BACKGROUND: The theory of Condition Dependent Sex predicts that - everything else being equal - less fit individuals would outcross at higher rates compared with fitter ones. Here we used the mixed mating plant Lamium amplexicaule, capable of producing both self-pollinating closed flowers (CL), alongside open flowers (CH) that allow cross pollination to test it. We investigated the effects of abiotic stress - salt solution irrigation - on the flowering patterns of plants and their offspring. We monitored several flowering and vegetative parameters, including the number and distribution of flowers, CH fraction, and plant size.
RESULTS: We found that stressed plants show an increased tendency for self-pollination and a deficit in floral and vegetative development. However, when parentally primed, stressed plants show a milder response. Un-stressed offspring of stressed parents show reversed responses and exhibit an increased tendency to outcross, and improve floral and vegetative development.
CONCLUSIONS: In summary, we found that stress affects the reproduction strategy in the plants that experienced the stress and in subsequent offspring through F2 generation. Our results provide experimental evidence supporting a transgenerational extension to the theories of fitness associate sex and dispersal, where an individual\'s tendency for sex and dispersal may depend on the stress experienced by its parents.

OBJECTIVE: The impact of urbanization on plant evolution, particularly the evolution of reproductive traits, remains largely unknown. In this study, we aimed to investigate the consequences of urbanization on the reproductive traits of Portulaca oleracea in the Kantō region of Japan. Portulaca oleracea has a unique cleistogamous reproductive system, which consists of genetically determined chasmogamous (open, CH) and cleistogamous (closed, CL) plants.
METHODS: We collected seeds of P. oleracea from ten populations in rural areas and ten populations in urban areas. In a common garden experiment, we recorded the type of flowers (CH or CL), reproductive phenology and seed production.
RESULTS: All individuals produced either CH or CL flowers, allowing us to classify them as either CH or CL plants. We observed a significant difference in the prevalence of CH and CL plants between rural and urban populations: the number of CH plants was generally low and was particularly low among urban individuals. Compared to CH plants, CL plants showed earlier phenology and produced heavier seeds, which is consistent with stress avoidance in response to heat and drought stress conditions in urban areas.
CONCLUSIONS: Our findings suggest that urbanization may drive an evolutionary change in the cleistogamous reproductive system of P. oleracea. CL plants with earlier phenology and larger seeds might be better adapted to urban environments, where they are subjected to harsh heat and drought stress.

Competition affects mixed-mating strategies by limiting available abiotic or biotic resources such as nutrients, water, space, or pollinators. Cleistogamous species produce closed (cleistogamous, CL), obligately selfed, simultaneously with open (chasmogamous, CH), potentially outcrossed flowers. The effects of intraspecific competition on fitness and cleistogamy variation can range from limiting the production of costly CH flowers because of resource limitation, to favouring CH production because of fitness advantages of outcrossed, CH offspring. Moreover, the effects of competition can be altered when it co-occurs with other environmental variations. We grew plants from seven populations of the ruderal Lamium amplexicaule, originating from different climates and habitats, in a common garden experiment combining drought, interspecific competition, and seasonal variation. All these parameters have been shown to influence the degree of cleistogamy in the species on their own. In spring, competition and drought negatively impacted fitness, but the CL proportion only increased when plants were exposed to both treatments combined. We did not observe the same results in autumn, which can be due to non-adaptive phenotypic variation, or to differences in soil compactness between seasons. The observed responses are largely due to phenotypic plasticity, but we also observed phenotypic differentiation between populations for morphological, phenological, and cleistogamy traits, pointing to the existence of different ecotypes. Our data do not support the hypothesis that CL proportion should decrease when resources are scarce, as plants with reduced growth had relatively low CL proportions. We propose that variation in cleistogamy could be an adaptation to pollinator abundance, or to environment-dependent fitness differences between offspring of selfed and outcrossed seeds, two hypotheses worth further investigation. This opens exciting new possibilities for the study of the maintenance of mixed-mating systems using cleistogamous species as models that combine the effects of inbreeding and reproductive costs.

CONCLUSIONS: Differentially expressed microRNAs were found associated with the development of chasmogamous and cleistogamous flowers in Viola prionantha, revealing potential roles of microRNAs in the developmental evolution of dimorphic flowers. In Viola prionantha, chasmogamous (CH) flowers are induced by short daylight, while cleistogamous (CL) flowers are triggered by long daylight. How environmental factors and microRNAs (miRNAs) affect dimorphic flower formation remains unknown. In this study, small RNA sequencing was performed on CH and CL floral buds at different developmental stages in V. prionantha, differentially expressed miRNAs (DEmiRNAs) were identified, and their target genes were predicted. In CL flowers, Viola prionantha miR393 (vpr-miR393a/b) and vpr-miRN3366 were highly expressed, while in CH flowers, vpr-miRN2005, vpr-miR172e-2, vpr-miR166m-3, vpr-miR396f-2, and vpr-miR482d-2 were highly expressed. In the auxin-activated signaling pathway, vpr-miR393a/b and vpr-miRN2005 could target Vpr-TIR1/AFB and Vpr-ARF2, respectively, and other DEmiRNAs could target genes involved in the regulation of transcription, e.g., Vpr-AP2-7. Moreover, Vpr-UFO and Vpr-YAB5, the main regulators in petal and stamen development, were co-expressed with Vpr-TIR1/AFB and Vpr-ARF2 and showed lower expression in CL flowers than in CH flowers. Some V. prionantha genes relating to the stress/defense responses were co-expressed with Vpr-TIR1/AFB, Vpr-ARF2, and Vpr-AP2-7 and highly expressed in CL flowers. Therefore, in V. prionantha, CH-CL flower development may be regulated by the identified DEmiRNAs and their target genes, thus providing the first insight into the formation of dimorphic flowers in Viola.

