These authors contributed equally to this work.
Populus CEN/TFL1 regulates first onset of flowering, axillary meristem identity and dormancy release in Populus
Article first published online: 26 FEB 2010
© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd
The Plant Journal
Volume 62, Issue 4, pages 674–688, May 2010
How to Cite
Mohamed, R., Wang, C.-T., Ma, C., Shevchenko, O., Dye, S. J., Puzey, J. R., Etherington, E., Sheng, X., Meilan, R., Strauss, S. H. and Brunner, A. M. (2010), Populus CEN/TFL1 regulates first onset of flowering, axillary meristem identity and dormancy release in Populus. The Plant Journal, 62: 674–688. doi: 10.1111/j.1365-313X.2010.04185.x
- Issue published online: 11 MAY 2010
- Article first published online: 26 FEB 2010
- Received 20 October 2009; revised 22 January 2010; accepted 9 February 2010; published online 6 April 2010.
- TERMINAL FLOWER 1;
- axillary meristem
- Top of page
- Experimental procedures
- Supporting Information
Members of the CENTRORADIALIS (CEN)/TERMINAL FLOWER 1 (TFL1) subfamily control shoot meristem identity, and loss-of-function mutations in both monopodial and sympodial herbaceous plants result in dramatic changes in plant architecture. We studied the degree of conservation between herbaceous and woody perennial plants in shoot system regulation by overexpression and RNA interference (RNAi)-mediated suppression of poplar orthologs of CEN, and the related gene MOTHER OF FT AND TFL 1 (MFT). Field study of transgenic poplars (Populus spp.) for over 6 years showed that downregulation of PopCEN1 and its close paralog, PopCEN2, accelerated the onset of mature tree characteristics, including age of first flowering, number of inflorescences and proportion of short shoots. Surprisingly, terminal vegetative meristems remained indeterminate in PopCEN1-RNAi trees, suggesting the possibility that florigen signals are transported to axillary mersitems rather than the shoot apex. However, the axillary inflorescences (catkins) of PopCEN1-RNAi trees contained fewer flowers than did wild-type catkins, suggesting a possible role in maintaining the indeterminacy of the inflorescence apex. Expression of PopCEN1 was significantly correlated with delayed spring bud flush in multiple years, and in controlled environment experiments, 35S::PopCEN1 and RNAi transgenics required different chilling times to release dormancy. Considered together, these results indicate that PopCEN1/PopCEN2 help to integrate shoot developmental transitions that recur during each seasonal cycle with the age-related changes that occur over years of growth.
- Top of page
- Experimental procedures
- Supporting Information
Many features that distinguish tree growth habit from that of herbaceous plants are the result of the pattern of activity and identity of shoot meristems. Trees typically postpone flowering, producing only vegetative meristems for several years. In Populus, terminal shoot meristems remain indeterminate throughout a tree’s life, but adult trees develop axillary inflorescence buds shortly after winter dormancy, and catkins complete development the following year (Boes and Strauss, 1994; Yuceer et al., 2003). In contrast to annual plants with monopodial shoot growth that undergo a single vegetative to reproductive phase transition, both phases coexist within adult poplar trees (Brunner and Nilsson, 2004). Adult poplars contain branches with and without inflorescence buds, and individual branches produce both axillary vegetative and reproductive buds.
In temperate- and boreal-zone trees, shoot meristems seasonally cycle between growth and winter dormancy (Howe et al., 2003; Welling and Palva, 2006; Rohde and Bhalerao, 2007). Decreases in day length and temperature during fall induce growth cessation, cold acclimation, endodormancy and, finally, maximal cold hardiness. Following an extended chilling period to release dormancy, the increasing temperatures and photoperiod in spring stimulate the resumption of growth. Genetic variation among populations in the timing of these seasonal-related traits is associated with climatic and geographic gradients, reflecting the adaptive importance of maximizing growth while minimizing cold injury.
In addition to seasonal shoot-growth patterns, trees exhibit maturation-related changes in shoot architecture (e.g. branch frequency) that occur over years (Greenwood, 1995; Brunner et al., 2003). The poplar crown is composed of shoot types ranging from extremely short shoots, which set buds very early in the growing season, to several meter-long shoots bearing both preformed and neoformed leaves, which continue growing until a critical day length for short day (SD)-induced bud set. There is considerable natural variation in the proportion of long shoots and a strong maturation trend in their frequency: over years, trees have proportionally fewer long shoots, and catkins tend to be most prevalent on short shoots in the upper crown (Critchfield, 1960; Dickmann et al., 2001).
There is abundant evidence that the genes and pathways regulating shoot meristem activity are highly conserved among plants (McSteen and Leyser, 2005; Kobayashi and Weigel, 2007). However, there is also evidence that signaling modules for shoot meristem regulation have been adapted for control of seasonal growth in perennial plants such as poplar (Bohlenius et al., 2006; Ruttink et al., 2007). In Arabidopsis, CONSTANS (CO) and FLOWERING LOCUS T (FT) control long-day (LD)-induced flowering (Kardailsky et al., 1999; Kobayashi et al., 1999; Samach et al., 2000). Poplar orthologs of FT also promote flowering in poplar, and, in addition, the PtCO2/PtFT1 regulon mediates SD-induced growth cessation and bud set (Bohlenius et al., 2006; Hsu et al., 2006).
