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.

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.

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.

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.

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.

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.

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.

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.

From theory to experiments for testing the proximate mechanisms of mast seeding: an agenda for an experimental ecology.

Highly variable and synchronised production of seeds by plant populations, known as masting, is implicated in many important ecological processes, but how it arises remains poorly understood. The lack of experimental studies prevents underlying mechanisms from being explicitly tested, and thereby precludes meaningful predictions on the consequences of changing environments for plant reproductive patterns and global vegetation dynamics. Here we review the most relevant proximate drivers of masting and outline a research agenda that takes the biology of masting from a largely observational field of ecology to one rooted in mechanistic understanding. We divide the experimental framework into three main processes: resource dynamics, pollen limitation and genetic and hormonal regulation, and illustrate how specific predictions about proximate mechanisms can be tested, highlighting the few successful experiments as examples. We envision that the experiments we outline will deliver new insights into how and why masting patterns might respond to a changing environment.

Gene regulatory network models in response to sugars in the plant circadian system.

Circadian entrainment is the process by which internal circadian oscillators staying in synchronization with the local environmental rhythms. Circadian clocks are entrained by adjusting phase and period in response to environmental and metabolic signals. In Arabidopsis thaliana, light and sugar signals differentially affect the circadian phase; the former advances the phase in the late of the subjective night and delays around dusk, while the latter advances the phase mainly in the morning, which is optimal to maintain sucrose homeostasis. We have proposed that the phase adjustment of the A. thaliana circadian oscillator by sugar signals contributes to the realization of carbon homeostasis and the increase of plant growth under fluctuating day-night cycles. However, which genes in the circadian oscillator are targets of sucrose signals and how the potential target genes should be regulated by sucrose to realize sucrose homeostasis has not been studied from the theoretical perspective. Here we investigate the effect of sugar on the phase response property of the plant circadian oscillator using clock gene-regulatory network models. We simulated phase response curves (PRCs) to sucrose pulses, which were compared with an experimental PRC. Our analyses of the gene-regulatory network model demonstrated that target genes of the sugar signal could be members of the PSEUDO-RESPONSE REGULATOR gene family and the evening complex components. We also examined the phase response property using a single feedback-loop model and elucidated how phase advance is induced in the subjective morning under certain conditions of a target clock gene of sucrose and its regulatory property.

Unravelling proximate cues of mass flowering in the tropical forests of South-East Asia from gene expression analyses.

Elucidating the physiological mechanisms of the irregular yet concerted flowering rhythm of mass flowering tree species in the tropics requires long-term monitoring of flowering phenology, exogenous and endogenous environmental factors, as well as identifying interactions and dependencies among these factors. To investigate the proximate factors for floral initiation of mast seeding trees in the tropics, we monitored the expression dynamics of two key flowering genes, meteorological conditions and endogenous resources over two flowering events of Shorea curtisii and Shorea leprosula in the Malay Peninsula. Comparisons of expression dynamics of genes studied indicated functional conservation of FLOWERING LOCUS T (FT) and LEAFY (LFY) in Shorea. The genes were highly expressed at least 1 month before anthesis for both species. A mathematical model considering the synergistic effect of cool temperature and drought on activation of the flowering gene was successful in predicting the observed gene expression patterns. Requirement of both cool temperature and drought for floral transition suggested by the model implies that flowering phenologies of these species are sensitive to climate change. Our molecular phenology approach in the tropics sheds light on the conserved role of flowering genes in plants inhabiting different climate zones and can be widely applied to dissect the flowering processes in other plant species.

Parameterisation and validation of a resource budget model for masting using spatiotemporal flowering data of individual trees.

Synchronised and fluctuating reproduction by plant populations, called masting, is widespread in diverse taxonomic groups. Here, we propose a new method to explore the proximate mechanism of masting by combining spatiotemporal flowering data, biochemical analysis of resource allocation and mathematical modelling. Flowering data of 170 trees over 13 years showed the emergence of clustering with trees in a given cluster mutually synchronised in reproduction, which was successfully explained by resource budget models. Analysis of resources invested in the development of reproductive organs showed that parametric values used in the model are significantly different between nitrogen and carbon. Using a fully parameterised model, we showed that the observed flowering pattern is explained only when the interplay between nitrogen dynamics and climatic cues was considered. This result indicates that our approach successfully identified resource type-specific roles on masting and that the method is suitable for a wide range of plant species.

Florigen distribution determined by a source-sink balance explains the diversity of inflorescence structures in Arabidopsis.

The ability to continue flowering after loss of inductive environmental cues that trigger flowering is termed floral commitment. Reversible transition involving a switch from floral development back to vegetative development has been found in Arabidopsis mutants and many plant species. Although the molecular basis for floral commitment remains unclear, recent studies suggest that the persistent activity of FLOWERING LOCUS T (FT) at inflorescences is required for floral commitment in Arabidopsis thaliana. Because FT encodes a mobile signal, florigen, which is generally transported from leaves to meristems through the phloem, understanding the transportation dynamics of FT is required to explore the role of FT on floral commitment. Here we developed a transportation model of leaf- and inflorescence-derived florigen and sucrose based on pressure-flow hypothesis. Depending on the demanded level of florigen supply for floral commitment of each floral meristem, the model predicted the change in inflorescence pattern from stable commitment to flower, transient flowering, and complete reversion. FT activity in inflorescence partly suppressed floral reversion, but complete suppression was achieved only when inflorescence became a source of sucrose. This finding highlights the importance of monitoring the spatio-temporal sucrose distribution and floral stimulus to understand inflorescence development mechanism.