Cleistogamy or closed flowering is a widely used trait in barley (Hordeum vulgare) breeding because it reduces the risk of fungal infection in florets at anthesis. Cleistogamy in barley is caused by a point mutation within the microRNA172 (miR172) target site of the Cly1 gene, which encodes the Apetala2 (AP2) transcription factor. Because cleistogamy is not apparent in cultivars of hexaploid wheat (Triticum aestivum), a strategy to develop cleistogamous wheat was proposed by inducing point mutations in all three AP2 homoeologs, which are the wheat orthologs of barley Cly1. In this study, we investigated the effects of miR172 target site mutations on wheat cleistogamy using double mutants by combining three previously obtained mutant alleles (AP2-A1, D1 and D2) in a near-isogenic background. The AP2-D2 allele had the greatest effect on reducing the anther extrusion rate and lodicule size compared with the other two mutant alleles. The double mutant containing the AP2-A1 and AP2-D2 alleles had a much greater suppression of anther extrusion by reducing the lodicule size than the single AP2-D2 mutant, suggesting cumulative effects of the two mutant alleles. In addition, both single and double mutants exhibited compact spikes and shorter plant heights due to reduced rachis and culm internodes in the upper parts. The presence or absence of the wild-type AP2-B homoeolog had no significant effect on phenotype. This study provides insights into the cumulative effects of mutant AP2 alleles in suppressing open flowering and provides a basis for further research on the development of complete cleistogamy in hexaploid wheat.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s11032-024-01458-9.

Plants display various forms of phenotypic plasticity in anticipation of changing conditions, many of which are influenced by information obtained from neighbouring plants. Here, we tested the hypothesis that cleistogamic Lamium amplexicaule plants can adaptively modify production of chasmogamous (CH) and cleistogamous (CL) flowers based on the perception of conspecific neighbours. The production and proportion of CH and CL flowers was examined in individual L. amplexicaule grown at varying densities or treated with root leachates from plants grown at different densities. When growing at high density or treated with root leachates from high-density pots, L. amplexicaule increased production of more expensive, potentially outcrossing CH flowers. In contrast, single plants or plants treated with root leachates from empty pots or single-source plants predominantly developed cheaper, self-pollinated CL flowers. The results demonstrate a novel root-based neighbour-perception modality that enables plants to adaptively adjust production of CH and CL flowers in response to the presence of potential reproductive partners. Further research is needed to explore the broader ecological implications of this novel interplant cueing on reproductive bet-hedging and plasticity in natural settings, as well as to identify the involved cues and their mode of operation.

Closed fertilization in flowers, or cleistogamy, reduces the risk of fungal infection in Triticeae crops. In barley (Hordeum vulgare), cleistogamy is determined by a single recessive gene, cly1, which results from a single nucleotide polymorphism within the microRNA172 target site of the Apetala2 (AP2) transcription factor gene. The recessive cly1 allele negatively regulates the development of lodicules, keeping florets closed at anthesis. However, cleistogamy is not evident in hexaploid wheat (Triticum aestivum) cultivars. This study aimed at identifying mutations in wheat AP2 orthologs by ethyl methane sulfonate-induced mutagenesis and high-resolution melt analysis. Although flowers of AP2 mutants induced in the A and D genomes opened at anthesis, their lodicule size was significantly smaller, especially in the direction of depth, than that of wild-type plants. One of the mutants that carried a nucleotide replacement in AP2 from the D genome produced a compact spike caused by a substantial decrease in rachis internode length, analogous to the barley dense spike. Cleistogamous hexaploid wheat might be generated by combining effective mutant alleles of AP2-homoeologous genes.

