Light on perenniality: Para-dormancy is based on ABA-GA antagonism and endo-dormancy on the shutdown of GA biosynthesis.

Perennial para- and endo-dormancy are seasonally separate phenomena. Whereas para-dormancy is the suppression of axillary buds (AXBs) by a growing shoot, endo-dormancy is the short-day elicited arrest of terminal and AXBs. In hybrid aspen (Populus tremula x P. tremuloides) compromising the apex releases para-dormancy, whereas endo-dormancy requires chilling. ABA and GA are implicated in both phenomena. To untangle their roles, we blocked ABA biosynthesis with fluridone (FD), which significantly reduced ABA levels, downregulated GA-deactivation genes, upregulated the major GA3ox-biosynthetic genes, and initiated branching. Comprehensive GA-metabolite analyses suggested that FD treatment shifted GA production to the non-13-hydroxylation pathway, enhancing GA4 function. Applied ABA counteracted FD effects on GA metabolism and downregulated several GA3/4 -inducible α- and γ-clade 1,3-ß-glucanases that hydrolyze callose at plasmodesmata (PD), thereby enhancing PD-callose accumulation. Remarkably, ABA-deficient plants repressed GA4 biosynthesis and established endo-dormancy like controls but showed increased stress sensitivity. Repression of GA4 biosynthesis involved short-day induced DNA methylation events within the GA3ox2 promoter. In conclusion, the results cast new light on the roles of ABA and GA in dormancy cycling. In para-dormancy, PD-callose turnover is antagonized by ABA, whereas in short-day conditions, lack of GA4 biosynthesis promotes callose deposition that is structurally persistent throughout endo-dormancy.

Lipid Body Dynamics in Shoot Meristems: Production, Enlargement, and Putative Organellar Interactions and Plasmodesmal Targeting.

Post-embryonic cells contain minute lipid bodies (LBs) that are transient, mobile, engage in organellar interactions, and target plasmodesmata (PD). While LBs can deliver γ-clade 1,3-ß-glucanases to PD, the nature of other cargo is elusive. To gain insight into the poorly understood role of LBs in meristems, we investigated their dynamics by microscopy, gene expression analyzes, and proteomics. In developing buds, meristems accumulated LBs, upregulated several LB-specific OLEOSIN genes and produced OLEOSINs. During bud maturation, the major gene OLE6 was strongly downregulated, OLEOSINs disappeared from bud extracts, whereas lipid biosynthesis genes were upregulated, and LBs were enlarged. Proteomic analyses of the LB fraction of dormant buds confirmed that OLEOSINs were no longer present. Instead, we identified the LB-associated proteins CALEOSIN (CLO1), Oil Body Lipase 1 (OBL1), Lipid Droplet Interacting Protein (LDIP), Lipid Droplet Associated Protein1a/b (LDAP1a/b) and LDAP3a/b, and crucial components of the OLEOSIN-deubiquitinating and degradation machinery, such as PUX10 and CDC48A. All mRFP-tagged LDAPs localized to LBs when transiently expressed in Nicotiana benthamiana. Together with gene expression analyzes, this suggests that during bud maturation, OLEOSINs were replaced by LDIP/LDAPs at enlarging LBs. The LB fraction contained the meristem-related actin7 (ACT7), "myosin XI tail-binding" RAB GTPase C2A, an LB/PD-associated γ-clade 1,3-ß-glucanase, and various organelle- and/or PD-localized proteins. The results are congruent with a model in which LBs, motorized by myosin XI-k/1/2, traffic on F-actin, transiently interact with other organelles, and deliver a diverse cargo to PD.

Dual Role of Gibberellin in Perennial Shoot Branching: Inhibition and Activation.

