Barley TAPETAL DEVELOPMENT and FUNCTION1 (HvTDF1) gene reveals conserved and unique roles in controlling anther tapetum development in dicot and monocot plants.

The anther tapetum helps control microspore release and essential components for pollen wall formation. TAPETAL DEVELOPMENT and FUNCTION1 (TDF1) is an essential R2R3 MYB tapetum transcription factor in Arabidopsis thaliana; however, little is known about pollen development in the temperate monocot barley. Here, we characterize the barley (Hordeum vulgare L.) TDF1 ortholog using reverse genetics and transcriptomics. Spatial/temporal expression analysis indicates HvTDF1 has tapetum-specific expression during anther stage 7/8. Homozygous barley hvtdf1 mutants exhibit male sterility with retarded tapetum development, delayed tapetum endomitosis and cell wall degeneration, resulting in enlarged, vacuolated tapetum surrounding collapsing microspores. Transient protein expression and dual-luciferase assays show TDF1 is a nuclear-localized, transcription activator, that directly activates osmotin proteins. Comparison of hvtdf1 transcriptome data revealed several pathways were delayed, endorsing the observed retarded anther morphology. Arabidopsis tdf1 mutant fertility was recovered by HvTDF1, supporting a conserved role for TDF1 in monocots and dicots. This indicates that tapetum development shares similarity between monocot and dicots; however, barley HvTDF1 appears to uniquely act as a modifier to activate tapetum gene expression pathways, which are subsequently also induced by other factors. Therefore, the absence of HvTDF1 results in delayed developmental progression rather than pathway failure, although inevitably still results in pollen degeneration.

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.

A single MYB transcription factor with multiple functions during flower development.

Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana.

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Little directional change in the timing of Arctic spring phenology over the past 25 years.

With the global change in climate, the Arctic has been pinpointed as the region experiencing the fastest rates of change. As a result, Arctic biological responses-such as shifts in phenology-are expected to outpace those at lower latitudes. 15 years ago, a decade-long dataset from Zackenberg in High Arctic Greenland revealed rapid rates of phenological change.1 To explore how the timing of spring phenology has developed since, we revisit the Zackenberg time series on flowering plants, arthropods, and birds. Drawing on the full 25-year period of 1996-2020, we find little directional change in the timing of events despite ongoing climatic change. We attribute this finding to a shift in the temporal patterns of climate conditions, from previous directional change to current high inter-annual variability. Additionally, some taxa appear to have reached the limits of their phenological responses, resulting in a leveling off in their phenological responses in warm years. Our findings demonstrate the importance of long-term monitoring of taxa from across trophic levels within the community, allowing for detecting shifts in sensitivities and responses and thus for updated inference in the light of added information.

Integrating analog and digital modes of gene expression at Arabidopsis FLC.

Quantitative gene regulation at the cell population level can be achieved by two fundamentally different modes of regulation at individual gene copies. A 'digital' mode involves binary ON/OFF expression states, with population-level variation arising from the proportion of gene copies in each state, while an 'analog' mode involves graded expression levels at each gene copy. At the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), 'digital' Polycomb silencing is known to facilitate quantitative epigenetic memory in response to cold. However, whether FLC regulation before cold involves analog or digital modes is unknown. Using quantitative fluorescent imaging of FLC mRNA and protein, together with mathematical modeling, we find that FLC expression before cold is regulated by both analog and digital modes. We observe a temporal separation between the two modes, with analog preceding digital. The analog mode can maintain intermediate expression levels at individual FLC gene copies, before subsequent digital silencing, consistent with the copies switching OFF stochastically and heritably without cold. This switch leads to a slow reduction in FLC expression at the cell population level. These data present a new paradigm for gradual repression, elucidating how analog transcriptional and digital epigenetic memory pathways can be integrated.

Body mass decline in a Mediterranean community of solitary bees supports the size shrinking effect of climatic warming.