FT belongs to the phosphatidylethanolamine-binding protein (PEBP) superfamily. The plant PEBP genes group into three major subfamilies –FT-like, TFL1-like and MFT-like – with the MFT-like clade being ancestral to the other clades (Kobayashi et al., 1999; Hedman et al., 2009). TFL1 acts in opposition to FT: tfl1 mutants flower earlier, and the inflorescence meristem converts to a terminal flower (Shannon and Meeks-Wagner, 1991; Bradley et al., 1997). Constitutive expression of TFL1 results in a longer vegetative phase, a larger and highly branched inflorescence and delayed flower formation (Ratcliffe et al., 1998). Study of TFL1 homologs in various plant species have revealed similar but also distinctive functions, and diversification of paralogous genes has also been shown. Whereas TFL1 is expressed in vegetative and inflorescence meristems, its paralog Arabiodopsis thaliana CEN (ATC) is expressed in the hypocotyl, and these expression differences have resulted in functional differences: atc mutants showed no effects on flowering, nor any obvious phenotype (Mimida et al. 2001). The snapdragon (Antirrhinum spp.) homolog, CEN, is only expressed in the inflorescence meristem, and cen mutants form terminal flowers, but flowering time is not altered (Bradley et al., 1996). In pea (Pisum sativum), flowering time and meristem determinacy functions are subdivided between two TFL1 homologs (Foucher et al., 2003). LATE FLOWERING (LF) acts only as a repressor of flowering, whereas DETERMINATE (DET) maintains the indeterminate inflorescence meristem. In tomato, a recessive mutation in SELF-PRUNING (SP) causes premature termination of sympodial units into inflorescences (Pnueli et al., 1998). In contrast to FT and TFL1, the function of MFT is unclear. No phenotypic changes were observed in the mft-1 mutant, but overexpression of MFT slightly accelerated flowering in Arabidopsis, suggesting it might act redundantly to control flowering time (Yoo et al., 2004).
To study the role of PopCEN1 and PopMFT in the age/size-related developmental changes that occur in conjunction with recurrent seasonal transitions in temperate-zone trees, we overexpressed and downregulated these genes in poplar. The effects of misexpression were then studied during the onset of flowering over six growing seasons in the field. Whereas we did not identify a function for PopMFT, we show that PopCEN1 and/or PopCEN2 share functional commonalities with their homologs in herbaceous plants, in control of flowering and inflorescence architecture, but also find differences that reflect unique features of the tree growth habit, including a role in the control of shoot phenology.
- Top of page
- Experimental procedures
- Supporting Information
The Populus trichocarpa TFL1/FT gene family
PopCEN1 and PopMFT were isolated from P. trichocarpa by homology-based cloning (Mohamed, 2006). Subsequent searches of the P. trichocarpa version 1.1 (poptr v1.1) and recently released poptr v2 genome assemblies (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html and http://www.phytozome.net/poplar) showed that poplar contains only one member of the MFT subfamily and three genes in the TFL1-like subfamily. PopCEN2 is 91% identical in both nucleotide and amino acid sequence to PopCEN1. Within the TFL1/CEN group, eudicot members form a well-supported subgroup with CEN, or weakly group with TFL1 (Figure S1). A number of eudicots, including Arabidopsis, grapevine (Vitis vinifera) and tomato (Solanum lycopersicum), contain members of both the CEN and TFL1 groups, but the Populus lineage apparantly has lost the TFL1-like gene (also see Igasaki et al., 2008). In addition, poplar has one ortholog of BROTHEROF FT AND TFL1 (BFT), which groups separately from the TFL1/CEN group. Whereas five members of the FT subfamily were identified in poptr v1.1, poptr v2 only contains three, including a possible pseudogene that lacks 5′ exons (Table S1). Resolution of some FT group members as alleles from unassembled haplotypes may explain this difference. However, Igasaki et al. (2008) cloned five different Populus nigra FT-like genes, indicating that additonal mapping is needed to resolve the number of FT homologs in Populus.
PopCEN1, PopCEN2 and PopMFT have distinct expression patterns
PopMFT was expressed in both vegetative and inflorescence buds (Figure S2a). The seasonal pattern of PopMFT expression was similar in terminal and axillary vegetative buds, and also in juvenile and adult trees (Figure S2b,c). Expression decreased as buds approached the time of bud flush, remained low in actively elongating shoot tips and was upregulated in fall buds. PopMFT expression in reproductive shoots was also highest in fall (Figure S2d). PopCEN1 and PopCEN2 had very different tissue-type expression patterns (Figure 1a). PopCEN1 was highly expressed in shoot tips and vegetative buds, whereas PopCEN2 showed highest expression in the stem, leaf blade, petiole and immature inflorescence. In contrast to the seasonal pattern of PopMFT expression, PopCEN1 was strongly upregulated in both terminal and axillary vegetative buds around the time of spring bud flush (Figures 1b–c and S3). PopCEN1 expression decreased markedly a month later, when shoots were still elongating, and was lowest in late summer and fall buds. Although PopCEN1 expression was relatively low during the summer, expression level was fourfold higher in actively growing long shoots compared with short shoots that had set bud, and threefold higher in newly formed axillary buds in the axils of neoformed leaves compared with older buds in the axils of preformed leaves (7/19 collection date; Figure 1b–c). The seasonal expression pattern of PopCEN1 was similar for both terminal and axillary vegetative buds, and for both juvenile and adult trees (Figures 1b–c and S3b–e).
