Plant Molecular Phenology and Climate Feedbacks Mediated by BVOCs.

Climate change profoundly affects the timing of seasonal activities of organisms, known as phenology. The impact of climate change is not unidirectional; it is also influenced by plant phenology as plants modify atmospheric composition and climatic processes. One important aspect of this interaction is the emission of biogenic volatile organic compounds (BVOCs), which link the Earth's surface, atmosphere, and climate. BVOC emissions exhibit significant diurnal and seasonal variations and are therefore considered essential phenological traits. To understand the dynamic equilibrium arising from the interplay between plant phenology and climate, this review presents recent advances in comprehending the molecular mechanisms underpinning plant phenology and its interaction with climate. We provide an overview of studies investigating molecular phenology, genome-wide gene expression analyses conducted in natural environments, and how these studies revolutionize the concept of phenology, shifting it from observable traits to dynamic molecular responses driven by gene-environment interactions. We explain how this knowledge can be scaled up to encompass plant populations, regions, and even the globe by establishing connections between molecular phenology, changes in plant distribution, species composition, and climate. Expected final online publication date for the Annual Review of Plant Biology, Volume 75 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

An ecological transcriptome approach to capture the molecular and physiological mechanisms of mass flowering in Shorea curtisii.

Climatic factors have commonly been attributed as the trigger of general flowering, a unique community-level mass flowering phenomenon involving most dipterocarp species that forms the foundation of Southeast Asian tropical rainforests. This intriguing flowering event is often succeeded by mast fruiting, which provides a temporary yet substantial burst of food resources for animals, particularly frugivores. However, the physiological mechanism that triggers general flowering, particularly in dipterocarp species, is not well understood largely due to its irregular and unpredictable occurrences in the tall and dense forests. To shed light on this mechanism, we employed ecological transcriptomic analyses on an RNA-seq dataset of a general flowering species, Shorea curtisii (Dipterocarpaceae), sequenced from leaves and buds collected at multiple vegetative and flowering phenological stages. We assembled 64,219 unigenes from the transcriptome of which 1,730 and 3,559 were differentially expressed in the leaf and the bud, respectively. Differentially expressed unigene clusters were found to be enriched with homologs of Arabidopsis thaliana genes associated with response to biotic and abiotic stresses, nutrient level, and hormonal treatments. When combined with rainfall data, our transcriptome data reveals that the trees were responding to a brief period of drought prior to the elevated expression of key floral promoters and followed by differential expression of unigenes that indicates physiological changes associated with the transition from vegetative to reproductive stages. Our study is timely for a representative general flowering dipterocarp species that occurs in forests that are under the constant threat of deforestation and climate change as it pinpoints important climate sensitive and flowering-related homologs and offers a glimpse into the cascade of gene expression before and after the onset of floral initiation.

Assessing the impacts of genetic defects on starch metabolism in Arabidopsis plants using the carbon homeostasis model.

Starch serves as an important carbon storage mechanism for many plant species, facilitating their adaptation to the cyclic variations in the light environment, including day-night cycles as well as seasonal changes in photoperiod. By dynamically adjusting starch accumulation and degradation rates, plants maintain carbon homeostasis, enabling continuous growth under fluctuating environmental conditions. To understand dynamic nature of starch metabolism at the molecular level, it is necessary to integrate empirical knowledge from genetic defects in specific regulatory pathways into the dynamical system of starch metabolism. To achieve this, we evaluated the impact of genetic defects in the circadian clock, sugar sensing and starch degradation pathways using the carbon homeostasis model that encompasses the interplay between these pathways. Through the collection of starch metabolism data from 10 Arabidopsis mutants, we effectively fitted the experimental data to the model. The system-level assessment revealed that genetic defects in both circadian clock components and sugar sensing pathway hindered the appropriate adjustment of the starch degradation rate, particularly under long-day conditions. These findings not only confirmed the previous empirical findings but also provide the novel insights into the role of each gene within the gene regulatory network on the emergence of carbon homeostasis.

Seasonal gene expression signatures of delayed fertilization in Fagaceae.