Ribosomes in a stacked array: elucidation of the step in translation elongation at which they are stalled during S-adenosyl-L-methionine-induced translation arrest of CGS1 mRNA.

Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.

Understanding circadian regulation of carbohydrate metabolism in Arabidopsis using mathematical models.

C3 plants assimilate carbon by photosynthesis only during the day, but carbon resources are also required for growth and maintenance at night. To avoid carbon starvation, many plants store a part of photosynthetic carbon in starch during the day, and degrade it to supply sugars for growth at night. In Arabidopsis, starch accumulation in the day and degradation at night occur almost linearly, with the shape of this diel starch profile adaptively changing to allow continuous supply of sugar even in long-night conditions. The anticipation of dawn required to ensure linear consumption of starch to almost zero at dawn presumably requires the circadian clock. We review the links between carbon metabolism and the circadian clock, and mathematical models aimed at explaining the diel starch profile. These models can be considered in two classes, those that assume the level of available starch is sensed and the system ensures linearity of starch availability, and those in which sugar sensing is assumed, yielding linearity of starch availability as an emergent property of sucrose homeostasis. In the second class of model the feedback from starch metabolism to the circadian clock is considered to be essential for adaptive response to diverse photoperiods, consistent with recent empirical data demonstrating entrainment of the circadian clock by photosynthesis. Knowledge concerning the mechanisms regulating the dynamics of starch metabolism and sugar homeostasis in plants is required to develop new theories about the limitations of growth and biomass accumulation.

In silico simulation modeling reveals the importance of the Casparian strip for efficient silicon uptake in rice roots.

Silicon (Si) uptake by the roots is mediated by two different transporters, Lsi1 (passive) and Lsi2 (active), in rice (Oryza sativa). Both transporters are polarly localized in the plasma membranes of exodermal (outer) and endodermal (inner) cells with Casparian strips. However, it is unknown how rice is able to take up large amounts of Si compared with other plants, and why rice Si transporters have a characteristic cellular localization pattern. To answer these questions, we simulated Si uptake by rice roots by developing a mathematical model based on a simple diffusion equation that also accounts for active transport by Lsi2. In this model, we calibrated the model parameters using in vivo experimental data on the Si concentrations in the xylem sap and a Monte Carlo method. In our simulation experiments, we compared the Si uptake between roots with various transporter and Casparian strip locations and estimated the Si transport efficiency of roots with different localization patterns and quantities of the Lsi transporters. We found that the Si uptake by roots that lacked Casparian strips was lower than that of normal roots. This suggests that the double-layer structure of the Casparian strips is an important factor in the high Si uptake by rice. We also found that among various possible localization patterns, the most efficient one was that of the wild-type rice; this may explain the high Si uptake capacity of rice.

A mathematical model of phloem sucrose transport as a new tool for designing rice panicle structure for high grain yield.

Rice (Oryza sativa) is one of the most important food crops in the world. Numerous quantitative trait loci or genes controlling panicle architecture have been identified to increase grain yield. Yet grain yield, defined as the product of the number of well-ripened grains and their weight, is a complex trait that is determined by multiple factors such as source, sink and translocation capacity. Mechanistic modelling capturing capacities of source, sink and transport will help in the theoretical design of crop ideotypes that guarantee high grain yield. Here we present a mathematical model simulating sucrose transport and grain growth within a complex phloem network. The model predicts that the optimal panicle structure for high yield shows a simple grain arrangement with few higher order branches. In addition, numerical analyses revealed that inefficient delivery of carbon to panicles with higher order branches prevails regardless of source capacity, indicating the importance of designing grain arrangement and phloem structure. Our model highlights the previously unexplored effect of grain arrangement on the yield, and provides numerical solutions for optimal panicle structure under various source and sink capacities.

Systematic genome sequence differences among leaf cells within individual trees.

BACKGROUND: Even in the age of next-generation sequencing (NGS), it has been unclear whether or not cells within a single organism have systematically distinctive genomes. Resolving this question, one of the most basic biological problems associated with DNA mutation rates, can assist efforts to elucidate essential mechanisms of cancer. RESULTS: Using genome profiling (GP), we detected considerable systematic variation in genome sequences among cells in individual woody plants. The degree of genome sequence difference (genomic distance) varied systematically from the bottom to the top of the plant, such that the greatest divergence was observed between leaf genomes from uppermost branches and the remainder of the tree. This systematic variation was observed within both Yoshino cherry and Japanese beech trees. CONCLUSIONS: As measured by GP, the genomic distance between two cells within an individual organism was non-negligible, and was correlated with physical distance (i.e., branch-to-branch distance). This phenomenon was assumed to be the result of accumulation of mutations from each cell division, implying that the degree of divergence is proportional to the number of generations separating the two cells.

Nitrogen as a key regulator of flowering in Fagus crenata: understanding the physiological mechanism of masting by gene expression analysis.

The role of resource availability in determining the incidence of masting has been widely studied, but how floral transition and initiation are regulated by the resource level is unclear. We tested the hypothesis that floral transition is stimulated by high resource availabiltiy in Fagus crenata based on a new technique, the expression analyses of flowering genes. We isolated F. crenata orthologues of FLOWERING LOCUS T, LEAFY and APETALA1, and confirmed their functions using transgenic Arabidopsis thaliana. We monitored the gene expression levels for 5 years and detected a cycle of on and off years, which was correlated with fluctuations of the shoot-nitrogen concentration. Nitrogen fertilisation resulted in the significantly higher expression of flowering genes than the control, where all of the fertilised trees flowered, whereas the control did not. Our findings identified nitrogen as a key regulator of mast flowering, thereby providing new empirical evidence to support the resource budget model.