Functional analysis of PopCEN1/PopCEN2 and PopMFT in transgenic poplars
We transformed poplar clone INRA 717-1B4 (Populus tremula × Populus alba) with 35S::PopMFT and 35S::PopCEN1 transgenes. RNAi transgenes were introduced to downregulate endogenous PopMFT or PopCEN1. None of the transgenics exhibited flowering or other obvious phenotypic effects while in tissue culture, or during 2 months in the glasshouse. Four ramets per event and 10 non-transgenic control ramets were planted in the field in June 2003. We only studied events showing detectable transgene expression in the overexpression transgenics (15–18 events per construct), and events showing target endogene expression levels below that of non-transgenic controls for RNAi transgenics (9–10 events per construct). The selected events differed widely in transgene expression level or degree of target endogene downregulation (Figure S4). Because of their high sequence similarity, both PopCEN1 and PopCEN2 were suppressed in PopCEN1-RNAi transgenics (Figure S4d); thus, transgenic phenotypes are not specific to PopCEN1, but indicate the combined function of PopCEN1/PopCEN2. We measured net growth for the 2004 growing season, when competition effects were still minor, and found no significant differences between the transgenic and control groups (Figure S4g). One event from the 35S::PopCEN1 trees grew slower than the non-transgenic trees (P < 0.0009, Dunnett’s test), but no other transgenic events were significantly different from control trees (Dunnett’s test, α = 0.05). However, in later years, 35S::PopCEN1 trees were noticeably smaller, presumably because their delayed spring bud flush phenotype (described below) increasingly put them at a competitive disadvantage, as the closely surrounding trees that exhibited WT bud phenology grew larger.
PopCEN1/PopCEN2 expression regulates first onset of flowering and the identity of axillary buds
The first time that any tree initiated inflorescence buds was 2 years after planting (2005). Two ramets from each of two PopCEN1-RNAi events showed an unusual phenotype, in that some of the floral buds flushed in August and rapidly elongated into mature catkins, thus completing flower development in the same season in which they were initiated (Figure 2a,d). The inflorescence buds formed in the leaf axils of current-year stems, and, unlike normal pendulous inflorescences of poplar, the catkins were more branch-like: long and upright (Figure 2a). On one catkin of event 191, new vegetative shoots formed near the terminus of the catkin (Figure 2a, inset), indicating a reversion to vegetative identity.
Most of the floral buds initiated in 2005 overwintered as buds, flushed the following spring and produced pendulous catkins bearing female flowers with morphology indistinguishable from the wild type (Figure 2b–c). One of the 10 control trees produced a few catkins and two or more trees from each of the transgenic populations flowered, but the PopCEN1-RNAi population showed a much higher occurrence and intensity of flowering (measured as flowering index; Figure 3a). Moreover, the flowering index was inversely correlated with the degree of downregulation of the PopCEN1 endogene (Figure 3b), indicating that PopCEN1 regulates the first onset of flowering and the intensity of flowering. No correlations were found between flowering and expression level of the PopMFT endogene or the overexpression transgenes (data not shown).
The field trial was thinned in 2006, leaving all control trees and at least two ramets per transgenic event. The summer floral bud flush that occurred on a few trees in 2005 did not occur in subsequent years; all inflorescences elongated after winter dormancy. For control trees, there was little change in the number of flowering trees between 2006 and 2007, but by 2008, eight of the 10 control trees were flowering, although most were in the weakest flowering category (Figure 3c). Both the 35S::PopMFT and PopMFT-RNAi transgenic populations showed an increase in the number of trees flowering between 2007 and 2008, although to a lesser degree than controls (Figure S5). Most PopCEN1-RNAi transgenics were flowering intensely by 2007. Whereas flowering showed an inverse linear correlation with PopCEN1 endogene expression in 2006 (Figure 3b), by 2007 the PopCEN1-RNAi transgenics appeared to form two clusters, with all ramets from events showing more than a 50% reduction in PopCEN1 endogene expression flowering heavily (Figure 3c,d and S4c). All ramets from 35::PopCEN1 transgenic events that had transgene expression levels in the top 50% remained non-flowering, except that one ramet from event 36 flowered in 2009 (Figure 3c,e).
By 2007, many of the catkins occurred on shorter, higher-order shoots in the intensely flowering PopCEN1-RNAi trees (Figure 2e), whereas control trees produced far fewer catkins (Figure 2f). Extremely short shoots with nearly all axillary buds giving rise to catkins became more frequent in the following years (compare Figure 4c with the catkin-bearing shoot of Figure 2b), a feature that is typically characteristic of older, larger trees. Terminal shoot apices always remained vegetative (Figure 4c), indicating that PopCEN1/PopCEN2 regulates axillary meristem identity. Moreover, PopCEN1/PopCEN2 downregulation affected the development of the axillary inflorescences, with RNAi transgenics having a significantly lower (P < 0.0001, Tukey–Kramer’s test) average catkin length, weight and flower number per catkin, compared with controls (Figure 4a,b). In mid-April, when catkins had elongated and no new flowers were developing, PopCEN1-RNAi transgenic events had 26–51% fewer flowers/catkins than controls. On average, control catkins were 19% longer and 34% heavier (data not shown) than PopCEN1-RNAi transgenics.
PopCEN1/PopCEN2 affects shoot phenology and crown architecture
Because of the seasonal expression patterns of PopCEN1 and PopMFT, we recorded the date of spring vegetative bud flush. In 2005, the 35S::PopCEN1 group flushed 9 days later than non-transgenic trees (P < 0.0001; Figure 5a). Seven out of 18 35S::PopCEN1 events flushed late, ranging from 8 to 22 days later than control trees. The level of the 35S::PopCEN1 transgene expression was significantly correlated with the extent of delay in bud burst (Figure 5b). Although shoot phenology varies among years, the transgene expression level was again significantly correlated (R = 0.838; P < 0.0001) with delayed bud flush in the following year (Figure 5c–e). There was no evidence that bud flush of the two groups of RNAi trees differed from the non-transgenic trees (Tukey–Kramer’s test, α = 0.05). Four out of 19 35S::PopMFT events flushed significantly later than non-transgenic trees in 2005 (P < 0.0009, Dunnett’s test), but bud burst date was not correlated with transgene expression level.