In the family Fagaceae, fertilization is delayed by several weeks to 1 year after pollination, leading to 1- or 2-year fruiting species depending on whether fruiting occurs in the same or the next year after flowering. To investigate physiological responses underlying the regulation of delayed fertilization, we monitored seasonal changes in genome-wide gene expression in tissues including leaves and buds over 2 years under natural conditions in one- (Quercus glauca) and 2-year fruiting species (Lithocarpus edulis). Genes associated with metabolic changes in response to winter cold, photosynthesis and cell proliferation, which are essential for survival and growth, showed highly conserved seasonal expression profiles between species. However, seasonal expression profiles diverged between species in genes associated with pollination, an important process contributing to the origin and maintenance of the reproductive barrier between plant species. By comparing seasonal progression of ovule development and gene expression in pistillate flowers, we revealed that ovules started developing after winter in the 2-year fruiting species, which could be linked to the activation of genes involved in fertilization and female gametophyte development after winter. These findings suggest that the 2-year fruiting species may have evolved a requirement of winter cold to prevent fertilization before winter and facilitate fertilization and embryo development in the following spring when temperature rises. This study offers new possibilities to explore the evolution of reproductive strategies in Fagaceae.

AGAMOUS regulates various target genes via cell cycle-coupled H3K27me3 dilution in floral meristems and stamens.

The MADS domain transcription factor AGAMOUS (AG) regulates floral meristem termination by preventing maintenance of the histone modification lysine 27 of histone H3 (H3K27me3) along the KNUCKLES (KNU) coding sequence. At 2 d after AG binding, cell division has diluted the repressive mark H3K27me3, allowing activation of KNU transcription prior to floral meristem termination. However, how many other downstream genes are temporally regulated by this intrinsic epigenetic timer and what their functions are remain unknown. Here, we identify direct AG targets regulated through cell cycle-coupled H3K27me3 dilution in Arabidopsis thaliana. Expression of the targets KNU, AT HOOK MOTIF NUCLEAR LOCALIZED PROTEIN18 (AHL18), and PLATZ10 occurred later in plants with longer H3K27me3-marked regions. We established a mathematical model to predict timing of gene expression and manipulated temporal gene expression using the H3K27me3-marked del region from the KNU coding sequence. Increasing the number of del copies delayed and reduced KNU expression in a polycomb repressive complex 2- and cell cycle-dependent manner. Furthermore, AHL18 was specifically expressed in stamens and caused developmental defects when misexpressed. Finally, AHL18 bound to genes important for stamen growth. Our results suggest that AG controls the timing of expression of various target genes via cell cycle-coupled dilution of H3K27me3 for proper floral meristem termination and stamen development.

Carbon use strategies in shoot and acorn growth of two evergreen broadleaf trees unraveled by seasonal carbohydrate measurements and carbon isotope analysis.

Woody species have evolved carbon (C) storage processes that meet needs for reserves associated with asynchronies between C supply and demand. However, our understanding of storage dynamics is still elusive in mature trees, especially when reproduction is involved. Integrated analyses of isotope ratios, concentrations, and biomass may enhance understanding of stored C fractions' dynamics and roles. Thus, we monitored starch and soluble sugars (SSs), C isotope ratios, and biomass, in leaves, twigs and reproductive organs of two mature evergreen broadleaf trees, Quercus glauca and Lithocarpus edulis, for two years. During the growing season, no starch was observed in twigs, while constant starch levels were observed in leaves. Increase in SSs for winter hardening was earlier in L. edulis than in Q. glauca, in line with L. edulis acorns' earlier ripening. Decrease in SSs and increase in starch occurred simultaneously in the next spring. In addition, sucrose accounted for less than 10% of total SSs in leaves of both species, whereas mannose accounted for up to 75% in Q. glauca and myo-inositol up to 23% in L. edulis, indicating species specific sugar composition. These results indicate that seasonal variation of SSs fraction was more reflective to climatic change and NSC storage was less influenced by reproduction. No starch was detected in acorn organs of either Q. glauca or L. edulis except in ripening seeds. The biomass of ripe acorns was 1.7- and 6.4-fold greater than that of current-year twigs in Q. glauca and L. edulis, respectively. Bulk twigs and reproductive organs were ca. 1.0‰ 13C enriched relative to bulk leaves, which was lower than in deciduous trees. These results indicate that new photo-assimilate is the predominant C source for reproductive growth. These findings provide new insights into the dynamics of C storage in relation to reproduction in evergreen broadleaf trees.

Modelling somatic mutation accumulation and expansion in a long-lived tree with hierarchical modular architecture.