Shoot branching from axillary buds (AXBs) is regulated by a network of inhibitory and promotive forces, which includes hormones. In perennials, the dwarfed stature of the embryonic shoot inside AXBs is indicative of gibberellin (GA) deficiency, suggesting that AXB activation and outgrowth require GA. Nonetheless, the role of GA in branching has remained obscure. We here carried out comprehensive GA transcript and metabolite analyses in hybrid aspen, a perennial branching model. The results indicate that GA has an inhibitory as well as promotive role in branching. The latter is executed in two phases. While the expression level of GA2ox is high in quiescent AXBs, decapitation rapidly downregulated it, implying increased GA signaling. In the second phase, GA3ox2-mediated de novo GA-biosynthesis is initiated between 12 and 24 h, prior to AXB elongation. Metabolite analyzes showed that GA1/4 levels were typically high in proliferating apices and low in the developmentally inactive, quiescent AXBs, whereas the reverse was true for GA3/6. To investigate if AXBs are differently affected by GA3, GA4, and GR24, an analog of the branch-inhibitor hormone strigolactone, they were fed into AXBs of single-node cuttings. GA3 and GA4 had similar effects on GA and SL pathway genes, but crucially GA3 induced AXB abscission whereas GA4 promoted outgrowth. Both GA3 and GA4 strongly upregulated GA2ox genes, which deactivate GA1/4 but not GA3/6. Thus, the observed production of GA3/6 in quiescent AXBs targets GA1/4 for GA2ox-mediated deactivation. AXB quiescence can therefore be maintained by GA3/6, in combination with strigolactone. Our discovery of the distinct tasks of GA3 and GA4 in AXB activation might explain why the role of GA in branching has been difficult to decipher. Together, the results support a novel paradigm in which GA3/6 maintains high levels of GA2ox expression and low levels of GA4 in quiescent AXBs, whereas activation and outgrowth require increased GA1/4 signaling through the rapid reduction of GA deactivation and subsequent GA biosynthesis.

Plant Lipid Bodies Traffic on Actin to Plasmodesmata Motorized by Myosin XIs.

Late 19th-century cytologists observed tiny oil drops in shoot parenchyma and seeds, but it was discovered only in 1972 that they were bound by a half unit-membrane. Later, it was found that lipid bodies (LBs) arise from the endoplasmic reticulum. Seeds are known to be packed with static LBs, coated with the LB-specific protein OLEOSIN. As shown here, apices of Populus tremula x P. tremuloides also express OLEOSIN genes and produce potentially mobile LBs. In developing buds, PtOLEOSIN (PtOLE) genes were upregulated, especially PtOLE6, concomitant with LB accumulation. To investigate LB mobility and destinations, we transformed Arabidopsis with PtOLE6-eGFP. We found that PtOLE6-eGFP fusion protein co-localized with Nile Red-stained LBs in all cell types. Moreover, PtOLE6-eGFP-tagged LBs targeted plasmodesmata, identified by the callose marker aniline blue. Pharmacological experiments with brefeldin, cytochalasin D, and oryzalin showed that LB-trafficking requires F-actin, implying involvement of myosin motors. In a triple myosin-XI knockout (xi-k/1/2), transformed with PtOLE6-eGFP, trafficking of PtOLE6-eGFP-tagged LBs was severely impaired, confirming that they move on F-actin, motorized by myosin XIs. The data reveal that LBs and OLEOSINs both function in proliferating apices and buds, and that directional trafficking of LBs to plasmodesmata requires the actomyosin system.

Strigolactone-Based Node-to-Bud Signaling May Restrain Shoot Branching in Hybrid Aspen.

The biosynthesis and roles of strigolactones (SLs) have been investigated in herbaceous plants, but so far, their role in trees has received little attention. In this study, we analyzed the presence, spatial/temporal expression and role of SL pathway genes in Populus tremula � Populus tremuloides. In this proleptic species, axillary buds (AXBs) become para-dormant at the bud maturation point, providing an unambiguous starting point to study AXB activation. We identified previously undescribed Populus homologs of DWARF27 (D27), LATERAL BRANCHING OXIDOREDUCTASE (LBO) and DWARF53-like (D53-like) and analyzed the relative expression of all SL pathway genes in root tips and shoot tissues. We found that, although AXBs expressed MORE AXILLARY GROWTH1 (MAX1) and LBO, they did not express MAX3 and MAX4, whereas nodal bark expressed high levels of all SL biosynthesis genes. By contrast, expression of the SL perception and signaling genes MAX2, D14 and D53 was high in AXBs relative to nodal bark and roots. This suggests that AXBs are reliant on the associated nodes for the import of SLs and SL precursors. Activation of AXBs was initiated by decapitation and single-node isolation. This rapidly downregulated SL pathway genes downstream of MAX4, although later these genes were upregulated coincidently with primordia formation. GR24-feeding counteracted all activation-related changes in SL gene expression but did not prevent AXB outgrowth showing that SL is ineffective once AXBs are activated. The results indicate that nodes rather than roots supply SLs and its precursors to AXBs, and that SLs may restrain embryonic shoot elongation during AXB formation and para-dormancy in intact plants.