The time of bud flush depends on fulfilling both the chilling requirement to release dormancy and the subsequent heat sum required to promote the resumption of growth (Rohde and Bhalerao, 2007). To find out if PopCEN1 affects dormancy release, we studied representative RNAi and overexpression transgenics in a growth chamber under a photoperiod and temperature regime previously shown to be effective for inducing, maintaining and releasing endodormancy in 717-1B4 (Figure 6a; Rohde et al., 2007). In WT trees, PopCEN1 expression in terminal buds increased during the chilling treatment phase (Figure 6b). All trees set bud during the SD treatment, with the 35S::PopCEN1 population completing bud set somewhat sooner than controls (Figure S6a). Ramets transferred to growth-promoting conditions on day 49 of the SD treatment were unable to resume growth (Figure 6d), indicating that dormancy was established in the controls and in both transgenic populations.
Monitoring dormancy release during chilling showed that the rate of release differed among the three groups (Figure 6c,d). Following 14 days of chilling, PopCEN1-RNAi transgenics resumed growth after an average of 37 days in LDs, but control and 35S::PopCEN1 terminal buds did not flush for 100 or more days (Figure 6c,d). After 28 days of chilling, RNAi and control plants flushed after an average of 18 and 21 days, respectively, but 35S::PopCEN1 trees required 94 days to flush. After 48 days of chilling, RNAi, control and 35S::PopCEN1 trees needed 10, 11 and 14 days, respectively, to resume growth. However, the flushed terminal shoots of 35S::PopCEN1 trees did not continue to elongate, and an axillary bud near the shoot apex grew out to become the dominant shoot (Figure 6d–f). 35S::PopCEN1trees that had received various chilling times also had a lower percentage of axillary buds that flushed compared with controls (Figure S6b).
We measured shoot characteristics in the upper crown of representative events in June 2006, 3 years after planting. The numbers of primary branches were similar for PopCEN1-RNAi, 35S::PopCEN1 and control trees, but secondary branching occurred rarely or not at all in the 35S::PopCEN1 transgenic trees (Figure 7a–b). For both primary and higher-order shoots, PopCEN1-RNAi trees had the highest proportion of short shoots, whereas 35S::PopCEN1 trees had a very high percentage of long shoots (Figure 7c–f). The high proportion of short shoots in the PopCEN1-RNAi trees in 2006 correlates with the occurrence of many catkins from 2007 onwards on short, higher-order shoots in these heavily flowering trees (Figures 2e and 4c).
- Top of page
- Experimental procedures
- Supporting Information
By studying PopCEN1/PopCEN2 function over several years, we have shown that it has a major role in both age/size-related maturation and seasonal shoot development (Figure 8). PopCEN1/PopCEN2 regulates the time of first flowering, and also the progression to increased proportion of short shoots and increased flowering intensity. Both these maturation trajectories typically continue for several years after first flowering. PopCEN1 downregulation is important for bud dormancy release and its upregulation in spring, when growth resumes, correlates with the time that newly formed axillary meristem identity is established. In addition PopCEN1/PopCEN2 appears to have a role in maintaining the indeterminacy of the terminal inflorescence meristem as floral meristems develop on the flanks.
PopCEN1/PopCEN2 regulates the first onset of flowering in poplar
Members of the TFL1/CEN subfamily vary with respect to their role in the regulation of flowering time. Downregulation of the apple ortholog, MdTFL1, and loss-of-function mutations in TFL1 and LF, but not CEN, SP or DET, result in early flowering (Bradley et al., 1996, 1997; Pnueli et al., 1998; Foucher et al., 2003; Kotoda et al., 2006). Poplar transgenics showing strong downregulation of PopCEN1 initiated floral buds after 2 years in the field, whereas most control trees first initiated floral buds at 4 years (Figure 3). In contrast, the eight 35S::PopCEN1 events showing the highest transgene expression levels never flowered, except for one ramet from event 36 that initiated floral buds during the sixth growing season (Figure 3). Because both PopCEN1 and PopCEN2 were suppressed in PopCEN1-RNAi transgenics (Figure S4d), it is possible that PopCEN2 rather than PopCEN1, or both genes, regulate flowering time.
Overexpression of poplar FT homologs induced flowering within months following transformation (Bohlenius et al., 2006; Hsu et al., 2006). The complete absence of PopCEN1/PopCEN2 activity might accelerate flowering to a similar degree as FT overexpression, but it is also possible that the more modest acceleration induced by PopCEN1/PopCEN2 downregulation is because other genes have a predominant role in maintaining the long juvenile phase of poplar. In Arabidopsis, FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) directly repress the flowering pathway integrators FT and SOC1 (Hepworth et al., 2002; Michaels et al., 2005; Helliwell et al., 2006; Li et al., 2008). In yeast two-hybrid studies, FT and TFL1 interact with the same proteins (Pnueli et al., 2001; Wigge et al., 2005), suggesting that TFL1 and FT might regulate flowering by competing for common interacting partners, and by having opposite effects on their partner’s activity (Ahn et al., 2006). In tomato, the local ratio of SP and the FT ortholog SINGLE FLOWERING TRUSS (SFT) regulates flowering as well as aspects of vegetative development (Shalit et al., 2009). The expression level of two poplar FT orthologs gradually increases with age (Bohlenius et al., 2006; Hsu et al., 2006), suggesting the possibility that downregulation of PopCEN1/PopCEN2 might have accelerated the time of first flowering by lowering the level of FT required to out-compete PopCEN1/PopCEN2.
