Potato CONSTANS is involved in photoperiodic tuberization in a graft-transmissible manner

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 [email protected]).

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.


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.



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.



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.



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.



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.



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.



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.



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



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.

REFERENCES

Amasino, R. (2010) Seasonal and developmental timing of flowering. Plant J.

61, 1001–1013.

An, H., Roussot, C., Suarez-Lopez, P. et al. (2004) CONSTANS acts in the

phloem to regulate a systemic signal that induces photoperiodic flowering

of Arabidopsis. Development, 131, 3615–3626.

Ayre, B.G. and Turgeon, R. (2004) Graft transmission of a floral stimulant

derived from CONSTANS. Plant Physiol. 135, 2271–2278.

Banerjee, A.K., Chatterjee, M., Yu, Y., Suh, S.-G., Miller, W.A. and Hannapel,

D.J. (2006a) Dynamics of a mobile RNA of potato involved in a long-dis-

tance signaling pathway. Plant Cell, 18, 3443–3457.

Banerjee, A.K., Prat, S. and Hannapel, D.J. (2006b) Efficient production of

transgenic potato (S. tuberosum L. ssp andigena) plants via Agrobacterium

tumefaciens-mediated transformation. Plant Sci. 170, 732–738.

Ben-Naim, O., Eshed, R., Parnis, A., Teper-Bamnolker, P., Shalit, A., Coupland,

G., Samach, A. and Lifschitz, E. (2006) The CCAAT binding factor can

mediate interactions between CONSTANS-like proteins and DNA. Plant J.

46, 462–476.

Bernier, G. (1988) The control of floral evocation and morphogenesis. Annu.

Rev. Plant Physiol. Plant Mol. Biol. 39, 175–219.

Bohlenius, H., Huang, T., Charbonnel-Campaa, L., Brunner, A.M., Jansson, S.,

Strauss, S.H. and Nilsson, O. (2006) CO/FT regulatory module controls

timing of flowering and seasonal growth cessation in trees. Science, 312,

1040–1043.

Carrera, E., Jackson, S.D. and Prat, S. (1999) Feedback control and diurnal

regulation of gibberellin 20-oxidase transcript levels in potato. Plant

Physiol. 119, 765–773.

Chen, H., Rosin, F.M., Prat, S. and Hannapel, D.J. (2003) Interacting tran-

scription factors from the three-amino acid loop extension superclass

regulate tuber formation. Plant Physiol. 132, 1391–1404.

Cheng, X.F. and Wang, Z.Y. (2005) Overexpression of COL9, a CONSTANS-

LIKE gene, delays flowering by reducing expression of CO and FT in Ara-

bidopsis thaliana. Plant J. 43, 758–768.

Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agro-

bacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,

735–743.

Corbesier, L., Vincent, C., Jang, S. et al. (2007) FT Protein Movement Con-

tributes to Long-Distance Signaling in Floral Induction of Arabidopsis.

Science, 316, 1030–1033.

Drobyazina, P.E. and Khavkin, E.E. (2006) A structural homolog of CONSTANS

in potato. Russ. J. Plant Physiol. 53, 698–701.

Drobyazina, P.E. and Khavkin, E.E. (2011) The structure of two CONSTANS-

LIKE1 genes in potato and its wild relatives. Gene, 471, 37–44.

Fan, C.-y., Yin, J.-m., Wang, B., Zhang, Y.-f. and Yang, Q. (2010) Molecular

cloning and expression analysis of a FT homologous gene from Solanum

tuberosum. Agr. Sci. China, 9, 1133–1139.

Gonzalez-Schain, N.D. and Suarez-Lopez, P. (2008) CONSTANS delays flow-

ering and affects tuber yield in potato. Biol. Plantarum, 52, 251–258.

Griffiths, S., Dunford, R.P., Coupland, G. and Laurie, D.A. (2003) The evolution

of CONSTANS-like gene families in barley, rice, and Arabidopsis. Plant

Physiol. 131, 1855–1867.

Guo, J.L., Yang, Q., Liang, F., Xing, Y.J. and Wang, Z. (2007) Molecular cloning

and expression analysis of a novel CONSTANS-like gene from potato.

Biochemistry, 72, 1241–1246.

Hayama, R., Yokoi, S., Tamaki, S., Yano, M. and Shimamoto, K. (2003)

Adaptation of photoperiodic control pathways produces short-day flow-

ering in rice. Nature, 422, 719–722.

Helliwell, C. and Waterhouse, P. (2003) Constructs and methods for high-

throughput gene silencing in plants. Methods, 30, 289–295.

Ishikawa, R., Tamaki, S., Yokoi, S., Inagaki, N., Shinomura, T., Takano, M.

and Shimamoto, K. (2005) Suppression of the floral activator Hd3a is

the principal cause of the night break effect in rice. Plant Cell, 17, 3326–

3336.



Jackson, S.D. and Prat, S. (1996) Control of tuberisation in potato by gibber-

ellins and phytochrome B. Physiol. Plantarum, 98, 407–412.

Jackson, S.D., Heyer, A., Dietze, J. and Prat, S. (1996) Phytochrome B medi-

ates the photoperiodic control of tuber formation in potato. Plant J. 9, 159–

166.

Jackson, S.D., James, P., Prat, S. and Thomas, B. (1998) Phytochrome B

affects the levels of a graft-transmissible signal involved in tuberization.

Plant Physiol. 117, 29–32.

Jaeger, K.E. and Wigge, P.A. (2007) FT Protein Acts as a Long-Range Signal in

Arabidopsis. Curr. Biol. 17, 1050–1054.

Johnson, C.M., Stout, P.R., Broyer, T.C. and Carlton, A.B. (1957) Comparative

chlorine requirements of different plant species. Plant Soil, 8, 337–353.