Decoupling photo- and thermoperiod by projected climate change perturbs bud development, dormancy establishment and vernalization in the model tree Populus.

BACKGROUND: The performance and survival of deciduous trees depends on their innate ability to anticipate seasonal change. A key event is the timely production of short photoperiod-induced terminal and axillary buds that are dormant and freezing-tolerant. Some observations suggest that low temperature contributes to terminal bud initiation and dormancy. This is puzzling because low temperatures in the chilling range universally release dormancy. It also raises the broader question if the projected climate instabilities, as well as the northward migration of trees, will affect winter preparations and survival of trees. RESULTS: To gauge the response capacity of trees, we exposed juvenile hybrid aspens to a 10-h short photoperiod in combination with different day/night temperature regimes: high (24/24 °C), moderate (18/18 °C), moderate-low (18/12 °C) and low (12/12 °C), and analysed bud development, dormancy establishment, and marker gene expression. We found that low temperature during the bud formation period (pre-dormancy) upregulated dormancy-release genes of the gibberellin (GA) pathway, including the key GA biosynthesis genes GA20oxidase and GA3oxidase, the GA-receptor gene GID1, as well as GA-inducible enzymes of the 1,3-ß-glucanase family that degrade callose at plasmodesmal Dormancy Sphincter Complexes. Simultaneously, this pre-dormancy low temperature perturbed the expression of flowering pathway genes, including CO, FT, CENL1, AGL14, LFY and AP1. In brief, pre-dormancy low temperature compromised bud development, dormancy establishment, and potentially vernalization. On the other hand, a high pre-dormancy temperature prevented dormancy establishment and resulted in flushing. CONCLUSIONS: The results show that pre-dormancy low temperature represents a form of chilling that antagonizes dormancy establishment. Combined with available field data, this indicates that natural Populus ecotypes have evolved to avoid the adverse effects of high and low temperatures by initiating and completing dormant buds within an approximate temperature-window of 24-12 °C. Global warming and erratic temperature patterns outside this range can therefore endanger the successful propagation of deciduous perennials.

Axillary buds are dwarfed shoots that tightly regulate GA pathway and GA-inducible 1,3-ß-glucanase genes during branching in hybrid aspen.

Axillary buds (AXBs) of hybrid aspen (Populus tremula×P. tremuloides) contain a developing dwarfed shoot that becomes para-dormant at the bud maturation point. Para-dormant AXBs can grow out after stem decapitation, while dormant AXBs pre-require long-term chilling to release them from dormancy. The latter is mediated by gibberellin (GA)-regulated 1,3-ß-glucanases, but it is unknown if GA is also important in the development, activation, and outgrowth of para-dormant AXBs. The present data show that para-dormant AXBs up-regulate GA receptor genes during their maturation, but curtail GA biosynthesis by down-regulating the rate-limiting GIBBERELLIN 3-OXIDASE2 (GA3ox2), which is characteristically expressed in the growing apex. However, decapitation significantly up-regulated GA3ox2 and GA4-responsive 1,3-ß-glucanases (GH17-family; α-clade). In contrast, decapitation down-regulated γ-clade 1,3-ß-glucanases, which were strongly up-regulated in maturing AXBs concomitant with lipid body accumulation. Overexpression of selected GH17 members in hybrid aspen resulted in characteristic branching patterns. The α-clade member induced an acropetal branching pattern, whereas the γ-clade member activated AXBs in recurrent flushes during transient cessation of apex proliferation. The results support a model in which curtailing the final step in GA biosynthesis dwarfs the embryonic shoot, while high levels of GA precursors and GA receptors keep AXBs poised for growth. GA signaling, induced by decapitation, reinvigorates symplasmic supply routes through GA-inducible 1,3-ß-glucanases that hydrolyze callose at sieve plates and plasmodesmata.

Long and short photoperiod buds in hybrid aspen share structural development and expression patterns of marker genes.