Overexpression of MFT and PopMFT in Arabidopsis induces a modest acceleration of flowering (Yoo et al., 2004; data not shown); however, overexpression of PopMFT did not detectably accelerate the year of flowering onset in poplar (Figures 3a and S5). MFT homologs in P. nigra and maize (Zea mays) were most strongly expressed in seeds, suggesting MFT may have a role in seed development (Igasaki et al., 2008; Danilevskaya et al., 2008). PopMFT expression in buds is also seasonally regulated, with the highest expression in fall buds (Figure S2). Both SD-induced bud set and seed maturation are regulated by ABSCISIC ACID-INSENSITIVE (ABI3), and PopMFT is upregulated in poplars overexpressing ABI3 just after the onset of SD (Rohde et al., 2002; Gutierrez et al., 2007; Ruttink et al., 2007). Although PopMFT poplar transgenics formed buds and became dormant in the field, additional study under different, controlled conditions could perhaps reveal a functional role in bud set.
PopCEN1/PopCEN2 regulates axillary meristem identity
The most striking phenotype induced by the strong downregulation of PopCEN1/PopCEN2 was an increase in inflorescence number. All 13 trees showing 50% or more downregulation had more than 100 catkins by 2007, whereas only one of 10 control trees reached this level of flowering, but not until 2008 (Figure 3). Moreover, PopCEN1/PopCEN2 downregulation did not alter terminal vegetative meristem identity: inflorescences only developed from axillary meristems (Figure 4c). In monopodial annual plants such as Arabidopsis, flowering signals are translocated to the shoot apex, which transitions to an inflorescence meristem that only then gives rise to axillary flowers and secondary inflorescence shoots. Inflorescence meristems are converted to terminal flowers in tfl1 mutants and growth ceases (Shannon and Meeks-Wagner, 1991). Thus, as discussed below, the PopCEN1-RNAi results shed light on the question of where florigen signals are translocated to and active in poplar (Hsu et al., 2006).
There is a seasonal window after growth resumes in spring when an adult poplar tree is able to specify inflorescence identity to some of the axillary meristems, and then initiation and development of flowers continue within the inflorescence bud during the growing season (Boes and Strauss, 1994; Yuceer et al., 2003). Overexpression of FT2 and LEAFY (LFY) induced terminal flowers in poplar, indicating that terminal shoot apices are competent to respond to flower-promoting signals (Weigel and Nilsson, 1995; Rottmann et al., 2000; Hsu et al., 2006). One possibility is that flowering signals are translocated to the poplar shoot apex, allowing the initiation of axillary inflorescence buds, but signals are not maintained and the shoot meristem reverts to producing vegetative buds. If this is the case, our results indicate that factors other than PopCEN1/PopCEN2 maintain the indeterminacy of the terminal shoot meristem, or that greatly reduced levels of PopCEN1/PopCEN2 are sufficient to maintain terminal but not axillary meristems.
In Arabidopsis, axillary meristems arise acropetally during the vegetative phase, but arise basipetally after the transition to flowering; thus, developing axillary meristems are subtended by leaves with well-developed vasculature only during the vegetative phase (Grbic and Bleecker, 2000; Long and Barton, 2000). TFL1 is not upregulated in the shoot apex until the inflorescence phase, but is strongly expressed in axillary meristems during the vegetative phase, perhaps to provide protection from florigen signals originating from their subtending leaves (Conti and Bradley, 2007). In contrast, axillary meristems arise acropetally during all phases of poplar development. If florigen signals are translocated to axillary meristems in poplar, the transition of an individual axillary meristem to an inflorescence meristem would be analogous to the reproductive transition of an entire Arabidopsis plant. In this case, PopCEN1 might not be needed to maintain terminal vegetative meristems, but by analogy, might be expected to maintain the identity of inflorescence apices.
Poplar inflorescences are thought to be indeterminate, but, to our knowledge, detailed SEM studies to conclusively show the absence of a terminal flower, such as those carried out for pea (Singer et al., 1999), have not yet been reported. Nonetheless, catkins on PopCEN1-RNAi trees had significantly fewer flowers than those on control trees (Figure 4), suggesting a possible role in maintaining the inflorescence meristem. In Arabidopsis, LFY and APETALA1 (AP1) repress TFL1 expression in floral meristems, and TFL1 represses LFY and AP1 in the inflorescence meristem (Liljegren et al., 1999; Ratcliffe et al., 1999). Consistent with the conservation of these regulatory interactions in poplar, poplar LFY (PTLF) and poplar AP1 were strongly expressed in initiating axillary floral meristems, but were not detected in the apical inflorescence meristem (Rottmann et al., 2000; Brunner et al., unpublished data). Although we could not differentiate the functions of PopCEN1 and PopCEN2, their different expression patterns (Figure 1a) suggest functional diversification. The higher expression of PopCEN2 in developing inflorescences in summer suggests that it might maintain inflorescence meristem identity.
Vegetative bud and shoot tip samples included embryonic and immature leaves, and thus, initiating axillary meristems. The expression of PopCEN1 in these samples (Figures 1 and S4) is, therefore, consistent with a role in regulating whether or not an axillary meristem transitions to an inflorescence meristem. Moreover, the striking upregulation of PopCEN1 as growth resumes in spring corresponds to the seasonal time when new axillary meristems are developing, and some are transitioning to inflorescences (Boes and Strauss, 1994; Yuceer et al., 2003). Thus, both expression and transgenic phenotypes indicate that a major role of PopCEN1 is to promote the vegetative identity of axillary meristems, and that in adult trees, signals promoting flowering are able to switch some axillary meristems to inflorescence meristems. In addition to age-related increases in expression, poplar FT1 and FT2 were markedly upregulated in leaves in late spring, and FT2 was also upregulated in axillary buds that subsequently formed inflorescences (Bohlenius et al., 2006; Hsu et al., 2006). Considered together, these results suggest that the local balance of PopCEN1/PopCEN2 and FT1/FT2 might regulate the seasonal determination of vegetative versus inflorescence identity of axillary meristems, as well as inflorescence versus floral meristem identity in the developing inflorescence.
