CENL1 Expression in the Rib Meristem Affects StemElongation and the Transition to Dormancy in Populus W OA

Raili Ruonala,a Paivi L.H. Rinne,b Jaakko Kangasjarvi,a and Christiaan van der Schootb,1

a Plant Biology, Department of Biological and Environmental Sciences, University of Helsinki, FI-00014 Helsinki, Finlandb Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, N-1432 As, Norway

We investigated the short day (SD)–induced transition to dormancy in wild-type hybrid poplar (Populus tremula 3 P. tremuloides)

and its absence in transgenic poplar overexpressing heterologous PHYTOCHROME A (PHYA). CENTRORADIALIS-LIKE1

(CENL1), a poplar ortholog of Arabidopsis thaliana TERMINAL FLOWER1 (TFL1), was markedly downregulated in the wild-type

apex coincident with SD-induced growth cessation. By contrast, poplar overexpressing a heterologous Avena sativa PHYA

construct (P35S:AsPHYA), with PHYA accumulating in the rib meristem (RM) and adjacent tissues but not in the shoot apical

meristem (SAM), upregulated CENL1 in the RM area coincident with an acceleration of stem elongation. In SD-exposed

heterografts, both P35S:AsPHYA and wild-type scions ceased growth and formed buds, whereas only the wild type assumed

dormancy and P35S:AsPHYA showed repetitive flushing. This shows that the transition is not dictated by leaf-produced

signals but dependent on RM and SAM properties. In view of this, callose-enforced cell isolation in the SAM, associated with

suspension of indeterminate growth during dormancy, may require downregulation of CENL1 in the RM. Accordingly,

upregulation of CENL1/TFL1 might promote stem elongation in poplar as well as in Arabidopsis during bolting. Together, the

results suggest that the RM is particularly sensitive to photoperiodic signals and that CENL1 in the RM influences transition to

dormancy in hybrid poplar.

INTRODUCTION

Morphogenesis at the shoot apical meristem (SAM) is a robust

process that results in the formation of leaf-stem units in orga-

nized patterns. The SAM has a high degree of autonomy, and iso-

lated SAMs can continue shoot formation (Smith and Murashige,

1970; Ball, 1980). This may require the regeneration of a rib mer-

istem (RM), a meristematic region immediately subjacent to the

SAM, as the continuous production of leaf-stem units during in-

determinate growth (Smith and Murashige, 1970; Esau, 1977) is a

coordinated effort of both SAM and RM. Although the SAM and

the RM were originally recognized as separate areas with distinct

morphogenetic tasks, this distinction gradually became unclear

due to the tendency to include the RM in the SAM (Esau, 1977).

Since then, the RM has received little attention (Sablowski, 2007).

The RM is composed of small densely cytoplasmic cells that

produce the ribs of vacuolated and elongated cells that make up

the rib zone (RZ). Together, the RM and the RZ push up the SAM

(Bernier et al., 1981; Harkess and Lyons, 1993).

Despite the robustness of the apical morphogenetic centers,

the apex is highly sensitive to stimuli contributed by the shoot

system (Steeves and Sussex, 1989). For example, in response to

a combination of endogenous and environmental cues, a SAM

can give up indeterminate growth, either permanently or tempo-

rarily. A SAM may transform itself into an inflorescence meristem

(IM) that continues indeterminate growth while producing branches

with determinate floral primordia. Alternatively, a SAM may dif-

ferentiate directly into a determinate flower (Bernier et al., 1993)

or suspend indeterminacy and transit to dormancy for survival

through winter (van der Schoot, 1996). How SAM and RM re-

spond to specific environmental cues to reorganize their activ-

ities is not fully understood.

Key environmental signals influencing SAM and RM behavior

are temperature and light. Photoperiod, in particular, changes

in a strictly predictable manner during the progression of the

season, making it a reliable source of information for the timing

of developmental events. Capitalizing on this situation, plants

evolved systems to monitor photoperiod by means of light-

sensitive pigments, including phytochromes (PHY), which play

central roles in leaf-to-apex signaling. In conjunction with physi-

ological clocks, they regulate the expression of key genes that

control the production of systemic signals (Zeevaart, 1976;

Bernier et al., 1993; Vince-Prue, 1994; Mouradov et al., 2002).

For example, poplar (Populus species) responds to short days

(SDs) with a PHYA-mediated cessation of elongation growth,

terminal bud set, and dormancy (Howe et al., 1995; Olsen et al.,

1997; Welling et al., 2002; Rohde and Bhalerao, 2007). It is un-

clear if PHYA within the apex plays any role in SD responses,

but considering that apices can directly respond to light, this

cannot be ruled out (Wareing, 1956; Gressel et al., 1980; Knapp

et al., 1986). In other species, photoperiod sensitivity can lead to

growth promotion. For example, in Arabidopsis thaliana long

days (LDs) induce floral transition at the apex (Yanovsky and Kay,

2002), accompanied by the emergence of a rapidly elongating

1 Address correspondence to chris.vanderschoot@umb.no.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Raili Ruonala (raili.ruonala@helsinki.fi).W Online version contains Web-only data.OA Open Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.107.056721

The Plant Cell, Vol. 20: 59–74, January 2008, www.plantcell.org ª 2008 American Society of Plant Biologists

inflorescence stem. Although end-of-season growth cessation

and flowering seem disparate processes, in terms of photoperiod-

controlled stem elongation at the RM/RZ, they might have

important commonalities.

In Arabidopsis, elongation of the inflorescence stem (bolting)

requires the clock-regulated gene CONSTANS (CO), which

produces a graft-transmissible signal in the companion cells of

source leaves (Takada and Goto, 2003; An et al., 2004; Ayre and

Turgeon, 2004). To produce sufficient signal, CO protein requires

the coincident perception of light by the photoreceptors PHYA

and cryptochrome2 (Putterill et al., 1995; Samach et al., 2000;

Suarez-Lopez et al., 2001). A direct target of CO is FLOWERING

LOCUS T (FT) (Kardailsky et al., 1999; Kobayashi et al., 1999),

whose mRNA accumulates at the end of the day (Yanovsky and

Kay, 2002). Overexpression of FT in companion cells or the apex

is sufficient to induce flowering (An et al., 2004), implicating FT

mRNA and FT protein as candidates for long-distance signaling.

Although both mRNAs and proteins may traffic in the phloem

(Ruiz-Medrano et al., 1999; Haywood et al., 2005), it is the FT/

Hd3a protein that can move from source leaves to the apex to

induce flowering, as was recently demonstrated in Arabidopsis

(Corbesier et al., 2007), cucurbit (Cucurbita maxima; Lin et al.,

2007), and rice (Oryza sativa; Tamaki et al., 2007). Interestingly,

the poplar FT orthologs Pt FT1 and Pt FT2 not only have a role in

the transition to flowering (Bohlenius et al., 2006; Hsu et al.,

2006), but their downregulation is required in early growth

cessation and the transition to dormancy (Bohlenius et al.,

2006). This suggests that parts of the regulatory networks that

underlie photoperiodically induced transitions, including those

that connect RM-mediated stem elongation to SAM transition-

ing, are conserved among different species.

In addition to the early downregulation of FT1/FT2 in poplar

leaves (Bohlenius et al., 2006; Hsu et al., 2006), the apex of

perennials responds to SDs by reducing symplasmic connectiv-

ity of SAM cells. In birch (Betula pubescens), this may start as

early as 4 d after the start of SDs. Eventually cells become

symplasmically isolated during dormancy by the formation of

callose-containing sphincters at the plasmodesmata (PD) (Rinne

and van der Schoot, 1998; Rinne et al., 2001). This may disrupt

metabolic coupling and signaling via PD and arrest patterning. By

contrast, LD-induced flowering is accompanied by a tripling of

PD frequencies in the apex of Sinapis (Ormenese et al., 2000), and

in both Sinapis (Ormenese et al., 2002) and Arabidopsis (Gisel

et al., 1999), symplasmic communication patterns may change.