What maintains mixed mating is an evolutionary enigma. Cleistogamy-the production of both potentially outcrossing chasmogamous and obligately selfing cleistogamous flowers on the same individual plant-is an excellent system to study the costs of selfing. Inbreeding depression can prevent the evolution of greater selfing within populations, and heterosis in crosses between populations may further tip the balance in favor of outcrossing. Few empirical estimates of inbreeding depression and heterosis in the same system exist for cleistogamous species.
We investigate the potential costs of selfing by quantifying inbreeding depression and heterosis in three populations of the cleistogamous perennial Ruellia humilis Nutt (Acanthaceae). We performed three types of hand-pollinations-self, outcross-within, and outcross-between populations-and measured seed number, germination, total flower production, and estimated cumulative fitness for the resulting progeny in a greenhouse experiment.
We found moderate inbreeding depression for cumulative fitness (<30%) in two populations, but outbreeding depression for crosses within a third population (-26%). For between-population crosses, there was weak to modest heterosis (11-47%) in two of the population combinations, but modest to strong outbreeding depression (-21 to -71%) in the other four combinations.
Neither inbreeding depression nor heterosis was of sufficient magnitude to explain the continued production of chasmogamous flowers given the relative energetic advantage of cleistogamous flowers previously estimated for these populations. Outbreeding depression either within or between populations makes the maintenance of chasmogamous flowers even harder to explain. More information is needed on the genetic basis of cleistogamy to resolve this conundrum.
Lo que mantiene los sistemas de apareamiento mixto aún es un enigma. La cleistogamia, la producción de flores con potencial de cruzamiento casmógamas, y de flores cleistógamas obligadamente autofecundadas en la misma planta, es un excelente sistema para estudiar los costos de la autofecundación. La depresión endogámica puede prevenir la evolución hacia una mayor autofecundación dentro de las poblaciones, y la heterosis de los cruces entre poblaciones puede inclinar aún más la balanza a favor del cruzamiento. Existen pocas estimaciones empíricas de depresión endogámica y heterosis en el mismo sistema para especies cleistógamas. MÉTODOS: Investigamos los costos potenciales de la autofecundación cuantificando la depresión endogámica y la heterosis en tres poblaciones de la perenne cleistógama Ruellia humilis Nutt (Acanthaceae). Realizamos autopolinizaciones manuales, y cruces dentro y entre poblaciones, Medimos el número de semillas, la germinación, la producción total de flores y estimamos la acumulación de fitness para la progenie resultante en un experimento de invernadero.
Encontramos depresión endogámica moderada para fitness acumulado (<30%) en dos poblaciones, pero depresión exogámica para cruces dentro de la tercera población (-26%). Entre cruces de población, hubo heterosis de débil a modesta (11-47%) en dos de las combinaciones de poblaciones, pero depresión exogámica moderada a fuerte (-21 a -71%) en las otras cuatro combinaciones.
Ni la depresión endogámica, ni la heterosis fueron de suficiente magnitud para explicar la producción continua de flores casmógamas dada la ventaja energética relativa de las flores cleistógamas previamente estimadas para estas poblaciones. La depresión exogámica, ya sea dentro o entre poblaciones, hace que el mantenimiento de las flores casmógamas sea aún más difícil de explicar. Se necesita más información sobre la base genética de la cleistogamia para resolver este enigma.

Crop improvement has focused on enhancing yield, nutrient content, harvestability, and stress resistance using a trait-centered reductionist approach. This has downplayed the fact that plants are developmentally integrated and respond coordinately and predictably to genetic and environmental variation, with potential consequences for food production. Crop yield, including both fruit/seed production and the possibility of generating hybrid crop varieties, is highly dependent on flower morphology and sex, which, in turn, can be profoundly affected by slight shifts in the timing and rate of flower organ development (i.e., flower heterochrony). We argue that understanding the genetic and environmental bases of flower heterochrony and their effect on flower morphology and sex in cultivated plants and in their wild relatives can facilitate crop improvement.

Environmental conditions during seed development and maturation can affect seed traits and germination behavior, yet systematic research on the effects of seed maturation time on seed traits, germination behavior and seedling emergence of cleistogamy plants is lacking. Here, we determined the difference in phenotypic characteristics of CH and CL (namely CL1, CL2 and CL3 based on maturation time, respectively) fruits/seeds that were collected from Viola prionantha Bunge, a cleistogamous perennial plant, and evaluated the effects of various environmental factors on seed germination and seedling emergence. The fruit mass, width, seed number per fruit and mean seed mass of CL1 and CL3 were greater than that of CH and CL2, while seed setting of CH was lower than that of CL1, CL2 and CL3. Germination of CH, CL1, CL2, and CL3 seeds was < 10% in the dark at 15/5 and 20/10 ℃, whereas germination (0%-99.2%) of CH, CL1, CL2, and CL3 seeds changed significantly under light conditions. In contrast, more than 71% (from 71.7 to 94.2%) germination of both CH, CL1, CL2 and CL3 seeds occurred under both light/dark conditions and continuous darkness at 30/20 ℃. Germination of CH, CL1, CL2 and CL3 seeds was sensitive to osmotic potential, but CL1 seeds were more resistant to osmotic stress, compared with CH, CL2 and CL3. Seedling emergence of CH seeds was more than 67% (from 67.8 to 73.3%) at a burial depth of 0 cm-2 cm, while all types of CL seeds were below 15% at a burial depth of 2 cm. Information gathered from this study indicates that CH and CL seeds of V. prionantha were different in fruit size, seed mass, thermoperiod and photoperiod sensitivity, osmotic potential tolerance and seedling emergence, especially, maturation time significantly affect phenotypic characteristics and germination behavior of CL seeds matured at different periods. These results indicate that V. prionantha adapts to unpredictable environmental conditions by developing a variety of adaptation strategies, and ensures the survival and reproduction of the populations.