Role of PopCEN1/PopCEN2 in vegetative growth
Plant growth and size was markedly reduced in tfl1, cen, det and sp mutants, and by the downregulation of MdTFL1 (Bradley et al., 1996, 1997; Pnueli et al., 1998; Foucher et al., 2003; Kotoda et al., 2006), but this phenotype was not induced by PopCEN1 downregulation, because terminal vegetative meristems remained indeterminate. However, PopCEN1-RNAi and 35S::PopCEN1 transgenics exhibited opposite trends in branching, with RNAi transgenics having a high proportion of short shoots 3 years after planting (Figure 7). Over the years, higher-order branching and the proportion of short shoots increase in poplars (Critchfield, 1960; Dickmann et al., 2001). Catkins occur on any length or order of shoot, but most commonly appear on higher-order short shoots. Hence, higher-order short shoots are not a prerequisite for flowering, but their occurrences might be coordinated; however, further studies are needed to determine if the onsets of heavy flowering and a skewed ratio of short/long shoots are significantly correlated. By reducing leaf display costs, the high leaf-to-stem mass of short shoots may be beneficial to mature trees that accumulate more non-photosynthetic organs (Suzuki, 2003). Perhaps coordination of these processes by PopCEN1/PopCEN2 is an important feature of the poplar growth habit, allowing a large, self-supporting tree to thrive while displaying high levels of reproductive effort over many decades.
Poplars constitutively expressing oat PHYA do not set bud under SDs, and, unlike WT trees, PopCEN1 expression was not downregulated in response to SDs (Ruonala et al., 2008). Our studies did not reveal a role for PopCEN1/PopCEN2 in dormancy induction, but indicate that downregulation of PopCEN1/PopCEN2 is important for dormancy (Figures 6 and S6). Spring bud flush was delayed in 35S::PopCEN1 transgenics, yet PopCEN1 transcripts increased during chilling, and showed a marked upregulation around the time of bud flush (Figures 1, 6 and S3). Under controlled conditions, PopCEN1-RNAi and 35S::PopCEN1 transgenics had different requirements for dormancy release, with 35S::PopCEN1 needing longer chilling than the WT. Thus, PopCEN1 downregulation might be needed to enable dormancy release, and then later, as dormancy is released and growth resumes, upregulation of PopCEN1 promotes meristem indeterminacy.
A chilling period sufficient to reduce the time to terminal bud flush for 35S::PopCEN1 transgenics was not able to sustain shoot elongation, and an axillary shoot became dominant (Figure 6). This suggests that resumption of shoot growth could have separable phases that require different signals or different levels of a signal, and that at least some axillary buds require less chilling than the terminal bud. In beech (Fagus spp.), chilling had a strong effect on time of bud flush for terminal but not axillary buds; however, chilling accelerated the progression of bud flush in both types of bud, although the effect was more pronounced for terminal buds (Falusi and Calamassi, 1990).
During dormancy induction, plasmodesmata are blocked, and chilling restores symplastic circuitry (Rinne et al., 2001). Moreover, storage vacuoles are produced during dormancy induction, and are distributed throughout the cytoplasm, but during chilling, they align along the plasma membrane. Delineating relationships between these cellular changes and PopCEN1 function are important because TFL1 moves among cells to become evenly distributed across the shoot meristem (Conti and Bradley, 2007), and trafficking of proteins to storage vacuoles is impaired in tfl1 mutants (Sohn et al., 2007). Ruonala et al. (2008) localized PopCEN1 expression to the rib meristiem (RM), suggesting that the dormancy-associated changes in symplastic circuity are relevant to PopCEN1 function. For example, when PopCEN1 is upregulated as growth resumes in spring, plasmodesmata are open, making it possible for the PopCEN1 protein to move from the RM into the SAM. Also important to determine is whether the functions of PopCEN1 in dormancy and flowering are distinct or part of a shared regulatory pathway. The effect of PopCEN1 expression level on dormancy release and its expression pattern suggest that it could have a role in measuring chilling accumulation, a process that might be shared between dormancy and vernalization (Horvath et al., 2003; Sung and Amasino, 2005; Rohde and Bhalerao, 2007). Although there is no conclusive evidence linking vernalization with flowering in poplar, the seasonal timing of the floral transition and expression patterns of FT1 and FT2 (Bohlenius et al., 2006; Hsu et al., 2006) suggest that this is possible.
FT, TFL1 and other meristem identity genes have been of interest as tools for modifying tree flowering (Brunner et al., 2007). They may be useful for the promotion of flowering, and for its prevention as a means of reducing unwanted gene flow. Our data suggests that, with continued technological development, PopCEN1 holds promise for both purposes. Its suppression caused large numbers of fertile flowers to form at least 2–3 years earlier and lower in the crown than would be expected in most poplars. In contrast, the more dramatic acceleration of the onset of flowering via overexpression of LFY or FT does not appear to give rise to normal catkins that are capable of completing the reproductive cycle to form viable seeds (Rottmann et al., 2000; Strauss et al., unpublished data). Overexpression of PopCEN1 caused a nearly complete absence of flowering, but also caused severely disturbed shoot phenology and crown architecture. However, our results suggest that it might be productive to study the use of PopCEN1 to prevent the transition to flowering via floral or inflorescence meristem-predominant promoters.
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- Experimental procedures
- Supporting Information
TFL1/FT family members were identified by BLASTP searches of predicted proteins in the Arabidopsis (TAIR, http://www.arabidopsis.org), rice (http://rice.plantbiology.msu.edu/index.shtml) and poplar (JGI, http://genome.jgi-psf.org) genome databases, and in the NCBI protein database (http://www.ncbi.nlm.nih.gov) (Table S1). Proteins were aligned using muscle (Edgar, 2004), and phylogenetic analysis was performed using the neighbor-joining method in mega 3.1 (Kumar et al., 2004).
