potato plants.
RESULTS
Isolation of a CO family member from potato
Following a cloning strategy based on sequence conserva-
tion among members of the CO family, we isolated a potato
gene, Solanum tuberosum CONSTANS (StCO), encoding a
predicted 410-amino acid protein with two B boxes and a
CCT domain (Figure 1a). A partial sequence of a CO-like
protein from potato, initially named StCO1 (Drobyazina and
Khavkin, 2006), and later renamed St-lCOL1 (Drobyazina and
Khavkin, 2011), shares 95% identity with StCO (Figure 1a).
StCO and St-lCOL1 probably represent different alleles of the
same gene, which corresponds to the gene encoding amino
acid sequence PGSC0003DMP400017796 of the recently
sequenced potato genome (Xu et al., 2011). StCO shows a
high degree of similarity to CO-like proteins involved in the
photoperiodic control of flowering, such as CO (50% iden-
tity) and Hd1 (43% identity; Figure 1a). The similarity is
especially high in the B-box and CCT domains, and also
encompasses four small conserved motifs of the middle
region that are present in the most CO-like proteins (Griffiths
et al., 2003), and the seven C-terminal amino acids that are
identical in StCO and CO (Figure 1a). StCO is also highly
similar to TCOL3 (86% identity) from tomato, which affects
flowering when expressed in Arabidopsis (Ben-Naim et al.,
2006), and PtCO2 (47% identity), involved in the photoperi-
odic regulation of growth cessation in aspen (Bohlenius
et al., 2006; Figure 1a).
A phylogenetic comparison of the StCO amino acid
sequence with other CO family members placed StCO within
the group that contains CO and its closest homologs
(Figure 1b).
Regulation of tuberization by potato CONSTANS 679
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
(a)
(b)
680 Nahuel D. Gonzalez-Schain et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
StCO mRNA shows a daily oscillation
All CO-like genes analyzed so far show cyclic oscillations of
mRNA abundance (e.g. Liu et al., 2001; Suarez-Lopez et al.,
2001; Kojima et al., 2002; Cheng and Wang, 2005; Ben-Naim
et al., 2006; Bohlenius et al., 2006). StCO mRNA levels were
analyzed every 4 h over a period of 24 h under three pho-
toperiods to determine whether this gene is under daily and
day-length control in S. tuberosum ssp. andigena, a potato
genotype with a strong photoperiodic response. Using an
RNA blot analysis, StCO mRNA showed a peak between 20 h
and dawn (0 h), and was almost undetectable for the rest of
the 24-h cycle under SD and SD + NB conditions
(Figure 2a,b; Appendix S1). Under LDs, StCO transcript
abundance peaked between dawn and 4 h, with levels
remaining below detection for the rest of the 24-h cycle
(Figure 2c). Essentially the same pattern was obtained when
StCO levels were examined by reverse transcription fol-
lowed by real-time quantitative PCR (RT-qPCR; Figure 2d).
Despite overall StCO mRNA abundance being lower under
LDs than SDs, 4 h after dawn StCO levels were higher under
LDs than SDs (Figure 2a,c,e). These results show that StCO
transcript levels, like those of CO and other CO family
members, follow a daily oscillation under all photoperiods
tested. The level and oscillation pattern of StCO mRNA are
very similar under SD and SD + NB conditions. Under LDs,
however, the peak is shifted 4 h later than under SDs and
SD + NB, indicating that day length affects the pattern of
StCO expression.
StCO has a weak effect on flowering time
To study the function of StCO, the gene was overexpressed
and silenced in andigena potato. The constitutive CaMV 35S
promoter was used for overexpression. To silence StCO, a
fragment of its middle region, which is highly divergent
among CO-like genes, was used for RNA interference. Four
35S::StCO and four StCO-RNAi lines showing increased and
reduced StCO mRNA levels, respectively (Figure S1), were cho-
(a) (b)
(c)
(e)
(d)
Figure 2. Daily oscillation of StCO transcript
levels in potato.
StCO RNA levels were analyzed by RNA blot
hybridization (a, b, c) and RT-qPCR (d, e). Leaf
samples were collected at the indicated times
after dawn (0 h) under short days (SD) (a), short
days with a night break (SD + NB) (b) and long
days (LD) (c, d), and 4 h after dawn under SDs
and LDs (e). All samples in (a–c) were analyzed
together in a single RNA blot, and quantification
of StCO levels is shown in the lower panels.
Values are represented relative to the highest
value after normalization to the 18S rDNA (a–c)
and StACT (d, e) controls. Values represent the
mean and standard deviation of two indepen-
dent experiments. Arrows indicate the expected
size for StCO mRNA. Sizes of bands of an RNA
ladder are shown. Statistically significant differ-
ences relative to SDs (two-way ANOVA):
**P < 0.01; ***P < 0.001.
Figure 1. Comparison of StCO with CO-like proteins.
(a) Alignment of amino acid sequences of StCO, rice Hd1, Populus trichocarpa PtCO2, Arabidopsis CO, potato cv. Bastoneza StCO1 (StCO1Ba) and tomato TCOL3.