In a long-lived organism with a modular architecture, such as trees, somatic mutations accumulate throughout the long lifespan and result in genetic mosaicism in each module within the same individual. In recent years, next-generation sequencing technology has provided a snapshot of such intra-organismal genetic variability. However, the dynamic processes underlying the accumulation and expansion of somatic mutations during the growth remain poorly understood. In this study, we constructed a model to describe these processes in a form that can be applied to a real tree. Given that the proliferation dynamics of meristematic cells vary across plant species, multiple possible processes for elongation and branching were comprehensively expressed in our model. Using published data from a poplar tree, we compared the prediction of the models with the observation and explained the cell lineage dynamics underlying somatic mutations accumulation that were not evident from the snapshot of the sequenced data. We showed that the somatic genetic drift during growth increases inter-meristem mosaicism, resulting in genetically distinct branches and less integrity within an individual tree. We also showed that the somatic genetic drift during branching leads to the mutation accumulation pattern that does not reflect the tree topology. Our modelling framework can help interpret and provide further insights into the empirical findings of genetic mosaicism in long-lived trees.

The genetic structure within a single tree is determined by the behavior of the stem cells in the meristem.

Genomic sequencing revealed that somatic mutations cause a genetic differentiation of the cells in a single tree. We studied a mathematical model for stem cell proliferation in the shoot apical meristem (SAM). We evaluated the phylogenetic distance between cells sampled from different portions of a shoot, indicating their genetic difference due to mutations accumulated during shoot elongation. The plant tissue has cell walls that suppress the exchange of location between cells. This leads to the genetic differentiation of cells according to the angle around the shoot and a larger genetic variance among cells in the body. The assumptions are as follows: stem cells in the SAM normally undergo asymmetric cell division, producing successor stem cells and differentiated cells. Occasionally, a stem cell fails to leave its successor stem cell and the vacancy is filled by the duplication of one of the nearest neighbor stem cells. A mathematical analysis revealed the following: the genetic diversity of cells sampled at the same position along the shoot increases with the distance from the base of the shoot. Stem cells hold a larger variation if they are replaced only by the nearest neighbors. The coalescent length between two cells increases not only with the difference in the position along the shoot but also in the angle around the shoot axis. The dynamics of stem cells at the SAM determine the genetic pattern of the entire shoot.

An External Coincidence Model for the Lunar Cycle Reveals Circadian Phase-Dependent Moonlight Effects on Coral Spawning.

Many marine organisms synchronously spawn at specific times to ensure the success of external fertilization in the ocean. Corals are famous examples of synchronized spawning at specific lunar phases, and two distinct spawning patterns have been observed in two dominant taxa: merulinid corals spawn at regular lunar phases, several days after the full moon, whereas Acropora corals spawn at more irregular lunar phases around the full moon. Although it has been suggested that the two coral taxa have different responses to moonlight and seawater temperature, their spawning times have never been analyzed by integrating the two environmental factors, resulting in an incomplete understanding of the regulatory mechanisms of spawning. In this study, we developed a new predictive model of coral spawning days by integrating moonlight and temperature effects based on the external coincidence model for the lunar cycle. We performed model fitting using a 10-year monitoring record of coral spawning time in Taiwan. Our model successfully demonstrated the synergistic effects of moonlight and temperature on coral spawning time (days) and provided two testable hypotheses to explain the different spawning patterns regarding the preparation (maturation) process for spawning and the sensitivity to moonlight at different circadian phases: (1) Acropora corals may have an earlier onset and longer period of preparation for spawning than merulinid corals; and (2) merulinid corals may use moonlight signals near sunset, while Acropora corals may have a similar onset at approximately midnight. This is the first study to indicate the difference in circadian phase-dependent moonlight sensitivities between coral taxa, providing a basis for underlying coral spawning mechanisms for rhythmic studies.

Impacts of climate change on reproductive phenology in tropical rainforests of Southeast Asia.

In humid forests in Southeast Asia, many species from dozens of plant families flower gregariously and fruit synchronously at irregular multi-year intervals1-4. Little is known about how climate change will impact these community-wide mass reproductive events. Here, we perform a comprehensive analysis of reproductive phenology and its environmental drivers based on a monthly reproductive phenology record from 210 species in 41 families in Peninsular Malaysia. We find that the proportion of flowering and fruiting species decreased from 1976 to 2010. Using a phenology model, we find that 57% of species in the Dipterocarpaceae family respond to both drought and low-temperature cues for flowering. We show that low-temperature flowering cues will become less available in the future in the RCP2.6 and 8.5 scenarios, leading to decreased flowering opportunities of these species in a wide region from Thailand to the island of Borneo. Our results highlight the vulnerability of and variability in phenological responses across species in tropical ecosystems that differ from temperate and boreal biomes.

