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Plant Cell. Jan 2008; 20(1): 59–74.
PMCID: PMC2254923

CENL1 Expression in the Rib Meristem Affects Stem Elongation and the Transition to Dormancy in Populus[W][OA]

Abstract

We investigated the short day (SD)–induced transition to dormancy in wild-type hybrid poplar (Populus tremula × 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 organized patterns. The SAM has a high degree of autonomy, and isolated SAMs can continue shoot formation (Smith and Murashige, 1970; Ball, 1980). This may require the regeneration of a rib meristem (RM), a meristematic region immediately subjacent to the SAM, as the continuous production of leaf-stem units during indeterminate 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 temporarily. 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 differentiate 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 respond to specific environmental cues to reorganize their activities 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 physiological 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 unclear 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 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; Suárez-López 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 (Böhlenius et al., 2006; Hsu et al., 2006), but their downregulation is required in early growth cessation and the transition to dormancy (Böhlenius 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 transitioning, are conserved among different species.

In addition to the early downregulation of FT1/FT2 in poplar leaves (Böhlenius et al., 2006; Hsu et al., 2006), the apex of perennials responds to SDs by reducing symplasmic connectivity 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 signaling (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 transitioning 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 × P. tremuloides) 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 overexpression in the RM/RZ area, but not in the SAM, correlates with a failure to downregulate CENL1 in its RM domain and an absence of transition to dormancy. Collectively, the results suggest that the apex is a semi-independent player in photoperiodic growth responses and that the roles of RM and SAM in photoperiod 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 × P. tremuloides) (Olsen et al., 1997). In line 22 (hereafter called P35S:AsPHYA), this defect is specific to the photoperiodic pathway as their capacity to cease elongation growth under adverse conditions is not impaired. Cold stress or inhibition of gibberellin (GA) biosynthesis, for example, can arrest elongation growth of both the wild type and P35S:AsPHYA independent of photoperiod (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 similar 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.

Figure 1.
Growth Habit of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA).

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 acceleration 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 simultaneously increased from 5.2 (±0.63) under LDs to 7.5 (±0.74) after 2 to 3 weeks of SDs, while in the wild type, no change was observed (LD 7.1 ± 0.74 versus SD 7.4 ± 0.97). Due to this SD-induced acceleration of internode elongation, the P35S:AsPHYA growth habit was restored almost to that of the wild type under LDs (Figure 2C).

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

We next investigated the pattern of bud dormancy development 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 conditions due to SAM-intrinsic constraints, for which reason it is also referred to as endodormancy (Horvath et al., 2003). When decapitated 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, decapitation 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 comparable to that of the decapitated wild type under LD (Figures 3B and 3C). This demonstrates that dormancy was completely prevented in P35S:AsPHYA, not only in the leading shoot but also in resting axillary buds.

Figure 3.
Bud Dormancy Development in Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA).

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 deformed 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 molecules, 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 morphogenetically active state is prevented by the formation of dormancy sphincter complexes (DSCs) composed of an extracellular ring of protein and callose that compresses the PD channel 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 cellular activities remained high as judged from the Em values (Table 1).

Figure 4.
Em Measurement, Iontophoretic Microinjection, Dye Coupling, and PD Ultratructure in the SAM of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA).
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

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 discriminate 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.

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

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 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. Surprisingly, 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. Subsequently, 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 accompanied 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 produced. 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 photoperiod 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 photoperiod. 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 response 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, respectively (see Supplemental Figures 1 and 2 online; Böhlenius et al., 2006). Pt CENL1 and At TFL1 appear to be functionally conserved, as overexpression in Arabidopsis reproduced the effects of TFL1 overexpression (Böhlenius et al., 2006).

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).

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).

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 decreased 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 downregulated ~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 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 subtending 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 = 10 apices).

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

This distinction between the meristematic and the differentiating 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 expressed mainly in the upper part of the RZ (i.e., in the RM), whereas CO2 is expressed more widely in the RZ and the underlying 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).

Figure 8.
Gene Expression in Distinct Areas at the Shoot Apex of Wild-Type and PHYA-Overexpressing Hybrid Poplar P35S:AsPHYA (P35) under LDs.

The relatively low expression level of As PHYA in the dissected SAM samples of P35S:AsPHYA was unexpected since the transferred 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 transcript accumulated at high levels in the tissues beneath the dome (i.e., in the RM/RZ, stem, and leaves) (Figure 9A; see Supplemental 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 vascular 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 hybridization of Pt PHYA was also detected in axillary buds (Figure 9H).

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

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 sink. As younger source leaves predominantly deliver photosynthates 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 predominantly by younger source leaves. To identify genes potentially involved in systemic signaling, RNA should therefore preferentially 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 (Böhlenius 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 continue 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 maturation (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, 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 produce 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 receptor gene etr1-1 can enter dormancy under SDs without forming a clear terminal bud. The distinct nature of both processes, 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 reversible 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 processes that orchestrate DSC formation.

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 formation, suggesting that FT2, transported via the phloem to the apex, assists the continuation of stem elongation and morphogenesis 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 transitions 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 elongation. For example, Arabidopsis mutants lacking PHYA produce elongated hypocotyls when grown under a canopy (Johnson et al., 1994; Yanovsky et al., 1995), whereas strong overexpression 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., 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 overexpressed 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, considering that Pt PHYA was expressed similarly in the SAM of P35S:AsPHYA and the wild type, it might be the high overexpression 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 and9A;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 and8B),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 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 (Böhlenius 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 implausible 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 formation (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 interactors, 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.

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 × P. tremuloides, clone T89) and transgenic line 22 (called P35S:AsPHYA) were investigated. Wild-type and P35S:AsPHYA ramets were micropropagated 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 18°C and 80% relative humidity. Natural light was supplemented to 200 μmol 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 longitudinal 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 defoliated 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) paraformaldehyde 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-μm-thick sections were cut with an ultramicrotome (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 microscopy, ultrathin sections (70 nm) were cut with an ultramicrotome, stained with 2% aqueous uranyl acetate and Reynolds' lead citrate, and examined 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 callose, 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 microscope (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, embryonic 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 transcriptase (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 manufacturer (Invitrogen). Gene-specific primer sequences (see Supplemental 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 using 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-μm 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 μg mL−1), and the hybridizations were performed at 43°C overnight with a probe concentration of 0.28 ng μL−1 kb−1. After hybridizations, slides were washed with 3× SSC and treated with RNase A. Final high-stringency washes consisted of a 30-min wash in 2× SSC at room temperature followed by a 30-min wash in 0.3× SSC at 43°C or in 0.2× SSC at 56°C, with similar results. Hybridizations of As PHYA were also performed at a hybridization temperature of 50°C, with consistent results. Detection was performed using the Dig Nucleic Acid Detection kit (Roche) supplemented with polyvinyl alcohol to increase 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).

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 × 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.

Supplementary Material

[Supplemental Data]

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 Research, and the Norwegian Research Council (155041 ClimaTree and 171970 ClimaDorm) are acknowledged for financial support.

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Raili Ruonala (if.iknisleh@alanour.iliar).

[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

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