Sequence PGSC0003DMP400017796 (17796) from the recently sequenced potato genome has also been included. Identical amino acids are indicated by asterisks.
Boxes indicate conserved regions: the B boxes near the N terminus, the four small conserved motifs (M1–M4) present in the middle region of the most CO-like
proteins and the CCT domain at the C terminus.
(b) Phylogenetic tree showing protein sequence relationships among CO family members. Complete amino acid sequences were used, except in cases in which only
partial sequences were available. Chlamydomonas reinhardtii CrCO was used as the out-group. Bootstrap values greater than 50% for 100 replicates are indicated at
the nodes. Accession numbers of the proteins used in this figure, as well as the species names and references, are listed in Table S1.
Regulation of tuberization by potato CONSTANS 681
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
sen for further analysis. Given the role of CO in flowering-time
control and the effect of CO expression on potato flowering
(Putterill et al., 1995; Gonzalez-Schain and Suarez-Lopez,
2008), the flowering time of the plants with altered levels of
StCO was examined. Both StCO-RNAi and 35S::StCO plants
transferred from LDs to SDs showed a slight early-flowering
phenotype (Figure S2). We had previously shown that
flowering of wild-type andigena potato occurs essentially at
the same time in plants grown under LDs as in plants
transferred from LDs to SDs (Gonzalez-Schain and Suarez-
Lopez, 2008). We further tested the response of flowering to
photoperiod by growing wild-type andigena plants under
LDs and SDs from the moment they were planted in soil.
Under SDs, wild-type plants needed more days to flower
than under LDs because they grew more slowly
(Figure S3a). However, they flowered with the same number
of leaves under both photoperiods (Figure S3b), indicating
that they flowered at the same developmental stage.
Therefore, under our growth conditions, andigena plants
flower in a photoperiod-insensitive manner. To confirm the
effect of StCO on flowering time, StCO-RNAi and 35S::StCO
plants were grown under LDs. All transgenic lines tested
flowered slightly earlier and after developing fewer leaves
than the wild type (Figure 3a,b). This shows that the proper
regulation of StCO expression is required for flowering-time
control in potato.
To determine whether StCO performs a similar function to
CO, we tested whether StCO can rescue the late-flowering
phenotype of an Arabidopsis co mutant. StCO was
expressed from the 35S promoter in wild-type Arabidopsis
and in the co-2 mutant. Figure S4 shows the transcript level
of StCO in several Arabidopsis 35S::StCO (At-35S::StCO)
plants. All the lines expressing significant levels of StCO in
an otherwise wild-type background flowered later than the
(a) (b)
(c)
(e)
(d)
Figure 3. Flowering time of transgenic plants
with altered levels of StCO.
(a, b) Flowering time of potato wild-type, StCO-
RNAi and 35S::StCO plants grown under long
days (LDs). The number of days from potting to
flowering (a), and the number of leaves at
flowering (b), are shown. Data represent means
and SEMs for at least 17 plants.
(c, d) Flowering time of Arabidopsis wild-type,
co-2 and At-35S::StCO plants under LDs (c) and
short days (SDs) (d). Seven independent
At-35S::StCO lines are shown. The total number
of leaves is shown. Data represent means and
SEMs for at least 14 plants.
(e) Flowering time of At-35S::StCO co-2 plants
under LDs. The total number of leaves is shown.
Data represent the mean and SEM for at least 19
plants.
Significant differences relative to wild type:
**P < 0.01; ***P < 0.001. Statistically significant
differences relative to co-2 plants (one-way ANO-
VA): ###P < 0.001. All data are representative of at
least two independent experiments.
682 Nahuel D. Gonzalez-Schain et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
wild type under LDs (Figure 3c), and three of them showed
early flowering under SDs (Figure 3d). Line At-35S::StCO-27
was crossed to the co-2 mutant and double At-35S::StCO co-
2 homozygous plants were selected. Figure 3e shows that
At-35S::StCO co-2 plants flowered earlier than the co-2
mutant under LD conditions, but still later than the wild
type, indicating that StCO partially rescues the late-flower-
ing phenotype of this mutant.
StCO is involved in the photoperiodic control
of tuberization
As CO family members affect several photoperiodic pro-
cesses (Putterill et al., 1995; Yano et al., 2000; Bohlenius
et al., 2006; Serrano et al., 2009), and expression of Arabi-
dopsis CO in potato affects tuberization time (Martınez-Garcıa
et al., 2002; Gonzalez-Schain and Suarez-Lopez, 2008), we
analyzed the effects of silencing and overexpressing StCO on
tuber induction in potato plants. We used four silenced
(StCO-RNAi-06, -08, -21 and -22) and four overexpressing
(35S::StCO-18, -19, -21 and -25) independent lines. Under
SDs, a strongly inductive photoperiod for tuberization, there
were no significant differences in tuberization time between
StCO-RNAi or 35S::StCO lines and the wild type (Figure 4a,b).