Populus trichocarpa tissues were collected from trees near Corvallis, OR, USA, as previously described (Kalluri et al., 2007). Buds and shoot tips were collected from juvenile and adult female hybrid poplar trees (P. trichocarpa × P. deltoides, clone 15–29) growing in plantations near Clatskanie, OR, USA, in 2001. Populus alba tissues were collected from a clonal group in Blacksburg, VA, USA, at different seasonal times. For Figure 1a, shoot tip, terminal and axillary vegetative buds, stems (primary/transitional growth region), leaves and petioles were collected from the upper crown of an adult tree on 19 July 2006. Flowers and inflorescence buds were collected from the same tree on 11 April 2006 and 30 August 2006, respectively. Roots were collected from a 1-year-old tree on 19 April 2007. Secondary xylem and phloem/cambium were collected from a 5-year-old tree on 9 May 2007.
Total RNA was extracted and treated with DNAse, as described previously (Brunner et al., 2004). cDNA was synthesized from 1 μg RNA using SuperScript™ II (Invitrogen, http://www.invitrogen.com), according to the manufacturer’s protocol.
Quantitative PCR was performed in a 25-μl final volume containing 12.5 μl of Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen), 0.4 μm each of forward and reverse primers, and 1 μl of a 1:5 dilution of the cDNA reaction mixture as template. Reactions were performed on an MX3000P™ Real-time PCR System (Stratagene, http://www.stratagene.com). The relative quantities were determined according to Pfaffl (2001), and were normalized to levels of UBQ. Normalization, calibration and standard deviation calculations were performed using qbase v1.2.2 (Hellemans et al., 2007). Experiments shown in Figures 1 and 6b were performed as described above, with the following exceptions: cDNA was synthesized from 2 μg RNA using the High Capacity cDNA kit (Applied Biosystems, http://www.appliedbiosystems.com), and qPCR was performed on an ABI PRISM™ 7500, according to the manufacturer’s protocol, with relative quantities determined according to Livak and Schmittgen (2001). Primer sequences are shown in Table S2. PCR efficiencies were at least 95%, and poplar UBQ was validated as an internal control for the various tissues analyzed (Mohamed, 2006; Wang et al., unpublished data), as previously described (Brunner et al., 2004; Gutierrez et al., 2008).
Vector construction and transgenic plant production
All sequences were amplified with primers introducing restriction sites suitable for subcloning (Table S2). For the PopCEN1-RNAi construct, a 147-bp fragment was amplified and inserted into pHANNIBAL to create an inverted repeat transgene (Wesley et al., 2001). The transgene was then excised with NotI and ligated into pART27 (Gleave, 1992). A 239-bp fragment was amplified to create the PopMFT-RNAi construct. Full-length coding regions of PopCEN1 and PopMFT were amplified and inserted into the 35S cassette (Hellens et al., 2000), and the fusions were excised with EcoRV and ligated into the filled-recessed termini of the SstI site of pART27.
INRA clone 717-1B4 (P. tremula × P. alba) was transformed, and DNA was isolated as described by Filichkin et al. (2006). The presence of the transgene was verified by PCR (see Table S2 for primers).
Phenotypic assessment of poplar transgenics
Plants were grown in the glasshouse for 2 months before being planted at a field site near Corvallis, Oregon, USA, in June 2003. Ten ramets of non-transgenic control and four ramets from independent transgenic events were transplanted in two pairs, with each pair being placed randomly on the site.
Height and diameter were measured in early spring 2004, and again in fall 2004, and the tree volume index (VI = height × diameter2) was calculated. Net growth was defined as the difference between ln(VI) at the end and beginning of the measurement period. The vegetative bud flush date was recorded in spring 2005 when any buds along the main stem began to open. Flowering index was measured in early spring 2006 as: (number of flowering ramets per event) × (mean catkin number for each event). Growth, bud flush and flowering data were analyzed in sas v9.1 (SAS Institute Inc., 2002–2003), using the MIXED procedures model to test the effects of constructs and the events within constructs. The response (Y-data) was the average of the two ramets in a pair, resulting in two independent data points for each transgenic event, and five data points for the control trees. To estimate and test differences between means, we used the LSMEANS protocol: Tukey–Kramer’s adjustment was used for all possible pairwise comparisons between transgenic group means; Dunnett’s adjustment was employed for comparisons between transgenic events and non-transgenic controls.
We assessed branching phenotypes in the upper third of the tree crown for representative events in June 2006. To measure higher-order branching, five primary or secondary long shoots with branches were randomly selected, and the percentage of higher-order long shoots was determined. We measured inflorescence number in 2007–2009 on all remaining trees. Between 10 and 27 catkins per PopCEN1-RNAi transgenic event for six events, or per four control ramets, were collected on 31 March 2009 and 17 April 2009, to measure average catkin length, weight and flower number. Data were analyzed using the MIXED procedures in sas v9.1: LSMEANS and the Tukey–Kramer’s test were used to estimate and detect differences between PopCEN1-RNAi and control groups.
Selected transgenic events and controls were propagated, grown for 10 weeks at 22°C with a 16-h photoperiod, and then subjected to a dormancy cycle regime (Rohde et al., 2007) in a growth chamber. At various time points during the treatment, four ramets per transgenic event and WT were transferred to a glasshouse (22°C day/20°C night, with an extension of the photoperiod to 16 h), and time of bud flush (scored when the tips of the first leaves had emerged) and extent of regrowth was measured.