Kojima, S., Takahashi, Y., Kobayashi, Y., Monna, L., Sasaki, T., Araki, T. and

Yano, M. (2002) Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes

transition to flowering downstream of Hd1 under short-day conditions.

Plant Cell Physiol. 43, 1096–1105.

Lang, A. (1952) Physiology of flowering. Annu. Rev. Plant Physiol. 3, 265–306.

Liu, J., Yu, J., McIntosh, L., Kende, H. and Zeevaart, J.A. (2001) Isolation of a

CONSTANS ortholog from Pharbitis nil and its role in flowering. Plant

Physiol. 125, 1821–1830.

Logemann, J., Schell, J. and Willmitzer, L. (1987) Improved method for the

isolation of RNA from plant tissues. Anal. Biochem. 163, 16–20.

Martin, A., Adam, H., Dıaz-Mendoza, M., _Zurczak, M., Gonzalez-Schain, N.D.

and Suarez-Lopez, P. (2009) Graft-transmissible induction of potato tub-

erization by the microRNA miR172. Development 136, 2873–2881.

Martınez-Garcıa, J.F., Virgos-Soler, A. and Prat, S. (2002) Control of photo-

period-regulated tuberization in potato by the Arabidopsis flowering-time

gene CONSTANS. Proc. Natl Acad. Sci. USA, 99, 15211–15216.

Mathieu, J., Warthmann, N., Kuttner, F. and Schmid, M. (2007) Export of FT

Protein from Phloem Companion Cells Is Sufficient for Floral Induction in

Arabidopsis. Curr. Biol. 17, 1055–1060.

Navarro, C., Abelenda, J.A., Cruz-Oro, E., Cuellar, C.A., Tamaki, S., Silva, J.,

Shimamoto, K. and Prat, S. (2011) Control of flowering and storage organ

formation in potato by FLOWERING LOCUS T. Nature, 478, 119–122.

Putterill, J., Robson, F., Lee, K., Simon, R. and Coupland, G. (1995) The CON-

STANS gene of Arabidopsis promotes flowering and encodes a protein

showing similarities to zinc finger transcription factors. Cell, 80, 847–857.

Robson, F., Costa, M.M.R., Hepworth, S.R., Vizir, I., Pineiro, M., Reeves, P.H.,

Putterill, J. and Coupland, G. (2001) Functional importance of conserved

domains in the flowering-time gene CONSTANS demonstrated by analysis

of mutant alleles and transgenic plants. Plant J. 28, 619–631.

Roden, L.C., Song, H.-R., Jackson, S., Morris, K. and Carre, I.A. (2002) Floral

responses to photoperiod are correlated with the timing of rhythmic

expression relative to dawn and dusk in Arabidopsis. Proc. Natl Acad. Sci.

USA, 99, 13313–13318.

Rodrıguez-Falcon, M., Bou, J. and Prat, S. (2006) Seasonal control of tuber-

ization in potato: conserved elements with the flowering response. Annu.

Rev. Plant Biol. 57, 151–180.

Serrano, G., Herrera-Palau, R., Romero, J.M., Serrano, A., Coupland, G. and

Valverde, F. (2009) Chlamydomonas CONSTANS and the Evolution of Plant

Photoperiodic Signaling. Curr. Biol. 19, 359–368.

Shimizu, M., Ichikawa, K. and Aoki, S. (2004) Photoperiod-regulated expres-

sion of the PpCOL1 gene encoding a homolog of CO/COL proteins in the

moss Physcomitrella patens. Biochem. Biophys. Res. Commun. 324, 1296–

1301.

Suarez-Lopez, P., Wheatley, K., Robson, F., Onouchi, H., Valverde, F. and

Coupland, G. (2001) CONSTANS mediates between the circadian clock and

the control of flowering in Arabidopsis. Nature, 410, 1116–1120.

Takada, S. and Goto, K. (2003) TERMINAL FLOWER2, an Arabidopsis homo-

log of HETEROCHROMATIN PROTEIN1, counteracts the activation of

FLOWERING LOCUS T by CONSTANS in the vascular tissues of leaves to

regulate flowering time. Plant Cell, 15, 2856–2865.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G.

(1997) The CLUSTAL_X windows interface: flexible strategies for multiple

sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25,

4876–4882.

Turck, F., Fornara, F. and Coupland, G. (2008) Regulation and Identity of Flo-

rigen: FLOWERING LOCUS T Moves Center Stage. Annu. Rev. Plant Biol.

59, 573–594.

Valverde, F., Mouradov, A., Soppe, W., Ravenscroft, D., Samach, A. and

Coupland, G. (2004) Photoreceptor regulation of CONSTANS protein in

photoperiodic flowering. Science, 303, 1003–1006.

Van de Peer, Y. and De Wachter, R. (1994) TREECON for Windows: a software

package for the construction and drawing of evolutionary trees for the

Microsoft Windows environment. Comput. Appl. Biosci. 10, 569–570.

Xu, X., Pan, S., Cheng, S. et al. (2011) Genome sequence and analysis of the

tuber crop potato. Nature, 475, 189–195.

Yano, M., Katayose, Y., Ashikari, M. et al. (2000) Hd1, a major photoperiod

sensitivity quantitative trait locus in rice, is closely related to the Arabid-

opsis flowering time gene CONSTANS. Plant Cell, 12, 2473–2483.

Yanovsky, M.J. and Kay, S.A. (2002) Molecular basis of seasonal time mea-

surement in Arabidopsis. Nature, 419, 308–312.



Potato CONSTANS is involved in photoperiodic tuberization in a graft-transmissible manner

Documents

Transcript of Potato CONSTANS is involved in photoperiodic tuberization in a graft-transmissible manner