The long-known, widely documented inverse relationship between body size and environmental temperature ("temperature-size rule") has recently led to predictions of body size decline following current climatic warming ("size shrinking effect"). For keystone pollinators such as wild bees, body shrinking in response to warming can have significant effects on pollination processes but there is still little direct evidence of the phenomenon because adequate tests require controlling for confounding factors linked to climate change (e.g., habitat change). This paper assesses the shrinking effect in a community of solitary bees from well-preserved habitats in the core of a large nature reserve experiencing climatic warming without disturbances or habitat changes. Long-term variation in mean body mass was evaluated using data from 1704 individual bees (137 species, 27 genera, 6 families) sampled over 1990-2023. Climate warmed at a fast rate during this period, annual mean of daily maximum temperature increasing 0.069°C/year on average during 2000-2020. Changes in bee body mass verified expectations from the size shrinking effect. The mean individual body mass of the community of solitary bees declined significantly, irrespective of whether the analysis referred to the full species sample or only to the subset of species that were sampled in both the old (1990-1997) and recent (2022-2023) periods. On average, body mass declined ~0.7%·year-1 , or an estimated average cumulative reduction of ~20 mg per individual bee from 1990 to 2023. Proportional size reduction was greatest among large-bodied species, ranging from around -0.6%·year-1 for the smallest species to -0.9%·year-1 for the largest ones. Declining rate was steeper for cavity-nesting than ground-nesting species. The pollination and mating systems of bee-pollinated plants in the study region are probably undergoing significant alterations as a consequence of supra-annual decline in bee body mass.

ZmSPL13 and ZmSPL29 act together to promote vegetative and reproductive transition in maize.

Flowering time is a key agronomic trait determining environmental adaptation and yield potential of crops. The regulatory mechanisms of flowering in maize still remain rudimentary. In this study, we combine expressional, genetic, and molecular studies to identify two homologous SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors ZmSPL13 and ZmSPL29 as positive regulators of juvenile-to-adult vegetative transition and floral transition in maize. We show that both ZmSPL13 and ZmSPL29 are preferentially expressed in leaf phloem, vegetative and reproductive meristem. We show that vegetative phase change and flowering time are moderately delayed in the Zmspl13 and Zmspl29 single knockout mutants and more significantly delayed in the Zmspl13/29 double mutants. Consistently, the ZmSPL29 overexpression plants display precocious vegetative phase transition and floral transition, thus early flowering. We demonstrate that ZmSPL13 and ZmSPL29 directly upregulate the expression of ZmMIR172C and ZCN8 in the leaf, and of ZMM3 and ZMM4 in the shoot apical meristem, to induce juvenile-to-adult vegetative transition and floral transition. These findings establish a consecutive signaling cascade of the maize aging pathway by linking the miR156-SPL and the miR172-Gl15 regulatory modules and provide new targets for genetic improvement of flowering time in maize cultivars.

Shifts in flowering phenology in response to spring temperatures in eastern Tennessee.

PREMISE: Plant phenological shifts are among the clearest indicators of the effects of climate change. In North America, numerous studies in the northeastern United States have demonstrated earlier spring flowering compared to historical records. However, few studies have examined phenological shifts in the southeastern United States, a highly biodiverse region of North America characterized by dramatic variations in abiotic conditions over small geographic areas. METHODS: We examined 1000+ digitized herbarium records along with location-specific temperature data to analyze phenological shifts of 14 spring-flowering species in two adjacent ecoregions in eastern Tennessee. RESULTS: Spring-flowering plant communities in the Blue Ridge and the Ridge and Valley ecoregions differed in their sensitivity to temperature; plants in the Ridge and Valley flower 0.73 days earlier/°C on average compared to 1.09 days/°C for plants in the Blue Ridge. Additionally, for the majority of species in both ecoregions, flowering is sensitive to spring temperature; i.e., in warmer years, most species flowered earlier. Despite this sensitivity, we did not find support for community-level shifts in flowering within eastern Tennessee in recent decades, likely because increases in annual temperature in the southeast are driven primarily by warming summer (rather than spring) temperatures. CONCLUSIONS: These results highlight the importance of including ecoregion as a predictor in phenological models for capturing variation in sensitivity among populations and suggest that even small shifts in temperature can have dramatic effects on phenology in response to climate in the southeastern United States.