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- Experimental procedures
- Supporting Information
We thank Dr Brian Stanton and GreenWood Resources, Inc. for permission to study their plantation trees. This research was supported by grants from the USDA Cooperative State Research, Education and Extension Service (2002-35301-12173 and 00-52100-9623), NSF Plant Genome Research Program (DBI-0501890), and the Tree Biosafety and Genomics Research Cooperative at Oregon State University.
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- Experimental procedures
- Supporting Information
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- Experimental procedures
- Supporting Information
Figure S1. Phylogenetic analysis of TFL1/FT family. Alignment of protein sequences and the neighbor-joining method was used to produce the tree. All full-length family members identified in the Populus trichocarpa (Poptr v2; black boxes), rice (light gray boxes) and Arabidopsis (dark gray boxes) genomes were included. Additional TFL1 and MFT homologs are from Aquilegia formosa (AfTFL1), Antirrhinum majus (CEN), Citrus sinensis (CsTFL), Glycine max (GmCETS1), Impatiens balsamina (IbTFL1), Lolium perenne (LpTFL1), Lotus japonicus (LjCEN1), Solanum lycopersicum (SP, SP9D, SP2I ), Malus × domestica (MdTFL1/2), Nicotiana tabacum (CET1/2/4), Picea abies (PaFT4), Pisum sativum (DET, LF), and Vitis vinifera (VvTFL1A/B/C, VvMFT). A list of sequence identifiers is provided in Table S1. Bootstrap support of 50% or higher is shown at nodes.
Figure S2.PopMFT expression in different tissues, ages and seasonal times. (a) Expression in different Populus trichocarpa tissues. P. trichocarpa female flowers were collected post-pollination, while male flowers were collected at anthesis in March. Newly-initiated female and male inflorescence buds were collected in June. Vegetative tissues were collected from clone Nisqually-1 in April and June, including shoot apices, new axillary buds, and mature leaves. Xylem and phloem/cambium were sampled from a 2-year-old, actively growing trees in August. FB, floral bud. (b) Terminal vegetative buds (TVB), lateral vegetative buds (LVB) and actively growing shoot-tips (ST) were collected from juvenile and adult trees of P. trichocarpa × P. deltoides clone 15-29 in Oregon, USA at the month/day indicated in 2001. Vegetative buds were newly flushed on 4/18. (c) Mature, elongated catkins (4/3) and inflorescence buds were collected from the same adult trees as in (b). Inflorescence bud flush occurred between the 3/20 and 4/3 collection dates. Error bars are standard deviations over 2 technical replicates using cDNA produced from pooled samples from 2 to 5 biological replicates.
Figure S3.PopCEN1 expression in juvenile and adult cottonwood trees at different seasonal times. (a) Expression in different Populus trichocarpa tissues. See figure legend for S3a for details on the tissues. FB, floral bud. (b) Juvenile terminal vegetative buds (TVB) and actively growing shoot tips (ST). (c) Juvenile lateral vegetative buds (LVB). (d) Adult TVB and ST. (e) Adult LVB. (f) Mature, elongated catkins (4/3) and inflorescence buds collected from the same adult trees as in (c) and (d). Error bars are standard deviations over two technical replicates using cDNA produced from pooled samples from 2 to 5 biological replicates. For (b–f) samples were collected from juvenile and adult (upper crown) trees of P. trichocarpa × P. deltoides clone 15–29 in Oregon, USA at the month/day indicated in 2001. Vegetative buds were newly flushed on 4/18 and buds were set on 8/7. Inflorescence bud flush occurred between the 3/20 and 4/3 collection dates.
Figure S4. Growth of transgenic poplars and expression levels of transgene and target endogene. (a) Expression level of 35S::PopCEN1 transgene. Black bars denote events for which all ramets have never flowered except 1 ramet of event 36 in 2009. (b) Expression level of 35S::PopCEN1 transgene in the lowest-expressing transgenic events that are not visible in the scale shown in (a). (c) Expression level of PopCEN1 endogene in PopCEN1-RNAi transgenics. Black bars denote events for which all ramets showed profuse flowering in 2007–2009. (d) Comparison of PopCEN1 and PopCEN2 downregulation in PopCEN1-RNAi event 178 (e) Expression level of 35S::PopMFT transgene. (f) Expression level of PopMFT endogene in PopMFT-RNAi transgenics. Values were normalized to UBQ and calibrated to the sample showing the lowest transgene expression for overexpression transgenics (a, b and e) or to target endogene expression in non-transgenic controls for RNAi transgenics (c, d and f). For (d), expression was analyzed in lateral buds collected in June, whereas in all other panels, expression was studied in recently flushed shoot tips. In (a) to (d) error bars are standard deviations over two real-time PCR runs using the same cDNA templates (produced from pooled samples from 22 biological replicates), with duplicate PCR reactions in each run. (g) Means and standard errors for average net growth in 2004 for control and transgenic populations. Black bar denotes population with mean statistically different from control population.
Figure S5. Flowering in PopMFT poplar transgenics. Flowering level of individual trees in 2007 to 2009. Flower scores were: 1 = no catkins, 2 = 1–50 catkins, 3 = 50–100 catkins, and 4 = >100 catkins.
Figure S6. SD-induced bud set and axillary bud flush in PopCEN1 poplar transgenics.
(a) Terminal bud set stage after 42 days of SD treatment (see Figure 6) following the protocol of Rohde et al. (2007). At least 25 trees for each transgenic and control population were scored for stage of bud set defined as (1) no bud scale apparent (bottom photo); (2) bud scale present (middle photo); and (3) fully closed bud (top photo). (b) Percent of lateral buds flushed after transfer to LD conditions. Lateral bud flush was measured 238 days after the start of the treatment for the top 15 nodes. Values are means and standard deviations over four biological replicates.
Table S1. Sequences used for phylogenetic analysis of the TFL1/FT family.
Table S2. Primer Sequences.
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