FT belongs to a group of proteins that share specific motifs that

are involved in ligand binding (Banfield and Brady, 2000) and sig-

naling (Yeung et al., 1999). In Arabidopsis, TERMINAL FLOWER1

(TFL1) belongs to the same group of proteins. The expression of

both FT and TFL1 is increased coincident with floral induction

(Bradley et al., 1997), but, remarkably, their effects on flowering

are opposite (Kobayashi et al., 1999) as tfl1 mutants flower early

(Shannon and Meeks-Wagner, 1991; Alvarez et al., 1992; Bradley

et al., 1997) and ft mutants flower late (Koornneef et al., 1991).

Interestingly, tfl1 mutants have a short stem and determinate

IM, whereas TFL1 overexpressors have expanded shoot and

inflorescence systems (Ratcliffe et al., 1998). Although the TFL1

expression domain in the Arabidopsis apex is just below the

dome (Bradley et al., 1997), corresponding closely to the RM, the

potential dual role of TFL1 in stem elongation and SAM transi-

tioning has received little attention.

We aimed to establish if the transition to dormancy is a mere

consequence of a decision taken in the leaves or whether the

apex is an active player and, second, if the roles of RM and SAM

are distinct. We used hybrid poplar (Populus tremula 3 P. trem-

uloides) and a transgenic line overexpressing heterologous PHYA

and lacking the typical wild-type responses to SDs (Olsen et al.,

1997; Welling et al., 2002). Our results show that PHYA over-

expression in the RM/RZ area, but not in the SAM, correlates with

a failure to downregulate CENL1 in its RM domain and an ab-

sence of transition to dormancy. Collectively, the results sug-

gest that the apex is a semi-independent player in photoperiodic

growth responses and that the roles of RM and SAM in photo-

period perception are distinct.

RESULTS

Dissection of Photoperiod-Dependent Growth in the Wild

Type and a PHYA Overexpressor

Overexpression of oat (Avena sativa) PHYA (As PHYA) under the

control of the cauliflower mosaic virus (CaMV) 35S promoter

considerably alters the growth habit of hybrid poplar (P. tremula 3

P. tremuloides) (Olsen et al., 1997). In line 22 (hereafter called

P35S:AsPHYA), this defect is specific to the photoperiodic path-

way as their capacity to cease elongation growth under adverse

conditions is not impaired. Cold stress or inhibition of gibber-

ellin (GA) biosynthesis, for example, can arrest elongation growth

of both the wild type and P35S:AsPHYA independent of photo-

period (Welling et al., 2002; Mølmann et al., 2005), while in

P35S:AsPHYA, a combination of cold, SDs, and inhibition of

GA biosynthesis were required to eventually obtain a bud-like

structure (Mølmann et al., 2005). To establish the specific roles of

the SAM and the RM/RZ in photoperiodic responses, we have

dissected the quantitative and qualitative changes in growth

and development of P35S:AsPHYA. Despite the fact that LD-

exposed P35S:AsPHYA had smaller leaves, downwards twisting

petioles, and a stunted growth habit (Figures 1A and 1B; Olsen

et al., 1997; Welling et al., 2002), the plastochron of 1.2 d was sim-

ilar to that of the wild type in our experimental conditions (Figure

1C). This shows that leaf production that is orchestrated by the

SAM was unaffected, while internode length, which is regulated by

the RM/RZ, was altered by overexpression of PHYA.

When exposed to SDs, the wild type ceased elongation growth

in 3 weeks time (Figure 1B) and subsequently developed a

terminal bud. P35S:AsPHYA did not respond to SDs (Figure 2A),

a feat shown earlier (Olsen et al., 1997; Welling et al., 2002), but

instead lost its stunted growth habit and gradually accelerated

elongation growth under SDs (Figures 2B and 2C). This accel-

eration did not result from any change in the activity of the SAM,

as the plastochron did not change (Figure 1C). Instead, it was

exclusively due to a prolonged elongation of internodes (Figure

2B). The number of internodes that were elongating simulta-

neously increased from 5.2 (60.63) under LDs to 7.5 (60.74) after

2 to 3 weeks of SDs, while in the wild type, no change was

observed (LD 7.1 6 0.74 versus SD 7.4 6 0.97). Due to this

60 The Plant Cell

SD-induced acceleration of internode elongation, the P35S:

AsPHYA growth habit was restored almost to that of the wild

type under LDs (Figure 2C).

We next investigated the pattern of bud dormancy develop-

ment in the wild type. The term dormancy denotes a state in

which apical growth (or axillary growth in case of decapitated

plants) does not resume even under growth-promoting condi-

tions due to SAM-intrinsic constraints, for which reason it is also

referred to as endodormancy (Horvath et al., 2003). When de-

capitated wild-type plants were transferred back from SD to LD

at various time points, axillary bud bursting was prevented

already after 3 weeks of SDs (Figure 3A). However, if the leaves

were removed after transfer to LDs (in week 6), it appeared that

bud dormancy required minimally 6 weeks to develop (Figure 3),

indicating that leaves can effectively prevent bud burst when

the bud is not yet dormant. After 7 to 8 weeks of SDs, all buds

were completely dormant (data not shown). By contrast, decap-

itation of P35S:AsPHYA consistently resulted in the bursting of

axillary buds (Figure 3A). These buds developed into side shoots

(branches), the number and length of which was independent of

SD exposure (Figures 3B and 3C), their total length being com-

parable to that of the decapitated wild type under LD (Figures 3B

and 3C). This demonstrates that dormancy was completely pre-

vented in P35S:AsPHYA, not only in the leading shoot but also in

resting axillary buds.

To assess the earliest point at which wild-type plants visibly

responded to SDs, intact plants were brought back to LDs at

regular intervals. The first changes occurred after 2 weeks in

primordia that had developed under SDs. They were often de-

formed and unable to elongate, indicating that SD had induced

changes in leaf development.

The Wild Type but Not P35S:AsPHYA Suspends

Communication under SDs

Because the morphogenetic activity of the SAM is suppressed

by an innate mechanism during SD-induced dormancy, we next

investigated cellular and ultrastructural aspects of SAMs. In

morphogenesis, symplasmic connectivity of SAM cells via PD is

crucial, permitting the symplasmic exchange of signaling mole-

cules, the formation of morphogen gradients, and the metabolic

coupling of SAM cells (Rinne and van der Schoot, 1998; Rinne

et al., 2001). By contrast, during dormancy, reversion to a mor-

phogenetically active state is prevented by the formation of

dormancy sphincter complexes (DSCs) composed of an extra-

cellular ring of protein and callose that compresses the PD chan-

nel onto an intracellular PD plug, thereby shutting off symplasmic

connections (Rinne and van der Schoot, 2003).

To examine if SAM cells were symplasmically connected, we

iontophoretically microinjected Lucifer Yellow CH (LYCH) into

individual SAM cells (Figures 4A and 4B). In the LD-exposed wild

type, cells in the SAM were united in symplasmic fields (Figures

4B to 4D). A time series revealed that in the wild type, the

plasmodesmal size exclusion limit decreased at the central zone

after 10 d of SDs, impeding cell-to-cell diffusion of LYCH. Cells

at the peripheral region may maintain symplasmic connectivity

somewhat longer as after 10 d of SDs, these cells could still show

some dye coupling (Figure 4E). In most cases, SAM cells were

symplasmically uncoupled after 15 d of SDs (data not shown).

After 7 weeks of SDs, when buds no longer bursted under growth-

promoting conditions, dye coupling between all SAM cells was

categorically prevented (Figure 4F) and membrane potentials

had fallen below the diffusion potential (Table 1). By contrast,

in the P35S:AsPHYA SAM, symplasmic fields similar to those in

LD (Figure 4G) were found after 7 weeks of SDs (Figure 4H) and

Figure 1. Growth Habit of Wild-Type and PHYA-Overexpressing Hybrid

Poplar (P35S:AsPHYA).