However, all StCO-RNAi lines with reduced levels of StCO
showed earlier tuberization than wild-type plants under
weakly inductive SD + NB conditions (Figure 4c,d). StCO-
RNAi plants tuberized in fewer days than the wild type, and
produced fewer leaves until they started to tuberize. This
indicates that StCO delays tuberization in potato under
SD + NB conditions. To verify this, plants overexpressing
StCO were studied. As expected, 35S::StCO tuberized later
than wild-type plants under SD + NB conditions, a pheno-
type that was more evident in terms of days than in terms of
leaves (Figure 4e,f). We also checked the tuberization of wild-
type, StCO-RNAi and 35S::StCO plants after 4 months of
growth under LDs, a repressive photoperiod. In these
experiments, two StCO-RNAi lines (StCO-RNAi-05 and -07)
with no significant reduction of StCO transcript levels (Fig-
ure S1) were used as additional controls. All the StCO-RNAi
lines with reduced levels of StCO (StCO-RNAi-06, -08, -21 and
-22) tuberized under LDs (Table 1), confirming that StCO has
an inhibitory effect on tuberization. As expected, wild-type
and 35S::StCO plants, as well as control StCO-RNAi-05 and -
07 lines, did not develop tubers for the duration of the LD
(a) (b)
(c) (d)
(e) (f)
Figure 4. Tuberization time of transgenic plants
with altered levels of StCO.
(a–f) Control (wild-type, grey bars), StCO-RNAi
(white bars) and 35S::StCO (black bars) plants
were grown under long days (LDs) for 4 weeks
and then transferred to either short days (SDs) (a,
b) or short days with a night break (SD + NB)
(c–f). Tuberization time was measured as days to
tuberization (a, c, e), or as the total number of
leaves produced before tuberization (b, d, f).
Means and SEMs for at least 10 plants per line
and condition are shown. **Significant differ-
ence relative to wild type at P < 0.01 (one-way
ANOVA). Data are representative of two indepen-
dent experiments.
Regulation of tuberization by potato CONSTANS 683
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
experiments (Table 1). Altogether, the results show that
StCO controls the tuberization response to photoperiod, as
StCO depletion affects tuber induction under SD + NB and
LDs, but not under SDs.
Graft-transmissible repression of tuberization by StCO
In Arabidopsis, CO triggers flowering by activating signals in
leaf phloem that travel through the vascular system to the
shoot apical meristem (An et al., 2004; Ayre and Turgeon,
2004; Corbesier et al., 2007; Jaeger and Wigge, 2007; Mat-
hieu et al., 2007). A similar mechanism has been postulated
in potato tuberization (Rodrıguez-Falcon et al., 2006). To
determine whether StCO controls long-distance signals reg-
ulating tuber induction, we tested whether the tuberization
phenotype of StCO transgenic plants is graft transmissible.
Grafting experiments were performed in the photoperiod
that caused the biggest difference in tuberization between
the wild-type and transgenic plants, i.e. SD + NB for
35S::StCO plants and LDs for StCO-RNAi plants. 35S::StCO/
WT grafts (35S::StCO scions grafted onto wild-type stocks)
tuberized as late as 35S::StCO/35S::StCO or ungrafted
35S::StCO controls under SD + NB conditions (Figure 5),
showing that StCO overexpression in aerial organs is suffi-
cient to delay tuberization. By contrast, the overexpression of
StCO in underground parts had no effect on this process, as
WT/35S::StCO grafts tuberized at the same time as ungrafted
wild-type (WT) or WT/WT controls (Figure 5). Furthermore,
StCO-RNAi/WT grafts tuberized under LDs, like ungrafted
StCO-RNAi or StCO-RNAi/StCO-RNAi controls, whereas, as
expected, ungrafted WT or WT/WT plants did not develop
tubers in this photoperiod (Table 2). Moreover, the depletion
of StCO in underground parts of WT/StCO-RNAi grafts is not
sufficient to release the inhibition of tuberization under LDs
(Table 2). Taken together, these data indicate that StCO is
required in the leaves to control the production and/or
movement of tuberizing signals in potato.
StCO negatively affects StBEL5
In order to understand the molecular mechanism underlying
tuberization control by StCO, we analyzed whether this gene
Table 1 Tuberization of StCO-RNAi and 35S::StCO plants under longdays (LDs)
Number of plants
Experiment 1 Experiment 2
With tubers Total With tubers Total
WT 0 12 0 14StCO-RNAi-05 0 12 ND NDStCO-RNAi-06 5 12 5 13StCO-RNAi-07 0 12 ND NDStCO-RNAi-08 7 12 12 12StCO-RNAi-21 12 12 14 14StCO-RNAi-22 6 12 6 13WT 0 18 0 1135S::StCO-18 0 17 0 1035S::StCO-21 0 15 0 10
ND, not determined. Plants were maintained under LDs for 4 months.
(a)
(b)
Figure 5. Tuberization of 35S::StCO grafts under short days with a night break
(SD + NB).
Five-week-old LD-grown 35S::StCO (line 18) and wild-type plants were
grafted. Ungrafted plants were also used as controls. Grafts and controls
were transferred to SD + NB conditions and tuberization time was measured
as days to tuberization (a), or as the total number of leaves produced before
tuberization (b). Means and SEMs for at least seven grafts of each type and 15
ungrafted control plants are shown. Data are representative of two indepen-
dent experiments.