Limited reproductive interference despite high rates of heterospecific pollen transfer among co-occurring bat-pollinated Burmeistera.

PREMISE: Many tropical plants are bat-pollinated, but these mammals often carry copious, multispecific pollen loads making bat-pollinated plants susceptible to heterospecific pollen deposition and reproductive interference. We investigated pollen transfer between sympatric bat-pollinated Burmeistera species and their response to heterospecific pollen deposition from each other. METHODS: We quantified conspecific and heterospecific pollen deposition for two populations of B. ceratocarpa, a recipient species in heterospecific pollen transfer interactions, that co-occur with different donor relatives (B. borjensis and B. glabrata). We then used a cross-pollination scheme using pollen mixtures to assess the species' responses to heterospecific pollen deposition in terms of fruit abortion and seed production. RESULTS: Burmeistera ceratocarpa received significantly more heterospecific pollen from its relatives at both sites than its own pollen was deposited on its relatives. However, heterospecific pollen deposition only affected seed production by B. borjensis and B. glabrata, but not by B. ceratocarpa, suggesting that early acting post-pollination barriers buffer the latter against reproductive interference. Crosses between sympatric and allopatric populations suggest that the study species are fully isolated in sympatry, while isolation between allopatric populations is strong but incomplete. CONCLUSIONS: We did not observe evidence of reproductive interference among our study species, because either heterospecific pollen deposition did not affect their seed production (B. ceratocarpa) or they receive heterospecific pollen only rarely (B. borjensis and B. glabrata). Frequent heterospecific pollen deposition might favor the evolution of barriers against foreign pollen (as in B. ceratocarpa) that alleviate the competitive costs of sharing low fidelity pollinators with co-occurring species.

Phenotype-fitness relationships and pollen-transfer efficiency of five orchid species with different pollination strategies.

PREMISE: Deceptive pollination, a fascinating mechanism that independently originated in several plant families for benefiting from pollinators without providing any reward, is particularly widespread among orchids. Pollination efficiency is crucial in orchids due to the aggregated pollen in a pollinarium, which facilitates pollen transfer and promotes cross-pollination as pollinators leave after being deceived. METHODS: In this study, we compiled data on reproductive ecology from five orchid species with different pollination strategies: three deceptive-strategy species (shelter imitation, food deception, sexual deception), one nectar-rewarding species, and one shelter-imitation but spontaneously selfing species. We aimed to compare the reproductive success (female fitness: fruit set; male fitness: pollinarium removal) and pollination efficiency of species representing these strategies. We also investigated pollen limitation and inbreeding depression among the pollination strategies. RESULTS: Male and female fitness were strongly correlated in all species but the spontaneously selfing species, which had high fruit set and low pollinarium removal. As expected, pollination efficiency was highest for the rewarding species and the sexually deceptive species. Rewarding species had no pollen limitation but did have high cumulative inbreeding depression; deceptive species had high pollen limitation and moderate inbreeding depression; and spontaneously selfing species did not have pollen limitation or inbreeding depression. CONCLUSIONS: Pollinator response to deception is critical to maintain reproductive success and avoid inbreeding in orchid species with non-rewarding pollination strategies. Our findings contribute to a better understanding of the trade-offs associated with different pollination strategies in orchids and highlight the importance of pollination efficiency in orchids due to the pollinarium.

Hidden worlds within flowers.