(A) Young plants after 3 weeks of LDs. P35S:AsPHYA plants show

characteristic stunting due to deficient internode elongation.

(B) and (C) Cumulative stem growth (B) and leaf formation (C) were

measured in the wild type and P35S:AsPHYA under LDs (time point 0)

and during the course of SD exposure. Values represent means 6 SD

(n ¼ 10).

Pt CENL1 and RM in Developmental Switches 61

cellular activities remained high as judged from the Em values

(Table 1).

We investigated if the arrest of the SAM in a dormant state was

due to formation of DSCs, as shown previously for birch (Rinne

et al., 2001). Using tannic acid staining and transmission electron

microscopy, DSCs were visualized at virtually all PD in dormant

SAMs (7 weeks of SDs) (Figure 4I) and some adjacent tissue. By

contrast, DSCs were not formed in apices of P35S:AsPHYA

(Figure 4J), where PD resembled those found in the wild-type

SAM under LD (data not shown), agreeing with the presence of

symplasmic fields under SDs (Figure 4H).

Grafts Show Deviant Behavior

The anomalous behavior of P35S:AsPHYA under SDs can be due

to a number of conditions. P35S:AsPHYA might fail to respond

due to (1) a compromised SD perception in the source leaves, (2)

an inadequate communication to the apex, or (3) the inability of

the apex to respond to SD-generated leaf signals. To discrim-

inate between these a priori scenarios, various types of graft

systems were produced and subsequently exposed to SDs.

When grafts of a P35S:AsPHYA scion and a wild-type stock,

or the reverse grafts, were exposed to SDs, they behaved as

nongrafts. P35S:AsPHYA scions continued growth for extended

periods (Figure 5A), whereas wild-type scions responded with

growth cessation and bud formation (Figure 5B). At first sight,

this may suggest that the apex determines the response to SDs

and not the leaves. However, it seems plausible that young sink

leaves developing at the scion gradually took over the source

function for the apical parts. Signaling from leaves to apex would

then be executed by the scion’s own source leaves.

In subsequent experiments, we therefore removed developing

scion leaves before they reached 25 to 30% of the final leaf

length (i.e., before they started to export) (Turgeon, 1989). In

defoliated P35S:AsPHYA scions, the apex responded to SDs

with growth cessation and terminal bud formation (Figure 5C),

although it could be delayed with 1 to 2 weeks compared with

wild-type plants. When a defoliated wild-type scion grafted onto

Figure 2. Internode Elongation of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA) under LDs and SDs.

(A) Wild-type and P35S:AsPHYA plants were grown under constant LD conditions, subsequently transferred to SDs, and photographed after 6 weeks of

SDs (SD6). For comparison, a collage is made with a picture at scale of a P35S:AsPHYA plant grown for a similar period under LDs only (LD6). Close-ups

show the terminal bud of the wild type and the growing apex of P35S:AsPHYA that remains active under both LDs and SDs. Note the red apical leaves

and the spontaneously developing branches (stippling) that form in P35S:AsPHYA exposed to LDs. Horizontal arrows indicate the approximate height of

the plants upon transfer to the indicated conditions.

(B) The average maximal length of internodes that reached their final length at the indicated time under SDs. Values represent means 6 SD (n ¼ 10

plants).

(C) The average length of the 10 youngest internodes in the wild type and P35S:AsPHYA under 0 to 5 weeks of SDs. The internodes are numbered

from the youngest to the oldest (i.e., internode 1 corresponds to the uppermost internode). SD2 and SD5 correspond to 2 and 5 weeks under

SDs, respectively. Values represent means 6 SD (n ¼ 10 plants).

62 The Plant Cell

a P35S:AsPHYA stock was transferred to SDs, the wild-type

scion also ceased growth and developed a bud (Figure 5D). This

resembled the reverse graft (P35S:AsPHYA on the wild type;

Figure 5C), although the onset of growth cessation and bud

formation could be delayed further, to up to 4 weeks. Surpris-

ingly, however, when the terminal bud of the P35S:AsPHYA scion

was monitored for another 2 to 4 weeks under SDs, it started to

elongate and burst (Figure 5C, inset) approximately at the same

time when wild-type buds entered dormancy in nongrafts. Sub-

sequently, the P35S:AsPHYA scion elongated with seven to nine

phytomers, after which the cycle of growth cessation, bud set,

flushing, and elongation repeated itself under SDs (Figure 5E) for

at least three times in plants that stayed healthy. This sequence

of events was daylength dependent, as return to LD restored

continuous growth of the P35S:AsPHYA scion. By contrast, the

defoliated wild-type scion on a P35S:AsPHYA stock usually did

not flush, and if it did, the flushing was not recurrent and accom-

panied by stem elongation. Instead, such flushing terminated

in the formation of a conglomerate of dormant buds consisting

of a terminal bud and seven to nine axillary ones (Figure 5F). To

confirm that the grafting procedure itself was not inducing

growth cessation, bud set, and flushing, autografts were pro-

duced. Autografts, with or without defoliation, responded to LDs

and SDs as nongrafted wild type or P35S:AsPHYA, supporting

the conclusion that the behavior of the heterografts was photo-

period induced.

Some of the P35S:AsPHYA scions that set buds were used to

investigate the symplasmic connectivity of SAM cells. Despite

the fact that the SAM within these buds failed to enter dormancy,

dye coupling between their cells was absent at the bud stage.

The fact that the wild-type scion on the P35S:AsPHYA stock

was able to form terminal buds and become dormant suggests

that the wild-type apex itself might respond directly to photope-

riod. If so, the apex might respond to a combination of signals,

including those generated in the leaves and others generated

within the apex itself. In conclusion, the graft responses indicate

that growth cessation and bud set may depend on the interplay

of source leaves and apex, while entry into dormancy requires

SAM-intrinsic programs that are compromised in the P35S:

AsPHYA SAM.

Source and Sink Differentially Express PHYA-Affected

Flowering Pathway Genes

Graft experiments showed that the apex may modulate the re-

sponse to SDs. To obtain a more detailed picture of the roles

of source leaves, sink leaves, and the apex in the transition to

dormancy, we analyzed by real-time RT-PCR the transcript levels

of Pt FT1/FT2, CO2, and CENL1 (Figure 6), poplar orthologs of

Arabidopsis floral pathway genes At FT, CO, and TFL1, respec-

tively (see Supplemental Figures 1 and 2 online; Bohlenius et al.,

2006). Pt CENL1 and At TFL1 appear to be functionally conserved,

as overexpression in Arabidopsis reproduced the effects of TFL1

overexpression (Bohlenius et al., 2006).

Plants were exposed to LDs and SDs for up to 6 weeks. In our

experiments, the expression levels of FT1 were very weak or

below the detection limit in all samples analyzed (data not

shown). FT2 mRNA was clearly detectable in source leaves

under LDs, both in the wild type and P35S:AsPHYA (Figures 6A

and 6B), although approximately threefold higher in P35S:

AsPHYA. Under SDs, the expression level started to diminish

rapidly in both the wild type and P35S:AsPHYA (Figure 6A). This

decrease was evident already after 1 week of SDs, while stem

elongation and leaf formation were not yet visibly affected

(Figures 1B and 1C). In contrast with the wild type, the levels of

FT2 mRNA in P35S:AsPHYA recovered subsequently, peaking at

3 weeks of SDs and then remaining at low but measurable levels

(Figure 6A).

Figure 3. Bud Dormancy Development in Wild-Type and PHYA-

Overexpressing Hybrid Poplar (P35S:AsPHYA).

Wild-type and P35S:AsPHYA plants were exposed to SDs for 0 to 6

weeks after which the apex was severed. After decapitation, the plants

were transferred to LDs for 4 weeks to promote bursting of axillary buds.