MASTREE+: Time-series of plant reproductive effort from six continents.

Significant gaps remain in understanding the response of plant reproduction to environmental change. This is partly because measuring reproduction in long-lived plants requires direct observation over many years and such datasets have rarely been made publicly available. Here we introduce MASTREE+, a data set that collates reproductive time-series data from across the globe and makes these data freely available to the community. MASTREE+ includes 73,828 georeferenced observations of annual reproduction (e.g. seed and fruit counts) in perennial plant populations worldwide. These observations consist of 5971 population-level time-series from 974 species in 66 countries. The mean and median time-series length is 12.4 and 10 years respectively, and the data set includes 1122 series that extend over at least two decades (≥20 years of observations). For a subset of well-studied species, MASTREE+ includes extensive replication of time-series across geographical and climatic gradients. Here we describe the open-access data set, available as a.csv file, and we introduce an associated web-based app for data exploration. MASTREE+ will provide the basis for improved understanding of the response of long-lived plant reproduction to environmental change. Additionally, MASTREE+ will enable investigation of the ecology and evolution of reproductive strategies in perennial plants, and the role of plant reproduction as a driver of ecosystem dynamics.

Aún existen importantes vacíos en la comprensión de la respuesta reproductiva de las plantas al cambio medioambiental, en parte, porque su monitoreo en especies de plantas longevas requiere una observación directa durante muchos años, y estos conjuntos de datos rara vez han estado disponibles. Aquí presentamos a MASTREE +, una base de datos que recopila series de tiempo de la reproducción de las plantas de todo el planeta, poniendo a disposición estos datos de libre acceso para la comunidad científica. MASTREE + incluye 73.828 puntos de observación de la reproducción anual georreferenciados (ej. conteos de semillas y frutos) en poblaciones de plantas perennes en todo el mundo. Estas observaciones consisten en 5971 series temporales a nivel de población provenientes de 974 especies en 66 países. La mediana de la duración de las series de tiempo es de 10 años (media = 12.4 años) y el conjunto de datos incluye 1.122 series de al menos dos décadas (≥20 años de observaciones). Para un subconjunto de especies bien estudiadas, MASTREE +incluye un amplio conjunto de series temporales replicadas en gradientes geográficos y climáticos. Describimos el conjunto de datos de acceso abierto disponible como un archivo.csv y presentamos una aplicación web asociada para la exploración de datos. MASTREE+ proporcionará la base para mejorar la comprensión sobre la respuesta reproductiva de plantas longevas al cambio medioambiental. Además, MASTREE+ facilitará los avances en la investigación de la ecología y la evolución de las estrategias reproductivas en plantas perennes y el papel de la reproducción vegetal como determinante de la dinámica de ecosistemas.

A cross-scale approach to unravel the molecular basis of plant phenology in temperate and tropical climates.

Plants have evolved to time their leafing, flowering and fruiting in appropriate seasons for growth, reproduction and resting. As a consequence of their adaptation to geographically different environments, there is a rich diversity in plant phenology from temperate and tropical climates. Recent progress in genetic and molecular studies will provide numerous opportunities to study the genetic basis of phenological traits and the history of adaptation of phenological traits to seasonal and aseasonal environments. Integrating molecular data with long-term phenology and climate data into predictive models will be a powerful tool to forecast future phenological changes in the face of global environmental change. Here, we review the cross-scale approach from genes to plant communities from three aspects: the latitudinal gradient of plant phenology at the community level, the environmental and genetic factors underlying the diversity of plant phenology, and an integrated approach to forecast future plant phenology based on genetically informed knowledge. Synthesizing the latest knowledge about plant phenology from molecular, ecological and mathematical perspectives will help us understand how natural selection can lead to the further evolution of the gene regulatory mechanisms in phenological traits in future forest ecosystems.

Delayed fertilization facilitates flowering time diversity in Fagaceae.