There is a growing realization that ecological interactions take place at many scales, from acorns to forests, and that formerly overlooked community members, particularly microbes, can play outsized ecological roles. Beyond their primary function as the reproductive organs of angiosperms, flowers constitute resource-rich, ephemeral habitats teeming with flower-loving symbionts, or 'anthophiles'. The physical, chemical, and structural properties of flowers combine to create a habitat filter, selectively determining which anthophiles can reside there, and how, and when they interact. The microhabitats within flowers can provide shelter from predators or inclement weather, places to eat, sleep, thermoregulate, hunt, mate or reproduce. In turn, floral microhabitats contain the full range of mutualists, antagonists and apparent commensals, whose complex interactions impact how flowers look and smell, how profitable they are to foraging pollinators, and how selection feeds back upon the traits shaping those interactions. Recent studies suggest coevolutionary paths by which floral symbionts might be co-opted as mutualists and provide compelling examples in which ambush predators or florivores are recruited as floral allies. Unbiased studies that include the full roster of floral symbionts are likely to reveal novel links and additional nuance in the rich ecological communities hidden within flowers.

Comparative transcriptomic analysis of transcription factors and hormones during flower bud differentiation in 'Red Globe' grape under red‒blue light.

Grape is a globally significant fruit-bearing crop, and the grape flower bud differentiation essential to fruit production is closely related to light quality. To investigate the regulatory mechanism of grape flower bud differentiation under red‒blue light, the transcriptome and hormone content were determined at four stages of flower bud differentiation. The levels of indole-3-acetic acid (IAA) and abscisic acid (ABA) in grape flower buds at all stages of differentiation under red‒blue light were higher than those in the control. However, the levels of cytokinins (CKs) and gibberellic acid (giberellins, GAs) fluctuated continuously over the course of flower bud differentiation. Moreover, many differentially expressed genes were involved in auxin, CK, GA, and the ABA signal transduction pathways. There were significant differences in the AUX/IAA, SAUR, A-RR, and ABF gene expression levels between the red‒blue light treatment and the control buds, especially in regard to the ABF genes, the expression levels of which were completely different between the two groups. The expression of GBF4 and AI5L2 in the control was always low, while the expression under red‒blue light increased. AI5L7 and AI5L5 expression levels showed an upwards trend in the control plant buds and gradually decreased in red‒blue light treatment plant buds. Through weighted gene coexpression network analysis, we determined that the transcription factors WRK48 (WRKY family), EF110 (ERF family), ABR1, CAMTA3 (CAMTA family), and HSFA3 (HSF family) may be involved in the regulation of the GBF4 gene. This study lays a foundation for further analysis of grape flower bud differentiation regulation under red‒blue light.

The P-body component DECAPPING5 and the floral repressor SISTER OF FCA regulate FLOWERING LOCUS C transcription in Arabidopsis.

Flowering is the transition from vegetative to reproductive growth and is critical for plant adaptation and reproduction. FLOWERING LOCUS C (FLC) plays a central role in flowering time control, and dissecting its regulation mechanism provides essential information for crop improvement. Here, we report that DECAPPING5 (DCP5), a component of processing bodies (P-bodies), regulates FLC transcription and flowering time in Arabidopsis (Arabidopsis thaliana). DCP5 and its interacting partner SISTER OF FCA (SSF) undergo liquid-liquid phase separation (LLPS) that is mediated by their prion-like domains (PrDs). Enhancing or attenuating the LLPS of both proteins using transgenic methods greatly affects their ability to regulate FLC and flowering time. DCP5 regulates FLC transcription by modulating RNA polymerase II enrichment at the FLC locus. DCP5 requires SSF for FLC regulation, and loss of SSF or its PrD disrupts DCP5 function. Our results reveal that DCP5 interacts with SSF, and the nuclear DCP5-SSF complex regulates FLC expression at the transcriptional level.

GSK3 regulates VRN1 to control flowering time in wheat.

GLYCOGEN SYNTHASE KINASE 3 physically interacts with VRN1 and regulates its accumulation to mediate flowering in wheat.