Since no buds had burst in the wild type pre-exposed to SDs for 3 weeks,

all plants that were exposed to SDs longer than 3 weeks and already

transferred to LDs were defoliated during week 6 to promote bud burst.

Bursting of axillary buds (A), number of branches developed (B), and

length of the branches (C) were recorded (n ¼ 8 to 18 in the wild type;

n ¼ 8 to 10 in P35S:AsPHYA). Vertical bars represent SD.

Pt CENL1 and RM in Developmental Switches 63

On the other hand, CO2 mRNA levels were somewhat lower in

all sampled tissues of P35S:AsPHYA compared with the wild

type under LDs and SDs, with a similar temporary reduction

during the first 2 weeks of SDs (Figures 6C and 6D). At the time

when growth cessation was initiated in the wild type, at around 3

weeks of SDs, morphological changes became apparent at the

shoot tip: sink leaves continued to expand while the emergence

of new sink leaves from the periphery of the SAM ceased when it

became enclosed by developing bud scales. The gradually

enlarging youngest sink leaves of the wild type considerably

increased their abundance of CO2 mRNA, reflecting loss of sink

status, while in the apex, the CO2 mRNA levels decreased

(Figure 6D). By contrast, in P35S:AsPHYA sink leaves, transcript

abundance of CO2 remained at the same level during SD

exposure (Figure 6D) due to the regular replacement of existing

sink leaves by new ones.

A low but constant level of CENL1 was expressed in the source

leaves of P35S:AsPHYA throughout the experiment, whereas the

equally low expression level in wild-type source leaves de-

creased even further after 3 weeks of SDs (Figure 6E). By

contrast, marked alterations in CENL1 transcript levels took

place in the apices. In the wild type, CENL1 expression increased

at 2 to 3 weeks under SDs and then rapidly decreased (Figure

6F), a trend similar to that in source leaves (Figure 6E), coinciding

with the initiation of growth cessation (Figure 1B). By contrast, in

P35S:AsPHYA, the expression of CENL1 continued to increase

under SDs from 2 weeks onward (Figure 6F), corresponding to

the acceleration of elongation growth (Figure 2B). The same

typical expression pattern of CENL1 was also found in the sink

leaves, although the magnitude of gene expression was lower

(Figure 6F).

In summary, in source leaves of SD-exposed plants, the initial

downregulation of CO2 and FT2 was similar for the wild type and

P35S:AsPHYA. Subsequently, the expression of CO2 and FT2

recovered in P35S:AsPHYA, while in the wild type, only CO2

recovered. At the gene expression level, the apex of P35S:

AsPHYA responded to SDs with a gradual upregulation of CENL1

transcript levels, which initially (LD) were only half of that of the

wild type. The wild-type apex also increased CENL1 during the

first 3 weeks of SDs, but subsequently it was strongly down-

regulated ;40 fold, coincident with the cessation of elongation

growth, bud set, and dormancy assumption.

Pinpointing the RM in the Apex of Poplar

The grafting experiments showed that P35S:AsPHYA is capable

of SD-induced growth cessation and bud set provided adequate

Figure 4. Em Measurement, Iontophoretic Microinjection, Dye Coupling,

and PD Ultratructure in the SAM of Wild-Type and PHYA-Overexpressing

Hybrid Poplar (P35S:AsPHYA).

(A) Typical stable Em recorded from a single tunica cell in the SAM of the

wild type under LD.

(B) Iontophoretic microinjection of LYCH into a single tunica cell of LD-

exposed wild type results in dye coupling of the cells that occupy the

SAM center. Blue excitation light, photographed through bathing me-

dium. Arrow indicates SAM. me, microelectrode; p, primordia. Bar ¼75 mm.

(C) and (D) Apex of (B) with medium removed. Low (C) and medium (D)

levels of white light mixed in with blue light reveal the position of the

central symplasmic field, the primordia (p), and a leaf buttress (lb). Arrow

indicates SAM. Bars ¼ 75 mm.

(E) Dye coupling is restricted to small cell groups in the SAM of the wild

type after 10 d of SDs (blue light, photographed through the medium).

me, microelectrode; p, primordia. Bar ¼ 75 mm.

(F) In the dormant SAM of the wild type, after 7 weeks of SDs, cells are

uncoupled (blue light, photographed through the medium). Bar ¼ 75 mm.

(G) and (H) In the P35S:AsPHYA SAM, cells are dye coupled into a

central symplasmic field under both LDs (G) and 7 weeks of SDs (H),

photographed as in (C) and (B), respectively. p, primordia. Bars¼ 75 mm.

(I) PD in SAM of dormant wild type (7 weeks of SDs) are equipped with

DSCs, containing tannic acid/protein deposits. The PD channel plug and

sphincter ring are indicated by the black and white arrows, respectively.

Bar ¼ 200 nm.

(J) PD in the P35S:AsPHYA SAM exposed to SDs (7 weeks) lack DSC,

typical of dormant wild-type SAM. Bar ¼ 200 nm.

64 The Plant Cell

signal is supplied. Yet, P35S:AsPHYA failed to enter dormancy,

as the arrest was not permanent, and new leaves developed

inside the bud (Figures 5C and 5E). To identify the potentially

distinct roles of the RM and the SAM at the molecular level, we

set out to surgically separate them. First it was determined, using

histological sections, where the RM was located in the apex of

poplar. The sections showed that the meristematic part of the

apex was composed of small, densely cytoplasmic cells, which

were subtended by ribs of elongated and vacuolated cells (Figure

7A). The delineation between the SAM and the RM was evident

from their distinct cell division patterns (Figure 7B). Cells in the

corpus of the SAM divide more or less randomly, following

space-filling requirements, while those recruited by the sub-

tending RM start to divide periclinally to produce the ribs of the

RZ. Following these established criteria (Sachs et al., 1960; Esau,

1977), the poplar SAM includes a two-cell layered tunica and a

corpus of five to six cell layers (Figures 7A and 7B). Immediately

adjacent to the corpus is the RM composed of about two to four

cell layers (Figure 7B). These figures were consistently found in

all samples of both the wild type and P35S:AsPHYA (n ¼ 10

apices).

This distinction between the meristematic and the differenti-

ating areas is also visible in longitudinally cut live apices, viewed

with white epi-illumination. The differentiating cells of the RZ

possessed more chlorophyll than the meristematic part of the

apex (Figure 7C), reflecting the fact that the meristematic part of

the apex and the youngest leaves in general do not possess fully

developed chloroplasts (Rinne et al., 2005). This made it possible

to identify the approximate site of the RM, at the lower edge of

the whitish zone in Figure 7C.

Iontophoretic microinjection of LYCH into a single cell of the

putative RM (cell layer 9 or 10) led to LYCH diffusion to a disc-like

group of symplasmically coupled cells that was separated from

the overlying corpus cells (Figure 7D). Diffusion of LYCH into the

ribs of the RZ was restricted (Figure 7E, inset). When injected into

the central corpus, LYCH distributed in a rectangular area

without entering the RM (data not shown). Together, these

experiments provided support for the classic notion that the

RM is a morphogenetic unit in its own right (Sachs et al., 1960;

Esau, 1977).

P35S:AsPHYA Overexpresses PHYA in the RM/RZ but

Not in the SAM

As the SAM in poplar appeared to be a small and shallow dome

(Figure 7), it was not possible to isolate the SAM and the RM

with great precision (Figure 8A). Nevertheless, we were able to

reconstruct the approximate expression domains of the selected

genes using quantitative real-time RT-PCR. As expected, FT1

and FT2 expression in the SAM and the RM/RZ were extremely

weak or below the detection limit in all samples (data not shown).