Fagaceae includes typical masting species that exhibit highly synchronized and fluctuating acorn production. Fagaceae shows an interesting feature in that fertilization is delayed by several weeks to more than 1 year after pollination. Although delayed fertilization was recorded over a century ago, the evolutionary advantage of delayed fertilization is still poorly understood. Here, we present a new hypothesis that delayed fertilization facilitates temporal niche differentiation via non-overlapping flowering times among species. Comparing flowering and fruiting times in 228 species from five genera in Fagaceae, we first show that there is a close association between a wider spread of flowering times and the likelihood of a 2-year fruiting habit in which there is a long delay from pollination to fertilization. To study the coevolution of flowering time and delayed fertilization, we developed a mathematical model that incorporates the effects of competition for pollinators, seed predator satiation and unfavourable season for reproduction on fitness. The model shows that delayed fertilization facilitates the diversification of flowering time in a population, which is advantageous for animal-pollinated trees that compete over pollinators. Our new hypothesis about the coevolution of delayed fertilization and flowering time will provide new insight into the evolution of masting. This article is part of the theme issue 'The ecology and evolution of synchronized seed production in plants'.

Studying the genetic basis of masting.

The mechanisms underlying mast seeding have traditionally been studied by collecting long-term observational data on seed crops and correlating seedfall with environmental variables. Significant progress in ecological genomics will improve our understanding of the evolution of masting by clarifying the genetic basis of masting traits and the role of natural selection in shaping those traits. Here, we summarize three important aspects in studying the evolution of masting at the genetic level: which traits govern masting, whether those traits are genetically regulated, and which taxa show wide variation in these traits. We then introduce recent studies on the molecular mechanisms of masting. Those studies measure seasonal changes in gene expression in natural conditions to quantify how multiple environmental factors combine to regulate floral initiation, which in many masting plant species is the single largest contributor to among-year variation in seed crops. We show that Fagaceae offers exceptional opportunities for evolutionary investigations because of its diversity at both the phenotypic and genetic levels and existing documented genome sequences. This article is part of the theme issue 'The ecology and evolution of synchronized seed production in plants'.

An Evolutionary Game Model of Sex-Dependent Antipredator Signaling.

AbstractVarious prey animals behave conspicuously to approaching predators. The conspicuous behavior is considered to be an antipredator signal, and the frequency of signaling individuals in a population differs between males and females in many species. We theoretically assessed the evolution of the inter- and intrasexual dimorphism in antipredator signaling by developing an evolutionary game model. We particularly focused on the Chinese grasshopper, Acrida cinerea, in which only a proportion of males and no females escape conspicuously. In our model, the antipredator signal was assumed to be costly and affect the probabilities of predation of both the signaling individual (individual effect) and the signaling or nonsignaling conspecifics around it (collective effect). The model indicates that (1) a positive individual effect is essential for the evolution of antipredator signaling; (2) sexual dimorphism in fecundity cost for signaling individuals or natural predation probability can produce intersexual dimorphism in the signaling where all individuals of one sex and no individuals of the other sex emit signal; and (3) a positive collective effect can explain the intrasexual dimorphism where only some individuals of one sex signal. This study provides the first model of intrasexual dimorphism in antipredator signaling and brings new testable predictions.

Why meals during resting time cause fat accumulation in mammals? Mathematical modeling of circadian regulation on glucose metabolism.

Synchronization of metabolic rhythms regulated by circadian clock and meal timing is essential for maintaining nutrient homeostasis in response to fluctuating food intake in animals. Despite numerous experimental findings on the involvement of circadian regulation of glucose and lipid metabolism, the optimal regulatory strategy for the maintenance of energy homeostasis remains poorly defined. A mathematical framework is useful to assess the circadian regulation of glycogen production/breakdown and de novo lipogenesis/lipolysis by evaluating the contribution of time of the day-dependent activation or the repression of each metabolic process in the maintenance of energy homeostasis. Here, we present a mathematical model that describes the dynamics of glycogen and triglyceride contents, two major forms of energy storage in the body that provide the fuel needed during different phases of food deprivation. By changing peak phases of glycogenesis and fat synthesis, we searched for the optimal phase set that minimizes the risks of two types of possible metabolic dysfunctions: (1) high blood glucose and (2) energy exhaustion. Based on the optimal phase set, we compared the level of fat accumulation between meal timing in the active and resting periods. Our results showed that an increased fat accumulation by food intake in the resting period can be the byproduct of minimizing energy homeostasis risks in the synchronized feeding schedule that animals adopt in nature. Our finding will be useful to schedule an optimal meal timing to prevent metabolic diseases caused by misalignment of biological and social time in modern society.

Copy number analyses of DNA repair genes reveal the role of poly(ADP-ribose) polymerase (PARP) in tree longevity.

