CENL1 expression in the rib meristem affects stem elongation and the transition to dormancy in Populus

Abstract

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

Figure 1.

Growth Habit of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA). (A) Young plants after 3 weeks of LDs. P35S:AsPHYA plants show characteristic stunting due to deficient internode elongation. (B) and (C) Cumulative stem growth (B) and leaf formation (C) were measured in the wild type and P35S:AsPHYA under LDs (time point 0) and during the course of SD exposure. Values represent means ± sd (n = 10).

Figure 2.

Internode Elongation of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA) under LDs and SDs. (A) Wild-type and P35S:AsPHYA plants were grown under constant LD conditions, subsequently transferred to SDs, and photographed after 6 weeks of SDs (SD6). For comparison, a collage is made with a picture at scale of a P35S:AsPHYA plant grown for a similar period under LDs only (LD6). Close-ups show the terminal bud of the wild type and the growing apex of P35S:AsPHYA that remains active under both LDs and SDs. Note the red apical leaves and the spontaneously developing branches (stippling) that form in P35S:AsPHYA exposed to LDs. Horizontal arrows indicate the approximate height of the plants upon transfer to the indicated conditions. (B) The average maximal length of internodes that reached their final length at the indicated time under SDs. Values represent means ± sd (n = 10 plants). (C) The average length of the 10 youngest internodes in the wild type and P35S:AsPHYA under 0 to 5 weeks of SDs. The internodes are numbered from the youngest to the oldest (i.e., internode 1 corresponds to the uppermost internode). SD2 and SD5 correspond to 2 and 5 weeks under SDs, respectively. Values represent means ± sd (n = 10 plants).

Figure 3.

Bud Dormancy Development in Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA). Wild-type and P35S:AsPHYA plants were exposed to SDs for 0 to 6 weeks after which the apex was severed. After decapitation, the plants were transferred to LDs for 4 weeks to promote bursting of axillary buds. Since no buds had burst in the wild type pre-exposed to SDs for 3 weeks, all plants that were exposed to SDs longer than 3 weeks and already transferred to LDs were defoliated during week 6 to promote bud burst. Bursting of axillary buds (A), number of branches developed (B), and length of the branches (C) were recorded (n = 8 to 18 in the wild type; n = 8 to 10 in P35S:AsPHYA). Vertical bars represent sd.

Figure 4.

E_m Measurement, Iontophoretic Microinjection, Dye Coupling, and PD Ultratructure in the SAM of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA). (A) Typical stable E_m recorded from a single tunica cell in the SAM of the wild type under LD. (B) Iontophoretic microinjection of LYCH into a single tunica cell of LD-exposed wild type results in dye coupling of the cells that occupy the SAM center. Blue excitation light, photographed through bathing medium. Arrow indicates SAM. me, microelectrode; p, primordia. Bar = 75 μm. (C) and (D) Apex of (B) with medium removed. Low (C) and medium (D) levels of white light mixed in with blue light reveal the position of the central symplasmic field, the primordia (p), and a leaf buttress (lb). Arrow indicates SAM. Bars = 75 μm. (E) Dye coupling is restricted to small cell groups in the SAM of the wild type after 10 d of SDs (blue light, photographed through the medium). me, microelectrode; p, primordia. Bar = 75 μm. (F) In the dormant SAM of the wild type, after 7 weeks of SDs, cells are uncoupled (blue light, photographed through the medium). Bar = 75 μm. (G) and (H) In the P35S:AsPHYA SAM, cells are dye coupled into a central symplasmic field under both LDs (G) and 7 weeks of SDs (H), photographed as in (C) and (B), respectively. p, primordia. Bars = 75 μm. (I) PD in SAM of dormant wild type (7 weeks of SDs) are equipped with DSCs, containing tannic acid/protein deposits. The PD channel plug and sphincter ring are indicated by the black and white arrows, respectively. Bar = 200 nm. (J) PD in the P35S:AsPHYA SAM exposed to SDs (7 weeks) lack DSC, typical of dormant wild-type SAM. Bar = 200 nm.

Figure 5.

Behavior of the Apices of Grafts of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA) under SDs. (A) and (B) A nondefoliated P35S:AsPHYA scion on a wild-type stock (A) and the reverse graft with a nondefoliated wild-type scion on a P35S:AsPHYA stock (B). The photographs were taken just before SD exposure. The insets show the status of the apex as indicated after 4 to 6 weeks of SDs. Letters refer to percentage of grafts showing the response: B, bud formation; D, dormancy; F, flushing; E, elongation. (C) and (D) A defoliated P35S:AsPHYA scion on a wild-type stock (C) and a defoliated wild-type scion on a P35S:AsPHYA stock (D). The photographs were taken just before SD exposure. The insets show the status of the apex as indicated after 6 to 7 weeks of SDs. Letters refer to percentage of grafts showing the response, as described above. (E) A defoliated P35S:AsPHYA scion on a wild-type stock after repeated bud set and flushing. The insets show the bud scales or their scars on the stem. The specimen was photographed after bud set and subsequent flushing under SDs. (F) A defoliated wild-type scion on a P35S:AsPHYA stock after one flush and successive bud set without elongation. The specimen was photographed after bud set and subsequent flushing under SDs.