Table 2 Tuberization of grafted StCO-RNAi plants under long days(LDs)
Number of plants
Experiment 1 Experiment 2
With tubers Total With tubers Total
WT 0 15 0 14StCO-RNAi 10 10 11 11WT/WTa 0 14 0 11WT/StCO-RNAia 0 9 0 10StCO-RNAi/WTa 9 11 8 8StCO-RNAi/StCO-RNAia 10 11 12 12
Plants were maintained under LDs for 4 months. aScion/stock.
684 Nahuel D. Gonzalez-Schain et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
affects the expression of StBEL5, which promotes tuberiza-
tion when it is overexpressed. Leaves of StCO-RNAi plants
displayed higher levels of StBEL5 mRNA than those of wild-
type plants under LDs (Figure 6a). In addition, StBEL5
transcript abundance increased in StCO-RNAi stolons when
these started to swell (Figure 6a; difference between swollen
and unswollen stolons statistically significant at P < 0.01),
showing a correlation between the ectopic induction of
tuberization in StCO-RNAi plants under LDs and the increase
in StBEL5 mRNA levels. These results indicate that silencing
of StCO upregulates StBEL5, and therefore that StCO is
involved in the negative regulation of StBEL5. This was
confirmed in 35S::StCO plants, which showed lower StBEL5
transcript levels than wild-type plants in stolons under
SD + NB conditions (Figure 6b), correlating with a delay in
tuberization.
Correlation of StFT/StSP6A mRNA abundance with tuber
induction
An FT homolog, named StFT/StSP6A, has recently been
cloned from potato and has been shown to promote tuber-
ization (Fan et al., 2010; Navarro et al., 2011). We studied
whether the levels of StSP6A mRNA correlate with tuber
induction. Wild-type plants showed a higher abundance of
StSP6A transcript in leaves than in stolons under SDs and
LDs (Figure 7a). StSP6A mRNA levels were much higher
under SDs than LDs in leaves, showing that high StSP6A
expression is associated with tuber-inducing conditions
(Figure 7a; note that under LDs the levels are so low that
they are not visible on the graph when SDs and LDs are
represented together). Moreover, there was at least a four-
fold increase from unswollen to swollen stolons under SDs
(Figure 7a; statistically significant difference at P < 0.01),
indicating an upregulation of StSP6A at the stolon-to-tuber
transition.
StCO negatively affects StFT/StSP6A
We then examined whether StSP6A is regulated by StCO.
We found that StSP6A mRNA levels are dramatically
upregulated in StCO-RNAi plants grown under LDs, espe-
cially in leaves (Figure 7b). Conversely, 35S::StCO plants
showed reduced StSP6A in all organs analyzed (Figure 7c).
These results demonstrate that StCO negatively regulates
StSP6A. In addition, there was a substantial increase of
StSP6A transcript abundance in stolons of all genotypes at
early stages of tuber induction, as swollen stolons show
considerably higher StSP6A levels than unswollen stolons
(Figure 7b,c; statistically significant difference at P < 0.01).
Therefore, high StSP6A expression correlates again with
tuber induction.
DISCUSSION
We have studied the function of a potato CO-like gene, StCO,
that belongs to group Ia, defined by Griffiths et al. (2003), in
the CO family phylogenetic tree (Figure 1). This group
includes several genes regulating photoperiodic processes,
e.g. Arabidopsis CO, rice Hd1 and Populus PtCO2 (Putterill
et al., 1995; Yano et al., 2000; Griffiths et al., 2003; Bohlenius
et al., 2006). Two potato CO-like genes of this group, short
and long COL1 (St-sCOL1 and St-lCOL1, respectively), have
recently been identified (Drobyazina and Khavkin, 2011).
StCO shows the highest similarity to St-lCOL1 (previously
named StCO1; Drobyazina and Khavkin, 2006); these two
sequences probably represent alleles of the same gene. The
other previously reported potato CO-like proteins, StCOL1
and StCOL (Martınez-Garcıa et al., 2002; Guo et al., 2007),
are much more distant from CO, Hd1 and PtCO2 (Figure 1b),
and are therefore less likely to have a similar function.
(a)
(b)
Figure 6. Effect of StCO on StBEL5 mRNA levels.
RT-qPCR analysis of StBEL5 mRNA in leaves, stems, stolons and swollen
stolons from wild-type and StCO-RNAi plants grown under long days (LDs) for
8 weeks (a), and from wild-type and 35S::StCO plants grown under short days
with a night break (SD + NB) for 4 weeks (b). All samples were collected 4 h
after dawn. Data were standardized to StACT mRNA levels. Relative StBEL5
levels are given, with levels in wild-type leaves set to 1. Note that wild-type
plants did not tuberize under LDs, and therefore did not produce swollen
stolons. Values represent the mean and standard deviation of two biological
samples analyzed in triplicate. *Statistically significant difference relative to
wild type at P < 0.05 (two-way ANOVA). Three independent experiments were
performed, and one is shown as being representative.