Both in the wild type and P35S:AsPHYA, CO2 mRNA amount

was higher in the RM/RZ than the SAM, whereas CENL1 mRNA

seemed to be present predominantly in the SAM sample (Figure

8B). In a second series of experiments, apices were dissected

into even smaller pieces, thereby enriching the RZ sample with

RM. When comparing these minuscule samples to the larger

samples, only CENL1 levels were increased (Figure 8B), whereas

CO2 levels remained at similar or slightly decreased levels (data

not shown). Together, the analysis of apices (Figures 6D and 6F)

and dissected apex parts (Figure 8) indicates that CENL1 is ex-

pressed mainly in the upper part of the RZ (i.e., in the RM),

whereas CO2 is expressed more widely in the RZ and the un-

derlying tissues. Transcripts of Pt PHYA, poplar’s endogenous

PHYA gene, were present at approximately equal low amounts

in all samples (Figure 8B). Interestingly, however, the transcript

level of the transgene As PHYA was markedly lower in the SAM

than in the RM/RZ (Figure 8B).

The relatively low expression level of As PHYA in the dissected

SAM samples of P35S:AsPHYA was unexpected since the trans-

ferred As PHYA gene is under the control of the constitutive

CaMV 35S promoter. In situ hybridization revealed that in P35S:

AsPHYA, the mRNA of As PHYA could not be detected in the

apical dome or the youngest primordia. By contrast, the tran-

script accumulated at high levels in the tissues beneath the dome

(i.e., in the RM/RZ, stem, and leaves) (Figure 9A; see Supple-

mental Figure 3 online). In negative controls, As PHYA transcripts

were not detected in the wild-type apex or in hybridizations

performed using a sense probe (Figure 9B; see Supplemental

Figure 3 online). In situ hybridization of endogenous Pt PHYA

showed weak expression in the SAM, young primordia, and vas-

cular tissue in both the wild type and P35S:AsPHYA, while in the

same samples, a hybridization signal with As PHYA was only

found in P35S:AsPHYA (Figures 9C to 9G). Similar weak hybrid-

ization of Pt PHYA was also detected in axillary buds (Figure 9H).

DISCUSSION

Source-Sink Relations and the Time Frame of

SD-Induced Events

The shoot apex responds to diurnal and seasonal rhythms in the

light environment, which are detected by phytochromes residing

in the leaves, and translated into graft-transmissible signals.

Signaling follows the paths of photosynthates from source to

Table 1. Membrane Potentials (Em), Dye Coupling, and Cell Isolation

in the Peripheral Zone and the Central Zone of Wild-Type and

PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA) during LDs

and after 7 Weeks of SDs

Plant Daylength SAM Area Em (-mV)a (n)b C/Ic (n)d

Wild type LD PZ 89 6 2.6 32 13/0 13

CZ 44 6 1.3 70

SD PZ/CZe 32 6 1.0 35 0/9 9

P35S:AsPHYA LD PZ 90 6 2.5 37 13/0 13

CZ 45 6 1.6 53

SD PZ 79 6 1.9 35 8/0 8

CZ 36 6 1.1 37

a Membrane potential 6 SE.b Number of cells from which membrane potentials were measured.c Dye coupling (C)/cell isolation (I); both zones are combined.d Total number of SAMs analyzed.e Em data combined as distinction between the central zone (CZ) and the

peripheral zone (PZ) were unclear due to cell isolation.

Pt CENL1 and RM in Developmental Switches 65

sink. As younger source leaves predominantly deliver photosyn-

thates to the upper shoot parts, whereas older source leaves

supply the lower stem and roots (Turgeon, 1989; Oparka et al.,

1999), signals destined for the shoot apex are supplied predom-

inantly by younger source leaves. To identify genes potentially

involved in systemic signaling, RNA should therefore preferen-

tially be isolated from young source leaves (Figure 6).

One of the earliest events in photoperiodic signaling in hybrid

poplar is the recently reported downregulation of FT1 (Bohlenius

et al., 2006) and FT2 (Hsu et al., 2006), which is supported by our

results (Figure 6). Downregulation of FT2 was evident already

after 1 week of SDs (Figure 6A) and thus separated from the late

events of growth cessation, bud set, and dormancy by a time lag

of several weeks (Figures 1B and 1C). Downregulation of FT1 and

FT2 might therefore predominantly serve the timing of events that

eventually lead up to growth cessation and bud set, rather than

the transition itself. The observation that poplar is able to con-

tinue normal growth when transferred back to LDs after 1 week of

SDs indicates that FT2 levels can normalize and that a point of no

return is not reached. The time lag between initial and late events

is a period in which the apex receives other signals that target

downstream genes that help direct its response at the cellular

level. For example, ethylene signaling, in addition to FT1 and FT2,

affects growth cessation and bud development (Ruonala et al.,

2006), whereas abscisic acid signaling is involved in bud matu-

ration (Rohde et al., 2002) as well as acclimation (Rinne et al.,

1998). Recent microarray data confirm that genes involved in

biosynthesis and perception of GA, ethylene, and abscisic acid,

Figure 5. Behavior of the Apices of Grafts of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA) under SDs.

(A) and (B) A nondefoliated P35S:AsPHYA scion on a wild-type stock (A) and the reverse graft with a nondefoliated wild-type scion on a P35S:AsPHYA

stock (B). The photographs were taken just before SD exposure. The insets show the status of the apex as indicated after 4 to 6 weeks of SDs. Letters

refer to percentage of grafts showing the response: B, bud formation; D, dormancy; F, flushing; E, elongation.

(C) and (D) A defoliated P35S:AsPHYA scion on a wild-type stock (C) and a defoliated wild-type scion on a P35S:AsPHYA stock (D). The photographs

were taken just before SD exposure. The insets show the status of the apex as indicated after 6 to 7 weeks of SDs. Letters refer to percentage of grafts

showing the response, as described above.

(E) A defoliated P35S:AsPHYA scion on a wild-type stock after repeated bud set and flushing. The insets show the bud scales or their scars on the stem.

The specimen was photographed after bud set and subsequent flushing under SDs.

(F) A defoliated wild-type scion on a P35S:AsPHYA stock after one flush and successive bud set without elongation. The specimen was photographed

after bud set and subsequent flushing under SDs.

66 The Plant Cell

as well as genes that are downstream targets, are regulated

during the early phases of SD exposure (Ruttink et al., 2007).

Eventually, the translocation of long-distance signals in the phloem

slows down and entirely ceases when the sieve tubes become

physically blocked by callose deposits, a phenomenon known as

winter phloem (Aloni et al., 1991).

Bud Set and Responses of the SAM to SDs

During SD induction of terminal buds in poplar, leaf primordia

develop into embryonic leaves and stipules into bud scales

(Rohde et al., 2002). There is some plasticity, however, as leaves

that started development under LDs can still be modified to pro-

duce bud scales (Goffinet and Larson, 1981; Howe et al., 1995).

The terminal bud undergoes a gradual enlargement due to the

growth of embryonic leaves, which fold up within the confines of

the bud scales. This gradual process of bud formation is distinct

from that of dormancy development, as suggested by work on

transgenic birch (Ruonala et al., 2006); birch (Betula pendula)

transgenically expressing the mutated Arabidopsis ethylene re-

ceptor gene etr1-1 can enter dormancy under SDs without

forming a clear terminal bud. The distinct nature of both pro-

cesses, bud development and dormancy, is further confirmed by

our finding that SD-exposed defoliated P35S:AsPHYA scions

temporarily formed a bud without entering dormancy.

The diminished symplasmic communication at 10 to 15 d of

SD exposure (Figure 4E) might have contributed to malformation

of leaves when plants were returned to LDs at this point. At this

stage, diminished cell–cell diffusion of LYCH was due to a re-

versible decrease in PD conductivity and not to obstruction of

PD by DSCs as dormancy was not yet established, a situation

comparable to that in birch (Rinne et al., 2001). Our data show

that absence of SD-induced growth cessation and transition to

dormancy in P35S:AsPHYA is in accordance with the absence

of cellular and ultrastructural features that characterized the

dormant state, such as low membrane potentials (Table 1), DSCs

at the PD (Figure 4I), and symplasmically isolated cells (Figure

4F). However, reversible narrowing of the PD diffusion channels

could take place during the temporary formation of terminal buds

in scions. Since P35S:AsPHYA was unable to enter dormancy,

PHYA overexpression may negatively interfere with the pro-

cesses that orchestrate DSC formation.