Figure 6.

Expression Patterns of Flowering-Related Genes in Source and Sink Tissues of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA) under LDs (Time Point 0) and SDs (Time Points 1 to 6). Expression levels of Pt FT2 (A) and (B), Pt CO2 (C) and (D), and Pt CENL1 (E) and (F) were analyzed in source ([A], [C], and [E]) and sink ([B], [D], and [F]) tissues of the wild type and P35S:AsPHYA during the course of a 0- to 6-week SD exposure using real-time RT-PCR. Inset in (D) represents the data of time points 0 and 1 at a stretched out scale. Inset in (E) represents the same data at a stretched out scale. Three replicate samples were analyzed of each sample type in each time point, and the average values are shown. For (B), one to two replicate samples were analyzed for each data point. Vertical bars represent sd. Independent experiments were repeated two times with similar results.

Figure 7.

Functional Zones in the Apex of Hybrid Poplar. (A) Light micrograph of a median-longitudinal section through a proliferating shoot apex showing the SAM and the RM, subtended by the RZ. Cells of the SAM and RM are densely cytoplasmic with large nuclei. The cells of the RZ, arranged in longitudinal ribs, are stretched, vacuolated, and differentiated. Cytoplasm and nucleoplasm are stained blue and cell walls red. (B) Detail of the area boxed in (A). Recent cell divisions, visible as thin walls crossing existing cells, are color coded on the backdrop of the image in (A). Cell division walls are anticlinal in the tunica (cell layers 1 and 2), random in the corpus (approximate cell layers 3 to 8), and periclinal in the RM (approximate cell layers 9 to 11) and upper RZ. PZ, peripheral zone; CZ, central zone; p₀, youngest leaf primordium; T, tunica; C, corpus. Arrows point to the approximate border between the central zone and peripheral zone. (C) to (E) Light micrographs of a life apex cut median-longitudinally. (C) White light epi-illumination reveals differences in chlorophyll content between the meristematic and the differentiating areas. Boxed area indicates microinjection site in (D) (arrow). (D) Iontophoretic microinjection of LYCH into a single cell in layer 10 or 11 at the SAM/RM boundary. LYCH diffuses intercellularly to a group of symplasmically coupled cells (situated in cell layers 8 to 11), which in position closely corresponds to the RM. (E) Final dye-coupling pattern. The coupling group is symplasmically separated from the overlying corpus, but small traces of LYCH (inset) diffuse into the cells that make up the ribs of the RZ. Bars = 50 μm.

Figure 8.

Gene Expression in Distinct Areas at the Shoot Apex of Wild-Type and PHYA-Overexpressing Hybrid Poplar P35S:AsPHYA (P35) under LDs. (A) A scheme of the microdissected apical areas, with estimated contributions of the SAM, the RM, and the RZ to each type of sample in two different series of experiments (exp1 and exp2). The sample of the second experiment is enriched for RM, whereas the uncolored SAM sample was not analyzed. p_1-3, primordia 1 to 3. (B) Expression levels of Pt CO2, Pt CENL1, Pt PHYA, and As PHYA were analyzed using quantitative real-time RT-PCR from samples collected in experiment 1 and 2 (exp1 and exp2; described in [A]). Experiments are separated by vertical stippling. Bar colors correspond to the color code used in (A). SAM and RM/RZ samples of 40 and 20 apices, respectively, were pooled for the analyses in experiment 1, and 10 apices were pooled for experiment 2.

Figure 9.

Localization of As PHYA and Pt PHYA Transcripts in the Apex of Wild-Type and PHYA-Overexpressing Hybrid Poplar (P35S:AsPHYA). (A) and (B) mRNA of As PHYA, which was transformed into hybrid poplar under the control of the CaMV 35S promoter, was localized in median longitudinal sections of P35S:AsPHYA (A) and wild-type (B) apices using in situ hybridization. Purple color indicates the presence of the transcript in (A). The inset displays a detail of P35S:AsPHYA apex showing high expression in RM and areas subjacent to it. (C) and (D) In situ hybridization of the native Pt PHYA in wild-type apex using antisense (C) and sense (D) hybridization probes. Purple color indicates the presence of the transcript in (C). (E) to (G) In situ hybridization using an antisense probe of Pt PHYA (E), As PHYA (F), and a control RNA (G) in successive sections of the P35S:AsPHYA apex. Purple color indicates the presence of the transcript in (E) and (F). (H) In situ hybridization using an antisense probe of Pt PHYA in an axillary bud of the wild type. Purple color indicates the presence of the transcript. RM is indicated by a double arrow in (A), (B), and (E). Bars = 100 μm.