Regulation of tuberization by potato CONSTANS 685
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
The high similarity of StCO to CO, Hd1 and PtCO2
suggested that StCO could play a role in photoperiodic
control in potato. Indeed, our results demonstrate that
StCO is involved in the photoperiodic regulation of tuber
induction. This is shown by the tuberization of StCO-RNAi
plants under non-inductive LDs and the early tuberization
of these plants under moderately inductive SD + NB
conditions, and further confirmed by the late tuberization
of 35S::StCO plants under this photoperiod (Figure 4;
Table 1). StCO silencing does not affect tuberization under
SDs, suggesting that StCO does not play any role in the
control of tuber formation under strongly inductive condi-
tions. The effect of StCO on tuberization is opposite to that
of CO on flowering. Whereas CO promotes flowering
under LDs, which are inductive for Arabidopsis, StCO
represses tuberization under LDs, which are inhibitory for
potato, a result also supported by Navarro et al. (2011).
Neither CO nor StCO seem to play a role under SDs.
Several CO family members repress SD responses under
LDs: Hd1 represses flowering in rice, PtCO2 represses bud
set in Populus trees and StCO represses tuberization in
potato (Yano et al., 2000; Bohlenius et al., 2006; Figure 4;
Table 1). Despite this similarity, differences among these SD
responses also exist. Hd1 has an opposite effect on flower-
ing under LDs and SDs, acting as a flowering promoter
under SDs (Yano et al., 2000), whereas StCO affects tuber-
ization under LDs, but not under SDs (Figure 4; Table 1).
Therefore, although the role of CO proteins in photoperiodic
control is conserved in several plant species, at least in three
of them – potato, rice and Arabidopsis – there are differences
in the mechanisms by which CO acts.
To understand the molecular mechanisms of tuberization
repression by StCO, we studied its effect on tuberization-
associated genes. Overexpression of StBEL5 induces tuber
formation (Chen et al., 2003; Banerjee et al., 2006a).
Recently, an FT homolog, StFT/StSP6A, has been identified
in potato and shown to act as a tuberization inducer (Fan
et al., 2010; Navarro et al., 2011). Consistent with this,
StSP6A mRNA levels in wild-type andigena plants correlate
with tuber induction (Figure 7a), in agreement with previous
analyses of StSP6A expression (Fan et al., 2010; Navarro
et al., 2011). The higher levels of this transcript in leaves than
in stolons (Figure 7a) are consistent with the transcription of
FT taking place mainly in leaves in Arabidopsis (Takada and
Goto, 2003; An et al., 2004). Moreover, StCO is involved in
regulating both StSP6A and StBEL5 mRNA levels (Figures 6
and 7), strongly suggesting that the role of StCO in the
photoperiodic control of tuberization is mediated by StSP6A
and StBEL5. Upregulation of StSP6A and StBEL in StCO-
RNAi plants and downregulation in 35S::StCO plants corre-
late with the de-repressed tuberization phenotype of
StCO-RNAi plants under LDs and the late tuberization of
35S::StCO plants under SD + NB conditions, respectively.
(a)
(b)
(c)
Figure 7. Correlation of StFT/StSP6A mRNA levels with tuber induction and
effect of StCO on StFT/StSP6A mRNA levels. StSP6A transcript levels were
analyzed by RT-qPCR in leaves, stolons and swollen stolons from wild-type,
StCO-RNAi and 35S::StCO plants.
(a) Wild-type plants grown under short days (SDs) and long days (LDs). Plants
were grown under LDs for 4 weeks and then either transferred to SDs or
maintained under LDs. Eleven days later, samples were collected. Relative
StSP6A levels are given, with levels in leaves under SDs set to 1. Note that
wild-type plants grown under LDs did not tuberize and therefore did not
produce swollen stolons. The inset shows LD-grown plants from the same
experiment represented alone, in order to see the difference between leaves
and stolons. ***Statistically significant difference relative to an SD leaf at
P < 0.001 (two-way ANOVA).
(b) Wild-type and StCO-NAi plants grown under LDs for 8 weeks.
(c) Wild-type and 35S::StCO plants grown under short days with a night break
(SD + NB) for 4 weeks.
In (b) and (c) the relative StSP6A levels are given, with levels in wild-type
leaves set to 1. Statistically significant differences relative to the wild type
(two-way ANOVA): *P < 0.05; **P < 0.01. All samples were collected 4 h after
dawn. Data were standardized to StACT mRNA levels. Values represent the
mean and standard deviation of two biological samples analyzed in triplicate.
Four independent experiments were performed and one is shown as being
representative.
686 Nahuel D. Gonzalez-Schain et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
The effect of StCO on StSP6A is in accordance with the
results of Navarro et al. (2011).
We propose a mechanism for StCO function under LDs
similar to that of rice Hd1 and opposite to that of Arabidopsis
CO, with StCO repressing StSP6A, and in addition StBEL5,
expression. In Arabidopsis and rice, CO/Hd1 transcript levels
are high during the light period only under LDs (Suarez-
Lopez et al., 2001; Hayama et al., 2003). Similarly, StCO
mRNA peaks during the light period under LDs and mainly
during the night under SDs (Figure 2). On the basis of what
has been found in Arabidopsis, we speculate that light is
required to stabilize or activate the StCO protein. Thus, StCO
would be active under LDs, leading to the repression of
StSP6A and StBEL5 transcription. Tuberization, therefore,
would not be induced. Under SDs, StCO would not coincide
with light and thus would remain inactive, enabling StSP6A
and StBEL5 expression and the induction of tuber formation.