Figure 6. Expression Patterns of Flowering-Related Genes in Source and Sink Tissues of Wild-Type and PHYA-Overexpressing Hybrid Poplar

(P35S:AsPHYA) under LDs (Time Point 0) and SDs (Time Points 1 to 6).

Expression levels of Pt FT2 (A) and (B), Pt CO2 (C) and (D), and Pt CENL1 (E) and (F) were analyzed in source ([A], [C], and [E]) and sink ([B], [D], and

[F]) tissues of the wild type and P35S:AsPHYA during the course of a 0- to 6-week SD exposure using real-time RT-PCR. Inset in (D) represents the data

of time points 0 and 1 at a stretched out scale. Inset in (E) represents the same data at a stretched out scale. Three replicate samples were analyzed of

each sample type in each time point, and the average values are shown. For (B), one to two replicate samples were analyzed for each data point.

Vertical bars represent SD. Independent experiments were repeated two times with similar results.

Pt CENL1 and RM in Developmental Switches 67

Although all plants showed an early downregulation of FT2 in

source leaves, the expression partly recovered in P35S:AsPHYA,

peaking at 3 weeks of SDs (Figure 6A). At this point in time, the

wild type ceases elongation (Figure 1B) and initiates bud for-

mation, suggesting that FT2, transported via the phloem to the

apex, assists the continuation of stem elongation and morpho-

genesis at a critical point in the SD trajectory. It is feasible that

the RM, which regulates elongation, is the target of FT2 and

possibly also FT1. In general, the role of the RM in SAM tran-

sitions might be underestimated due to the tendency to view this

area as part of the SAM (Esau, 1977). Clearly, the symplasmic

separation of the RM from the overlying corpus of the SAM

(Figures 7D and 7E) supports the notion that the RM is a distinct

morphogenetic area, if not an independent unit (Esau, 1977).

Interestingly, recently published photographs of FT-induced

floral transition tend to show that green fluorescent protein–

tagged FT is delivered to the RM rather than to the SAM, that is

the dome, in Arabidopsis (Corbesier et al., 2007) and rice (Tamaki

et al., 2007). It is tempting to speculate that PD trafficking

between the RM and the corpus, as between symplasmic fields

in the SAM (Rinne and van der Schoot, 1998; van der Schoot and

Rinne, 1999), requires PD gating by factors that assist proteins

like FT to enter the SAM.

PHYA Abundance and Spatial Distribution Affect

Stem Growth

Earlier work has shown that PHYA plays a role in stem elonga-

tion. For example, Arabidopsis mutants lacking PHYA produce

elongated hypocotyls when grown under a canopy (Johnson

et al., 1994; Yanovsky et al., 1995), whereas strong overexpres-

sion reduces internode elongation in several species. This is

often attributed to PHYA inhibition of GA biosynthesis in vascular

tissue (Boylan and Quail, 1991; Jordan et al., 1995; Casal et al.,

Figure 7. Functional Zones in the Apex of Hybrid Poplar.

(A) Light micrograph of a median-longitudinal section through a proliferating shoot apex showing the SAM and the RM, subtended by the RZ. Cells of

the SAM and RM are densely cytoplasmic with large nuclei. The cells of the RZ, arranged in longitudinal ribs, are stretched, vacuolated, and

differentiated. Cytoplasm and nucleoplasm are stained blue and cell walls red.

(B) Detail of the area boxed in (A). Recent cell divisions, visible as thin walls crossing existing cells, are color coded on the backdrop of the image in (A).

Cell division walls are anticlinal in the tunica (cell layers 1 and 2), random in the corpus (approximate cell layers 3 to 8), and periclinal in the RM

(approximate cell layers 9 to 11) and upper RZ. PZ, peripheral zone; CZ, central zone; p0, youngest leaf primordium; T, tunica; C, corpus. Arrows point to

the approximate border between the central zone and peripheral zone.

(C) to (E) Light micrographs of a life apex cut median-longitudinally.

(C) White light epi-illumination reveals differences in chlorophyll content between the meristematic and the differentiating areas. Boxed area indicates

microinjection site in (D) (arrow).

(D) Iontophoretic microinjection of LYCH into a single cell in layer 10 or 11 at the SAM/RM boundary. LYCH diffuses intercellularly to a group of

symplasmically coupled cells (situated in cell layers 8 to 11), which in position closely corresponds to the RM.

(E) Final dye-coupling pattern. The coupling group is symplasmically separated from the overlying corpus, but small traces of LYCH (inset) diffuse into

the cells that make up the ribs of the RZ.

Bars ¼ 50 mm.

68 The Plant Cell

1996; Olsen et al., 1997; Eriksson and Moritz, 2002). Our results

show that the transgene As PHYA was strongly expressed in the

RM/RZ area of P35S:AsPHYA but not in the SAM itself (Figure 9A;

see Supplemental Figure 3 online). The fact that PHYA is over-

expressed in conjunction with CENL1 in the RM/RZ area, which

serves stem elongation, suggests that PHYA and CENL1 may be

part of the same regulatory circuit in the RM. Why As PHYA

transcripts were absent from the apical dome remains an open

question, but it could be envisaged that the 35S promoter is not

activated in the SAM. On the other hand, as the 35S can drive

gene expression in the SAM of Arabidopsis (Conti and Bradley,

2007), it seems equally possible that As PHYA is locally silenced

by SAM intrinsic mechanisms that detect and destroy foreign

gene products (Van Blokland et al., 1994). In both cases, con-

sidering that Pt PHYA was expressed similarly in the SAM of

P35S:AsPHYA and the wild type, it might be the high over-

expression in the RM/RZ area of P35S:AsPHYA (Figure 9; see

Supplemental Figure 3 online), which is particularly decisive in

generating this phenotype. It can also not be ruled out that the

strong imbalance in PHYA expression in the SAM and in the RM/

RZ might break down potential feedback mechanisms between

the SAM and the RM, thereby impairing the ability of P35S:

AsPHYA to transit to dormancy. Such blockade could function at

the level of signal production as well as signal transfer through

PD or the membranes of adjacent RM and corpus cells (Rinne

and van der Schoot, 2003).

Permanent Arrest of the RM/RZ Is Required for Dormancy

Grafting experiments confirmed that overexpression of PHYA in

the apex shaped the apical responses to SDs (Figure 5). This only

became apparent when the scions of the heterografts were

defoliated because without this procedure, stock signals were

probably not sufficiently channeled toward the apex of the scion.

Only when P35S:AsPHYA scions were defoliated could they

produce a bud. However, they failed to enter dormancy and

flushed repeatedly under SDs (Figures 5C and 5E), showing that

RM activity remained high in these scions. This is congruent with

the overexpression of PHYA in the RM/RZ area (Figures 8 and 9A;

see Supplemental Figure 3 online). Furthermore, the lack of

elongation growth in SD-exposed wild-type plants and wild-type

scions (Figure 5) suggests lack of cell division activity in the RM.

Indeed, a complete lack of cell division in the RM/RZ area in

transgenic tobacco entirely prevented stem elongation and

spectacularly forced them to grow as a rosette, while flowering

rescued elongation growth much like Arabidopsis (Rinne et al.,

2005). Collectively, this indicates that the two meristematic

regions of the apex, RM and SAM (Figures 7A and 7B), might

function as semi-independent but interacting units (Rinne and

van der Schoot, 2003), an interpretation that is supported by the

finding that the RM and the SAM are symplasmically distinct

(Figures 7D and 7E).