How consistently do species leaf-out or flower in the same order? Understanding the factors that shape this characteristic of plant communities.

Plant species are frequently reported to undergo leaf-out and flowering in a consistent order from 1 year to the next; however, only a limited number of these findings arise from studies encompassing many species or sites. Here, we evaluate the consistency in the order species leafed out in the northeastern United States using observations contributed to the USA National Phenology Network's Nature's Notebook platform. We repeated this analysis for flowering, evaluating a total of 132 species across 84 sites. We documented a relatively high degree of consistency in the order of both events among individual plants, with higher consistency in flowering. A small number of species pairs exhibited very high consistency in phenological order across several sites. The majority of species pairs exhibited variability in how consistently they underwent either leaf-out or flowering from site to site, which could be the result of either plastic or locally adaptive responses. Our investigation revealed that neither functional type nor seasonal position played a major role in shaping how consistently species leafed out or flowered in the same order. Instead, we found the number of days separating the events and interannual variability in timing to be the most influential factors driving the consistency in ordering.

Complex cross-incompatibility in morning glories is consistent with a role for mating system in plant speciation.

Reproductive isolation between selfing and outcrossing species can arise through diverse mechanisms, some of which are directly associated with differences in mating system. We dissected cross-incompatibility between the highly selfing morning glory Ipomoea lacunosa and its mixed-mating sister species Ipomoea cordatotriloba. We found that cross-incompatibility is complex, with contributions acting both before and after fertilization. We then investigated whether the transition in mating system may have facilitated the evolution of these reproductive barrier components through mismatched floral morphology, differences in reproductive context, or both. We found evidence that morphological mismatch likely contributes to reproductive isolation in at least one cross-direction and that other pollen-pistil interactions are present. We also identified hybrid seed inviability consistent with the predictions of the weak-inbreeder, strong-outbreeder hypotheses, suggesting endosperm misregulation plays an important role in cross-incompatibility. In contrast, we did not find evidence consistent with the prezygotic weak-inbreeder, strong-outbreeder hypothesis. Our study highlights the complexity of reproductive isolation between outcrossing and selfing species and the extent to which evolutionary consequences of mating system transitions can facilitate speciation.

The AP2/ERF transcription factor TOE4b regulates photoperiodic flowering and grain yield per plant in soybean.

Photoperiod-mediated flowering determines the phenological adaptability of crops including soybean (Glycine max). A genome-wide association study (GWAS) identified a new flowering time locus, Time of flowering 13 (Tof13), which defined a gene encoding an AP2/ERF transcription factor. This new transcription factor, which we named TOE4b, is localized in the nucleus. TOE4b has been selected for soybean latitude adaptability. The existing natural variant TOE4bH4 was rare in wild soybean accessions but occurred more frequently in landraces and cultivars. Notably, TOE4bH4 improved high-latitude adaptation of soybean to some extent. The gene-edited TOE4b knockout mutant exhibited earlier flowering, conversely, TOE4b overexpression delayed flowering time. TOE4b is directly bound to the promoters and gene bodies of the key flowering integration factor genes FT2a and FT5a to inhibit their transcription. Importantly, TOE4b overexpression lines in field trials not only showed late flowering but also altered plant architecture, including shorter internode length, more internodes, more branches and pod number per plant, and finally boosted grain yield per plant by 60% in Guangzhou and 87% in Shijiazhuang. Our findings therefore identified TOE4b as a pleiotropic gene to increase yield potential per plant in soybean, and these results provide a promising option for breeding a soybean variety with an idealized plant architecture that promotes high yields.

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.

CONSTANS, a key-player connecting day length to seed size.

Plants sense oscillation in the day length as a reliable seasonal cue to drive optimal vegetative and reproductive growth. A recent study by Yu et al. has revealed how day length regulates seed size through CONSTANS. The CONSTANS-APETALA2 module enables plants to optimize their reproductive growth based on their photoperiod response type.