Long-lived organisms are exposed to the risk of accumulating mutations due to DNA damage. Previous studies in animals have revealed the positive relationship between the copy number of DNA repair genes and longevity. However, the role of DNA repair in the lifespan of plants remains poorly understood. Using the recent accumulation of the complete genome sequences of diverse plant species, we performed systematic comparative analyses of the copy number variations of DNA repair genes in 61 plant species with different lifespans. Among 121 DNA repair gene families, PARP gene family was identified as a unique gene that exhibits significant expansion in trees compared to annual and perennial herbs. Among three paralogs of plant PARPs, PARP 1 showed a close association with growth rate. PARPs catalyze poly(ADP-ribosyl)ation and play pivotal roles in DNA repair and antipathogen defense. Our study suggests the conserved role of PARPs in longevity between plants and animals.

Gene co-expression network analysis identifies BEH3 as a stabilizer of secondary vascular development in Arabidopsis.

In plants, vascular stem cells located in the cambium continuously undergo self-renewal and differentiation during secondary growth. Recent advancements in cell sorting techniques have enabled access to the transcriptional regulatory framework of cambial cells. However, mechanisms underlying the robust control of vascular stem cells remain unclear. Here, we identified a new cambium-related regulatory module through co-expression network analysis using multiple transcriptome datasets obtained from an ectopic vascular cell transdifferentiation system using Arabidopsis cotyledons, Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL). The cambium gene list included a gene encoding the transcription factor BES1/BZR1 Homolog 3 (BEH3), whose homolog BES1 negatively affects vascular stem cell maintenance. Interestingly, null beh3 mutant alleles showed a large variation in their vascular size, indicating that BEH3 functions as a stabilizer of vascular stem cells. Genetic analysis revealed that BEH3 and BES1 perform opposite functions in the regulation of vascular stem cells and the differentiation of vascular cells in the context of the VISUAL system. At the biochemical level, BEH3 showed weak transcriptional repressor activity and functioned antagonistically to other BES/BZR members by competing for binding to the brassinosteroid response element. Furthermore, mathematical modeling suggested that the competitive relationship between BES/BZR homologs leads to the robust regulation of vascular stem cells.

H3K27me3 demethylases alter HSP22 and HSP17.6C expression in response to recurring heat in Arabidopsis.

Acclimation to high temperature increases plants' tolerance of subsequent lethal high temperatures. Although epigenetic regulation of plant gene expression is well studied, how plants maintain a memory of environmental changes over time remains unclear. Here, we show that JUMONJI (JMJ) proteins, demethylases involved in histone H3 lysine 27 trimethylation (H3K27me3), are necessary for Arabidopsis thaliana heat acclimation. Acclimation induces sustained H3K27me3 demethylation at HEAT SHOCK PROTEIN22 (HSP22) and HSP17.6C loci by JMJs, poising the HSP genes for subsequent activation. Upon sensing heat after a 3-day interval, JMJs directly reactivate these HSP genes. Finally, jmj mutants fail to maintain heat memory under fluctuating field temperature conditions. Our findings of an epigenetic memory mechanism involving histone demethylases may have implications for environmental adaptation of field plants.

Plant stem cell research is uncovering the secrets of longevity and persistent growth.

Plant stem cells have several extraordinary features: they are generated de novo during development and regeneration, maintain their pluripotency, and produce another stem cell niche in an orderly manner. This enables plants to survive for an extended period and to continuously make new organs, representing a clear difference in their developmental program from animals. To uncover regulatory principles governing plant stem cell characteristics, our research project 'Principles of pluripotent stem cells underlying plant vitality' was launched in 2017, supported by a Grant-in-Aid for Scientific Research on Innovative Areas from the Japanese government. Through a collaboration involving 28 research groups, we aim to identify key factors that trigger epigenetic reprogramming and global changes in gene networks, and thereby contribute to stem cell generation. Pluripotent stem cells in the shoot apical meristem are controlled by cytokinin and auxin, which also play a crucial role in terminating stem cell activity in the floral meristem; therefore, we are focusing on biosynthesis, metabolism, transport, perception, and signaling of these hormones. Besides, we are uncovering the mechanisms of asymmetric cell division and of stem cell death and replenishment under DNA stress, which will illuminate plant-specific features in preserving stemness. Our technology support groups expand single-cell omics to describe stem cell behavior in a spatiotemporal context, and provide correlative light and electron microscopic technology to enable live imaging of cell and subcellular dynamics at high spatiotemporal resolution. In this perspective, we discuss future directions of our ongoing projects and related research fields.