CENL1 Expression Correlates with RM Activity

The data show that CENL1 was differentially regulated in the

apices of the wild type and P35S:AsPHYA (Figures 6F and 8B),

corresponding to the proliferation activity of the RM under a

given photoperiod. The decrease in CENL1 expression in wild-

type apices at the time of growth cessation and bud set (Figure

6F) is also the trend reported in a recent microarray study (Ruttink

et al., 2007), but the current real-time RT-PCR study shows a

much larger, 40-fold decrease. The fact that CENL1 expression in

P35S:AsPHYA plants increases during SD exposure (Figure 6F)

indicates that it is involved in the continuation of growth. Because

internode elongation was affected in P35S:AsPHYA, but not the

plastochron, CENL1 is most likely modifying the activity of the RM.

This is supported by the observation that short internodes in

P35S:AsPHYA possess fewer cells (Olsen et al., 1997).

In Arabidopsis, TFL1 is generally linked to SAM identity. This

role is based on LEAFY (LFY)–mediated movement of TFL1

Figure 8. Gene Expression in Distinct Areas at the Shoot Apex of Wild-

Type and PHYA-Overexpressing Hybrid Poplar P35S:AsPHYA (P35)

under LDs.

(A) A scheme of the microdissected apical areas, with estimated con-

tributions of the SAM, the RM, and the RZ to each type of sample in two

different series of experiments (exp1 and exp2). The sample of the

second experiment is enriched for RM, whereas the uncolored SAM

sample was not analyzed. p1-3, primordia 1 to 3.

(B) Expression levels of Pt CO2, Pt CENL1, Pt PHYA, and As PHYA were

analyzed using quantitative real-time RT-PCR from samples collected in

experiment 1 and 2 (exp1 and exp2; described in [A]). Experiments are

separated by vertical stippling. Bar colors correspond to the color code

used in (A). SAM and RM/RZ samples of 40 and 20 apices, respectively,

were pooled for the analyses in experiment 1, and 10 apices were pooled

for experiment 2.

Pt CENL1 and RM in Developmental Switches 69

protein beyond its expression domain into the IM (Conti and

Bradley, 2007). However, as the upregulation of TFL1 expression

during the transition from SAM to IM occurs locally in the RM

domain (Bradley et al., 1997; Kardailsky et al., 1999; Ratcliffe

et al., 1999; Conti and Bradley, 2007), it is conceivable that TFL1

has two functions, protecting IM identity and promoting stem

elongation. If so, TFL1/CENL1 might play a similar role in the RM,

both in Arabidopsis and poplar. In support of this, the TFL1

homolog Os CEN2 in rice is expressed mainly in intercalary

meristem (Zhang et al., 2005), a file meristem that corresponds to

the RM of dicotyledons (Esau, 1977). Because elongation and

flowering in juvenile poplar are separated by a multiple-year

phase (Bohlenius et al., 2006; Hsu et al., 2006), poplar might be

an excellent model to investigate these processes independently

from each other. The putative role of CENL1 in RM activity might

be supported by the finding that CENL1 is also highly expressed

at the time of bud burst in poplar (Mohamed, 2006; P.L.H. Rinne,

unpublished data).

Putative Role of CENL1 Beyond Its mRNA

Expression Domain

Is it conceivable that CENL1 protein in poplar moves from the RM

to the SAM? In view of the fact that TFL1 protein is present in the

SAM of (para-)dormant axillary buds of Arabidopsis that do not

express LFY (Conti and Bradley, 2007), it doesn’t seem implau-

sible that CENL1 would move from the RM to the SAM in poplar.

In Arabidopsis, TFL1 protein is proposed to safeguard the

indeterminacy of the IM by restricting LFY and APETALA1

(AP1) expression to floral primordia (Conti and Bradley, 2007).

In poplar, CENL1 protein might have a similar nonautonomous

role, moving via PD much alike LFY in the Arabidopsis SAM

(Sessions et al., 2000), as AP1 is upregulated during bud forma-

tion (Ruttink et al., 2007). Such import into the SAM would require

gating of PD at the boundary between symplasmic fields, as

suggested earlier (Rinne and van der Schoot, 1998, 2003; van der

Schoot and Rinne, 1999), thereby protecting SAM indeterminacy

during the early phases of the transition to dormancy. Within the

SAM, CENL1 could potentially associate with multiple interac-

tors, including bZIP transcription factors, as suggested for TFL1

in Arabidopsis (Conti and Bradley, 2007). The time frame for

putative movement of CENL1 in poplar would be the first 3

weeks, when its expression levels are considerably elevated,

while PD are not yet locked by DSCs and can still be gated. In this

scenario, the downregulation of CENL1 in the RM and the

reduced export of its protein to the SAM would be compensated

by the formation of DSCs, protecting the indeterminate state of

the SAM during dormancy.

Figure 9. Localization of As PHYA and Pt PHYA Transcripts in the Apex of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA).

(A) and (B) mRNA of As PHYA, which was transformed into hybrid poplar under the control of the CaMV 35S promoter, was localized in median

longitudinal sections of P35S:AsPHYA (A) and wild-type (B) apices using in situ hybridization. Purple color indicates the presence of the transcript in (A).

The inset displays a detail of P35S:AsPHYA apex showing high expression in RM and areas subjacent to it.

(C) and (D) In situ hybridization of the native Pt PHYA in wild-type apex using antisense (C) and sense (D) hybridization probes. Purple color indicates the

presence of the transcript in (C).

(E) to (G) In situ hybridization using an antisense probe of Pt PHYA (E), As PHYA (F), and a control RNA (G) in successive sections of the P35S:AsPHYA

apex. Purple color indicates the presence of the transcript in (E) and (F).

(H) In situ hybridization using an antisense probe of Pt PHYA in an axillary bud of the wild type. Purple color indicates the presence of the transcript. RM

is indicated by a double arrow in (A), (B), and (E).

Bars ¼ 100 mm.

70 The Plant Cell

METHODS

Plant Material and Growth Conditions

Transgenic hybrid poplar lines expressing As PHYA under the control of

the CaMV 35S promoter were described previously (Olsen et al., 1997).

For our experiments, wild-type hybrid poplar (Populus tremula 3 P.

tremuloides, clone T89) and transgenic line 22 (called P35S:AsPHYA)

were investigated. Wild-type and P35S:AsPHYA ramets were micro-

propagated in vitro, planted in a mixture of sterilized peat and perlite

(4:1, v/v), and fertilized with 4 g L�1 Osmocote (Substral; Scotts). Prior to

SDs, plants were grown in a phytotron for 4 weeks under LDs (18 h light)

at 188C and 80% relative humidity. Natural light was supplemented to

200 mmol m�2 s�1 at 400 to 750 nm (Osram). For the SD treatments, the

daylength was shortened to 10 h.

Generating Grafts

Grafts were made from the wild type and P35S:AsPHYA grown for 6

weeks under LDs. Stems of stock donors were cut back at a node, leaving

;10 mature source leaves on each stock plant. Scions were excised

2 cm below the apex, and the stem was trimmed into a wedge. A lon-

gitudinal split was made in the cut stem of the stock, the stem halves

slightly forced apart, and the trimmed scion inserted. The stock-scion

interface was secured with parafilm, and the scion was enclosed in a

transparent plastic bag to prevent drying. The bags were ventilated daily

and removed when graft leaves enlarged. Part of the scions was defo-

liated to promote transport from stock to scion. Properly growing grafts

(40 out of 100) were selected for SD experiments.