Tree architecture develops over time through the collective activity of apical and axillary meristems. Although the capacity of both meristems to form buds is crucial for perennial life, a comparative analysis is lacking. As shown here for hybrid aspen, axillary meristems engage in an elaborate process of axillary bud (AXB) formation, while apical dominance prevents outgrowth of branches. Development ceased when AXBs had formed an embryonic shoot (ES) with a predictable number of embryonic leaves at the bud maturation point (BMP). Under short days, terminal buds (TBs) formed an ES similar to that of AXBs, and both the TB and young AXBs above the BMP established dormancy. Quantitative PCR and in situ hybridizations showed that this shared ability and structural similarity was reflected at the molecular level. TBs and AXBs similarly regulated expression of meristem-specific and bud/branching-related genes, including CENTRORADIALIS-LIKE1 (CENL1), BRANCHED1 (BRC1), BRC2, and the strigolactone biosynthesis gene MORE AXILLARY BRANCHES1 (MAX1). Below the BMP, AXBs maintained high CENL1 expression at the rib meristem, suggesting that it serves to maintain poise for growth. In support of this, decapitation initiated outgrowth of CENL1-expressing AXBs, but not of dormant AXBs that had switched CENL1 off. This singles out CENL1 as a rib meristem marker for para-dormancy. BRC1 and MAX1 genes, which may counterbalance CENL1, were down-regulated in decapitation-activated AXBs. The results showed that removal of apical dominance shifted AXB gene expression toward that of apices, while developing TBs adopted the expression pattern of para-dormant AXBs. Bud development thus follows a shared developmental pattern at terminal and axillary positions, despite being triggered by short days and apical dominance, respectively.

Mapping symplasmic fields at the shoot apical meristem using iontophoresis and membrane potential measurements.

Microinjections of fluorescent dyes have revealed that the shoot apical meristem (SAM) is dynamically partitioned into symplasmic fields (SFs), implying that plasmodesmata (Pd) are held shut at specific locations in the proliferating cellular matrix. The SFs are integrated into a coherent morphogenetic unit by exchange of morphogens and transcription factors via gating Pd between adjacent SFs, and by ligand-receptor interactions that operate across the extracellular space. We describe a method for the real-time mapping of SF in the SAM by iontophoresis and membrane potential measurements.

Refurbishing the plasmodesmal chamber: a role for lipid bodies?

Lipid bodies (LBs) are universal constituents of both animal and plant cells. They are produced by specialized membrane domains at the tubular endoplasmic reticulum (ER), and consist of a core of neutral lipids and a surrounding monolayer of phospholipid with embedded amphipathic proteins. Although originally regarded as simple depots for lipids, they have recently emerged as organelles that interact with other cellular constituents, exchanging lipids, proteins and signaling molecules, and shuttling them between various intracellular destinations, including the plasmamembrane (PM). Recent data showed that in plants LBs can deliver a subset of 1,3-ß-glucanases to the plasmodesmal (PD) channel. We hypothesize that this may represent a more general mechanism, which complements the delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to the PD exterior via the secretory pathway. We propose that LBs may contribute to the maintenance of the PD chamber and the delivery of regulatory molecules as well as proteins destined for transport to adjacent cells. In addition, we speculate that LBs deliver their cargo through interaction with membrane domains in the cytofacial side of the PM.

Shoot meristems of deciduous woody perennials: self-organization and morphogenetic transitions.

Shoot apical meristems of deciduous woody perennials share gross structural features with other angiosperms, but are unique in the seasonal regulation of vegetative and floral meristems. Supporting longevity, flowering is postponed to the adult phase, and restricted to some axillary meristems. In cold climates, photoperiodic timing mechanisms and chilling are recruited to schedule end-of-season growth arrest, dormancy cycling and flowering. We review recently uncovered generic meristem properties, perennial meristem fate, and the role of CENL1, FT1 and FT2 in bud formation and flowering. We also highlight novel findings, suggesting that dormancy release is mediated by mobile lipid bodies that deliver enzymes to plasmodesmata to recover symplasmic communication and meristem function.

The embryonic shoot: a lifeline through winter.

