In trees, production of intercellular signals and accessibility of signal conduits jointly govern dormancy cycling at the shoot apex. We identified 10 putative cell wall 1,3-b-glucanase genes (glucan hydrolase family 17 [GH17]) in Populus that could turn over 1,3-b-glucan (callose) at pores and plasmodesmata (PD) and investigated their regulation in relation to FT and CENL1 expression. The 10 genes encode orthologs of Arabidopsis thaliana BG_ppap, a PD-associated glycosylphosphatidylinositol (GPI) lipid-anchored protein, the Arabidopsis PD callose binding protein PDCB, and a birch (Betula pendula) putative lipid body (LB) protein. We found that these genes were differentially regulated by photoperiod, by chilling (58C), and by feeding of gibberellins GA 3 and GA 4 . GA 3 feeding upregulated all LB-associated GH17s, whereas GA 4 upregulated most GH17s with a GPI anchor and/or callose binding motif, but only GA 4 induced true bud burst. Chilling upregulated a number of GA biosynthesis and signaling genes as well as FT, but not CENL1, while the reverse was true for both GA 3 and GA 4 . Collectively, the results suggest a model for dormancy release in which chilling induces FT and both GPI lipid-anchored and GA 3 -inducible GH17s to reopen signaling conduits in the embryonic shoot. When temperatures rise, the reopened conduits enable movement of FT and CENL1 to their targets, where they drive bud burst, shoot elongation, and morphogenesis.

SummaryThe shoot apex of overwintering perennials ceases its morphogenetic activity at the end of the growing season and transforms into a bud which is dormant and freezing-tolerant. In birch (Betula pubescens) these events are triggered by short photoperiod, and involve the production of 1,3-b-D-glucan containing sphincters on the plasmodesmata. As a result, all symplasmic pathways shut down. Here we show that breakage of bud dormancy by chilling involves restoration of the symplasmic organization of the meristem. This restoration is likely to be mediated by 1,3-b-D-glucanase, which was present in small spherosome-like vacuoles that arose de novo during dormancy induction. During chilling these vacuoles were displaced from the bulk cytoplasm to the cortical cytoplasm where they became aligned with the plasma membrane, often associated with plasmodesmata. At this stage the enzyme also appeared outside the vacuoles. During chilling, 1,3-b-D-glucan disappeared from the plasmodesmal channels and wall sleeves, and the plasmodesmata regained the capacity for cell±cell transport, as demonstrated by microinjection of Lucifer Yellow CH and Fluorescein-tagged gibberellic acid. Collectively, the present experiments demonstrate that restoration of the symplasmic organization of the meristem is indispensable for the release of buds from dormancy and the assumption of a proliferation-competent state, and implicate 1,3-b-D-glucanase action at the plasmodesmata. Based on these ®ndings we propose a model for`dormancy cycling' which depicts the meristem as passing through three sequential states of cellular communication with characteristic sensitivities to distinct environmental cues.

We investigated the short day (SD)-induced transition to dormancy in wild-type hybrid poplar (Populus tremula 3 P. tremuloides) and its absence in transgenic poplar overexpressing heterologous PHYTOCHROME A (PHYA). CENTRORADIALIS-LIKE1 (CENL1), a poplar ortholog of Arabidopsis thaliana TERMINAL FLOWER1 (TFL1), was markedly downregulated in the wild-type apex coincident with SD-induced growth cessation. By contrast, poplar overexpressing a heterologous Avena sativa PHYA construct (P35S:AsPHYA), with PHYA accumulating in the rib meristem (RM) and adjacent tissues but not in the shoot apical meristem (SAM), upregulated CENL1 in the RM area coincident with an acceleration of stem elongation. In SD-exposed heterografts, both P35S:AsPHYA and wild-type scions ceased growth and formed buds, whereas only the wild type assumed dormancy and P35S:AsPHYA showed repetitive flushing. This shows that the transition is not dictated by leaf-produced signals but dependent on RM and SAM properties. In view of this, callose-enforced cell isolation in the SAM, associated with suspension of indeterminate growth during dormancy, may require downregulation of CENL1 in the RM. Accordingly, upregulation of CENL1/TFL1 might promote stem elongation in poplar as well as in Arabidopsis during bolting. Together, the results suggest that the RM is particularly sensitive to photoperiodic signals and that CENL1 in the RM influences transition to dormancy in hybrid poplar.

Dehydrins accumulate in various plant tissues during dehydration. Their physiological role is not well understood, but it is commonly assumed that they assist cells in tolerating dehydration. Since in perennials the ability of the shoot apex to withstand dehydration is pivotal for survival through winter, we investigated if and how dehydrins may be involved. A first step in assessing such a role is the identification of their subcellular location. We therefore mapped the location of dehydrin homologues, abscisic acid-responsive (RAB 16-like) polypeptides, in the apex of birch (Betula pubescens Ehrh.). In non-cold-acclimated plants a single low-abundant RAB 16-member (a 33-kDa polypeptide) was produced, and localized in the cytoplasm only. During cold acclimation two additional members were produced (24 and 30 kDa) and accumulated in nuclei, storage protein bodies and starch-rich amyloplasts. Western blots of proteins isolated from purified starch granules and from protein bodies revealed the presence of the 24-kDa dehydrin. Since starch and protein reserves are gradually consumed during winter, serving cell maintenance, starch- and protein-degrading enzymes must remain locally active. We therefore investigated the hypothesis that dehydrins might create local pools of water in otherwise dehydrated cells, thereby maintaining enzyme function. In agreement with our hypothesis, enzyme assays showed that under conditions of low water activity a partially purified dehydrin fraction was able to improve the activity of alpha-amylase (EC 3.2.1.1.) relative to fractions from which dehydrin was removed by immunoprecipitation. The results confirm the general belief that dehydrins serve desiccation tolerance, and suggest that a major function is to rescue the metabolic processes that are required for survival and re-growth.

The overwintering of trees in northern areas depends on processes regulated by photoperiod and temperature. To identify the physiological and genetic factors involved in this environmental control, three latitudinal ecotypes of pubescent birch (Betula pubescens Ehrh.) growing in a common garden experiment were used. Each ecotype responded to the shortening of the photoperiod according to its specific critical daylength, resulting in the induction of freezing tolerance and dehydration of buds first in the northern ecotype, followed by the central and southern ecotypes, respectively. By contrast, there was no clear difference in the timing of dormancy release, bud rehydration, and deacclimation in the spring, suggesting that these traits were controlled mainly by temperature. To elucidate the role of dehydrins (DHN) in the overwintering process, two DHN genomic clones were isolated from pubescent birch and expression of the corresponding genes, both in field and under controlled conditions, was characterized. BpuDhn1 was found to encode an Y(n)K(n)-type of basic DHN, while BpuDhn2 encoded an acidic, SK(n)-type of DHN. In field-grown trees the level of BpuDhn1 increased in buds during the autumn, while the level of BpuDhn2 was highest during the coldest winter months. Under controlled conditions BpuDhn1 increased in response to the combined effect of short daylength and low, non-freezing temperatures whereas the expression of BpuDhn2 was mainly controlled by low temperature while photoperiod had less effect on its expression. These results suggest that DHNs participate in the sensitive environmental regulation of the overwintering process in birch.