Light Microscopy and Transmission Electron Microscopy

Apices were fixed for 4 h in 2% (v/v) glutaraldehyde and 3% (v/v) para-

formaldehyde in 100 mM phosphate buffer, pH 7.2, with 1% (w/v) tannic

acid, postfixed for 2 h in 1% (w/v) OsO4, and embedded in LR White

(Sigma-Aldrich) as described (Rinne and van der Schoot, 1998). For light

microscopy, longitudinal 1-mm-thick sections were cut with an ultrami-

crotome (Leica), stained with Methylene Blue, Azure, and Basic Fuchsin

(Rinne et al., 2005), investigated microscopically (Nikon Optiphot II),

and recorded with a digital camera (Nikon Coolpix 995). Pictures were

processed with Adobe Photoshop. For transmission electron micros-

copy, ultrathin sections (70 nm) were cut with an ultramicrotome, stained

with 2% aqueous uranyl acetate and Reynolds’ lead citrate, and exam-

ined in a JEOL 1200 EXII electron microscope at 80 kV.

Membrane Potentials and Dye-Coupling Experiments

Symplasmic coupling and membrane potentials of SAM cells were

investigated by a combination of electrophysiology and iontophoresis

(Rinne and van der Schoot, 1998; Ormenese et al., 2002). Apices were

isolated from the wild type and P35S:AsPHYA growing under LDs and

SDs and from heterografts (P35S:AsPHYA on the wild type) under SDs.

Microelectrodes were made from borosilicate glass tubes with inner

filament (World Precision Instruments) on a horizontal puller (PN-3;

Narishige Scientific Instruments Laboratory). The tip was backfilled with

1% (w/v) LYCH (MW 457 D; Molecular Probes) and the remainder with

100 mM KCl as an electrolyte. The microelectrode was inserted into a

holder with an Ag/AgCl bridge, mounted on a pre-amp, and attached to a

hydraulic micromanipulator (MO-203; Narishige). Membrane potentials

were recorded with a WPI Duo 773 Dual Microprobe System and a chart

recorder (BD111; Kip and Zonen). LYCH was microinjected into single

tunica cells after the recording of a stable membrane potential by

applying a low intermittent current (�1 to �5 nA) to the microelectrode

tip. In a set of additional experiments, apices were first incubated for 30

min and subsequently cut longitudinally in a medium of 10 mM 2-deoxy-

D-glucose (Sigma-Aldrich) to prevent formation of wound-induced cal-

lose, attached to microscope slides, and fixed in a bathing chamber as

described previously (Rinne et al., 2005). In both types of experiments,

LYCH diffusion to other cells was examined with a fluorescence micro-

scope (Nikon Optiphot II) equipped with standard filter set combinations

(Rinne and van der Schoot, 1998) and recorded with a digital camera

(Nikon Coolpix 995) or a conventional camera (Nikon FX 35 WA/uFX-II)

with Ektachrome 400 super slide film.

RNA Preparation and Real-Time RT-PCR Analysis

RNA was analyzed separately from source leaves, sink leaves, and

apices. Source tissue was obtained from the tip part of the youngest fully

expanded leaf, and sink leaves were very young unexpanded leaves just

below the apex, whereas apex tissues contained SAM, primordia, em-

bryonic leaves, and the nonelongated internodes containing the RM/RZ.

Sampling occurred during the last hour of the light period, and plant

material from five to six individuals was pooled before further processing.

RNA was extracted using the Spectrum Plant Total RNA kit according to

manufacturer’s instructions (Sigma-Aldrich). RNA was DNase treated,

reverse-transcribed using RevertAid H Minus M-MuLV reverse transcrip-

tase (Fermentas), and analyzed using real-time RT-PCR as described

(Ruonala et al., 2006). Transcript levels were normalized against poplar

ACTIN. SAM and RM/RZ samples were microsurgically isolated using

sapphire blades (World Precision Instruments) and processed as above,

except that RNA was treated using on-column DNase (Sigma-Aldrich),

and maximally 500 ng of DNase-treated RNA was reverse transcribed

using SuperScriptIII reverse transcriptase as recommended by the man-

ufacturer (Invitrogen). Gene-specific primer sequences (see Supplemen-

tal Table 1 online) for the real-time RT-PCR analysis were designed using

Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).

In Situ Hybridization

Digoxigenin-labeled antisense and sense RNA probes from a partial oat

(Avena sativa) PHYA (As PHYA) cDNA-clone pAPSX2.7 (Seeley et al.,

1992) and a full-length poplar PHYA (Pt PHYA) cDNA clone were prepared

and hydrolyzed using the Dig RNA labeling kit (Roche). As an additional

control, digoxigenin-labeled RNA supplied in the Dig RNA labeling kit

was used for some hybridizations. Apices were dissected and fixed us-

ing 4% (w/v) paraformaldehyde and 0.25% (v/v) glutaraldehyde in 10 mM

phosphate buffer, pH 7.2, for 4 h at room temperature, dehydrated in

ethanol, exchanged with xylene, and embedded in paraffin. From the

paraffin blocks, 7-mm sections were cut and attached to silane-coated

microscopy slides (Electron Microscopy Sciences). Prehybridization and

hybridization protocols were based on those described by De Block and

Debrouwer (1993), with some modifications. Briefly, the tissue sections

were treated with proteinase K (20 mg mL�1), and the hybridizations were

performed at 438C overnight with a probe concentration of 0.28 ng mL�1

kb�1. After hybridizations, slides were washed with 33 SSC and treated

with RNase A. Final high-stringency washes consisted of a 30-min wash

in 23 SSC at room temperature followed by a 30-min wash in 0.33 SSC

at 438C or in 0.23 SSC at 568C, with similar results. Hybridizations of

As PHYA were also performed at a hybridization temperature of 508C,

with consistent results. Detection was performed using the Dig Nucleic

Acid Detection kit (Roche) supplemented with polyvinyl alcohol to in-

crease sensitivity as described (De Block and Debrouwer, 1993). The

color reaction to visualize hybridized probe was done for 2.5 to 4.5 h at

room temperature. Sections were examined and photographed with a

Zeiss Axioplan2 or an Olympus AX70 equipped with an Olympus digital

camera (DP70).

Pt CENL1 and RM in Developmental Switches 71

Accession Numbers

The poplar gene model identifiers (Tuskan et al., 2006) and/or sequence

accessions used for real-time RT-PCR analysis are as follows: Pt FT1

(fgenesh1_pm.C_LG_VIII000284), Pt FT2 (eugene3.14090001), Pt CO2

(estExt_fgenesh4_pm.C_LG_IV0339), and Pt CENL1 (AY383600; estExt_

fgenesh4_pg.C_660171) from Populus trichocarpa; As PHYA (M18822)

from Avena sativa; Pt PHYA (AJ001318) from Populus tremula 3 P.

tremuloides; and Pt ACTIN (DQ099740) from Populus tremula.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Phylogenetic Analysis of TFL1/FT-Like

Proteins.

Supplemental Figure 2. Protein Sequence Alignment Used for

Phylogenetic Analysis.

Supplemental Figure 3. Localization of As PHYA Transcripts in the

Apex of PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA).

Supplemental Table 1. Primer Sequences Used for Real-Time RT-

PCR Analyses.

ACKNOWLEDGMENTS

We thank Olavi Junttila for providing plant material, Jim Colbert for a gift

of pAPSX2.7, Maria Eriksson for a gift of poplar PHYA cDNA clone,

Sissel Haugslien for advice on grafting, and Linda Ripel for work with

tissue cultures. The Academy of Finland (Finnish Centre of Excellence

program 2000 to 2005, and 2006 to 2011), SNS-Nordic Forest Re-

search, and the Norwegian Research Council (155041 ClimaTree and

171970 ClimaDorm) are acknowledged for financial support.

Received November 7, 2007; revised November 7, 2007; accepted

December 19, 2007; published January 11, 2008.

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74 The Plant Cell

DOI 10.1105/tpc.107.056721; originally published online January 11, 2008; 2008;20;59-74Plant Cell

Raili Ruonala, Päivi L.H. Rinne, Jaakko Kangasjärvi and Christiaan van der SchootPopulusin

Expression in the Rib Meristem Affects Stem Elongation and the Transition to DormancyCENL1

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