Although Navarro et al. (2011) hypothesize that StCO acti-
vates StSP6A under SDs to promote tuberization, our data
indicate that StCO does not affect tuberization under SDs,
consistent with the fact that StSP6A levels are not altered in
StCO-overexpressing plants after six SDs (Navarro et al.,
2011).
In line with the hypothesis that StCO may be regulated by
light, plants with reduced levels of the photoreceptor phy-
tochrome B (PHYB) show a tuberization phenotype similar to
that of StCO-RNAi plants (Jackson et al., 1996; Figure 4;
Table 1). In rice, the effect of SD + NB is mediated by PHYB
through the downregulation of Hd3a, an FT homolog, in a
manner that is partially dependent on Hd1, but without
affecting Hd1 mRNA levels (Ishikawa et al., 2005). In potato,
StCO transcript levels are also very similar under SD and
SD + NB. It would be interesting to test whether the regu-
lation of tuberization by StCO under SD + NB is caused by
the effects of PHYB on StCO protein stability, as has been
proposed for rice (Ishikawa et al., 2005).
The effect of StCO on tuberization is graft transmissible
(Figure 5; Table 2), suggesting the involvement of StCO in
the long-distance signaling mechanisms that control tuber-
ization. In Arabidopsis and rice, CO/Hd1 regulates the
production of graft-transmissible signals, i.e. FT/Hd3a, to
modulate flowering in response to photoperiod (Turck et al.,
2008). In potato, StCO regulates the levels of StSP6A and
StBEL5 mRNAs. The upregulation of these two transcripts in
StCO-RNAi leaves is in agreement with CO acting in leaves
to control FT expression in Arabidopsis (An et al., 2004).
Given that the StBEL5 mRNA has been shown to be mobile
(Banerjee et al., 2006a), the high StBEL5 levels in swollen
stolons of StCO-RNAi plants might be the result of StBEL5
mRNA movement from leaves to stolons, although tran-
scriptional regulation of StBEL5 by StCO in stolons is also
possible. It had previously been suggested that PHYB
induces the production of a systemic inhibitor of tuberiza-
tion (Jackson et al., 1998). Our results indicate that StCO
represses mobile tuber-promoting molecules. Therefore, it
is likely that the long-distance tuberization signals include
both positive and negative regulators, as had been proposed
for flowering decades ago (Lang, 1952).
The results of Navarro et al. (2011) suggest that in stocks
of grafted plants StCO has a slight effect on tuberization and
StSP6A mRNA levels, but this has only been shown when
StSP6A-overexpressing scions are grafted onto StCO-over-
expressing stocks. By contrast, when wild-type scions are
used, neither overexpression nor silencing of StCO in stocks
affects tuberization (Figure 5; Table 2), suggesting that the
effect of StCO in stocks only occurs when StSP6A levels are
constitutively increased in the scion.
Despite flowering time not being responsive to photope-
riod in andigena potato, it is affected by StCO (Figure 3;
Figures S2 and S3), indicating that a CO gene influences
floral induction in a plant that is day-length neutral for
flowering. Unexpectedly, both silencing and overexpression
of StCO cause early flowering. This can be explained by the
fact that StSP3D, a potato FT homolog that promotes
flowering, is upregulated in StCO-RNAi and StCO-over-
expressing plants (Navarro et al., 2011). These findings
suggest that a CO/FT module can act in the control of
flowering in a day-length neutral plant. The effect of StCO on
potato flowering is significant, but much weaker than its
effect on tuber induction. The late flowering of an Arabid-
opsis co mutant is only partially complemented by StCO,
indicating that this gene is not a strong flowering-time
regulator (Figure 3c–e; Appendix S1).
Therefore, the main role of StCO is to regulate tuberiza-
tion, a photoperiodic process, but this gene still plays some
role in flowering-time control in potato. Given that potato
plants can perpetuate through tuberization, it is possible that
the evolutionary pressure to maintain the role of StCO in
flowering has diminished. On the other hand, the involve-
ment of other potato CO-like genes, e.g. St-sCOL1, in
flowering has not been ruled out. Perhaps StCO/St-lCOL1
and St-sCOL1 have diverged and subfunctionalized in
potato, in a similar way as StFT/StSP6A and StSP3D.
Taking together the results presented here, we conclude
that StCO is the potato functional counterpart of CO, Hd1
and PtCO2 in terms of day-length regulation. The ancestral
role of this key regulatory protein in photoperiodic control is
conserved, as has been proposed previously (Serrano et al.,
2009), but different species have recruited it for diverse
developmental responses, adapting its mechanism of action
in slightly different ways. In addition, similarly to Arabidopsis
CO and rice Hd1, StCO participates in regulating the
expression of mobile molecules affecting photoperiodic
processes. Finally, our findings, together with those of
Navarro et al. (2011), indicate that a CO/FT module is
involved in the control of tuber induction by day length,
and suggest that a second CO/FT module affects day-neutral
flowering in potato.