The tiny vascular axis of the embryo emerges post-embryonically as an elaborate and critical infrastructure, pervading the entire plant system. Its expansive nature is especially impressive in trees, where growth and development continue for extended periods. While the shoot apical meristem (SAM) orchestrates primary morphogenesis, the vascular system is mapped out in its wake in the provascular cylinder, situated just below the emerging leaf primordia and surrounding the rib meristem. Formation of leaf primordia and provascular tissues is incompatible with the harsh conditions of winter. Deciduous trees of boreal and temperate climates therefore enter a survival mode at the end of the season. However, to be competitive, they need to maximize their growth period while avoiding cellular frost damage. Trees achieve this by monitoring photoperiod, and by timely implementation of a survival strategy that schedules downstream events, including growth cessation, terminal bud formation, dormancy assumption, acquisition of freezing tolerance, and shedding of leaves. Of central importance are buds, which contain an embryonic shoot that allows shoot development and elongation in spring. The genetic and molecular processes that drive the cycle in synchrony with the seasons are largely elusive. Here, we review what is known about the signals and signal conduits that are involved, the processes that are initiated, and the developmental transitions that ensue in a terminal bud. We propose that addressing dormancy as a property of the SAM and the bud as a unique shoot type will facilitate our understanding of winter dormancy.

Plant lipid bodies and cell-cell signaling: a new role for an old organelle?

Plant lipid droplets are found in seeds and in post-embryonic tissues. Lipid droplets in seeds have been intensively studied, but those in post-embryonic tissues are less well characterised. Although known by a variety of names, here we will refer to all of them as lipid bodies (LBs). LBs are unique spherical organelles which bud off from the endoplasmic reticulum, and are composed of a single phospholipid (PL) layer enclosing a core of triacylglycerides. The PL monolayer is coated with oleosin, a structural protein that stabilizes the LB, restricts its size, and prevents fusion with adjacent LBs. Oleosin is uniquely present at LBs and is regarded as a LB marker. Although initially viewed as simple stores for energy and carbon, the emerging view is that LBs also function in cytoplasmic signalling, with the minor LB proteins caleosin and steroleosin in a prominent role. Apart from seeds, a variety of vegetative and floral structures contain LBs. Recently, it was found that numerous LBs emerge in the shoot apex of perennial plants during seasonal growth arrest and bud formation. They appear to function in dormancy release by reconstituting cell-cell signalling paths in the apex. As apices and orthodox seeds proceed through comparable cycles of dormancy and dehydration, the question arises to what degree LBs in apices share functions with those in seeds. We here review what is known about LBs, particularly in seeds, and speculate about possible unique functions of LBs in post-embryonic tissues in general and in apices in particular.

Dormancy cycling at the shoot apical meristem: transitioning between self-organization and self-arrest.

To survive winter deciduous perennials of the temperate zones cease growth and acquire a cold-acclimated state. Timing of these events is guided by sensory systems in the leaves that register critical alterations in photoperiod. Growth cessation on its own is not sufficient to develop adequate freezing tolerance, which requires entry of the shoot apical meristem (SAM) into dormancy. To fully appreciate perennial dormancy as a precondition for cold acclimation it is necessary to assess how it is brought about in a timely fashion, what the nature of it is, and how it is released. Short day (SD) exposure results in growth cessation, bud set, dormancy establishment at the SAM, and a moderate to high level of freezing tolerance. Subsequent chilling releases the SAM from dormancy and enhances freezing tolerance further. Recent investigations indicate that dormancy is a state of self-arrest that is brought about by an enzyme-based system which disrupts the intrinsic signal network of the SAM. Release from this state requires a complimentary enzyme-based system that is preformed during SD and mobilized by chilling. These findings are in agreement with the paradigm of dormancy cycling, which defines the seasonal alternations at the SAM as transitions between states of self-organization and self-arrest. The success of this survival strategy is based on the adequate scheduling of a complex array of events. The appreciation is growing that this involves signal cascades that are, mutatis mutandis, also recruited in floral evocation in many annuals, including Arabidopsis. A heuristic model is presented of dormancy cycling at the SAM, which depicts crucial molecular and cellular events that drive the cycle.

Chilling of dormant buds hyperinduces FLOWERING LOCUS T and recruits GA-inducible 1,3-beta-glucanases to reopen signal conduits and release dormancy in Populus.

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-ß-glucanase genes (glucan hydrolase family 17 [GH17]) in Populus that could turn over 1,3-ß-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 (5°C), 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.

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

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.