Annual dormancy-growth cycle is a developmental and physiological process essential for the survival of deciduous trees in temperate and boreal forests. Seasonal control of shoot growth in woody perennials requires specific genetic programmes responding to environmental signals. The environmental-controlled mechanisms that regulate the shift between winter dormancy and the growth-promoting genetic programmes are still unknown. Here, we show that dynamics in genomic DNA methylation levels are involved in the regulation of dormancy-growth cycle in poplar. The reactivation of growth in the apical shoot during bud break process in spring is preceded by a progressive reduction of genomic DNA methylation in apex tissue. The induction in apex tissue of a chilling-dependent poplar DEMETER-LIKE 10 (PtaDML10) DNA demethylase precedes shoot growth reactivation. Transgenic poplars showing downregulation of PtaDML8/10 caused delayed bud break. Genome-wide transcriptome and methylome analysis and data mining revealed that the gene targets of DEMETER-LIKE-dependent DNA demethylation are genetically associated with bud break. These data point to a chilling-dependent DEMETER-like DNA demethylase mechanisms being involved in the shift from winter dormancy to a condition that precedes shoot apical vegetative growth in poplar.

Root nodule symbiosis (RNS) allows plants to access atmospheric nitrogen converted into usable forms through a mutualistic relationship with soil bacteria. RNS is a complex trait requiring coordination from both the plant host and the bacterial symbiont, and pinpointing the evolutionary origins of root nodules is critical for understanding the genetic basis of RNS. This endeavor is complicated by data limitations and the intermittent presence of RNS in a single clade of ca. 30,000 species of flowering plants, i.e., the nitrogen-fixing clade (NFC). We developed the most extensive de novo phylogeny for all major lineages of the NFC and an enhanced root nodule trait database to reconstruct the evolution of RNS. Through identification of the evolutionary pathway to RNS gain, we show that shifts among heterogeneous evolutionary rates can explain how a complex trait such as RNS can arise many times across a large phylogeny. Our analysis identifies a two-step process in which an ancestral precursor state gave rise to a more labile state from which RNS was quickly gained at specific points in the NFC. Our rigorous reconstruction of ancestral states illustrates how a two-step pathway could have led to multiple independent gains and losses of RNS, contrary to recent hypotheses invoking just a single gain and numerous losses. RNS may be an example of multi-level convergent evolution, thus requiring a broader phylogenetic and genetic scope for genome-phenome mapping to elucidate mechanisms enabling fully functional RNS.

The transition from active growth to dormancy is critical for the survival of perennial plants. We identified a DEMETER-like (CsDML) cDNA from a winter-enriched cDNA subtractive library in chestnut (Castanea sativa Mill.), an economically and ecologically important species. Next, we characterized this DNA demethylase and its putative ortholog in the more experimentally tractable hybrid poplar (Populus tremula × alba), under the signals that trigger bud dormancy in trees. We performed phylogenetic and protein sequence analysis, gene expression profiling, and 5-methyl-cytosine methylation immunodetection studies to evaluate the role of CsDML and its homolog in poplar, PtaDML6. Transgenic hybrid poplars overexpressing CsDML were produced and analysed. Short days and cold temperatures induced CsDML and PtaDML6. Overexpression of CsDML accelerated short-day-induced bud formation, specifically from Stages 1 to 0. Buds acquired a red-brown coloration earlier than wild-type plants, alongside with the up-regulation of flavonoid biosynthesis enzymes and accumulation of flavonoids in the shoot apical meristem and bud scales. Our data show that the CsDML gene induces bud formation needed for the survival of the apical meristem under the harsh conditions of winter.

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Single-cell transcriptome analysis has been extensively applied in humans and animal models to uncover gene expression heterogeneity between the different cell types of a tissue or an organ. It demonstrated its capability to discover key regulatory elements that determine cell fate during developmental programs. Single-cell analysis requires the isolation and labeling of the messenger RNA (mRNA) derived from each cell. These challenges were primarily addressed in mammals by developing microfluidic-based approaches. For plant species whose cells contain cell walls, these approaches have generally required the generation of isolated protoplasts. Many plant tissues’ secondary cell wall hinders enzymatic digestion required for individual protoplast isolation, resulting in an unequal representation of cell types in a protoplast population. This limitation is especially critical for cell types located in the inner layers of a tissue or the inner tissues of an organ. Consequently, single-cell RNA sequencing (scRNA-seq) studies using microfluidic approaches in plants have mainly been restricted to Arabidopsis roots, for which well-established procedures of protoplast isolation are available. Here we present a simple alternative approach to generating high-quality protoplasts from plant tissue by characterizing the mRNA extracted from individual nuclei instead of whole cells. We developed the protocol using two different plant materials with varying cellular complexity levels and cell wall structure, Populus shoot apices, and more lignified stems. Using the 10× Genomics Chromium technology, we show that this procedure results in intact mRNA isolation and limited leakage, with a broad representation of individual cell transcriptomes.

In temperate and boreal regions, perennial trees arrest cell division in their meristematic tissues during winter dormancy until environmental conditions become appropriate for their renewed growth. Release from the dormant state requires exposure to a period of chilling temperatures similar to the vernalization required for flowering in Arabidopsis . Over the past decade, genomic DNA (gDNA) methylation and transcriptome studies have revealed signatures of chromatin regulation during active growth and winter dormancy. To date, only a few chromatin modification genes, as candidate regulators of these developmental stages, have been functionally characterized in trees. In this work, we summarize the major findings of the chromatin-remodeling role during growth-dormancy cycles and we explore the transcriptional profiling of vegetative apical bud and stem tissues during dormancy. Finally, we discuss genetic strategies designed to improve the growth and quality of forest trees.

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Perennial species in the boreal and temperate regions are subject to extreme annual variations in light and temperature. They precisely adapt to seasonal changes by synchronizing cycles of growth and dormancy with external cues. Annual dormancy–growth transitions and flowering involve factors that integrate environmental and endogenous signals. MADS-box transcription factors have been extensively described in the regulation of Arabidopsis flowering. However, their participation in annual dormancy–growth transitions in trees is minimal. In this study, we investigate the function of MADS12, a Populus tremula × alba SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1)-related gene. Our gene expression analysis reveals that MADS12 displays lower mRNA levels during the winter than during early spring and mid-spring. Moreover, MADS12 activation depends on the fulfillment of the chilling requirement. Hybrid poplars overexpressing MADS12 show no differences in growth cessation and bud set, while ecodormant plants display an early bud break, indicating that MADS12 overexpression promotes bud growth reactivation. Comparative expression analysis of available bud break-promoting genes reveals that MADS12 overexpression downregulates the GIBBERELLINS 2 OXIDASE 4 (GA2ox4), a gene involved in gibberellin catabolism. Moreover, the mid-winter to mid-spring RNAseq profiling indicates that MADS12 and GA2ox4 show antagonistic expression during bud dormancy release. Our results support MADS12 participation in the reactivation of shoot meristem growth during ecodormancy and link MADS12 activation and GA2ox4 downregulation within the temporal events that lead to poplar bud break.