Regulation of tuberization by potato CONSTANS 687
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
EXPERIMENTAL PROCEDURES
Plant material and growth conditions
Solanum tuberosum L. ssp. andigena line 7540 (Jackson and Prat,1996) was used as the wild-type potato. Plants were vegetativelypropagated and grown in vitro, as previously described (Martinet al., 2009). Two-week-old plants were planted in soil and growneither in the glasshouse at 23�C under LDs or in controlled-envi-ronment chambers at 23�C under SDs (8-h light and 16-h dark) orSDs supplemented with a 30-min white-light night break, given 8 hafter the start of the dark period (SD + NB), as previously described(Martin et al., 2009).
Arabidopsis thaliana ecotype Landsberg erecta (Ler) was used asthe wild-type Arabidopsis. The co-2 mutant, which also contains thetransparent testa 4 (tt4) mutation, was kindly provided by GeorgeCoupland (John Innes Centre, Norwich, UK). Seeds were incubatedon moist filter paper in darkness at 4�C for 4–7 days prior to sowingon soil. Soil composition was identical for Arabidopsis and potato.Plants were grown in the glasshouse at 22�C under LDs. Plants werewatered three times per week and fertilized weekly with modifiedHoagland’s solution (Johnson et al., 1957) diluted 1/240.
Cloning of StCO
Degenerate primers were designed from the regions encoding theconserved B-box and CCT domains of the CO family, and used in aPCR reaction with andigena genomic DNA as the template. A 1.16-kb PCR product was purified, cloned into pGEM�-T Easy (Promega,http://www.promega.com) and sequenced. The nucleotidesequences of several clones showed high similarity with CO, PnCOand Hd1. A cDNA library from the ga1 dwarf mutant of S. tubero-sum ssp. andigena (Carrera et al., 1999) was screened using one ofthese clones as a probe, labeled with digoxigenin, according to themanufacturer’s instructions (Roche Diagnostics, http://www.rochediagnostics.es). Eighteen clones were obtained and eightwere sequenced. Among these, clone 10.1.1 contained a completeopen reading frame (ORF), sharing high similarity with CO andPnCO. We initially named this gene StCOL3, but later we renamed itStCO based on its inclusion in the CO/Hd1 clade of the CO familyphylogenetic tree, and its function in flowering and photoperiodictuberization (see Results). This cDNA sequence has been depositedin the EMBL Nucleotide Sequence Database, with accession numberAM888389.
Phylogenetic analysis
A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) using theStCO protein sequence as a query was performed. Sixty-sevenrepresentative protein sequences with E values below 10)20 werealigned with CLUSTALX (Thompson et al., 1997). A preliminary phy-logenetic tree was constructed using the neighbor-joining algo-rithm of TREECON (Van de Peer and De Wachter, 1994). Afterchoosing 35 representative proteins from the different clades andadding 17 new CO-like sequences from the potato genome (Xu etal., 2011), with E values also below 10)20, a new phylogenetic treewas constructed, with Chlamydomonas reinhardtii CrCO (Serranoet al., 2009) as the out-group. The number of bootstrap replicateswas 100.
Generation of transgenic lines with increased or reduced
levels of StCO
A fusion of the StCO coding sequence to the CaMV 35S promoter(35S::StCO) was constructed into vectors pCHF3 and pBINAR, gen-
erating plasmids pCHF3-StCO and pBINAR-StCO, respectively.Details of the cloning procedures are given in Appendix S1. pBIN-AR-StCO was introduced by electroporation into Agrobacteriumtumefaciens strain C58 GV2260, which was used to transformandigena potato essentially as described by Banerjee et al. (2006b).Twenty-six kanamycin-resistant plants were regenerated and thenpropagated from single-node stem cuttings on MS medium con-taining 20 g l)1 sucrose, 50 mg l)1 kanamycin, 250 mg l)1 cefotaximeand 2 g l)1 Gelrite� (Duchefa Biochemie, http://www.duchefa.com).
Agrobacterium tumefaciens C58 GV2260 carrying the pCHF3-StCO construct was used to transform Arabidopsis by the floral-dipmethod (Clough and Bent, 1998). Thirty-five kanamycin-resistantlines were obtained and self-pollinated. Lines containing a singleinsertion of the transgene were selected based on kanamycin-resistance segregation in the T2 generation. Homozygous lines wereidentified in the T3 generation and used for all experiments shown.
Four transgenic potato lines (35S::StCO) and seven Arabidopsislines overexpressing StCO (At-35S::StCO) were selected for furtheranalysis.
At-35S::StCO plants (line 27) were crossed with the Arabidopsisco-2 mutant to obtain At-35S::StCO co-2. Double homozygoteswere identified in the F2 population by segregation analysis ofkanamycin resistance, the yellow color of the seeds carrying the tt4mutation linked to co-2, and the presence of a CAPS marker for co-2.Details of this marker and its use for genotyping the F2 populationare described in Appendix S1.
To reduce StCO levels in potato plants, a StCO-RNAi constructwas cloned into pHELLSGATE12 (Helliwell and Waterhouse, 2003)by Gateway� recombination, generating plasmid pHELL12-StCOi.Details of the cloning procedure are given in Appendix S1.pHELL12-StCOi was used to transform andigena potato afterintroducing it into A. tumefaciens, as described above. Twenty-eight kanamycin-resistant plants were regenerated and propagated,as described above. The transgenic plants obtained were analyzedfor StCO mRNA levels by RT-PCR, as described below. Fourtransgenic lines showing reduced levels of StCO mRNA wereselected for further analysis.
Measurement of flowering and tuberization time
To analyze flowering time, potato plants were grown under LDs inthe glasshouse. Flowering time was determined as previouslydescribed (Martin et al., 2009).
Arabidopsis plants were grown in controlled-environment cham-bers under SDs as described above, or under LDs (16-h light and 8-hdark) at 22�C. The lighting in the LD chamber was provided byfluorescent lamps TLD840 and TLD827 (Philips Iberica, http://www.philips.es). Flowering time was measured as the total numberof rosette and cauline leaves produced on the main stem.
For tuberization experiments, potato plants grown in the glass-house were transferred to SD or SD + NB controlled-environmentchambers approximately 4 weeks after potting. LD experimentswere performed in the glasshouse. Tuberization was analyzed onceper week under SD and SD + NB conditions, and after 4 months ofgrowth under LDs. Tuberization time was measured as the numberof days from transfer to SD or SD + NB conditions to the appear-ance of tubers. Leaf number was also recorded when tubers werefirst visible.
Grafting experiments
Five-week-old LD-grown StCO-RNAi (line 8), 35S::StCO (line 18)and wild-type plants were used. V-shape grafts were made as pre-viously described (Martin et al., 2009). Ten days after grafting, whengraft unions healed, the stock leaves were removed and plants were
688 Nahuel D. Gonzalez-Schain et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690
either transferred to SD + NB conditions (for 35S::StCO and wild-type grafts) or maintained in the LD glasshouse (for StCO-RNAi andwild-type grafts). Tuberization time was determined as describedabove.
Analysis of mRNA levels
Plants were grown under LDs and transferred to either SD orSD + NB conditions 4 weeks after potting. For StCO analysis, leafsamples were harvested 1 week after transfer, starting at dawn,every 4 h over a period of 24 h. Leaf 8 (counted from the shootapex) was used and two leaves, each from a different plant, werecombined for each sample. For StBEL5 and StFT/StSP6A analysis,leaf, stolon and swollen stolon samples were collected underSD + NB and LD conditions, when the plants started to tuberize.Total RNA was isolated as described by Logemann et al. (1987), orusing the Real ARNzol Spin kit (+PVP; Durviz, http://www.durviz.com). Any remaining DNA was removed using the DNA-freeTM
kit (Ambion, http://www.ambion.com), following the manufac-turer’s instructions.
For RNA blot analysis, the StCO probe was the 234-bp fragmentused to make the StCO-RNAi construct described above. The 18SrRNA probe was described previously (Chen et al., 2003). Valueswere represented relative to the lowest value after normalization tothe 18S control. Further details are given in Appendix S1.
For reverse transcription followed by real-time quantitative PCR(RT-qPCR), the following primers were used. StBEL5 and StACTIN(StACT) qPCR primers have been described (Martin et al., 2009).Primers used for StCO and StFT/StSP6A qPCR were StCO forward(5¢—AGGCCAAGAATCAAAGGC-3¢), StCO reverse (5¢—ACTGCTGTAGTACATTTCTC-3¢), StFTfor (5¢-GACGATCTTCGCAACTTTTACA-3¢)and StFTrev (5¢—CCTCAAGTTAGGGTCGCTTG-3¢). Details of theRT-qPCR protocol are given in Appendix S1.
ACKNOWLEDGEMENTS
We thank Salome Prat for the sequence of the StSP6A primers andadvice on the cloning of StCO; George Coupland for the Arabidopsisco-2 mutant; David Hannapel for the plasmid used to generate the18S probe; Jose Castresana for advice on phylogenetic analysis;and Marta Casado, Eva Saavedra, Leire Etxarri, Menanna Cherkaoui,Cristina Mejıa and Cristina Valdivieso for technical assistance. Thiswork was supported by the Spanish Ministry of Science and Inno-vation (grants BIO2002-00933, BIO2005-00717, BIO2008-00760, allco-financed by the European Regional Development Fund, andCSD2007-00036) and the Generalitat de Catalunya (2005SGR-00182and Xarxa de Referencia en Biotecnologia). PS-L was supported by aRamon y Cajal contract and the I3 Program from the Spanish Min-istry of Education and Science. M _Z was a recipient of a JAE PhDfellowship from CSIC.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the onlineversion of this article:Figure S1. StCO mRNA levels in potato 35S::StCO and StCO-RNAilines.Figure S2. Flowering time of transgenic plants with altered levels ofStCO under short days.Figure S3. Flowering time of wild-type potato plants grown undershort days and long days.Figure S4. StCO mRNA levels in Arabidopsis 35S::StCO plants.Table S1. Sequence accession numbers of proteins used forFigure 1.Appendix S1. Supporting text and experimental procedures.
Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.
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ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 70, 678–690